A century ago, Alfred Nissle discovered that intentional intake of particular strains of Escherichia coli could treat patients suffering from infectious diseases. Since then, one of these strains became the most frequently used probiotic E. coli in research and was applied to a variety of human conditions. Here, properties of that E. coli Here, Escherichia coli Nissle 1917 (ECN), a kind of oral probiotic, was genetically engineered to overexpress catalase and superoxide dismutase (ECN-pE) for the treatment of intestinal inflammation. To improve the bioavailability of ECN-pE in the gastrointestinal tract, chitosan and sodium alginate, effective biofilms, were used to coat ECN-pE In 1917, as war was tearing its way across Europe, a fascinating scientific observation was being made. to isolate a strain of bacteria that came to be known as Escherichia coli Nissle 1917 Escherichia coli strain Nissle 1917 has been widely used as a probiotic for the treatment of inflammatory bowel disorders and shown to have immunomodulatory effects. Nissle 1917 expresses a K5 capsule, the expression of which often is associated with extraintestinal and urinary tract isolates of E. … Escherichia coli Nissle 1917 (Mutaflor) is one of the most investigated probiotic bacteria. While the number of reports discussing the underlying mechanisms of Mutaflor has increased rapidly in recent years, novel clinical studies are missing. Escherichia coli Nissle 1917 (EcN) is a genetically controlled probiotic with an excellent human safety record for improving gut microbiome metabolic disorders and immune system disorders. Here we focused to explore the application and effect of probiotic EcN on the gut microbiota-metabolism-IL-22-mitochondrial damage axis in PCOS. However, E. coli Nissle 1917 (EcN), the unique probiotic strain, has seldom been precisely adapted to the T7 sys … Lac operon is the standard regulator used to control the orthogonality of T7RNA polymerase (T7RNAP) and T7 promoter inEscherichia coli BL21(DE3) strain for protein expression. Patients and methods: In a post-marketing-surveillance study with the probiotic Escherichia Coli strain Nissle 1917 (EcN) data on the range of indications as well as on efficacy and tolerance were gathered prospectively in 446 centres. The intended treatment duration was limited to a maximum of 12 weeks. Escherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. Nonpathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1) is used as a probiotic agent in medicine, mainly for the treatment of various gastroenterological diseases. To gain insight on the genetic level into its properties of colonization and commensalism, this strain's genome structure has been … nrr5xC. Loading metrics Open Access Peer-reviewed Research Article Francine C. Paim, Ayako Miyazaki, Stephanie N. Langel, David D. Fischer, Juliet Chepngeno, Steven D. Goodman, Gireesh Rajashekara, Linda J. Saif , Anastasia Nickolaevna Vlasova Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota Husheem Michael, Francine C. Paim, Ayako Miyazaki, Stephanie N. Langel, David D. Fischer, Juliet Chepngeno, Steven D. Goodman, Gireesh Rajashekara, Linda J. Saif, Anastasia Nickolaevna Vlasova x Published: February 16, 2021 Figures AbstractHuman rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in low- and middle-income countries (LMICs), where HRV vaccine efficacy is low. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of enteric diseases in humans. However, repeated doses of EcN are required to achieve maximum beneficial effects. Administration of EcN on a microsphere biofilm could increase probiotic stability and persistence, thus maximizing health benefits without repeated administrations. Our aim was to investigate immune enhancement by the probiotic EcN adhered to a dextranomar microsphere biofilm (EcN biofilm) in a neonatal, malnourished piglet model transplanted with human infant fecal microbiota (HIFM) and infected with rotavirus. To create malnourishment, pigs were fed a reduced amount of bovine milk. Decreased HRV fecal shedding and protection from diarrhea were evident in the EcN biofilm treated piglets compared with EcN suspension and control groups. Moreover, EcN biofilm treatment enhanced natural killer cell activity in blood mononuclear cells (MNCs). Increased frequencies of activated plasmacytoid dendritic cells (pDC) in systemic and intestinal tissues and activated conventional dendritic cells (cDC) in blood and duodenum were also observed in EcN biofilm as compared with EcN suspension treated pigs. Furthermore, EcN biofilm treated pigs had increased frequencies of systemic activated and resting/memory antibody forming B cells and IgA+ B cells in the systemic tissues. Similarly, the mean numbers of systemic and intestinal HRV-specific IgA antibody secreting cells (ASCs), as well as HRV-specific IgA antibody titers in serum and small intestinal contents, were increased in the EcN biofilm treated group. In summary EcN biofilm enhanced innate and B cell immune responses after HRV infection and ameliorated diarrhea following HRV challenge in a malnourished, HIFM pig model. Citation: Michael H, Paim FC, Miyazaki A, Langel SN, Fischer DD, Chepngeno J, et al. (2021) Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota. PLoS ONE 16(2): e0246193. Nicholas J. Mantis, New York State Department of Health, UNITED STATESReceived: October 18, 2020; Accepted: January 14, 2021; Published: February 16, 2021Copyright: © 2021 Michael et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are Availability: All relevant data are within the manuscript and its Supporting Information filesFunding: This work was supported by the Bill and Melinda Gates Foundation (OPP 1117467), the NIAID, NIH (R01 A1099451), federal and state funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University and from the NIH Office of Dietary Supplements (ODS) supplemental grant interests: The authors have declared that no competing interests exist. IntroductionHuman rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in developing countries [1]. Malnutrition is a major contributor of high mortality due to viral gastroenteritis, including HRV, in countries with low socioeconomic status [2–4]. A number of studies have shown that malnutrition triggers immune dysfunction, including altered innate and adaptive immune responses, impairment of epithelial cell barrier function and/or dysfunction of intestinal epithelial cells [5–10]. Probiotics are increasingly used to enhance oral vaccine responses and to treat enteric infections [11] and ulcerative colitis in children [12]. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of ulcerative colitis in humans [13]. EcN lacks virulence factors and possesses unique health-promoting properties [14]. The long term persistence of EcN in humans suggests adaption to a host with an established gut microbiome [15]. Our research group has shown that EcN protected gnotobiotic (Gn) piglets against HRV infection and decreased the severity of diarrhea by modulating innate and adaptive immunity, and protecting the intestinal epithelium [16–18]. Oral administration of probiotics is associated with a number of challenges, such as low pH of gastric acid and bile salts in the stomach, effector functions of the host immune system, and competition with commensal and pathogenic bacteria [19]. These factors adversely influence adherence and persistence of probiotics within the host and thus reduce the beneficial effects [20]. Probiotics must survive in gastric acids to reach the small intestine and colonize the host to confer beneficial effects of preventing or moderating gastrointestinal diseases [21]. Encapsulation of lyophilized probiotics have resulted in enhanced bacterial viability [22, 23]. Navarro and his colleagues (2017) have formulated a new synbiotic formulation that employed porous semi-permeable, biocompatible and biodegradable microspheres (dextranomer microspheres) containing readily diffusible prebiotic cargo [24]. Adherence of the probiotic bacteria to the microsphere has a two-fold effect; it facilitates the more formidable biofilm state of probiotics as well as a creates a directed means to provide a high concentration gradient of prebiotics via diffusion of the microsphere cargo. However, currently there are no strategies for improved EcN probiotic efficacy and stability within the malnourished host. Previously we have established a deficient HIFM-transplanted neonatal pig model that recapitulates major aspects of malnutrition seen in children in impoverished countries [5, 6]. The purpose of this study was to investigate a novel probiotic delivery method to prolong the persistence of probiotics in the gut and to enhance their beneficial effects. We hypothesized that oral administration of EcN attached to the surface of biocompatible dextranomar microspheres in a biofilm state will protect against harsh conditions of the stomach and improve gut stability, thus enhancing their beneficial effects with a single administration compared with the repetitive administration of probiotics in the suspension form, which results in transient and often inconsistent outcomes. In addition, administration of probiotics in their suspension state has modest impact on the host’s microbiome [25]. High doses and repeated administration of probiotics are needed to achieve potential health benefits; however, in impoverished countries this poses challenges due to lack of product availability, the limited health care system, and resources [26–28]. Whether the use of the biofilm microsphere can overcome this remains to be established. The multifactorial pathobiology of malnutrition is associated with a vicious cycle of intestinal dysbiosis, epithelial breaches, altered metabolism, impaired immunity, intestinal inflammation, and malabsorption [29, 30]. Malnutrition increases the risk of diarrheal diseases caused by some, but not all, entero-pathogens. Malnutrition can result in impaired immune defenses that compromise gut integrity, and dybiosis that can influence defense against intestinal pathogens in the malnourished host [31]. This in turn limits the ability of probiotics to repair the intestinal epithelium and establish healthy microbiota. These concerns necessitate further research to enhance the stability and persistence of probiotics in malnourished hosts. Probiotics are generally considered safe, however there are some associated risks. These risks are increased if there are chronic medical conditions that weaken the immune system or if there are gut barrier breeches. Possible risks can include: developing an infection, developing resistance to antibiotics, and developing harmful byproducts from the probiotic supplement. Also, in malnourished hosts due to increased intestinal motility, probiotics can be eliminated from the gut faster limiting their beneficial effects [32, 33]. Furthermore, we aimed to investigate whether a single dose of EcN biofilm microspheres enhances immune responses after HRV infection in a malnourished Gn pig model. Previous transplantation of Gn pigs with probiotic bacteria demonstrated upregulated innate and adaptive immune responses following HRV infection [16, 17, 34–37]. In this preliminary study, we report increased innate immune and B cell responses after EcN biofilm treatment that were associated with protection against HRV disease and infection in a neonatal malnourished, HIFM pig model. Materials and methods Human Infant Fecal Microbiota (HIFM) The collection and use of HIFM was approved by The Ohio State University Institutional Review Board (IRB). With parental consent, sequential fecal samples were collected from a healthy, two-month-old, exclusively breastfed, vaginally delivered infant. Samples were pooled and diluted to 1:20 (wt/vol) in PBS containing (vol/vol) cysteine and 30% glycerol and stored at -80°C as described previously [5, 6]. Virus HRV (VirHRV) Wa strain passaged 25–26 times in Gn piglets was used to orally inoculate piglets at a dose of 1 × 106 fluorescent focus units (FFU) as described previously [5, 6]. Preparation of biofilm dextrananomer microspheres Anhydrous dextranomer microspheres (Sephadex, GE Healthcare Life Sciences, Pittsburgh, PA) were used. Anhydrous microspheres were hydrated in growth medium at 50 mg per ml and autoclaved for 20 min. Autoclaved microspheres were removed from solution on a vacuum filter apparatus and collected via sterile loop into a filter-sterilized 1M solution of sucrose. The microsphere mixture was vortexed and incubated for 24 hours at room temperature (RT). Sugar was removed from solution on a vacuum filter apparatus and collected via sterile loop. The microspheres were then added to EcN [1 × 109 colony-forming unit (CFU) per ml], pelleted, washed, and re-suspended in sterile saline. EcN was allowed to incubate with the microspheres for 1h at RT to facilitate binding and stored in -80°C in 30% glycerol. Prior to use, microspheres were thawed, mixed 1:1 with Natrel and administered orally. For EcN administered as a suspension, 1 × 109 CFU per ml was pelleted and re-suspended in sterile saline in preparation for oral inoculation. Animal experiments The animal experiments were approved by the Institutional Animal Care and Use Committee at The Ohio State University (OSU). Piglets were derived from near-term sows (purchased from OSU specific pathogen-free swine herd) by hysterectomy and maintained in sterile isolators as described previously [38]. For preliminary investigations, neonatal pigs were randomly assigned to three groups: 1) EcN biofilm (n = 3); 2) EcN suspension (n = 4); and 3) control pigs (n = 3). Pigs were fed a deficient diet of 50% ultra-high temperature pasteurized bovine milk diluted with 50% sterile water which contained half of the recommended protein levels ( that met or exceeded the National Research Council Animal Care Committee’s guidelines for calories, fat, protein and carbohydrates in suckling pigs. All pigs were confirmed free from bacterial and fungal contamination prior to HIFM transplantation by aerobic and anaerobic cultures of rectal swabs. Pigs were orally inoculated with 2ml of diluted HIFM stock at 4 days of age (post-HIFM transplantation day, PTD 0). The pigs were colonized orally with EcN biofilm or EcN suspension at PTD 11. Pigs were then challenged with VirHRV [1 × 106 FFU, post challenge day (PCD) 0] at PTD 13 and euthanized at PTD 27/PCD 14. Post-VirHRV challenge, rectal swabs were collected daily to assess HRV shedding. Blood, spleen, duodenum, and ileum were collected to isolate mononuclear cells (MNCs) as described previously (31, 35, 36). Jejunum was collected to isolate intestinal epithelial cells (IECs) using modified protocols [18, 39–41]. Serum and small intestinal contents (SIC) were collected to determine the HRV specific and total antibody responses [6, 17, 34, 42, 43]. Assessment of clinical signs and detection of HRV shedding Rectal swabs were collected daily post-VirHRV challenge. Fecal consistency was scored as follows; 0, normal; 1, pasty; 2, semi-liquid; and 3, liquid, and pigs with fecal score more than 1 were considered as diarrheic. Rectal swabs were suspended in 2 ml of minimum essential medium (MEM) (Life technologies, Waltham, MA, USA), clarified by centrifugation for 800 × g for 10 minutes at 4°C, and stored at -20°C until quantification of infectious HRV by a cell culture immunofluorescence (CCIF) assay as previously described [44]. Isolation of mononuclear cells (MNCs) Systemic (blood and spleen) and intestinal (duodenum and ileum) tissues were collected to isolate MNCs as described previously [36, 45, 46]. The purified MNCs were re-suspended in E-RPMI 1640. The viability of each MNCs preparation was determined by trypan blue exclusion (≥95%). Flow cytometry analysis Freshly isolated MNCs were stained to assess frequencies of conventional dendritic cells (DCs) (cDCs, SWC3a+CD4-CD11R1+) and plasmacytoid DCs (pDCs, SWC3a+CD4+CD11R1-), MHC II and CD103 marker expression on DCs were used in our experiments. Frequencies of IgA+ B lymphocytes were determined by identifying CD79β and IgA expression in MNCs as reported previously [34]. Similarly, frequencies of memory/resting (CD79β+CD2-CD21-) and activated (CD79β+CD2+CD21-) B cells among systemic and intestinal MNCs were determined as described previously [34]. Appropriate isotype matched control antibodies were included. Subsequently, 50,000 events were acquired per sample using BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using C6 flow sampler software. NK cytotoxicity assay Total blood MNCs and K562 cells were used as effector and target cells, respectively. Effector: target cell ratios of 10:1, 5:1, 1:1 and were used and the assay was done as described previously [47, 48]. HRV-specific and total antibody responses The HRV specific and total antibody titers in serum and SIC were detected by enzyme-linked immunosorbent assay (ELISA) as described previously [6, 17, 34, 42, 43]. To determine the intestinal antibody responses, small intestinal contents (SIC) were collected with protease inhibitors in the medium. HRV-specific Antibody Secreting Cells (ASCs) responses HRV and isotype-specific antibody secretion in MNCs isolated from blood, spleen, duodenum and ileum were analyzed by ELISPOT assay as described previously [17, 34, 42, 43]. Isolation of Intestinal Epithelial Cells (IECs) and extraction of RNA The IECs were isolated from jejunum (mid gut) using a modified protocol adapted from Paim et al. [18, 49]. The viability and numbers of IECs were determined by the trypan blue exclusion method (70–80%). IECs were stored at −80°C in 500 μl of RNAlater tissue collection buffer (Life technologies, Carlsbad, CA, USA) until further analysis. Total RNA from IECs was extracted using Direct-Zol RNA Miniprep (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. The RNA concentrations and purity were measured using NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Real-time quantitative RT-PCR (qRT-PCR) of CgA, MUC2, PCNA, SOX9 and villin gene mRNA levels in Intestinal Epithelial Cells (IECs) qRT-PCR was performed using equal amounts of total RNA (75 ng) with Power SYBR Green RNA-to-CT 1 step RT-PCR kit (Applied Biosystems, Foster, CA, USA). The primers for enteroendocrine cells chromogramin A (CgA), goblet cells mucin 2 (MUC2), transient amplifying progenitor cells proliferating cell nuclear antigen (PCNA), intestinal epithelial stem cells transcription factor SRY-box9 (SOX9), enterocytes (villin) and β-actin were based on previously published data [18, 39–41]. Relative gene expression of CgA, MUC2, PCNA, SOX9 and villin were normalized to β-actin and expressed as fold change using the 2-ΔΔCt method [50]. Statistical analysis All statistical analyses were performed using GraphPad Prism version 6 (GraphPad software, Inc., La Jolla, CA). Log10 transformed isotype ELISA antibody titers that were analyzed using one-way ANOVA followed by Duncan’s multiple range test. Data represent the mean numbers of HRV specific antibody secreting cells per 5 × 105 mononuclear cells and analyzed using non-parametric t-test (Mann-Whitney). HRV shedding and diarrheal analysis were performed using two way ANOVA followed by Bonferroni posttest. *P values < **P values < and ***P values < Error bars indicate the standard error of mean. Results EcN biofilm treatment reduced fecal HRV shedding and protected malnourished pigs from diarrhea post HRV challenge Analysis revealed that EcN biofilm treated malnourished pigs had shorter and delayed onset of HRV shedding as compared with the EcN suspension and the control group pigs (Table 1). A significant reduction in fecal virus peak titers shed was observed both in EcN biofilm (GMT = FFU/ml) and EcN suspension groups (GMT = FFU/ml), as compared with the control pigs (GMT = FFU/ml). In addition, EcN biofilm and EcN suspension groups had decreased peak shedding titers at PCD 2 as compared with that of control pigs (S1 Fig). EcN biofilm treatment shortened the mean duration of viral shedding to days as compared with and days in EcN suspension treated and control pigs, respectively (Table 1). Control pigs developed diarrhea ( at days post HRV challenge, continuing for days with mean cumulative fecal score (Table 1). Single administration of EcN biofilm microspheres completely protected the pigs from diarrhea (Table 1). However, administration of EcN suspension protected only 50% of the pigs from diarrhea. No significant differences were observed for mean days to diarrheal onset ( days), mean cumulative fecal score ( and the mean duration of diarrhea ( days) when they are compared with those in the control group (Table 1). These findings suggest that administration of EcN biofilm suppressed HRV infection greater than EcN administered in suspension. EcN biofilm treatment enhanced natural killer (NK) cell cytotoxicity in blood mononuclear cells (MNCs), increased the frequencies of activated pDCs in systemic and intestinal tissues, and increased activated cDCs in the blood and duodenum NK cell cytotoxicity in blood MNCs was significantly enhanced in EcN biofilm treatment compared with control pigs (Fig 1A). On the other hand, frequency of apoptotic MNCs were marginally decreased in EcN biofilm (3%) compared with EcN suspension (5%) and control ( pigs in blood (S2 Fig). Fig 1. EcN biofilm enhanced NK cell activity in blood mononuclear cells (MNCs) and significantly increased the frequencies of activated pDCs in systemic and intestinal tissues and increased activated cDCs in blood and duodenum (significantly).(a) Blood MNCs and carboxyfluorescein diacetate succinimidyl ester (CFSE) stained K562 tumor cells were used as effector and target cells, respectively, and co-cultured at set ratios to assess the NK cytotoxic function, (EcN biofilm vs control group). The effector: target cell co-cultures were stained with 7-Aminoactinomycin D (7AAD) after 12 hours of incubation at 37°C, and the frequencies of CFSE-7AAD double positive cells (lysed K562 target cells) were assessed by flow cytometry. Mean frequencies of activated (b) pDCs and (c) cDCs in systemic and intestinal tissues. Data represent means ± SEM. Significant differences (*p < **p < ***p < are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to the respective groups at PTD 11, followed by challenge with virulent human rotavirus (HRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. biofilm treatment significantly increased the frequencies of activated pDC in systemic and intestinal tissues as compared with EcN suspension and the control pigs (Fig 1B). Moreover, EcN biofilm treatment significantly increased the frequencies of activated cDC in duodenum while numerically in blood (Fig 1C). There were no differences observed in other tissues. CD103+ cDC were increased (numerically) in spleen and intestinal tissues in EcN biofilm treated group as compared with EcN suspension and control pigs (S3 Fig). There were no differences observed in blood. EcN biofilm treatment significantly increased the frequencies of activated antibody secreting B cells in systemic tissues, resting antibody forming B cells in blood, and IgA+ B cells in spleen EcN biofilm treated malnourished pigs had significantly increased frequencies of activated antibody forming B cells in systemic tissues as compared with EcN suspension or the control pigs (S4A and S4B Fig). The frequency of IgA+ B cells in the spleen (significantly, S4C Fig) and blood (numerically, S4D Fig) increased in EcN biofilm treatment compared with EcN suspension and control pigs. Moreover, the frequency of resting/memory antibody forming B cells was significantly increased in blood in EcN biofilm compared with EcN suspension treated pigs (S4E Fig). These findings suggest that EcN biofilm treatment enhanced B cell immune response in systemic tissues, although no significant trends were observed in intestinal tissues. EcN biofilm treatment increased the number of HRV-specific Antibody Secreting Cells (ASCs) in systemic and intestinal tissues, and increased HRV-specific IgA antibody titers in serum and Small Intestinal Contents (SIC) Coinciding with decreased HRV shedding and protection from diarrhea, the mean numbers of HRV-specific IgA ASCs were increased in systemic and intestinal tissues of EcN biofilm treatment compared with EcN suspension and control group pigs (Fig 2A and 2B). A similar trend was observed with HRV-specific IgG ASCs (S5 Fig). HRV-specific IgM ASC numbers were below the detection limit in systemic and intestinal tissues. HRV-specific IgA antibody titers were increased in serum (significantly) and SIC (numerically) of EcN biofilm treated pigs compared with EcN suspension and control group pigs, coinciding with increased HRV-specific IgA ASCs (Fig 2C and 2D). Similar trends were observed with HRV-specific IgG antibody titers in serum (S6 Fig). In addition, total IgA concentration was increased (numerically) in serum samples of EcN biofilm treated pigs compared with EcN suspension or control group pigs (S7 Fig). No significant trends were observed in total and HRV-specific IgG in SIC (S8 Fig). These results indicate that EcN biofilm treatment enhanced B cell formation and clonal expansion of antibody producing cells in malnourished, HIFM transplanted pigs infected with HRV. Fig 2. EcN biofilm significantly increased HRV-specific IgA Antibody Secreting Cells (ASCs) in systemic and intestinal tissues and increased HRV-specific IgA antibody titers in serum and Small Intestinal Contents (SIC).(a) HRV-specific IgA ASCs in systemic cells; (b) HRV-specific IgA ASCs in intestinal cells; (c) HRV-specific IgA antibody titers in serum and (d) SIC. No significant differences were observed in intestinal tissues. Data represent means ± SEM. Significant differences (*p < **p < ***p < are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to respective groups at PTD 11, followed by challenge with virulent human rotavirus (HRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. EcN biofilm treatment significantly upregulated the expression of CgA and SOX9 mRNA levels in jejunal epithelial cells Gene expression levels of CgA, SOX9, villin, MUC2, and PCNA were assessed from jejunal epithelial cells. The relative mRNA levels of CgA, SOX9, and villin genes were increased in jejunal epithelial cells of EcN biofilm compared with EcN suspension and control treated malnourished pigs (Fig 3A–3C). This coincided with the decreased severity of HRV shedding and diarrhea. There were no differences in gene expression levels for MUC2 and PCNA in jejunal epithelial cells of EcN biofilm and EcN suspension treated pigs (S9 Fig). Fig 3. EcN biofilm upregulated the expression of various cell components in jejunal epithelial cells.(a) Relative mRNA levels of enteroendocrine cells chromogramin A (CgA), (b) intestinal epithelial stem cells (SOX9), and (c) enterocytes (villin) in EcN biofilm, EcN suspension groups measured by real-time quantitative RT-PCR (RT-PCR), normalized to β-actin gene. Graphs represent means ± SEM. Significant difference (*p < **p < relative to control) are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to respective groups at PTD 11, followed by challenge with virulent human rotavirus (VirHRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. DiscussionUsing a malnourished and HIFM transplanted pig model, we showed that compared with EcN administered as suspension, EcN administered as a biofilm on dextranomer microspheres enhanced multiple aspects of the immune response. EcN biofilm treated pigs had significantly reduced titers of virus shedding and diarrhea following VirHRV challenge compared with EcN suspension treated and control pigs. The presence of HRV-specific IgA antibodies in pigs is strongly correlated with protection from HRV infection [46, 51, 52]. Moreover, our study demonstrated for the first time that EcN biofilm treatment enhanced HRV specific-IgA and IgG ASCs in circulation and gut, enhanced HRV-specific IgA and IgG antibody titers in serum and HRV-specific IgA antibody titers in SIC, which collectively coincided with reduced diarrhea and virus shedding. Total IgA concentration was marginally increased in serum of EcN biofilm treated malnourished pigs (data not shown). Although not examined in this study, EcN biofilm treatment might have increased colonization in the gut, inhibiting competition by other members of the gut microbiota [53, 54]. It is possible that the observed effects of EcN biofilm treatment on systemic IgA responses could be mediated by direct modulation of host immune responses, suggesting that biofilm microspheres maybe more stable and persistent compared to probiotics in suspension in the host’s gastrointestinal system. Innate immune responses are critical as a first line of defense, limiting RV replication and disease severity in the host [16, 55]. EcN biofilm treatment enhanced innate immune responses. For example, blood NK cell cytotoxicity was higher in EcN biofilm treatment compared to EcN suspension treated and control groups. This suggests that EcN as a biofilm promoted innate immune responses, improving protection against HRV infection in vivo. Also the frequency of apoptotic blood MNCs was slightly reduced in EcN biofilm treated pigs compared with EcN suspension treatment and control pigs (data not shown). DCs play a key role in probiotic bacteria stimulation of the innate immune system [56, 57] and pDCs were shown to contribute to RV clearance in a murine model [58]. Moreover, DC MHC II expression is a marker for maturation [59]. In our study, higher frequencies of activated pDCs in systemic and intestinal tissues and activated cDCs in the blood and duodenum were observed in EcN biofilm treated pigs compared with EcN suspension treated piglets. These results suggest that the biofilm provided stability to the probiotic and thus enhanced maturation of systemic and intestinal activated DC, promoting pDC development and increased IgA antibody responses in probiotic biofilm treated piglets compared with probiotic suspension treated pigs [60, 61]. Enhancing the protective effects of pDCs via an EcN biofilm may be critical for protection against enteric pathogens [16]. Expression of CD103 (αEβ7 integrin) has been demonstrated to influence cellular intraepithelial morphogenesis and motility [62], which are critical for the proper communication among pathogen, DCs, and T and B lymphocytes. We observed that EcN biofilm treatment increased CD103 expression by DCs and this could have further enhanced innate immune responses against HRV and reduced HRV infection. Consequently, enhancement of signaling between DCs and T/B lymphocytes could have contributed to improved antigen presentation to the lymphocytes resulting in increased HRV-specific IgA ASCs, IgA antibody titers, and increased NK cell activity in EcN biofilm treated pigs. The increased frequencies of activated and resting/memory B cells were enhanced in EcN biofilm treated pigs that coincided with increased frequencies of pDCs in the intestine. These results are similar to our previous studies where EcN protected against HRV infection [34, 37]. The frequency of IgA+ B cells were increased in EcN biofilm treated pigs in systemic tissues, suggesting that EcN as a biofilm may potentiate systemic IgA responses. These responses and the increased HRV-specific IgA antibody responses in serum and SIC coincided with reduced HRV diarrhea and shedding. An upregulation of the enteroendocrine CgA gene in EcN biofilm treated piglets could be reflective of greater protection of the epithelial intestinal barrier. Other studies have shown that enteroendocrine cells that produce hormones promoting repair of intestinal epithelium are activated after treatment with probiotics [63, 64]. In our investigations, we observed an upregulation of stem cell specific-gene SOX9 in the EcN biofilm treated pigs greater than in EcN suspension treated pigs. SOX9 plays an important role in the proliferative capacity of stem cells to replenish different lineages of IECs [65]. Moreover, we demonstrated that EcN biofilm treatment increased mRNA levels of the enterocyte-specific gene villin. It is likely that biofilm microspheres supported a greater number of villin cells and epithelial cells and probiotic adherence. This likely modulated the effects of HRV infection by increasing villin gene expression of enterocytes, repairing/restoring functional enterocytes and increasing barrier and absorptive functions during HRV-induced diarrhea. Our results suggest that using a microsphere biofilm as a novel delivery system for EcN compared to EcN as a suspension may have increased survival of the probiotics at low pH in the stomach and supported increased adherence to intestinal epithelial cells [24], thereby promoting probiotic longevity, survival, and persistence in the malnourished host. Additionally, the EcN biofilm enhanced innate and B cell immune responses in the HRV infected HIFM neonatal pigs. Our results support previous work demonstrating protection against experimental necrotizing enterocolitis in a rat model after treatment with Lactobacillus reuteri adhered to dextranomer microspheres [66]. Recently, Shelby et al. 2020 and colleagues have demonstrated that a single dose of Lactobacillus reuteri in its biofilm state reduces the severity and incidence of experimental C. difficile infection and necrotizing enterocolitis when administered as both prophylactic and treatment therapy [67, 68]. Moreover, Navarro and colleagues demonstrated that probiotic bacterium L. reuteri delivered in association with dextranomar microspheres adhered in greater numbers, conferred resistance to clearance, transported nutrients that promote bacterial growth, promoted the production of the antimicrobial reuterin or histamine, resisted acid-mediated killing, and better supported adherence to intestinal epithelial cells, thereby promoting persistence in the gut [24]. Thus, we this agreed with our hypothesis that EcN adhered to dextranomer microspheres acted similarly during HRV infection in the neonatal malnourished HIFM pig model. In the future, we have plan to increase to number of piglets and study different age groups to further investigate the biofilm impact. Thus, our results suggest that low cost, stable, and efficient dietary supplementation of EcN coupled with a dextranomer microsphere biofilm can protect against HRV infection in a physiologically relevant malnourished HIFM pig model. Similar studies are warranted in children to moderate the symptoms of other gastrointestinal infections and disorders including gastritis and chronic inflammatory bowel disease. Supporting information Acknowledgments We thank Marcia Lee and Rosario Candelero-Rueda for their technical assistance and Dr. Juliette Hanson, Ronna Wood, Jeffery Ogg, Megan Strother and Sara Tallmadge for animal care assistance. References1. Tate JE, Burton AH, Boschi-Pinto C, Steele AD, Duque J, et al. (2012) 2008 estimate of worldwide rotavirus-associated mortality in children younger than 5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and meta-analysis. Lancet Infect Dis 12: 136–141. pmid:22030330 View Article PubMed/NCBI Google Scholar 2. Clark A, Black R, Tate J, Roose A, Kotloff K, et al. (2017) Estimating global, regional and national rotavirus deaths in children aged <5 years: Current approaches, new analyses and proposed improvements. 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The protease allergen papain disrupts the airway epithelium triggering a rapid eosinophilic inflammation by innate lymphoid cell type 2 (ILC2) activation, leading to a Th2 immune response. Here we asked whether the daily oral administrations of the probiotic Escherichia coli strain Nissle 1917 (ECN) might affect the outcome of the papain protease induced allergic lung inflammation in BL6 mice. We find that ECN gavage significantly prevented the severe allergic response induced by repeated papain challenges and reduced lung inflammatory cell recruitment, Th2 and Th17 response and respiratory epithelial barrier disruption with emphysema and airway hyperreactivity. In conclusion, ECN administration attenuated severe protease induced allergic inflammation, which may be beneficial to prevent allergic asthma. IntroductionAllergic asthma is one of the most common chronic respiratory diseases with a significant impact on public health1,2. In recent years, the incidence of allergic asthma in developed countries has dramatically increased and it is predicted that the number of affected people worldwide will increase by 100 million by 20253. Risk alleles have been identified for the development of asthma4 but the rapidity of its increased incidence does not support solely a genetic basis and suggest the involvement of environmental factors. Long-term observations support the notion that urban life is associated with increased prevalence of chronic immunological disorders including asthma incidence as compared to children living in farms5. Early in life microbial exposure might modulate allergic disorders6. In addition, such favorable socioeconomic factors, like enriched dietary habits or increased level of hygiene are presumably important factors for a considerable shift in the gut microbiota and increased asthma susceptibility. Epidemiological and clinical studies indicate an association between alteration of intestinal microbial communities and increased incidence of allergic asthma7. Several studies revealed changes in gut microbiota composition in adults suffering from allergic diseases at distant body sites (eczema, rhinitis, asthma)8,9, which precede the development of allergic diseases10,11. Gut bacteria outnumber the human body cells and the microbiome encode approximately 100 times more genes than the human genome12. This impressive genetic capacity contribute to essential functions for the host including nutrients supply like short-chain fatty acids (SCFAs)13,14, vitamins and hormones15, energy balance16,17,18, metabolic signaling19, resistance to pathogens colonization20,21,22 and has a key role in promoting the postnatal maturation of the intestinal mucosal barrier23,24, etiology is complex, but exposure to allergens or air pollution, are clearly important factors for the pathogenesis5. Sensitization to allergen is one of the first steps involved in asthma. Various allergens, including house dust mite (HDM), fungi, cockroach and pollen have proteolytic activities26. Protease properties of allergens cause injury of the airway epithelium with increased permeability, airway remodeling, type 2 cytokine and chemokine production and cell recruitment27. Papain, a cysteine protease, induces a type 2 response characterized by interleukin (IL)-5 and IL-13 production, mediated by an IL-2-dependent IL-9 production28 and specific IgE production29,30. There is evidence that the commensal microflora is critical in the maintenance of systemic immune tolerance, which is instrumental in protecting against allergic asthma. Escherichia coli strain Nissle 1917 (Mutaflor®, ECN) is successfully used for the treatment of intestinal inflammation, especially in patients suffering from ulcerative colitis31. In the present study, we investigated the impact of the colonization by ECN on the allergic lung inflammatory response induced by single or repeated challenges to the protease allergen papain. We show here that chronic ECN administration reduces severe allergic lung inflammation, improves the respiratory epithelial barrier function and modulates emphysema in response to repeated papain colonization has a dual effect in acute papain-induced lung inflammationTo study the impact of the administration of the ECN strain on the development of allergic inflammation, we compared the susceptibility ECN treated mice to acute papain-induced lung inflammation in comparison to non-treated controls according to the protocol shown in Fig. 1a. ECN was administered by gavage over 6 days (108 cfu of live ECN/day) then the mice were challenged twice by intranasal instillation ( of the protease allergen papain (25 µg on day 7 and 8 and the inflammatory response was analyzed 24 h later as described before32. Microscopic examinations of the lungs revealed focal inflammatory cell infiltration around bronchi, capillaries and in alveoli, as well as mucus hypersecretion (Fig. 1b). The lung inflammation as assessed by a semi-quantitative score of microscopic lesions was not reduced in ECN fed mice (Fig. 1b,c), except for the production of mucus (Fig. 1d).Figure 1ECN colonization as a dual effect in acute papain-induced lung inflammation. (a) Experimental settings of acute papaïn-induced lung inflammation and ECN treatment. (b) Lung tissues were histologically examined 24 h after the last papaïn challenge. Lung sections stained with HE from controls (NaCl/NaCl), papaïn (NaCl/Papaïn) and ECN (ECN/Papaïn)-treated mice are represented. (c) Histological score of lung inflammation infiltration was performed on paraffin embedded section after HE staining. (d) Histological score of lung mucus production was performed on paraffin embedded section after PAS staining. (e) Total cells and differential cell count of eosinophils, neutrophils, lymphocytes and macrophages were determined in BALF by numeration of MGG stained cytospin. Lung homogenate level of (F) CCL11, (g) CCL17 and (h) CXCL1 were measured by ELISA. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imagePapain-induced lung inflammation is associated with enhanced cell recruitment in the lung, involving especially eosinophils32. Cell recruitment into the broncho-alveolar lavage fluid (BALF) was modulated with increased total cells, especially neutrophils upon ECN treatment as compared to control mice (Fig. 1e) with increased myeloperoxidase (MPO) (Supplementary Figure 1) and neutrophil chemoattractant CXCL1 levels (Fig. 1h). By contrast, the recruitment of eosinophils in the BALF was significantly decreased in ECN-treated animals as compared to papain controls (Fig. 1e). This was correlated with a lowered production of CCL17 (Fig. 1g) while CCL11 levels was not modified (Fig. 1f).Interestingly, mice treated with a non-probiotic K12 E. coli strain MG1655 and tested in the acute papain model (Supplementary Figure 2A) develop a similar lung neutrophilia as compared to ECN-treated animals (Supplementary Figure 2B–D), suggesting that this effect is probably mediated an E. coli genus dependent molecular determinant. On the contrary, MG1655 treatment has no protective effect on eosinophilia as observed with cell count and chemokine production (Supplementary Figure 2B,E,F). Taken together, these results suggest that gut colonization by ECN may modulate lung inflammation by enhancing neutrophil, but importantly reducing eosinophil cell recruitment in BALF and tissue. This data motivated studies in a chronic model of lung allergic lung inflammation induced by repeated papain challenges is attenuated by ECN administrationTo determine whether ECN modulates chronic airway inflammation induced by a protease allergen papain, BL6 mice were immunized with papain (25 µg on days 6, 7 by intranasal route), followed by two intranasal challenges at day 20 and 25 (25 µg). Control mice received vehicle (NaCl). In addition, mice were orally administered with 108 cfu of live ECN (Fig. 2a). 24 h after the last papain challenge, the mice were sacrificed and the extent of the lung inflammation was assessed. Histological analysis revealed a prominent lung inflammation characterized by perivascular, peribronchial and alveolar infiltration of eosinophils, neutrophils and air space enlargement with epithelial damage and disruption of alveolar septa, a hallmark of emphysema upon papain challenge (Fig. 2b,c). ECN-treated mice largely prevented lung inflammation, epithelial injury and emphysema (Fig. 2b–d). Finally, the extensive goblet cell hyperplasia and mucus production observed in primed/challenged mice was lowered in ECN probiotic treated mice (Fig. 2b,e). Diminished mucus expression was confirmed at the mRNA level for Muc5ac in lung (Fig. 2f). Interestingly, mice treated with E. coli strain MG1655 and tested in the chronic papain model develop a similar lung inflammation as compared to untreated animals, as revealed by the histological analysis (Supplementary Figure 3A–E), suggesting that the protective effect observed with ECN is due to intrinsic probiotic properties rather than a non-specific effect due to daily gavage E. coli species on the gut microbiota. The absence of protection with MG1655 is unlikely related to the lack of gut colonization, as we quantified equivalent Enterobacteria and E. coli colony counts in both ECN- and MG1655-treated animals along the treatment (Supplementary Figure 4).Figure 2Repeated papain challenges causing severe lung inflammation is attenuated by ECN administration. (a) Experimental settings of chronic papaïn-induced lung inflammation and ECN treatment. (b) Lung tissues were histologically examined 24 h after the last papaïn challenge. Lung sections stained with HE from controls (NaCl/NaCl), papaïn (NaCl/Papaïn) and ECN (ECN/Papaïn)-treated mice are represented. (c) Histological score of lung inflammation infiltration was performed on paraffin embedded section after HE staining. (d) Histological score of airway remodeling was performed on paraffin embedded section after HE staining. (e) Histological score of lung mucus production was performed on paraffin embedded section after PAS staining. (f) Muc5ac relative gene expression levels in lung tissues was measured by qPCR. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageECN-treated mice develop reduced airway eosinophilia and Th2-driven airway inflammation upon papain chronic challengesPapain-induced chronic inflammation is characterized by a type 2 inflammatory response28. To determine whether ECN inhibited inflammatory cell recruitment, BALF cell counts were assessed for cell phenotyping. Saline sensitized and challenged mice present negligible leukocyte numbers in BALF, whereas papain-treated mice presented a dramatic increase of total cells, eosinophils and fewer neutrophils and macrophages (Fig. 3a). By contrast, ECN-treated mice had ~ less total BALF cell counts with a 2-fold reduction in eosinophils, neutrophils and macrophages. This was consistent with significant lower levels of eosinophils attracting chemokines CCL24 and CCL11 (Fig. 3b,d), EPO levels (Supplementary Figure 5) and neutrophils/monocytes chemoattractant CXCL1 (Fig. 3e), while CCL17 was unchanged in the lungs of ECN-treated mice as compared to controls. Moreover, Th2 cytokines such as IL-5 and to a lesser extent IL4 were significantly reduced in the lung of ECN-treated mice as compared to papain controls (Fig. 3f,g). The production of IFNγ was reduced, while IL17A level was unchanged in ECN probiotic-treated mice (Fig. 3h,i).Figure 3ECN-treated mice develop reduced airway eosinophilia and Th2-driven airway inflammation upon papaïn chronic challenges. (a) Total cells and differential cell count of eosinophils, neutrophils, lymphocytes and macrophages were determined in BALF by numeration of MGG stained cytospin. Lung homogenate level of (b) CCL24, (C) CCL17, (D) CCL11, (e) CXCL1, (f) IL-4, (g) IL-5, (h) IL-17 and (i) IFNγ were measured by ELISA. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageTaking together, these data indicate that ECN gut colonization reduces papain induced Th2 immune airways hyperreactivity and respiratory barrier injury is attenuatedA hallmark of allergic lung inflammation is airways hyperreactivity (AHR), which is due functional changes of the respiratory barrier. AHR was assessed by invasive plethysmography in untreated and ECN-treated mice upon chronic papain exposure. Airway resistance and compliance in response to methacholine as a measure of AHR and were increased upon papain challenge. ECN administration reduced airway resistance and compliance indicating a significant amelioration of the lung function (Fig. 4a,b).Figure 4Papaïn-induced pulmonary dysfunction is attenuated by ECN. (a) Airway hyper-responsiveness to increasing doses of methacholine (Mch; 0−200 mg/ml) was measured by recording changes in lung resistance and (b) airway compliance. The pulmonary epithelial integrity was assessed by the leak of (c) Evans blue and (d) total protein in BAL. (e) Immunofluorescent staining for E-cadherin (green) on lung cryosections. (f) Quantitative evaluation of E-cadherin expression on lung sections. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageThe protease papain induces inflammation and injury of the lung epithelium and capillaries with increased vascular permeability. The probiotic ECN has the ability to strengthen the epithelial barrier33. We used Evans Blue (EB), which binds to serum albumin, as a tracer of the capillary leak of macromolecules from the circulation into the BALF. Our data reveal that ECN treatment reduced the acute lung capillary/epithelial leak of intravenous administered EB upon papain exposure (Fig. 4c). Furthermore, total protein in BALF was also reduced (Fig. 4d). To get further insights into the role of ECN in the improvement of lung epithelial barrier function during allergic asthma, lung histological sections were analyzed for the expression of E-cadherin, a critical component of the epithelial barrier, which is crucial in the maintenance of the immunologic tolerance during airway allergic sensitization34. Immunofluorescence analysis revealed reduced E-cadherin expression concomitant with epithelial cell injury upon papain exposure, while ECN feeding attenuated the reduction of E-cadherin expression (Fig. 4e), which was confirmed by a semi-quantitative assessment of E-cadherin immunostaining (Fig. 4f).Therefore ECN colonization attenuated papain protease induced allergic lung inflammation with reduced Th2 response and airways hyperreactivity. Importantly the protease induced injury of the alveolar septae reflected by emphysema and of the respiratory barrier were significantly diminished by the probiotic strain mice has reduced Th2 lymphocytes and ILC2 activation upon papain chronic challengesTh2 lymphocytes and ILC2 accumulate in lungs after papaïn exposure and produce IL-5 and IL-1335. We determine the relative contribution of ECN on Th2 and ILC2 activation 24 h after the last allergen challenge. Lung cells were restimulated by papain and the production of cytokines was analyzed. IL-5 (Fig. 5a) and to a lesser extent IL-13 (Fig. 5b) was significantly reduced upon ECN treatment while IL-33 levels remain unchanged (Fig. 5c). Total Th2 and ILC2 producing IL-5 and IL-13 were analyzed by flow cytometry (Supplementary Figures 6 and 7). The frequency of CD3+ CD4+ IL5+ or IL13+ cells were significantly reduced in ECN-treated mice as compared to untreated controls (Fig. 5d–f). This was associated with a similar decrease of ILC2+ and ILC2+ IL13+ (Fig. 5g–i). These data indicate that ECN was able to dampen Th2 and ILC2 activation and the production of the prototypal pro-allergenic IL-5 and 5ECN-treated mice has reduced Th2 lymphocytes and ILC2 activation upon papain chronic challenges. IL-5 (a), IL-13 (b) and IL-33 (c) levels after lung mononuclear cell restimulation with papaïn for 72 h. Frequency of CD3+ CD4+ lymphocytes (d) producing IL-5 (e) or IL-13 (f) are shown. Frequency of ILC2 (g) producing IL-5 (h) or IL-13 (i) are shown. Data are expressed as mean + SEM from a single experiment with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. * and ** refer to P < and P < size imageDiscussionAllergic asthma is a major health issue with increasing incidence especially in developed countries with an epidemic feature36. Asthma etiology is complex including both genetic and environmental factors, such as exposure to allergens and/or air pollution, are important for the pathogenesis5. Data regarding the use of probiotics in the prevention of allergic diseases and asthma are conflicting37. Several different bacterial strains or combinations have been used in clinical trials to assess protective effects in the context of allergic asthma with significant reduction of both incidence and severity of allergic diseases38 which were not confirmed by others39. A meta-analysis concluded that probiotic are not efficient for the prevention of allergy40. This discrepancy may be related to the dose and duration of probiotic administration, immunomodulatory differences41 among strains, mostly Lactobacillus or Bifidobacterium probiotics42. Here we evaluated the probiotic potential of the Gram negative ECN to prevent allergic lung inflammatory allergic response induced by the protease papain. ECN drastically reduced the severity of chronic lung inflammation through the modulation of the Th2 inflammatory response, injury of the respiratory barrier and airways hyperreactivity. The beneficial effects of ECN has been demonstrated before in intestinal inflammatory disorders, especially in ulcerative colitis43. Two previous studies investigated ECN in experimental asthma. Bickert et al. using the inert protein allergen OVA observed a protection upon oral administration of ECN, but no inhibition of the Th2 immune response44. Adam et al. evaluated the prophylactic potential of ECN on recombinant house mite antigen Derp1 as mucosal antigen. ECN strongly reduced the antigen specific humoral response45. Here, using oral prophylactic administration of ECN we demonstrate for the first time a reduction of papain-induced lung inflammation and amelioration of AHR. In contrast, mice administered K12 E. coli strain MG1655 were as sensitive to lung inflammation as untreated papain challenged mice suggesting that the genetic background of the strain is of particular importance and determines its ability to act as a probiotic. Nevertheless, we observed that both E. coli strains has the ability to induce a potent lung neutrophilia. These results are in line with several papers demonstrating that ECN capsule antigen K5 was an important contributor the recruitment of neutrophil46,47. More generally, it has also been suggested that the presence of capsular antigen may induce an increased influx of pulmonary neutrophils48,49. The mechanisms by which capsular antigen modulate neutrophil response are not completely understood but may include direct effect such an upregulation of shed bacterial formylmethionyl-leucyl-phenylalanine50, a potent neutrophil chemotactic factor; or indirect by modulating the host’s generation of chemokines, including CXCL1 or IL-8 which was observed upon ECN or MG1655 of the best-characterized features contributing to the effectiveness of ECN is its ability to strengthen the epithelial barrier function51. This probiotic property of ECN has been extensively demonstrated in the context of intestinal inflammatory diseases. Asthma is often associated with mucosal barrier dysfunction52. We found that respiratory barrier dysfunction due to papain-induced inflammation and injury is alleviated by ECN with reduced protein leak and upregulation of E-cadherin. Recent studies suggests that this adhesion molecule contributes to the structural and immunological function of the airway epithelium, acting as a rheostat through the regulation of epithelial junctions and production of pro-inflammatory mediators34. Alterations of the airway epithelium enhance both allergic sensitization and airway remodeling including goblet cell hyperplasia, mucus hyperproduction and subepithelial fibrosis53 thus contributing to severe airways hyperreactivity. ECN conferred a significant reduction of inflammatory cell recruitment in BALF, lung tissue inflammation and disruption of alveolar septa with epithelial cells participate in the innate immune response of the lung and have barrier function. Barrier dysfunction favors the access of noxious or immunogenic protein or chemicals to the mucosa-associated lymphoid tissues. Thus, regulation of airway epithelial barrier function is an important checkpoint of the immune response during asthma54. In the present study, we show that ECN treatment affects a prevalent Th2 response known for papain induced lung inflammation28. We observed a significant reduction of eosinophils and eosinophil-related chemokines/cytokines associated with diminished recruitment of neutrophils and CXCL1 and IFN-γ levels. The data are consistent with previous studies showing that colonization by ECN lead to a modification of the cytokines repertoire55,56. In addition, we show for the first time that ECN treatment reduce Th2 CD4+ lymphocytes as well as ILC2 activation, resulting in decreased IL-5 and IL-13 production. The latter population is known to precede Th2 activation which is the cardinal feature of allergic asthma, culminating in airway hyperresponsiveness and Th2 cytokines and chemokines. In this setting, we investigated IL-33, which is known to be involved in ILC2 activation35 but we did not find any difference upon ECN treatment, which was also the case in another reduced allergic asthma molecular rationale behind the immunomodulatory properties of ECN has not yet been elucidated and is under investigation58. The beneficial effect of ECN could rely on the improvement of the intestinal barrier function and the resulting prevention of a continuous stimulation of the host innate immune system by the gut components. Indeed, we have recently demonstrated that ECN was able to prevent CNS inflammation through the improvement of the intestinal permeability59 showing that modulation of the gut microbiota with ECN exerts remote immunological imprinting. ECN genome encodes the production of specialized molecules that may modulate immune functions60,61,62. The intestinal mucosa represents an interface between bacterial-derived metabolites and mucosal immune processes that will influence immunological processes on the host conclusion, our findings indicate that ECN is able to prevent papain-induced lung inflammation after high dose per os administration supporting a gut-lung mucosal communication64. In addition, our results suggest that the prevention of the respiratory barrier dysfunction by probiotic treatment may be important to control allergic lung inflammation. Therefore, ECN might be considered as a valuable prophylactic or diet supplement to prevent allergic (B6) mice were bred in our specific pathogen free animal facility at TAAM-CNRS, Orleans, France (agreement D-45-234-6 delivered on March, 10 of 2014). Mice were maintained in a temperature-controlled (23 °C) facility with a strict 12 h light/dark cycle and were given free access to food and water. The experiments were performed with female mice aged 8–10 weeks using 5 mice per group, and the experiments were repeated at least twice. All animal experimental protocols were carried out in accordance with the French ethical and animal experiments regulations (see Charte Nationale, Code Rural R 214-122, 214-124 and European Union Directive 86/609/EEC) and were approved by the “Ethics Committee for Animal Experimentation of CNRS Campus Orleans” (CCO), registered (N°3) by the French National Committee of Ethical Reflexion for Animal Experimentation (CLE CCO 2013-1006).Bacterial preparation, growth conditions and administrationThe strains used in this study are the probiotic Escherichia coli Nissle 1917 (ECN) and the archetypal K12 E. coli strain MG1655. Both strains were engineered to exhibit a mutation in the rpsL gene, which is known to confer resistance to streptomycin62. Before oral administrations, ECN and MG1655 strains were grown for 6 h in LB broth supplemented with streptomycin (50 µg/mL) at 37 °C with shaking. This culture was diluted 1:100 in LB broth without antibiotics and cultured overnight at 37 °C with shaking. Bacterial pellets from this overnight culture were diluted in sterile PBS to the concentration of 109 colony forming units (cfu)/ml. Mice were treated by oral gavage with 108 cfu of ECN or MG1655 in 100 µl of PBS or 100 µl of PBS as negative lung inflammation model in miceMice were anesthetized by an iv injection of ketamine/xylazine followed by an intranasal administration of 25 µg of papain (Calbiochem, Darmstadt, Germany) in 40 µL of saline solution. Mice were euthanized by CO2 inhalation 24 h after papain administration and BALF was collected. After a hearth perfusion with ISOTON II (Acid free balanced electrolyte solution Beckman Coulter, Krefeld, Germany) lung were collected and sampled for alveolar lavage (BAL)BAL was performed by 4 lavages of lung with 500 µL of saline solution via a cannula introduced into mice trachea. BAL fluids were centrifuged at 400 g for 10 min at 4 °C, the supernatants were stored at −20 °C for ELISA analysis and pellets were recovered to prepare cytospin (Thermo scientific, Waltham, USA) glass slides followed by a Diff-Quik (Merz & Dade Dudingen, Switzerland) staining. Differential cell counts were performed with at least 400 eosinophil peroxidase (EPO) activityEPO activity was determined in order to estimate the recruitment of eosinophil counts in lung parenchyma as expressionTotal RNA was isolated from homogenized mouse lung using Tri Reagent (Sigma) and quantified by NanoDrop (Nd-1000). Reverse transcription was performed withSuperScript III Kit according to manufacturers’ instructions (Invitrogen). cDNA was subjected to quantitative PCR using primers for Muc5ac (sense 5′-CAGCCGAGAGGAGGGTTTGATCT-3′ and anti-sense 5′-AGTCTCTCTCCGCTCCTCTCA-3′; Sigma). Relative transcript expression of a gene is given as 2−ΔCt(ΔCt = Cttarget−Ctreference), and relative changes compared with control are 2−ΔΔCtvalues (ΔΔCt = ΔCttreated−ΔCtcontrol) {John, 2014 #340}.Enzyme-linked Immunosorbent assay (ELISA)Homogenized lung or cell supernatant were tested for MPO, CXCL1, CCL24, CCL11, CCL17, IL-4, IL17A and IFNγ (R&D systems Abingdon, UK), IL-13, IL-5, IL-33 (eBiosciences, San-5, Diego, USA) using commercial ELISA kits according to the manufacturer’s left lobe of lung was fixed in 4% buffered formaldehyde and paraffin embedded under standard conditions. Tissue sections (3 µm) were stained with PAS. Histological changes such as inflammation and emphysema were assessed by a semi-quantitative score from 0 to 5 for cell infiltration (with increasing severity) as described before66. The slides were examined by two blinded investigators with a Leica microscope (Leica, Germany).Determination of bronchial hyperresponsiveness (AHR)For invasive measurement of dynamic resistance, mice were anesthetized with intra-peritoneal injection of solution containing ketamine (100 mg/kg, Merial) and xylasine (10 mg/kg, Bayer), paralyzed using D-tubocuranine ( Sigma), and intubated with an 18-gauge catheter. Respiratory frequency was set at 140 breaths per min with a tidal volume of ml and a positive end-expiratory pressure of 2 ml H2O. Increasing concentrations of aerosolized methacholine ( 75 and 150 mg/ml) were administered. Resistance was recorded using an invasive plethysmograph (Buxco, London, UK). Baseline resistance was restored before administering the subsequent doses of immunofluorescence stainingLungs were fixed for 3 days in 4% PFA and submerged in 20% sucrose for 1 week. Lungs were embedded in OCT (Tissue-Teck) and 10 µM sections were prepared with cryotome (Leica). Slides were incubated 30 min in citrate buffer at 80 °C, washed in TBS-Tween and then incubated overnight with mouse-anti-mouse-E-cadherin (1 µg/ml, ab76055, Abcam). After washing with slides were treated with 0,05% pontamin sky blue (Sigma) for 15 min and then incubated with secondary AF-546 goat anti-mouse antibody (Abcam) for 30 min at room temperature. After washing, slides were incubated with DAPI (Fisher Scientific) and mounted in fluoromount® (SouthernBiotech). Lung sections were observed on a fluorescence microscope Leica (Leica, CTR6000) at x200 magnification. The slides were analyzed and semi-quantitatively scored and the MFI was epithelial barrier functionTotal protein in BAL fluid and Evans blue EB leak in BAL fluid was determined as described mononuclear cell isolation and stimulationLung mononuclear cells were isolated from mice 24 h after the last challenge as described previously67. Briefly the aorta and the inferior vena cava were sectioned and the lungs were perfused with 10 mL of saline. The lobes of the lungs were sliced into small cubes and then incubated for 45 min in 1 ml of RPMI 1640 solution and digested in 1,25 mg/ml of Liberase TL (Roche Diagnostics) and 1 mg/ml DNAse 1 (Sigma) during 1 h at 37 °C. Red blood cells were lysed with lysing buffer (BD Pharm LyseTM – BD Pharmingen). Isolated lung mononuclear single live cells were plated in round bottom 96-well plates (1 × 106/ml) and restimulated 3 h at 37 °C with PMA (50 ng/mL) and ionomicyn (750 ng/mL) in the presence of Brefeldin A (1 μl/1 × 106 cells, BD Biosciences) for intracellular flow cytometry analysis. Lung mononuclear cell (1 × 106 cells) were restimulated with 25 µg of papain in RPMI and 10% SVF at 37 °C in a 96 well plate for 3 days. Supernatants were analyzed for the presence of IL-5, IL-13 and IL-33 by ELISA (invitrogen).Flow cytometry analysis on lung mononuclear cellsLung mononuclear cells were stained with V450-conjugated anti-CD45 (clone 30F11), PerCp anti-CD3e (clone 145-2C11), FITC-conjugated anti-CD4 (clone RM4-5), PE-Cy7 -conjugated anti-ICOS (clone FITC-conjugated anti-ST2 (clone U29-93), anti B220 (clone RA3-6B2), anti FcεRI (clone MAR-1), anti CD11b (clone M1/70), anti Siglec-F (clone E50-2440) and Fixable Viability Dye eFluor™ 780 (eBioscience). 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The authors are grateful to Dieudonnée Togbé for helpful discussions and suggestions. This work was supported by ANR (ANR-GUI-AAP-06-Coliforlife), le Centre National de la Recherche Scientifique, the University of Orléans, la Région Centre (2013-00085470), European funding in Region Centre-Val de Loire (FEDER N° 2016-00110366), le Ministère de l’Education Nationale, de la Recherche et de la Technologie to RA as PhD fellowship, l’Institut National de la Santé et de la Recherche Médicale to ACM as a postdoctoral informationAuthor notesThomas SecherPresent address: INSERM, UMR 1100, Research Center for Respiratory Diseases, and University of Tours, Tours, FranceAuthors and AffiliationsIRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, FranceThomas Secher, Michèle Boury & Eric OswaldCNRS, UMR7355, Experimental and Molecular Immunology and Neurogenetics, Orleans, FranceIsabelle Maillet, Claire Mackowiak, Jessica Le Bérichel, Amandine Philippeau, Corinne Panek, Francois Erard, Marc Le Bert, Valérie Quesniaux, Aurélie Couturier-Maillard & Bernhard RyffelCHU Toulouse, Hôpital Purpan, Service de Bactériologie-Hygiène, Toulouse, FranceEric OswaldCentre de Physiopathologie de Toulouse Purpan (CPTP), Université de Toulouse, UPS, Inserm, CNRS, Toulouse, FranceAbdelhadi SaoudiUniversity of Orleans, Orleans, FranceValérie Quesniaux & Bernhard RyffelUniversity of Cape Town, IDM, Cape Town, Republic of South AfricaBernhard RyffelAuthorsThomas SecherYou can also search for this author in PubMed Google ScholarIsabelle MailletYou can also search for this author in PubMed Google ScholarClaire MackowiakYou can also search for this author in PubMed Google ScholarJessica Le BérichelYou can also search for this author in PubMed Google ScholarAmandine PhilippeauYou can also search for this author in PubMed Google ScholarCorinne PanekYou can also search for this author in PubMed Google ScholarMichèle BouryYou can also search for this author in PubMed Google ScholarEric OswaldYou can also search for this author in PubMed Google ScholarAbdelhadi SaoudiYou can also search for this author in PubMed Google ScholarFrancois ErardYou can also search for this author in PubMed Google ScholarMarc Le BertYou can also search for this author in PubMed Google ScholarValérie QuesniauxYou can also search for this author in PubMed Google ScholarAurélie Couturier-MaillardYou can also search for this author in PubMed Google ScholarBernhard RyffelYou can also search for this author in PubMed Google ScholarContributionsConceived and designed the experiments: and Performed the experiments: and Analyzed the data: Wrote the paper: and authorsCorrespondence to Thomas Secher or Bernhard declarations Competing Interests The authors declare no competing interests. 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To view a copy of this license, visit Reprints and PermissionsAbout this articleCite this articleSecher, T., Maillet, I., Mackowiak, C. et al. The probiotic strain Escherichia coli Nissle 1917 prevents papain-induced respiratory barrier injury and severe allergic inflammation in mice. Sci Rep 8, 11245 (2018). citationReceived: 12 September 2017Accepted: 16 July 2018Published: 26 July 2018DOI: CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Skip Nav Destination Imaging, Diagnosis, Prognosis| April 15 2008 Peter Brader; 1Department of Radiology, Search for other works by this author on: Jochen Stritzker; 6Genelux Corporation, San Diego Science Center, San Diego, California; and 7Institute for Biochemistry, Biocenter; Institute for Molecular Infectious Biology; and Search for other works by this author on: Pat Zanzonico; 2Department of Medical Physics, Search for other works by this author on: Shangde Cai; 3Cyclotron and Radiochemistry Core Facility, Search for other works by this author on: Eva M. Burnazi; 3Cyclotron and Radiochemistry Core Facility, Search for other works by this author on: Hedvig Hricak; 1Department of Radiology, Search for other works by this author on: Aladar A. Szalay; 6Genelux Corporation, San Diego Science Center, San Diego, California; and 7Institute for Biochemistry, Biocenter; Institute for Molecular Infectious Biology; and 8Virchow Center for Biomedical Research, School of Medicine, University of Wuerzburg, Wuerzburg, Germany Search for other works by this author on: Yuman Fong; 5Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York; Search for other works by this author on: Ronald Blasberg 1Department of Radiology, 4Nuclear Pharmacy, and Search for other works by this author on: Requests for reprints: Ronald G. Blasberg, Departments of Neurology and Radiology, MH (Box 52), Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 646-888-2211; Fax: 646-422-0408; E-mail: blasberg@ Received: September 14 2007 Revision Received: December 03 2007 Accepted: December 04 2007 Online Issn: 1557-3265 Print Issn: 1078-0432 American Association for Cancer Research2008 Clin Cancer Res (2008) 14 (8): 2295–2302. Article history Received: September 14 2007 Revision Received: December 03 2007 Accepted: December 04 2007 Split-Screen Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review PDF Tools Icon Tools Search Site Article Versions Icon Versions Version of Record April 15 2008 Proof March 27 2008 Citation Peter Brader, Jochen Stritzker, Christopher C. Riedl, Pat Zanzonico, Shangde Cai, Eva M. Burnazi, Ghani, Hedvig Hricak, Aladar A. Szalay, Yuman Fong, Ronald Blasberg; Escherichia coli Nissle 1917 Facilitates Tumor Detection by Positron Emission Tomography and Optical Imaging. Clin Cancer Res 15 April 2008; 14 (8): 2295–2302. Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex Abstract Purpose: Bacteria-based tumor-targeted therapy is a modality of growing interest in anticancer strategies. Imaging bacteria specifically targeting and replicating within tumors using radiotracer techniques and optical imaging can provide confirmation of successful colonization of malignant Design: The uptake of radiolabeled pyrimidine nucleoside analogues and [18F]FDG by Escherichia coli Nissle 1917 (EcN) was assessed both in vitro and in vivo. The targeting of EcN to 4T1 breast tumors was monitored by positron emission tomography (PET) and optical imaging. The accumulation of radiotracer in the tumors was correlated with the number of bacteria. Optical imaging based on bioluminescence was done using EcN bacteria that encode luciferase genes under the control of an l-arabinose–inducible PBAD promoter We showed that EcN can be detected using radiolabeled pyrimidine nucleoside analogues, [18F]FDG and PET. Importantly, this imaging paradigm does not require transformation of the bacterium with a reporter gene. Imaging with [18F]FDG provided lower contrast than [18F]FEAU due to high FDG accumulation in control (nontreated) tumors and surrounding tissues. A linear correlation was shown between the number of viable bacteria in tumors and the accumulation of [18F]FEAU, but not [18F]FDG. The presence of EcN was also confirmed by bioluminescence can be imaged by PET, based on the expression of endogenous E. coli thymidine kinase, and this imaging paradigm could be translated to patient studies for the detection of solid tumors. Bioluminescence imaging provides a low-cost alternative to PET imaging in small animals. In recent years, successful targeting of viruses and bacteria to solid tumors has been shown (1, 2) and such oncolytic therapy is receiving renewed interest. Tumor-targeting bacteria have been studied and they showed preferential accumulation in tumors compared with normal organs; studies have included the use of Bifidobacterium spp. (3), Listeria monocytogenes (1, 4), Clostridium spp. (5), Salmonella spp. (6–8), Shigella flexneri (6), Vibrio cholerae (2), and Escherichia coli (6). A number of different oncolytic viruses have already entered into clinical trials and adenovirus H101 has been approved in China for the treatment of head and neck cancer (8). However, only a single phase I human clinical trial using bacteria, Salmonella VNP20009, has been initiated (7). In this trial, a lower percentage of tumor-targeting efficacy was observed compared with the previously investigated rodent models in which tumor-colonization was high (7). The authors stated that this discrepancy could be the result of inadequate sampling that was inherent in their use of fine-needle biopsies. In an excisional biopsy done on one patient, bacteria were found to colonize the tumor, whereas a previous needle biopsy of the same tumor did not detect the microorganisms. Currently, biopsy is the only clinical method available for determining the presence of bacteria. Future clinical studies will require the ability to accurately detect the presence of bacteria in tumors (and also in other organs and tissues) without excision of the respective tissue. To address this issue, noninvasive imaging of bacteria-colonized tumors has several advantages compared with biopsy. In contrast to biopsies, imaging can be done repeatedly, provides a much wider assessment of the entire tumor as well as other tissues and body organs ( minimizes sampling errors), and can provide both a spatial and time dimension from sequential tomographic images. Different imaging modalities [positron emission tomography (PET), single-photon emission computed tomographyy, and optical imaging] in combination with reporter genes have been used to visualize the distribution of microorganisms and to confirm their presence within experimental tumors. Most studies on bacterial tumor colonization in tumor-bearing mice have used luciferase and/or fluorescence (green fluorescent protein) imaging for bacterial detection (2, 4, 6, 9). However, current optical imaging modalities using fluorescent proteins or luciferases are restricted to small animals and cannot be readily translated to patient studies. Therefore, radiotracer or magnetic resonance imaging techniques need to be used to track bacteria in human subjects. The best known and most widely used radiotracer for PET imaging is fluorine-18 (18F)–labeled fluorodeoxyglucose ([18F]FDG), which is accumulated by metabolically active cells. On entry into the cell, [18F]FDG is phosphorylated by hexokinase; the phosphorylated FDG can neither exit the cell nor be further metabolized and is therefore trapped within the cell in relation to the level of glycolytic activity. FDG uptake in many malignant tumors is high because glucose metabolism in the tumors is high. In addition, any inflammatory processes associated with the tumor contribute to the high FDG uptake because granulocytes and macrophages also have high rates of glucose metabolism (10). Although tumor tissue targeted by bacteria is likely to have high levels of FDG accumulation, baseline (before bacterial administration) is also likely to be high, and the difference between baseline and tumor-targeted FDG uptake may be difficult to image and quantitate. Another powerful imaging strategy is the use of reporter genes in to identify the location and number of tissue-targeted bacteria. Among the PET-based reporter genes, herpes simplex virus 1 thymidine kinase (HSV1-TK) has been used most extensively. The expression of HSV1-TK can be imaged and monitored using specific radiolabeled substrates that are selectively phosphorylated by HSV1-TK and trapped within transfected cells. [18F]-2′-Fluoro-2′deoxy-1β-d-arabionofuranosyl-5-ethyl-uracil ([18F]FEAU) and [124I]-2′-fluoro-1-β-d-arabino-furanosyl-5-iodo-uracil ([124I]FIAU) are radiopharmaceuticals for imaging HSV1-TK gene expression (11) and are used widely by many investigators (12–15). HSV1-TK–expressing Salmonella VNP20009 have recently been shown to localize in tumors, including C-38 colon carcinoma and B16-F10 murine melanoma, and were successfully imaged with [124I]FIAU and PET (16). In contrast to using an exogenous reporter gene such as HSV1-TK, we investigated the feasibility of using the endogenous thymidine kinase of probiotic E. coli Nissle 1917 (EcN) to phosphorylate [18F]FEAU and [124I]FIAU for noninvasive PET imaging of EcN-colonized tumors. We show that the uptake of [18F]FEAU by the tumors is dependent on the presence of EcN and that the magnitude of radioactivity accumulation correlates with the number of bacteria that colonize the tumor. We also compared [18F]FEAU and [124I]FIAU images to those obtained with [18F]FDG. Bioluminescence images of EcN were also obtained and the optical signal shown to colocalize with the [124I]FIAU activity distribution in the same animals, showing the feasibility of using EcN for identifying tumors by both bioluminescence and PET imaging in small animals. Materials and Methods Cell culture and animal experiments The murine mammary carcinoma cell line 4T1 (ATCC CRL-2539) was cultured in RPMI containing 10% FCS. The cells were maintained at 37°C with 5% CO2 in air, and subcultured twice weekly. For tumor cell implantation, 6- to 8-wk-old athymic nu/nu mice (National Cancer Institute) were used, housed five per cage, and allowed food and water ad libitum in the Memorial Sloan Kettering Cancer Center facility for 1 wk before tumor cell implantation. The 4T1 cells were removed by trypsinization, washed in PBS, and × 104 cells (resuspended in 50-μL PBS) were implanted into the right and left shoulders. Two weeks postimplantation (tumor diameter >5 mm), bacteria were administered systemically by tail vein injection. Animal studies were done in compliance with all applicable policies, procedures, and regulatory requirements of the Institutional Animal Care and Use Committee, the Research Animal Resource Center of Memorial Sloan Kettering Cancer Center, and the NIH Guide for the Care and Use of Laboratory Animals. All animal procedures were done by inhalation of 2% isofluorane. After the studies, all animals were sacrificed by CO2 inhalation. Bacteria E. coli Nissle 1917 (EcN), a probiotic, non–protein-toxin-expressing strain, was used to specifically colonize tumors and harbored a pBR322DEST PBAD-DUAL-term, a luxABCDE-encoding plasmid that enables the bacteria to be detected with bioluminescence imaging when induced with l-arabinose (6). The light is emitted from the bacteria as a result of a heterodimeric luciferase (encoded by luxAB) catalyzing the oxidation of reduced flavin mononucleotide and a long-chain fatty aldehyde (synthesized by a fatty acid reductase complex encoded by luxCDE; ref. 17). For injection, bacteria were grown in LB broth supplemented with 100 μg/mL ampicillin until reaching an absorbance at 600 nm (A600 nm) of [corresponding to 2 × 108 colony-forming units (CFU)/mL] and washed twice in PBS. The suspension was then diluted to 4 × 107 CFU/mL and 100 μL were injected into the lateral tail vein of tumor-bearing mice. Vehicle control mice were injected with 100-μL PBS via tail vein. Radiopharmaceuticals [18F]FEAU was synthesized by coupling the radiolabeled fluoro sugar with the silylated pyrimidine derivatives following a procedure previously reported by Serganova et al. (12). The specific activity of the product was ∼37 GBq/μmol (∼1 Ci/μmol); radiochemical purity was >95% following purification by high-pressure liquid chromatography. [124I]FIAU was synthesized by reacting the precursor of 5-trimethylstannyl-1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)uracil (FTAU) with carrier-free [124I]NaI. I-124 was produced on the Memorial Sloan-Kettering Ebco cyclotron using the 124Te(p,n) 124I nuclear reaction on an enriched 124TeO2/Al2O3 solid target. Radiosynthesis was done as previously described (13, 14) with minor modifications. The specific activity of the product was >1,000 GBq/μmol (>27 Ci/μmol); radiochemical purity was >95% and was determined by radio TLC (Rf using silica gel plates and a mobile phase of ethyl acetate/acetone/water (14:8:1, v/v/v). [18F]FDG (clinical grade) was obtained from IBA Molecular with a specific activity >41 MBq/μmol (>11 mCi/μmol) and a radiochemical purity of 99% by TLC and 98% by high-pressure liquid chromatography. In vitro uptake of [18F]FDG and [18F]FEAU An overnight culture of EcN was diluted 1:50 into 5-mL fresh LB broth containing either MBq (25 μCi) of [18F]FDG or [18F]FEAU and grown at 37°C for 4 h. The bacteria were then harvested by centrifugation, washed twice with PBS, and the radioactivity in the pelleted bacteria and medium was measured in a gamma counter (Packard, United Technologies). MicroPET imaging FDG. In the first group of six animals, each animal was injected via the tail vein with MBq (250 μCi) of [18F]FDG before and 16 or 72 h after administration of EcN. [18F]FDG PET scanning was done 1 h after tracer administration using a 10-min list mode acquisition. Animals were fasted 12 h before tracer administration and kept under anesthesia between FDG injection and imaging. FEAU. In the second group of 24 animals, three subgroups of eight animals each were studied; each animal was injected via tail vein with MBq (250 μCi) of [18F]FEAU. Subgroup 1 (control) was not injected with bacteria (they received 100-μL PBS); subgroups 2 and 3 were injected with EcN-bacteria 16 and 72 h before [18F]FEAU administration. [18F]FEAU PET scanning was done 2 h after tracer administration using a 10-min list mode acquisition. FIAU. In a third set of six mice, three were injected with EcN-bacteria and three with PBS (control). [124I]FIAU [37 MBq (1 mCi)] was injected in each animal 72 h after bacterial injection. Potassium iodide was used to block the uptake of radioactive iodine by the thyroid. [124I]FIAU PET was obtained 4, 8, 12, 24, 48, and 72 h after tracer administration with 10-min list acquisition at the 4- and 8-h imaging time points, 15 min at the 12-h time point, 30 min at 24 h, and 60 min at the 48- and 72-h time points. After tracer administration and between imaging time points, the animals were allowed to wake up and maintain normal husbandry. Imaging was done using a Focus 120 microPET dedicated small-animal PET scanner (Concorde Microsystems, Inc.). Mice were maintained under 2% isofluorane anesthesia with an oxygen flow rate of 2 L/min during the entire scanning period. Three-dimensional list mode data were acquired using an energy window of 350 to 700 keV for 18F and 410 to 580 keV for 124I and a coincidence timing window of 6 ns. These data were then sorted into two-dimensional histograms by Fourier rebinning using a span of 3 and a maximum ring difference of 47. Transverse images were reconstructed by filtered back-projection using a ramp filter with a cutoff frequency equal to the Nyquist frequency in a 128 × 128 × 94 matrix composed of × × voxels. The image data were corrected for (a) nonuniformity of scanner response using a uniform cylinder source-based normalization, (b) dead time count losses using a singles count rate–based global correction, (c) physical decay to the time of injection, and (d) the 124I branching ratio. There was no correction applied for attenuation, scatter, or partial-volume averaging. The count rates in the reconstructed images were converted to activity concentration [percent of injected dose per gram of tissue (%ID/g)] using a system calibration factor (μCi/mL/cps/voxel) derived from imaging of a rat-size phantom filled with a uniform aqueous solution of 18F. PET image analysis was done using ASIPro software (Concorde Microsystems, Inc.). For each PET scan, regions of interest were manually drawn over tumor, liver, skeletal muscle, and heart. For each tissue and time point postinjection, the measured radioactivity was expressed as %ID/g. The maximum pixel value was recorded for each tissue and tumor-to-organ ratios for liver, skeletal muscle, and heart were then plotted versus time. Bacterial and radioactivity quantification of tissue samples Euthanized mice were rinsed with 100% ethanol before tissue removal. Organs such as liver, lung, spleen, and heart were sampled and weighed before radioactivity measurements. Tumor tissue was weighed and homogenized in 1-mL PBS. Serial dilutions of the homogenized sample were plated on l-arabinose–containing LB agar plates and growing colonies were counted and confirmed to be EcN harboring a pBR322DEST PBAD-DUAL-term by bioluminescence imaging using an IVIS 100 Imaging system (Caliper). The remaining tumor suspension was assayed for radioactivity in a gamma counter (Packard, United Technologies); [18F]FEAU radioactivity (%ID/g) in the samples was determined and tumor-to-organ ratios were calculated. To assess the correlation between radioactivity and scintillation counter measurements, the Pearson correlation coefficient was computed. In vivo optical imaging of bioluminescence The same animals were imaged for localization of bioluminescence after the 72-h [124I]FIAU PET scans. Each animal was injected with 200-μL l-arabinose (25% w/v) to induce transcriptional expression of the luciferase reporter for bioluminescence imaging. Images were acquired for 60 s, 4 h after l-arabinose injection, using an IVIS 100 Imaging System (Caliper). The photon emissions (photons/cm2/s/steradian) from the animals and cell samples were analyzed using the LIVINGIMAGE software (Caliper). Statistics A two-tailed unpaired t test was applied to determine the significance of differences between values using the MS Office 2003 Excel statistical package (Microsoft). Results In vitro [18F]FDG and [18F]FEAU uptake into EcN. The in vitro uptakes of [18F]FDG and of [18F]FEAU by the tumor-colonizing strain E. coli Nissle 1917 were compared. There was a 120-fold higher concentration of [18F]FDG and a higher concentration of [18F]FEAU activity in EcN-bacteria compared with that in the LB broth, suggesting that [18F]FDG would be a better imaging agent than [18F]FEAU. Distribution of EcN in tumor-bearing mice. Following EcN injection into the tail vein of 4T1 tumor–bearing mice, most bacteria (>99%) are quickly cleared from the animals and only a small percentage of the administered bacteria colonize the tumor (6). These tumor-colonizing bacteria started to grow exponentially for ∼24 hours before reaching a plateau of ∼1 × 109 CFU/g of tumor tissues. During the growth phase, the bacteria are metabolically active and rapidly proliferate. For our studies, we elected to use tumor-bearing mice that were injected with EcN at 16 hours (lower CFU per gram but in rapid growth phase) and at 72 hours (higher numbers of bacteria in a slower phase) before administration of [18F]FDG or [18F]FEAU. The number of bacteria per gram of tumor tissue at 16 and 72 hours postinjection is shown in (Fig. 1). Fig. colonization of EcN at 16 and 72 h after bacterial injection. Columns, mean of eight analyzed tumors; bars, colonization of EcN at 16 and 72 h after bacterial injection. Columns, mean of eight analyzed tumors; bars, SD. Close modal In vivo PET imaging of EcN colonized tumors. [18F]FDG PET imaging was done before and at 16 and 72 hours after tail vein injection of EcN in the same animals (Fig. 2A). The [18F]FDG tumor-to-organ ratios (mean ± SD) before injection of EcN bacteria were high in liver ( ± and muscle ( ± and low in heart ( ± At 16 hours after EcN injection, tumor-to-organ ratios were significantly increased for liver, muscle, and heart ( ± ± and ± respectively). At 72 hours after EcN injection, the tumor-to-organ ratios were lower for the same tissues ( ± ± and ± respectively). This represents a ∼ enhancement at 16 hours (P 5 in the EcN-treated animals (Fig. 5B). However, the control (non–EcN-treated) animals also show some [124I]FIAU retention in the 4T1 xenografts. This reduces the specificity of the radioactivity measured in the EcN-treated tumors and results in only a enrichment of [124I]FIAU in the bacteria-treated tumors (Fig. 5B). Fig. axial and coronal views of [124I]FIAU microPET images of representative EcN-treated and nontreated (control) 4T1 xenograft–bearing animals at different times (12, 24, 48, and 72 h; X-axis) after tracer injection. B, [124I]FIAU uptake of tumors compared with background as calculated from region of interest measurements; six tumors in each group (FIAU uptake ratio; left Y-axis). Data from the EcN colonized group are shown in green and the control group in blue. The mean tumor uptake ratios in EcN colonized animals normalized to the mean values obtained for the control animals are indicated in red (relative FIAU uptake; right Y-axis). C, bioluminescence images of the same animals in A 4 h after injection of l-arabinose; l-arabinose induces the expression of luciferase genes in EcN × pBR322DEST PBAD-DUAL-term bacteria. Tumors are axial and coronal views of [124I]FIAU microPET images of representative EcN-treated and nontreated (control) 4T1 xenograft–bearing animals at different times (12, 24, 48, and 72 h; X-axis) after tracer injection. B, [124I]FIAU uptake of tumors compared with background as calculated from region of interest measurements; six tumors in each group (FIAU uptake ratio; left Y-axis). Data from the EcN colonized group are shown in green and the control group in blue. The mean tumor uptake ratios in EcN colonized animals normalized to the mean values obtained for the control animals are indicated in red (relative FIAU uptake; right Y-axis). C, bioluminescence images of the same animals in A 4 h after injection of l-arabinose; l-arabinose induces the expression of luciferase genes in EcN × pBR322DEST PBAD-DUAL-term bacteria. Tumors are encircled. Close modal Colocalization of bioluminescence and [124I]FIAU uptake. To further verify that the increased [124I]FIAU PET signal reflected bacterial localization in 4T1 xenografts, we took advantage of the l-arabinose–inducible luciferase reporter plasmid pBR322DEST PBAD-DUAL-term (6). l-Arabinose was injected into each mouse following [124I]FIAU PET imaging, and bioluminescence imaging was done 4 hours later when the expression of luciferase is at its maximum (6). The l-arabinose–induced bioluminescence signal was readily detected at the site of the 4T1 xenografts (Fig. 5C). Control tumors did not show any such signal. The bioluminescence images of EcN-treated mice also indicated no bacterial presence in other tissues of mice. Discussion EcN is one of the best studied probiotic bacterial strains and it has been successfully used in humans as an oral treatment for a number of intestinal disorders ( diarrhea, inflammatory bowel diseases, and ulcerative colitis) for more than 90 years (18, 19). Although the genome of EcN shows high similarity to the uropathogenic E. coli CFTR073 (20), the probiotic strain lacks any known protein toxins or mannose-resistant hemagglutinating adhesins (21). Furthermore, EcN was not found to colonize any organs other than tumor when administered systemically to tumor-bearing mice (6). Thus, EcN seems to be a good candidate for human application, although it still produces lipopolysaccharide (endotoxin), which could result in adverse effects. Because deletion of genes responsible for lipopolysaccharide biosynthesis ( msbB) has been shown to be successful for Salmonella typhimurium, a similar strategy could be adopted with EcN to insure its clinical safety. A noninvasive, clinically applicable method for imaging bacteria in target tissue or specific organs of the body would be of considerable value for monitoring and evaluating bacterial-based therapy in human subjects. This imaging system could also be used for monitoring the targeting and proliferation of the bacterial vector, such as EcN, to identify sites of occult tumor and to identify sites of bacterial proliferation in occult infectious disease. EcN imaging provides the following benefits: Following systemic administration of the bacteria, imaging can (a) confirm successful targeting to known tumor sites, (b) potentially identify additional sites of tumor metastases, and (a) assess whether the number (concentration) of EcN in tumor tissue is adequate to deliver a sufficient dose of a “therapeutic gene.” In our study, we assessed the feasibility of detecting EcN-colonized tumors with [18F]FDG, [18F]FEAU, and [124I]FIAU PET imaging. We showed that EcN accumulate and trap radiolabeled [18F]FDG, [18F]FEAU, and [124I]FIAU using endogenous enzyme systems ( bacterial hexokinase and thymidine kinase). It was previously shown that tumor targeting of HSV1-TK–transformed Salmonella VNP20009 could be successfully imaged with [124I]FIAU and that [124I]FIAU accumulation was HSV1-TK dependent (16). Here, the expression of the endogenous bacterial thymidine kinase of EcN and phosphorylation of [18F]FEAU and [124I]FIAU are sufficient to result in selective accumulation of these radiotracers in tissue colonized by EcN. In contrast to the marked structural specificity of mammalian thymidine kinase for thymidine alone (resulting in little or no phosphorylation of thymidine analogues), the thymidine kinase of bacteria has been shown by Bettegowda et al. (5) to be less specific for thymidine than the mammalian enzyme. Bacterial as well as viral thymidine kinase will phosphorylate thymidine analogues such as FIAU and FEAU. This study opens up new possibilities for future investigations and for the use of alternative pyrimidine nucleoside derivatives such as FEAU that can be selectively phosphorylated by endogenous bacterial thymidine kinase ( E. coli, Salmonella, or Clostridium). The tumor-selective replication of EcN in live animals allowed us to distinguish tumors from other tissues by PET imaging following administration of radiolabeled [18F]FEAU or [124I]FIAU. By using tumors in different stages of bacterial colonization ( 16 and 72 hours after bacterial administration), we showed a linear relationship between the number of viable bacteria in tumor tissue and the uptake of radiolabeled [18F]FEAU. This result is similar to that found with HSV1-TK–transformed Salmonella VNP20009 and [124I]FIAU accumulation (16). Comparing the Salmonella VNP20009 and EcN data shows that the HSV1-TK–transformed Salmonella accumulate more radiopharmaceutical per viable bacteria than EcN bacteria over the dose ranges that were studied (Fig. 4B). These results, for several reasons, are not unexpected and indicate that there is a role for reporter-transformed bacteria when higher imaging sensitivity is required: In addition to the genomic thymidine kinase gene of Salmonella VNP20009, HSV1-TK was present in multiple copies under control of a constitutive promoter. In contrast, only the genomic copy of the EcN thymidine kinase gene under control of its own promoter was present in EcN bacteria. Therefore, higher expression of thymidine kinase is achieved in VNP20009 Salmonella. Furthermore, [124I]FIAU and [18F]FEAU were developed to specifically image HSV1-TK, and not mammalian TK1, to achieve low background activity, and these tracer substrates may not be an ideal substrate for bacterial thymidine kinases (5). There was no correlation between the level of [18F]FDG uptake and number of viable bacteria in the tumors, and the signal-to-background ratio was not as high with [18F]FDG as with [18F]FEAU and [124I]FIAU. This clearly reflects the high baseline uptake (%ID/g) of [18F]FDG by the tumor compared with that of [18F]FEAU and [124I]FIAU. However, [18F]FDG imaging in combination with EcN (or other bacteria) might show better results in tumors with a low baseline level of [18F]FDG uptake. The absence of a correlation between number of viable bacteria and [18F]FDG uptake might also be due to the presence of necrosis induced by the bacteria or to the presence of glucose-metabolizing macrophages in the tumors (6). For example, on day 1 after bacterial injection, a high number of metabolically active bacteria were present and only very small patches of necrosis were observed. Two days later, the number of bacteria increases, but the number of living cells in the tumor decreases dramatically because the necrotic region takes up 30% to 50% of the tumor volume (6). It should also be noted that 4T1 xenografts in the absence of bacteria accumulate [124I]FIAU to low levels above background (48-and 72-hour images in Fig. 5B) in comparison with the near-background levels of [18F]FEAU accumulation (Fig. 2D) in non–bacteria-treated animals. This is consistent with similar observations in other tumor systems (12–14, 22, 23). Thus, [18F]FEAU may be a better bacterial-imaging probe than [124I]FIAU. The current study showed the feasibility of noninvasive imaging of bacteria based on the expression of genomic bacterial thymidine kinase. The potential for monitoring patients that have received tumor-colonizing bacteria without the inclusion of an exogenous ( viral) reporter gene has previously been shown (5) and is confirmed here. Imaging should be able to determine whether bacterial tumor colonization has occurred successfully and whether previously undetected metastases or specific organs are colonized by the bacteria. We have shown that the level of radioactivity can also be taken as an indicator of the number of bacteria that are present in the target tissue and whether therapeutic effects ( by administration of prodrugs or induction of toxic genes) can be expected. In addition, the presence of pathogenic bacteria in localized infections may also be identifiable, and it may also be possible to differentiate bacterial infections from nonmicrobial inflammations by [18F]FEAU or [124I]FIAU PET imaging. In conclusion, the results of our study indicate that EcN (or other bacteria expressing endogenous thymidine kinase) can be imaged with pyrimidine nucleoside analogues that are selectively phosphorylated and trapped in the bacteria. The advantage of using EcN over many other bacteria is their probiotic character. It is therefore a relatively safe “imageable vector” that could also include genes conferring therapeutic potential. We show that the PET images for EcN-colonized tumors were better ( resulted in higher signal-to-background ratios) with [18F]FEAU than with [18F]FDG, and this was mainly due to the low baseline (pre-bacterial) activity in the tumors and surrounding tissue. Most importantly, a linear relationship between the number of viable bacteria and level of [18F]FEAU activity in the xenografts was found, an essential component of the imaging paradigm. Other pyrimidine nucleoside analogues that have been developed for PET imaging of HSV1-TK, such as [124I]FIAU and [18F]FHBG, could also be further evaluated for noninvasive monitoring of bacterial tumor colonization because both positron-emitting radiopharmaceuticals have already been successfully administered to patients in gene imaging studies (15, 23–26). Grant support: NIH grants R25-CA096945 and P50 CA86438, Department of Energy grant FG03-86ER60407, R&D Division of Genelux Corporation San Diego, and a Service contract awarded to the University of Würzburg, Germany ( Szalay). Technical services were provided by the Memorial Sloan Kettering Cancer Center Small-Animal Imaging Core Facility, supported in part by NIH Small-Animal Imaging Research Program grant R24 CA83084 and NIH Center grant P30 CA08748. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 Section 1734 solely to indicate this fact. Note: P. Brader and J. Stritzker contributed equally to this work. Acknowledgments We thank Dr. Steven Larson (Memorial Sloan Kettering Cancer Center, New York, NY) for his help and support. References 1Liu TC, Kirn D. Systemic efficacy with oncolytic virus therapeutics: clinical proof-of-concept and future directions. 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