Vitamin C alleviates acute enterocolitis in Campylobacter jejuni infected mice.

Human foodborne infections with the zoonotic pathogen Campylobacter jejuni are on the rise and constitute a significant socioeconomic burden worldwide. The health-beneficial, particularly anti-inflammatory effects of vitamin C (ascorbate) are well known. In our preclinical intervention study, we assessed potential anti-pathogenic and immunomodulatory effects of ascorbate in C. jejuni-infected secondary abiotic IL-10-/- mice developing acute campylobacteriosis similar to humans. Starting 4 days prior peroral C. jejuni-infection, mice received synthetic ascorbate via the drinking water until the end of the experiment. At day 6 post-infection, ascorbate-treated mice harbored slightly lower colonic pathogen loads and suffered from less severe C. jejuni-induced enterocolitis as compared to placebo control animals. Ascorbate treatment did not only alleviate macroscopic sequelae of infection, but also dampened apoptotic and inflammatory immune cell responses in the intestines that were accompanied by less pronounced pro-inflammatory cytokine secretion. Remarkably, the anti-inflammatory effects of ascorbate pretreatment in C. jejuni-infected mice were not restricted to the intestinal tract but could also be observed in extra-intestinal compartments including liver, kidneys and lungs. In conclusion, due to the potent anti-inflammatory effects observed in the clinical murine C. jejuni-infection model, ascorbate constitutes a promising novel option for prophylaxis and treatment of acute campylobacteriosis.

Carvacrol ameliorates acute campylobacteriosis in a clinical murine infection model.

BACKGROUND: The prevalence of human infections with the zoonotic pathogen Campylobacter jejuni is rising worldwide. Therefore, the identification of compounds with potent anti-pathogenic and anti-inflammatory properties for future therapeutic and/or preventive application to combat campylobacteriosis is of importance for global health. Results of recent studies suggested carvacrol (4-isopropyl-2-methylphenol) as potential candidate molecule for the treatment of campylobacteriosis in humans and for the prevention of Campylobacter colonization in farm animals. RESULTS: To address this in a clinical murine infection model of acute campylobacteriosis, secondary abiotic IL-10-/- mice were subjected to synthetic carvacrol via the drinking water starting 4 days before peroral C. jejuni challenge. Whereas at day 6 post-infection placebo treated mice suffered from acute enterocolitis, mice from the carvacrol cohort not only harbored two log orders of magnitude lower pathogen loads in their intestines, but also displayed significantly reduced disease symptoms. Alleviated campylobacteriosis following carvacrol application was accompanied by less distinct intestinal apoptosis and pro-inflammatory immune responses as well as by higher numbers of proliferating colonic epithelial cells. Remarkably, the inflammation-ameliorating effects of carvacrol treatment were not restricted to the intestinal tract, but could also be observed in extra-intestinal organs such as liver, kidneys and lungs and, strikingly, systemically as indicated by lower IFN-γ, TNF, MCP-1 and IL-6 serum concentrations in carvacrol versus placebo treated mice. Furthermore, carvacrol treatment was associated with less frequent translocation of viable C. jejuni originating from the intestines to extra-intestinal compartments. CONCLUSION: The lowered C. jejuni loads and alleviated symptoms observed in the here applied clinical murine model for human campylobacteriosis highlight the application of carvacrol as a promising novel option for both, the treatment of campylobacteriosis and hence, for prevention of post-infectious sequelae in humans, and for the reduction of C. jejuni colonization in the intestines of vertebrate lifestock animals.

Resveratrol Alleviates Acute Campylobacter jejuni Induced Enterocolitis in a Preclinical Murine Intervention Study.

The polyphenolic compound resveratrol has been shown to exert health-beneficial properties. Given globally emerging Campylobacter infections in humans, we addressed potential anti-pathogenic, immuno-modulatory and intestinal epithelial barrier preserving properties of synthetic resveratrol in the present preclinical intervention study applying a murine acute campylobacteriosis model. Two days following peroral C. jejuni infection, secondary abiotic IL-10-/- mice were either subjected to resveratrol or placebo via the drinking water. Whereas placebo mice suffered from acute enterocolitis at day 6 post-infection, resveratrol treatment did not only lead to improved clinical conditions, but also to less pronounced colonic epithelial apoptosis as compared to placebo application. Furthermore, C. jejuni induced innate and adaptive immune cell responses were dampened in the large intestines upon resveratrol challenge and accompanied by less colonic nitric oxide secretion in the resveratrol versus the placebo cohort. Functional analyses revealed that resveratrol treatment could effectively rescue colonic epithelial barrier function in C. jejuni infected mice. Strikingly, the disease-alleviating effects of resveratrol could additionally be found in extra-intestinal and also systemic compartments at day 6 post-infection. For the first time, our current preclinical intervention study provides evidence that peroral resveratrol treatment exerts potent disease-alleviating effects during acute experimental campylobacteriosis.

Peptidase PepP is a novel virulence factor of Campylobacter jejuni contributing to murine campylobacteriosis.

Mechanisms of host-pathogen interactions resulting in immunopathological responses upon human Campylobacter jejuni infection are not completely understood, but the recent availability of murine infection models mimicking key features of campylobacteriosis helps solving this dilemma. During a screen for proteases expressed by C. jejuni, we identified a peptidase of the M24 family as a potential novel virulence factor, which was named PepP. The gene is strongly conserved in various Campylobacter species. A constructed deletion mutant ΔpepP of C. jejuni strain 81-176 grew as efficiently compared to isogenic wild-type (WT) or pepP complemented bacteria. To shed light on the potential role of this protease in mediating immunopathological responses in the mammalian host, we perorally challenged microbiota-depleted IL-10-/- mice with these strains. All strains stably colonized the murine gastrointestinal tract with comparably high loads. Remarkably, pepP deficiency was associated with less severe induced malaise, with less distinct apoptotic and innate immune cell responses, but also with more pronounced proliferative/regenerative epithelial cell responses in the large intestine at d6post-infection. Furthermore, pro-inflammatory mediators were lower in the colon, ileum, and mesenteric lymph nodes of mice that had been challenged with the ΔpepP mutant compared to the WT or pepP complemented strains. This also held true for extra-intestinal organs including liver, kidneys, and lungs, and, strikingly, to systemic compartments. Taken together, protease PepP is a novel virulence determinant involved in mediating campylobacteriosis. The finding that apoptosis in the colon is significantly diminished in mice infected with the pepP mutant highlights the epithelial layer as the first and main target of PepP in the intestine.

Multidrug-Resistant Pseudomonas aeruginosa Accelerate Intestinal, Extra-Intestinal, and Systemic Inflammatory Responses in Human Microbiota-Associated Mice With Subacute Ileitis.

The globally rising incidences of multidrug-resistant (MDR) Pseudomonas aeruginosa (Psae) in humans and live-stock animals has prompted the World Health Organization to rate MDR Psae as serious threat for human health. Only little is known, however, regarding factors facilitating gastrointestinal Psae-acquisition by the vertebrate host and subsequently induced inflammatory sequelae. In the present study, we addressed whether subacute ileitis predisposed mice harboring a human gut microbiota for intestinal MDR Psae carriage and whether inflammatory responses might be induced following peroral challenge with the opportunistic pathogen. To accomplish this, secondary abiotic mice were associated with a human gut microbiota by fecal microbiota transplantation. Ten days later (i.e., on day 0), subacute ileitis was induced in human microbiota associated (hma) mice by peroral low-dose Toxoplasma gondii infection. On day 5 post-infection, mice were perorally challenged with 109 colony forming units of a clinical MDR Psae isolate by gavage and the fecal bacterial loads surveyed thereafter. Four days post-peroral challenge, only approximately one third of mice with a human gut microbiota and subacute ileitis harbored the opportunistic pathogen in the intestinal tract. Notably, the gut microbiota composition was virtually unaffected by the Psae-carriage status during subacute ileitis of hma mice. The Psae challenge resulted, however, in more pronounced intestinal epithelial apoptotic cell and T lymphocyte responses upon ileitis induction that were not restricted to the ileum, but also affected the large intestines. Higher Psae-induced abundances of T cells could additionally be observed in extra-intestinal compartments including liver, kidney, lung, and heart of hma mice with subacute ileitis. Furthermore, higher apoptotic cell numbers, but lower anti-inflammatory IL-10 concentrations were assessed in the liver of Psae as compared to mock treated mice with ileitis. Remarkably, Psae-challenge was accompanied by even more pronounced systemic secretion of pro-inflammatory cytokines such as TNF and IL-6 at day 9 post ileitis induction. In conclusion, whereas in one third of hma mice with subacute ileitis Psae could be isolated from the intestines upon peroral challenge, the opportunistic pathogen was responsible for inflammatory sequelae in intestinal, extra-intestinal, and even systemic compartments and thus worsened subacute ileitis outcome irrespective of the Psae-carrier status.

Pituitary Adenylate Cyclase-Activating Polypeptide-A Neuropeptide as Novel Treatment Option for Subacute Ileitis in Mice Harboring a Human Gut Microbiota.

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) is well-known for its important functions in immunity and inflammation. Data regarding anti-inflammatory properties of PACAP in the intestinal tract are limited, however. In our present preclinical intervention study we addressed whether PACAP treatment could alleviate experimental subacute ileitis mimicking human gut microbiota conditions. Therefore, secondary abioitic mice were subjected to human fecal microbiota transplantation (FMT) and perorally infected with low-dose Toxoplasma gondii to induce subacute ileitis on day 0. From day 3 until day 8 post-infection, mice were either treated with synthetic PACAP38 or placebo. At day 9 post-infection, placebo, but not PACAP treated mice exhibited overt macroscopic sequelae of intestinal immunopathology. PACAP treatment further resulted in less distinct apoptotic responses in ileal and colonic epithelia that were accompanied by lower T cell numbers in the mucosa and lamina propria and less secretion of pro-inflammatory cytokines in intestinal ex vivo biopsies. Notably, ileitis-associated gut microbiota shifts were less distinct in PACAP as compared to placebo treated mice. Inflammation-ameliorating effects of PACAP were not restricted to the intestines, but could also be observed in extra-intestinal including systemic compartments as indicated by lower apoptotic cell counts and less pro-inflammatory cytokine secretion in liver and lungs taken from PACAP treated as compared to placebo control mice, which also held true for markedly lower serum TNF and IL-6 concentrations in the former as compared to the latter. Our preclinical intervention study provides strong evidence that synthetic PACAP alleviates subacute ileitis and extra-intestinal including systemic sequelae of T cell-driven immunopathology. These findings further support PACAP as a novel treatment option for intestinal inflammation including inflammatory bowel diseases (IBD).

Protease Activity of Campylobacter jejuni HtrA Modulates Distinct Intestinal and Systemic Immune Responses in Infected Secondary Abiotic IL-10 Deficient Mice.

Even though human Campylobacter jejuni infections are progressively increasing worldwide, the underlying molecular mechanisms of pathogen-host-interactions are still not fully understood. We have recently shown that the secreted serine protease HtrA plays a key role in C. jejuni cellular invasion and transepithelial migration in vitro, and is involved in the onset of intestinal pathology in murine infection models in vivo. In the present study, we investigated whether the protease activity of HtrA had an impact in C. jejuni induced acute enterocolitis. For this purpose, we perorally infected secondary abiotic IL-10-/- mice with wildtype C. jejuni strain NCTC11168 (11168WT) or isogenic bacteria carrying protease-inactive HtrA with a single point mutation at S197A in the active center (11168HtrA-S197A). Irrespective of the applied pathogenic strain, mice harbored similar C. jejuni loads in their feces and exhibited comparably severe macroscopic signs of acute enterocolitis at day 6 postinfection (p.i.). Interestingly, the 11168HtrA-S197A infected mice displayed less pronounced colonic apoptosis and immune cell responses, but enhanced epithelial proliferation as compared to the 11168WT strain infected controls. Furthermore, less distinct microscopic sequelae in 11168HtrA-S197A as compared to parental strain infected mice were accompanied by less distinct colonic secretion of pro-inflammatory cytokines such as MCP-1, IL-6, TNF, and IFN-γ in the former as compared to the latter. Strikingly, the S197A point mutation was additionally associated with less pronounced systemic pro-inflammatory immune responses as assessed in serum samples. In conclusion, HtrA is a remarkable novel virulence determinant of C. jejuni, whose protease activity is not required for intestinal colonization and establishment of disease, but aggravates campylobacteriosis by triggering apoptosis and pro-inflammatory immune responses.

Immunopathological properties of the Campylobacter jejuni flagellins and the adhesin CadF as assessed in a clinical murine infection model.

BACKGROUND: Campylobacter jejuni infections constitute serious threats to human health with increasing prevalences worldwide. Our knowledge regarding the molecular mechanisms underlying host-pathogen interactions is still limited. Our group has established a clinical C. jejuni infection model based on abiotic IL-10-/- mice mimicking key features of human campylobacteriosis. In order to further validate this model for unraveling pathogen-host interactions mounting in acute disease, we here surveyed the immunopathological features of the important C. jejuni virulence factors FlaA and FlaB and the major adhesin CadF (Campylobacter adhesin to fibronectin), which play a role in bacterial motility, protein secretion and adhesion, respectively. METHODS AND RESULTS: Therefore, abiotic IL-10-/- mice were perorally infected with C. jejuni strain 81-176 (WT) or with its isogenic flaA/B (ΔflaA/B) or cadF (ΔcadF) deletion mutants. Cultural analyses revealed that WT and ΔcadF but not ΔflaA/B bacteria stably colonized the stomach, duodenum and ileum, whereas all three strains were present in the colon at comparably high loads on day 6 post-infection. Remarkably, despite high colonic colonization densities, murine infection with the ΔflaA/B strain did not result in overt campylobacteriosis, whereas mice infected with ΔcadF or WT were suffering from acute enterocolitis at day 6 post-infection. These symptoms coincided with pronounced pro-inflammatory immune responses, not only in the intestinal tract, but also in other organs such as the liver and kidneys and were accompanied with systemic inflammatory responses as indicated by increased serum MCP-1 concentrations following C. jejuni ΔcadF or WT, but not ΔflaA/B strain infection. CONCLUSION: For the first time, our observations revealed that the C. jejuni flagellins A/B, but not adhesion mediated by CadF, are essential for inducing murine campylobacteriosis. Furthermore, the secondary abiotic IL-10-/- infection model has been proven suitable not only for detailed investigations of immunological aspects of campylobacteriosis, but also for differential analyses of the roles of distinct C. jejuni virulence factors in induction and progression of disease.

Peroral Low-Dose Toxoplasma gondii Infection of Human Microbiota-Associated Mice - A Subacute Ileitis Model to Unravel Pathogen-Host Interactions.

Within 1 week following high-dose Toxoplasma gondii infection, mice develop lethal necrotizing ileitis. However, data from a subacute T. gondii-induced ileitis model are scarce. Therefore, mice harboring a human gut microbiota were perorally infected with one cyst of T. gondii. Within 9 days post-infection, the intestinal microbiota composition shifted towards higher loads of commensal enterobacteria and enterococci. Following T. gondii infection, mice were clinically only mildly affected, whereas ≈60% of mice displayed fecal blood and mild-to-moderate ileal histopathological changes. Intestinal inflammation was further characterized by increased apoptotic intestinal epithelial cells, which were accompanied by elevated proliferating gut epithelial cell numbers. As compared to naive controls, infected mice displayed elevated numbers of intestinal T lymphocytes and regulatory T-cells and increased pro-inflammatory mediator secretion. Remarkably, T. gondii-induced apoptotic and pro-inflammatory immune responses were not restricted to the gut, but could also be observed in extra-intestinal compartments including kidney, liver, and lung. Strikingly, low-dose T. gondii infection resulted in increased serum levels of pro- and anti-inflammatory cytokines. In conclusion, the here presented subacute ileitis model following peroral low-dose T. gondii infection of humanized mice allows for detailed investigations of the molecular mechanism underlying the "ménage à trois" of pathogens, human gut microbiota, and immunity.

Anti-inflammatory Effects of the Octapeptide NAP in Human Microbiota-Associated Mice Suffering from Subacute Ileitis.

The octapeptide NAP is well known for its neuroprotective properties. We here investigated whether NAP treatment could alleviate pro-inflammatory immune responses during experimental subacute ileitis. To address this, mice with a human gut microbiota were perorally infected with one cyst of Toxoplasma gondii (day 0) and subjected to intraperitoneal synthetic NAP treatment from day 1 until day 8 postinfection (p.i.). Whereas placebo (PLC) control animals displayed subacute ileitis at day 9 p.i., NAP-treated mice exhibited less pronounced pro-inflammatory immune responses as indicated by lower numbers of intestinal mucosal T and B lymphocytes and lower interferon (IFN)-γ concentrations in mesenteric lymph nodes. The NAP-induced anti-inflammatory effects were not restricted to the intestinal tract but could also be observed in extra-intestinal including systemic compartments, given that pro-inflammatory cytokines were lower in liver, kidney, and lung following NAP as compared to PLC application, whereas at day 9 p.i., colonic and serum interleukin (IL)-10 concentrations were higher in the former as compared to the latter. Remarkably, probiotic commensal bifidobacterial loads were higher in the ileal lumen of NAP as compared to PLC-treated mice with ileitis. Our findings thus further support that NAP might be regarded as future treatment option directed against intestinal inflammation.

Function of Serine Protease HtrA in the Lifecycle of the Foodborne Pathogen Campylobacter jejuni.

Campylobacter jejuni is a major food-borne zoonotic pathogen, responsible for a large proportion of bacterial gastroenteritis cases, as well as Guillian-Barré and Miller-Fisher syndromes. During infection, tissue damage is mainly caused by bacteria invading epithelial cells and traversing the intestinal barrier. C. jejuni is able to enter the lamina propria and the bloodstream and may move into other organs, such as spleen, liver, or mesenteric lymph nodes. However, the involved molecular mechanisms are not fully understood. C. jejuni can transmigrate effectively across polarized intestinal epithelial cells mainly by the paracellular route using the serine protease high-temperature requirement A (HtrA). However, it appears that HtrA has a dual function, as it also acts as a chaperone, interacting with denatured or misfolded periplasmic proteins under stress conditions. Here, we review recent progress on the role of HtrA in C. jejuni pathogenesis. HtrA can be transported into the extracellular space and cleaves cell-to-cell junction factors, such as E-cadherin and probably others, disrupting the epithelial barrier and enabling paracellular transmigration of the bacteria. The secretion of HtrA is a newly discovered strategy also utilized by other pathogens. Thus, secreted HtrA proteases represent highly attractive targets for anti-bacterial treatment and may provide a suitable candidate for vaccine development.

Toll-Like Receptor-4 Dependent Inflammatory Responses Following Intestinal Colonization of Secondary Abiotic IL10-Deficient Mice with Multidrug-Resistant Pseudomonas Aeruginosa.

The rising incidences of infections with multidrug-resistant (MDR) Gram-negative bacteria including Pseudomonas aeruginosa (PA) have gained increasing attention in medicine, but also in the general public and global health politics. The mechanisms underlying opportunistic pathogen-host interactions are unclear, however. To address this, we challenged secondary abiotic IL10-/- mice deficient for Toll-like receptor-4 (TLR4-/- × IL10-/-), the main receptor of the Gram-negative cell wall constituent lipopolysaccharide, with a clinical MDR PA isolate. Despite higher intestinal colonization densities, apoptotic colonic epithelial cell numbers were lower in TLR4-/- × IL10-/- mice as compared to IL10-/- controls at day 14 postinfection (p.i.), whereas proliferating/regenerating cells had increased in the latter only. Furthermore, PA-colonized TLR4-/- × IL10-/- mice displayed less distinct innate and adaptive immune cell responses in the colon as compared to IL10-/- counterparts that were accompanied by lower nitric oxide concentrations in mesenteric lymph nodes in the former at day 14 p.i. Conversely, splenic NO levels were higher in both naive and PA-colonized TLR4-deficient IL10-/- mice versus IL10-/- controls. Remarkably, intestinal MDR PA was able to translocate to extra-intestinal including systemic compartments of TLR4-/- × IL10-/- mice only. Hence, MDR PA-induced intestinal and systemic immune responses observed in secondary abiotic IL10-/- mice are TLR4-dependent.

Toll-like receptor-4 differentially mediates intestinal and extra-intestinal immune responses upon multi-drug resistant Pseudomonas aeruginosa association of IL10-/- mice with chronic colitis.

BACKGROUND: Infections with multi-drug resistant (MDR) Gram-negative bacteria including Pseudomonas aeruginosa (PA) have become a serious threat particularly in hospitalized patients with immunopathological co-morbidities. The well-balanced interplay between immune cells, pattern recognition receptors such as Toll-like receptor (TLR)-4 sensing lipopolysaccharide from Gram-negative bacteria including PA, and evolving pathways is crucial to prevent the host from invading (opportunistic) pathogens. Information regarding the molecular mechanisms underlying the interactions between intestinal carriage of MDR PA and host immunity during chronic large intestinal inflammation is scarce, however. METHODS AND RESULTS: We therefore perorally challenged conventionally colonized TLR4-deficient IL10-/- mice and IL10-/- counterparts displaying comparably severe chronic colitis with a clinical MDR PA strain. PA could more sufficiently establish in the intestinal tract of TLR4-deficient IL10-/- mice until day 14 postinfection (p.i.), whereas within 48 h the majority of IL10-/- mice had already expelled the opportunistic pathogen from their guts. Intestinal colonization properties of PA in TLR4-deficient IL10-/- mice were associated with distinct genotype-dependent differences in gut microbiota compositions before challenge given that TLR4-deficient IL10-/- mice harbored more fecal enterobacteria and enterococci, but lower Clostridium/Eubacterium burdens. At day 14 p.i., PA-induced increases in colonic immune cells such as macrophages, monocytes and T-lymphocytes could be observed in TLR4-deficient IL10-/- mice, but not IL10-/- counterparts, that were accompanied by a more distinct secretion of IFN-γ in the colon and TNF in the mesenteric lymph nodes (MLN) of the former as compared to the latter. Conversely, splenic TNF levels were lower in TLR4-deficient IL10-/- mice as compared to IL10-/- controls at day 14 p.i. Interestingly, more pronounced apoptotic responses could be assessed in colonic epithelia of PA-challenged IL10-/- mice only. This was paralleled by enhanced pro-inflammatory cytokine secretion not only in the intestines, but also in extra-intestinal compartments of IL10-/- mice as indicated by increased concentrations of nitric oxide in the colon, IFN-γ in the MLN and IL-12p70 in the spleen at day 14 p.i. CONCLUSIONS: Under chronic intestinal inflammatory conditions including IL10-/- colitis MDR PA-association results in well-orchestrated TLR4-dependent immune responses both in intestinal and extra-intestinal compartments. Further studies should unravel the underlying molecular mechanisms in more detail.

Lactobacillus johnsonii ameliorates intestinal, extra-intestinal and systemic pro-inflammatory immune responses following murine Campylobacter jejuni infection.

Campylobacter jejuni infections are progressively increasing worldwide. Probiotic treatment might open novel therapeutic or even prophylactic approaches to combat campylobacteriosis. In the present study secondary abiotic mice were generated by broad-spectrum antibiotic treatment and perorally reassociated with a commensal murine Lactobacillus johnsonii strain either 14 days before (i.e. prophylactic regimen) or 7 days after (i.e. therapeutic regimen) peroral C. jejuni strain 81-176 infection. Following peroral reassociation both C. jejuni and L. johnsonii were able to stably colonize the murine intestinal tract. Neither therapeutic nor prophylactic L. johnsonii application, however, could decrease intestinal C. jejuni burdens. Notably, C. jejuni induced colonic apoptosis could be ameliorated by prophylactic L. johnsonii treatment, whereas co-administration of L. johnsonii impacted adaptive (i.e. T and B lymphocytes, regulatory T cells), but not innate (i.e. macrophages and monocytes) immune cell responses in the intestinal tract. Strikingly, C. jejuni induced intestinal, extra-intestinal and systemic secretion of pro-inflammatory mediators (such as IL-6, MCP-1, TNF and nitric oxide) could be alleviated by peroral L. johnsonii challenge. In conclusion, immunomodulatory probiotic species might offer valuable strategies for prophylaxis and/or treatment of C. jejuni induced intestinal, extra-intestinal as well as systemic pro-inflammatory immune responses in vivo.

Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice.

Secondary abiotic mice generated by broad-spectrum antibiotic treatment provide a valuable tool for association studies with microbiota derived from different vertebrate hosts. We here generated human microbiota-associated (hma) mice by human fecal microbiota transplantation of secondary abiotic mice and performed a comprehensive survey of the intestinal microbiota dynamics in offspring of hma mice over 18 weeks following weaning as compared to their mothers applying both cultural and molecular methods. Mice were maintained under standard hygienic conditions with open cages, handled under aseptic conditions, and fed autoclaved chow and water. Within 1 week post weaning, fecal loads of commensal enterobacteria and enterococci had decreased, whereas obligate anaerobic bacteria such as Bacteroides/Prevotella species and clostridia were stably colonizing the intestines of hma offspring at high loads. Lactobacilli numbers were successively increasing until 18 weeks post weaning in both hma offspring and mothers, whereas by then, bifidobacteria were virtually undetectable in the former only. Interestingly, fecal lactobacilli and bifidobacteria were higher in mothers as compared to their offspring at 5 and 18 weeks post weaning. We conclude that the intestinal microbiota composition changes in offspring of hma mice, but also their mothers over time particularly affecting aerobic and microaerobic species.

Immune Responses to Broad-Spectrum Antibiotic Treatment and Fecal Microbiota Transplantation in Mice.

Compelling evidence demonstrates the pivotal role of the commensal intestinal microbiota in host physiology and the detrimental effects of its perturbations following antibiotic treatment. Aim of this study was to investigate the impact of antibiotics induced depletion and subsequent restoration of the intestinal microbiota composition on the murine mucosal and systemic immunity. To address this, conventional C57BL/6j mice were subjected to broad-spectrum antibiotic treatment for 8 weeks. Restoration of the intestinal microbiota by peroral fecal microbiota transplantation (FMT) led to reestablishment of small intestinal CD4+, CD8+, and B220+ as well as of colonic CD4+ cell numbers as early as 7 days post-FMT. However, at d28 following FMT, colonic CD4+ and B220+ cell numbers were comparable to those in secondary abiotic (ABx) mice. Remarkably, CD8+ cell numbers were reduced in the colon upon antibiotic treatment, and FMT was not sufficient to restore this immune cell subset. Furthermore, absence of gut microbial stimuli resulted in decreased percentages of memory/effector T cells, regulatory T cells, and activated dendritic cells in the small intestine, colon, mesenteric lymph nodes (MLN), and spleen. Concurrent antibiotic treatment caused decreased cytokine production (IFN-γ, IL-17, IL-22, and IL-10) of CD4+ cells in respective compartments. These effects were, however, completely restored upon FMT. In summary, broad-spectrum antibiotic treatment resulted in profound local (i.e., small and large intestinal), peripheral (i.e., MLN), and systemic (i.e., splenic) changes in the immune cell repertoire that could, at least in part, be restored upon FMT. Further studies need to unravel the distinct molecular mechanisms underlying microbiota-driven changes in immune homeostasis subsequently providing novel therapeutic or even preventive approaches in human immunopathologies.