Ursula Grundmann

Campylobacter jejuni infection of infant mice: acute enterocolitis is followed by asymptomatic intestinal and extra-intestinal immune responses.

Campylobacter (C.) jejuni is among the leading bacterial agents causing enterocolitis worldwide. Despite the high prevalence of C. jejuni infections and its significant medical and economical consequences, intestinal pathogenesis is poorly understood. This is mainly due to the lack of appropriate animal models. In the age of 3 months, adult mice display strong colonization resistance (CR) against C. jejuni. Previous studies underlined the substantial role of the murine intestinal microbiota in maintaining CR. Due to the fact that the host-specific gut flora establishes after weaning, we investigated CR against C. jejuni in 3-week-old mice and studied intestinal and extra-intestinal immunopathogenesis as well as age dependent differences of the murine colon microbiota. In infant animals infected orally immediately after weaning C. jejuni strain B2 could stably colonize the gastrointestinal tract for more than 100 days. Within six days following infection, infant mice developed acute enterocolitis as indicated by bloody diarrhea, colonic shortening, and increased apoptotic cell numbers in the colon mucosa. Similar to human campylobacteriosis clinical disease manifestations were self-limited and disappeared within two weeks. Interestingly, long-term C. jejuni infection was accompanied by distinct intestinal immune and inflammatory responses as indicated by increased numbers of T- and B-lymphocytes, regulatory T-cells, neutrophils, as well as apoptotic cells in the colon mucosa. Strikingly, C. jejuni infection also induced a pronounced influx of immune cells into extra-intestinal sites such as liver, lung, and kidney. Furthermore, C. jejuni susceptible weaned mice harbored a different microbiota as compared to resistant adult animals. These results support the essential role of the microflora composition in CR against C. jejuni and demonstrate that infant mouse models resemble C. jejuni mediated immunopathogenesis including the characteristic self-limited enterocolitis in human campylobacteriosis. Furthermore, potential clinical and immunological sequelae of chronic C. jejuni carriers in humans can be further elucidated by investigation of long-term infected infant mice. The observed extraintestinal disease manifestations might help to unravel the mechanisms causing complications such as reactive arthritis or Guillain-Barré syndrome.

The role of serine protease HtrA in acute ulcerative enterocolitis and extra-intestinal immune responses during Campylobacter jejuni infection of gnotobiotic IL-10 deficient mice

Campylobacter jejuni infections have a high prevalence worldwide and represent a significant socioeconomic burden. C. jejuni can cross the intestinal epithelial barrier as visualized in biopsies derived from human patients and animal models, however, the underlying molecular mechanisms and associated immunopathology are still not well understood. We have recently shown that the secreted serine protease HtrA (high temperature requirement A) plays a key role in C. jejuni cellular invasion and transmigration across polarized epithelial cells in vitro. In the present in vivo study we investigated the role of HtrA during C. jejuni infection of mice. We used the gnotobiotic IL-10−/− mouse model to study campylobacteriosis following peroral infection with the C. jejuni wild-type (WT) strain NCTC11168 and the isogenic, non-polar NCTC11168ΔhtrA deletion mutant. Six days post infection (p.i.) with either strain mice harbored comparable intestinal C. jejuni loads, whereas ulcerative enterocolitis was less pronounced in mice infected with the ΔhtrA mutant strain. Moreover, ΔhtrA mutant infected mice displayed lower apoptotic cell numbers in the large intestinal mucosa, less colonic accumulation of neutrophils, macrophages and monocytes, lower large intestinal nitric oxide, IFN-γ, and IL-6 as well as lower TNF-α and IL-6 serum concentrations as compared to WT strain infected mice at day 6 p.i. Notably, immunopathological responses were not restricted to the intestinal tract given that liver and kidneys exhibited mild histopathological changes 6 days p.i. with either C. jejuni strain. We also found that hepatic and renal nitric oxide levels or renal TNF-α concentrations were lower in the ΔhtrA mutant as compared to WT strain infected mice. In conclusion, we show here that the C. jejuni HtrA protein plays a pivotal role in inducing host cell apoptosis and immunopathology during murine campylobacteriosis in the gut in vivo.

Pituitary adenylate cyclase-activating polypeptide ameliorates experimental acute ileitis and extra-intestinal sequelae

Background The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis. Methodology/Principal Findings Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner. Conclusion/Significance Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases.

The role of gelatinases in Campylobacter jejuni infection of gnotobiotic mice

Matrix metalloproteinases (MMP)-2 and -9 (also referred to gelatinases-A and -B, respectively) are upregulated in the inflamed gut of mice and men. We recently demonstrated that synthetic gelatinase blockage reduced large intestinal pro-inflammatory immune responses and apoptosis following murine Campylobacter (C.) jejuni infection. In order to address which gelatinase mediates C. jejuni-induced immune responses, gnotobiotic MMP-2–/–, MMP-9–/–, and wildtype (WT) mice were generated by broadspectrum antibiotic treatment and perorally infected with C. jejuni strain 81-176. The pathogen stably colonized the murine intestinal tract irrespective of the genotype but did not translocate to extra-intestinal compartments. At days 8 and 14 postinfection (p.i.), less pronounced colonic histopathological changes were observed in infected MMP-2–/– mice, less distinct epithelial apoptosis, but more epithelial proliferation in both MMP-2–/– and MMP-9–/– mice, as compared to WT controls. Reduced immune responses in gelatinase-deficient mice were characterized by lower numbers of effector as well as innate and adaptive immune cells within the colonic mucosa and lamina propria. The expression of IL-22, IL-18, IL-17A, and IL-1β mRNA was higher in the colon of MMP-2–/– as compared to WT mice. In conclusion, both MMP-2 and MMP-9 are differentially involved in mediating C. jejuni-induced intestinal immunopathology.

Matrix Metalloproteinase-2 Mediates Intestinal Immunopathogenesis in Campylobacter Jejuni-Infected Infant Mice

Increased levels of the matrix metalloproteinases (MMPs)-2 and -9 (also referred to gelatinase-A and -B, respectively) can be detected in the inflamed gut. We have recently shown that synthetic gelatinase blockage reduces colonic apoptosis and pro-inflammatory immune responses following murine Campylobacter (C.) jejuni infection. In order to dissect whether MMP-2 and/or MMP-9 is involved in mediating C. jejuni-induced immune responses, infant MMP-2–/–, MMP-9–/–, and wildtype (WT) mice were perorally infected with the C. jejuni strain B2 immediately after weaning. Whereas, at day 2 postinfection (p.i.), fecal C. jejuni B2 loads were comparable in mice of either genotype, mice expelled the pathogen from the intestinal tract until day 4 p.i. Six days p.i., colonic MMP-2 but not MMP-9 mRNA was upregulated in WT mice. Remarkably, infected MMP-2–/– mice exhibited less frequent abundance of blood in feces, less distinct colonic histopathology and apoptosis, lower numbers of effector as well as innate and adaptive immune cells within the colonic mucosa, and higher colonic IL-22 mRNA levels as compared to infected WT mice. In conclusion, these results point towards an important role of MMP-2 in mediating C. jejuni-induced intestinal immunopathogenesis.

Colonic Expression of Genes Encoding Inflammatory Mediators and Gelatinases During Campylobacter Jejuni Infection of Conventional Infant Mice

Within 1 week following peroral Campylobacter jejuni infection, infant mice develop acute enteritis resolving thereafter. We here assessed colonic expression profiles of mediators belonging to the IL-23/IL-22/IL-18 axis and of matrix-degrading gelatinases MMP-2 and MMP-9 at day 6 post C. jejuni strain 81-176 infection. Whereas the pathogen readily colonized the intestines of infant IL-18–/– mice only, colonic mucin-2 mRNA, a pivotal mucus constituent, was downregulated in IL-22–/– mice and accompanied by increased expression of pro-inflammatory cytokines including IFN-γ, TNF, IL-17A, and IL-1β. Furthermore, in both naive and infected IL-22–/– mice, colonic expression of IL-23p19 and IL-18 was lower as compared to wildtype mice, whereas, conversely, colonic IL-22 mRNA levels were lower in IL-18–/– and colonic IL-18 expression lower in IL-23p19–/– as compared to wildtype mice. Moreover, colonic expression of MMP-2 and MMP-9 and their endogenous inhibitor TIMP-1 were lower in IL-22–/– as compared to wildtype mice at day 6 postinfection. In conclusion, mediators belonging of the IL-23/IL-22/IL-18 axis as well as the gelatinases MMP-2 and MMP-9 are involved in mediating campylobacteriosis of infant mice in a differentially regulated fashion.

The Role of IL-23, IL-22, and IL-18 in Campylobacter Jejuni Infection of Conventional Infant Mice

We have recently shown that, within 1 week following peroral Campylobacter jejuni infection, conventional infant mice develop self-limiting enteritis. We here investigated the role of IL-23, IL-22, and IL-18 during C. jejuni strain 81-176 infection of infant mice. The pathogen efficiently colonized the intestines of IL-18–/– mice only, but did not translocate to extra-intestinal compartments. At day 13 postinfection (p.i.), IL-22–/– mice displayed lower colonic epithelial apoptotic cell numbers as compared to wildtype mice, whereas, conversely, colonic proliferating cells increased in infected IL-22–/– and IL-18–/– mice. At day 6 p.i., increases in neutrophils, T and B lymphocytes were less pronounced in gene-deficient mice, whereas regulatory T cell numbers were lower in IL-23p19–/– and IL-22–/– as compared to wildtype mice, which was accompanied by increased colonic IL-10 levels in the latter. Until then, colonic pro-inflammatory cytokines including TNF, IFN-γ, IL-6, and MCP-1 increased in IL-23p19–/– mice, whereas IL-18–/– mice exhibited decreased cytokine levels and lower colonic numbers of T and B cell as well as of neutrophils, macrophages, and monocytes as compared to wildtype controls. In conclusion, IL-23, IL-22, and IL-18 are differentially involved in mediating C. jejuni-induced immunopathology of conventional infant mice.

Interleukin-18 Mediates Immune Responses to Campylobacter jejuni Infection in Gnotobiotic Mice

Background Human Campylobacter jejuni infections are progressively rising worldwide. Information about the molecular mechanisms underlying campylobacteriosis, however, are limited. In the present study we investigated whether cytokines such as IL-23, IL-22 and IL-18, which share pivotal functions in host immunity, were involved in mediating intestinal and systemic immunopathological responses upon C. jejuni infection. Methodology/Principal Findings To assure stable infection, gnotobiotic (i.e. secondary abiotic) IL-23p19-/-, IL-22-/- and IL-18-/- mice were generated by broad-spectrum antibiotic treatment. Following peroral C. jejuni strain 81–176 infection, mice of all genotypes harbored comparably high pathogenic loads in their intestines. As compared to wildtype controls, however, IL-18-/- mice displayed less distinct C. jejuni induced sequelae as indicated by less pronounced large intestinal shrinkage and lower numbers of apoptotic cells in the colonic epithelial layer at day 8 postinfection (p.i.). Furthermore, lower colonic numbers of adaptive immune cells including regulatory T cells and B lymphocytes were accompanied by less distinct secretion of pro-inflammatory cytokines such as TNF and IFN-γ and lower IL-17A mRNA expression levels in colonic ex vivo biopsies of infected IL-18-/- as compared to wildtype mice. Upon C. jejuni infection, colonic IL-23p19 expression was up-regulated in IL-18-/- mice only, whereas IL-22 mRNA levels were lower in uninfected and infected IL-23p19-/- as well as infected IL-18-/- as compared to respective wildtype control mice. Remarkably, not only intestinal, but also systemic infection-induced immune responses were less pronounced in IL-18-/- mice as indicated by lower TNF, IFN-γ and IL-6 serum levels as compared to wildtype mice. Conclusion/Significance We here show for the first time that IL-18 is essentially involved in mediating C. jejuni infection in the gnotobiotic mouse model. Future studies need to further unravel the underlying regulatory mechanisms orchestrating pathogen-host interaction.

Selective gelatinase inhibition reduces apoptosis and pro-inflammatory immune cell responses in Campylobacter jejuni-infected gnotobiotic IL-10 deficient mice

Increased levels of the matrix metalloproteinases-2 and -9 (also referred to gelatinase-A and -B, respectively) can be detected in intestinal inflammation. We have recently shown that selective gelatinase blockage by the synthetic compound RO28-2653 ameliorates acute murine ileitis and colitis. We here investigated whether RO28-2653 exerts anti-inflammatory effects in acute Campylobacter jejuni-induced enterocolitis of gnotobiotic IL-10(-/-) mice generated following antibiotic treatment. Mice were perorally infected with C. jejuni (day 0) and either treated with RO28-2653 (75 mg/kg body weight/day) or placebo from day 1 until day 6 post infection (p.i.) by gavage. Irrespective of the treatment, infected mice displayed comparable pathogen loads within the gastrointestinal tract. Following RO28-2653 administration, however, infected mice exhibited less severe symptoms such as bloody diarrhea as compared to placebo controls. Furthermore, less distinct apoptosis but higher numbers of proliferating cells could be detected in the colon of RO28-2653-treated as compared to placebo-treated mice at day 7 p.i. Remarkably, gelatinase blockage resulted in lower numbers of T- and B-lymphocytes as well as macrophages and monocytes in the colonic mucosa of C. jejuni-infected gnotobiotic IL-10(-/-) mice. Taken together, synthetic gelatinase inhibition exerts anti-inflammatory effects in experimental campylobacteriosis.

Microbiota composition and immune responses during Campylobacter jejuni infection in conventionally colonized IL-10−/− mice lacking nucleotide oligomerization domain 2

Host immune responses are pivotal for combating enteropathogenic infections. We here assessed the impact of the innate receptor nucleotide oligomerization domain protein 2 (NOD2) in murine Campylobacter jejuni-infection. Conventionally colonized IL-10–/– mice lacking NOD2 and IL-10–/– controls were perorally challenged with C. jejuni strain 81-176 and displayed comparable pathogenic colonization of intestines until day 14 postinfection (p.i.). Whereas overall intestinal microbiota compositions were comparable in naive mice, NOD2–/– IL-10–/– mice exhibited less fecal bifidobacteria and lactobacilli than IL-10–/– counterparts after infection. Interestingly, NOD2–/– IL-10–/– mice were clinically more compromised during the early phase of infection, whereas, conversely, IL-10–/– animals exhibited more frequently bloody feces lateron. While colonic apoptotic cell and T lymphocyte numbers were comparable in either C. jejuni-infected mice, B lymphocytes were lower in the colon of infected NOD2–/– IL-10–/– mice versus controls. At day 14 p.i., colonic TNF and IL-23p19 mRNA levels were upregulated in NOD2–/– IL-10–/– mice only. Translocation rates of intestinal commensals to mesenteric lymphnodes and extra-intestinal compartments including liver and kidney were comparable, whereas viable bacteria were more frequently detected in spleens derived from IL-10–/– as compared to NOD2–/– IL-10–/– mice. In conclusion, NOD2 is involved during C. jejuni infection in conventionally colonized IL-10–/– mice in a time-dependent manner.