Absence of Nucleotide-Oligomerization-Domain-2 Is Associated with Less Distinct Disease in Campylobacter jejuni Infected Secondary Abiotic IL-10 Deficient Mice.

Human Campylobacter jejuni-infections are progressively increasing worldwide. Despite their high prevalence and socioeconomic impact the underlying mechanisms of pathogen-host-interactions are only incompletely understood. Given that the innate immune receptor nucleotide-oligomerization-domain-2 (Nod2) is involved in clearance of enteropathogens, we here evaluated its role in murine campylobacteriosis. To address this, we applied Nod2-deficient IL-10-/- (Nod2-/- IL-10-/-) mice and IL-10-/- counterparts both with a depleted intestinal microbiota to warrant pathogen-induced enterocolitis. At day 7 following peroral C. jejuni strain 81-176 infection, Nod2 mRNA was down-regulated in the colon of secondary abiotic IL-10-/- and wildtype mice. Nod2-deficiency did neither affect gastrointestinal colonization nor extra-intestinal and systemic translocation properties of C. jejuni. Colonic mucin-2 mRNA was, however, down-regulated upon C. jejuni-infection of both Nod2-/- IL-10-/- and IL-10-/- mice, whereas expression levels were lower in infected, but also naive Nod2-/- IL-10-/- mice as compared to respective IL-10-/- controls. Remarkably, C. jejuni-infected Nod2-/- IL-10-/- mice were less compromised than IL-10-/- counterparts and displayed less distinct apoptotic, but higher regenerative cell responses in colonic epithelia. Conversely, innate as well as adaptive immune cells such as macrophages and monocytes as well as T lymphocytes and regulatory T-cells, respectively, were even more abundant in large intestines of Nod2-/- IL-10-/- as compared to IL-10-/- mice at day 7 post-infection. Furthermore, IFN-γ concentrations were higher in ex vivo biopsies derived from intestinal compartments including colon and mesenteric lymph nodes as well as in systemic tissue sites such as the spleen of C. jejuni infected Nod2-/- IL-10-/- as compared to IL10-/- counterparts. Whereas, at day 7 postinfection anti-inflammatory IL-22 mRNA levels were up-regulated, IL-18 mRNA was down-regulated in large intestines of Nod2-/- IL-10-/- vs. IL-10-/- mice. In summary, C. jejuni-infection induced less clinical signs and apoptosis, but more distinct colonic pro- and (of note) anti-inflammatory immune as well as regenerative cell responses in Nod2 deficient IL-10-/- as compared to IL-10-/- control mice. We conclude that, even though colonic Nod2 mRNA was down-regulated upon pathogenic challenge, Nod2-signaling is essentially involved in the well-balanced innate and adaptive immune responses upon C. jejuni-infection of secondary abiotic IL-10-/- mice, but does neither impact pathogenic colonization nor translocation.

Small Intestinal Pro-Inflammatory Immune Responses Following Campylobacter Jejuni Infection of Secondary Abiotic IL-10-/- Mice Lacking Nucleotide-Oligomerization-Domain-2.

Host immune responses are crucial for combating enteropathogenic infections including Campylobacter jejuni. Within 1 week following peroral C. jejuni infection, secondary abiotic IL-10-/- mice develop severe immunopathological sequelae affecting the colon (ulcerative enterocolitis). In the present study, we addressed whether pathogen-induced pro-inflammatory immune responses could also be observed in the small intestines dependent on the innate receptor nucleotide-oligomerization-domain-protein 2 (Nod2). Within 7 days following peroral infection, C. jejuni stably colonized the gastrointestinal tract of both IL-10-/- mice lacking Nod2 (Nod2-/- IL-10-/-) and IL-10-/- controls displaying bloody diarrhea with similar frequencies. Numbers of apoptotic and regenerating epithelial cells increased in the small intestines of C. jejuni-infected mice of either genotype that were accompanied by elevated ileal T and B lymphocyte counts. Notably, ileal T cell numbers were higher in C. jejuni-infected Nod2-/- IL-10-/- as compared to IL-10-/- counterparts. Furthermore, multifold increased concentrations of pro-inflammatory cytokines including IFN-γ, TNF, and MCP-1 could be measured in small intestinal ex vivo biopsies derived from C. jejuni-infected mice of either genotype. In conclusion, C. jejuni-induced pro-inflammatory immune responses affected the small intestines of both Nod2-/- IL-10-/- and IL-10-/- mice, whereas ileal T lymphocyte numbers were even higher in the former.

Immune responses upon Campylobacter jejuni infection of secondary abiotic mice lacking nucleotide-oligomerization-domain-2.

BACKGROUND: Campylobacter jejuni infections are of rising importance worldwide. Given that innate immune receptors including nucleotide-oligomerization-domain-2 (Nod2) are essentially involved in combating enteropathogenic infections, we here surveyed the impact of Nod2 in murine campylobacteriosis. METHODS AND RESULTS: In order to overcome physiological colonization resistance preventing from C. jejuni infection, we generated secondary abiotic Nod2-/- and wildtype (WT) mice by broad-spectrum antibiotic treatment. Mice were then perorally infected with C. jejuni strain 81-176 on 2 consecutive days and could be stably colonized by the pathogen at high loads. Notably, Nod2 deficiency did not affect gastrointestinal colonization properties of C. jejuni. Despite high intestinal pathogenic burdens mice were virtually uncompromised and exhibited fecal blood in single cases only. At day 7 postinfection (p.i.) similar increases in numbers of colonic epithelial apoptotic cells could be observed in mice of either genotype, whereas C. jejuni infected Nod2-/- mice displayed more distinct regenerative properties in the colon than WT controls. C. jejuni infection was accompanied by increases in distinct immune cell populations such as T lymphocytes and regulatory T cells in mice of either genotype. Increases in T lymphocytes, however, were less pronounced in large intestines of Nod2-/- mice at day 7 p.i. when compared to WT mice, whereas colonic numbers of B lymphocytes were elevated in WT controls only upon C. jejuni infection. At day 7 p.i., colonic pro-inflammatory mediators including nitric oxide, TNF, IFN-γ and IL-22 increased more distinctly in Nod2-/- as compared to WT mice, whereas C. jejuni induced IL-23p19 and IL-18 levels were lower in the large intestines of the former. Converse to the colon, however, ileal concentrations of nitric oxide, TNF, IFN-γ, IL-6 and IL-10 were lower in Nod2-/- as compared to WT mice at day 7 p.i. Even though MUC2 was down-regulated in C. jejuni infected Nod2-/- mice, this did not result in increased pathogenic translocation from the intestinal tract to extra-intestinal compartments. CONCLUSION: In secondary abiotic mice, Nod2 signaling is involved in the orchestrated host immune responses upon C. jejuni infection, but does not control pathogen loads in the gastrointestinal tract.

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.

Campylobacter jejuni infection of conventionally colonized mice lacking nucleotide-oligomerization-domain-2.

BACKGROUND: The nucleotide-binding oligomerisaton protein 2 (NOD2) constitutes a pivotal sensor of bacterial muramyl dipeptide and assures expression of distinct antimicrobial peptides and mediators produced by enterocytes and immune cells directed against pathogens including Campylobacter jejuni. We here elucidated the role of NOD2 during murine C. jejuni infection in more detail. RESULTS: Conventionally colonized NOD2 deficient (NOD2-/-) mice and corresponding wildtype (WT) counterparts were perorally infected with C. jejuni strain 81-176 on three consecutive days. The pathogen colonized both WT and NOD2-/- mice only sporadically until day 14 post infection (p.i.). However, the slightly higher prevalence of C. jejuni in NOD2-/- mice was accompanied by higher intestinal Escherichia coli loads known to facilitate C. jejuni colonization. Neither overt macroscopic (clinical) nor microscopic sequelae (such as colonic epithelial apoptosis) could be observed upon murine C. jejuni infection of either genotype. Innate immune responses were less distinctly induced in C. jejuni infected NOD2-/- versus WT mice as indicated by lower colonic numbers of neutrophils in the former. Conversely, adaptive immune cell counts including T lymphocytes were higher in large intestines of NOD2-/- as compared to WT mice that were paralleled by increased colonic IL-6 secretion and higher TNF and IL-18 mRNA expression levels in large intestines of the former. Only in NOD2-/- mice, however, colonic IL-22 mRNA expression was down-regulated at day 14 p.i. Whereas viable commensal intestinal bacteria could exclusively be detected in mesenteric lymph nodes and livers of NOD2-/- mice, bacterial translocation rates to kidneys and spleen were NOD2 independent. Notably, large intestinal mRNA expression levels of mucin-2, constituting a pivotal factor involved in epithelial barrier integrity, were comparable in naive and C. jejuni infected mice of either genotype. CONCLUSION: NOD2 is involved in the well-balanced regulation of innate and adaptive pro-inflammatory immune responses of conventional mice upon C. jejuni infection.

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-1ß. 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.

The IL-23/IL-22/IL-18 axis in murine Campylobacter jejuni infection.

BACKGROUND: Human Campylobacter jejuni infections are worldwide on the rise. Information about the distinct molecular mechanisms underlying campylobacteriosis, however, are scarce. In the present study we investigated whether cytokines including IL-23, IL-22 and IL-18 sharing pivotal functions in host immunity were involved in mediating immunopathological responses upon C. jejuni infection. RESULTS: To address this, conventionally colonized IL-23p19(-/-), IL-22(-/-) and IL-18(-/-) mice were perorally infected with C. jejuni strain ATCC 43431. Respective gene-deficient, but not wildtype mice were susceptible to C. jejuni infection and could be readily colonized with highest pathogenic loads in the terminal ileum and colon at day 14 postinfection (p.i.). In IL-23p19(-/-), IL-22(-/-) and IL-18(-/-) mice viable C. jejuni were detected in MLNs, but did not translocate to spleen, liver, kidney and blood in the majority of cases. Susceptible IL-22(-/-), but neither IL-23p19(-/-), nor IL-18(-/-) mice harbored higher intestinal commensal E. coli loads when compared to resistant wildtype mice. Alike C. jejuni, commensal E. coli did not translocate from the intestinal to extra-intestinal tissue sites. Despite C. jejuni infection, mice lacking IL-23p19, IL-22 or IL-18 exhibited less apoptotic cells, but higher numbers of proliferating cells in their colonic epithelium as compared to wildtype mice at day 14 p.i. Less pronounced apoptosis was parallelled by lower abundance of neutrophils within the colonic mucosa and lamina propria of infected IL-23p19(-/-) and IL-22(-/-) as compared to wildtype control mice, whereas less distinct colonic TNF secretion could be measured in IL-22(-/-) and IL-18(-/-) than in wildtype mice at day 14 p.i. Notably, in infected IL-22(-/-) mice, colonic IL-23p19 mRNA levels were lower, whereas the other way round, colonic IL-22 expression rates were lower in IL-23p19(-/-) mice as compared to wildtype controls. Moreover, IL-18 mRNA was less distinctly expressed in large intestines of naive and infected IL-22(-/-) mice, but not vice versa, given that IL-22 mRNA levels did not differ between in IL-18(-/-) and wildtype mice. CONCLUSION: Cytokines belonging to the IL-23/IL-22/IL-18 axis mediate immunopathological responses upon murine C. jejuni infection in a differentially orchestrated manner. Future studies need to further unravel the underlying regulatory mechanisms orchestrating pathogenic-host interaction.

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.

Ly6C(hi) Monocytes Provide a Link between Antibiotic-Induced Changes in Gut Microbiota and Adult Hippocampal Neurogenesis.

Antibiotics, though remarkably useful, can also cause certain adverse effects. We detected that treatment of adult mice with antibiotics decreases hippocampal neurogenesis and memory retention. Reconstitution with normal gut flora (SPF) did not completely reverse the deficits in neurogenesis unless the mice also had access to a running wheel or received probiotics. In parallel to an increase in neurogenesis and memory retention, both SPF-reconstituted mice that ran and mice supplemented with probiotics exhibited higher numbers of Ly6C(hi) monocytes in the brain than antibiotic-treated mice. Elimination of Ly6C(hi) monocytes by antibody depletion or the use of knockout mice resulted in decreased neurogenesis, whereas adoptive transfer of Ly6C(hi) monocytes rescued neurogenesis after antibiotic treatment. We propose that the rescue of neurogenesis and behavior deficits in antibiotic-treated mice by exercise and probiotics is partially mediated by Ly6C(hi) monocytes.

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-1ß 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.

Survey of small intestinal and systemic immune responses following murine Arcobacter butzleri infection.

BACKGROUND: Arcobacter (A.) butzleri has been described as causative agent for sporadic cases of human gastroenteritis with abdominal pain and acute or prolonged watery diarrhea. In vitro studies revealed distinct adhesive, invasive and cytotoxic properties of A. butzleri. Information about the underlying immunopathological mechanisms of infection in vivo, however, are scarce. The aim of this study was to investigate the immunopathological properties of two different A. butzleri strains in a well-established murine infection model. RESULTS: Gnotobiotic IL-10(-/-) mice, in which the intestinal microbiota was depleted by broad-spectrum antibiotic treatment, were perorally infected with two different A. butzleri strains isolated from a diseased patient (CCUG 30485) or fresh chicken meat (C1), respectively. Eventhough bacteria of either strain could stably colonize the intestinal tract at day 6 and day 16 postinfection (p.i.), mice did not exert infection induced symptoms such as diarrhea or wasting. In small intestines of infected mice, however, increased numbers of apoptotic cells could be detected at day 16, but not day 6 following infection with either strain. A strain-dependent influx of distinct immune cell populations such as T and B cells as well as of regulatory T cells could be observed upon A. butzleri infection which was accompanied by increased small intestinal concentrations of pro-inflammatory cytokines such as TNF, IFN-γ, MCP-1 and IL-6. Remarkably, inflammatory responses following A. butzleri infection were not restricted to the intestinal tract, given that the CCUG 30485 strain induced systemic immune responses as indicated by increased IFN-γ concentrations in spleens at day 6, but not day 16 following infection. CONCLUSION: Upon peroral infection A. butzleri stably colonized the intestinal tract of gnotobiotic IL-10(-/-) mice. The dynamics of distinct local and systemic inflammatory responses could be observed in a strain-dependent fashion pointing towards an immunopathogenic potential of A. butzleri in vivo. These results indicate that gnotobiotic IL-10(-/-) mice are well suited to further investigate the molecular mechanisms underlying arcobacteriosis in vivo.

Arcobacter butzleri Induce Colonic, Extra-Intestinal and Systemic Inflammatory Responses in Gnotobiotic IL-10 Deficient Mice in a Strain-Dependent Manner.

BACKGROUND: The immunopathological impact of human Arcobacter (A.) infections is under current debate. Episodes of gastroenteritis with abdominal pain and acute or prolonged watery diarrhea were reported for A. butzleri infected patients. Whereas adhesive, invasive and cytotoxic capacities have been described for A. butzleri in vitro, only limited information is available about the immunopathogenic potential and mechanisms of infection in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Gnotobiotic IL-10-/- mice were generated by broad-spectrum antibiotic treatment and perorally infected with the A. butzleri strains CCUG 30485 and C1 shown to be invasive in cell culture assays. Bacterial colonization capacities, clinical conditions, intestinal, extra-intestinal and systemic immune responses were monitored at day six and 16 postinfection (p.i.). Despite stable intestinal A. butzleri colonization at high loads, gnotobiotic IL-10-/- mice were virtually unaffected and did not display any overt symptoms at either time point. Notably, A. butzleri infection induced apoptosis of colonic epithelial cells which was paralleled by increased abundance of proliferating cells. Furthermore A. butzleri infection caused a significant increase of distinct immune cell populations such as T and B cells, regulatory T cells, macrophages and monocytes in the colon which was accompanied by elevated colonic TNF, IFN-γ, nitric oxide (NO), IL-6, IL-12p70 and MCP-1 concentrations. Strikingly, A. butzleri induced extra-intestinal and systemic immune responses as indicated by higher NO concentrations in kidney and increased TNF, IFN-γ, IL-12p70 and IL-6 levels in serum samples of infected as compared to naive mice. Overall, inflammatory responses could be observed earlier in the course of infection by the CCUG 30485 as compared to the C1 strain. CONCLUSION/SIGNIFICANCE: Peroral A. butzleri infection induced not only intestinal but also extra-intestinal and systemic immune responses in gnotobiotic IL-10-/- mice in a strain-dependent manner. These findings point towards an immunopathogenic potential of A. butzleri in vertebrate hosts.

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.

Nucleotide-oligomerization-domain-2 affects commensal gut microbiota composition and intracerebral immunopathology in acute Toxoplasma gondii induced murine ileitis.

BACKGROUND: Within one week following peroral high dose infection with Toxoplasma (T.) gondii, susceptible mice develop non-selflimiting acute ileitis due to an underlying Th1-type immunopathology. The role of the innate immune receptor nucleotide-oligomerization-domain-2 (NOD2) in mediating potential extra-intestinal inflammatory sequelae including the brain, however, has not been investigated so far. METHODOLOGY/PRINCIPAL FINDINGS: Following peroral infection with 100 cysts of T. gondii strain ME49, NOD2-/- mice displayed more severe ileitis and higher small intestinal parasitic loads as compared to wildtype (WT) mice. However, systemic (i.e. splenic) levels of pro-inflammatory cytokines such as TNF-α and IFN-γ were lower in NOD2-/- mice versus WT controls at day 7 p.i. Given that the immunopathological outcome might be influenced by the intestinal microbiota composition, which is shaped by NOD2, we performed a quantitative survey of main intestinal bacterial groups by 16S rRNA analysis. Interestingly, Bifidobacteria were virtually absent in NOD2-/- but not WT mice, whereas differences in remaining bacterial species were rather subtle. Interestingly, more distinct intestinal inflammation was accompanied by higher bacterial translocation rates to extra-intestinal tissue sites such as liver, spleen, and kidneys in T. gondii infected NOD2-/- mice. Strikingly, intracerebral inflammatory foci could be observed as early as seven days following T. gondii infection irrespective of the genotype of animals, whereas NOD2-/- mice exhibited higher intracerebral parasitic loads, higher F4/80 positive macrophage and microglia numbers as well as higher IFN-γ mRNA expression levels as compared to WT control animals. CONCLUSION/SIGNIFICANCE: NOD2 signaling is involved in protection of mice from T. gondii induced acute ileitis. The parasite-induced Th1-type immunopathology at intestinal as well as extra-intestinal sites including the brain is modulated in a NOD2-dependent manner.

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.

The impact of serine protease HtrA in apoptosis, intestinal immune responses and extra-intestinal histopathology during Campylobacter jejuni infection of infant mice.

BACKGROUND: Campylobacter jejuni has emerged as a leading cause of bacterial enterocolitis. The serine protease HtrA has been shown to be a pivotal, novel C. jejuni virulence factor involved in cell invasion and transmigration across polarised epithelial cells in vitro. However, the functional relevance of the htrA gene for the interaction of C. jejuni with the host immune system in the infant mouse infection model has not been investigated so far. RESULTS: Here we studied the role of C. jejuni htrA during infection of 3-weeks-old infant mice. Immediately after weaning, conventional wild-type mice were perorally infected with the NCTC11168∆htrA mutant (∆htrA) or the parental wild-type strain. Approximately one third of infected infant mice suffered from bloody diarrhea until day 7 post infection (p.i.), whereas colonic histopathological changes were rather moderate but comparable between the two strains. Interestingly, parental, but not ∆htrA mutant infected mice, displayed a multifold increase of apoptotic cells in the colonic mucosa at day 7 p.i., which was paralleled by higher colonic levels of pro-inflammatory cytokines such as TNF-α and IFN-γ and the matrix-degrading enzyme matrixmetalloproteinase-2 (MMP-2). Furthermore, higher numbers of proliferating cells could be observed in the colon of ∆htrA infected mice as compared to the parental wild-type strain. Remarkably, as early as 7 days p.i. infant mice also exhibited inflammatory changes in extra-intestinal compartments such as liver, kidneys and lungs, which were less distinct in kidneys and lungs following ∆htrA versus parental strain infection. However, live C. jejuni bacteria could not be found in these organs, suggesting the induction of systemic effects during intestinal infection. CONCLUSION: Upon C. jejuni ∆htrA strain infection of infant mice, intestinal and extra-intestinal pro-inflammatory immune responses were ameliorated in the infant mouse model system. Future studies will shed further light onto the molecular mechanisms of host-pathogen interactions.

The impact of Toll-like-receptor-9 on intestinal microbiota composition and extra-intestinal sequelae in experimental Toxoplasma gondii induced ileitis.

BACKGROUND: Following peroral Toxoplasma (T.) gondii infection, susceptible mice develop acute ileitis due to a microbiota-dependent Th1 type immunopathology. Toll-like-receptor (TLR)-9 is known to recognize bacterial DNA and mediates intestinal inflammation, but its impact on intestinal microbiota composition and extra-intestinal sequelae following T. gondii infection has not yet been elucidated. METHODS AND RESULTS: Seven days following peroral infection (p.i.) with 100 cysts of T. gondii ME49 strain, TLR-9(-/-) and wildtype (WT) mice suffered from comparable ileitis, whereas ileal parasitic loads as well as IFN-γ and nitric oxide levels were higher in TLR-9(-/-) compared to WT mice. Locally, TLR-9(-/-) mice exhibited increased ileal CD3+, but not FOXP3+ cell numbers at day 7 p.i.; in mesenteric lymph nodes IFN-γ-producing CD4+ cell numbers and TNF-α and IFN-γ concentrations were also increased in TLR-9(-/-) compared to WT mice. T. gondii DNA levels, however, did not differ in mice of either genotype. Differences in intestinal microbiota were rather subtle except for bifidobacteria that were virtually absent in both, naïve and T. gondii infected TLR-9(-/-), but not WT mice. Extra-intestinally, TLR-9(-/-) mice displayed less distinct systemic immune responses as indicated by lower serum IL-6, and splenic TNF-α and IFN-γ levels as compared to WT mice despite higher translocation rates of intestinal bacteria to extra-intestinal compartments such as liver, spleen, kidney, and cardiac blood. Most importantly, brains were also affected in this inflammatory scenario as early as day 7 p.i. Remarkably, TLR-9(-/-) mice exhibited more pronounced inflammatory infiltrates with higher numbers of F4/80+ macrophages and microglia in the cortex and meninges as compared to WT mice, whereas T. gondii DNA levels did not differ. CONCLUSION: We here show that TLR-9 is not required for the development of T. gondii induced ileitis but mediates distinct inflammatory changes in intestinal and extra-intestinal compartments including the brain.

Impact of Campylobacter jejuni cj0268c knockout mutation on intestinal colonization, translocation, and induction of immunopathology in gnotobiotic IL-10 deficient mice.

BACKGROUND: Although Campylobacter jejuni infections have a high prevalence worldwide and represent a significant socioeconomic burden, the underlying molecular mechanisms of induced intestinal immunopathology are still not well understood. We have recently generated a C. jejuni mutant strain NCTC11168::cj0268c, which has been shown to be involved in cellular adhesion and invasion. The immunopathological impact of this gene, however, has not been investigated in vivo so far. METHODOLOGY/PRINCIPAL FINDINGS: Gnotobiotic IL-10 deficient mice were generated by quintuple antibiotic treatment and perorally infected with C. jejuni mutant strain NCTC11168::cj0268c, its complemented version (NCTC11168::cj0268c-comp-cj0268c), or the parental strain NCTC11168. Kinetic analyses of fecal pathogen loads until day 6 post infection (p.i.) revealed that knockout of cj0268c did not compromise intestinal C. jejuni colonization capacities. Whereas animals irrespective of the analysed C. jejuni strain developed similar clinical symptoms of campylobacteriosis (i.e. enteritis), mice infected with the NCTC11168::cj0268c mutant strain displayed significant longer small as well as large intestinal lengths indicative for less distinct C. jejuni induced pathology when compared to infected control groups at day 6 p.i. This was further supported by significantly lower apoptotic and T cell numbers in the colonic mucosa and lamina propria, which were paralleled by lower intestinal IFN-γ and IL-6 concentrations at day 6 following knockout mutant NCTC11168::cj0268c as compared to parental strain infection. Remarkably, less intestinal immunopathology was accompanied by lower IFN-γ secretion in ex vivo biopsies taken from mesenteric lymphnodes of NCTC11168::cj0268c infected mice versus controls. CONCLUSION/SIGNIFICANCE: We here for the first time show that the cj0268c gene is involved in mediating C. jejuni induced immunopathogenesis in vivo. Future studies will provide further deep insights into the immunological and molecular interplays between C. jejuni and innate immunity in human campylobacteriosis.