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Pulmonary mucosal immune response is critical for preventing opportunistic Aspergillus fumigatus infections. Although fungus‐specific CD4+ T cells in blood are described to reflect the actual host–pathogen interaction status, little is known about Aspergillus‐specific pulmonary T‐cell responses. Here, we exploit the domestic pig as human‐relevant large animal model and introduce antigen‐specific T‐cell enrichment in pigs to address Aspergillus‐specific T cells in the lung compared to peripheral blood. In healthy, environmentally Aspergillus‐exposed pigs, the fungus‐specific T cells are detectable in blood in similar frequencies as observed in healthy humans and exhibit a Th1 phenotype. Exposing pigs to 106 cfu/m3 conidia induces a long‐lasting accumulation of Aspergillus‐specific Th1 cells locally in the lung and also systemically. Temporary immunosuppression during Aspergillus‐exposure showed a drastic reduction in the lung‐infiltrating antifungal T‐cell responses more than 2 weeks after abrogation of the suppressive treatment. This was reflected in blood, but to a much lesser extent. In conclusion, by using the human‐relevant large animal model the pig, this study highlights that the blood clearly reflects the mucosal fungal‐specific T‐cell reactivity in environmentally exposed as well as experimentally exposed healthy pigs. But, immunosuppression significantly impacts the mucosal site in contrast to the initial systemic immune response. [ABSTRACT FROM AUTHOR]

NKp46+ innate lymphoid cells (ILC) modulate tissue homeostasis and anti-microbial immune responses. ILC development and function are regulated by cytokines such as Interleukin (IL)−7 and IL-15. However, the ILC-intrinsic pathways translating cytokine signals into developmental programs are largely unknown. Here we show that the anti-apoptotic molecule cellular FLICE-like inhibitory protein (c-FLIP) is crucial for the generation of IL-7/IL-15-dependent NKp46+ ILC1, including conventional natural killer (cNK) cells, and ILC3. Cytokine-induced phosphorylation of signal transducer and activator of transcription 5 (STAT5) precedes up-regulation of c-FLIP, which protects developing NKp46+ ILC from TNF-induced apoptosis. NKp46+ ILC-specific inactivation of c-FLIP leads to the loss of all IL-7/IL-15-dependent NKp46+ ILC, thereby inducing early-onset chronic colitis and subsequently microbial dysbiosis; meanwhile, the depletion of cNK, but not NKp46+ ILC1/3, aggravates experimental colitis. In summary, our data demonstrate a non-redundant function of c-FLIP for the generation of NKp46+ ILC, which protect T/B lymphocyte-sufficient mice from intestinal inflammation. Innate lymphoid cells (ILC) are important immune cells for maintaining the gut homeostasis. Here the authors show that c-FLIP, an anti-apoptotic molecule, is important for the development of NKP46+ ILC1, including conventional natural killer (cNK) cells, and ILC3, with cNK being more critical for ameliorating intestinal inflammation. [ABSTRACT FROM AUTHOR]

Currently, methods for monitoring changes of gut barrier integrity and the associated immune response via non-invasive means are limited. Therefore, we aimed to develop a novel non-invasive technique to investigate immunological host responses representing gut barrier changes in response to infection. We identified the mucous layer on feces from mice to be mainly composed of exfoliated intestinal epithelial cells. Expression of RELM-β, a gene prominently expressed in intestinal nematode infections, was used as an indicator of intestinal cellular barrier changes to infection. RELM-β was detected as early as 6 days post-infection (dpi) in exfoliated epithelial cells. Interestingly, RELM-β expression also mirrored the quality of the immune response, with higher amounts being detectable in a secondary infection and in high dose nematode infection in laboratory mice. This technique was also applicable to captured worm-infected wild house mice. We have therefore developed a novel non-invasive method reflecting gut barrier changes associated with alterations in cellular responses to a gastrointestinal nematode infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 matrixdegrading 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 proinflammatory immune responses were ameliorated in the infant mouse model system. Future studies will shed further light onto the molecular mechanisms of host-pathogen interactions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The stable lineage commitment of naïve T helper cells to a hybrid Th1/2 phenotype reveals the cell-intrinsic reconciliation of two opposing T cell differentiation programs and provides a self-limiting mechanism to dampen immunopathology. [ABSTRACT FROM AUTHOR]

After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor-mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo. Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell-mediated immune responses and open new therapeutic possibilities. [ABSTRACT FROM AUTHOR]

Parasitic nematodes are potent modulators of immune reactivity in mice and men. Intestinal nematodes live in close contact with commensal gut bacteria, provoke biased Th2 immune responses upon infection, and subsequently lead to changes in gut physiology. We hypothesized that murine nematode infection is associated with distinct changes of the intestinal bacterial microbiota composition. We here studied intestinal inflammatory and immune responses in mice following infection with the hookworm Heligmosomoides polygyrus bakeri and applied cultural and molecular techniques to quantitatively assess intestinal microbiota changes in the ileum, cecum and colon. At day 14 post nematode infection, mice harbored significantly higher numbers of γ-Proteobacteria/Enterobacteriaceae and members of the Bacteroides/Prevotella group in their cecum as compared to uninfected controls. Abundance of Gram-positive species such as Lactobacilli, Clostridia as well as the total bacterial load was not affected by worm infection. The altered microbiota composition was independent of the IL-4/-13 – STAT6 signaling axis, as infected IL-4Rα-/- mice showed a similar increase in enterobacterial loads. In conclusion, infection with an enteric nematode is accompanied by distinct intestinal microbiota changes towards higher abundance of gram-negative commensal species at the small intestinal site of infection (and inflammation), but also in the parasite-free large intestinal tract. Further studies should unravel the impact of nematode-induced microbiota changes in inflammatory bowel disease to allow for a better understanding of how theses parasites interfere with intestinal inflammation and bacterial communities in men. [ABSTRACT FROM AUTHOR]

IL-17–producing Th17 cells mediate immune responses against a variety of fungal and bacterial infections. Signaling via NF-κB has been linked to the development and maintenance of Th17 cells. We analyzed the role of the unusual inhibitor of NF-κB, IκBNS, in the proliferation and effector cytokine production of murine Th17 cells. Our study demonstrates that nuclear IκBNS is crucial for murine Th17 cell generation. IκBNS is highly expressed in Th17 cells; in the absence of IκBNS, the frequencies of IL-17A–producing cells are drastically reduced. This was measured in vitro under Th17-polarizing conditions and confirmed in two colitis models. Mechanistically, murine IκBNS−/− Th17 cells were less proliferative and expressed markedly reduced levels of IL-2, IL-10, MIP-1α, and GM-CSF. Citrobacter rodentium was used as a Th17-inducing infection model, in which IκBNS−/− mice displayed an increased bacterial burden and diminished tissue damage. These results demonstrate the important function of Th17 cells in pathogen clearance, as well as in inflammation-associated pathology. We identified IκBNS to be crucial for the generation and function of murine Th17 cells upon inflammation and infection. Our findings may have implications for the therapy of autoimmune conditions, such as inflammatory bowel disease, and for the treatment of gut-tropic infections. [ABSTRACT FROM AUTHOR]

Summary: Forkhead box P3 positive (Foxp3+) regulatory T (Treg) cells suppress immune responses and regulate peripheral tolerance. Here we show that the atypical inhibitor of NFκB (IκB) IκBNS drives Foxp3 expression via association with the promoter and the conserved noncoding sequence 3 (CNS3) of the Foxp3 locus. Consequently, IκBNS deficiency leads to a substantial reduction of Foxp3+ Treg cells in vivo and impaired Foxp3 induction upon transforming growth factor-β (TGF-β) treatment in vitro. Moreover, fewer Foxp3+ Treg cells developed from IκBNS-deficient CD25−CD4+ T cells adoptively transferred into immunodeficient recipients. Importantly, IκBNS was required for the transition of immature GITR+CD25+Foxp3− thymic Treg cell precursors into Foxp3+ cells. In contrast to mice lacking c-Rel or Carma1, IκBNS-deficient mice do not show reduced Treg precursor cells. Our results demonstrate that IκBNS critically regulates Treg cell development in the thymus and during gut inflammation, indicating that strategies targeting IκBNS could modulate the Treg cell compartment. [ABSTRACT FROM AUTHOR]