Autophagy proteins suppress protective type I interferon signalling in response to the murine gut microbiota.

As a conserved pathway that lies at the intersection between host defence and cellular homeostasis, autophagy serves as a rheostat for immune reactions. In particular, autophagy suppresses excess type I interferon (IFN-I) production in response to viral nucleic acids. It is unknown how this function of autophagy relates to the intestinal barrier where host-microbe interactions are pervasive and perpetual. Here, we demonstrate that mice deficient in autophagy proteins are protected from the intestinal bacterial pathogen Citrobacter rodentium in a manner dependent on IFN-I signalling and nucleic acid sensing pathways. Enhanced IFN-stimulated gene expression in intestinal tissue of autophagy-deficient mice in the absence of infection was mediated by the gut microbiota. Additionally, monocytes infiltrating into the autophagy-deficient intestinal microenvironment displayed an enhanced inflammatory profile and were necessary for protection against C. rodentium. Finally, we demonstrate that the microbiota-dependent IFN-I production that occurs in the autophagy-deficient host also protects against chemical injury of the intestine. Thus, autophagy proteins prevent a spontaneous IFN-I response to microbiota that is beneficial in the presence of infectious and non-infectious intestinal hazards. These results identify a role for autophagy proteins in controlling the magnitude of IFN-I signalling at the intestinal barrier.

Fasting metabolism modulates the interleukin-12/interleukin-10 cytokine axis.

A crucial role of cell metabolism in immune cell differentiation and function has been recently established. Growing evidence indicates that metabolic processes impact both, innate and adaptive immunity. Since a down-stream integrator of metabolic alterations, mammalian target of rapamycin (mTOR), is responsible for controlling the balance between pro-inflammatory interleukin (IL)-12 and anti-inflammatory IL-10, we investigated the effect of upstream interference using metabolic modulators on the production of pro- and anti-inflammatory cytokines. Cytokine release and protein expression in human and murine myeloid cells was assessed after toll-like receptor (TLR)-activation and glucose-deprivation or co-treatment with 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators. Additionally, the impact of metabolic interference was analysed in an in-vivo mouse model. Glucose-deprivation by 2-deoxy-D-glucose (2-DG) increased the production of IL-12p40 and IL-23p19 in monocytes, but dose-dependently inhibited the release of anti-inflammatory IL-10. Similar effects have been observed using pharmacological AMPK activation. Consistently, an inhibition of the tuberous sclerosis complex-mTOR pathway was observed. In line with our in vitro observations, glycolysis inhibition with 2-DG showed significantly reduced bacterial burden in a Th2-prone Listeria monocytogenes mouse infection model. In conclusion, we showed that fasting metabolism modulates the IL-12/IL-10 cytokine balance, establishing novel targets for metabolism-based immune-modulation.

The Tumor Suppressor Hace1 Is a Critical Regulator of TNFR1-Mediated Cell Fate.

The HECT domain E3 ligase HACE1 has been identified as a tumor suppressor in multiple cancers. Here, we report that HACE1 is a central gatekeeper of TNFR1-induced cell fate. Genetic inactivation of HACE1 inhibits TNF-stimulated NF-κB activation and TNFR1-NF-κB-dependent pathogen clearance in vivo. Moreover, TNF-induced apoptosis was impaired in hace1 mutant cells and knockout mice in vivo. Mechanistically, HACE1 is essential for the ubiquitylation of the adaptor protein TRAF2 and formation of the apoptotic caspase-8 effector complex. Intriguingly, loss of HACE1 does not impair TNFR1-mediated necroptotic cell fate via RIP1 and RIP3 kinases. Loss of HACE1 predisposes animals to colonic inflammation and carcinogenesis in vivo, which is markedly alleviated by genetic inactivation of RIP3 kinase and TNFR1. Thus, HACE1 controls TNF-elicited cell fate decisions and exerts tumor suppressor and anti-inflammatory activities via a TNFR1-RIP3 kinase-necroptosis pathway.

Gastrointestinal dissemination and transmission of Staphylococcus aureus following bacteremia.

Mutations that alter virulence and antibiotic susceptibility arise and persist during Staphylococcus aureus bacteremia. However, an experimental system demonstrating transmission following bacteremia has been lacking, and thus implications of within-host adaptation for between-host transmission are unknown. We report that S. aureus disseminates to the gastrointestinal tract of mice following intravenous injection and readily transmits to cohoused naive mice. Both intestinal dissemination and transmission were linked to the production of virulence factors based on gene deletion studies of the sae and agr two-component systems. Furthermore, antimicrobial selection for antibiotic-resistant S. aureus displaced susceptible S. aureus from the intestine of infected hosts, which led to the preferential transmission and dominance of antibiotic-resistant bacteria among cohoused untreated mice. These findings establish an animal model to investigate gastrointestinal dissemination and transmission of S. aureus and suggest that adaptation during the course of systemic infection has implications beyond the level of a single host.

An enteric virus can replace the beneficial function of commensal bacteria.

Intestinal microbial communities have profound effects on host physiology. Whereas the symbiotic contribution of commensal bacteria is well established, the role of eukaryotic viruses that are present in the gastrointestinal tract under homeostatic conditions is undefined. Here we demonstrate that a common enteric RNA virus can replace the beneficial function of commensal bacteria in the intestine. Murine norovirus (MNV) infection of germ-free or antibiotic-treated mice restored intestinal morphology and lymphocyte function without inducing overt inflammation and disease. The presence of MNV also suppressed an expansion of group 2 innate lymphoid cells observed in the absence of bacteria, and induced transcriptional changes in the intestine associated with immune development and type I interferon (IFN) signalling. Consistent with this observation, the IFN-α receptor was essential for the ability of MNV to compensate for bacterial depletion. Importantly, MNV infection offset the deleterious effect of treatment with antibiotics in models of intestinal injury and pathogenic bacterial infection. These data indicate that eukaryotic viruses have the capacity to support intestinal homeostasis and shape mucosal immunity, similarly to commensal bacteria.

Autophagy, viruses, and intestinal immunity.

PURPOSE OF REVIEW: To highlight recent findings that identify an essential role for the cellular degradative pathway of autophagy in governing a balanced response to intestinal pathogens and commensals. RECENT FINDINGS: Following the genetic association of autophagy with inflammatory bowel disease susceptibility, increasing evidence indicates that this pathway functions in various epithelial lineages to support the intestinal barrier. New studies are also revealing that autophagy proteins dictate the quality and magnitude of immune responses. Mouse models, in particular, suggest that autophagy and inflammatory bowel disease susceptibility genes regulate inflammatory responses to viruses, a finding that coincides with an increasing appreciation that viruses have intricate interactions with the host and the microbiota beyond the obvious host-pathogen relationship. SUMMARY: Autophagy and other immunological or stress response pathways intersect in mucosal immunity to dictate the response to pathogenic and commensal agents. The development of novel treatment strategies, as well as prognostic and diagnostic tools for gastrointestinal disorders, will be greatly facilitated by a deeper understanding of these interactions at the cell type and microbe-specific manner, which includes less appreciated components of the microbiota, such as eukaryotic and prokaryotic viruses.

Route of Infection Determines the Impact of Type I Interferons on Innate Immunity to Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen which causes mild to life threatening disease in humans. Ingestion of contaminated food delivers the pathogen to the gastrointestinal tract, where it crosses the epithelial barrier and spreads to internal organs. Type I interferons (IFN-I) are produced during infection and decrease host resistance after systemic delivery of L. monocytogenes. Here we show that mice benefit from IFN-I production following infection with L. monocytogenes via the gastrointestinal route. Intragastric infection lead to increased lethality of IFN-I receptor chain 1-deficient (Ifnar1-/-) animals and to higher bacterial numbers in liver and spleen. Compared to infection from the peritoneum, bacteria infecting via the intestinal tract localized more often to periportal and pericentral regions of the liver and less frequently to the margins of liver lobes. Vigorous replication of intestine-borne L. monocytogenes in the livers of Ifnar1-/- mice 48 h post infection was accompanied by the formation of large inflammatory infiltrates in this organ and massive death of surrounding hepatocytes. This was not observed in Ifnar1-/- mice after intraperitoneal infection. The inflammatory response to infection is shaped by alterations in splenic cytokine production, particularly IFNγ, which differs after intragastric versus intraperitoneal infection. Taken together, our data suggest that the adverse or beneficial role of a cytokine may vary with the route of infection and that IFN-I are not harmful when infection with L. monocytogenes occurs via the natural route.

Sustained generation of nitric oxide and control of mycobacterial infection requires argininosuccinate synthase 1.

Nitric oxide (NO) defends against intracellular pathogens, but its synthesis must be regulated due to cell and tissue toxicity. During infection, macrophages import extracellular arginine to synthesize NO, generating the byproduct citrulline. Accumulated intracellular citrulline is thought to fuel arginine synthesis catalyzed by argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl), which would lead to abundant NO production. Instead, we find that citrulline is exported from macrophages during early stages of NO production with <2% retained for recycling via the Ass1-Asl pathway. Later, extracellular arginine is depleted, and Ass1 expression allows macrophages to synthesize arginine from imported citrulline to sustain NO output. Ass1-deficient macrophages fail to salvage citrulline in arginine-scarce conditions, leading to their inability to control mycobacteria infection. Thus, extracellular arginine fuels rapid NO production in activated macrophages, and citrulline recycling via Ass1 and Asl is a fail-safe system that sustains optimum NO production.

Conditional Stat1 ablation reveals the importance of interferon signaling for immunity to Listeria monocytogenes infection.

Signal transducer and activator of transcription 1 (Stat1) is a key player in responses to interferons (IFN). Mutations of Stat1 cause severe immune deficiencies in humans and mice. Here we investigate the importance of Stat1 signaling for the innate and secondary immune response to the intracellular bacterial pathogen Listeria monocytogenes (Lm). Cell type-restricted ablation of the Stat1 gene in naïve animals revealed unique roles in three cell types: macrophage Stat1 signaling protected against lethal Lm infection, whereas Stat1 ablation in dendritic cells (DC) did not affect survival. T lymphocyte Stat1 reduced survival. Type I IFN (IFN-I) signaling in T lymphocytes reportedly weakens innate resistance to Lm. Surprisingly, the effect of Stat1 signaling was much more pronounced, indicating a contribution of Stat1 to pathways other than the IFN-I pathway. In stark contrast, Stat1 activity in both DC and T cells contributed positively to secondary immune responses against Lm in immunized animals, while macrophage Stat1 was dispensable. Our findings provide the first genetic evidence that Stat1 signaling in different cell types produces antagonistic effects on innate protection against Lm that are obscured in mice with complete Stat1 deficiency. They further demonstrate a drastic change in the cell type-dependent Stat1 requirement for memory responses to Lm infection.

Generation of mice with a conditional Stat1 null allele.

Interferons (IFNs) are key cytokines in the innate immune response that also bridge the gap to adaptive immunity. Signaling upon stimulation by IFN type I, II and III is mediated by the Jak-Stat pathway. STAT1 is activated by all three IFN receptor complexes and absence of STAT1 from mice increases their susceptibility to pathogens. In addition, depending on the setting, STAT1 can act as tumor suppressor or oncogene. Here we report the generation and detailed functional characterization of a conditional Stat1 knockout mouse. We show the integrity of the conditional Stat1 locus and report successful in vivo deletion by means of a ubiquitous and a tissue-specific Cre recombinase. The conditional Stat1 null allele represents an important tool for identifying novel and cell-autonomous STAT1 functions in infection and cancer.

LipA, a tyrosine and lipid phosphatase involved in the virulence of Listeria monocytogenes.

Intracellular bacterial pathogens manipulate host cell functions by producing enzymes that stimulate or antagonize signal transduction. The Listeria monocytogenes genome contains a gene, lmo1800, encoding a protein with a conserved motif of conventional tyrosine phosphatases. Here, we report that the lmo1800-encoded protein LipA is secreted by Listeria and displays tyrosine as well as lipid phosphatase activity in vitro. Bacteria lacking LipA are severely attenuated in virulence in vivo, thus revealing a so-far-undescribed enzymatic activity involved in Listeria infection.

Characterization of the interferon-producing cell in mice infected with Listeria monocytogenes.

Production of type I interferons (IFN-I, mainly IFNalpha and IFNbeta) is a hallmark of innate immune responses to all classes of pathogens. When viral infection spreads to lymphoid organs, the majority of systemic IFN-I is produced by a specialized "interferon-producing cell" (IPC) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pDC). It is unclear whether production of systemic IFN-I is generally attributable to pDC irrespective of the nature of the infecting pathogen. We have addressed this question by studying infections of mice with the intracellular bacterium Listeria monocytogenes. Protective innate immunity against this pathogen is weakened by IFN-I activity. In mice infected with L. monocytogenes, systemic IFN-I was amplified via IFN-beta, the IFN-I receptor (IFNAR), and transcription factor interferon regulatory factor 7 (IRF7), a molecular circuitry usually characteristic of non-pDC producers. Synthesis of serum IFN-I did not require TLR9. In contrast, in vitro-differentiated pDC infected with L. monocytogenes needed TLR9 to transcribe IFN-I mRNA. Consistent with the assumption that pDC are not the producers of systemic IFN-I, conditional ablation of the IFN-I receptor in mice showed that most systemic IFN-I is produced by myeloid cells. Furthermore, results obtained with FACS-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pDC is responsible for bulk IFN-I synthesis. The amount of IFN-I produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. Based on these data, we propose that the engagement of pDC, the mode of IFN-I mobilization, as well as the shaping of the antimicrobial innate immune response by IFN-I differ between intracellular pathogens.