Gasdermin D is the only Gasdermin that provides protection against acute Salmonella gut infection in mice.

Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1ß (IL1ß) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we used epithelium-specific ablation, bone marrow chimeras, and mouse lines lacking individual Gasdermins, combinations of Gasdermins or even all Gasdermins (GSDMA1-3C1-4DE) at once and performed littermate-controlled oral S.Tm infections in streptomycin-pretreated mice to investigate the impact of all murine Gasdermins. While GSDMA, C, and E appear dispensable, we show that GSDMD i) restricts S.Tm loads in the gut tissue and systemic organs, ii) controls gut inflammation kinetics, and iii) prevents epithelium disruption by 72 h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments as well as 3D-, 2D-, and chimeric enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding IECs, affects extrusion kinetics, and promotes overall mucosal barrier capacity. As such, this work identifies a unique multipronged role of GSDMD among the Gasdermins for mucosal tissue defense against a common enteric pathogen.

Caspase-8-dependent gasdermin D cleavage promotes antimicrobial defense but confers susceptibility to TNF-induced lethality.

Gasdermin D (GSDMD) is a pore-forming protein that promotes pyroptosis and release of proinflammatory cytokines. Recent studies revealed that apoptotic caspase-8 directly cleaves GSDMD to trigger pyroptosis. However, the molecular requirements for caspase-8-dependent GSDMD cleavage and the physiological impact of this signaling axis are unresolved. Here, we report that caspase-8-dependent GSDMD cleavage confers susceptibility to tumor necrosis factor (TNF)-induced lethality independently of caspase-1 and that GSDMD activation provides host defense against Yersinia infection. We further demonstrate that GSDMD inactivation by apoptotic caspases at aspartate 88 (D88) suppresses TNF-induced lethality but promotes anti-Yersinia defense. Last, we show that caspase-8 dimerization and autoprocessing are required for GSDMD cleavage, and provide evidence that the caspase-8 autoprocessing and activity on various complexes correlate with its ability to directly cleave GSDMD. These findings reveal GSDMD as a potential therapeutic target to reduce inflammation associated with mutations in the death receptor signaling machinery.

Interleukin-35-Producing CD8α+ Dendritic Cells Acquire a Tolerogenic State and Regulate T Cell Function.

Dendritic cells (DCs) play a central role in shaping immunogenic as well as tolerogenic adaptive immune responses and thereby dictate the outcome of adaptive immunity. Here, we report the generation of a CD8α+ DC line constitutively secreting the tolerogenic cytokine interleukin (IL)-35. IL-35 secretion led to impaired CD4+ and CD8+ T lymphocyte proliferation and interfered with their function in vitro and also in vivo. IL-35 was furthermore found to induce a tolerogenic phenotype on CD8α+ DCs, characterized by the upregulation of CD11b, downregulation of MHC class II, a reduced costimulatory potential as well as production of the immunomodulatory molecule IL-10. Vaccination of mice with IL-35-expressing DCs promoted tumor growth and reduced the severity of autoimmune encephalitis not only in a preventive but also after induction of encephalitogenic T cells. The reduction in experimental autoimmune encephalitis severity was significantly more pronounced when antigen-pulsed IL-35+ DCs were used. These findings suggest a new, indirect effector mechanism by which IL-35-responding antigen-presenting cells contribute to immune tolerance. Furthermore, IL-35-transfected DCs may be a promising approach for immunotherapy in the context of autoimmune diseases.

Cross-presenting dendritic cells are required for control of Leishmania major infection.

Leishmania major infection induces self-healing cutaneous lesions in C57BL/6 mice. Both IL-12 and IFN-γ are essential for the control of infection. We infected Jun dimerization protein p21SNFT (Batf3(-/-) ) mice (C57BL/6 background) that lack the major IL-12 producing and cross-presenting CD8α(+) and CD103(+) DC subsets. Batf3(-/-) mice displayed enhanced susceptibility with larger lesions and higher parasite burden. Additionally, cells from draining lymph nodes of infected Batf3(-/-) mice secreted less IFN-γ, but more Th2- and Th17-type cytokines, mirrored by increased serum IgE and Leishmania-specific immunoglobulin 1 (Th2 indicating). Importantly, CD8α(+) DCs isolated from lymph nodes of L. major-infected mice induced significantly more IFN-γ secretion by L. major-stimulated immune T cells than CD103(+) DCs. We next developed CD11c-diptheria toxin receptor: Batf3(-/-) mixed bone marrow chimeras to determine when the DCs are important for the control of infection. Mice depleted of Batf-3-dependent DCs from day 17 or wild-type mice depleted of cross-presenting DCs from 17-19 days after infection maintained significantly larger lesions similar to mice whose Batf-3-dependent DCs were depleted from the onset of infection. Thus, we have identified a crucial role for Batf-3-dependent DCs in protection against L. major.

Interferon-γ induces expression of MHC class II on intestinal epithelial cells and protects mice from colitis.

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4(+) T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4(+) T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+) T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+) T cells and forkhead box P3 (FoxP3)(+) regulatory T (Treg) cells. IFN-γ produced mainly by CD4(+) T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.