Summary Intestinal epithelial cells (IECs) form a critical barrier against pathogen invasion. By generation of mice in which inflammasome expression is restricted to IECs, we describe a coordinated epithelium-intrinsic inflammasome response in vivo. This response was sufficient to protect against Salmonella tissue invasion, and involved a previously reported IEC expulsion that was coordinated with lipid mediator and cytokine production, and lytic IEC death. Excessive inflammasome activation in IECs was sufficient to result in diarrhea and pathology. Experiments with IEC organoids demonstrated that IEC expulsion did not require other cell types. IEC expulsion was accompanied by a major actin rearrangement in neighboring cells that maintained epithelium integrity, but, did not absolutely require Caspase-1 or Gasdermin D. Analysis of Casp1−/−Casp8−/− mice revealed a functional Caspase-8 inflammasome in vivo. Thus, a coordinated IEC-intrinsic, Caspase-1 and -8 inflammasome response plays a key role in intestinal immune defense and pathology.

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The NAIP/NLRC4 inflammasome is a cytosolic sensor of bacteria that activates caspase-1 and initiates potent immune responses. Structural, biochemical, and genetic data demonstrate that NAIP proteins are receptors for bacterial ligands, while NLRC4 is a downstream adaptor that multimerizes with NAIPs to form an inflammasome. NLRC4 has also been proposed to suppress tumor growth, though the underlying mechanism is unknown. Further, NLRC4 is phosphorylated on serine 533, which was suggested to be critical for its function. In the absence of S533 phosphorylation, it was proposed that another inflammasome protein, NLRP3, can induce NLRC4 activation. We generated a new Nlrc4-deficient mouse line and mice with S533D phosphomimetic or S533A nonphosphorylatable NLRC4. Using these models in vivo and in vitro, we fail to observe a requirement for phosphorylation in NLRC4 inflammasome function. Furthermore, we find no role for NLRP3 in NLRC4 function, or for NLRC4 in a model of melanoma. These results clarify our understanding of the mechanism and biological functions of NAIP/NLRC4 activation.

Peripheral CD4 T cells in Vβ5 transgenic (Tg) C57BL/6J mice undergo tolerance to an endogenous superantigen encoded by mouse mammary tumor virus 8 (Mtv-8) by either deletion or T-cell receptor (TCR) revision. Revision is a process by which surface expression of the Vβ5 + TCR is down-regulated in response to Mtv-8 and recombination activating genes are expressed to drive rearrangement of the endogenous TCRβ locus, effecting cell rescue through the expression of a newly generated, non-self-reactive TCR. In an effort to identify the microenvironment in which revision takes place, we show here that the proportion of T follicular helper cells (Tfh) and production of high-affinity antibody during a primary response are increased in Vβ5 Tg mice in an Mtv-8-dependent manner. Revising T cells have a Tfh-like surface phenotype and transcription factor profile, with elevated expression of B-cell leukemia/lymphoma 6 (Bcl-6), CXC chemokine receptor 5, programmed death-1, and other Tfh-associated markers. Efficient revision requires Bcl-6 and is inhibited by B lymphocyte-induced maturation protein-1. Revision completes less efficiently in the absence of signaling lymphocytic activation molecule-associated protein although initiation proceeds normally. These data indicate that Tfh formation is required for the initiation of revision and germinal-center interactions for its completion. The germinal center is known to provide a confined space in which B-cell antigen receptors undergo selection. Our data extend the impact of this selective microenvironment into the arena of T cells, suggesting that this fluid structure also provides a regulatory environment in which TCR revision can safely take place.Rag-mediated recombination | T-cell tolerance

The youngest peripheral T cells (recent thymic emigrants or RTEs) are functionally distinct from naïve T cells that have completed post-thymic maturation. We now assess the RTE memory response, and find that RTEs produced less granzyme B than their mature counterparts during infection, but proliferated more and therefore generated equivalent target killing in vivo. After infection, RTE numbers contracted less dramatically than those of mature T cells, but RTEs were delayed in their transition to central memory, displaying impaired expression of CD62L, IL-2, Eomesodermin, and CXCR4, which resulted in impaired bone marrow localization. RTE-derived and mature memory cells expanded equivalently during rechallenge, indicating the robust proliferative capacity of RTEs was maintained independently of central memory phenotype. Thus, the diminished effector function and delayed central memory differentiation of RTE-derived memory cells are counterbalanced by their increased proliferative capacity, driving the efficacy of the RTE response to that of mature T cells.

The innate immune system detects pathogens and initiates adaptive immune responses. Inflammasomes are central components of the innate immune system, but whether inflammasomes provide sufficient signals to activate adaptive immunity is unclear. In intestinal epithelial cells (IECs), inflammasomes activate a lytic form of cell death called pyroptosis, leading to epithelial cell expulsion and the release of cytokines. Here we employed a genetic system to show that simultaneous antigen expression and inflammasome activation specifically in IECs is sufficient to activate CD8+ T cells. By genetic elimination of direct T cell priming by IECs, we found that IEC-derived antigens are cross-presented to CD8+ T cells. However, cross-presentation of IEC-derived antigen to CD8+ T cells only partially depended on IEC pyroptosis. In the absence of inflammasome activation, cross-priming of CD8+ T cells required Batf3+ dendritic cells (cDC1), whereas cross-priming in the presence of pyroptosis required a Zbtb26+ but Batf3-independent cDC population. These data suggest the existence of parallel pyroptosis-dependent and pyroptosis-independent pathways for cross-presentation of IEC-derived antigens.

The NAIP/NLRC4 inflammasome is a cytosolic sensor of bacteria that activates Caspase-1 and initiates potent downstream immune responses. Structural, biochemical, and genetic data all demonstrate that the NAIP proteins act as receptors for specific bacterial ligands, while NLRC4 is a downstream adaptor protein that multimerizes with NAIPs to form a macromolecular structure called an inflammasome. However, several aspects of NLRC4 biology remain unresolved. For example, in addition to its clear function in responding to bacteria, NLRC4 has also been proposed to initiate anti-tumor responses, though the underlying mechanism is unknown. NLRC4 has also been shown to be phosphorylated on serine 533, and this modification was suggested to be important for NLRC4 function. In the absence of S533 phosphorylation, it was further proposed that another inflammasome component, NLRP3, can induce NLRC4 activation. We generated a new Nlrc4deficient mouse line as well as mice encoding phosphomimetic S533D and nonphosphorylatable S533A NLRC4 proteins. Using these genetic models in vivo and in vitro, we fail to observe a role for phosphorylation in NLRC4 inflammasome function.Furthermore, we find no role for NLRP3 in NLRC4 function, or for NLRC4 in a model of melanoma. These results simplify and clarify our understanding of the mechanism of NAIP/NLRC4 activation and its biological functions.

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