Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection.

Cell death and inflammation are intimately linked during Yersinia infection. Pathogenic Yersinia inhibits the MAP kinase TGFß-activated kinase 1 (TAK1) via the effector YopJ, thereby silencing cytokine expression while activating caspase-8-mediated cell death. Here, using Yersinia pseudotuberculosis in corroboration with costimulation of lipopolysaccharide and (5Z)-7-Oxozeaenol, a small-molecule inhibitor of TAK1, we show that caspase-8 activation during TAK1 inhibition results in cleavage of both gasdermin D (GSDMD) and gasdermin E (GSDME) in murine macrophages, resulting in pyroptosis. Loss of GsdmD delays membrane rupture, reverting the cell-death morphology to apoptosis. We found that the Yersinia-driven IL-1 response arises from asynchrony of macrophage death during bulk infections in which two cellular populations are required to provide signal 1 and signal 2 for IL-1α/ß release. Furthermore, we found that human macrophages are resistant to YopJ-mediated pyroptosis, with dampened IL-1ß production. Our results uncover a form of caspase-8-mediated pyroptosis and suggest a hypothesis for the increased sensitivity of humans to Yersinia infection compared with the rodent reservoir.

Cutting Edge: Novel Tmem173 Allele Reveals Importance of STING N Terminus in Trafficking and Type I IFN Production.

With the stimulator of IFN genes (STING) C terminus being extensively studied, the role of the N-terminal domain (NTD) of STING remains an important subject of investigation. In this article, we identify novel mutations in NTD of Sting of the MOLF strain in response to HSV and Listeria monocytogenes both in vitro and in vivo. These mutations are responsible for low levels of IFN-ß caused by failure of MOLF STING to translocate from the endoplasmic reticulum. These data provide evidence that the NTD of STING affects DNA responses via control of trafficking. They also show that the genetic diversity of wild-derived mice resembles the diversity observed in humans. Several human alleles of STING confer attenuated IFN-I production similar to what we observe with the MOLF Sting allele, a crucial functional difference not apparent in classical inbred mice. Thus, understanding the functional significance of polymorphisms in MOLF STING can provide basic mechanistic insights relevant to humans.

CD209a expression on dendritic cells is critical for the development of pathogenic Th17 cell responses in murine schistosomiasis.

In murine schistosomiasis, immunopathology and cytokine production in response to parasite eggs are uneven and strain dependent. CBA/J (CBA) mice develop severe hepatic granulomatous inflammation associated with prominent Th17 cell responses driven by dendritic cell (DC)-derived IL-1ß and IL-23. Such Th17 cells fail to develop in low-pathology C57BL/6 (BL/6) mice, and the reasons for these strain-specific differences in APC reactivity to eggs remain unclear. We show by gene profiling that CBA DCs display an 18-fold higher expression of the C-type lectin receptor CD209a, a murine homolog of human DC-specific ICAM-3-grabbing nonintegrin, compared with BL/6 DCs. Higher CD209a expression was observed in CBA splenic and granuloma APC subpopulations, but only DCs induced Th17 cell differentiation in response to schistosome eggs. Gene silencing in CBA DCs and overexpression in BL/6 DCs demonstrated that CD209a is essential for egg-elicited IL-1ß and IL-23 production and subsequent Th17 cell development, which is associated with SRC, RAF-1, and ERK1/2 activation. These findings reveal a novel mechanism controlling the development of Th17 cell-mediated severe immunopathology in helminthic disease.

Lessons for the Next Generation of Scientists from the Second Annual Arthur and Sandra Irving Cancer Immunology Symposium.

The Arthur and Sandra Irving Cancer Immunology Symposium has been created as a platform for established cancer immunologists to mentor trainees and young investigators as they launch their research career in the field. By sharing their different paths to success, the senior faculty mentors provide an invaluable resource to support the development of the next generation of leaders in the cancer immunology community. This Commentary describes some of the key topics that were discussed during the 2022 symposium: scientific and career trajectory, leadership, mentoring, collaborations, and publishing. For each of these topics, established investigators discussed the elements that facilitate success in these areas as well as mistakes that can hinder progress. Herein, we outline the critical points raised in these discussions for establishing a successful independent research career. These points are highly relevant for the broader scientific community.

Constitutive Interferon Attenuates RIPK1/3-Mediated Cytokine Translation.

Receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) are well known for their capacity to drive necroptosis via mixed-lineage kinase-like domain (MLKL). Recently, RIPK1/3 kinase activity has been shown to drive inflammation via activation of MAPK signaling. However, the regulatory mechanisms underlying this kinase-dependent cytokine production remain poorly understood. In the present study, we establish that the kinase activity of RIPK1/3 regulates cytokine translation in mouse and human macrophages. Furthermore, we show that this inflammatory response is downregulated by type I interferon (IFN) signaling, independent of type I IFN-promoted cell death. Specifically, low-level constitutive IFN signaling attenuates RIPK-driven activation of cap-dependent translation initiation pathway components AKT, mTORC1, 4E-BP and eIF4E, while promoting RIPK-dependent cell death. Altogether, these data characterize constitutive IFN signaling as a regulator of RIPK-dependent inflammation and establish cap-dependent translation as a crucial checkpoint in the regulation of cytokine production.

Constitutive interferon signaling maintains critical threshold of MLKL expression to license necroptosis.

Interferons (IFNs) are critical determinants in immune-competence and autoimmunity, and are endogenously regulated by a low-level constitutive feedback loop. However, little is known about the functions and origins of constitutive IFN. Recently, lipopolysaccharide (LPS)-induced IFN was implicated as a driver of necroptosis, a necrotic form of cell death downstream of receptor-interacting protein (RIP) kinase activation and executed by mixed lineage kinase like-domain (MLKL) protein. We found that the pre-established IFN status of the cell, instead of LPS-induced IFN, is critical for the early initiation of necroptosis in macrophages. This pre-established IFN signature stems from cytosolic DNA sensing via cGAS/STING, and maintains the expression of MLKL and one or more unknown effectors above a critical threshold to allow for MLKL oligomerization and cell death. Finally, we found that elevated IFN-signaling in systemic lupus erythematosus (SLE) augments necroptosis, providing a link between pathological IFN and tissue damage during autoimmunity.

Host-Intrinsic Interferon Status in Infection and Immunity.

Most genetic ablations of interferon (IFN) signaling abolish both the experimentally induced IFN response and constitutive IFN, whose effects are well established in autoimmunity but understudied during infection. In host-pathogen interactions, most IFN-mediated responses are attributed to infection-driven IFN. However, IFNs confer their activity by regulating networks of interferon-stimulated genes (ISGs), a process that requires de novo transcription and translation of both IFN and downstream ISGs through feedback of IFN receptor signaling. Due to the temporal requirement for IFN activity, many rapid antimicrobial responses may instead result from pre-established IFN signature stemming from host-intrinsic processes. Addressing the permeating effects of constitutive IFN is therefore needed to accurately describe immunity as host intrinsic or pathogen induced.

Constitutive Interferon Maintains GBP Expression Required for Release of Bacterial Components Upstream of Pyroptosis and Anti-DNA Responses.

Legionella pneumophila elicits caspase-11-driven macrophage pyroptosis through guanylate-binding proteins (GBPs) encoded on chromosome 3. It has been proposed that microbe-driven IFN upregulates GBPs to facilitate pathogen vacuole rupture and bacteriolysis preceding caspase-11 activation. We show here that macrophage death occurred independently of microbial-induced IFN signaling and that GBPs are dispensable for pathogen vacuole rupture. Instead, the host-intrinsic IFN status sustained sufficient GBP expression levels to drive caspase-1 and caspase-11 activation in response to cytosol-exposed bacteria. In addition, endogenous GBP levels were sufficient for the release of DNA from cytosol-exposed bacteria, preceding the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway for Ifnb induction. Mice deficient for chromosome 3 GBPs were unable to mount a rapid IL-1/chemokine (C-X-C motif) ligand 1 (CXCL1) response during Legionella-induced pneumonia, with defective bacterial clearance. Our results show that rapid GBP activity is controlled by host-intrinsic cytokine signaling and that GBP activities precede immune amplification responses, including IFN induction, inflammasome activation, and cell death.

The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms.

The T cell receptor (TCR) triggers the assembly of "SLP-76 microclusters," which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase-associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A "tandem dimer" containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP-interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and "inside-out" signaling to ß1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and ß1 integrins.