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RIPK3 kinase activity triggers cell death via necroptosis, while scaffold function supports protein binding and cytokine production. To determine if RIPK3 kinase or scaffold domains mediate pathology during Pseudomonas aeruginosa infection, control mice and those with deletion or mutation of RIPK3 and associated signaling partners were subjected to Pseudomonas pneumonia and followed for survival or sacrificed for biologic assays. Murine immune cells were studied in vitro for Pseudomonas-induced cytokine production and cell death, and RIPK3 binding interactions were blocked with the viral inhibitor M45. Human tissue effects were assayed by infecting airway epithelial cells with Pseudomonas and measuring cytokine production after siRNA inhibition of RIPK3. Deletion of RIPK3 reduced inflammation and decreased animal mortality following Pseudomonas pneumonia. RIPK3 kinase inactivation did neither. In cell culture, RIPK3 was dispensable for cell killing by Pseudomonas and instead drove cytokine production that required the RIPK3 scaffold domain but not kinase activity. Blocking the RIP homotypic interaction motif (RHIM) with M45 reduced the inflammatory response to infection in vitro. Similarly, siRNA knockdown of RIPK3 decreased infection-triggered inflammation in human airway epithelial cells. Thus, the RIPK3 scaffold drives deleterious pulmonary inflammation and mortality in a relevant clinical model of Pseudomonas pneumonia. This process is distinct from kinase-mediated necroptosis, requiring only the RIPK3 RHIM. Inhibition of RHIM signaling is a potential strategy to reduce lung inflammation during infection.

Programmed cell death pathways eliminate infected cells and regulate infection-associated inflammation during pathogen invasion. Cytomegaloviruses encode several distinct suppressors that block intrinsic apoptosis, extrinsic apoptosis, and necroptosis, pathways that impact pathogenesis of this ubiquitous herpesvirus. Here, we expanded the understanding of three cell autonomous suppression mechanisms on which murine cytomegalovirus relies: (i) M38.5-encoded viral mitochondrial inhibitor of apoptosis (vMIA), a BAX suppressor that functions in concert with M41.1-encoded viral inhibitor of BAK oligomerization (vIBO), (ii) M36-encoded viral inhibitor of caspase-8 activation (vICA), and (iii) M45-encoded viral inhibitor of RIP/RHIM activation (vIRA). Following infection of bone marrow-derived macrophages, the virus initially deflected receptor-interacting protein kinase (RIPK)3-dependent necroptosis, the most potent of the three cell death pathways. This process remained independent of caspase-8, although suppression of this apoptotic protease enhances necroptosis in most cell types. Second, the virus deflected TNF-mediated extrinsic apoptosis, a pathway dependent on autocrine TNF production by macrophages that proceeds independently of mitochondrial death machinery or RIPK3. Third, cytomegalovirus deflected BCL-2 family protein-dependent mitochondrial cell death through combined TNF-dependent and -independent signaling even in the absence of RIPK1, RIPK3, and caspase-8. Furthermore, each of these cell death pathways dictated a distinct pattern of cytokine and chemokine activation. Therefore, cytomegalovirus employs sequential, non-redundant suppression strategies to specifically modulate the timing and execution of necroptosis, extrinsic apoptosis, and intrinsic apoptosis within infected cells to orchestrate virus control and infection-dependent inflammation. Virus-encoded death suppressors together hold control over an intricate network that upends host defense and supports pathogenesis in the intact mammalian host.

The squalene-based oil-in-water emulsion (SE) vaccine adjuvant MF59 has been administered to more than 100 million people in more than 30 countries, in both seasonal and pandemic influenza vaccines. Despite its wide use and efficacy, its mechanisms of action remain unclear. In this study we demonstrate that immunization of mice with MF59 or its mimetic AddaVax (AV) plus soluble antigen results in robust antigen-specific antibody and CD8 T cell responses in lymph nodes and non-lymphoid tissues. Immunization triggered rapid RIPK3-kinase dependent necroptosis in the lymph node which peaked at 6 hr, followed by a sequential wave of apoptosis. Immunization with alum plus antigen did not induce RIPK3-dependent signaling. RIPK3-dependent signaling induced by MF59 or AV was essential for cross-presentation of antigen to CD8 T cells by Batf3-dependent CD8+ DCs. Consistent with this, RIPK3 deficient or Batf3 deficient mice were impaired in their ability to mount adjuvant-enhanced CD8 T cell responses. However, CD8 T cell responses were unaffected in mice deficient in MLKL, a downstream mediator of necroptosis. Surprisingly, antibody responses were unaffected in RIPK3-kinase or Batf3 deficient mice. In contrast, antibody responses were impaired by in vivo administration of the pan-caspase inhibitor Z-VAD-FMK, but normal in caspase-1 deficient mice, suggesting a contribution from apoptotic caspases, in the induction of antibody responses. These results demonstrate that squalene emulsion-based vaccine adjuvants induce antigen-specific CD8 T cell and antibody responses, through RIPK3-dependent and-independent pathways, respectively.

Z-nucleic acid binding protein (ZBP)1 (also known as DAI and DLM1) is a pathogen sensor activated by double-strand character RNA to recruit receptor-interacting protein (RIP) kinase via a RIP homotypic interaction motif. The activation of receptor-interacting protein kinase (RIPK)3 and initiation of virus-induced necroptosis were initially reported in a landmark publication Upton et al. (Cell Host Microbe 11: 290, 2012) employing the DNA virus murine cytomegalovirus (MCMV). M45-encoded viral inhibitor of RIP activation prevents virus-induced necroptosis. Additional virus-encoded suppressors of necroptosis were then identified, including herpes simplex virus ICP6 and vaccinia virus E3L. Caspase-8 suppressors encoded by these DNA viruses block apoptosis, unleashing necroptosis mediated through Z-nucleic acid binding protein 1 (ZBP1) recruitment of RIPK3. These studies all utilized ZBP1-deficient mice generated by the Akira Lab (Zbp1-/- AK ) to bring the significance of virus-induced necroptosis to light. C57BL/6 mice were chosen as controls based on the assumption that mutant mice were congenic; however, these mice were recently found to display an unexpected innate immune deficit, lacking C57BL/6-specific NK1.1 and Ly49H natural killer cell subpopulations important in the early control of MCMV infection. Short nucleotide polymorphism analysis of Zbp1-/- AK breeders revealed a mixed genetic background (~ 71% C57BL/6 DNA and ~ 29% 129). Even though this level of 129 strain background does not alter ZBP1 cell-autonomous function as a sensor and mediator of necroptosis, it confounds innate immune response characteristics. In the future, genetic background must be carefully controlled before implicating ZBP1 function in response characteristics that shape immunity, inflammation, metabolism, and pathogenesis.

Idiopathic Pulmonary Fibrosis (IPF) is a deadly disease with limited therapies. Tissue fibrosis is associated with Type 2 immune response, although the causal contribution of immune cells is not defined. The AP-1 transcription factor Fra-2 is upregulated in IPF lung sections and Fra-2 transgenic mice (Fra-2tg) exhibit spontaneous lung fibrosis. Here we show that Bleomycin-induced lung fibrosis is attenuated upon myeloid-inactivation of Fra-2 and aggravated in Fra-2tg bone marrow chimeras. Type VI collagen (ColVI), a Fra-2 transcriptional target, is up-regulated in three lung fibrosis models, and macrophages promote myofibroblast activation in vitro in a ColVI- and Fra-2-dependent manner. Fra-2 or ColVI inactivation does not affect macrophage recruitment and alternative activation, suggesting that Fra-2/ColVI specifically controls the paracrine pro-fibrotic activity of macrophages. Importantly, ColVI knock-out mice (KO) and ColVI-KO bone marrow chimeras are protected from Bleomycin-induced lung fibrosis. Therapeutic administration of a Fra-2/AP-1 inhibitor reduces ColVI expression and ameliorates fibrosis in Fra-2tg mice and in the Bleomycin model. Finally, Fra-2 and ColVI positively correlate in IPF patient samples and co-localize in lung macrophages. Therefore, the Fra-2/ColVI pro-fibrotic axis is a promising biomarker and therapeutic target for lung fibrosis, and possibly other fibrotic diseases.

Summary The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-β-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.