Sharma, B. R. | Search bioRxiv

bioRxiv2022

Determining distinct roles of IL-1α through generation of an IL-1α knockout mouse with no defect in IL-1β expression

Interleukin 1 (IL-1) and IL-1{beta} are the founding members of the IL-1 cytokine family, and these innate immune inflammatory mediators are critically important in health and disease. Early studies on these molecules suggested that their expression was interdependent, with an initial genetic model of IL-1 depletion, the IL-1 KO mouse (Il1a-KOline1), showing reduced IL-1{beta} expression. However, studies using this line in models of infection and inflammation resulted in contrasting observations. To overcome the limitations of this genetic model, we have generated and characterized a new line of IL-1 KO mice (Il1a-KOline2) using CRISPR-Cas9 technology. In contrast to cells from the Il1a-KOline1, where IL-1{beta} expression was drastically reduced, bone marrow-derived macrophages (BMDMs) from Il1a-KOline2 mice showed normal induction and activation of IL-1{beta}. Additionally, Il1a-KOline2 BMDMs showed normal inflammasome activation and IL-1{beta} expression in response to multiple innate immune triggers, including both pathogen-associated molecular patterns and pathogens. Moreover, using Il1a-KOline2 cells, we confirmed that IL-1, independent of IL-1{beta}, is critical for the expression of the neutrophil chemoattractant KC/CXCL1. Overall, we report the generation of a new line of IL-1 KO mice and confirm functions for IL-1 independent of IL-1{beta}. Future studies on the unique functions of IL-1 and IL-1{beta} using these mice will be critical to identify new roles for these molecules in health and disease and develop therapeutic strategies.

cell biology↗

bioRxiv2020

COVID-19 cytokines and the hyperactive immune response: Synergism of TNF-α and IFN-γ in triggering inflammation, tissue damage, and death

The COVID-19 pandemic has caused significant morbidity and mortality. Currently, there is a critical shortage of proven treatment options and an urgent need to understand the pathogenesis of multi-organ failure and lung damage. Cytokine storm is associated with severe inflammation and organ damage during COVID-19. However, a detailed molecular pathway defining this cytokine storm is lacking, and gaining mechanistic understanding of how SARS-CoV-2 elicits a hyperactive inflammatory response is critical to develop effective therapeutics. Of the multiple inflammatory cytokines produced by innate immune cells during SARS-CoV-2 infection, we found that the combined production of TNF- and IFN-{gamma} specifically induced inflammatory cell death, PANoptosis, characterized by gasdermin-mediated pyroptosis, caspase-8-mediated apoptosis, and MLKL-mediated necroptosis. Deletion of pyroptosis, apoptosis, or necroptosis mediators individually was not sufficient to protect against cell death. However, cells deficient in both RIPK3 and caspase-8 or RIPK3 and FADD were resistant to this cell death. Mechanistically, the JAK/STAT1/IRF1 axis activated by TNF- and IFN-{gamma} co-treatment induced iNOS for the production of nitric oxide. Pharmacological and genetic deletion of this pathway inhibited pyroptosis, apoptosis, and necroptosis in macrophages. Moreover, inhibition of PANoptosis protected mice from TNF- and IFN-{gamma}-induced lethal cytokine shock that mirrors the pathological symptoms of COVID-19. In vivo neutralization of both TNF- and IFN-{gamma} in multiple disease models associated with cytokine storm showed that this treatment provided substantial protection against not only SARS-CoV-2 infection, but also sepsis, hemophagocytic lymphohistiocytosis, and cytokine shock models, demonstrating the broad physiological relevance of this mechanism. Collectively, our findings suggest that blocking the cytokine-mediated inflammatory cell death signaling pathway identified here may benefit patients with COVID-19 or other cytokine storm-driven syndromes by limiting inflammation and tissue damage. The findings also provide a molecular and mechanistic description for the term cytokine storm. Additionally, these results open new avenues for the treatment of other infectious and autoinflammatory diseases and cancers where TNF- and IFN-{gamma} synergism play key pathological roles.

immunology↗