Return of the first inflammasome

Abstract

The assembly of inflammasomes is linked to the detection of pathogens and other danger signals by intracellular pattern-recognition receptors of the mammalian innate immune system. The human inflammasome sensor NLRP1 is activated by N-terminal proteolytic cleavage and subsequent degradation, causing the release of the C-terminal NLRP1^UPA-CARD fragment and the recruitment of the adaptor protein ASC and caspase-1, resulting in the processing of IL-1ß/IL-18 and pyroptotic cell death.<br />To study NLRP1 inflammasomes, I characterized HEK 293T and N/TERT-1 keratinocyte inflammasome reporter cell lines, and I identified two NLRP1^PYD-specific nanobodies which, combined with the E3 ligase receptor VHL, allowed the precise stimulation of endogenous NLRP1 by targeted NLRP1^PYD ubiquitination and subsequent N-terminal degradation.<br />Using the reporter cell lines, I found that various stimuli of the ribotoxic stress response activate human NLRP1 in a p38-dependent manner. In addition, infection with alphaviruses, including Semliki Forrest virus and Chikungunya virus, caused p38-dependent NLRP1 activation. p38 kinases directly phosphorylate the N-terminal linker region of the inflammasome sensor, in which serine 107 represents a critical phosphorylation site. I propose that phosphorylation of the N-terminal linker generates a phospho-degron which is recognized by cullin RING E3 ligases, causing the ubiquitination of NLRP1^PYD, N-terminal degradation, and inflammasome assembly.<br />Using the HEK 293T reporter cell lines, I further discovered two simian rotavirus strains that activate NLRP1 in a p38-independent manner. For the rotavirus A SA11-4F strain, I found that expression of the viral host antagonist NSP1 is necessary and sufficient for inflammasome activation. Since rotavirus NSP1 is known to induce host protein degradation, I hypothesize that NSP1 binds the N-terminus of NLRP1, leading to the activation of the inflammasome by N-terminal degradation. This would make rotavirus NSP1 the first pathogen protein to cause targeted degradation of human NLRP1.<br />Finally, I analyzed NLRP1 inflammasome assembly in primary blood cells and found that T and B lymphocytes can form NLRP1 inflammasomes. Moreover, NLRP1-specific activation by nanobody-mediated N-terminal degradation induced strong inflammasome formation and pyroptosis in stimulated primary T cells, confirming that T cells have functional NLRP1 inflammasomes. The identification of lymphocytes as NLRP1 inflammasome-forming cells might provide new insights into the development of autoimmune diseases that are associated with NLRP1 gain-of-function mutations.<br />Altogether, I extended the knowledge about human NLRP1 inflammasomes by delineating p38-mediated NLRP1 activation, identifying several novel viral NLRP1 stimuli, and establishing lymphocytes as NLRP1-competent cell types.