Regulation Of Immune Responses In Humans and in Experimental Animals

National Institute Of Allergy And Infectious Diseases

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Abstract

During this period we continued to conduct studies of the role of phosphorylation of NLRC4 at serine 533 vis-a-vie NLRC4 inflammasome activation, a previously unresolved and fraught question with respect to NLRC4 inflammasome-mediated cytokine secretion. In a series of studies we showed that NLRC4 is not phosphorylated in cells lacking the capacity to synthesize LRRK2 (LRRK2 KO cells) when stimulated with established NLRC4 activators, either LFn-Flagellin or LFn-Needle (N terminus of lethal factor fused to L.pneumophila flagellin or Needle protein); in addition, there is no NLRC4 phosphorylation of serine 533 in cells in which the phosphorylation of LRRK2 and thus its kinase function is totally blocked by an LRRK2 inhibitor. These results strongly suggest that, at least in dendritic cells and macrophages, phosphorylation of NLRC4 at serine 533 is dependent on LRRK2 kinase activity and there are no other kinases with this function at play. In further studies now focusing on the relation of ASC to LRRK2-mediated phosphorylation of NLRC4 we evaluated NLRC4 phosphorylation in cells lacking ASC expression (ASC KO THP1 cells) or cells with efficient ASC mRNA knock-down. Here we found that NLRC4 phosphorylation during NLRC4 activation does not occur in the absence of ASC, indicating that ASC binding to NLRC4 is necessary for NLRC4 interaction with and phosphorylation by LRRK2. In addition, whereas ASC binding to NLRC4 occurs in the absence of LRRK2 (and thus in the absence of NLRC4 phosphorylation) it does not undergo oligomerization in the absence of LRRK2-mediated NLRC4 phosphorylation. Finally, we found that whereas activation of the NLRC4 inflammasome and its production of mature (cleaved) IL-1b is greatly reduced in ASC-KO cells or ASC knock-down cells, its production of mature, cleaved IL-18 is only slightly reduced in such cells; furthermore, whereas inhibition of NLRC4 phosphorylation by LRRK2 inhibitor reduces NLRC4 inflammasome production of mature IL-1b by at least 40%, such inhibition has little effect on production of mature IL-18. Given the differential effect of ASC oligomerization on IL-1b and IL-18, these results strongly suggested that for optimal mature IL-1b production the NLRC4 inflammasome utilizes both the ASC and NLRC4 CARD domains for caspase activation whereas for mature IL-18 production the NLRC4 CARD domain is sufficient. In subsequent molecular studies to very this conclusion we showed that NLRC4 inflammasome activity in HEK293 cells transfected with WT NLRC4 and NLRC4 bearing mutations that negated ASC^CARD binding to NLRC4^CARD. We thus concluded that whereas pro-IL-1b cleavage is partially dependent on LRRK2-mediated ASC binding and cleavage function, pro-IL-18 is independent of such ASC function. In a series of functional studies exploring the significance of the above conclusion with regard to LRRK2 inhibitor function we subjected mice to IP injection of LFn-Flagellin or LFn-Needle IP in the presence or absence of LRRK2 inhibitor and determined effects of inhibitor on NLRC4 inflammsome activation on gut permeability as measured by labelled albumin translocation into the circulation. We found that both stimuli greatly increased gut permeability and that this effect was completely neutralized by inhibitor. Thus, the partial reduction of IL-1b production due to inhibition of NLRC4 phosphorylation coupled with the nil effect of the latter on IL-18 production was effective in reversing the untoward effect of NLRC4 inflammasome activation on gut permeability. In on-going studies of the effect of inhibitors on cells from patients with Crohn's disease conducted during this period we conducted extensive studies to optimize our protocol for evaluation of LRRK2 effects on NLRC4 inflammasome activation in peripheral macrophages or dendritic cells. In particular we sought methods of avoiding the confounding effect of inhibitors on pro-IL-1b and IL-18 production due to inhibitor effects on TLR ligands routinely used to stimulate such production. Ultimately we found that inhibitor function is best evaluated in the absense of TLR stimulation. With this optimized method in hand we corroborated previous studies showing that stimulation of patient cells with flagellin or needle proteligand led to higher NLRC4 inflammasome activation as indicated by higher IL-1b and IL-18 production than stimulation of control cells, possibly reflecting the presence of higher LRRK2 levels. However, whereas IL-1b production by patient cells was very substantially decreased by the presence of LRRK2-inhibitor, increased IL-18 production was unaffected by inhibitor. These data indicate that elevated NLRC4 inflammasome-induced IL-1b production in Crohn's disease is highly LRRK2-mediated NLRC4 phosphorylation dependent whereas NLRC4 inflammasome induced IL-18 production is independent of LRRK2-mediated phosphorylation. In related studies related to the use of LRRK2 inhibitors as a possible treatment of Crohn's disease we have continued assessment of the capacity of candidate inhibitors to ameliorate DSS-colitis. We have confirmed earlier findings that LRRK2 inhibition leads to amelioration of DSS-colitis. WE are now in a position to test candidate inhibitors in other gut inflammation models.