Regulation Of Immune Responses In Humans and in Experimental Animals

National Institute Of Allergy And Infectious Diseases

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Abstract

Our studies of LRRK2 function has centered around "gut-restricted" LRRK2 inhibitors of LRRK2 that are presumed to have less lung or kidney toxicity than previous inhibitors while retaining LRRK2 inhibitory potency and specificity. These inhibitors, synthesized by collaborating investigators at Mt. Sinai Medical Center, are analogs of known (already developed) LRRK2 kinase inhibitors (such as CZC-54252) that have previously been synthesized for the treatment of Parkinson's Disease. In essence, the modifications of known inhibitor involves the addition of chemical groups (called kinetophores)that have previously been shown to increase blood clearance of a compound while its enhancing intestinal retention. The modified inhibitors will be subjected to intensive testing by the Mt. Sinai investigators to define their pharmacokinetic properties, their kinase specificity and their toxicity with the intention of ultimately using them to treat Crohn's disease. We conducted extensive studies of the comparative capacity of modified inhibitor (CS-82) to suppress TLR4 (LPS), TLR2 (Zymosan) and Dectin-1 (Zymosan-depleted S.cerevisciae, ZymD) stimulated cytokine production at a non-toxic concentration. We found first that whereas CS-82 had little or no effect on LRRK2 expression it greatly inhibited LRRK2 phosphorylation and ability to phosphorylate known LRRK2 targets, Rab-10 and Rab-12; it was thus shown to be a potent inhibitor of LRRK2 kinase function. With respect to effect on macrophage cytokine production, CS-82 manifested equal or marginally greater ability to suppress TLR-induced TNF-a and IL-6 production than commercially available inhibitor. In contrast, it inhibited IL-10 production to a lesser extent than commercially available inhibitor. These inhibitory qualities were manifest in vivo in that administration of CS-82 to mice subjected to DSS-colitis proved able to ameliorate colitis and to reduce both IL-12 and TNF-a mRNA lamina propria production in the inflamed colon. In studies of the relation of LRRK2 to NLRC4 inflammasome activity we first established culture systems that could potentially be used to assess such activity. To this end, we set up cultures of human DCs or macrophages (or THP-1 cells) in which the cells are stimulated with LPS and Bacillus anthracis protective antigen (PA)(the latter to facilitate antigen entry) plus LFn-Flagellin or LFn-Needle (N terminus of lethal factor fused to L.pneumophila flagellin or Needle protein), two bacterial substances that act as activating ligands of NLRC4 via NAIP interaction upon cell entry. Indeed, we found that cultured cells exposed to these ligands secrete substantial amounts of IL-1b and IL-18, i,e., products of the NLRC4 inflammasome and thus that such cultured cells could serve to assess NLRC4 activation. With this assessment system in hand we then conducted extensive studies to determine the effect of LRRK2 inhibition on NLRC4 inflammasome activation using a spectrum of inhibitors including CS-82 mentioned above as well as an inhibitor thought to be a more specific inhibitor of LRRK2 kinase function, MLi-2. In initial in vitro studies we found that inhibitors of LRRK2 kinase activity completely inhibit both its binding to NLRC4 and the latter's phosphorylation upon activation of the NLRC4 inflammasome with purified needle protein, thus establishing that LRRK2 has an indispensable role in NLRC4 phosphorylation. In further studies using THP-1 cells with ASC deletion or human PBMC-derived dendritic cells with efficient shRNA ASC-knock-down we showed that ASC deficiency retards NLRC4 phosphorylation and that inhibition of NLRC4 phosphorylation prevents ASC oligomerization; thus, while phosphorylation requires NLRC4 by association with ASC, lack of phosphorylation inhibits ASC function. Finally, we found that activation of the NLRC4 inflammasome by either needle protein or live Salmonella organisms when its phosphorylation is blocked by LRRK2 inhibitor leads to reduced IL-1 cleavage and expression, but, surprisingly, preserved IL-18 cleavage and expression. This indicates that full IL-1b expression requires ASC-caspase binding whereas IL-18 expression does not and the latter is most likely due to NLRC4/CARD domain-dependent caspase cleavage. In accompanying in vivo studies, we found that systemic administration of an NLRC4 inflammasome activator (needle protein) caused increased intestinal permeability that was reversed by co-administration of LRRK2 inhibitor. This shows that inhibition of inflammasome production of IL-1b coupled with the preservation of production of IL-18 via LRRK2 inhibition in intestinal macrophages blocks the potentially harmful effects of NLRC4 inflammasome activation on intestinal permeability during intestinal inflammation. In studies of the effect of inhibitors on cells from patients with Crohn;s disease we found that stimulation of patient cells with flagellin ligand 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 (CS-82) whereas IL-1b production by control cells was not; at the same time, 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 LRRK2-mediated phosphorylation independent.