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 have 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 stimulated human DCs with LPS plus LFn-Fla or LFn-needle (N terminus of lethal factor fused to L.pneumophila flagellin or Needle protein) in the presence of PA (Bacillus anthracis protective antigen), two bacterial substances that enter cells and activate NLRC4 via NAIP interaction. We found that both stimuli induced secretion of IL-1b and IL-18, i,e., products of the NLRC4 inflammasome. In addition, we found that two modified inhibitors (CS-82 and CS-190)suppressed NLRC4-induced IL-1b secretion whereas the parent inhibitor had a much more marginal inhibitory effect. The modified inhibitor also inhibited NLRC4-induced IL-18 secretion but to a lesser extent. Control studies showed that the inhibitors had little effect on NLRP3-induced IL-1b secretion and therefore their inhibition of the NLRC4 inflammasome was an inflammasome-specific effect. Finally, with Western blot studies we showed that NLRC4 stimulation of cells in the presence of all inhibitors (both parent and modified) led to loss of NLRC4 phosphorylation at Serine 533 as well as LRRK2 phosphorylation. These studies imply that NLRC4 phosphorylation at S533 by LRRK2 kinase is indeed important for NLRC4 induction of IL-1b but less so for induction of IL-18. However, it should be noted that IL-1b production was observed (albeit at a reduced level) in the face of suppressed NLRC4 phosphorylation suggesting that inflammasome activity can proceed at some level without phosphorylation. In the limited studies of the effect of inhibitors on cells from patients with Crohn;s disease we found that stimulation of patient cells with flagellin led to higher inflammasome stimulation than in control cells, probably reflecting higher LRRK2 levels. This was accompanied by greater IL-1b production, but not greater IL-18 production.