Regulation Of Immune Responses In Humans and in Experimental Animals

National Institute Of Allergy And Infectious Diseases

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Abstract

Collaborating investigators at Mt. Sinai Medical Center have been developing 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 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, a disease frequently accompanied by gain of function LRRK2 mutations or polymorphisms. In essence, the modifications of known inhibitor consist of the addition of chemical groups (called kinetophores)that increase blood clearance of the compound while enhancing intestinal retention. The modified inhibitors will be subjected to intensive testing to define their pharmacokinetic properties, their kinase specificity and their toxicity with the intention of ultimately using them to treat Crohn's disease. Several of these inhibitors have been sent to this Laboratory and have undergone in vitro testing to determine their inhibitory properties. We found that human dendritic cells (DCs)stimulated by Zymogen-depleted S.cerevisciae (ZymD, a Dectin-1 ligand)and exposed to inhibitor exhibited decreased phosphorylated LRRK2 and decreased phosphorylated Rab 10 and Rab12 (GTP-binding proteins that are known LRRK2 kinase targets). These results were similar to those obtained with the parent inhibitor (CZC54252) and indicate that the modified inhibitors had retained LRRK2 inhibitory properties. In subsequent studies of cytokine secretion inhibition by two modified inhibitors (CS 190 and CS 82)were clearly more potent than parent inhibitor in their capacities to inhibit PMA/IFN-g-stimulated DC secretion of TNF-a and IL-1b and were equal to parent inhibitor in their capacity to inhibit ZymD-stimulated TNF-a production. Finally, administration of one of the inhibitors (CS82) to mice subjected to DSS-colitis proved able to ameliorate colitis. These studies provide evidence that the modified inhibitors retain characteristics of the parent inhibitor from which they were derived. 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 the two modified inhibitors (CS82 and CS190)almost completely abrogated 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. These studies also suggest that increased levels of LRRK2 such as that in some patients with Crohn's disease causes increased inflammation as a result of more potent NLRC4 inflammasome activity.