Inactivation of NF-κB and MAPKs confers the potent therapeutic effect of carboxyamidotriazole on Blau syndrome.

BACKGROUND AND OBJECTIVES: Blau syndrome (BS) is a rare autosomal dominant autoinflammatory disease with limited treatment options, driven by the nucleotide-binding oligomerization domain containing 2 (NOD2) mutations that constitutively activate RIP2-mediated nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling. We have reported the potent anti-inflammatory activity of carboxyamidotriazole (CAI), originally developed as a non-cytotoxic anticancer drug. In this study, we investigated the therapeutic potential and possible mechanism of CAI for BS. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with BS. Various BS-specific cellular models and animal models were established. Immunoblot, immunofluorescence and immunohistochemistry were utilized to examine the NOD2-mediated inflammatory signaling pathways. NF-κB activity was measured with a dual-luciferase reporter assay system, and cytokines were detected by ELISA. RESULTS: In BS patient-derived PBMCs, CAI did not affect RIP2 activation under unstimulated conditions or following stimulation with muramyl dipeptide (MDP) plus lipopolysaccharide (LPS), but it suppressed NF-κB activation by reducing IKK phosphorylation and p65 nuclear translocation, ultimately leading to a marked attenuation of pro-inflammatory cytokines production. In cellular models, CAI inhibited MDP-induced activation of both NF-κB and MAPKs pathways in RAW264.7 cells, as well as the production of cytokines triggered by MDP alone or in combination with LPS. This anti-inflammatory activity was also confirmed in immortalized bone marrow-derived macrophage (iBMDM) and THP-1 cell models. Furthermore, CAI effectively suppressed constitutive NF-κB activity driven by two prevalent BS-associated NOD2 mutants (R334W and R334Q) in HEK293T cells. Finally, in animal models, CAI treatment effectively alleviated L18-MDP-induced systemic inflammation in a mouse BS model. In a rat model of BS-associated uveitis, CAI’s efficacy was further validated by its ability to ameliorate ocular pathological damage, inhibit the overactivation of NF-κB and MAPKs, and decrease the levels of pro-inflammatory cytokines in ocular tissues. CONCLUSIONS: Our findings suggest that CAI may have therapeutic value in BS by targeting the core pathogenic NF-κB/MAPK signaling axes and reducing cytokines production, which highlights CAI as a new potential medication for BS.