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

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. 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. 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. 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.