CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

Purpose— This study assessed the pharmacological effect of a novel selective C-C chemokine receptor (CCR) 2 antagonist (GSK1344386B) on monocyte/macrophage infiltration into atherosclerotic plaque using magnetic resonance imaging (MRI) in an atherosclerotic mouse model. Methods and Results— Apolipoprotein E −/− mice expressing human CCR2 were fed a Western diet (vehicle group) or a Western diet plus10 mg/kg per day of GSK1344386B (GSK1344386B group). After the baseline MRI, mice were implanted with osmotic pumps containing angiotensin II, 1000 ng/kg per minute, to accelerate lesion formation. After five weeks of angiotensin II administration, mice received ultrasmall superparamagnetic iron oxide, an MRI contrast agent for the assessment of monocyte/macrophage infiltration to the plaque, and underwent imaging. After imaging, mice were euthanized, and the heart and aorta were harvested for ex vivo MRI and histopathological examination. After 5 weeks of dietary dosing, there were no significant differences between groups in body or liver weight or plasma cholesterol concentrations. An in vivo MRI reflected a decrease in ultrasmall superparamagnetic iron oxide contrast agent uptake in the aortic arch of the GSK1344386B group ( P <0.05). An ex vivo MRI of the aortic root also reflected decreased ultrasmall superparamagnetic iron oxide uptake in the GSK1344386B group and was verified by absolute iron analysis ( P <0.05). Although there was no difference in aortic root lesion area between groups, there was a 30% reduction in macrophage area observed in the GSK1344386B group ( P <0.05). Conclusion— An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.