Preventing MALT1-mediated CYLD cleavage induces intestinal dysbiosis and reduces EAE severity.

The paracaspase MALT1 is essential for lymphocyte activation and also plays roles in non-immune cells and cancer. Its protease activity regulates immune signaling by cleaving specific substrates, making it a promising therapeutic target. However, broad inhibition of MALT1 protease activity causes multiorgan inflammation in mice, highlighting the need to understand the effects of individual substrate cleavage. We generated CYLD(R321A) knock-in mice expressing a MALT1-resistant form of the deubiquitinase CYLD. These mice are healthy, with normal lymphocyte development and preserved immune signaling. Unlike MALT1 protease-dead mice, they do not develop spontaneous inflammation. Notably, they exhibit altered gut microbiota and reduced disease severity in a model of multiple sclerosis. Together, our work shows that blocking cleavage of a single MALT1 substrate is sufficient to modulate microbiota and neuroinflammation without causing overt defects in lymphocyte cell development or activation, providing in vivo evidence for substrate-specific targeting of MALT1 as a refined therapeutic strategy.