Immune dysregulation in SHARPIN-deficient mice is dependent on CYLD-mediated cell death

SHARPIN, together with RNF31/HOIP and RBCK1/HOIL1, form the linear ubiquitin chain assembly complex (LUBAC) E3 ligase that catalyzes M1-linked poly-ubiquitination. Mutations in RNF31/HOIP and RBCK/HOIL1 in humans and Sharpin in mice lead to auto-inflammation and immunodeficiency but the mechanism underlying the immune dysregulation remains unclear. We now show that the phenotype of the Sharpin-/- mice is dependent on CYLD, the deubiquitinase that removes K63-linked poly-ubiquitin chains. The dermatitis, disrupted splenic architecture, and loss of Peyer9s patches in the Sharpin-/- mice were fully reversed in Sharpin-/-Cyld-/- mice. There is enhanced association of RIPK1 with the death-inducing signaling complex (DISC) following TNF stimulation in Sharpin-/- cells, and this is dependent on CYLD since it is reversed in Sharpin-/-Cyld-/- cells. Enhanced RIPK1 recruitment to the DISC in Sharpin-/- cells correlated with impaired phosphorylation of CYLD at serine 418, a modification reported to inhibit its enzymatic activity. The dermatitis in the Sharpin-/- mice was also ameliorated by the conditional deletion of Cyld using LysM-cre or Cx3cr1-cre indicating that CYLD-dependent death of myeloid cells is inflammatory. Our studies reveal that under physiological conditions, TNF- and RIPK1-dependent cell death is suppressed by the linear ubiquitin-dependent inhibition of CYLD. The Sharpin-/- phenotype illustrates the pathological consequences when CYLD inhibition fails.