Optimal autophagic activity is crucial to maintain muscle integrity, with either reduced or excessive levels leading to specific myopathies. LGMD2H is a muscle dystrophy caused by mutations in the ubiquitin ligase TRIM32, whose function in muscles remains not fully understood. Here, we show that TRIM32 is required for the induction of muscle autophagy in atrophic conditions using both in vitro and in vivo mouse models. Trim32 inhibition results in a defective autophagy response to muscle atrophy, associated with increased ROS and MuRF1 levels. The proautophagic function of TRIM32 relies on its ability to bind the autophagy proteins AMBRA1 and ULK1 and stimulate ULK1 activity via unanchored K63-linked polyubiquitin. LGMD2H-causative mutations impair TRIM32’s ability to bind ULK1 and induce autophagy. Collectively, our study revealed a role for TRIM32 in the regulation of muscle autophagy in response to atrophic stimuli, uncovering a previously unidentified mechanism by which ubiquitin ligases activate autophagy regulators.
Altered innate immunity is a feature of certain skin inflammatory diseases such as psoriasis and atopic dermatitis (AD). In this study, we provide evidence that deficiency in Trim32 (a tripartite motif (TRIM) protein with innate antiviral activity) contributes to a Th2 biased response and predisposes to features of AD in mice. Upon treatment with the TLR7 agonist imquimod (IMQ), Trim32 knockout (KO) mice displayed compromised psoriasiform phenotypes and defective Th17 response. Instead, IMQ treatment of Trim32 KO mice induced AD-like phenotypes with enhanced skin infiltration of eosinophils and mast cells, elevation of Th2 cytokines/chemokines expression, and reduced expression of filaggrin protein expression. Furthermore, while the induction of phosphorylated Stat3 and RelA were compromised following IMQ treatment in the KO mice, phosphorylated Stat6 was elevated. CCL20 induction by TNFα and IL-17A was reduced in Trim32 deficient keratinocytes whereas CCL5 induction by TNFα and IL-4 was enhanced. In addition, Trim32 protein levels were elevated in mice treated with IMQ. Unlike Trim32 overexpression in psoriasis, TRIM32 levels were low in AD patients. Based on Trim32 induction by IMQ, the lower levels of TRIM32 in AD skin compared to healthy control and psoriatic skin suggest a defective TRIM32 pathway in AD pathogenesis.
Psoriasis is a T cell and IL-17 dependent inflammatory skin disease. Helper T cells have been assumed to be the major source of IL-17 in psoriasis but other cell types can also produce this cytokine. We immunostained human psoriatic lesions (n¼15) and healthy skin (n¼10) for IL-17A, T cells (CD4, CD8) and neutrophils (myeloperoxidase, MPO). We found that 32% of MPO+ neutrophils in psoriasis samples produced IL-17A, compared with less than 1% of total CD4 + and CD8 + cells. There were on average 10.6 IL-17A producing neutrophils per 10 high power fields in psoriasis, compared to 2.7 IL-17A producing T cells (p¼0.008). IL-17A producing neutrophils outnumbered IL-17A producing T cells four-fold demonstrating that neutrophils are an important source of IL-17A in psoriasis. To explore potential interactions between keratinocytes and neutrophils in psoriasis, we co-cultured these cells in vitro and studied neutrophil cytokine production by quantitative RT-PCR and intracellular immunostaining and flow cytometry analysis. Neutrophils co-cultured with keratinocytes upregulated production of IL-17A, IL-17F and IL-22 at both the protein and RNA levels. Neutrophils cultured with keratinocytes lost CD62L and upregulated CD11b, consistent with activation. In summary, this study suggests that neutrophils, known for long to be a part of the histologic landscape of psoriasis, are a major source of IL-17A production and have the potential to contribute to inflammation by producing IL-17F and IL-22. This is, to our knowledge, the first report that human neutrophils can produce IL-17F and IL-22.
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