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.
Autoimmune disease is the leading cause of morbidity among women. For largely unknown reasons, many autoimmune diseases show a striking female bias. Fibrosis is a common feature of female-biased autoimmune diseases. This is exemplified by systemic sclerosis (SSc), a debilitating disease marked by progressive skin hardening and organ damage that affects women at ninefold the rate of men. We previously identified the transcriptional cofactor VGLL3 as an immune regulator enriched in female skin whose targets overlap significantly with genes dysregulated in SSc. We further showed that excess epidermal VGLL3 causes cutaneous and systemic autoimmune disease in mice. However, how VGLL3 promotes autoimmunity in the skin remains entirely unexplored. By combining IP-mass spectrometry, RNA-seq, and ChIP-seq approaches in cell culture and transgenic mice with epidermal VGLL3 overexpression, we have found that VGLL3 binds key factors in the Hippo signaling pathway to modulate both immune genes and established Hippo pathway targets. These targets include the pro-fibrotic factor CTGF and members of the TGF-b pathway, both of which have been implicated in SSc pathogenesis. Consistent with this, transgenic mice with epidermal VGLL3 overexpression show gross and microscopic features of skin fibrosis. These findings elucidate the molecular mechanisms by which VGLL3 promotes autoimmunity and leads to the hallmark fibrosis of many autoimmune diseases such as SSc. Furthermore, this reveals a previously unexplored connection between autoimmune disease and the Hippo signaling pathway, which has recently been linked to organ fibrosis.
Pellagra, acrodermatitis enteropathica and biotin deficiency are caused by the deficiency of specific nutrients. These patients commonly present with similar skin dermatosis and show shared histological alterations, including vacuolization and necrosis of keratinocytes in epidermis. Recent studies have shown that number of epidermal Langerhans cells decreased at these skin lesions. However, the precise reasons, why Langerhans cells decrease or disappeared, have not yet been elucidated. In this study, we generated several nutrition deficient model mice including pellagra. After BALB/c mice were fed with niacin-deficient diet or control diet for 4 to 12 weeks (n¼3-6), we performed microarray analysis using RNA extracted from mice skin. We found Langerhans cells determined by CD207 were substantially decreased or eventually disappeared in pellagra model mice skin even without any pathological change. The microarray data also shows the decreased expression of CD209 (DC-SIGN), which expressed in dermal and mucous tissue, lymphoid tissue, as well as on monocyte-derived dendritic cells. In addition, the expressions of CCR2, CCL7 and CCL8 were also significantly downregulated in pellagra mice. In the inflamed skin, the activation of CCR2 by CCL2 or CCL7 is known to be essential for the recruitment of blood-borne precursor cells of Langerhans cells. Our observations suggest that the lack of niacin disturbs the crucial expression of CCL7, CCL8 and the subsequent absence of Langerhans cells is caused by the loss of recruitment of precursor cells to the lesional skin after prolonged inflammation.
Atopic dermatitis is a common cutaneous disorder characterized by severe itch, chronic inflammation and increased nerve fiber density. It has been assumed that the neural changes are in response to ongoing inflammation. We used in vivo imaging of fluorescently labeled peripheral sensory nerves over time during epicutaneous sensitization to ovalbumin in an allergic mouse model of atopic dermatitis. Visualization of the same cutaneous nerve branches and blood vessels sequentially over months revealed that peripheral sensory nerves begin to pathfind within 48 hours of antigen exposure and results in higher innervation density and arbor complexity of neuropeptidergic fibers in the skin within days. Neural sprouting preceded changes in vascularization, vascular permeability, and immune infiltration. Blocking neural activation during periods of sensitization prevented ovalbumin-induced changes in neural recruitment and pattern reorganization as well as subsequent inflammatory infiltrate, scratching behavior. These data implicate different roles for recently identified itch molecules in modulating various steps in the inflammatory response. Thus, in contrast to the traditional view that neural changes are reactive to inflammation and scratching, our data suggest that allergic stimulation in a chronic eczema model actually requires neural recruitment and activation for the elaboration and maintenance of the inflammatory cascade.
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