Probes for INS

Detection of individual cytokines in routine biopsies from patients with inflammatory skin diseases has the potential to personalize diagnosis and treatment selection, but this approach has been limited by technical feasibility. We evaluate whether a chromogen-based RNA in situ hybridization approach can be used to detect druggable cytokines in psoriasis and atopic dermatitis. A series of psoriasis (n = 20) and atopic dermatitis (n = 26) biopsies were stained using RNA in situ hybridization for IL4, IL12B (IL-12/23 p40), IL13, IL17A, IL17F, IL22, IL23A (IL-23 p19), IL31, and TNF (TNF-α). NOS2 and IFNG, canonical psoriasis biomarkers, were also included. All 20 of the psoriasis cases were positive for IL17A, which tended to be the predominant cytokine, although some cases had relatively higher levels of IL12B, IL17F, or IL23A. The majority of cytokine expression in psoriasis was epidermal. A total of 22 of 26 atopic dermatitis cases were positive for IL13, also at varying levels; a subset of cases had significant IL4, IL22, or IL31 expression. Patterns were validated in independent bulk RNA-sequencing and single-cell RNA-sequencing datasets. Overall, RNA in situ hybridization for cytokines appears highly specific with virtually no background staining and may allow for individualized evaluation of treatment-relevant cytokine targets in biopsies from patients with inflammatory skin disorders.

The peroxisome proliferator-activated receptor (PPAR) nuclear receptor has been an interesting target for the treatment of chronic diseases. Although the efficacy of PPAR pan agonists in several metabolic diseases has been well studied, the effect of PPAR pan agonists on kidney fibrosis development has not been demonstrated. To evaluate the effect of the PPAR pan agonist MHY2013, a folic acid (FA)-induced in vivo kidney fibrosis model was used. MHY2013 treatment significantly controlled decline in kidney function, tubule dilation, and FA-induced kidney damage. The extent of fibrosis determined using biochemical and histological methods showed that MHY2013 effectively blocked the development of fibrosis. Pro-inflammatory responses, including cytokine and chemokine expression, inflammatory cell infiltration, and NF-κB activation, were all reduced with MHY2013 treatment. To demonstrate the anti-fibrotic and anti-inflammatory mechanisms of MHY2013, in vitro studies were conducted using NRK49F kidney fibroblasts and NRK52E kidney epithelial cells. In the NRK49F kidney fibroblasts, MHY2013 treatment significantly reduced TGF-β-induced fibroblast activation. The gene and protein expressions of collagen I and α-smooth muscle actin were significantly reduced with MHY2013 treatment. Using PPAR transfection, we found that PPARγ played a major role in blocking fibroblast activation. In addition, MHY2013 significantly reduced LPS-induced NF-κB activation and chemokine expression mainly through PPARβ activation. Taken together, our results suggest that administration of the PPAR pan agonist effectively prevented renal fibrosis in both in vitro and in vivo models of kidney fibrosis, implicating the therapeutic potential of PPAR agonists against chronic kidney diseases.

Abstract BACKGROUND: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4 T-cell function. METHODS: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4 T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. RESULTS: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4 T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1β leads to enhanced production of IL17A. CONCLUSIONS: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

A high-fat diet (HFD) is a major risk factor for chronic kidney disease. Although HFD promotes renal injury, characterized by increased inflammation and oxidative stress leading to fibrosis, the underlying mechanism remains elusive. Here, we investigated the role and mechanism of protease-activating receptor 2 (PAR2) activation during HFD-induced renal injury in C57/BL6 mice. HFD for 16 weeks resulted in kidney injury, manifested by increased blood levels of blood urea nitrogen, increased levels of oxidative stress with inflammation, and structural changes in the kidney tubules. HFD-fed kidneys showed elevated PAR2 expression level in the tubular epithelial region. To elucidate the role of PAR2, PAR2 knockout mice and their littermates were administered HFD. PAR2 deficient kidneys showed reduced extent of renal injury. PAR2 deficient kidneys showed significantly decreased levels of inflammatory gene expression and macrophage infiltration, followed by reduced accumulation of extracellular matrix proteins. Using NRK52E kidney epithelial cells, we further elucidated the mechanism and role of PAR2 activation during renal injury. Palmitate treatment increased PAR2 expression level in NRK52E cells and scavenging of oxidative stress blocked PAR2 expression. Under palmitate-treated conditions, PAR2 agonist-induced NF-κB activation level was higher with increased chemokine expression level in the cells. These changes were attenuated by the depletion of oxidative stress. Taken together, our results suggest that HFD-induced PAR2 activation is associated with increased levels of renal oxidative stress, inflammatory response, and fibrosis.

Demodex mites are commensal parasites of hair follicles (HFs). Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction, and aging, but mechanisms restricting Demodex outgrowth are not defined. Here, we show that control of mite HF colonization in mice required group 2 innate lymphoid cells (ILC2s), interleukin-13 (IL-13), and its receptor, IL-4Ra-IL-13Ra1. HF-associated ILC2s elaborated IL-13 that attenuated HFs and epithelial proliferation at anagen onset; in their absence, Demodex colonization led to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammation, leading to the loss of barrier function and HF exhaustion. Humans with rhinophymatous acne rosacea, an inflammatory condition associated with Demodex, had increased HF inflammation with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a key role for skin ILC2s and IL-13, which comprise an immune checkpoint that sustains cutaneous integrity and restricts pathologic infestation by colonizing HF mites.