Dynamic pathological analysis reveals a protective role against skin fibrosis for TREM2-dependent macrophages.

Rationale: Systemic sclerosis (SSc) is a chronic and incurable autoimmune disease with high mortality rates, and skin fibrosis is one of distinguishing hallmarks in the pathogenesis. However, macrophage heterogeneity regulating skin fibrosis remain largely unknown. Methods: We established mouse disease model and performed single-cell RNA-sequencing (scRNA-seq) to resolve the dynamic and heterogenous characteristics of macrophages in skin fibrosis, and the role of TREM2-dependent macrophages in the pathological process was investigated using knockout mice and intraperitoneal transferring TREM2+ macrophages combining with functional assays. Results: We show that TREM2-expressing macrophages (TREM2+ MФs) accumulate in injured skin of mice treated by bleomycin (BLM) and human SSc, and their gene signatures and functional pathways are identified in the course of disease. Genetic ablation of Trem2 in mice globally accelerates and aggravates skin fibrosis, whereas transferring TREM2hi macrophages improves and alleviates skin fibrosis. Amazingly, we found that disease-associated TREM2+ MФs in skin fibrosis exhibit overlapping signatures with fetal skin counterparts in mice and human to maintain skin homeostasis, but each has merits in skin remodeling and development respectively. Conclusion: This study identifies that TREM2 acts as a functional molecule and a major signaling by which macrophage subpopulations play a protective role against fibrosis, and disease-associated TREM2+ MФs in skin fibrosis might undergo a fetal-like reprogramming similar to fetal skin counterparts.

JAK1/2 Inhibitor Baricitinib Improves Skin Fibrosis and Digital Ulcers in Systemic Sclerosis.

Background: Systemic sclerosis (SSc) is a rare disabling connective tissue disease with few available treatment options. Diffuse cutaneous systemic sclerosis (dcSSc) is associated with high mortality. A previous experiment has shown that JAK2 inhibitor can significantly improve skin fibrosis in bleomycin (BLM)-induced murine model, including reducing dermal thickening and collagen accumulation. We aimed to describe the efficacy of oral JAK1/2 inhibitor baricitinib in SSc patients, especially focusing on skin fibrosis and microvascular manifestations. Methods: We described the different effects of oral selective JAK1, JAK2, or JAK3 inhibitor treatment in a BLM-induced skin fibrosis mouse model. Furthermore, 10 adult patients with dcSSc were treated with baricitinib. We assessed the changes in modified rodman skin score (mRSS) and digital ulcer net burden at week 12 and 24 from baseline. We also compared the absolute changes in scores on the Scleroderma Health Assessment Questionnaire (SHAQ) and a total score on the St. George's Respiratory Questionnaire (SGRQ) over a 24-week period. Results: In the experimental mouse model of skin fibrosis, a JAK1 and JAK2 inhibitor ameliorated skin fibrosis, and a JAK2 inhibitor had the most obvious effect. Treatment with the JAK2 inhibitor also blunted the capillary rarefaction. We demonstrated that skin fibrosis and digital ulcers were significantly relieved in 10 SSc patients treated with baricitinib. The mRSS significantly improved at week 12 from baseline, with a mean change in mRSS of -8.3 [95% confidence interval (CI), -12.03 to -4.574; p = 0.0007] and improved greater at week 24 to -11.67 (95% CI, -16.84 to -6.496; p = 0.0008). Among the four patients with digital ulcers (DU), three were completely healed at week 24, the number of ulcers in another patient was significantly reduced, and there was no patient with new ulcers. Only one adverse event (AE) of herpes zoster was observed. Conclusions: Our results indicate that selective JAK1 and JAK2 inhibitor alleviates skin fibrosis, and oral JAK1/2 inhibitor baricitinib is a potentially effective treatment for dcSSc patients with skin fibrosis and DU. Baricitinib was well-tolerated by most patients in this study. Additional large clinical trials are needed to confirm our pilot findings. Chinese Clinical Trial Registry Number: ChiCTR2000030995.

Corrigendum: JAK1/2 inhibitor baricitinib improves skin fibrosis and digital ulcers in systemic sclerosis.

[This corrects the article DOI: 10.3389/fmed.2022.859330.].

Mesenchymal Stem Cells Activate the MEK/ERK Signaling Pathway and Enhance DNA Methylation via DNMT1 in PBMC from Systemic Lupus Erythematosus.

The defective MEK/ERK signaling pathway and downstream hypomethylation pattern of lymphocytes are crucial for the pathogenesis of systemic lupus erythematosus (SLE). However, the role that the mesenchymal stem cells play in the MEK/ERK signaling pathway and DNA methylation of peripheral blood mononuclear cells (PBMC) from SLE patients remains unknown. In this study, we found that the MEK/ERK signaling pathway of PBMC from SLE patients was activated after the coculture with bone marrow-derived mesenchymal stem cells (BM-MSC) compared with that from the control group. In addition, the expression level of DNA methyltransferase 1 (DNMT1) increased while the levels of CD70, integrin, alpha L (ITGAL), selectin-l, and IL-13 were reduced in PBMC from SLE patients. However, no obvious effect of BM-MSC on PBMC from healthy controls was observed. These findings revealed that BM-MSC might downregulate the expression of methylation-sensitive genes and then suppress the autoactivated PBMC via the MEK/ERK signaling pathway. And it may be one of the mechanisms that BM-MSC ameliorated SLE.

MCP-1 Priming Enhanced the Therapeutic Effects of Human Mesenchymal Stromal Cells on Contact Hypersensitivity Mice by Activating the COX2-PGE2/STAT3 Pathway.

Mesenchymal stromal cells (MSCs) have become a promising treatment for inflammation-related diseases, and their therapeutic efficacy mainly depends on crosstalk between MSCs and inflammation. However, methods to improve the immunosuppressive efficiency of MSCs in different diseases still need to be developed. In this study, we investigated whether preconditioning MSCs with a disease-related inflammatory cytokine could increase their immunosuppressive properties and improve therapeutic efficacy. In a contact hypersensitivity (CHS) mouse model, inflammatory profile screening revealed that among all tested cytokines, monocyte chemotactic protein-1 (MCP-1) exhibited the most significantly increased level in the local microenvironment. As expected, MSCs preconditioned with MCP-1 (P-MSCs) exhibited an enhanced ability to downregulate proinflammatory cytokine secretion, induce regulatory T cells, inhibit T cell proliferation, and polarize M2-type macrophages. In vivo experiments showed that P-MSCs alleviated ear swelling and local proinflammatory cytokine production more effectively than control MSCs. Mechanistically, MCP-1 could significantly activate the signal transducer and activator of transcription 3 (STAT3) signaling pathway and induce the expression of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2) in MSCs. STAT3 inhibitor reversed the MCP-1-mediated enhancing of their immunosuppressive ability. Collectively, our findings demonstrate that CHS-related MCP-1 preconditioning enhanced the immunomodulatory effects of MSCs and improved their therapeutic efficacy in CHS. Enhancing the immunosuppressive efficacy of MSCs by preconditioning with certain disease-related inflammatory cytokines may provide a new strategy for MSC-based therapies for inflammatory diseases.

Keratin 14-dependent disulfides regulate epidermal homeostasis and barrier function via 14-3-3σ and YAP1.

The intermediate filament protein keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Recent studies evidenced a role for K14-dependent disulfide bonding in the organization and dynamics of keratin IFs in skin keratinocytes. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at Krt14's codon 373 (C373A) exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in epidermis. A proteomics screen identified 14-3-3 as K14 interacting proteins. Follow-up studies showed that YAP1, a transcriptional effector of Hippo signaling regulated by 14-3-3sigma in skin keratinocytes, shows aberrant subcellular partitioning and function in differentiating Krt14 C373A keratinocytes. Residue C373 in K14, which is conserved in a subset of keratins, is revealed as a novel regulator of keratin organization and YAP function in early differentiating keratinocytes, with an impact on cell mechanics, homeostasis and barrier function in epidermis.

Research Techniques Made Simple: Single-Cell RNA Sequencing and its Applications in Dermatology.

RNA sequencing is one of the most highly reliable and reproducible methods of assessing the cell transcriptome. As high-throughput RNA sequencing libraries at the single cell level have recently developed, single cell RNA sequencing has become more feasible and popular in biology research. Single cell RNA sequencing allows investigators to evaluate cell transcriptional profiles at the single cell level. It has become a very useful tool to perform investigations that could not be addressed by other methodologies, such as the assessment of cell-to-cell variation, the identification of rare populations, and the determination of heterogeneity within a cell population. So far, the single cell RNA sequencing technique has been widely applied to embryonic development, immune cell development, and human disease progress and treatment. Here, we describe the history of single cell technology development and its potential application in the field of dermatology.

Formaldehyde Inhibits Sexual Behavior and Expression of Steroidogenic Enzymes in the Testes of Mice.

BACKGROUND: Formaldehyde, a ubiquitous environmental pollutant, is used extensively and has been proved to impair male reproduction in mammals. However, no trials have explored whether formaldehyde affects sexual function. AIM: To evaluate the effect of long-term formaldehyde exposure on sexual behavior and to investigate the potential mechanism. METHODS: Forty C57BL/6 male mice were randomly allocated to four equally sized groups. Mice were exposed to formaldehyde at a dose of 0 (control), 0.5, 5.0, or 10.0 mg/m3 by inhalation for 60 days. OUTCOMES: Sexual behavior, body and reproductive organ weights, testosterone concentration in serum and testicular tissue, expression of steroidogenic enzymes, quality of sperm, and testicular structure were measured. RESULTS: Formaldehyde inhibited sexual behavior and decreased reproductive organ weights in mice. Serum testosterone levels and intratesticular testosterone concentrations were decreased in the formaldehyde-treated groups. Expression levels of steroidogenic enzymes, including steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase (3ß-HSD), also were decreased in the testes of mice exposed to formaldehyde. Moreover, the structure of seminiferous tubules was destroyed and sperm quality decreased after formaldehyde exposure. In addition, the results indicated that the effects of formaldehyde were dose dependent. CLINICAL IMPLICATIONS: Efforts should be undertaken to decrease impairment of sexual function caused by formaldehyde exposure. STRENGTHS AND LIMITATIONS: The relatively small sample might have affected the outcomes. Further experiments are needed to study the mechanism of action of formaldehyde. CONCLUSION: Exposure to formaldehyde gas inhibited sexual behavior, caused reproductive organ atrophy, and impaired spermatogenesis in male mice, which might have been induced by suppressed expression of steroidogenic enzymes in Leydig cells and decreased testosterone synthesis. Zang Z-J, Fang Y-Q, Ji S-Y, et al. Formaldehyde Inhibits Sexual Behavior and Expression of Steroidogenic Enzymes in the Testes of Mice. J Sex Med 2017;14:1297-1306.

Effects of Saikokaryukotsuboreito on Spermatogenesis and Fertility in Aging Male Mice.

BACKGROUND: Aspermia caused by exogenous testosterone limit its usage in late-onset hypogonadism (LOH) patients desiring fertility. Saikokaryukotsuboreito (SKRBT) is reported to improve serum testosterone and relieve LOH-related symptoms. However, it is unclear whether SKRBT affects fertility. We aimed to examine the effects of SKRBT on spermatogenesis and fertility in aging male mice. METHODS: Thirty aging male mice were randomly assigned to three groups. Mice were orally administered with phosphate-buffer solution or SKRBT (300 mg/kg, daily) or received testosterone by subcutaneous injections (10 mg/kg, every 3 days). Thirty days later, each male mouse was mated with two female mice. All animals were sacrificed at the end of 90 days. Intratesticular testosterone (ITT) levels, quality of sperm, expression of synaptonemal complex protein 3 (SYCP3), and fertility were assayed. RESULTS: In the SKRBT-treated group, ITT, quality of sperm, and expression of SYCP3 were all improved compared with the control group (ITT: 85.50 ± 12.31 ng/g vs. 74.10 ± 11.45 ng/g, P = 0.027; sperm number: [14.94 ± 4.63] × 106 cells/ml vs. [8.79 ± 4.38] × 106 cells/ml, P = 0.002; sperm motility: 43.16 ± 9.93% vs. 33.51 ± 6.98%, P = 0.015; the number of SYCP3-positive cells/tubule: 77.50 ± 11.01 ng/ml vs. 49.30 ± 8.73 ng/ml, P < 0.001; the expression of SYCP3 protein: 1.23 ± 0.09 vs. 0.84 ± 0.10, P < 0.001), but fertility was not significantly changed (P > 0.05, respectively). In the testosterone-treated group, ITT, quality of sperm, and expression of SYCP3 were markedly lower than the control group (ITT: 59.00 ± 8.67, P = 0.005; sperm number: [4.34 ± 2.45] × 106 cells/ml, P = 0.018; sperm motility: 19.53 ± 7.69%, P = 0.001; the number of SYCP3-positive cells/tubule: 30.00 ± 11.28, P < 0.001; the percentage of SYCP3-positive tubules/section 71.98 ± 8.88%, P = 0.001; the expression of SYCP3 protein: 0.71 ± 0.09, P < 0.001), and fertility was also suppressed (P < 0.05, respectively). CONCLUSION: SKRBT had no adverse effect on fertility potential in aging male mice.

Effects of velvet antler polypeptide on sexual behavior and testosterone synthesis in aging male mice.

Twenty-four-month-old male C57BL/6 mice with low serum testosterone levels were used as a late-onset hypogonadism (LOH) animal model for examining the effects of velvet antler polypeptide (VAP) on sexual function and testosterone synthesis. These mice received VAP for 5 consecutive weeks by daily gavage at doses of 100, 200, or 300 mg kg-1 body weight per day (n = 10 mice per dose). Control animals (n = 10) received the same weight-based volume of vehicle. Sexual behavior and testosterone levels in serum and interstitial tissue of testis were measured after the last administration of VAP. Furthermore, to investigate the mechanisms of how VAP affects sexual behavior and testosterone synthesis in vivo, the expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in Leydig cells was also measured by immunofluorescence staining and quantitative real-time PCR. As a result, VAP produced a significant improvement in the sexual function of these aging male mice. Serum testosterone level and intratesticular testosterone (ITT) concentration also increased in the VAP-treated groups. The expression of StAR, P450scc, and 3ß-HSD was also found to be enhanced in the VAP-treated groups compared with the control group. Our results suggested that VAP was effective in improving sexual function in aging male mice. The effect of velvet antler on sexual function was due to the increased expression of several rate-limiting enzymes of testosterone synthesis (StAR, P450scc, and 3ß-HSD) and the following promotion of testosterone synthesis in vivo.

Mesenchymal stem cell transplantation inhibits abnormal activation of Akt/GSK3ß signaling pathway in T cells from systemic lupus erythematosus mice.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by activation and proliferation of autoreactive T cells and B cells. We examined changes in cell cycle progression of T cells from MRL/lpr mice with or without allogenic bone marrow mesenchymal stem cells (BMMSCs) treatment and analyzed the expression of cell cycle associated proteins. In addition, the Akt/GSK3ß protein kinase cascade was studied. We demonstrated that high-dose MSCs transplantation effectively ameliorated disease activity in MRL/lpr mice. BMMSCs treatment inhibited G1/S transition of the abnormal lupus T lymphocytes. Moreover, it increased the expression of p21(WAF1/CIP1) and p27(Kip1) and decreased the expression of CDK2. Furthermore, high-dose MSCs inhibited abnormal activation of the Akt/GSK3ß signaling pathway of T cells from MRL/lpr mice. Our results suggest that high-dose BMMSCs transplantation successfully treated MRL/lpr lupus mice by inhibiting abnormal activation of Akt/GSK3ß signaling pathway of T cells.

Predictive factors and unfavourable prognostic factors of interstitial lung disease in patients with polymyositis or dermatomyositis: a retrospective study.

BACKGROUND: Interstitial lung disease (ILD) is a serious lung complication in polymyositis (PM) and dermatomyositis (DM) which affects prognosis and requires a more aggressive approach in therapy. This study investigated the prevalence, characteristics, predictive factors and unfavourable prognostic factors of ILD in newly diagnosed PM, DM and amyopathic DM (ADM). METHODS: From January 2000 to December 2008, the medical records of 197 consecutive PM and DM patients at the Second Affiliated Hospital of Sun Yat-Sen University were reviewed excluding overlapping, juvenile, and malignancy-associated cases. The patients were assigned to an ILD (69 patients) and a non-ILD group (128 patients). The clinical features, laboratory findings, and prognosis were compared. RESULTS: The multivariate analysis indicated that older age at onset (OR 1.033, 95%CI 1.009 - 1.058, P = 0.007), fever (OR 4.109, 95%CI 1.926 - 8.767, P < 0.001) and arthritis/arthralgia (OR 2.274, 95%CI 1.101 - 4.695, P = 0.026) were the independent predictive factors for developing ILD in PM/DM after excluding anti-Jo-1. Regarding anti-Jo-1, fever (OR 4.912, 95%CI 2.121 - 11.376, P < 0.001) was associated with ILD. Poor survival in ILD patients was associated with ILD clinical subset (RR 0.122, 95%CI 0.049 - 0.399, P < 0.001), ADM/DM/PM-ILD (RR 0.140, 95%CI 0.031 - 0.476, P = 0.002), cardiac involvement (RR 4.654, 95%CI 1.391 - 15.577, P = 0.013) and serum albumin level (RR 0.910, 95%CI 0.831 - 0.997, P = 0.042). CONCLUSIONS: Patients who presented with fever tended to have a higher frequency of PM/DM-associated ILD. A Hamman-Rich-like presentation, ADM-ILD, cardiac involvement and hypoalbuminemia were poor prognostic factors in ILD-PM/DM.