Ox40-Cre-mediated deletion of BRD4 reveals an unexpected phenotype of hair follicle stem cells in alopecia.

BRD4 is a bromodomain extraterminal domain family member and functions primarily as a chromatin reader regulating genes involved in cell-fate decisions. Here, we bred Brd4fl/fl Ox40-Cre mice in which Brd4 was conditionally deleted in OX40-expressing cells to examine the role of BRD4 in regulating immune responses. We found that the Brd4fl/fl Ox40-Cre mice developed profound alopecia and dermatitis, while other organs and tissues were not affected. Surprisingly, lineage-tracing experiments using the Rosa26fl/fl-Yfp mice identified a subset of hair follicle stem cells (HFSCs) that constitutively express OX40, and deletion of Brd4 specifically in such HFSCs resulted in cell death and a complete loss of skin hair growth. We also found that death of HFSCs triggered massive activation of the intradermal γδ T cells, which induced epidermal hyperplasia and dermatitis by producing the inflammatory cytokine IL-17. Interestingly, deletion of Brd4 in Foxp3+ Tregs, which also constitutively express OX40, compromised their suppressive functions, and this, in turn, contributed to the enhanced activation of γδ T cells, as well as the severity of dermatitis and hair follicle destruction. Thus, our data demonstrate an unexpected role of BRD4 in regulating skin follicle stem cells and skin inflammation.

Suppression of FOXP3 expression by the AP-1 family transcription factor BATF3 requires partnering with IRF4.

FOXP3 is the lineage-defining transcription factor for Tregs, a cell type critical to immune tolerance, but the mechanisms that control FOXP3 expression in Tregs remain incompletely defined, particularly as it relates to signals downstream of TCR and CD28 signaling. Herein, we studied the role of IRF4 and BATF3, two transcription factors upregulated upon T cell activation, to the conversion of conventional CD4+ T cells to FOXP3+ T cells (iTregs) in vitro. We found that IRF4 must partner with BATF3 to bind to a regulatory region in the Foxp3 locus where they cooperatively repress FOXP3 expression and iTreg induction. In addition, we found that interactions of these transcription factors are necessary for glycolytic reprogramming of activated T cells that is antagonistic to FOXP3 expression and stability. As a result, Irf4 KO iTregs show increased demethylation of the critical CNS2 region in the Foxp3 locus. Together, our findings provide important insights how BATF3 and IRF4 interactions integrate activating signals to control CD4+ cell fate decisions and govern Foxp3 expression.

Neutrophil-to-Lymphocyte Ratio as a Predictive Biomarker for Stroke Severity and Short-Term Prognosis in Acute Ischemic Stroke With Intracranial Atherosclerotic Stenosis.

Background: Neutrophil-to-lymphocyte ratio (NLR) is an indicator of poor prognosis in acute ischemic stroke (AIS), but associations between NLR with stroke severity and prognosis of intracranial atherosclerotic stenosis (ICAS)-related ischemic events have not been well-elucidated; therefore, we aimed to evaluate whether admission NLR levels correlate with the early stroke severity and short-term functional prognosis in patients with symptomatic intracranial atherosclerotic stenosis (sICAS). Methods: This retrospective study enrolled 899 consecutive patients with AIS attributed to ICAS at Xiangya Hospital stroke center between May 2016 and September 2020. The initial stroke severity was rated by the admission National Institutes of Health Stroke Scale (NIHSS) scores, and the short-term prognosis was evaluated using the 14-day modified Rankin Scale (mRS) scores after stroke onset. A severe stroke was defined as NIHSS >8; an unfavorable functional outcome was defined as mRS scores of 3-6. Admission NLR was determined based on circulating neutrophil and lymphocyte counts. Results: The median admission NLR of all patients was 2.80 [interquartile range (IQR), 2.00-4.00]. In univariate analysis, admission NLR was significantly elevated in patients with severe stroke and poor short-term prognosis. After multivariate adjustment, admission NLR levels were significantly correlated with severe stroke [odds ratio (OR), 1.132; 95% confidence interval (95% CI), 1.038-1.234; P = 0.005] and unfavorable short-term prognosis (OR, 1.102; 95% CI, 1.017-1.195; P = 0.018) in Model 1. In Model 2, the highest NLR tertile (≥3.533) remained an independent predictor of severe stroke (OR, 2.736; 95% CI, 1.590-4.708; P < 0.001) and unfavorable functional outcome (OR, 2.165; 95% CI, 1.416-3.311; P < 0.001) compared with the lowest NLR tertile (<2.231). The receiver operating characteristic (ROC) curves showed the predictability of NLR regarding the stroke severity [area under the curve (AUC), 0.659; 95% CI, 0.615-0.703; P < 0.001] and short-term prognosis (AUC, 0.613; 95% CI, 0.575-0.650; P < 0.001). The nomograms were constructed to create the predictive models of the severity and short-term outcome of sICAS. Conclusions: Elevated admission NLR levels were independently associated with the initial stroke severity and could be an early predictor of severity and poor short-term prognosis in AIS patients with ICAS, which might help us identify a target group timely for preventive therapies.

Molecular Biomarkers Associated with Early-Onset Symptomatic Intracranial Atherosclerosis.

PURPOSE: Previous studies have shown a rising incidence of early-onset symptomatic intracranial atherosclerosis (sICAS), which has brought a severe economic burden to social development. This study aimed to evaluate the molecular biomarkers associated with early-onset sICAS and to seek possible intervention strategies for early prevention. PATIENTS AND METHODS: We consecutively recruited patients with sICAS and divided them into two groups based on age: early-onset sICAS group as age ≤60 years old and late-onset sICAS group as age >60 years old. We collected and compared the demographic data and laboratory results of each group. A bivariate logistic regression model was applied to evaluate the independent molecular biomarkers of early-onset sICAS. RESULTS: A total of 1007 subjects with sICAS were enrolled in this study, comprising 519 patients in the early-onset sICAS group and 488 patients in the late-onset sICAS group. Bivariate logistic regression analysis demonstrated an increased level of white blood cell, platelet, albumin globulin ratio, free triiodothyronine, and a decreased level of total bile acid, urea nitrogen, high-density lipoprotein, homocysteine, and fibrinogen in the early-onset sICAS group when compared to the late-onset group. CONCLUSION: Our study showed the relevance between early sICAS and circulating levels of different molecular biomarkers. Detection of these related molecular biomarkers may provide a simple way for early sICAS preventions in the future.

The transcription factor RelB restrains group 2 innate lymphoid cells and type 2 immune pathology in vivo.

The exact relationships between group 2 innate lymphoid cells (ILC2s) and Th2 cells in type 2 pathology, as well as the mechanisms that restrain the responses of these cells, remain poorly defined. Here we examined the roles of ILC2s and Th2 cells in type 2 lung pathology in vivo using germline and conditional Relb-deficient mice. We found that mice with germline deletion of Relb (Relb-/-) spontaneously developed prominent type 2 pathology in the lung, which contrasted sharply with mice with T-cell-specific Relb deletion (Relbf/fCd4-Cre), which were healthy with no observed autoimmune pathology. We also found that in contrast to wild-type B6 mice, Relb-deficient mice showed markedly expanded ILC2s but not ILC1s or ILC3s. Moreover, adoptive transfer of naive CD4+ T cells into Rag1-/-Relb-/- hosts induced prominent type 2 lung pathology, which was inhibited by depletion of ILC2s. Mechanistically, we showed that Relb deletion led to enhanced expression of Bcl11b, a key transcription factor for ILC2s. We concluded that RelB plays a critical role in restraining ILC2s, primarily by suppressing Bcl11b activity, and consequently inhibits type 2 lung pathology in vivo.

Epigenetically modifying the Foxp3 locus for generation of stable antigen-specific Tregs as cellular therapeutics.

Foxp3+ regulatory T cells (Tregs) are potent immunoregulatory cells, prompting strong interests in manipulating them for therapeutic purposes. However, significant challenges remain, including their heterogeneity and functional instability. Here we focused on the inducible Tregs (iTregs) and studied whether the Foxp3 locus can be epigenetically edited ex vivo to produce stable therapeutic iTregs. Under iTreg-inducing condition where activated CD4+ T effector cells were converted to Foxp3+ Tregs, we tested approximately 30 compounds and identified 3 chromatin-modifying chemical compounds (3C) consisting of sodium butyrate (a broad histone deacetylase inhibitor), UNC0646 (a histone methyltransferase inhibitor), and vitamin C (a TET dioxygenase co-activator), that together produced complete demethylation at the conserved noncoding sequence 2 (CNS2) region of Foxp3 locus. We found that iTregs induced in the presence of 3C (3C-iTregs) are stable, even after exposure to inflammatory cytokines. They expressed high levels of Foxp3 and exhibited potent suppressive activities both in vitro and in vivo. We showed that in models of autoimmunity and transplant rejection, adoptive transfer of antigen-specific 3C-iTregs prevented the induction of experimental autoimmune encephalitis and enabled long-term skin allograft survival. Our data demonstrate that the Foxp3 locus can be epigenetically edited ex vivo to generate stable therapeutic iTregs.