Analysis of LAP+ and GARP+ Treg subsets in peripheral blood of patients with neuromyelitis optica spectrum disorders.

INTRODUCTION: Neuromyelitis optica spectrum disorder (NMOSD) is a group of antibody-mediated inflammatory demyelinating central nervous system diseases. T lymphocytes participate in NMOSD pathogenesis, with regulatory T cells (Treg) being the core in maintaining immune homeostasis. Studies have revealed that different Treg subsets play different roles in autoimmune diseases. The distribution of LAP+ or GARP+ Treg subsets in NMOSD may help us deeply understand their immune mechanism. METHODS: This study reviewed 22 NMOSD patients and 20 normal controls. Flow cytometric analysis was utilized to detect subsets of Treg cells expressing Foxp3, Helios, LAP, or GARP in peripheral blood. ELISA was used to detect plasma TGF-ß1 and IL-10. In addition, changes in the proportion of Treg cell subsets before and after glucocorticoid treatment in 10 patients were analyzed. RESULTS: Compared with healthy controls, LAP and GARP expressions were significantly downregulated in the peripheral blood of NMOSD patients. TGF-ß1 expression in NMOSD patients was lower and was positively correlated with the ratio of CD4+GARP+ Treg cells. After treatment with glucocorticoid, LAP and GARP expressions in the peripheral blood of NMOSD patients were upregulated. CONCLUSIONS: The proportion of Treg cells expressing LAP and GARP is downregulated, implying that Treg cells with the best inhibitory function are insufficient to maintain autoimmune homeostasis in NMOSD patients. Upregulation of Treg cells expressing LAP and GARP in NMOSD patients may be one of the mechanisms of glucocorticoid treatment.

Vitamin D3 Supplementation Promotes Regulatory T-Cells to Maintain Immune Homeostasis After Surgery for Early Stages of Colorectal Cancer.

BACKGROUND/AIM: Vitamin D3 (VD3) affects the regulation of the immune system, including the differentiation and function of regulatory T-cells (Tregs). Tregs play an important role in maintaining immune homeostasis in patients with colorectal cancer (CRC). The effects of VD3 on Treg-associated immune function were investigated in Thai patients in the early stages of CRC. MATERIALS AND METHODS: Twenty-eight patients were randomized to one of two groups: Untreated or treatment with VD3 for 3 months. Whole blood samples were collected at baseline, and at 1 and 3 months. Peripheral blood mononuclear cells were isolated and the populations of forkhead box P3-positive Treg cells was analyzed by flow cytometry. The levels of Treg-associated cytokines, interleukin 10 (IL-10) and transforming growth factor beta 1 (TGF-ß1), were measured by enzyme-linked immunosorbent assays. RESULTS: Serum VD3 levels of the VD3-treated group were significantly increased at 1 (p=0.017) and 3 months (p<0.001) compared to the untreated control group. The mean percentage of Tregs was maintained between 1 and 3 months in the VD3-treated group. At 3 months, the untreated group had significantly lower Treg levels than the VD3-treated group (p=0.043). Serum IL-10 levels of the VD3-treated group were statistically increased at 1 month compared to the control group (p=0.032). No significant difference in serum TGF-ß1 levels was observed between the two groups. However, the TGF-ß1 level in the VD3-treated group at 1 month was lower than that of the control. CONCLUSION: Our findings suggest that VD3 supplementation can maintain immune responses in the early stages of CRC, helping to control Treg function. Therefore, VD3 should be supplemented to maintain immune homeostasis, especially in patients with vitamin D deficiency.

CTRP3 regulates NF-kB and TGFB1/Smad3 pathways to alleviate airway inflammation and remodeling in asthmatic mice induced by OVA

Background: Asthma is a common illness with chronic airway inflammation. C1q/tumor necrosis factor (TNF)-related protein 3 (CTRP3) plays a vital role ininflammatory response, but its effect on asthma is imprecise. Herein, we analyzed the functions of CTRP3 in asthma. Methods: The BALB/c mice were randomized into four groups: control, ovalbumin (OVA), OVA+vector, and OVA+CTRP3. The asthmatic mice model was established by OVA stimulation. Overexpression of CTRP3 was implemented by the transfection of corresponding adeno-associated virus 6 (AAV6). The contents of CTRP3, E-cadherin, N-cadherin, smooth muscle alpha-actin (α-SMA), phosphorylated (p)-p65/p65, transforming growth factor-beta 1 (TGFβ1), and p-Smad3/Smad3 were determined by Western blot analysis. The quantity of total cells, eosinophils, neutrophils, and lymphocytes in bronchoalveolar lavage fluid (BALF) was assessed by using a hemocytometer. The contents of tumor necrosis factor-α and interleukin-1β in BALF were examined by enzyme-linked immunesorbent serologic assay. The lung function indicators and airway resistance (AWR) were measured. The bronchial and alveolar structures were evaluated by hematoxylin and eosin staining and sirius red staining. Results: The CTRP3 was downregulated in mice of OVA groups; however, AAV6-CTRP3 treatment markedly upregulated the expression of CTRP3. Upregulation of CTRP3 diminished asthmatic airway inflammation by decreasing the number of inflammatory cells and the contents of proinflammatory factors. CTRP3 markedly lessened AWR and improved lung function in OVA-stimulated mice. Histological analysis found that CTRP3 alleviated OVA-induced airway remodeling in mice. Moreover, CTRP3 modulated NF-κB and TGFβ1/Smad3 pathways in OVA-stimulated mice. Conclusion: CTRP3 alleviated airway inflammation and remodeling in OVA-induced asthmatic mice via regulating NF-κB and TGFβ1/Smad3 pathways (AU)

TIM-4 in macrophages contributes to nasal polyp formation through the TGF-ß1-mediated epithelial to mesenchymal transition in nasal epithelial cells.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is caused by prolonged inflammation of the paranasal sinus mucosa. The epithelial to mesenchymal transition (EMT) is involved in the occurrence and development of CRSwNP. The T-cell immunoglobulin domain and the mucin domain 4 (TIM-4) is closely related to chronic inflammation, but its mechanism in CRSwNP is poorly understood. In our study, we found that TIM-4 was increased in the sinonasal mucosa of CRSwNP patients and, especially, in macrophages. TIM-4 was positively correlated with α-SMA but negatively correlated with E-cadherin in CRS. Moreover, we confirmed that TIM-4 was positively correlated with the clinical parameters of the Lund-Mackay and Lund-Kennedy scores. In the NP mouse model, administration of TIM-4 neutralizing antibody significantly reduced the polypoid lesions and inhibited the EMT process. TIM-4 activation by stimulating with tissue extracts of CRSwNP led to a significant increase of TGF-ß1 expression in macrophages in vitro. Furthermore, coculture of macrophages and human nasal epithelial cells (hNECs) results suggested that the overexpression of TIM-4 in macrophages made a contribution to the EMT process in hNECs. Mechanistically, TIM-4 upregulated TGF-ß1 expression in macrophages via the ROS/p38 MAPK/Egr-1 pathway. In conclusion, TIM-4 contributes to the EMT process and aggravates the development of CRSwNP by facilitating the production of TGF-ß1 in macrophages. Inhibition of TIM-4 expression suppresses nasal polyp formation, which might provide a new therapeutic approach for CRSwNP.

Optimization strategies for expression of a novel bifunctional anti-PD-L1/TGFBR2-ECD fusion protein.

The novel anti-PD-L1/TGFBR2-ECD fusion protein (BR102) comprises an anti-PD-L1 antibody (HS636) which is fused at the C terminus of the heavy chain to a TGF-ß1 receptor Ⅱ ectodomain (TGFBR2-ECD), and which can sequester the PD-1/PD-L1 pathway and TGF-ß bioactivity in the immunosuppressive tumor microenvironment. In the expression of TGFBR2-ECD wild-type fused protein (BR102-WT), a 50 kDa clipped species was confirmed to be induced by proteolytic cleavage at a "QKS" site located in the N-terminus of the ectodomain, which resulted in the formation of IgG-like clipping. The matrix metalloproteinase-9 was determined to be associated with BR102-WT digestion. In addition, it was observed that the N-glycosylation modifications of the fusion protein were tightly involved in regulating proteolytic activity and the levels of cleavage could be significantly suppressed by MMP-inhibitors. To avoid proteolytic degradation, eliminating protease-sensitive amino acid motifs and introducing potential glycosylation were performed. Three sensitive motifs were mutated, and the levels of clipping were strongly restrained. The mutant candidates exhibited similar binding affinities to hPD-L1 and hTGF-ß1 as well as highly purified BR102-WT2. Furthermore, the mutants displayed more significant proteolytic resistance than that of BR102-WT2 in the lysate incubation reaction and the plasma stability test. Moreover, the bifunctional candidate Mu3 showed an additive antitumor effect in MC38/hPD-L1 bearing models as compared to that of with anti-PD-L1 antibody alone. In conclusion, in this study, the protease-sensitive features of BR102-WT were well characterized and efficient optimization was performed. The candidate BR102-Mutants exhibited advanced druggability in drug stability and displayed desirable antitumor activity.

Direct and Indirect Effect of TGFß on Treg Transendothelial Recruitment in HCC Tissue Microenvironment.

The balance between anti-tumor and tumor-promoting immune cells, such as CD4+ Th1 and regulatory T cells (Tregs), respectively, is assumed to dictate the progression of hepatocellular carcinoma (HCC). The transforming growth factor beta (TGFß) markedly shapes the HCC microenvironment, regulating the activation state of multiple leukocyte subsets and driving the differentiation of cancer associated fibroblasts (CAFs). The fibrotic (desmoplastic) reaction in HCC tissue strongly depends on CAFs activity. In this study, we attempted to assess the role of TGFß on transendothelial migration of Th1-oriented and Treg-oriented CD4+ T cells via a direct or indirect, CAF-mediated mechanisms, respectively. We found that the blockage of TGFß receptor I-dependent signaling in Tregs resulted in impaired transendothelial migration (TEM) of these cells. Interestingly, the secretome of TGFß-treated CAFs inhibited the TEM of Tregs but not Th1 cells, in comparison to the secretome of untreated CAFs. In addition, we found a significant inverse correlation between alpha-SMA and FoxP3 (marker of Tregs) mRNA expression in a microarray analysis involving 78 HCCs, thus suggesting that TGFß-activated stromal cells may counteract the trafficking of Tregs into the tumor. The apparent dual behavior of TGFß as both pro- and anti-tumorigenic cytokines may add a further level of complexity to the mechanisms that regulate the interactions among cancerous, stromal, and immune cells within HCC, as well as other solid tumors, and contribute to better manipulation of the TGFß signaling as a therapeutic target in HCC patients.

Role of Transforming Growth Factor-ß1 in Regulating Fetal-Maternal Immune Tolerance in Normal and Pathological Pregnancy.

Transforming growth factor-ß (TGF-ß) is composed of three isoforms, TGF-ß1, TGF-ß2, and TGF-ß3. TGF-ß1 is a cytokine with multiple biological functions that has been studied extensively. It plays an important role in regulating the differentiation of immune cells and maintaining immune cell functions and immune homeostasis. Pregnancy is a carefully regulated process. Controlled invasion of trophoblasts, precise coordination of immune cells and cytokines, and crosstalk between trophoblasts and immune cells play vital roles in the establishment and maintenance of normal pregnancy. In this systematic review, we summarize the role of TGF-ß1 in regulating fetal-maternal immune tolerance in healthy and pathological pregnancies. During healthy pregnancy, TGF-ß1 induces the production of regulatory T cells (Tregs), maintains the immunosuppressive function of Tregs, mediates the balance of M1/M2 macrophages, and regulates the function of NK cells, thus participating in maintaining fetal-maternal immune tolerance. In addition, some studies have shown that TGF-ß1 is dysregulated in patients with recurrent spontaneous abortion or preeclampsia. TGF-ß1 may play a role in the occurrence and development of these diseases and may be a potential target for the treatment of these diseases.

Transplantation of mesenchymal stem cells ameliorates systemic lupus erythematosus and upregulates B10 cells through TGF-ß1.

BACKGROUND: Considerable experimental and clinical evidences have proved that human umbilical cord mesenchymal stem cells (UC-MSCs) transplantation was powerful in systemic lupus erythematosus (SLE) treatment. MSCs could upregulate regulatory B cells (Bregs) in the mice model of the other immune disease. However, the regulation of MSCs on Bregs in SLE environment remains unclear. METHODS: To assess the abilities of UC-MSCs to treat SLE, MSCs were transferred intravenously to 17- to 18-week-old MRL/lpr mice. Four weeks later, mice were sacrificed. Survival rates, anti-dsDNA antibodies and renal histology were evaluated. CD4+ T helper (Th) cell subgroups and interleukin (IL)-10+ Bregs (B10) in the spleen were quantitated by flow cytometry. The changes of transforming growth factor (TGF)-ß1, IL-6 and indoleamine 2,3-dioxyenase (IDO) mRNAs expressed by MSCs after co-cultured with B cells were detected using real-time polymerase chain reaction (RT-PCR). MSCs were infected by lentivirus carrying TGF-ß1 shRNAs, then MSCs with low expression of TGF-ß1 were conducted for co-culture in vitro and transplantation experiments in vivo. RESULTS: UC-MSCs transplantation could efficiently downregulate 24 h proteinuria and anti-dsDNA antibodies, correct Treg/Th17/Th1 imbalances and increase the frequency of B10 cells. The expression of TGF-ß1 in MSCs was significantly increased after co-culture with B cells. Downregulation of TGF-ß1 in MSCs could significantly attenuate the upregulation of B10 by MSCs in vitro and in vivo. Downregulation of TGF-ß1 also compromised the immunomodulation effects of MSCs on Th17 and Treg cells and the therapeutic effects of MSC transplantation. CONCLUSIONS: UC-MSCs could protect against SLE in mice and upregulate IL-10+ Bregs via TGF-ß1.

Antibody and cytokine levels in visceral leishmaniasis patients with varied parasitemia before, during, and after treatment in patients admitted to Arba Minch General Hospital, southern Ethiopia.

BACKGROUND: Visceral leishmaniasis is a disease caused by disseminated Leishmania donovani infection which affects almost half a million people annually. Most of the patients are reported from the Indian sub-continent, Eastern Africa and Brazil. In this study, we aimed to determine the levels of antibodies and cytokines in visceral leishmaniasis patients and to examine associations of parasitemia with the clinical states of patients. A prospective study was carried out, enrolling a total of 48 active VL patients who were evaluated before, during different time points and, three months after treatment. Serum cytokine concentrations, antibody levels, parasitemia, laboratory (hematologic and biochemical) measurements, and clinical parameters were assessed. RESULTS: Counts of WBC and platelets, and measurements of hemoglobin (Hb) increased during treatment (P ≤ 0.05). Elevated levels of circulating IL-10, IFN-γ, and TGF-ß1 were measured before treatment. The observed increase in serum IL-10 remarkably declined within 7 days after the start of treatment. Anti-leishmanial antibody index (AI) was high in all VL patients irrespective of spleen aspirate parasite grade before treatment and at different times during treatment. However, a significant (P ≤ 0.05) decrease of AI was observed 120 days post-treatment. IL-2 serum levels were below the detection limit at all sampling points. CONCLUSIONS: The present results suggest that IL-10, IFN-γ, and TGF-ß1 can be used as markers of active visceral leishmaniasis. In addition, measuring circulating cytokines concentrations, particularly IL-10, in combination with other clinical evaluations, could be used as criteria for the cure. The observation that a high serum concentration of IFN-gamma at baseline was associated with low parasitemia deserves further investigations.

Combined Blockade of GARP:TGF-ß1 and PD-1 Increases Infiltration of T Cells and Density of Pericyte-Covered GARP+ Blood Vessels in Mouse MC38 Tumors.

When combined with anti-PD-1, monoclonal antibodies (mAbs) against GARP:TGF-ß1 complexes induced more frequent immune-mediated rejections of CT26 and MC38 murine tumors than anti-PD-1 alone. In both types of tumors, the activity of anti-GARP:TGF-ß1 mAbs resulted from blocking active TGF-ß1 production and immunosuppression by GARP-expressing regulatory T cells. In CT26 tumors, combined GARP:TGF-ß1/PD-1 blockade did not augment the infiltration of T cells, but did increase the effector functions of already present anti-tumor T cells. Here we show that, in contrast, in MC38, combined GARP:TGF-ß1/PD-1 blockade increased infiltration of T cells, as a result of increased extravasation of T cells from blood vessels. Unexpectedly, combined GARP:TGF-ß1/PD-1 blockade also increased the density of GARP+ blood vessels covered by pericytes in MC38, but not in CT26 tumors. This appears to occur because anti-GARP:TGF-ß1, by blocking TGF-ß1 signals, favors the proliferation of and expression of adhesion molecules such as E-selectin by blood endothelial cells. The resulting densification of intratumoral blood vasculature probably contributes to increased T cell infiltration and to the therapeutic efficacy of GARP:TGF-ß1/PD-1 blockade in MC38. We conclude from these distinct observations in MC38 and CT26, that the combined blockades of GARP:TGF-ß1 and PD-1 can exert anti-tumor activity via multiple mechanisms, including the densification and normalization of intratumoral blood vasculature, the increase of T cell infiltration into the tumor and the increase of the effector functions of intratumoral tumor-specific T cells. This may prove important for the selection of cancer patients who could benefit from combined GARP:TGF-ß1/PD-1 blockade in the clinics.

Lactiplantibacillus plantarum 22A-3-induced TGF-ß1 secretion from intestinal epithelial cells stimulated CD103+ DC and Foxp3+ Treg differentiation and amelioration of colitis in mice.

In the present study, we evaluated the anti-inflammatory properties of Lactiplantibacillus plantarum 22A-3 (LP22A3) and attempted to elucidate the underlying molecular mechanism. The oral administration of LP22A3 significantly inhibited body weight reduction and decreased colon shortening and colitis score in mice with dextran sulfate sodium (DSS)-induced colitis. It was demonstrated that the production of the active-form of TGF-ß tended to increase in both the intestinal epithelial cells (IECs) of the ileum and serum but not in the colon of non-DSS-treated mice by LP22A3. IL-10 level in serum was also elevated by LP22A3-treatment. The mRNA expression of TGF-ß, IL-10 and Foxp3 increased only in the small intestines of LP22A3-treated mice. Both the aldehyde dehydrogenase 1 family member A2 (Aldh1a2) mRNA expression and population of CD103+ dendritic cells (DCs) in the small intestine significantly increased in the LP22A3-treated group. LP22A3 induced TGF-ß secretion from the IECs of the small intestine with retinoic acid production probably through TLR2, resulting in an increase in CD103+ DCs and the Foxp3+ Treg population. Both cells secrete a high level of anti-inflammatory cytokines, TGF-ß and IL-10 contributing to the protective condition in the intestine and thus making it less susceptible to inflammation. This suggested that oral administration of LP22A-3 may be an alternative therapeutic strategy for IBD.

Targeting immunosuppression by TGF-ß1 for cancer immunotherapy.

The TGF-ß1 cytokine is a key mediator of many biological processes. Complex regulatory mechanisms are in place that allow one single molecule to exert so many distinct indispensable activities. The complexity of TGF-ß1 biology is further illustrated by the opposing dual roles it plays during cancer progression. Risks of toxicities combined with lack of convincing therapeutical efficacy explain at least in part why therapies targeting TGF-ß1 have lagged behind in past decades. However, recent successes of immunostimulatory antibodies for the immunotherapy of cancer and findings that TGF-ß1 activity associates with resistance to immunotherapeutic drugs have revived the field. In this review, we discuss the biology of TGF-ß1 with a special focus on its roles in regulating immune responses in the context of cancer. We describe the various therapeutic approaches available to inhibit TGF-ß signalling, and more recent findings that allow selective targeting of specific sources of TGF-ß activity, which may prove relevant to increase the efficacy and reduce the toxicity of cancer immunotherapy.

Human Keratinocytes Inhibit CD4+ T-Cell Proliferation through TGFB1 Secretion and Surface Expression of HLA-G1 and PD-L1 Immune Checkpoints.

Human skin protects the body against infection and injury. This protection involves immune and epithelial cells, but their interactions remain largely unknown. Here, we show that cultured epidermal keratinocytes inhibit allogenic CD4+ T-cell proliferation under both normal and inflammatory conditions. Inhibition occurs through the secretion of soluble factors, including TGFB1 and the cell-surface expression of HLA-G1 and PD-L1 immune checkpoints. For the first time, we here describe the expression of the HLA-G1 protein in healthy human skin and its role in keratinocyte-driven tissue immunomodulation. The overexpression of HLA-G1 with an inducible vector increased the immunosuppressive properties of keratinocytes, opening up perspectives for their use in allogeneic settings for cell therapy.

The Role of Renal Macrophage, AIM, and TGF-ß1 Expression in Renal Fibrosis Progression in IgAN Patients.

Objective: To analyze the expression of macrophages, AIM, TGF-ß1 in the kidney of IgAN patients, and to explore the role of macrophages, AIM, TGF-ß1 in the progression of renal fibrosis in IgAN patients. Methods: The paraffin specimens of renal tissue from 40 IgAN patients were selected as the observation group. At the same time, paraffin specimens of normal renal tissue from 11 patients treated by nephrectomy were selected as the normal control group. We observed the distribution of macrophages, the expression of AIM and TGF-ß1 by immunohistochemical staining and/or immunofluorescence. Result: The number of M0, M1, M2 macrophages could be found increased in IgAN patients. M0 macrophages are mainly polarized towards M2 macrophages. The expression of AIM and TGF-ß1 were significantly higher in IgAN patients than in NC. M2 macrophage, AIM and TGF-ß1 were positively correlated with serum creatinine and 24-hour proteinuria, but negatively correlated with eGFR. M2 macrophages, AIM, TGF-ß1 were positively correlated with fibrotic area. Conclusion: M2 macrophages, AIM and TGF-ß1 play important roles in the process of IgAN fibrosis, and the three influence each other.

The VDR gene confers a genetic predisposition to Graves' disease and Graves' ophthalmopathy in the Southwest Chinese Han population.

OBJECTIVE: Graves' disease (GD) is a common autoimmune disease manifesting with diffuse symmetric thyroid gland enlargement, pretibial myxedema, and Graves' ophthalmopathy (GO). Recently, the vitamin D receptor (VDR) gene has been linked to various autoimmune diseases. This study aimed to investigate the association of VDR gene polymorphisms with susceptibility to GD and GO in the Southwest Chinese Han population. METHODS: A two-stage association study was performed in 1,209 controls and 650 GD patients by PCR-RFLP assay. Real-time PCR and ELISA were carried out to quantify gene expression and cytokine production. RESULTS: The first-stage study showed that the frequency of VDR/Apa I AA genotype was significantly increased in GD (Pc = 1.67 × 10-2, OR = 1.98). The second-stage and combined studies confirmed the association of VDR/Apa I with GD (AA genotype: Pc = 3.45 × 10-4, OR = 1.87; A allele: Pc = 2.62 × 10-2, OR = 1.20). The stratification analysis showed that GO patients had a higher frequency of the VDR/Apa I AA genotype (Pc = 8.69 × 10-5, OR = 2.84). Functional experiments showed a decreased VDR expression and TGF-ß1 production as well as an increased IL-17 production in VDR/Apa I AA genotype carriers. CONCLUSION: The VDR/Apa I polymorphism is significantly associated with GD and GO, and it may be involved in the development of GD and GO by influencing VDR mRNA expression levels and the secretion levels of cytokines.

PM2.5 and water-soluble components induce airway fibrosis through TGF-ß1/Smad3 signaling pathway in asthmatic rats.

Epidemiological studies have suggested that fine particulate matter (PM2.5) and asthma have been independently associated with pulmonary fibrosis but rarely studied together. Furthermore, it is unknown whether airway fibrosis in asthma is more attributable to water-soluble ions of PM2.5. Our current study was to explore the potential mechanism of PM2.5 and water-soluble components on airway fibrosis in ovalbumin (OVA)-sensitized asthmatic rats. Rats were intratracheally instilled with PM2.5 and water-soluble components every 3 days for 4 times or 8 times. Histopathological examination demonstrated that lung inflammatory and airway fibrosis were induced after PM2.5 and water-soluble components exposure. Meanwhile, PM2.5, in particular water-soluble extracts, increased expression of collagen 1 (COL-1), connective tissue growth factor (CTGF), interleukin-6 (IL-6), transforming growth factor-ß1 (TGF-ß1), Smad family member 3 (Smad3), and p-Smad3, whereas decreased secretion of heme oxygenase-1 (HO-1). However, pretreating asthmatic rats with SB432542, the inhibitor of TGF-ß1, and SIS3 HCl, the antagonist of Smad3, both reversed the activation of airway fibrosis induced by water-soluble extracts. Therefore, TGF-ß1/Smad3 signaling pathway may be responsible for the pathological process of airway fibrosis in asthmatic rats following PM2.5 and water-soluble components exposure.

Genetic polymorphisms of regulatory T cell-related genes modulate systemic inflammation induced by viral hepatitis.

Viral hepatitis is a devastating disease with the risk for cirrhosis and carcinogenicity. Regulatory T cells (Tregs) play important roles in the disease course of viral hepatitis via maintaining the balance between overt-immune responses and viral replications. We hypothesized that genetic polymorphisms of Treg-related genes, such as interleukin-2, transforming growth factor-ß 1 (TGF-ß1), forkhead box P3 (FOXP3), and adenylyl cyclase type 9 modulate the hosts' immune regulation under circumstances of viral hepatitis. We examined the effect of five single nucleotide polymorphisms (SNPs) of Treg-related genes on the levels of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), alanine aminotransferase, and non-invasive hepatic fibrosis marker (Fibrosis-4 index) in a total of 138 participants with viral hepatitis. The rs1800469 (a TGF-ß1 SNP) GG genotype is associated with higher serum CRP levels, and the rs3761547 (a FOXP3 SNP) C allele in the females is associated with higher ESR levels. Besides, female participants carrying the rs3761547 C allele had a significantly higher Fibrosis-4 (FIB-4) index than the females carrying the TT genotype, while the rs3761547 C allele had the opposite effect in males. With linear-regression moderation analysis, we found that sex moderated the impact of the FOXP3 SNP on the levels of FIB-4, whereas the FOXP3 SNP caused the opposite effect between males and females on the severity of hepatic fibrosis. These results provide evidence for the participation of TGF-ß1 and FOXP3 in the inflammatory responses associated with viral hepatitis, where FOXP3 function may be moderated by sex.

Silicon, an important exposure marker in vivo in silicosis research.

PURPOSE: The degree of silicosis exposure is closely related to the progress of silicosis. At present, we use animal and human studies to explore whether silicon can be an important exposure marker in the development of silicosis. METHODS: Rats were randomly divided into 2 groups: (1) controls; and (2) silicosis. Rats in the silicosis group were killed at 4, 8, 12, 16, 24 h, 3, 7, 14, 21, and 28 days. Hematoxylin-eosin (HE) and immunohistochemistry (IHC) were performed to observe the histomorphology of lung tissue. The expression levels of CC16 and SP-D were detected using ELISA kits. In addition, we conducted a population study. Workers who have been selected to work in an iron mine for more than 1 year as research objects. The population was divided into four groups: silicosis exposure group (workers exposed to silica dust for more than 1 year in an iron mine were selected); patients group (silicosis patients); observation group (evidence of disease not meeting formal diagnostic criteria) and control group. Both the levels of trace silicon in the urine and blood of rats and human subjects were measured with ICP-MS. RESULTS: Serum levels of silicon were immediately increased in rats exposed to silicon dust. Similarly, our population study revealed that the silicon level in the silica exposure group and the observing group (exposed but no obvious symptoms) were significantly increased over that of the control group (P < 0.05). In subjects with extended exposure to silica, the serum and urine silicon level in exposed workers appeared to rapidly increase, reaching its peak in 1-5 years, followed by a gradual decline thereafter. Workers exposed to dust for less than 10 years were divided into subgroups by 2-year limit. The levels of serum silicon, urine silicon, TGF-ß1, and TNF-α were significantly higher than that of control group. CONCLUSION: Changes of the serum levels of silicon occurred earlier than the expression of cytokines such as TNF-α, TGF-ß1, CC16, and SP-D. The level of silicon in workers rapidly increased after exposure to silica, and the change occurred before the expression of TGF-ß1 and TNF-α. As a whole, the findings suggest that determining the level of silicon in vivo might be an effective exposure marker in the diagnosis and pathogenesis of silicosis.

Benzo(a)pyrene Enhanced Dermatophagoides Group 1 (Der f 1)-Induced TGFß1 Signaling Activation Through the Aryl Hydrocarbon Receptor-RhoA Axis in Asthma.

We have previously demonstrated that benzo(a)pyrene (BaP) co-exposure with dermatophagoides group 1 allergen (Der f 1) can potentiate Der f 1-induced airway inflammation. The underlying mechanism, however, remains undetermined. Here we investigated the molecular mechanisms underlying the potentiation of BaP exposure on Der f 1-induced airway inflammation in asthma. We found that BaP co-exposure potentiated Der f 1-induced TGFß1 secretion and signaling activation in human bronchial epithelial cells (HBECs) and the airways of asthma mouse model. Moreover, BaP exposure alone or co-exposure with Der f 1-induced aryl hydrocarbon receptor (AhR) activity was determined by using an AhR-dioxin-responsive element reporter plasmid. The BaP and Der f 1 co-exposure-induced TGFß1 expression and signaling activation were attenuated by either AhR antagonist CH223191 or AhR knockdown in HBECs. Furthermore, AhR knockdown led to the reduction of BaP and Der f 1 co-exposure-induced active RhoA. Inhibition of RhoA signaling with fasudil, a RhoA/ROCK inhibitor, suppressed BaP and Der f 1 co-exposure-induced TGFß1 expression and signaling activation. This was further confirmed in HBECs expressing constitutively active RhoA (RhoA-L63) or dominant-negative RhoA (RhoA-N19). Luciferase reporter assays showed prominently increased promoter activities for the AhR binding sites in the promoter region of RhoA. Inhibition of RhoA suppressed BaP and Der f 1 co-exposure-induced airway hyper-responsiveness, Th2-associated airway inflammation, and TGFß1 signaling activation in asthma. Our studies reveal a previously unidentified functional axis of AhR-RhoA in regulating TGFß1 expression and signaling activation, representing a potential therapeutic target for allergic asthma.