MBL Binding with AhR Controls Th17 Immunity in Silicosis-Associated Lung Inflammation and Fibrosis

Abstract

Objective: Mannan-binding lectin (MBL), a soluble pattern recognition molecule of the innate immune system, is primarily synthesized in the liver and secreted into the circulation. Low serum level of MBL has been reported to be related to an increased risk of lung diseases. Herein, we aimed to investigate the function of MBL in silicosis-associated pulmonary inflammation.

Methods: Serum collected from silicosis patients was tested for correlation between serum MBL levels and Th17 immunity. In vitro studies were performed to further demonstrated the effect of MBL on Th17 polarization. Silica was intratracheally injected in wild type (WT) or MBL-deficient (MBL^-/-) mice to induce silicosis-associated lung inflammation and fibrosis. Th17 response was evaluated to explore the effect of MBL on silicosis in vivo.

Results: Silicosis patients with high serum MBL levels displayed ameliorative lung function. We demonstrated that serum MBL levels negatively correlated to Th17 cell frequency in silicosis patients. MBL protein markedly reduced expression of IL-17 but enhanced expression of Foxp3 in CD4⁺ T cells in vitro when subjected to Th17 or Treg polarizing conditions, respectively. The presence of MBL during Th17 cell polarization significantly limited aryl hydrocarbon receptor (AhR) expression and suppressed the signal transducer and activator of transcription 3 (STAT3) phosphorylation. Treatment with the AhR antagonist abolished the effect of MBL on Th17 response. Strikingly, MBL directly bound to AhR and affected its nuclear translocation. Furthermore, MBL^-/- mice displayed elevated Th17 cell levels compared with WT mice in response to the silica challenge. The CD4⁺ T lymphocytes from silica-administrated MBL^-/- mice exhibited more AhR expression than the wild-type counterparts.

Conclusion: Our study suggested that MBL limited the Th17 immunity via controlling the AhR/STAT3 pathway, thus providing new insight into silicosis and other inflammatory diseases in patients with MBL deficiency.

Keywords: Th17 cell; aryl hydrocarbon receptor; mannan-binding lectin; signal transducer and activator of transcription 3; silicosis.

Figure 1

MBL level positively correlated with lung function and Th17/Treg cell balance in silicosis patients. (A and B) Serum MBL levels were detected by ELISA analysis. (A) The correlation between serum MBL levels and DLCO%, TLC%, VC%, FEV1/FVC%, PEF%, FEF75%, FEF50%, FEF25%, MVV% in silicosis patients was determined. (B) The correlation of serum MBL levels with different DLCO%, TLC% levels, pulmonary ventilation or small airway function condition was analyzed. (C) The frequency of Th17 and Treg in PBMCs from silicosis patients or healthy counterparts was determined by FCM assay. (D) RORγt and Foxp3 mRNA expression were measured by RT-qPCR analysis in CD4⁺ T cells isolated from silicosis patients and healthy controls. (E) The correlation between serum MBL levels and Th17 or Treg frequency in silicosis patients was analyzed. (F) The correlation between serum MBL levels and RORγt or Foxp3 mRNA expression in CD4⁺ T cells from silicosis patients. *p < 0.05, **p < 0.01. The data represent three independent experiments with similar results.

Figure 2

MBL inhibited Th17 cells polarization in vitro. (A and C) Naïve CD4⁺ T cells were cultured under Th17 differentiation conditions in the presence or absence of MBL protein (5μg/mL). (A) The expression of IL-17a and RORγt in CD4⁺ T cells were determined by FCM analysis. (B) The levels of IL-17a and IL-21 in the culture medium were evaluated by ELISA assay. (C) The mRNA levels of RORγt, IL-17a, and IL-21 were detected by RT-qPCR analysis. (D–F) Naïve CD4⁺ T cells were cultured under Treg differentiation conditions with or without MBL protein (5μg/mL). (D) The expression of Foxp3 in CD4⁺ T cells was determined by FCM analysis. (E) The levels of TGF-β and IL-10 in the culture medium were evaluated by ELISA assay. (F) The mRNA transcription levels of Foxp3, TGF-β, and IL-10 were detected by RT-qPCR analysis. *p < 0.05, **p < 0.01. The data represent three independent experiments with similar results.

Figure 3

MBL limited AhR expression and its nuclear translocation in CD4⁺ T lymphocytes. (A and C) Naïve CD4⁺ T cells were cultured under Th17 differentiation conditions with or without MBL protein (5μg/mL). (A) The expression of AhR in CD4+ T cells was determined by FCM analysis. (B) The protein levels of AhR were determined by Western blot. (C) The level of phosphorylated STAT3 was assessed by FCM analysis. (D–E) Naïve CD4⁺ T cells were pretreated with 10μM Sc144 for 2h, then cultured under Th17 differentiation conditions with or without MBL protein (5μg/mL). (D) The mRNA expression of AhR in CD4⁺ T cells was determined by RT-qPCR analysis. (E) The protein level of AhR was assessed by Western blot analysis. (F–H) CD4⁺ T cells were treated with 200nM FICZ for 6h in the presence or absence of 5μg/mL MBL. (F) The fraction of cytoplasm and nucleus protein were separated, and the expression of AhR was determined by Western blot. (G) The mRNA levels of CYP1A1, CYP1A2, and CYP1B1 were detected by RT-qPCR analysis. (H) The mRNA levels of RORγt and IL-17 were detected by RT-qPCR analysis. *p < 0.05, **p < 0.01. The data represent three independent experiments with similar results.

Figure 4

MBL interacted with AhR and regulated Th17 cell polarization through AhR signaling. (A and B) CD4⁺ T cells were stimulated with 200nM FICZ with 5μg/mL MBL for 6 h. (A) The association of AhR with MBL was analyzed by immunoprecipitation assay. (B) Colocalization of AhR and internalized MBL in T cells was determined by immunofluorescence analysis. (C–E) Naïve CD4⁺ T cells were cultured under Th17 differentiation conditions in the presence or absence of MBL protein (5μg/mL) combined with 1μM CH-223191 for 7d, t. (C) The expression of IL-17a and RORγt in CD4⁺ T cells was determined by FCM analysis. (D) The levels of IL-17a and IL-21 in the culture medium were evaluated by ELISA assay. (E) The mRNA levels of RORγt, IL-17a, and IL-21 were detected by RT-qPCR analysis. *p < 0.05, **p < 0.01. The data represent three independent experiments with similar results.

Figure 5

MBL deficiency aggravated silica-induced pulmonary inflammation in mice. (A–G) WT and MBL^−/− mice were intratracheally injected with 50μL silica suspension containing 5mg silica for 3 d. The lung tissues and sera were collected at the end of the experiment. (A) Histological analysis of mouse lungs was determined by H&E staining. (B) Cell counts in BALF were calculated under the microscope. (C) Total protein level was detected in BALF by BCA analysis. (D) FCM experiments were performed to measure Th17 or Treg frequency in lung tissues, HLN, and BALF from silica-challenged mice. (E) The levels of IL-17a, IL-6, TNFα, TGF-β, and IL-10 levels in BALF were evaluated by ELISA analysis. (F) The mRNA expression levels of IL-17a, IL-6, TNFα, RORγt, TGF-β, IL-10, and Foxp3 in lung tissues were determined by RT-qPCR analysis. (G) AhR expression in CD4⁺ T cells was evaluated by FCM assay. *p < 0.05, **p < 0.01. The data represent three independent experiments with similar results.

Figure 6

MBL-deficient mice exhibited aggravated silica-induced pulmonary fibrosis. (A–G) Mice were intratracheally injected with silica suspension (5mg silica in 50μL sterile PBS) for 35 d. The lung tissues and sera were collected at the end of the experiment. (A) Histological analysis of lung tissues was detected by H&E staining. (B) Ashcroft scores of lung tissues were determined under a microscope. (C) Hydroxyproline levels in lung tissues were tested. (D) Th17 or Treg frequency in lung tissues, HLN, and BALF were assessed by FCM analysis. (E) ELISA analysis was performed to evaluate IL-17a, IL-6, TNFα, TGF-β, and IL-10 levels in BALF collected from silica-treated mice at the last day. (F) The mRNA expression levels of IL-17a, IL-6, TNFα, RORγt, TGF-β, IL-10, and Foxp3 in lung tissues were determined by RT-qPCR analysis (G) Expression of AhR in CD4⁺ T cells was assessed by FCM assay. *p < 0.05, **p < 0.01. The data represent three independent experiments with similar results.

Figure 7

MBL restoration ameliorated silica-induced pulmonary fibrosis in mice. (A–D) MBL^−/− mice were tail vein injected with MBL-expressed AAV or control AAV combined with intratracheally injected with silica suspension (5mg silica in 50μL sterile PBS) for 35 d. The lung tissues and sera were collected at the end of the experiment. (A) Histological analysis of lung tissues was determined by H&E staining. (B) Hydroxyproline levels in lung tissues were detected. (C) FCM analysis was performed to evaluate Th17 or Treg frequency in lung tissues, HLN, and BALF. (D) Expression of AhR in CD4⁺ T cells was detected by FCM assay. *p < 0.05, **p < 0.01. The data represent three independent experiments with similar results.

Figure 8

Schematic representation of the mechanism by which MBL regulates Th17 cell differentiation through AhR signaling. Upon activation, AhR translocates to the nucleus and induces the expression of Th17-related transcripts. Besides, activated AhR promotes phosphorylation of STAT3. Phosphorylated STAT3 not only induces expression of Th17-related transcripts, but also promotes AhR expression. MBL interacts with AhR and inhibits AhR translocation, thereby limiting Th17 cell differentiation.