IL-37 Confers Protection against Mycobacterial Infection Involving Suppressing Inflammation and Modulating T Cell Activation

<div><p>Interleukin-37 (IL-37), a novel member of the IL-1 family, plays fundamental immunosuppressive roles by broadly reducing both innate inflammation and acquired immunity, but whether it is involved in the pathogenesis of tuberculosis (TB) has not been clearly elucidated. In this study, single nucleotide polymorphism (SNP) analysis demonstrated an association of the genetic variant rs3811047 of <i>IL-37</i> with TB susceptibility. In line with previous report, a significant elevated IL-37 abundance in the sera and increased expression of IL-37 protein in the peripheral blood mononuclear cells (PBMC) were observed in TB patients in comparison to healthy controls. Moreover, release of IL-37 were detected in either macrophages infected with <i>Mycobacterium tuberculosis</i> (Mtb) or the lung of BCG-infected mice, concurrent with reduced production of proinflammatory cytokines including IL-6 and TNF-α. Furthermore, in contrast to wild-type mice, BCG-infected IL-37-Tg mice manifested with reduced mycobacterial burden and tissue damage in the lung, accompanied by higher frequency of Th1 cell and less frequencies of regulatory T cells and Th17 cells in the spleen. Taken together, our findings demonstrated that IL-37 conferred resistance to Mtb infection possibly involving suppressing detrimental inflammation and modulating T cell responses. These findings implicated that IL-37 may be employed as a new molecular target for the therapy and diagnosis of TB.</p></div

Frequencies of IL-37 genotype in pulmonary tuberculosis patients and controls.

<p>Frequencies of IL-37 genotype in pulmonary tuberculosis patients and controls.</p

IL-37 protects mice from BCG infection.

<p><b>(A)</b> BCG burden in lung, measured in CFUs, in WT mice and IL-37-Tg mice at every week following i.n. infection with 4×10⁷ viable BCG. Data are presented as the median ± the interquartile range (iQR) and are representative of two independent experiments; Mann-Whitney <i>U</i> test was used for statistical analysis (n = 4 for each time point). <b>(B)</b> Peritoneal macrophages isolated from WT and IL-37-Tg were infected with H37Rv for 1 h at MOI = 5, washed and cultured for indicated time, followed by CFU assay. <b>(C)</b> Peritoneal macrophages were infected with H37Rv for 1 h at MOI = 5, washed and cultured in the absence or presence of different concentrations of recombinant IL-37 for indicated time followed by CFU assay. Data <b>(B-C)</b> are presented as the median ± the interquartile range (iQR) and representative of three independent experiments, with four duplicates each; Mann-Whitney <i>U</i> test was used for statistical analysis. <b>(D)</b> Representative H&E staining of lungs from BCG-infected WT (n = 4) and IL-37-Tg mice (n = 4) at 4 weeks p.i. of two independent experiments with comparable results. Scale bar, 100 μm. *, p<0.05; **, p<0.01.</p

Elevated IL-37 in TB patients and induction of IL-37 in response to Mtb infection.

<p><b>(A)</b> ELISA detection the abundance of IL-37 in the sera from healthy donors (HD) (n = 24) and TB patients (TB) (n = 24). Data are mean±SEM. Student’ <i>t</i> test was used for statistical analysis. <b>(B)</b> Western blot detection of IL-37 in the lysates of PBMC from HD (n = 7) and TB (n = 7). <b>(C)</b> Quantification and statistical analysis of the relative density (mean gray value) of IL-37/β-actin as shown in <b>(B)</b>. Data are mean±SEM. Student’ <i>t</i> test was used for statistical analysis. <b>(D)</b> Induction of IL-37 transcripts measured by qRT-PCR in human THP-1 cells infected with H37Rv at MOI 5 for indicated time. <b>(E)</b> Secretion of IL-37 protein measured by ELISA in PMA-differentiated human THP-1 cells infected with H37Rv at indicated MOI for 24 h. Data <b>(D-E)</b> are mean±SEM and representative of 3 independent experiments. <b>(F)</b> Kinetics of IL-37 production measured by ELISA in the lung of WT and IL-37-Tg mice infected with BCG. Results are mean±SEM and are pooled from two independent experiments (n = 7 or 8 for each time point). *, p<0.05; **, p<0.01.</p

IL-37 suppresses inflammatory responses in mycobacterial-infected macrophages and mice.

<p><b>(A- B)</b> ELISA detection of IL-6 <b>(A)</b> and TNF-α <b>(B)</b> in WT and IL-37-Tg macrophages infected with H37Rv at MOI = 5 for indicated times. Results are the mean±SEM and representative of 3 independent experiments with triplicates each. <b>(C-D)</b> Kinetics of IL-6 <b>(C)</b> and TNF-α <b>(D)</b> production detected by ELISA in the lung of WT and IL-37-Tg mice infected with BCG. Results are the mean±SEM and are pooled from two independent experiments. n = 7 or 8 for each time point; two way ANOVA with Bonferroni’s post test. *, p<0.05; **, p<0.01.</p

Allelic distribution and frequencies of <i>IL-37</i> in pulmonary tuberculosis patients and controls.

<p>Allelic distribution and frequencies of <i>IL-37</i> in pulmonary tuberculosis patients and controls.</p

Enhanced Th1 and reduced Treg and Th17 responses in IL-37 transgenic mice during mycobacterial infection.

<p>Kinetics of Treg <b>(A)</b>, Th1 <b>(B)</b> and Th17 <b>(C)</b> cells in the spleens of WT and IL-37-Tg mice infected with BCG. The frequencies of CD4⁺CD25⁺FoxP3⁺ <b>(A)</b>, CD4⁺IFNγ⁺ <b>(B)</b> and CD4⁺IL-17⁺ <b>(C)</b> cells among gated lymphocytes were determined at indicated times p.i. Results are the mean±SEM and are pooled from two independent experiments; n = 4 or 5 for each time point; two way ANOVA with Bonferroni’s post test. **, p<0.01.</p

High MCP-2/CCL8 abundance in TB-PEs.

<p>(A) Diagram showed that PEs from both TB and non-TB patients were applied to protein chip assays to screen differentially expressed cytokines. Data were subsequently confirmed by ELISA. (B) Differentially expressed proteins in PEs from TB patients and non-TB patients. (C–D) ELISA of IFN-γ (C) or MCP-2/CCL8(D) in the PEs from TB or non-TB patients. (E) ELISA of MCP-2/CCL8 protein in the peripheral blood from TB patients with or without PEs and healthy donors. (F) Receiver operating characteristic curve showing the area under curve (AUC) of MCP-2/CCL8 and IFN-γ. **, <i>p</i><0.001, *, <i>p</i><0.05.</p

Higher percentage of CCR5-expressing CD4⁺ T cells in TB-PEs.

<p>(A) Representative FACS detection of CD3⁺, CD4⁺ and CD8⁺ cells in PEs from TB patients. (B–D) Representative FACS detection of the expression of CCR5(B), CCR1 (C) or CCR2 (D) on CD3⁺, CD4⁺ and CD8⁺ cells from TB-PEs. (E–G) Graph shows the comparison of the percentage of CCR5 (E), CCR1 (F) or CCR2 (G) expressing CD3⁺,CD4⁺ and CD8⁺ cells between TB-PEs and malignant PEs (n = 6). All the fluorescent antibodies are purchased from ebioscience (San Diego, CA). The fluorescent antibodies are used according to the manual.*, <i>p</i><0.05</p

Mycobacteria-induced expression of MCP-2/CCL8 through TLR2.

<p>(A-B) Real-time PCR of <i>mcp-2/ccl8</i> mRNA in primary peritoneal macrophages from wildtype, TLR2^−/−, TLR3^−/−, or TLR4^−/− mice infected with either <i>M. bovis</i> BCG (A) or <i>M. tuberculosis</i> H37Rv (B) at MOI 5 for 24 h. (B) Western blot of phosphorylation of Akt and p38 in primary peritoneal macrophages from wildtype or TLR2 KO mice infected with <i>M. bovis</i> BCG at MOI 5 for indicated times. Data shown are representative of three independent experiments. (D) Densitometric analysis of three independent experiments as in (C). Data shown are the mean±sem of three independent experiments. **, <i>p</i><0.001, *, <i>p</i><0.05.</p