Background
Tuberculosis (TB) caused by infection of
Mycobacterium tuberculosis (MTB) is one of the top 10 causes of death in developing countries [
1]. The risk factors for TB include crowding, poor nutrition, alcoholism, race/ethnicity, socioeconomic status, diabetes mellitus, human immunodeficiency virus (HIV) infection and cigarette smoking [
2,
3]. Cigarette smoking (CS) has been reported to be harmful to human lungs and associated with an increase in both mortality and morbidity of TB. It has been shown that the risk of death in patients with long-term cigarette smoking-related pulmonary tuberculosis (LCS-PTB) is approximately 4.5 times higher than those with nonsmoking pulmonary tuberculosis (N-PTB) [
4]. Meanwhile, LCS-PTB patients always present more severe pulmonary lesions than N-PTB patients [
5]. Additionally, previous studies have shown that CS triggers an intense inflammatory response in lungs mediated by increased infiltration of alveolar and blood macrophages, monoc
ytes, and neutrophils that release large amounts of various inflammatory cytokines and chemokines [
6‐
8]. However, the molecular mechanisms underlying such pulmonary inflammatory responses in the setting of LCS have not been well documented.
Host immune system plays a critical role in the containment and cure of TB infection. The major types of innate immune cells involved in TB infection include macrophages, dendritic cells (DCs), neutrophils and natural killer (NK) cells, whereas classically activated (M1) macrophages play a central role in eliminating MTB via multiple mechanisms, such as increasing the components of free reactive oxygen and nitrogen species, production of various pro-inflammatory cytokines, phagosome acidification, autophagy induction, and among others [
9]. Although M1 macrophages are known as professional killers, MTB has adopted remarkable strategies to control phagosomal processing via inhibiting phagolysosome biogenesis and acidification processes [
10]. Notably, MTB survival is greatly enhanced when macrophage polarity is shifted toward alternatively activated (M2) macrophage [
11]. It has been reported that treatment of monocyte-derived macrophages (MDM) by cigarette smoke extract (CSE) results in the enrichment of IL-6, TNF-α, IL-1β and MMP-9, which are commonly associated with M1 macrophages, and simultaneously, the reduction of IL-4, IL-10, and IL-13, which are associated with M2 macrophages [
12‐
15]. It has been shown that the signal transducer and activator of transcription 1 (STAT1) plays a major role in dictating M1 macrophage phenotype, whereas STAT3/STAT6 direct M2 macrophage polarization [
16‐
18].
MicroRNAs (miRNAs), small noncoding RNAs of length 22 nucleotides, are important regulatory molecules and have been shown to be involved in the regulating development and functions of monocytes/macrophages [
17]. Also, they have been found to be valuable novel biomarkers for cigarette smoking [
19]. MiR-196b-5p has been identified in the most primitive hematopoietic stem cells (HSCs), the precursors of of monocytes/macrophage [
20]. Zhang et al. has reported that the level of miR-196b in the serum of patients with active TB was 1285.93-fold higher than that in latent TB infection (LTBI),
Bacillus Calmette‐
Guérin (BCG)-inoculated and un-inoculated individuals [
21]. Our previous results demonstrated that miR-196b-5p promotes colorectal cancer stemness and chemo-resistance via activating STAT3 signaling [
22]. Accumulating data have suggested that STAT3 is a major controller of the outcome of MTB infection [
23]. However, the expression of miR-196b-5p in monocytes from LCS-PTB patients and its role in the modulation of macrophage-mediated inflammatory responses during MTB infection in humans is yet to be fully elucidated. In the present study, we found that miR-196b-5p was upregulated in monocytes from LCS-PTB patients and treatment of MDM with CSE significantly increased miR-196b-5p expression. Moreover, overexpressing miR-196b-5p attenuated the uptake of BCG by MDM via targeting SOCS3 and subsequent activation of STAT3 signaling. These findings suggest that miR-196b-5p might be a valuable novel biomarker and therapeutic target for LCS-PTB patients.
Methods
Subjects
28 cases long-term cigarette smoking PTB (LCS-PTB) patients, 22 cases nonsmoking PTB (N-PTB) patients and 20 cases healthy volunteers (HV) from Dongguan sixth People’s Hospital (Dongguan, China) were enrolled in this study based on clinical symptoms, chest X radiography, acid fast bacilli (AFB) staining of sputum smears, as we previously reported [
24,
25]. Subjects with HIV infection, autoimmune diseases, diabetes, cancer, immunosuppressive treatment, or pulmonary tuberculosis history were excluded.
Monocytes sorting
Peripheral blood mononuclear cells (PBMCs) were prepared to isolate monocytes using the MojoSort™ Human CD14 Nanobeads (BioLegend) according to the manufacturer’s instructions. The sorted monocytes were primary monocytes for RNA extraction.
Cultures of human MDM and U937 cells
Human MDM and U937 (a monocytic cell line, obtained from Shanghai Chinese Academy of Sciences cell bank, China) cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (heat-inactivated, mycoplasma- and endotoxin-free), 10% M-CSF, 50 U/mL penicillin, 50 µg/mL streptomycin and 2 mM
l-glutamine. Monocytes were isolated from PBMC as we previously described [
24‐
26]. PBMC were seeded on 24-well plates at a density of 1.5 × 10
6 cells/well and differentiated for 7 days, non-adherent cells were removed, and adherent cells were obtained as differentiated MDM (more than 95% of cells were CD14
+, CD3
−). Before experiments were performed, the cells were cultured at 5 × 10
5 cells/well on 24-well plates, and 50 nM phorbol myristate acetate (PMA) was used to differentiate U937 cells for 24 h.
RNA extraction and qRT- PCR
Total RNA was extracted using RNA Isolation Kit (Qiagen, USA), and reverse transcribed using the Revert Aid First Strand cDNA Synthesis Kit (Thermo, USA) according to the manufacturer’s protocol. This cDNA was amplified and quantified on CFX96 system (BIO-RAD, USA) using iQ SYBR Green (BIO-RAD, USA). The primers were provided in Additional file
1: Table S1. Primers for U6 and miR-196b-5p (Cat#: miRQ0001080) were synthesized and purified by RiboBio (Guangzhou, China) (
http://www.sirna.cn/siteen/Products.aspx?id=181). QRT-PCR was performed according to a standard method, as we previously described [
22]. U6 or GAPDH was used as endogenous controls. Relative expressions were calculated with the comparative threshold cycle (2
−ΔΔCt) method.
Flow cytometric analysis
The isolated PBMCs were resuspended in 2% FBS-PBS, and then stained with indicated antibodies against human CD3 (HIT3α, BioLegend), CD14 (63D3, BioLegend), IL-6 (UV4, BioLegend), IL-10 (JES5-16E3, eBioscience), TNF-α (Mab11, BioLegend), and detected by flow cytometry (BD FACS Calibur II, San Jose, CA, USA) and analyzed using FlowJo 7.6 software (TreeStar Inc., USA) as we previously described [
24‐
26]. Mouse IgG1 (MOPC-21), IgG2b (27–35), IgG2a (G155–178), and rat IgG2b (κ) were used as isotype control.
Exposure of cells to BCG
For testing the effects of miR-196b-5p on mycobacterial uptake in vitro, MDM or U937 macrophages were infected with green fluorescent protein (GFP) tagged BCG at the multiplicity of infection (MOI) of 10 for 6 h, then washed and cultivated for 5 days. Bacterial uptake was evaluated by measuring the percentage and median fluorescence intensity (MFI) of GFP+ MDM (BCG uptake macrophages) by flow cytometric analysis.
MiRNAs, siRNA, lentiviral particles and transfection
The agomir-196b-5p (Cat#: miR40001080), antagomir-196b-5p (Cat#: miR30001080), small interfering RNA (siRNA) for SOCS3 (SOCS3-RNAi) and respective control RNA were synthesized and purified by RiboBio, as we previously described [
22]. SOCS-3 lentiviral activation particles (SOCS3-LAC, sc-400455-LAC) was purchased from Santa Cruz. Transfection of miRNAs, siRNA were performed using Lipofectamine 3000 (Life Technologies, USA) according to the manufacturer’s instructions.
Western blotting analysis
Nuclear/cytoplasmic fractionation was separated using Cell Fractionation Kit (Cell Signaling Technology, USA) and the whole cell lysates were extracted using RIPA Buffer (Cell Signaling Technology) according to the manufacturer’s instructions. Western blot was performed according to a standard method, as we previously described [
22]. Proteins were visualized using ECL reagents (Pierce, USA). Antibodies against SOCS1, SOCS3, STAT3 and pSTAT3 were purchased from Cell Signaling Technology.
Dual-luciferase reporter assay
For dual-luciferase reporter assay, the SOCS3 3′-UTR segments, containing the binding elements of miR-196b-5p or its mutant versions were synthesized as sense and antisense linkers. The wild-type and mutated 3′-UTR fragments were then cloned into the downstream of luciferase reporter gene of pmirGLO vectors (Promega, Madison, WI, USA). PmirGLO-Report-WT-SOCS1 (harboring wild-type 3′-UTR) and pmirGLO-Report-Mut-SOCS1 (harboring mutant 3′-UTR) were generated. The specificity of miR-196b-5p targeting SOCS3 mRNA was ascertained by co-transfection of miR-196b-5p mimic or inhibitor (RiboBio, Guangzhou, China) and pmirGLO-Report-SOCS1/Mut into U937 cells. Additionally, as we previously described [
22], relative luciferase activity of STAT3 was detected using pSTAT3 reporter luciferase plasmid (Promega). Luciferase and Renville signals were measured 36 h after transfection using a Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol as we previously described [
22].
Enzyme-linked immunosorbent assay (ELISA)
Cytokines were measured in the plasma and cell culture supernatant samples using human IL-6, IL-10 and TNF-α ELISA Kit (Ray Biotech, Atlanta, USA) according to the manufacturer’s instructions. Cell culture supernatant was diluted 1:10. Plasma was used as an undiluted specimen.
Statistical analysis
All analyses were performed using GraphPad Prism version 5.0 software (GraphPad Software Inc., San Diego, CA, USA). Data are presented as mean ± SEM (standard error of the mean, SEM). Student’s t-test or ANOVA (analysis of variance, ANOVA) was employed to compare the differences of measured data. A P value of less than 0.05 (95% confidence interval) was considered with statistical significance.
Discussion
Inflammatory responses are an essential part of innate immune responses to pathogens. Monocytes or macrophages were important effectors and regulators in innate immunity. Numerous studies, including our previous reports, showed an increased number of circulating blood monocytes in TB patients [
25,
30,
37,
38]. However, blood monocyte-derived macrophages in PTB patients exhibit impaired phagocytic capacity [
39]. MTB infection recruited the inflammatory monocytes or macrophages into lungs, which contribute to granuloma formation [
40,
41] whereby IL-6, and TNF-α play an important role [
25,
42‐
44]. Herein, we also found that IL-6- and TNF-α-positive CD14
+ monocytes and their serum levels were significantly increased in peripheral blood of both LCS-PTB and N-PTB patients as compared with those in HV volunteers, which is in consistent with the previous results found in adult but not childhood TB patients [
25,
43,
45‐
48]. However, the percentage of IL-6- and TNF-α-positive CD14
+ monocytes were relatively lower in peripheral blood of LCS-PTB patients (cigarette smoking > 7 years) than that of N-PTB patients, although the absolute count and percentage of monocytes increased in both LCS-PTB and N-PTB patients. In parallel, we found an elevated level of IL-6 and TNF-α in serum of both LCS-PTB and N-PTB patients as compared to that of HV volunteers, while the elevation of IL-6 and TNF-α in serum of LCS-PTB was less pronounced than that of N-PTB patients. Functionally, we showed that MDMs of LCS-PTB patients exhibited a lower uptake of BCG or impaired phagocytosis of BCG. These results support the notion that long-term cigarette smoking may lead to impaired phagocytosis of BCG by macrophages due to attenuated pro-inflammatory responses.
Aberrant activation of STAT3 signaling pathway was linked to immune disorders and inflammatory response of monocytes or macrophages. Human STAT family currently consisted of seven members, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6. In this study, we showed that
STAT3,
STAT5A,
STAT5B mRNAs were upregulated in peripheral CD14
+ monocytes from LCS- and N-PTB patients as compared with those of HV controls. Moreover, we have demonstrated, for the first time to our knowledge, that the increase in
STAT3 mRNA expression and pSTAT3 expression were less pronounced in CD14
+ monocytes and MDM from LCS-PTB patients than in those from N-PTB patients, which were inversely correlated with the expression levels of
SOCS3 mRNA and protein. Emerging evidence indicates that deregulation of negative regulators of STAT3 signaling pathway including tyrosine phosphatase and SOCS families plays a crucial role in the activation of STAT3 signaling [
49‐
51]. Therefore, our results suggest that SOCS3 is involved in the regulation of STAT3 activation in peripheral CD14
+ monocytes or MDMs from LCS-PTB patients.
Most recently, the biological role and clinical significance of miR-196b-5p have been extensively studied. Overexpression of miR-196-5p has been reported in the hematologic malignancies, solid tumors, coronary artery disease and Ebola virus infection [
22,
52‐
55]. Zhang et al. has reported that the serum level of miR-196b in active TB patients was higher than that in latent TB infection (LTBI), BCG-inoculated and un-inoculated individuals [
21]. In the present study, we demonstrated that miR-196b-5p was upregulated in peripheral monocytes from LCS-PTB patients as compared with that in those from N-PTB patients. Additionally, we showed CSE and PM2.5 (unpublished data) increased miR-196b-5p expression in MDMs and differentiated U937 cells. Overexpression of miR-196b-5p downregulated the expression of SOCS3 mRNA while simultaneously activated STAT3 signaling pathway in macrophages.
Conclusions
In summary, our findings support the notion that long-term cigarette smoking may lead to upregulated expression of miR-196b-5p, which then contributes to targeted inhibition of SOCS3 and activation of STAT3 signaling pathway, and subsequently, the impaired phagocytosis or elimination of BCG by macrophages due to their attenuated pro-inflammatory responses. Therefore, miR-196b-5p may represent a novel biomarker and therapeutic target for the treatment of LCS-PTB patients.
Authors’ contributions
JCZ, TC, and YQY conceived and designed the experiments; DZL, LF, YM, ZYY and HMY performed the experiments; MYH and BHL performed data analysis; YML, XLY, and YZM contributed to sample collection; JCZ wrote the paper; KYZ and QZZ assisted with writing and proofreading. All authors read and approved the final manuscript.