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Clinical Infection & Immunity

Original Article

Volume 6, Number 2, June 2021, pages 40-46

Metformin Regulates the Inflammatory Response of Human Macrophages to Mycobacterium tuberculosis Through a Reduction of M1 Polarization

Despite a steady decline in tuberculosis (TB) incidence worldwide, its global prevalence and burden of disease continuous to be a major public health problem worldwide [1]. Type 2 diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia and insulin resistance. Among the many comorbidities associated with DM, an increased risk for developing active TB is of great medical and epidemiological concern [2]. Clinically, more severe forms of TB and poorer outcomes, along with therapeutic challenges in anti-tuberculous treatment are present in the diabetic patient [3].

An immune dysregulation is observed in DM, which not only brings susceptibility to Mycobacterium tuberculosis (Mtb) infection, but also to latent TB reactivation. DM increases the risk for active pulmonary TB via multiple mechanisms, including those directly related to hyperglycemia, as well as an indirect effect leading to immune dysfunction involving the innate [4, 5] and the adaptive immune system [6].

Metformin (MTF) is an insulin sensitizer medication used predominantly in the treatment of type 2 DM, and it is the drug of choice in lowering glucose in diabetic patients with TB [7]. In Mtb-infected mice, MTF has shown to ameliorate lung pathology, reducing chronic inflammation, and enhancing specific immune responses and the efficacy of conventional TB drugs [8]. Various epidemiological studies have shown that MTF decreases the risk of developing active TB [9-12]. In combination with anti-TB regimens for DM-TB patients, MTF lowers relapse rates [13], improves the sputum conversion rate in patients with cavitary pulmonary TB [8, 14], and most notably, reduces the mortality in diabetic patients [8, 15].

It has been shown that MTF also has effects on immune cell function macrophage and lymphocyte functions that are keys to controlling TB infection [16]. Although recent reports have demonstrated a reduction in the intracellular growth of Mtb in human macrophages treated with MTF [8, 17], it is still unknown its effects on Mtb phagocytosis and on macrophage polarization. In this study, we aimed to better understand the effects of MTF on the response of human macrophages to Mtb.

THP1-Dual monocytes (Invivogen) were differentiated into macrophages with 5 ng/mL of phorbol 12-myristate 13-acetate (PMA, Sigma), and incubated (37 °C, 5% CO₂) for 72 h. Cells were then treated with 2 mM of MTF and incubated for 4 h. Cells were then infected with gamma-irradiated Mtb cells, strains HN878, Indo-Oceanic, CDC1551, East Africa, and H37Rv (Bei Resources) at multiplicity of infection (MOI) of 10:1, and incubated for 24 h. Detection of nuclear factor-kappa B (NF-κB) and interferon regulatory factor (IRF) activity was done according to manufacturers’ instructions. Plates were read in a Synergy Microplate reader (Biotek).

Supernatants from the cell assay were collected and analyzed with a multiplex immunoassay panel (Bio-Plex Pro Human Cytokine 17-plex assay, BioRad Laboratories) using a Bio-plex^® 200 instrument that was equipped with Bio-Plex Manager software, version 6.0 (Bio-Rad Laboratory, Hercules, CA, USA). This methodology allowed for simultaneous detection and quantification of 17 different analytes per sample, including M1 macrophage polarization (interferon (IFN)-γ, interleukin (IL)-17, tumor necrosis factor (TNF)-α, IL-1β, IL-6, IL-12 (70), granulocyte macrophage colony-stimulating factor (GM-CSF)), inflammatory mediators (monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β), IL-2, IL-7, IL-8, granulocyte colony-stimulating factor (G-CSF), IL-5), and anti-inflammatory M2-associated cytokines (IL-4, IL-10, and IL-13). The levels of inflammatory markers are expressed in pg/mL after correcting for four-fold dilution using the standards provided in the kit (Bio-Rad Laboratory, Hercules, CA, USA).

Cells were plated and differentiated on chamber slides under similar conditions described for the cell assay. After completion of the Mtb stimulation, cells were washed with phosphate buffered saline (PBS) and then fixed with 2% paraformaldehyde (PFA), permeabilized with 0.2% saponin, blocked, and stained with primary antibodies anti-Mtb NR-13802, and anti-LAMP (H4B4), followed by secondary antibody staining. Mounting media with 4’,6-diamidino-2-phenylindole (DAPI) was then added to slides. Fluorescence microscopy was conducted on an IX81 Olympus microscope. Images were the processed on Image J software (NIH).

In order to discriminate macrophage polarization phenotypic characteristics, cells were stained for the following markers indicating M1 polarization (cluster of differentiation (CD)86, CD11c, inducible nitric oxide synthase (iNOS)), and M2 polarization (CD206, CD163, and Cx3CR1) [18-20]. A BD AccuriTM C6 Flow Cytometer was used to collect the data, and CFlow software was used for analysis. Mean fluorescence intensity (MFI) values were determined after subtracting background fluorescence [21].

General statistical analysis was performed using GraphPad Prism 9.0. Phagocytosis levels were compared among the different stimuli using either a t-test or the equivalent nonparametric method if a Gaussian distribution could not be assumed. Comparison of cell activation and cytokine concentrations between untreated vs. MTF-treated cells was done using a two-way analysis of variance (ANOVA). A P value < 0.05 was considered significant.

The Institutional Review Board approval was not applicable; and the ethical compliance was not applicable as this is a research article, no human subjects were involved.

We first evaluated if MTF would have any effect on the internalization of Mtb by human macrophages. Since MTF can also directly inhibit key metabolic processes of Mtb [7], we utilized gamma-irradiated mycobacteria. In this away, we would avoid variations due to any possible differential effect of MTF in the various Mtb strains utilized.

Monocyte-derived macrophages treated with MTF demonstrated an increase in phagocytosis for most of the Mtb strains used as stimuli, regardless of the strain lineage. An increase in the number of macrophages with internalized Mtb was observed upon MTF treatment (Fig. 1a), as well as an increase in the number of internalized mycobacteria (Fig. 1b) for most of the strains. Only stimulation with HN878, and CDC 1551 showed no difference in the number of cells with internalized mycobacteria and the amount of internalized microorganisms in MTF-treated cells respectively.

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Figure 1. Metformin enhances phagocytosis of various Mtb strains by human macrophages. (a) Number of cells with internalized mycobacteria. (b) Number of mycobacteria per cell. *P < 0.05. Mtb: Mycobacterium tuberculosis.

To test if the observed increase in phagocytosis was accompanied by an increase in the inflammatory response to Mtb, we evaluated NF-κB and IRF activation in untreated vs. MTF-treated cells upon Mtb challenge. MTF-treated macrophages showed a decrease in NF-κB activation upon Mtb stimulation. No changes were observed in the IRF activity (Fig. 2).

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Figure 2. Metformin exerts an immunomodulatory effect on human macrophages response to Mtb. (a) NF-κB activation and (b) IRF activation in Untreated (white bars) vs. MTF-treated (gray bars) upon stimulation with Mtb from various lineages. NF-κB: nuclear factor-kappa B; IRF: interferon regulatory factor.

We then evaluated the secretion of pro-inflammatory and anti-inflammatory mediators by human macrophages upon Mtb stimulation. Overall, we observed that MTF treatment reduced the secretion of pro-inflammatory cytokines (Fig. 3a, b), with the exception of IL-12 and IL-7. This observed reduction applied to cells stimulated with any of the Mtb strains. MTF had no major effect in the secretion of anti-inflammatory cytokines IL-10 and IL-13 (Fig. 3c).

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Figure 3. Secretion of (a) M1-associated, (b) pro-inflammatory, and (c) M2-associated anti-inflammatory cytokines by human macrophages upon treatment with MTF. Bars represent mean ± SE. Concentration of each analyte was determined and is expressed in pg/mL. P values shown after two-way ANOVA analysis. NS: non-significant; MTF: metformin; SE: standard error; IFN: interferon; IL: interleukin; TNF: tumor necrosis factor; GM-CSF: granulocyte macrophage colony-stimulating factor; G-CSF: granulocyte colony-stimulating factor; MCP-1: monocyte chemoattractant protein 1; MIP-1β: macrophage inflammatory protein-1β; ANOVA: analysis of variance.

Since the reduction occurred in M1 polarization mediators [18-20], our next step was to assess the occurrence of a macrophage polarization shift upon treatment with MTF.

We observed M1 polarization markers iNOS and CD86 to be increased at baseline upon MTF treatment, only to experience a marked decrease after stimulation with Mtb. Untreated cells, on the other hand, showed a marked increase in all evaluated M1 markers and M2 marker CX3CR1 after Mtb stimulation (Table 1). No change in M2 marker CD206 was observed in MTF-treated cells upon Mtb stimulation.

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Table 1. Macrophage Phenotypic Changes

DM is known to affect chemotaxis, phagocytosis, activation and antigen presentation by phagocytes in response to Mtb [5]. It is also known that MTF can reduce the intracellular growth of Mtb in human macrophages in an adenosine monophosphate-activated protein kinase (AMPK)-dependent manner, facilitating phagosome-lysosome fusion and reactive oxygen species [8, 17].

A recent report has shown that MTF increases the phagocytosis of zymosan particles by human peripheral mononuclear cells, and that this phenomenon occurs along with a decreased cytokine production upon stimulation of Mtb lysates in vitro [22]. Here we show that MTF not only increases the uptake of Mtb by THP-1-derived macrophages, but that the number of internalized mycobacteria is also increased in MTF-treated cells.

Increased phagocytosis of zymosan particles by human peripheral blood mononuclear cells (PBMCs) stimulated with Mtb lysates have been shown to present lowered ex vivo production of TNF-α, IFN-γ, IL-1β, and reactive oxygen species production upon MTF treatment [22]. After initially observing a decrease in NF-κB in our system upon Mtb inoculation, we also observed a decrease in the secretion of inflammatory cytokines. This is concordant with previous studies of MTF showing a reduction of inflammatory gene activation (IL-1β, TNF-α, MCP-1, IFN-γ, IL-6, CXCL5, and CXCL10) in mice [8]. In the case of IFN-γ, an indicator of successful anti-TB treatment, the results are still debatable as an increase in IFN-γ has been reported in DM patients with TB after MTF administration along with insulin [23].

Type I IFNs are induced in patients with active Mtb infection [24], possibly interfering with IFN-γ-dependent immunity [25]. Although a down-regulation of type I IFN pathways in humans has been reported after MTF treatment [22], we did not observe any changes in the IRF reporter system used in this study. Also, MTF had no major effect on the secretion of anti-inflammatory factors (i.e., IL-10 and IL-13).

The anti-inflammatory effects of MTF [26] seem to be independent of its glucose-lowering effect [27]. Data from studies in animals have been rather contradictory. On one side MTF shows to confer CD8⁺CXCR3⁺ T cell-mediated protection in mice [28]. Despite persistent glucose intolerance MTF-treated guinea pigs showed a reduction in lung lesions [29]. On the other hand, in contrast to diabetic mice, non-diabetic mice do not benefit from MTF treatment [30].

Since many of the secreted cytokines that were reduced are associated with macrophage M1 polarization [18, 20], we decided to explore any phenotypic changes in macrophages after MTF treatment. We observed that although many M1 polarization markers were increased at baseline upon MTF treatment, these markers decreased markedly in MTF-treated cells after stimulation with Mtb. The opposite, i.e., a marked increase in M1 markers after Mtb stimulation, was observed in untreated cells. One particular aspect was the increase in M2 marker CX3CR1 expression upon inoculation with Mtb, which differs to CX3CR1 gene downregulation described in THP-1 derived macrophages infected with Mtb [31]. CX3CR1 macrophages in the lung display an M2 phenotype, and lack of this receptor skews the phenotype towards M1 polarization [32]. The expression of CX3CR1 and CD206 differs between resident and recruited macrophages in the skin of diabetic mice [33]. Both types of macrophage polarization can be seen in Mtb granulomas [34, 35], and their ratio follows a temporal dynamic [36]. The clinical significance of macrophage polarization may not only be related to the preservation of the granuloma architecture, with subsequent Mtb infection containment, but also to anti-tuberculous treatment outcomes and control of multi-drug and extensively-drug resistant [37].

Harnessing macrophage polarization may be a new target for TB therapeutic approaches [38]. Our findings were obtained through an in vitro system. More human studies will be needed to clarify if MTF, and its effect on macrophage polarization, could serve as an adjunct therapy in all TB patients. Nevertheless, the regulation of inflammation in TB-DM patients with MTF containing treatment regimens could dramatically impact the reduction of TB disease in an important part of the global population.

Acknowledgments

None to declare.

Financial Disclosure

None to declare.

Conflict of Interest

Authors declare no conflict of interest.

Informed Consent

Not applicable.

Author Contributions

JC conceptualized the study. AS, JB, PK, JVM, VDP, AC, and BG performed the experiments. JC, AS, JB, and BG wrote the paper.

Data Availability

The authors declare that data supporting the findings of this study are available within the article.

Abbreviations

DM: diabetes mellitus; Mtb: Mycobacterium tuberculosis; MTF: metformin; PMA: phorbol 12-myristate 13-acetate; TB: tuberculosis

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