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Clinical Infection & Immunity

Original Article

Volume 8, Number 1, March 2023, pages 31-36

Macrophage Polarization Leads to Differential Inflammatory Responses to Live and Dead Mycobacterium leprae

Leprosy is a chronic infectious disease, caused by the fastidious, slow-growing, and obligate intracellular Mycobacterium leprae (M. leprae) complex, which includes both M. leprae and M. lepromatosis. Its clinical presentation has been described as early as 600 BC, and the diagnosis has been associated with fear and prejudice throughout its history due to its ability to cause physical disability and deformities [1]. It was not until 1873 when G.A. Hansen first discovered the bacterium that caused this detrimental disease state [2]. The disease is still current and continues to affect individuals around the world. The World Health Organization (WHO) estimated 127,558 new leprosy cases globally in 2020, with 159 of those cases reported within the United States [3].

The clinical presentation of leprosy varies widely, generally falling within a spectrum between tuberculoid leprosy (TT) and lepromatous leprosy (LL) [4, 5]. Many patients with TT have a less severe disease course, consisting of hypopigmented well-defined macules often with surrounding edema and pain, which can progress to peripheral nervous system damage [6]. The lepromatous end, LL, on the other hand, initially presents similarly with multiple pale flat skin lesions accompanied by regional hair loss and limb weakness, but eventually progress to disfigurement. Where a patient lands on this spectrum largely depends on the individual’s immune response to the infection [2, 7, 8].

When M. leprae infection occurs, the body responds with both innate and adaptive immune functions. Macrophages are part of the innate process, defending the host through phagocytosis and other pathways, such as inducing the release of cytokines that modulate inflammation. Macrophages become activated and polarized according to the environment surrounding them. Activated macrophages are generally divided into M1, or pro-inflammatory, and M2, or anti-inflammatory, phenotypes [9]. M1 macrophages induce a pro-inflammatory state through the release of interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF). M2 macrophages, on the other hand, induce an anti-inflammatory state, often contributing to repair after inflammation [10]. These act in conjunction with cell-mediated Th1 and humoral Th2 responses, which involve different inflammatory phenotypes of T cells [11]. Several studies suggest that macrophage phenotypes have a role in the pathogenesis and recovery of disease states in leprosy [12, 13]. A Th1 response of the TT pole of the disease spectrum is associated with mycobacterial elimination or containment of the organism in granulomas, hence the term paucibacillary. However, an ineffective humoral Th2 response at the LL pole allows the proliferation of mycobacteria both outside and inside macrophages, referred to as multibacillary [4].

While this bacterium has been around for millennia, there is still much to discover about its transmission and pathophysiology. We aimed to study how macrophage polarization directly affects the inflammatory response towards live and irradiated M. leprae.

Live M. leprae, strain Lombardo-Pellegrino (ATCC 4243) were cultivated using OADC enrichment agar and Middlebrook 7H9 broth. Gamma-irradiated M. leprae was obtained from BEI-Resources (NR-19326). Whole cell sonicate M. leprae (NR-19329) was used as cell lysate.

Human monocytic cell line dual THP-1 (Invivogen) was differentiated into four different macrophage polarization categories: phorbol-12-myristate-13-acetate (PMA)-differentiated, M1, M1-activated, and M2. These phenotypic states were induced using standard methodology consisting of 5 ng/mL of PMA, 50 ng/mL of granulocyte colony-stimulating factor (G-CSF) for M1, 50 ng/mL of G-CSF with lipopolysaccharide (LPS) and interferon (IFN)-gamma for M1-activated macrophages, and 50 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) for M2 macrophages [14].

Macrophages were then stimulated with live and gamma-irradiated M. leprae at 6 days of growth, at a multiplicity of infection (MOI) of 10:1.

Immunofluorescence assay was used to observe M. leprae phagocytosis by macrophages. After M. leprae infection, cells were fixed using 2% paraformaldehyde for 10 min, then washed with phosphate-buffered saline (PBS), blocked with human immunoglobulin G (IgG), and permeabilized with 0.2% saponin in PBS. Cells were then incubated with primary antibodies against M. leprae MLMA-LAM (BEI-Resources), and against lysosomal LAMP-1 (DHSHB) overnight, followed by secondary antibodies (Alexa 488, Green, to visualize mycobacteria, and Texas Red for LAMP-1) (ThermoFisher). Imaging acquisition was done using a Motic fluorescence microscope, and images were processed with ImageJ. Both phagocytic rate and the number of bacteria per cell were then counted.

Supernatants were obtained for colorimetric and luciferase measurements, according to the maker (Invivogen), and analyzed using a microplate reader. Briefly, reporter plasmids were analyzed using Quanti-Blue, which measures transcription factor nuclear factor kappa B (NF-κB) activity, and Quanti-Luc which measures interferon regulatory factor (IRF) activity. Response under each condition was compared to unstimulated wells and expressed as a response ratio.

Supernatants from the cell assay were collected and analyzed with a multiplex immunoassay panel (Bio-Plex Pro Human Inflammation Panel 1, 37-Plex). This methodology allows for the simultaneous detection and quantification of 37 different analytes (APRIL/TNFSF13, BAFF/TNFSF13B, soluble CD30 (sCD30)/tumor necrosis factor receptor superfamily member 8 (TNFRSF8), sCD163, chitinase-3-like 1, gp130/sIL-6Rβ, IFN-α2, IFN-β, IFN-γ, IL-2, sIL-6Rα, IL-8, IL-10, IL-11, IL-12 (p40), IL-12 (p70), IL-19, IL-20, IL-22, IL-26, IL-27 (p28), IL-28A/IFN-λ2, IL-29/IFN-λ1, IL-32, IL-34, IL-35, LIGHT/TNFSF14, matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, osteocalcin, osteopontin, pentraxin-3, sTNF-R1, sTNF-R2, thymic stromal lymphopoietin (TSLP), TWEAK/TNFSF12).

General statistical analysis was conducted using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). A comparison of responses across the different cell polarization conditions was performed utilizing the t-test or a non-parametric test, whenever the data followed or not a normal distribution respectively. Non-parametric tests used were the Wilcoxon matched-pair signed rank test for paired data, or Mann-Whitney test for non-paired data. A P value < 0.05 was considered statistically significant.

Institutional Review Board approval and ethical compliance with human/animal study are not applicable for this study, as the study is not a human/animal study.

M1-activated macrophages showed a higher NF-κB and IRF-activation to the different stimuli, i.e., live M. leprae, dead M. leprae, or M. leprae lysate, compared to M1, M2, or baseline PMA-differentiated macrophages (Fig. 1a, b). The difference was remarkably higher on the IRF-activation, while the M2 macrophage NF-κB response upon live M. leprae was comparable compared to M1-activated macrophages. Overall, a lesser NF-κB and IRF-activation was observed on M1 and M2 macrophages compared to M1-activated macrophages upon stimulation with either irradiated M. leprae, or M. leprae lysate.

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Figure 1. M1 macrophage activation leads to an increased inflammatory response. (a) NF-κB and (b) IRF activation in response to live and dead M. leprae across PMA, M1, M2, and M1-activated macrophage polarizations. Bars represent means, and standard error. N = 3. *P < 0.05. M. leprae: Mycobacterium leprae; PMA: phorbol-12-myristate-13-acetate; NF-κB: nuclear factor kappa B; IRF: interferon regulatory factor.

We first explored the number of phagocytic cells upon stimulation with live or gamma-irradiated M. leprae. There was no difference in the phagocytic response across the different macrophage polarization conditions, although a slightly higher number of phagocytic cells were observed upon stimulation with dead M. leprae (Fig. 2a). The ratios range from 38% to 52% of cells with internalized M. leprae.

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Figure 2. M1 macrophage activation shows increased phagocytosis of dead M. leprae. (a) Cells with phagocytized bacteria were compared to the total amount of cells in the slide. (b) The number of bacteria was counted per cell in macrophages that had phagocytized M. leprae. Bars represent means, and standard error. N = 3. *P < 0.05. M. leprae: Mycobacterium leprae; PMA: phorbol-12-myristate-13-acetate.

When comparing the number of internalized mycobacteria per cell, we observed that M1-activated cells had the highest rate of phagocytized mycobacteria per cell upon stimulation with gamma-irradiated M. leprae (Fig. 2b).

M1 macrophages showed increased secretion of particular mediators upon stimulation with live M. leprae. Cytokine analysis revealed that secretion of IFN-beta was highest in M1 cells upon stimulation with live M. leprae, suggesting that active transcription in viable organisms is needed for induction of type I IFNs.

Curiously, the soluble form of M2 marker sCD163, and of TSLP, which promotes a shift of M1 to M2 [15, 16], were highest in M1-activated macrophages upon stimulation with either live or dead M. leprae (Fig. 3).

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Figure 3. M1 macrophages showed increased secretion of particular mediators upon stimulation with live M. leprae. Graphs show secreted values for IFN-beta, TSLP, and sCD163 in PMA-differentiated, M2, M1, and M1-activated macrophages infected with live or dead M. leprae. Bars represent means, and standard error. N = 3. *P < 0.05. TSLP: thymic stromal lymphopoietin; IFN: interferon; M. leprae: Mycobacterium leprae; PMA: phorbol-12-myristate-13-acetate; sCD163: soluble CD163.

Multiple studies have found that M. leprae preferentially invades macrophages for survival and replication, also attacking keratinocytes, dendritic cells, and Schwann cells [17, 18]. The effect on these cells helps explain why individuals suffering from leprosy will often have manifestations in the skin, peripheral nervous system, and immune system. Macrophages are found in almost every tissue within the body. Among their major functions is to protect tissues by clearing foreign bodies and is therefore, considered the first line of defense, activating the immune system if a threat is detected. However, different metabolic states can influence how and when macrophages respond, determine the strength of the immune response, and induce further pathophysiologic changes [19].

Macrophage polarization has been discussed in the pathophysiology of multiple diseases, including gastritis, tuberculosis, keratitis, and leprosy, although its specific effects in mediating infectious disease responses remain undiscovered [20, 21]. Macrophages respond to different immune cues, available nutrients, and pathogen phagocytosis by releasing different chemicals to induce pro-inflammatory, anti-inflammatory, or tissue repair states. These states are similar to T-helper cell phenotypes, Th1 and Th2. Th1 cells release cytokines, such as IFN-gamma. This, when combined with LPS has classically induced an M1-activation state in macrophages, ramping up the killing of intracellular pathogens and creating tissue damage as a side effect. Inversely, Th2 cytokines, such as IL-4, induce an M2 polarization, dampening inflammation and promoting remodeling of tissues [22].

Inflammatory markers generally act as a positive feedback mechanism, further promoting macrophage activation and phagocytosis. When activated through these cytokines, macrophages upregulate phagocytosis to help clear the offending pathogen, with hemoglobin receptor CD163 expressed on macrophage surfaces in response to bacterial shedding [23]. We observed an increase in sCD163 in M1-activated macrophages and also showed an increased number of phagocytized M. leprae.

Activation of transcription factor NF-κB is key in the inflammatory response following an insult by bacterial cell wall components (e.g., LPS) or induced by inflammatory cytokines, triggering a subsequent cascade of pro-inflammatory cytokines and chemokines. NF-κB can upregulate other transcription factors, including the IRF genes (IFN-regulatory family). NF-κB and IRF function as a sort of yin-yang, where each system regulates the other [24-27]. This elevated IRF-mediated response could represent an increased type 1 IFN, as IFN-beta secretion was highest in M1 and activated M1-activated cells upon stimulation with live M. leprae. This elevated secretion, suggests that active transcription in viable mycobacteria is needed for induction of a type I IFN response [28]. IFN-B has been identified as a marker for resolution macrophages during resolving bacterial inflammation [29].

Overall, we observed an increase in NF-κB and IRF activation in M1-activated macrophages irrespective of the stimuli. Nevertheless, such inflammatory response needs to be regulated, which may be explained by an increase in sCD163 and TSLP secretion. TSLP has been shown to promote M1 to M2 polarization, enhancing resolution of the inflammation [15, 16].

Variables affecting the host, pathogen, and cellular environment modulate disease states and must be assessed. In the case of leprosy, the immune function of the host has been shown to be among the most important factors in its pathogenesis. Why certain individuals are more prone to be in a predominantly M1 or M2 macrophage state is still unknown. As discoveries are made regarding the interplay and balance between pro- and anti-inflammatory responses, new treatment approaches can be designed to ameliorate and control disease severity in individuals suffering from leprosy.

Acknowledgments

None to declare.

Financial Disclosure

No funding available.

Conflict of Interest

Authors do not have conflict of interest to declare.

Informed Consent

Not applicable.

Author Contributions

JC conceptualized the study. AM, JB, JV, and KVH performed the experiment. JC analyzed the data. JC, AM, EB, and JV wrote the manuscript.

Data Availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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