Mycobacterium leprae-Infected Macrophages Preferentially Primed Regulatory T Cell Responses and Was Associated with Lepromatous Leprosy

Abstract

Background: The persistence of Mycobacterium leprae (M. leprae) infection is largely dependent on the types of host immune responses being induced. Macrophage, a crucial modulator of innate and adaptive immune responses, could be directly infected by M. leprae. We therefore postulated that M. leprae-infected macrophages might have altered immune functions.

Methodology/principal findings: Here, we treated monocyte-derived macrophages with live or killed M. leprae, and examined their activation status and antigen presentation. We found that macrophages treated with live M. leprae showed committed M2-like function, with decreased interleukin 1 beta (IL-1beta), IL-6, tumor necrosis factor alpha (TNF-alpha) and MHC class II molecule expression and elevated IL-10 and CD163 expression. When incubating with naive T cells, macrophages treated with live M. leprae preferentially primed regulatory T (Treg) cell responses with elevated FoxP3 and IL-10 expression, while interferon gamma (IFN-gamma) expression and CD8+ T cell cytotoxicity were reduced. Chromium release assay also found that live M. leprae-treated macrophages were more resistant to CD8+ T cell-mediated cytotoxicity than sonicated M. leprae-treated monocytes. Ex vivo studies showed that the phenotype and function of monocytes and macrophages had clear differences between L-lep and T-lep patients, consistent with the in vitro findings.

Conclusions/significance: Together, our data demonstrate that M. leprae could utilize infected macrophages by two mechanisms: firstly, M. leprae-infected macrophages preferentially primed Treg but not Th1 or cytotoxic T cell responses; secondly, M. leprae-infected macrophages were more effective at evading CD8+ T cell-mediated cytotoxicity.

Fig 1. Viable M. leprae induced M2-type macrophage differentiation.

Peripheral blood monocytes were obtained from healthy volunteers and were differentiated into macrophages in vitro. Viable or heat-killed M. leprae were added to the macrophage culture for 6 days. (A) Cytokine expression by macrophages during coculture, as measured by Luminex assay. N = 6. (B) Mean fluorescence intensity (MFI) of MHC class II and CD163 expression on macrophages after 6-day incubation in all healthy volunteers. N = 6. *: p < 0.05. **: p < 0.01. ***: p < 0.001.

Fig 2. Macrophage treated with different M. leprae antigens showed committed functional differentiation.

Live M. leprae-infected macrophages or killed M. leprae-treated macrophages were either restimulated with killed M. leprae, or cultured in plain culture medium for 6 days. Cytokine expression during the restimulation period was measured by Luminex assay. **: p < 0.01. ***: p < 0.001.

Fig 3. Live M. leprae-infected macrophages preferentially primed Treg cell responses.

Autologous CD45RA⁺ naive T cells were incubated with live M. leprae-infected macrophages or killed M. leprae-treated macrophages for 6 days. The T cells were then negatively selected and incubated separately in anti-CD3/CD28-stimulated media for an additional 72 hours, after which the supernatant was collected for ELISA and cells for flow cytometry. (A) Summary of IFN-gamma and IL-10 concentration from all healthy volunteers in the supernatant. N = 6. (B) Ratio of IFN-gamma-to-IL-10 in each individual. (C) Mean fluorescence intensity (MFI) of FoxP3 expression in T cells from all treatment conditions. N = 6. *: p < 0.05. **: p < 0.01.

Fig 4. Macrophages incubated with viable M. leprae suppressed CD8⁺ T cell cytotoxicity.

Viable or killed M. leprae were incubated with T cells for 6 days, after which the CD8⁺ T cells were negatively purified from the coculture and were added to chromium-51-labeled target cells at the indicated effector-to-target ratio. (A) Percentage specific lysis using purified autologous monocyte loaded with sonicated M. leprae antigen as the target cells. (B) Percentage specific lysis using in vitro derived, M. leprae-infected macrophages as the target cells. The CD8⁺ T cell-induced cytotoxicity (isolated CD8⁺ T cell + target cell culture minus naive T cell + target cell culture) was shown in (C) and (D), at 27-to-1 effector-to-target ratio. The effectors are (C) CD8⁺ T cells incubated with killed M. leprae-stimulated macrophages and (D) CD8⁺ T cells incubated with live M. leprae-infected macrophages. N = 6, with two independent repetitions. *: p < 0.05. **: p < 0.01. ***: p < 0.001.

Fig 5. Peripheral blood monocytes and lesion site macrophages in L-lep patients exhibited high IL-10 and low TNF-alpha and MHC class II expressions.

(A) The cytokine expression profile by peripheral blood monocytes from L-lep (N = 13) and T-lep (N = 12) patients, as well as healthy volunteers (N = 6) following a 6-day incubation. (B) The MHC class II expression of peripheral blood monocytes from L-lep and T-lep patients and healthy volunteers. (C) The cytokine expression profile by lesion site macrophage from L-lep (N = 7) patients, following a 6-day incubation. (D) The MHC class II molecule expression of lesion site macrophages L-lep patients. *: p < 0.05.