Tuberculosis is associated with expansion of a motile, permissive and immunomodulatory CD16(+) monocyte population via the IL-10/STAT3 axis

Abstract

The human CD14(+) monocyte compartment is composed by two subsets based on CD16 expression. We previously reported that this compartment is perturbed in tuberculosis (TB) patients, as reflected by the expansion of CD16(+) monocytes along with disease severity. Whether this unbalance is beneficial or detrimental to host defense remains to be elucidated. Here in the context of active TB, we demonstrate that human monocytes are predisposed to differentiate towards an anti-inflammatory (M2-like) macrophage activation program characterized by the CD16(+)CD163(+)MerTK(+)pSTAT3(+) phenotype and functional properties such as enhanced protease-dependent motility, pathogen permissivity and immunomodulation. This process is dependent on STAT3 activation, and loss-of-function experiments point towards a detrimental role in host defense against TB. Importantly, we provide a critical correlation between the abundance of the CD16(+)CD163(+)MerTK(+)pSTAT3(+) cells and the progression of the disease either at the local level in a non-human primate tuberculous granuloma context, or at the systemic level through the detection of the soluble form of CD163 in human sera. Collectively, this study argues for the pathogenic role of the CD16(+)CD163(+)MerTK(+)pSTAT3(+) monocyte-to-macrophage differentiation program and its potential as a target for TB therapy, and promotes the detection of circulating CD163 as a potential biomarker for disease progression and monitoring of treatment efficacy.

Figure 1

The secretome of Mtb-infected macrophages induces the differentiation of CD16⁺ monocytes towards an M2-like phenotype. Monocytes were cultured with conditioned media from Mtb-infected (cmMTB, black) or non-infected (cmCTR, white) macrophages. (A) Flow cytometry gating strategy of a representative donor and the percentage of CD14⁺CD16⁺ cells. (B) Vertical scatter plots showing the median fluorescent intensity (MFI) of cell-surface markers during cmCTR or cmMTB treatment. (C) Vertical scatter plots showing the MFI of CD163, MerTK or CD206 in the CD14⁺CD16⁻ (white circles) and CD14⁺CD16⁺ (black circles) cell populations during cmCTR or cmMTB treatment. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Each circle within vertical scatter plots represents a single donor.

Figure 2

The IL-10/STAT3 pathway drives the predisposition of CD16⁺ monocytes towards an M2-like phenotype. (A) Vertical scatter plots showing the MFI of cell-surface markers in monocytes cultured with IL-10-depleted cmMTB (cmMTBαIL-10) or the mock depletion control (cmMTBαIgG). (B) Immunoblot images of pY705-STAT3, STAT3 and actin (upper panel); quantification of pY705-STAT3 versus STAT3 on monocytes after 1 h treatment with cmCTR, cmMTB, cmMTBαIL-10 or cmMTBαIgG (n = 6 donors; lower panel). (C) Vertical scatter plots showing the MFI of cell-surface markers in monocytes transfected with STAT3 (siSTAT3-black) or non-targeting control (siCTR-white) siRNAs, and conditioned with cmCTR or cmMTB. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Each circle within vertical scatter plots represents a single donor.

Figure 3

The CD16⁺ monocyte expansion is predisposed towards an M2-like phenotype in patients with active TB. (A) Vertical scatter plots showing the percentage of CD14⁺CD16⁺ cells in the peripheral blood of healthy subjects (PB-HS), TB patients (PB-TB) or subjects with latent TB (PB-LTB). (B) Immunoblot images of pY705-STAT3, STAT3 and actin (left), and quantification of pY705-STAT3 versus STAT3 (right) on monocytes from healthy subjects (HS) or TB patients (TB) (n = 5 donors). (C) Vertical scatter plots showing the MFI of cell-surface markers in HS or TB monocyte-derived macrophages differentiated in vitro for 7 days. (D) Analysis of soluble form of CD163 (sCD163) level in sera of TB patients according to disease severity, as compared with HS. (E) Vertical scatter plots showing the serum level of sCD163 in TB patients before and 12 months after start of treatment (TB-M12), as compared with contact donors. (F) ROC (receiver operating characteristic) curve of sCD163 concentration in TB patients compared with HS. (G) ROC curve of sCD163 concentration in TB patients before versus after treatment (TB versus TB-M12). The red circle represents the optimal cut point. (H) Monocytes from HS were treated with pleural effusion (PE) from TB (PE-TB) or non-TB (PE-nonTB) patients, or culture medium. Representative images of western blot illustrating the expression of pY⁷⁰⁵-STAT3, STAT3 and actin (n = 3). (I) Vertical scatter plots showing the MFI of CD16, CD163 or MerTK on CD14⁺ monocytes present in PE-TB or PE-nonTB from patients. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. Each circle within vertical scatter plots represents a single donor. AUC, area under the curve.

Figure 4

The abundance of the CD16⁺CD163⁺MerTK⁺pSTAT3⁺ cell population correlates with pathology severity in non-human primate (NHP) pulmonary TB. (A) Representative immunohistochemical images demonstrate expression and distribution of CD68, CD16, CD163 and MerTK in lung granulomas from non-vaccinated Mtb-infected NHPs exhibiting different severity of pulmonary TB. NHP numbers refer to Supplementary information, Table S6. (B) Correlation analysis between the number of CD16⁺, CD163⁺ or MerTK⁺ cells in lung tissue and lung pathology score in BCG-vaccinated (red circles) or -non-vaccinated (black circles) Mtb-infected NHPs. (C, D) Immunohistochemistry stainings of lung biopsies from NHP n°51 (intermediate). (C) Upper panel: co-localization of CD163 (green; Alexa-488) and CD16 (red; Alexa-555) in lung tissue; lower panel: co-localization of CD163 (green; Alexa-488) and MerTK (red; Alexa-555) in lung tissue. White arrows indicate double-positive cells that are magnified in 1 and 2 (inset scale bar = 10 μm). (D) Upper panel: nuclear localization of STAT3 (green; Alexa-488) in CD16 (red; Alexa-555)-positive cells in lung tissue; lower panel: nuclear localization of STAT3 (green; Alexa-488) in CD163 (red; Alexa-555)-positive cells in lung tissue. White arrows indicate CD16- or CD163-positive cells with STAT3 translocated in the nucleus stained with DAPI (blue; inset scale bar = 10 μm).

Figure 5

The CD16⁺CD163⁺MerTK⁺pSTAT3⁺ monocyte-macrophages display a strong ability to migrate in dense matrices. (A) Human monocytes were seeded on the top of fibrillar collagen I or Matrigel matrices, and allowed to migrate in response to cmCTR (white) or cmMTB (black). Quantification of the percentage of migrating cells (n = 7 donors). (B) Effect of protease (Pimix), matrix metalloprotease (GM6001), Rho-associated kinase (Y27632) inhibitors or DMSO on cell migration in Matrigel (n = 9 donors). (C) Representative scanning electron microscopy images showing the matrix remodeling activity of cells on Matrigel (n = 3 donors, scale bar = 10 μm). (D) Quantification of gelatin zymogram gels for MMP9 (left) and MMP1 (right) activities (n > 4 donors). (E) Monocytes transfected with SMARTpool targeting STAT3 (siSTAT3) or non-targeting control siRNAs (siCTR) were seeded on Matrigel and allowed to migrate in response to cmMTB (n = 3 donors). (F) Monocytes from HS were seeded on Matrigel and allowed to migrate in response to PE-TB, PE-nonTB or culture medium (n = 3 donors). Results are expressed as mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 6

The CD16⁺CD163⁺MerTK⁺pSTAT3⁺ monocyte-macrophages are associated with a permissive phenotype to Mtb infection. (A) Representative confocal microscopy images of CD16⁺ cells (red, Alexa-555) in lung granulomas from NHP N°51 infected by Mtb (green, Alexa-488). White arrow indicates CD16⁺ cells containing Mtb bacilli (inset, scale bar = 10 μm). (B) Monocytes conditioned with cmCTR (white circles) or cmMTB (black circles) were infected with Mtb. On day 0 and day 5 after infection, the intracellular growth of Mtb was scored by colony-forming unit (CFU) assay. (C) Monocytes transfected with SMARTpool targeting STAT3 (siSTAT3) or non-targeting control siRNAs (siCTR) were conditioned with cmMTB, and then infected with Mtb. The CFU scoring was measured on day 0 and day 5 after infection. Results are expressed as before-and-after plot; ^*P < 0.05.

Figure 7

The CD16⁺CD163⁺MerTK⁺pSTAT3⁺ monocyte-macrophages display immunomodulatory properties. (A-C) Monocytes were stimulated with killed Mtb or PBS (mock). Top panels: vertical scatter plots showing the ratio of MFI obtained for PD-L1 and CD86. Results are expressed as mean ± SEM. Bottom panels: allogeneic human T lymphocytes labeled with CFSE were co-cultured with monocytes. Before-and-after plots showing T cell proliferation illustrated as the percentage of CFSE-dividing cells (left) and the production of IFNγ by proliferating T cells quantified by (A-B) ELISA in co-culture supernatants or (C) flow cytometry (right). (A) cmCTR- or cmMTB-conditioned monocytes were stimulated with PFA-killed Mtb or PBS (mock). (B) cmMTB conditioning of monocytes was performed in the presence of the STAT3 inhibitor, cucurbitacin I (CCB) or DMSO as control. Cells were then stimulated with PFA-killed Mtb or mock. (C) Monocytes from HS or TB patients were stimulated with irradiated Mtb or mock. ^*P < 0.05; ^**P < 0.01. Each circle within vertical scatter plots represents a single donor.

Figure 8

Model illustrating how the environment induced by Mtb infection predisposes human monocyte differentiation towards an M2-like macrophage, altering host defense during infection. Alveolar macrophages are one of the first leukocytes able to recognize and phagocytose Mtb upon entry in the respiratory system. At this site, infected macrophages reshape their microenvironment by secreting many soluble factors including cytokines and chemokines, which for the most part are responsible for the leukocyte infiltration during the earliest stages of infection. However, (1) Mtb has the capacity to modulate the macrophage response and to induce the secretion of anti-inflammatory cytokines, such as IL-10. IL-10, tilts, through a bystander effect, monocytes towards an M2-like macrophage program (CD16⁺CD163⁺MerTK⁺pSTAT3⁺) in a STAT3-dependent manner. In the blood, CD163 and MerTK receptors are cleaved off, and concentration of their soluble form correlates positively with disease severity. The CD16⁺CD163⁺MerTK⁺pSTAT3⁺ phenotype acquisition is accompanied by (2) an enhanced protease-dependent motility through matrix metalloprotease activity (e.g., MMP-1), which allows extracellular matrix remodeling, and hypothetically, trans-tissular migration. This phenotype acquisition also renders (3) monocyte-macrophages permissive to Mtb intracellular growth, and (4) immunomodulatory in terms of their reduced ability to secrete pro-inflammatory cytokines (e.g., low TNFα) and activate the T-helper 1 (Th1) response via co-stimulatory signaling (e.g., high ratio PD-L1/CD86). Collectively, the Mtb-derived bystander activation of STAT3 in monocytes predisposes their differentiation program towards a macrophage population that ultimately shifts the microenvironment (e.g., tuberculous granulomas) in favor of microbial resilience in the host.