Tuberculosis Exacerbates HIV-1 Infection through IL-10/STAT3-Dependent Tunneling Nanotube Formation in Macrophages

Abstract

The tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), and HIV-1 act synergistically; however, the mechanisms by which Mtb exacerbates HIV-1 pathogenesis are not well known. Using in vitro and ex vivo cell culture systems, we show that human M(IL-10) anti-inflammatory macrophages, present in TB-associated microenvironment, produce high levels of HIV-1. In vivo, M(IL-10) macrophages are expanded in lungs of co-infected non-human primates, which correlates with disease severity. Furthermore, HIV-1/Mtb co-infected patients display an accumulation of M(IL-10) macrophage markers (soluble CD163 and MerTK). These M(IL-10) macrophages form direct cell-to-cell bridges, which we identified as tunneling nanotubes (TNTs) involved in viral transfer. TNT formation requires the IL-10/STAT3 signaling pathway, and targeted inhibition of TNTs substantially reduces the enhancement of HIV-1 cell-to-cell transfer and overproduction in M(IL-10) macrophages. Our study reveals that TNTs facilitate viral transfer and amplification, thereby promoting TNT formation as a mechanism to be explored in TB/AIDS potential therapeutics.

Keywords: AIDS; HIV-1; IL-10; Mycobacterium tuberculosis; STAT3; biomarker; co-infection; macrophage; monocyte; tuberculosis; tunneling nanotubes; viral spread.

Figure 1. (Related to Figure S1, S2, S3). TB-induced microenvironments exacerbate HIV-1 infection of M(IL-10) macrophages.

(A) Representation of the experimental design of two in vitro models. Briefly, monocytes from healthy subjects were treated either with conditioned medium from mock- (CmCTR) or Mtb-infected macrophages (CmMTB), or with pleural effusions (PE) from TB (PE-TB) or non-TB (PE-nonTB) patients for 3 days. Cells were then infected with HIV-1 ADA strain at MOI of 0.1 and kept in culture for at least 10 more days. (B) Vertical scatter plot showing p24 concentration from day 13 supernatants of HIV-1 infected macrophages treated with CmCTR or CmMTB. (C) Vertical scatter plot showing the infection index of day 13 HIV-1 infected macrophages treated with CmCTR or CmMTB. Infection index was calculated as 100 x the ratio of the area covered by Gag+ cells over the total cell area, measured from immunofluorescence (IF) images. (D) Representative IF images of day 13 HIV-1 infected macrophages treated with CmCTR or CmMTB. HIV-1 Gag (red), F-actin (green), DAPI (blue). Scale bar, 500μm. Insets are 4x zooms (lower panels). (E) Vertical scatter plot showing p24 concentration from day 13 supernatants of HIV-1 infected macrophages treated with PE-TB or PE-nonTB. (F) Vertical scatter plot showing the infection index of day 13 HIV-1 infected macrophages treated with PE-TB or PE-nonTB. (G) Representative IF images of day 13 HIV-1 infected macrophages treated with PE-nonTB or PE-TB. HIV-1 Gag (red), F-actin (green), DAPI (blue). Scale bar, 500μm. Insets are 4x zooms (lower panels). Each circle within vertical scatter plots represents a single donor. Mean value is represented as a dark grey line. In this figure, PE-nonTB are parapneumonic PE. Statistical analyses: Two-tailed, Wilcoxon matched-paired signed rank test (B,C), paired t-test (E,F). ** p≤0.005; *** p≤0.0005; **** p≤0.0001.

Figure 2. (Related to Table S2, S3). Accumulation of M(IL-10) macrophages in the lung of co-infected non-human primates (NHPs) correlates with pathology.

(A)Representative immunohistochemical images illustrate higher number of CD163⁺ cells and p^Y705-STAT3⁺ cells in lung biopsies of SIV-Mtb infected NHPs compared to Mtb or SIV mono-infected NHPs. Scale bar, 100μm. Insets are 4x zooms. (B) Quantification of the number of CD163⁺ cells (left) and p^Y705-STAT3⁺ cells (right) per mm² of lung tissue of healthy (H), SIV-infected, Mtb-infected and SIV-Mtb-co-infected NHPs. (C) Immunohistochemistry staining of lung biopsy of SIV-Mtb co-infected NHP showing the nuclear localization of p^Y705-STAT3 (red, center) in CD163 alveolar macrophages (green, top). Nuclei are stained using DAPI (blue, bottom). Scale bar, 50 μm. Insets are 1.3x zooms. (D) Vertical scatter plot showing the pathological scoring of NHPs used in this study (See Table S3). (E) Correlation between CD163⁺ cells (left) or p^Y705-STAT3⁺ cells (right) per mm² of lung tissue and pathological score in the indicated NHPs. Each symbol within vertical scatter plots represents a single animal. Mean value is represented as a dark grey line. Statistical analyses: Two-tailed Mann-Whitney (B,D). * p≤0.05; ** p≤0.005; ns, not significant.

Figure 3. (Related to Figure S4 and Table S1). Systemic expansion of the M(IL-10) monocyte population in co-infected patients.

(A) Vertical scatter plots showing the median fluorescence intensity (MFI) of cell-surface marker CD163 on CD14⁺ monocytes from peripheral blood of healthy subjects, TB patients (TB) and HIV-1/Mtb co-infected patients (HIV/TB). (B) Vertical scatter plots showing the amount of sCD163, the cleaved form of CD163 in the serum of healthy subjects, TB, HIV and HIV/TB patients. (C) Vertical scatter plots showing the MFI of cell-surface marker MerTK on CD14⁺ monocytes from PB of healthy subjects, TB and HIV/TB patients. (D) Vertical scatter plots showing the amount of sMer, the cleaved form of MerTK in the serum of healthy subjects, TB, HIV and HIV/TB patients. Each circle within vertical scatter plots represents a single donor. Mean value is represented as a dark grey line. Statistical analyses: Two-tailed, Mann-Whitney (A-C), unpaired t-test (D). * p≤0.05; ** p≤0.005; *** p≤0.0005; ns, not significant.

Figure 4. (Related to Figure S5). The TB-driven exacerbation of HIV-1 infection in macrophages is not due to modulation in viral entry, HIV-1related activation or restriction factors, or autophagy.

(A) Vertical scatter plots showing the Median Fluorescence Intensity (MFI) of cell-surface receptors involved in HIV-1 entry (CD4, CCR5, CXCR4) on monocytes differentiated for 3 days under the presence of CmCTR and CmMTB. Each circle within vertical scatter plots represents a single donor. Mean value is represented as a dark grey line. (B) Histogram showing the percentage of HIV-1 fusion with CmCTR- (white) or CmMTB (black)-pre-treated cells, as determined using the Blam-Vpr assay in the presence of entry inhibitor Maraviroc (dashed bars). (C) Left: Representative images of Western Blot analysis illustrating the expression of IFITM1/2/3 and Actin as loading control. Right: Quantification of IFITM1/2/3 expressed as a ratio related to actin of monocytes differentiated for 3 days into macrophages under the presence of CmCTR (white) and CmMTB (black). n = 6 donors. (D) Representative images of Western Blot analysis illustrating the expression of SAMHD1 and its phosphorylated version (pSAMHD1), and Actin as loading control (left). Quantification of SAMHD1 (center) pSAMHD1 (right) expressed as a ratio related to actin of monocytes differentiated for 3 days into macrophages under the presence of CmCTR and CmMTB. n = 11 donors. (E) Representative images of Western Blot analysis illustrating the expression of C/EBP-β (LAP), C/EBP-β (LIP), and Actin as loading control (left). Quantification of C/EBP-β (LAP, center left) and C/EBP-β (LIP, center right) expressed as a ratio related to actin, and LAP expressed as a ratio related to LIP (right), of monocytes differentiated for 3 days into macrophages under the presence of CmCTR and CmMTB. n = 9 donors. LAP is an activator of HIV-1-LTR whereas LIP is a repressor of HIV-1-LTR. (F) Representative images of Western Blot analysis illustrating the expression of CUGBP1 and Actin as loading control (left). Quantification of CUGBP1 expressed as a ratio related to actin (right) of monocytes differentiated for 3 days into macrophages under the presence of CmCTR and CmMTB. n = 9 donors. (G) Quantification of LC3-II expression as a ratio to actin of monocytes differentiated for 3 days under the presence of CmCTR and CmMTB at the indicated time points after 2h treatment with Bafilomycin A1 (BafA1) or DMSO as control, as measured by western blot analysis. Uninfected cells at day 3 of the experiment (left, n = 6 donors), and HIV-infected cells at 1 (day 4, center, n = 4 donors) and 3 (day 6, right, n = 6 donors) days post-infection. Each circle within vertical scatter plots represents a single donor. Mean value is represented as a dark grey line. Data in histograms are represented as mean ± SD. * p≤0.05; ** p≤0.005; *** p≤0.0005; **** p≤0.0001.

Figure 5. (Related to Movie 3). TB enhances HIV-1-induced tunneling nanotube (TNT) formation.

(A)Representative immunofluorescences image of macrophages interconnected through thick (left) and thin (right) TNTs. F-Actin (red), Tubulin (green), DAPI (blue). Scale bar, 20 μm. The arrows point at the thin TNT without microtubules. (B) Stacked bars showing the percentage of cells with thick (grey) and thin (white) TNTs of day 6 uninfected or HIV-1-infected macrophages treated with CmCTR or CmMTB. Data in histograms are represented as mean ± SD. (C) Representative wide field Immunofluorescence (IF) images showing F-actin staining in day 6 HIV-1 infected macrophages, treated with CmCTR or CmMTB. Arrows: thick TNTs; arrowheads: thin TNTs. Scale bar, 20μm. (D) Scanning electron microscopy images showing TNTs of day 6 HIV-1 infected macrophages, treated with CmMTB. Arrows : thick TNTs ; arrowheads : thin TNTs. Scale bar, 20μm. (E) Deconvolution microscopy images showing HIV-1 Gag intra-TNT distribution in day 6 HIV-1 infected macrophages, treated with CmMTB. HIV-1 Gag (red), F-actin (green), DAPI (blue). Scale bar, 20μm. Insets are 3x zooms. (F) Immunofluorescence (IF) images showing viral particles in TNT of day 6 HIV-1-Gag-iGFP infected macrophages previously treated with CmMTB. HIV-1-Gag-GFP (green), F-actin (red) and DAPI (blue). Scale bar, 10μm. Inset is 4x zoom. Statistical analyses: Two-tailed paired t-test (B). * p≤0.05; ** p≤0.005; *** p≤0.0005; **** p≤0.0001; ns, not significant.

Figure 6. (Related to Figure S2, S6). TB-driven tunneling nanotube (TNT) formation and increased of HIV-1 infection are dependent on the IL10/STAT3 axis.

(A)Stacked bars showing the percentage of cells with thick (grey) and thin (white) TNTs of day 6 HIV-1 infected macrophages untreated or treated with recombinant IL-10 (10ng/mL). (B) Vertical scatter plots showing p24 concentration (left) and infection index (right) of day 13 HIV-1 infected macrophages untreated or treated with recombinant IL-10 (10ng/mL). (C) Stacked bars showing the percentage of cells with thick (grey) and thin (white) TNTs of day 6 HIV-1 infected macrophages treated with IL-10-depleted (α-IL-10) CmMTB and mock depletion controls (α-IgG) of CmCTR and CmMTB. (D) Vertical scatter plots showing p24 concentration (left) and infection index (right) of day 13 HIV-1 infected macrophages treated with IL-10-depleted (α-IL-10) CmMTB and mock depletion controls (α-IgG) of CmCTR and CmMTB. (E) Stacked bars showing the percentage of cells with thick (grey) and thin (white) TNTs of day 6 HIV-1 infected macrophages treated with CmCTR, CmMTB or CmMTB in the presence of the STAT3 activation inhibitor, Stattic (1μM). (F) Vertical scatter plots showing p24 release (left) and infection index (right) of day 13 HIV-1 infected macrophages treated with CmCTR, CmMTB or CmMTB in the presence of the STAT3 activation inhibitor, Stattic (1μM). Statistical analyses: Two-tailed, Wilcoxon matched-paired signed rank test (B right, D right, F), paired t-test (A, B left, C, D left, E). * p≤0.05; ** p≤0.005; *** p≤0.0005; ns, not significant.

Figure 7. (Related to Figure S7 and Movies 5 and 6). TB-driven tunneling nanotube (TNT) formation is necessary for increased HIV-1 infection.

(A) Stacked bars showing the percentage of cells with thick (grey) and thin (white) TNTs of day 6 HIV-1 infected macrophages treated with CmCTR, CmMTB or CmMTB in the presence of TNT inhibitor (TNTi, 20μM). (B) Vertical scatter plots showing p24 release (left) and infection index (right) of day 13 HIV-1 infected macrophages treated with CmCTR, CmMTB or CmMTB in the presence of TNTi. (C) Experimental set-up used for the co-culture (also referred to as a transfer assay), transwell and cell-free HIV-1 experiments. IL-10-treated MDM were either infected with HIV-1 NLAD8-VSVG pseudotyped strain at a MOI 5 for 48h (donor cells) or stained with CellTracker (acceptor cells). For the co-culture experiment, donor cells were mixed with acceptor cells at a 1:1 ratio. The transwell assay was designed to separate donor and acceptor cells to investigate the contribution of cell contact to transfer of HIV-1. The experiment was performed as described in the co-culture assay, with the exception that acceptor cells were plated in the well, and then a transwell filter was placed containing the donor cells. Finally, to assess the contribution of cell-free infection in the propagation of HIV-1, the supernatant of donor cells (containing the virions produced during 48h of infection) was incubated with acceptor cells. To assess the contribution of TNTs in HIV-1 transfer, all 3 experiments were performed in the presence or not of TNTi (20μM), and fixed after 24h. The cells were stained for HIV-1 Gag protein and the percentage of CellTracker⁺ cells among Gag⁺ cells was quantified. (D) Deconvolution microscopy image showing HIV-1 Gag transfer into CellTracker+ acceptor cell after 24h co-culture. HIV-1 Gag (red), Cell Tracker (green), F-actin (grey), DAPI (blue). Scale bar, 10μm. (E) Stacked bars showing the percentage of CellTracker⁺ cells among Gag⁺ cells after 24h co-culture with or without TNTi (20μM). Data in histograms are represented as mean ± SD. Each circle within vertical scatter plots represents a single donor. Mean value is represented as a dark grey line. Statistical analyses: Two-tailed, paired t-test (A, E), Wilcoxon matched-paired signed rank test (B). * p≤0.05; *** p≤0.0005; **** p≤0.0001; ns, not significant.