High Turnover of Tissue Macrophages Contributes to Tuberculosis Reactivation in Simian Immunodeficiency Virus-Infected Rhesus Macaques

Abstract

Background: Tuberculosis (TB) and human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) profoundly affect the immune system and synergistically accelerate disease progression. It is believed that CD4+ T-cell depletion by HIV is the major cause of immunodeficiency and reactivation of latent TB. Previous studies demonstrated that blood monocyte turnover concurrent with tissue macrophage death from virus infection better predicted AIDS onset than CD4+ T-cell depletion in macaques infected with simian immunodeficiency virus (SIV).

Methods: In this study, we describe the contribution of macrophages to the pathogenesis of Mycobacterium tuberculosis (Mtb)/SIV coinfection in a rhesus macaque model using in vivo BrdU labeling, immunostaining, flow cytometry, and confocal microscopy.

Results: We found that increased monocyte and macrophage turnover and levels of SIV-infected lung macrophages correlated with TB reactivation. All Mtb/SIV-coinfected monkeys exhibited declines in CD4+ T cells regardless of reactivation or latency outcomes, negating lower CD4+ T-cell levels as a primary cause of Mtb reactivation.

Conclusions: Results suggest that SIV-related damage to macrophages contributes to Mtb reactivation during coinfection. This also supports strategies to target lung macrophages for the treatment of TB.

Figure 1.

Increasing levels of serum C-reactive protein (CRP) correlated with the Mycobacterium tuberculosis (Mtb) burden in lung tissues of infected macaques. Spearman’s correlation analysis was performed to relate the Mtb bacterial load of the lung (CFU/gram) and the level of serum CRP (mg/dL) of the Mtb singly-infected macaques with different clinical manifestations (n = 23).

Figure 2.

Latent Mycobacterium tuberculosis (Mtb) infection is reactivated by coinfection with simian immunodeficiency virus (SIV). (A) The majority of macaques (7 of 9) administered low-dose aerosol Mtb remained latent and exhibited extremely low levels of serum C-reactive protein (CRP) (n = 7). (B) Macaques with signs of active tuberculosis (TB) had high levels of serum CRP (n = 15). (C and D) Nonhuman primate (NHPs) latently infected with Mtb as in group A with no clinical signs of TB were infected with SIV (arrow). Despite SIV infection, 4 Mtb/SIV-coinfected NHPs in group C did not show signs of TB reactivation in contrast to animals in group D (n = 10) that required euthanasia after presenting with active TB.

Figure 3.

Increased turnover levels of blood monocytes in the Mycobacterium tuberculosis (Mtb)/simian immunodeficiency virus (SIV)-coinfected rhesus macaques correlate with reactivation of latent tuberculosis (TB). (A) Analyses were performed to compare peak plasma viral loads and set point viral loads among 3 groups of SIV-infected rhesus macaques (SIV infection alone, n = 8; latent Mtb/SIV infection, n = 4; and reactivated Mtb/SIV coinfection, n = 6) during the acute stage viral peak (left) and after viral set point (right). (B) Depletion of CD4⁺ T cells in the lung was evaluated using CD4/CD8 ratios (Mtb infection alone, n = 17; latent Mtb/SIV coinfection, n = 4; and reactivated Mtb/SIV coinfection, n = 6). (C) The absolute number of CD4⁺ T cells in blood was calculated based on complete blood count and flow cytometry data (latent Mtb/SIV coinfection, n = 4; and reactivated Mtb/SIV coinfection, n = 6). Arrows depict time of SIV infection. (D) Monocyte turnover was measured by detecting 5-bromo-2’-deoxyuridine (BrdU)-labeled monocytes (blood) 24 hours after in vivo BrdU injection. Abbreviations: AIDS, acquired immune deficiency syndrome; n.s., not significant; st dev, standard deviation.

Figure 4.

Increased turnover of lung macrophages in the Mycobacterium tuberculosis (Mtb)/simian immunodeficiency virus (SIV)-coinfected rhesus macaques correlates with reactivation of latent tuberculosis (TB). (A–F) Confocal microscopy was used to analyze lung tissue sections stained with anti-CD206 (mature macrophages; green), anti-5-bromo-2’-deoxyuridine (BrdU) (turnover; red), and anti-CD163 (macrophages; blue), in Mtb/SIV-coinfected macaques with signs of TB reactivation (A and D, n = 2), Mtb/SIV-coinfected animals that remained latent (B and E, n = 2), as well as from the lungs of macaques infected only with Mtb that remained latent (C and F, n = 2). Images were captured from the capsid region of granuloma with a Leica TCS SP2 confocal microscope equipped with 3 lasers (Leica Microsystems) at ×200 (A, B, C) or ×630 (D, E, F) magnification. Orange arrows indicate recently immigrated macrophages ([24]), and yellow arrows indicate macrophages without BrdU label.

Figure 5.

Increased turnover of intestinal macrophages in Mycobacterium tuberculosis (Mtb)/simian immunodeficiency virus (SIV)-coinfected rhesus macaques also correlates with reactivation of latent tuberculosis (TB). Paraffin-embedded colon tissues collected during necropsy were stained as described in Figure 4 with anti-CD206 (mature macrophages; green), anti-5-bromo-2’-deoxyuridine (BrdU) (turnover; red), and anti-CD163 (macrophages; blue) antibodies and examined by confocal imaging. Images of intestinal tissues are shown from Mtb/SIV-coinfected macaques with signs of TB reactivation (A and D, n = 2), Mtb/SIV-coinfected animals that remained latent (B and E, n = 2), and from macaques infected only with Mtb that remained latent (C and F, n = 2). Images were captured at ×200 (A, B, C) or ×630 (D, E, F) magnification. Orange arrows indicate recently immigrated macrophages, and yellow arrows indicate macrophages without BrdU label.

Figure 6.

Increased simian immunodeficiency virus (SIV) levels in lung tissue of rhesus macaques with Mycobacterium tuberculosis (Mtb) (reactivated)/SIV. (A) Confocal microscopy was used to analyze stained lung tissue sections from Mtb/SIV-coinfected macaques with antibodies specific to Mtb (green), SIV RNA (red), macrophage marker CD68 (blue), and BoPro1 for cellular nuclei (gray) to identify Mtb/SIV-coinfected lung macrophages (n = 3). (B) The number of SIV deoxyribonucleic acid (DNA) copy equivalents in lung tissue was quantified with TaqMan real-time polymerase chain reaction. Student’s t test was applied for comparing the SIV DNA copy number in the lung tissue from macaques with active TB/SIV (n = 5), from macaques with latent TB/SIV infection (n = 3), and from SIV-infected macaques exhibiting low monocyte turnover rate (≤30%, n = 4) and high monocyte turnover rate (>30%, n = 5). P < .05 was considered significant (*).

Figure 7.

Proposed mechanism of tuberculosis (TB) reactivation of SIV-infected macaques. Mycobacterium tuberculosis (Mtb) organisms are known to be contained by the formation of granulomas that mainly comprise macrophages and T cells including CD4⁺ T cells (latent TB). Soon after simian immunodeficiency virus (SIV) infection, CD4⁺ T cells declined regardless of the TB latency or reactivation status. In this model, massive infection of macrophages by SIV and a high rate of cell death correlating with increased blood monocyte turnover were associated with TB reactivation in the Mtb/SIV-coinfected macaques. Macrophage destruction from SIV infection thus appears to be critical for TB reactivation more than CD4⁺ T-cell decline in human immunodeficiency virus-infected individuals with latent TB.