Increased monocyte turnover is associated with interstitial macrophage accumulation and pulmonary tissue damage in SIV-infected rhesus macaques

Abstract

We recently reported that increasing blood monocyte turnover that was associated with tissue macrophage death better predicts terminal disease progression in adult SIV-infected macaques than does declining CD4(+) T cell levels. To understand better mechanisms of pathogenesis, this study relates severity of lung-tissue damage to the ratio, distribution, and inflammatory responses of lung macrophage subsets during SIV infection in rhesus macaques exhibiting varying rates of monocyte turnover. In vivo BrdU incorporation was used to evaluate kinetics of monocyte/tissue macrophage turnover. Tissue damage was scored microscopically from H&E-stained lung-tissue sections, and cytokine expression was examined via immunohistochemistry and confocal microscopy. Increased monocyte turnover in SIV-infected rhesus macaques significantly correlated with severity of lung-tissue damage, as exhibited by perivasculitis, vasculitis, interstitial pneumonia, alveolar histiocytosis, foamy macrophages, multinucleated giant cells, fibrin, and edema in the alveoli. In addition, the higher monocyte turnover correlated with declining AI ratio, increased accumulation of IM in the perivascular region of the lung, and higher expression of IL-6 in the IM of the lung tissue exposed to a LPS, calcium ionophore, and tumor promoter combination stimulation ex vivo. Accumulation of IM associated with increasing monocyte turnover during SIV infection appears to contribute to chronic pulmonary inflammation and tissue damage during disease progression to AIDS.

Keywords: HIV/AIDS; lung; pathogenesis.

Figure 1.. Severity of pulmonary lesions correlates with increasing monocyte turnover in SIV-infected monkeys progressing to AIDS.

Pulmonary lesions in SIV-infected monkeys were evaluated histologically by use of a scoring system modified from Baskin et al. [10] as follows: 0 (normal), 1 (minimal), 2 (mild), 3 (moderate), and 4 (severe). Spearman correlation analysis was used to relate severity of pulmonary lesions with blood monocyte turnover rate. The 9 categories of pulmonary lesions described by Baskin et al. [10] were evaluated, and numbers of animals affected are shown in Supplemental Table 1B. The types of pulmonary lesions that correlated significantly with increasing blood monocyte turnover during SIV infection in rhesus macaques included; pulmonary perivasculitis (A; r = 0.7029, P = 0.0016), vasculitis (B; r = 0.6014, P = 0.0107), interstitial pneumonia (C; r = 0.6842, P = 0.0025), syncytial cell formation (D; r = 0.4971, P = 0.0423), AM accumulation (E; r = 0.6484, P = 0.0049), fibrin and edema (F; r = 0.5666, P = 0.0177). (G–J) Normal lung (G), moderate perivasculitis (H; score = 3), moderate vasculitis (I; score = 3), moderate interstitial pneumonia (J; score = 3), mild multinucleate giant cell pneumonia and alveolar histiocytosis (J inset; score = 2). BR, Bronchioles; BV, blood vessel; AL, alveoli; PV, perivascular interstitium; II, hyperplastic type II pneumocytes; >, AMs; *, alveolar septum; open triangle, multinucleated giant cells; closed triangle, mononuclear cells. Original magnification, 100×; J, inset, 400×.

Figure 2.. The AI ratio in the lung inversely correlates with monocyte turnover in SIV-infected monkeys.

Student’s t-test was used to compare lung tissues from uninfected (n = 5) and SIV-infected (n = 19) rhesus macaques (A) and demonstrated a significant decreased AI ratio in SIV-infected animals. Blood monocyte turnover rates were measured by detecting BrdU or EdU incorporation and plotted against the AI ratios in lung tissues from the corresponding SIV-infected monkeys. A signification correlation was measured by Spearman analysis (B; n = 19) between increasing monocyte turnover and declining AI ratio.

Figure 3.. Accumulation of IM in the lung interstitium correlates with monocyte turnover in SIV-infected monkeys.

Lung-tissue sections from 2 uninfected rhesus macaques and 7 SIV-infected monkeys were stained for the macrophage scavenger receptor CD163 (green), endothelium caveolin-1 (red), and nuclear ToPro-3 (blue) and examined by confocal microscopy at 400× original magnification. The blood monocyte turnover rates of the animals from which the lung tissues were examined were 1.61% (A), 22.7% (B), 31% (C), 40.5% (D), and 61.5% (E). Note the increasing numbers of CD163⁺ IM in the peribronchovascular region of lung tissue from monkeys exhibiting increasing blood monocyte turnover rates. A Leica TCS SP2 confocal microscope equipped with 3 lasers (Leica Microsystems) was used to capture the images.

Figure 4.. IL-6 expression in IM following ex vivo stimulation of lung tissue correlates with monocyte turnover rates in SIV-infected and -uninfected monkeys.

Lung-tissue sections were incubated with a cell-stimulation mixture containing LPS, PHA, PMA, and calcium ionophore for 4 h. Tissues were stained for IL-6 (green), macrophage CD163 marker (red), and nuclear ToPro-3 (blue), and images were captured with a Leica TCS SP2 confocal microscope (Leica Microsystems) at 1260× original magnification. Representative images are shown from an uninfected monkey (A and D; n = 5) and from SIV-infected monkeys with lower (≤30%) blood monocyte turnover (B and E; n = 4) and higher (>30%) blood monocyte turnover (C and F), demonstrating relatively higher IL-6 production in IM of SIV-infected monkeys. Student’s t-test was used to compare mean intensity levels of IL-6-staining cells in the ex vivo-stimulated tissues via Image-Pro Plus (Version 6.0; Media Cybernetics), and significantly higher IL-6 expression was observed from tissues of the SIV-infected macaques (G). Furthermore, there was a significant correlation between IL-6 expression and monocyte turnover rates in corresponding to lung tissues of uninfected (closed stars) and SIV-infected (closed circles) macaques by Spearman analysis (H).