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. 2023 Apr;193(4):380-391.
doi: 10.1016/j.ajpath.2022.12.016.

Inflammation-Associated Lung Tissue Remodeling and Fibrosis in Morphine-Dependent SIV-Infected Macaques

Divya T Chemparathy  1 , Susmita Sil  1 , Shannon Callen  1 , Hitendra S Chand  2 , Mohan Sopori  3 , Todd A Wyatt  4 , Arpan Acharya  1 , Siddappa N Byrareddy  1 , Howard S Fox  5 , Shilpa Buch  6
Affiliations

Inflammation-Associated Lung Tissue Remodeling and Fibrosis in Morphine-Dependent SIV-Infected Macaques

Divya T Chemparathy et al. Am J Pathol. 2023 Apr.

Abstract

With the advent of antiretroviral therapy, improved survival of people with HIV (PWH) is accompanied with increased prevalence of HIV-associated comorbidities. Chronic lung anomalies are recognized as one of the most devastating sequelae in PWH. The limited available data describing the lung complications in PWH with a history of opioid abuse warrants more research to better define the course of disease pathogenesis. The current study was conducted to investigate the progression of lung tissue remodeling in a morphine (Mor)-exposed rhesus macaque model of SIV infection. Pathologic features of lung remodeling, including histopathologic changes, oxidative stress, inflammation, and proliferation of fibroblasts, were investigated in archival lung tissues of SIVmac-251/macaque model with or without Mor dependence. Lungs of Mor-exposed, SIV-infected macaques exhibited significant fibrotic changes and collagen deposition in the alveolar and the bronchiolar region. There was increased oxidative stress, profibrotic transforming growth factor-β, fibroblast proliferation and trans-differentiation, epithelial-mesenchymal transition, and matrix degradation in SIV-infected macaques, which was further exacerbated in the lungs of Mor-exposed macaques. Interestingly, there was decreased inflammation-associated remodeling in Mor-dependent SIV-infected macaques compared with SIV-infected macaques that did not receive Mor. Thus, the current findings suggest that SIV independently induces fibrotic changes in macaque lungs, which is further aggravated by Mor.

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Figures

Figure 1
Figure 1
Pathologic analysis of the lung tissue. A: Hematoxylin and eosin (H&E) staining to examine lung pathology in saline, SIV, morphine (Mor), and SIV + Mor groups of macaques. Top panels: H&E-stained sections. Bottom panels: H&E-stained sections. B: Histopathologic scores of the different treatment groups. Immune cell infiltration was scored in macaque lungs stained with H&E. One-way analysis of variance, followed by Tukey multiple comparison test, was used to determine the statistical significance. C: Masson trichrome staining in lung tissues of experimental macaques. Top panels: Masson trichrome–stained sections. Bottom panels: Masson trichrome–stained sections. D: Masson trichrome staining in the proximal bronchioles (top panels) and distal bronchioles (bottom panels) of the experimental macaque lung tissues. Values represent the means ± SEM (B). ∗P < 0.05 versus saline; †P < 0.05 versus Mor; ‡P < 0.05 versus SIV + Mor. Scale bars: 1000 μm (A and C, top panels, and D); 100 μm (A and C, bottom panels). Original magnification: ×4 (A and C, top panels, and D); ×20 (A and C, bottom panels).
Figure 2
Figure 2
Increased oxidative stress in lungs of morphine (Mor)–exposed, SIV-infected macaques. A: Representative immunohistochemistry photomicrographs showing expression of 4-hydroxynonenal (4HNE) in lung tissues of morphine-exposed, SIV-infected macaques. B: Quantitative analysis of mean fluorescence intensity was performed in minimum 10 fields from four macaques. C: Western blot analysis showing expression of 4HNE in lung tissues of Mor-exposed, SIV-infected macaques. D: Quantitative analysis of relative fold change of 4HNE expression. E: Western blot analysis showing expression of superoxide dismutase 2 (MnSOD2), catalase, and glutathione peroxidase 1 (GPX1) in lung tissues of morphine-exposed, SIV-infected macaques. F: Quantitative analysis of relative fold change of protein expression. One-way analysis of variance, followed by Tukey multiple comparison test, was used to determine the statistical significance (B, D, and F). Values represent the means ± SEM (B, D, and F). ∗P < 0.05 versus saline (Sal); †P < 0.05 versus SIV; ‡P < 0.05 versus Mor. Scale bars = 1000 μm (A). Original magnification, ×4 (A).
Figure 3
Figure 3
Increased inflammation and matrix degradation in lungs of morphine (Mor)–exposed, SIV-infected macaques. A and B: Enzyme-linked immunosorbent assay showing expression of tumor necrosis factor (TNF)-α and IL-1β in lung tissues of Mor-exposed, SIV-infected macaques. C: Western blot analysis showing expression of matrix metalloproteinase (MMP)-2 and MMP-9 in lung tissues of morphine-exposed, SIV-infected macaques. D and E: Quantitative analysis of relative fold change of MMP-2 and MMP-9 expression. F: Gelatin zymography analysis showing the expression of MMP-2 and MMP-9 in lung tissues of morphine-exposed, SIV-infected macaques. G: Western blot analysis showing expression of MMP-7 in lung tissues of morphine-exposed, SIV-infected macaques. H: Quantitative analysis of relative fold change of MMP-7 expression. One-way analysis of variance, followed by Tukey multiple comparison test, was used to determine the statistical significance (A, B, D, E, and H). Values represent the means ± SEM (A, B, D, E, and H). ∗P < 0.05 versus saline (Sal); †P < 0.05 versus Mor; ‡P < 0.05 versus SIV + Mor.
Figure 4
Figure 4
Increased expression of transforming growth factor (TGF)-β1 in lungs of morphine (Mor)–exposed, SIV-infected macaques. A: Western blot showing expression of TGF-β1 in lung tissues of Mor-exposed, SIV-infected macaques. B: Quantitative analysis of relative fold change of TGF-β1 expression. C: Representative immunohistochemistry photomicrographs showing expression of TGF-β1 and CD64 in lung tissues of Mor-exposed, SIV-infected macaques. D and E: Quantitative analysis of mean fluorescence intensity was performed in minimum 10 fields from four macaques. One-way analysis of variance, followed by Tukey multiple comparison test, was used to determine the statistical significance (B, D, and E). Values represent the means ± SEM (B, D, and E). ∗P < 0.05 versus saline (Sal); †P < 0.05 versus SIV; ‡P < 0.05 versus Mor. Scale bars = 100 μm (C). Original magnification, ×20 (C).
Figure 5
Figure 5
Increased fibroblast proliferation and myofibroblast differentiation in morphine (Mor)–exposed, SIV-infected rhesus macaques. A and C: Representative immunohistochemistry photomicrographs showing expression of Ki-67 (A) and α-smooth muscle actin (α-SMA; C) in lung tissues of morphine-exposed, SIV-infected macaques. B and D: Quantitative analysis of mean fluorescence intensity was performed in minimum 10 fields from four macaques. E: Western blot showing expression of α-SMA in lung tissues of Mor-exposed, SIV-infected macaques. F: Quantitative analysis of relative fold change of α-SMA expression. G: Representative immunohistochemistry photomicrographs showing the colocalization of Ki-67 and α-SMA in lung tissues of macaques from SIV + Mor group. H: Representative immunohistochemistry photomicrographs showing the colocalization of vimentin, Ki-67, and α-SMA in lung tissues of macaques from SIV + Mor group. One-way analysis of variance, followed by Tukey multiple comparison test, was used to determine the statistical significance (B, D, and F). Values represent the means ± SEM (B, D, and F). ∗P < 0.05 versus saline (Sal); †P < 0.05 versus SIV; ‡P < 0.05 versus Mor. Scale bars = 100 μm (A, C, G, and H). Original magnification, ×20 (A, C, G, and H).
Figure 6
Figure 6
Increased epithelial-mesenchymal transition in the lungs of morphine (Mor)–exposed, SIV-infected rhesus macaque. A: Western blot analysis showing expression of E-cadherin, N-cadherin, and vimentin in lung tissues of morphine-exposed, SIV-infected macaques. B–D: Quantitative analysis of relative fold change of protein expression. E: Representative immunohistochemistry photomicrographs showing expression of claudin 5 in lung tissues of morphine-exposed, SIV-infected macaques. F: Quantitative analysis of mean fluorescence intensity was performed in minimum 10 fields from four macaques. One-way analysis of variance, followed by Tukey multiple comparison test, was used to determine the statistical significance (B–D and F). Values represent the means ± SEM (B–D and F). ∗P < 0.05 versus saline (Sal); †P < 0.05 versus SIV; ‡P < 0.05 versus Mor. Scale bars = 100 μm (E). Original magnification, ×20 (E).
Figure 7
Figure 7
Schematic representation of the pathologic role of SIV and morphine in macaque lungs. SIV was shown to induce oxidative stress and inflammation with up-regulated expression of matrix-degrading enzymes matrix metalloproteinase (MMP)-2/MMP-9/MMP-7 in macaque lungs. These pathologic events may result in increased production of transforming growth factor (TGF)-β1 by alveolar macrophages, which further facilitate the induction of epithelial-mesenchymal transition (EMT) in lungs. Increased fibroblast proliferation and trans-differentiation of fibroblasts into myofibroblasts may result in the massive accumulation of interstitial collagen and fibrosis in experimental macaque lungs. Morphine was found to potentiate SIV-mediated pathologic changes in the lungs. ECM, extracellular matrix; GPX1, glutathione peroxidase 1; 4HNE, 4-hydroxynonenal; α-SMA, α-smooth muscle actin; SOD, superoxide dismutase; TNF-α, tumor necrosis factor-α.
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