To effectively evaluate prospective mechanisms

To effectively evaluate prospective mechanisms by which chronic HIV infection induces pulmonary fibrotic changes in humans, we confirmed that predisposition to fibrotic change in older individuals could be mimicked in a mouse model. Hydroxyproline is a nonessential amino Phosphatase Inhibitor Cocktail 3 (100X in DMSO) required for the synthesis of collagen, which in turn, is abundant in pulmonary fibrosis. We chose to quantify hydroxyproline as a surrogate of collagen and predisposition to fibrotic-like change in order to detect any difference in collagen content that may predate true fibrosis and could be potentially missed on histology. Additionally, we chose to measure α-SMA expression in whole lung in order to determine if any difference in hydroxyproline content could be attributed to increased myofibroblast activity. Our results confirm that in the lungs of older HIV-1 TG as compared to WT mice, there is higher collagen content as assessed by a hydroxyproline assay. This finding is likely due to an increase in the myofibroblast pool in the HIV-1 TG lung as evidenced by increased α-SMA expression in HIV-1 TG PLFs. gp120-mediated activation of CXCR4 could potentially explain these in vivo results; however, this is purely speculative as HIV-TG mice express multiple other viral genes, including tat, nef, rev, vif, vpr, and vpu, all of which may contribute. Within this limitation, these results are the first of their kind and provide a potential explanation as to why chronic infection with HIV may lead to pulmonary fibrotic changes in humans. There are a number of limitations to this study. We chose a mouse model rather than human cell line or human PLFs in order to better examine the putative mechanistic pathway by which HIV-1 predisposes to pulmonary fibrotic changes, and to readily discern the relevance of this pathway in vivo; as such, the applicability of our findings in humans is speculative and these experimental findings need to be extended to the clinical setting with the obvious caveat that it is not feasible to isolate primary lung fibroblasts and lung tissue from individuals living with HIV. In addition, although we determined by PCR and Western immunoblot that CXCR4 was expressed in mouse primary lung fibroblasts, cell-surface presence of CXCR4 was not confirmed. We did not feel the need to confirm this because prior studies have demonstrated that CXCR4 is expressed on the surface of both mouse and human lung fibroblasts.8, 28 AMD3100 limits fibroblast-to-myofibroblast differentiation and could potentially limit fibrotic changes attributed to X4-tropic HIV-1; however, it is unlikely to have any effect on R5 or dual-tropic strains of the virus. The significance of this limitation is currently unknown, as prior studies evaluating the risk of pulmonary fibrosis in patients with HIV-1 did not account for viral tropism, and it is not clear what proportion of patients infected with X4-tropic, R5-tropic, and dual-tropic HIV-1 are at risk for fibrosis. Finally, while we demonstrated that direct activation of CXCR4 by gp120 in the lung fibroblast may provide at least one explanation for why individuals infected with HIV are prone to pulmonary fibrotic changes, we have not excluded other potential mechanisms; in particular, it remains unknown if an indirect effect from recurrent pulmonary infections secondary to immunosuppression may also be a risk factor. In conclusion, we determined that gp120-mediated activation of the CXCR4-ERK1/2 signaling pathway, with resultant fibroblast-to-myofibroblast differentiation, is a previously unrecognized mechanism by which HIV-1 might promote pulmonary fibrotic changes in older individuals living with HIV. Further, we identified that this effect can be inhibited by treatment with the CXCR4-specific antagonist AMD3100. Finally, we correlated our in vitro findings with increased markers of profibrotic changes in the lungs of HIV-1 TG mice in which gp120 and other HIV-1-related proteins are chronically expressed in vivo, consistent with the pulmonary fibrotic changes associated with chronic HIV infection in older humans. Although these findings in a relevant preclinical mouse model need to be confirmed in human lung fibroblasts and, eventually, in individuals living with HIV, our experimental findings raise the intriguing possibility that treatment of HIV-infected individuals with a CXCR4-specific antagonist such as AMD3100 may be efficacious in preventing and/or limiting pulmonary fibrosis.