The HIV genome encodes for a variety of viral proteins

The HIV-1 genome encodes for a variety of viral proteins necessary for target cell entry and viral replication. gp120 is an HIV-1 envelope glycoprotein that mediates viral particle entry by binding to cell surface receptor CD4 and co-receptors CXCR4 and/or CCR5 depending on viral tropism. While CXCR4 is a chemokine receptor classically associated with leukocytes, its presence has also been noted on a variety of other cell types, including hepatic stellate herpes simplex virus type 1 and circulating fibrocytes.7, 8, 9 In addition to aiding viral cell entry, gp120 can serve as an agonist of CXCR4 in human CD4+ T-lymphocytes, promoting a variety of potentially profibrotic intracellular signaling pathways, which in the lung fibroblast have been associated with fibroblast-to-myofibroblast transdifferentiation.10, 11, 12 Finally, CXCR4-tropic (X4-tropic) gp120 has been shown to activate hepatic stellate cells with resultant increased expression of α-SMA and collagen type I. However, no study to date has evaluated if gp120 has any effect on the lung fibroblast. While the possibility of CXCR4 blockade has been extensively studied, there are no FDA-approved anti-CXCR4 medications indicated for HIV management; however, AMD3100, one of the early CXCR4 antagonists, is approved in individuals with multiple myeloma and non-Hodgkin lymphoma to aid in mobilization of hematopoietic stem cells to the peripheral blood.14, 15 AMD3100 is a bicyclam that is a specific CXCR4 antagonist and does not affect any other chemokine receptor, nor does it act as a CXCR4 agonist. Its ability to block HIV-1 viral entry via prevention of gp120 binding to CXCR4 prompted a phase I trial with promising results; however, further studies regarding its therapeutic use in HIV have been limited due to inability to block CCR5-tropic (R5) viral entry and limited oral bioavailability.14, 17, 18 In the present study, we hypothesized that HIV-1 directly mediates an increase in pulmonary fibrotic changes, at least in part, through gp120-mediated activation of CXCR4 on the lung fibroblast, with resultant increase in fibroblast-to-myofibroblast transdifferentiation and increased deposition of matrix proteins, such as collagen, over time. Further, we asked whether or not gp120-mediated fibroblast-to-myofibroblast transdifferentiation could be inhibited by treatment with the CXCR4 antagonist AMD3100. To test our hypothesis, we used the well-characterized HIV-1 transgenic mouse model that allowed us to examine the effect of HIV-1 infection in primary lung fibroblasts ex vivo in addition to the in vivo effect of HIV-1 on collagen deposition in lung tissue.
METHODS
RESULTS
DISCUSSION In the present study, mouse PLFs treated with gp120 had increased α-SMA expression. In parallel, direct visualization of gp120-treated PLFs confirmed there was a significantly higher percentage undergoing myofibroblast transdifferentiation. We further showed that gp120 induced activation of ERK1/2 but not PI3K-Akt intracellular signaling pathway. Additionally, the CXCR4 antagonist AMD3100 inhibited gp120-mediated augmentation of α-SMA and gp120-induced ERK1/2 phosphorylation. Finally, HIV-1 TG mice had age-dependent increased hydroxyproline concentration in their lungs and greater α-SMA expression in their PLFs. Taken together, these results suggest that activation of the chemokine receptor CXCR4 by the HIV-1-related protein gp120 mediates lung fibroblast-to-myofibroblast transdifferentiation with resultant increased pulmonary collagen deposition and potentially reveal a novel mechanism by which older individuals living with HIV are susceptible to pulmonary fibrotic changes1, 3; this pathophysiological pathway is shown schematically in Figure 7. Further, this study raises the provocative possibility that antagonizing CXCR4, such as with the FDA-approved AMD3100, could prevent and/or limit the development of fibrotic changes in these vulnerable individuals. We discovered that gp120 directly induces lung fibroblast-to-myofibroblast differentiation by activating CXCR4. This pathway was discerned in vitro by demonstrating that PLFs treated with gp120 had greater α-SMA expression and myofibroblastic phenotype as compared to untreated cells and the effect of gp120 on α-SMA could be inhibited by a CXCR4-specific antagonist. While speculative, a potential in vivo scenario by which this mechanism occurs in individuals infected with HIV is via direct contact of either free of virion-bound gp120 with circulating fibrocytes recruited to injured lung.24, 6 We were drawn to study the association of gp120 and CXCR4 in the lung fibroblast because activation of CXCR4 by stromal cell-derived factor 1 (SDF-1) can induce PI3K-Akt and ERK1/2 signaling pathways, both of which have been independently associated with increased fibroblast-to-myofibroblast transdifferentiation via noncanonical TGFβ1 signaling.12, 25, 26 In our current study, we identified that gp120 activates the ERK1/2 but not the PI3K-Akt signaling pathway. These results are consistent with previous findings which showed that gp120 activates CXCR4 in the human hepatic stellate cell with resultant increase in α-SMA and collagen type I via increase in ERK signaling. In a similar fashion, our results are compatible with prior studies which showed that gp120 is a partial agonist of chemokine receptors CXCR4 and CCR5 and activates intracellular signaling comparable to the receptors’ natural ligands in multiple cell types outside of the lung fibroblast.10, 13, 27 gp120-mediated fibroblast-to-myofibroblast transdifferentiation could be inhibited by pretreatment with the CXCR4 antagonist AMD3100. In addition, inhibition of CXCR4 signaling with AMD3100 also attenuated activation of ERK1/2 by gp120. To confirm there was no dose-dependent response, we performed a dose titration at concentrations of 5 μM and 10 μM; Figure 3 showed no difference between the 2 doses examined in our study. Additionally, we chose to treat cells with AMD3100 at 10 μM because this concentration was previously shown to limit SDF-1-induced stress fiber formation in human lung fibroblasts. Our results not only confirm that the effect of gp120 on the lung fibroblast was due to activation of CXCR4, but this effect could be entirely inhibited by a medication shown to be safe in humans.18, 29 AMD3100 is a bicylcam that was first studied as a potential inhibitor of HIV-1 target cell fusion and entry via CXCR4-selective antagonism, limiting binding of gp120 to the coreceptor.16, 30 In fact, there was a phase 1 trial evaluating AMD3100 as a potential treatment for HIV-1 with a promising side effect profile and intravenous pharmacokinetics; however, further studies of the drug for HIV-1 treatment were not pursued primarily due to its inability to inhibit R5-tropic HIV-1 fusion and poor oral bioavailability.14, 18 Despite limitations to its use, we believe the results of our current study argue for further analysis of AMD3100 treatment in individuals infected with HIV-1 in order to attenuate or prevent HIV-associated pulmonary fibrotic changes.