論文の公開元へMechanisms of HIV-induced peripheral neuropathic pain by focusing on Schwann cell-macrophage interaction
概要
Distal sensory neuropathy (DSN) with symptoms including spontaneous pain, hyperalgesia, allodynia and numbness occurs in 50% of patients infected with human immunodeficiency virus (HIV). HIV strains are divided into macrophage-tropic R5 strain and T-cell line tropic X4 strain. Accumulating evidence suggest that X4 strain is primarily involved in HIV DSN pathogenesis via the CD4-CXCR4 chemokine receptor complex. However, it remains controversial whether HIV can infect neurons that do not express CD4. Recently, it is reported that macrophage accumulation around peripheral nerves is observed in patients with HIV DSN, and the HIV envelope protein glycoprotein 120 (HIV gp120) is involved in the development of pain. In this study, I examined the roles of macrophage-mediated neuroinflammation in the HIV DSN pathogenesis by using X4 gp120-induced neuropathic pain mouse model (Chapter I), and determined the regulators of X4 gp120-induced macrophage recruitment to peripheral nerves by focusing on interactions between macrophages and Schwann cells (Chapter II).
Chapter I. Roles of macrophages in pain-like behaviors in X4 gp120-induced HIV DSN mouse model
For the mouse model of HIV DSN, the right sciatic nerve (ipsilateral) of C57BL/6J mice was exposed topically to X4 strain gp120 IIIB (13 nM) or gp120 MN (13 nM) by loosely wrapping oxidized cellulose gauze. Perineural application of gp120 IIIB or MN induced mechanical hypersensitivity in the ipsilateral hindpaw 7–28 days after the application. Likewise, perineural application of gp120 IIIB elicited spontaneous pain-like behaviors (flinching, scratching, biting and licking) against the ipsilateral hindpaw 7 days after the application. Flow cytometry and immunohistochemical studies revealed increased infiltration of bone marrow-derived macrophages into the parenchyma of the ipsilateral sciatic nerves and dorsal root ganglia (DRG) 7 days after gp120 IIIB or MN application. Neither pain-like behaviors nor macrophage infiltration was observed in the contralateral hindpaw.
Next, the effects of clodronate liposomes, a well-characterized macrophage depleting compound, on gp120 IIIB-induced pain-like behaviors were investigated. Flow cytometry and immunohistochemical studies confirmed that clodronate treatment markedly decreased macrophages on the ipsilateral sciatic nerve and DRG in gp120 IIIB-treated mice. Under these conditions, chemical deletion of circulating macrophages by clodronate treatment significantly inhibited gp120 IIIB-induced mechanical hypersensitivity and spontaneous pain-like behaviors.
These findings suggest that recombinant X4 HIV-1 gp120 delivered directly to the mouse sciatic nerve induced both mechanical hypersensitivity and spontaneous pain-like behaviors, and infiltration of macrophages into the peripheral nerves is associated with the development of pain-like behaviors.
Chapter II. Roles of Schwann cell-derived CXCL1 in X4 HIV gp120-induced macrophage infiltration and pain-like behaviors
We investigated the mechanisms underlying X4 HIV gp120-induced macrophage infiltration into peripheral neurons using primary cultured cells and HIV DSN mouse model. A cell migration assay using the macrophage cell line RAW 264.7 showed no chemotactic response in culture medium containing gp120 IIIB or gp120 MN (5 nM), and toward conditioned medium collected from rat primary cultured DRG neurons treated with gp120 IIIB or MN (5 nM) for 3 days. By contrast, RAW 264.7 cells demonstrated significant chemotactic responses toward conditioned medium from rat primary cultured Schwann cells treated with either gp120 IIIB or MN, although gp120 IIIB or MN did not affect Schwann cell viability, morphology, or differentiation state, despite expression of CXCR4. These results suggest that Schwann cell-derived soluble factors may recruit macrophages in response to X4 gp120 exposure. Thus, alterations in gene expression for cytokines and chemokines were analyzed by DNA array analysis. Among the 89 genes examined, six genes were upregulated in gp120 IIIB-treated Schwann cells, and then verified with a real-time PCR assay. Among them, significant mRNA upregulation of CXCL1, a chemoattractant of macrophages and neutrophils, was confirmed. Furthermore, the chemotactic response of RAW 264.7 cells toward culture medium containing CXCL1 (50 nM) was confirmed. Similar to gp120 IIIB or MN, perineural application of recombinant CXCL1 elicited both mechanical hypersensitivity and spontaneous pain-like behaviors accompanied by macrophage infiltration to the peripheral nerves. Furthermore, the repeated injection of CXCR2 (receptor for CXCL1) antagonist SB220052 (4 mg/kg) or CXCL1 neutralizing antibody prevented both pain-like behaviors and macrophage infiltration in gp120 IIIB-treated mice.
These results suggest that Schwann cell-derived CXCL1, secreted in response to X4 gp120 exposure, is responsible for macrophage infiltration into peripheral nerves, and is thereby associated with pain-like behaviors in mice.
Taken together, the present study suggests that the HIV envelope protein X4 gp120 elicits a neuroinflammatory response by promoting CXCL1-mediated cell-cell interactions between Schwann cells and macrophages. These macrophages ultimately converged on neurons to induce mechanical hypersensitivity and spontaneous pain-like behaviors. These new findings will lead to the development of novel therapeutic drugs for HIV DSN.
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