Tax and Semaphorin 4D Released from Lymphocytes Infected with Human Lymphotropic Virus Type 1 and Their Effect on Neurite Growth

Abstract

Human lymphotropic virus type 1 (HTLV-1) is a retrovirus causing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a neurodegenerative central nervous system (CNS) axonopathy. This virus mainly infects CD4(+) T lymphocytes without evidence of neuronal infection. Viral Tax, secreted from infected lymphocytes infiltrated in the CNS, is proposed to alter intracellular pathways related to axonal cytoskeleton dynamics, producing neurological damage. Previous reports showed a higher proteolytic release of soluble Semaphorin 4D (sSEMA-4D) from CD4(+) T cells infected with HTLV-1. Soluble SEMA-4D binds to its receptor Plexin-B1, activating axonal growth collapse pathways in the CNS. In the current study, an increase was found in both SEMA-4D in CD4(+) T cells and sSEMA-4D released to the culture medium of peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients compared to asymptomatic carriers and healthy donors. After a 16-h culture, infected PBMCs showed significantly higher levels of CRMP-2 phosphorylated at Ser(522). The effect was blocked either with anti-Tax or anti-SEMA-4D antibodies. The interaction of Tax and sSEMA-4D was found in secreted medium of PBMCs in patients, which might be associated with a leading role of Tax with the SEMA-4D-Plexin-B1 signaling pathway. In infected PBMCs, the migratory response after transwell assay showed that sSEMA-4D responding cells were CD4(+)Tax(+) T cells with a high CRMP-2 pSer(522) content. In the present study, the participation of Tax-sSEMA-4D in the reduction in neurite growth in PC12 cells produced by MT2 (HTLV-1-infected cell line) culture medium was observed. These results lead to the participation of plexins in the reported effects of infected lymphocytes on neuronal cells.

FIG. 1.

Proposed hypothetical model. Human lymphotropic virus type 1 (HTLV-1)-infected lymphocytes have increased CRMP-2 phosphorylation facilitating their migration and recruitment into the central nervous system (CNS). The higher MT1-MMP expression increases the cleavage of Semaphorin 4D (SEMA-4D) from the membrane releasing its soluble form soluble Semaphorin 4D (sSEMA-4D) that interacts with the secreted viral protein Tax. Tax-sSEMA-4D may bind Plexin-B1, impairing axon growth.

FIG. 2.

Western blot analysis of the expression of Tax, SEMA-4D, and MT1-MMP in peripheral blood mononuclear cells (PBMCs) of an HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patient. (A) Detection in 20 μl of PBMC culture medium and (B) in PBMC lysates. GAPDH is used as loading control. Lane HAM, HAM/TSP, patient sample; lane Control, healthy donor sample (n = 3).

FIG. 3.

Flow cytometry analysis of the CD4⁺SEMA-4D⁺ population in HAM/TSP patients, asymptomatic carriers, and healthy donors and SEMA-4D proteolytically shed from PBMCs. (A) Dot-plot representation of selected lymphocyte population of PBMCs, (B) CD4⁺SEMA-4D⁺ population in an HAM/TSP patient, (C) CD4⁺SEMA-4D⁺ population in a healthy donor, and (D) CD4⁺SEMA-4D⁺ population in an asymptomatic carrier. (E) Data analysis of the percentage of CD4⁺SEMA-4D⁺ cells obtained by flow cytometry shows statistical differences between HAM/TSP patients and both asymptomatic carriers (carriers) and healthy donors (controls). (F) sSEMA-4D analysis by Western blot also shows significant differences between HAM/TSP patients and the other two groups. Statistical significance with *p < 0.05.

FIG. 4.

Tax effect on SEMA-4D shedding and interaction of both proteins in PBMC culture media from HAM/TSP patients. (A) Representative Western blot of sSEMA-4D using PBMC culture medium from an HAM/TSP patient cultured with (upper panel) or without (lower panel) anti-Tax antibody. Lanes 1, 2, 3, 4, 5, and 6 represent 0, 0.5, 1, 1.5, 2, and 2.5 h, respectively, after anti-Tax antibody addition to the culture medium. (B) Analysis of sSEMA-4D data expressed in arbitrary units (AU) of pixel intensity (n = 3). Statistical significance with *p < 0.05; **p < 0.01; ***p < 0.001 when each time is compared with the initial time. (C) Coimmunoprecipitation of sSEMA-4D and Tax from PBMC culture medium from an HAM/TSP patient detected by Western blot (sSEMA-4D in lane 1 and Tax in lane 2).

FIG. 5.

CRMP-2 phosphorylation at Ser⁵²² in infected lymphocytes and effect of culture medium containing Tax and sSEMA-4D on noninfected lymphocytes. (A) CRMP-2 phosphorylation levels in PBMCs from HAM/TSP patients and asymptomatic carriers. Data are expressed as arbitrary units (AU) of pixel intensity of the ratio CRMP-2 pS⁵²²/total CRMP-2 obtained by Western blot. Effect of the inclusion of anti-sSEMA-4D (B) or anti-Tax (C) antibody in the culture medium of PBMCs from an HAM/TSP patient. (D) Representative Western blot of CRMP-2 pS⁵²² (upper panel) and total CRMP-2 (lower panel). PBMC lysates from healthy controls were cultured in standard conditions (lanes 1, 2), including PBMC culture medium from HAM/TSP patients (lanes 4, 5), including Tax immunodepleted culture medium (lanes 6, 8) or SEMA-4D immunodepleted culture medium (lanes 3, 7). (E) Data analysis of the CRMP-2 phosphorylation of PBMC lysates treated as described in (D) showing a significant reduction in both immunodepleted conditions. Statistical significance with *p < 0.05; **p < 0.01; ***p < 0.001.

FIG. 6.

Transmigration analysis of lymphocytes in response to sSEMA-4D. (A) Times of changes in the number of transmigrated cells from the control condition in response to 1.0 ng/ml of sSEMA-4D after 1 and 6 h of the assay. The control does not include sSEMA-4D in the lower chamber. (B) Flow cytometry analysis of the PBMC populations that migrated after 1 and 6 h. (C) Data analysis of CRMP-2 phosphorylation observed in the lower chamber and upper chamber in response to sSEMA-4D. Control condition corresponds to the values found in the upper chamber of the transwell system using PBMCs from a healthy donor (n = 4). Data are represented as arbitrary units (AU) of pixel intensity of the ratio CRMP-2 pS⁵²²/total CRMP-2 obtained by Western blot. Statistical significance with **p < 0.01; ***p < 0.001.

FIG. 7.

Detection of Tax, SEMA-4D, and MT1-MMP in MT2 cells and in their culture medium. (A) Detection in 20 μl of infected MT2 cell culture medium and (B) in 50 μg MT2 cell lysate. GAPDH was used as loading control.

FIG. 8.

Tax effect on neurite length extension in PC12 cells. (A) Representative microphotographs of PC12 cells after 3 days of differentiation incubated with MT2 culture medium, K562 culture medium, MT2 medium with anti-Tax antibody, MT2 medium with anti-SEMA-4D antibody, and MT2 medium with an irrelevant antibody as control. (B) Data analysis of the neurite length expressed as mean values of μm ± SEM, showing a significantly lower values of neurite length in the presence of Tax protein and sSEMA-4D. Significance with respect to MT2 condition with *p < 0.05.