Figure 1.
Effect of HIV p17 on stellate cell activation.
(A–B) Serum starved LX2 cells were stimulated with 0.1, 1 and 10 µg/ml p17 for 18 hours. (A) Immunoblot of syndecan-2 (Syn-2), CXCR2, αSMA, pro-col1α1, endothelin-1 (ET-1) and Tubulin. (B) qRT-PCR of syn-2, CXCR2, αSMA, collagen-I and ET-1. Data represent the mean of 4 experiments. Values are normalized relative to HPRT mRNA and are expressed relative to those of untreated cells, which were arbitrarily set to 1. *P<0.05 versus not treated cells. (C) Serum starved human primary hepatic stellate cells were stimulated with 0.1, 1 and 10 µg/ml p17 for 18 hours. At the end of stimulations relative mRNA expression of syn-2, CXCR2, αSMA, collagen-I and ET-1 was evaluated by Real-Time PCR. Data represent the mean of 4 experiments. Values are normalized relative to HPRT mRNA and are expressed relative to those of untreated cells, which were arbitrarily set to 1. *P<0.05 versus not treated cells.
Figure 2.
Effect of p17 on expression and distribution of cytoskeleton fibers in LX2 cells.
Confocal immunofluorescence of vimentin (A) and α-SMA (B) in LX2 cells left not treated (a) or stimulated with 2 µg/ml p17 for 24 hours (b–c).
Figure 3.
Blocking antibodies against p17 inhibit LX2 activation.
(A–C) Serum starved LX2 cells were pre-incubated 2 h with the serum taken from and healthy donor (HD) or with the serum of an HIV patient taken before (preV) and after (postV) the vaccination with a recombinant p17 peptide, diluted 1∶100 in culture medium, followed by additional treatment with 2 µg/ml p17 for 18 hours. qRT-PCR of Col1α1 (A), αSMA (B) and ET-1. Values are normalized relative to HPRT mRNA and are expressed relative to those of untreated cells, which were arbitrarily set to 1. *P<0.05 versus not treated cells. #P<0.05 versus p17 stimulated cells. (D) Hydrated collagen lattices, photographed 18 hours after stimulation of LX2 with 10 µg/ml p17 alone (a) or in the presence of the serum of an HIV patient taken before (b) and after (c) the vaccination with a recombinant p17 peptide, diluted 1∶100 in culture medium. Contraction of LX2 cells in response to 10% FBS was also evaluated after 6 h incubation. (E) Mean percent gel surface area of collagen gels compared with initial surface area. Data are mean ± SE of 3 experiments. *P<0.05 versus control cells. #P<0.05 versus p17 stimulated cells. (nt: not treated cells).
Figure 4.
p17 activates CXCR2/Rho pathway on LX2 cells.
(A) Serum starved LX2 cells were pre-incubated for 2 h with 100 nM SB265610 followed by additional treatment with 2 µg/ml p17 for 18 hours. qRT-PCR of collagen-I, αSMA and ET-1. Values are normalized relative to HPRT mRNA and are expressed relative to those of untreated cells, which were arbitrarily set to 1. *P<0.05 versus not treated cells. #P<0.05 versus p17 stimulated cells. (B) Serum starved LX2 cells were pre-incubated for 2 h with 4 µg anti-syndecan-2 antibody followed by additional treatment with 2 µg/ml p17 for 18 hours. qRT-PCR of collagen-I, αSMA and ET-1. Values are normalized relative to HPRT mRNA and are expressed relative to those of untreated cells, which were arbitrarily set to 1. *P<0.05 versus not treated cells. #P<0.05 versus p17 stimulated cells. (C) Time course of phosphoMLC and total MLC expression in LX-2 cells treated with 2 µg/ml of p17. (C) LX2 cells were pre-incubated for 2 h with indicated concentrations of Rho kinase inhibitor Y-27632 prior to stimulation with 2 µg/ml p17 for 18 hour. qRT-PCR of collagen-I, αSMA and ET-1. Values are normalized relative to HPRT mRNA and are expressed relative to those of untreated cells, which were arbitrarily set to 1. *P<0.05 versus not treated cells. #P<0.05 versus p17 stimulated cells.
Figure 5.
HIV matrix protein p17 activates JAK/STAT pathway on LX2 cells.
(A) Time course of phosphoSTAT1 and total STAT1 expression in LX-2 cells treated with 2 µg/ml of p17. (B) Time course of phosphoSTAT3 and total STAT3 expression in LX-2 cells treated with 2 µg/ml of p17. (C–E) LX2 cells were pre-incubated for 2 h with the STAT1 inhibitor Fludarabine (5 µM) or with the STAT3 inhibitor 5,15 DPP (5 µM) prior to stimulation with 2 µg/ml p17 for 18 hour. qRT-PCR of collagen-I (C), αSMA (D) and ET-1 (E). Values are normalized relative to HPRT mRNA and are expressed relative to those of untreated cells, which were arbitrarily set to 1. *P<0.05 versus not treated cells. #P<0.05 versus p17 stimulated cells.
Figure 6.
p17 drives the formation of a multiprotein complex containing CXCR-2, syn-2, RACK-1 and JAK-1 on LX2 cells.
LX2 cells were serum starved and stimulated for 5, 15, 30 and 60 minutes with 2 µg/ml of p17. (A) Co-immuneprecipitation of CXCR2 with syndecan-2 and RACK-1. (B) Co-immuneprecipitation of RACK1 with syndecan-2, CXCR2 and JAK1. (C) Confocal immunofluorescence of LX2 cells left untreated (a) or exposed to p17 for 5 minutes and stained with anti-CXCR2 antibody. (D) Immunofluorescence analysis of syndecan-2 and CXCR2 localization on LX2 cells left untreated (NT) or stimulated 5 minutes with p17. Cells were stained with the indicated antibodies. Red: syn-2. Green: CXCR2. The red and green signals were electronically merged for co-localization analysis.
Figure 7.
Progression of liver fibrosis is associated with a down-regulation of CXCR2 receptor.
Liver biopsies from HIV/HCV coinfected and HCV monoinfected patients were serially sectioned and stained with the following antibodies: αSMA, syndecan-2 and CXCR2. (A) Patient n. 3 HCV positive. Diffusely and intensely sinusoidal walls staining with αSMA (Magnification 200x). Patient n.6 HIV/HCV positive. Focally and weakly sinusoidal walls staining with αSMA (Magnification 200x). (B) Patient n. 3 HCV positive and patient n. 6 HIV/HCV positive. In both cases strongly cytoplasmatic staining of stellate cells with syndecan-2 (Magnification 200x) (C) Patient n. 3 HCV positive. Focally and weakly sinusoidal walls staining with CXCR2 (Magnification 200x; inset 400x). Patient n.5 HIV/HCV positive. Absence of staining with CXCR2 (Magnification 200x)
Table 1.
Fibrosis: Comparison of HCV and HIV/HCV Patients.
Table 2.
Primers used for Real-Time PCR.