Bystander Effects and Profibrotic Interactions in Hepatic Stellate Cells during HIV and HCV Coinfection

Figure 2

Evaluation of the susceptibility and permissiveness of LX-2 cells to HIV infection by flow cytometry. (a) Representative flow cytometry dot plots showing the relative percentages of LX-2 infection after 72 hr. On the left, LX-2 cells exposed to HIV-X4-GFP showed undetectable GFP expression depicting them as not susceptible or permissive to HIV infection after cell-free virus challenge. On the right, the dot plot shows that LX-2 cells are permissive to pseudotyped HIV replication, after exposure to free HIV-VSV-GFP. (b) Bar graph representing the percentage of infection obtained (0.0744 ± 0.05) and (10.85% ± 1.14%), respectively. (c) Viral infection was evaluated by measuring p24 antigen in culture supernatants from LX-2 cells exposed to free HIV-X4 and HIV-VSV. After 72 hr (1.63 ± 011 ng/mL and 21.67 ± 3.61, respectively). (d) Bar graph exhibiting poor coexpression of CD4 receptor, CXCR4, and CCR5 coreceptors measured on LX-2: 0.47% ± 0.13%, and CD4/CXCR4: 0.33% ± 0.15%, respectively. These determinations were performed by flow cytometry. (e) Representative flow cytometry dot plots showing the efficiency of Jurkat cell infection at 72 hr according to the method used: HIV-X4-GFP-free virus infection (low), HIV-X4-GFP spinoculation infection (high), and HIV-VSV-GFP-free virus infection. (f) Bar graphs depicting the corresponding infection percentages for “low” (6.1% ± 1.7%), “high” (29.5% ± 6.4%), and HIV-VSV-GFP (85.1% ± 7.1%) respectively. (g) Programed cell death levels (PCD) (positive staining for annexin V and/or 7-AAD) in Jurkat control nonexposed (Ctrl), Jurkat (HIV) (low), and Jurkat HIV (high). The PCD was significantly higher in Jurkat (HIV) (high) relating to Jurkat (HIV) (low) . (h) Cocultivation between VPD (−) LX-2 as target cells and VPD (+)-labeled HIV-exposed Jurkat (inoculum: 1 pg of p24/cell) as donor cells. Representative flow cytometry dot plots showing the LX-2 VPD (−) and Jurkat VPD (+) after 72 hr post-cocultivation. In addition, the dot plots depicting the HIV infection level for both populations reveal that LX-2 cells remain uninfected at 72 hr post-cocultivation, regardless of the infection efficiency of the cocultivated Jurkat cells. (i) Percentage of cells expressing HIV—GFP for LX-2 VPD (−) HIV low: 0.047% ± 0.015%; LX-2 VPD (−) HIV high: 0.238% ± 0.31%; Jurkat VPD (+) HIV low: 10.2% ± 1%; and Jurkat VPD (+) HIV high: 21% ± 2.5% and HIV replication in Jurkat cells (measured as p24 antigen in supernatants). Low-grade: 4.68 ± 1.51 ng/mL; high-grade: 632.83 ± 34 ng/mL. (j) Programed cell death levels (PCD) (positive staining for annexin V and/or 7-AAD) in Jurkat control nonexposed (Ctrl), Jurkat (HIV) (low), and Jurkat HIV (high) after 72 hr of coculture. The PCD was significantly higher in Jurkat (HIV) (high) relating to noninfected Jurkat (Ctrl) . (k) Bar graph showing an increase in the IL-2, CD25, and TGF-β expression in HIV-infected Jurkat cells vs. uninfected Jurkat (14.27 ± 4.47, 2.71 ± 0.96, and 5.76 ± 1.16, respectively), measured by RT-qPCR at 72 hr postinfection. (l) Bar graphs showing flow cytometry measurement of the CD4, CXCR4, and CXCR5 percentage expression on LX-2 at 72 hr post-cocultivation or conditioned medium exposition. CXCR4 levels in Jurkat (HIV): LX-2 cocultivation vs. Jurkat (NI): LX-2 cocultivation ; CCR5 levels in Jurkat (HIV): LX-2 cocultivation vs. Jurkat (NI): LX-2 cocultivation ; CD4 levels remained unchanged for any treatment. NS, not significant. Graphics are showing values obtained from three independent experiments. Data are given as the mean ± SD, , , , and .

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)