Intrahepatic CXCL10 is strongly associated with liver fibrosis in HIV-Hepatitis B co-infection

Abstract

In HIV-hepatitis B virus (HBV) co-infection, adverse liver outcomes including liver fibrosis occur at higher frequency than in HBV-mono-infection, even following antiretroviral therapy (ART) that suppresses both HIV and HBV replication. To determine whether liver disease was associated with intrahepatic or circulating markers of inflammation or burden of HIV or HBV, liver biopsies and blood were collected from HIV-HBV co-infected individuals (n = 39) living in Bangkok, Thailand and naïve to ART. Transient elastography (TE) was performed. Intrahepatic and circulating markers of inflammation and microbial translocation were quantified by ELISA and bead arrays and HIV and HBV infection quantified by PCR. Liver fibrosis (measured by both transient elastography and liver biopsy) was statistically significantly associated with intrahepatic mRNA for CXCL10 and CXCR3 using linear and logistic regression analyses adjusted for CD4 T-cell count. There was no evidence of a relationship between liver fibrosis and circulating HBV DNA, qHBsAg, plasma HIV RNA or circulating cell-associated HIV RNA or DNA. Using immunohistochemistry of liver biopsies from this cohort, intrahepatic CXCL10 was detected in hepatocytes associated with inflammatory liver infiltrates in the portal tracts. In an in vitro model, we infected an HBV-infected hepatocyte cell line with HIV, followed by interferon-γ stimulation. HBV-infected cells lines produced significantly more CXCL10 than uninfected cells lines and this significantly increased in the presence of an increasing multiplicity of HIV infection. Conclusion: Enhanced production of CXCL10 following co-infection of hepatocytes with both HIV and HBV may contribute to accelerated liver disease in the setting of HIV-HBV co-infection.

Fig 1. Correlations between HIV, Hepatitis B and inflammatory markers in the liver and blood.

Heatmap showing the significant associations by Spearman correlation between intrahepatic CXCL10, CXCR3 and HIV with liver related outcomes including fibrosis and liver enzymes (left hand panel) and between the related intrahepatic markers CXCL10, CXCR3 and IFN-γ and liver and HIV in the liver (right hand panel). P-value < 0.05 if absolute value of Spearman’s correlation is at least 0.30, < 0.01 if at least 0.44 (n = 39). HIV human immunodeficiency virus, HBV hepatitis B virus, CA cell associated, sAg surface antigen, ALP alkaline phosphatase; GGT γ-glutamyl transferase, ALT Alanine transaminase, AST Aspartate transaminase, HMGB1 high mobility group box-1, CCL-2 C-C motif chemokine 2, sCD14 soluble CD14, LPS lipopolysaccharide, CXCL10 C-X-C motif chemokine 10, CXCR3 C-X-C motif chemokine receptor 3, IFN interferon, MPO myeloperoxidase; cccDNA covalently closed circular DNA, rcDNA relaxed circular DNA; Geq genome equivalent, TE transient elastography.

Fig 2

(A) Liver fibrosis was significantly correlated with intrahepatic HIV DNA and with mRNA for CXCL10 and CXCR3. Plots showing correlations between liver fibrosis by TE (kPa) with intrahepatic HIV DNA, CXCL10, CXCR3 (left side panels) and between liver CXCL10 levels with intrahepatic HIV DNA, CXCR3 and peripheral AST (right side panels). The lower limit of detection for HIV RNA and HIV DNA was one copy per well. If there was no HIV PCR signal, this was recorded as zero and if there was a detectable signal but <1, this was recorded as 0.5 copies. All data points were included in the Spearman correlation. r = Spearman rank correlation coefficient. HIV human immunodeficiency virus, CXCL10 C-X-C motif chemokine 10, CXCR3 C-X-C motif chemokine receptor 3, TE transient elastography, AST Aspartate transaminase. (B) Liver fibrosis (kPa) and AST were higher in participants with detectable HIV DNA in the liver. Each symbol represents values from each participant. The lines represent the median and IQR. Comparisons were made using Wilcoxon Rank Sum test (* p<0.05).

Fig 3. Intrahepatic CXCL10 staining is localised in hepatocytes and increased in HIV-HBV co-infected liver biopsies.

(A) Biopsies from people living with HIV-HBV coinfection and controls who were HIV and HBV negative were examined using immunohistochemistry (IHC) for CXCL10 (red-AP). The percentage area positive for CXCL10 staining (dark pink stain) was quantified using Photoshop CS5 and Fovea tools. Comparisons were made using the Wilcoxon Rank Sum test (* p<0.05). (i) The median and IQR for percentage area positive for CXCL10 is shown as well as (ii) representative pictures. CXCL10 staining was found within inflammatory Infiltrates, close to portal regions or blood vessels. (B) Fluorescence microscopy was performed using an RNAscope approach with a probe targeting CXCL10 (red), a nuclear stain DAPI (blue or grey) and antibodies to either (i) Myeloid cells (CD68+CD163), (ii) myeloperoxidase (MPO) or (iii) Hepatocyte (green). This demonstrates CXCL10 is predominantly located in hepatocytes. Scale bars: 100𝜇m.

Fig 4. CXCL10 production is increased with HIV infection and is higher in HBV-producing cell lines.

(A) A representative flow cytometry plot showing eGFP expression (indicating infection with VSV-G-pseudotyped NL4.3Δenv eGFP HIV (MOI 0.5)) in live cells after gating on singlets and live cells, in uninfected hepatocytes (left plot) and hepatocytes infected with VSV-G-pseudotyped NL4.3Δenv eGFP HIV (MOI 0.5). (B) CXCL10 production measured by ELISA following stimulation of HepG2 (open symbols), and HBV-producing AD38 (filled symbols) hepatocyte cell lines with 500ng/ml IFN-γ plus1000ng/ml P3CSK4 and infected with VSV G-pseudotyped NL4.3Δenv eGFP HIV (MOI 0.5). HIV infection is indicated by symbols having a red border. Individual symbols represent the mean of replicates from a single experiment. The median+/-SEM for each stimulus from multiple experiments is shown. Comparisons between conditions were made using Wilcoxon Rank Sum test (* p<0.05). VSV-G-pseudotyped NL4.3Δenv eGFP HIV Vesicular stomatitis virus (VSV) glycoprotein G-pseudotyped NL4.3 virus with an envelope deletion expressing green fluorescent protein, MOI multiplicity of infection, HIV human immunodeficiency virus, CXCL10 C-X-C motif chemokine 10, IFN interferon, P3CSK4 Pam3CysSerLys4.

Fig 5. CXCL10 production is enhanced in the setting of HIV and HBV co-infection in vitro and decreased with the addition of antiretroviral agents efavirenz or raltegravir.

CXCL10 production was measured by ELISA (absolute and fold change, middle and lower panel) and eGFP expression measured by flow cytometry (upper panel) following stimulation of HepG2 (open symbols), and HBV-producing AD38 (filled symbols) cells, with 500ng/ml IFN-γ plus1000ng/ml P3CSK4 and infected with VSV-G-pseudotyped NL4.3Δenv eGFP HIV. The left sided panels show eGFP expression and CXCL10 production after cells were stimulated with 500ng/ml IFN-γ plus1000ng/ml P3CSK4 and infected with VSV-G-pseudotyped NL4.3Δenv eGFP HIV with a MOI of 0.0625, 0.125, 0.25 and 0.5. Increasing MOI is indicated by the triangle at the bottom of the Fig. The right sided panels show these same parameters, following infection with VSV-G-pseudotyped NL4.3Δenv eGFP HIV (red border) with MOI of 0.5 in the presence and absence of the antiretroviral agents efavirenz (EFV), raltegravir (RAL) and the fusion inhibitor T20. Individual symbols represent the mean of replicates from a single experiment. The median+/-SEM for each stimulus from multiple experiments is shown. Comparisons between conditions were made using Wilcoxon Rank Sum test (* p<0.05). HIV human immunodeficiency virus, CXCL10 C-X-C motif chemokine 10, GFP green fluorescent protein, IFN interferon, VSV-G-pseudotyped NL4.3Δenv eGFP HIV Vesicular stomatitis virus (VSV) glycoprotein G-pseudotyped NL4.3 virus with an envelope deletion expressing green fluorescent protein, MOI multiplicity of infection, P3CSK4 Pam3CysSerLys4, efavirenz (EFV), raltegravir (RAL).

Fig 6

Factors driving liver disease pathogenesis in HIV-HBV co-infection In HIV-HBV co-infection we propose a model where liver fibrosis is driven by an increase in production of CXCL10 by hepatocytes (left hand side of diagram) and products of microbial translocation (right hand side of diagram). Hepatocytes produce CXCL10 and production is enhanced following infection with HBV and HIV in the presence of IFN-γ. CXCL10 recruits activated T-cells and NK cells that express CXCR3 to the liver. In the absence of antiretroviral therapy, this is an ideal environment for HIV replication which produces IFN-γ and drives further CXCL10 production. In addition, altered GI tract permeability in the setting of HIV infection leads to an increase in circulating microbial products, including LPS which binds to TLR-4 and P3S4K4 which binds to TLR-2 expressed on hepatocytes, Kupffer cells and hepatic stellate cells. This cycle of CXCL10-mediated inflammation, together with elevated LPS directly activates hepatic stellate cells (HSC) driving fibrosis. HIV human immunodeficiency virus, HBV hepatitis B, CXCL10 C-X-C motif chemokine 10, IFN interferon, NK natural killer, CXCR3 C-X-C motif chemokine receptor 3, GI gastrointestinal tract, LPS lipopolysaccharide, TLR toll like receptor, HSC hepatic stellate cells.