Profiling Circulating Inflammation and Microbial Translocation Biomarkers Associated with Advanced Liver Fibrosis in HIV/HCV Coinfected Persons

Background : HIV/HCV coinfection leads to accelerated liver fibrosis, in which microbial translocation and systemic inflammation might play important roles. This study aimed to provide an extensive profile of plasma microbial translocation and inflammation biomarkers associated with advanced liver fibrosis among HIV/HCV coinfected patients. Methods: This cross-sectional study recruited 343 HIV/HCV coinfected patients on combination antiretroviral therapy (cART) from a rural prefecture of Yunnan province in Southwest China. Plasma concentrations of sCD14 and 27 cytokines/chemokines were assayed, and were compared against advanced or mild levels of liver fibrosis. Results: Of the 343 HIV/HCV coinfected patients, 188 (54.8%) had severe or advanced liver fibrosis (FIB-4 >3.25). Patients with advanced liver fibrosis (FIB-4 >3.25 vs. FIB-4 ≤3.25) had higher plasma levels of IL-1β, IL-6, IL-7, IL-9, IL-12, IL-15, IL-17, GM-CSF, IFN-γ, TNF-α, IL-4, IL-10, IL-13, FGF-basic and MCP-1. Multivariable logistic regression models showed that advanced liver fibrosis was associated with increased plasma level of IL-1β, IL-6, IL-7, IL-12, IL-17, GM- CSF, IFN-γ, IL-4, IL -10, MCP-1, Eotaxin and FGF-basic, with FGF-basic remained to be positively and significantly associated with advanced liver fibrosis after Bonferroni correction for multiple comparisons (aOR=1.82; 95%CI: 1.26-2.66; p =0.002). Plasma sCD14 was also significantly associated with advanced liver fibrosis (aOR=1.14; 95%CI: 1.01-1.30; p =0.048). Conclusions : HIV/HCV coinfected patients are living with high prevalence of advanced liver fibrosis which coexists with a mixture of elevated plasma inflammation and microbial translocation Bonferroni correction for multiple comparisons. FGF-basic or Fibroblast Growth Factor-basic (FGF-basic/FGF-2/FGF-β) is a single-chain polypeptide growth factor that plays a significant role in the process of wound healing, is a potent inducer of angiogenesis, and has potent heparin-binding activity. Recent studies have suggested potential yet controversical associations of FGF-basic with disease status among HIV which coexists with a mixture of elevated plasma inflammation and microbial translocation biomarkers. The significant associations of advanced liver fibrosis with FGF-basic and sCD14 may reveal pathogenic mechanisms and potential clinical intervention targets of liver fibrosis in HCV/HIV coinfection.

stage liver diseases (ESLD) including cirrhosis and hepatocellular carcinoma (HCC) compared to HCV monoinfected patients [2]. This is a serious public health challenge in China given the fact that China has a large number of HIV/HCV coinfected patients who were most likely to be former plasma donors or injection drug users [3]. However, different from western countries, direct-acting antivirals (DAAs) or even peg-interferon plus ribavirin (PR) for HCV treatment are not widely used in China especially in rural areas because of limited access to health care and high prices of the drugs, which leads to a heavier disease burden [4]. Pathogenesis of progressive liver fibrosis in HIV/HCV coinfection is complex [5], and is mainly characterized by higher HCV viral load, altered cellular immunity, elevated pro-inflammatory and profibrogenic cytokines. More specifically, the immune suppression and CD4 Tcell depletion induced by HIV infection cause persistent immune activation, and Kupffer cells and hepatic stellate cells (HSCs) are directly stimulated to secrete pro-fibrotic cytokines or type 1 collagen [6]. In addition, HIV infection causes weakened CD4 and CD8 T cell responses, leading to the persistence of HCV infection [7], which in turn contributes to the increased rates of liver fibrosis [8].
Moreover, microbial translocation (MT), featured by dysfunction of mucosal barrier and increased intestinal permeability due to immune dysregulation and intestinal microbiome change, has been commonly observed among HIV-infected individuals [9]. Microbial components including, lipopolysaccharide (LPS) lipoteichoic acid and flagellin may promote liver fibrosis by stimulation of HSCs and/or Kupffer cells with LPS binding protein (LBP) and soluble CD14 (sCD14). These microbial components may enhance hepatic inflammation, immune response and liver cell death [5].
Thus, the above-mentioned mechanisms jointly emphasize the important role played by inflammation in liver fibrosis among HIV/HCV coinfected patients. Cirrhosis-associated dysregulation of immune responses can be reflected by increased production and elevated circulatinglevels of proinflammatory cytokines [10]. Kupffer cells generate IL-1b, IL-6, IL-12, IL-18 and also release antiinflammatory cytokines, including IL-10 stimulated by LPS [11]. Upregulation of IL-4, IL-13, TGF-b1 and platelet-derived growth factor (PDGF) was observed during fibrogenesis, while gp120 induces HSC accumulation by secretion of monocyte chemoattractant protein-1 (MCP-1) by HSCs among HIVinfected patients [12]. Furthermore, HIV suppression by combination antiretroviral therapy (cART) may decrease inflammation and immune activation, and may slow down the progression of liver disease [13], however, the levels of plasma inflammatory biomarkers may remain abnormal in many individuals [14]. So far, the inflammatory profile among HIV/HCV coinfected patients with different levels of liver fibrosis has not been well established, especially under the circumstance of combination antiretroviral therapy (cART). To fill this gap, the present study aimed to examine the association of advanced liver fibrosis with microbial translocation and inflammation cytokines among HIV/HCV coinfected patients in the era of cART.

Study sample
The present cross-sectional study was conducted in Dehong Prefecture of Yunnan Province at China's southwest border, with injection drug use (IDU) as the predominant mode of HIV transmission through the early 2000s and continues to be an important mode of HIV and HCV infection [15]. By March 2016, 1,017 HIV/HCV coinfected patients with 170 (16.7%) deaths had been reported to and registered in the Comprehensive Response Information Management System (CRIMS) for HIV/AIDS in China [16].
Out of the remaining 847 patients, 463 (54.7%) were identifiable during the study period from April to October in 2016. Of them, 390 (84.2%) were receiving cART and gave informed consent to participate in the present study. Forty-seven patients were further excluded from the study due to missing data on biochemical measures for FIB-4 calculation including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and platelet count (PLT). Thus, a total of 343 HIV/HCV coinfected patients were included in the final analysis. These participants showed no significant differences with the 120 identified yet excluded patients in the distribution of age, sex, marital status, HIV transmission route, HBsAg serostatus, baseline CD4 cells count, years on cART, current HCV RNA and HIV RNA levels, while showed difference in ethnicity (Table S1). The study was approved by the Institutional Review Board of Fudan University School of Public Health, Shanghai, China. All subjects provided informed consent prior to enrollment.

Data Extraction
Demographical and clinical epidemiological data were extracted from the CRIMS. The data included age, sex, marital status, ethnicity, HIV transmission route, date of cART initiation, cART regimen, CD4+ T cell counts at cART initiation and follow-up visits.

Biochemical tests for liver fibrosis assessment
Liver biopsy is an invasive technique with complications and costly, though it is a gold reference standard for determining liver fibrosis. In the light of these limitations , we assessed liver fibrosis by formula [17], which is an internationally recognized well-established noninvasive indicator for liver fibrosis [18]. Biochemical measurements of AST, ALT and PLT were performed using an automatic biochemistry analyzer (Beckman Coultner, USA), according to the manufacturer's protocol.

HCV RNA Quantification and genotyping
Plasma HCV viral RNA was extracted (Roche diagnostic products (Shanghai) Co., Ltd., China), and quantified by a real-time polymerase chain reaction (RT-PCR) technique using commercially available kits for the quantification of HCV RNA (PCR-Fluorescent Probing, PG Biotech Ltd., Shenzhen, China).
The limit of detection was 500 copies/ml, and the linear range of HCV RNA quantification was from 1.0 × 10 3 to 5.0 × 10 7 copies/ml. Amplification was completed by a nested PCR with E1-or NS5B-specific primers. Splicing, proofreading, and aligning sample sequences were performed using ChromasPro 1.5 and BioEdit7.0.9.0 software. HCV genotype reference sequences were retrieved from the HCV database (http://hcv.lanl.gov/content/sequence/HCV/ToolsOutline.html). The HCV gene subtype was determined according to the phylogenetic tree,which established by the Neighbor-joining method using MEGA 7.0 software [19].

Multiplex cytokine bead assay and sCD14
Twenty-seven cytokines, chemokines and growth factors cytokines in plasma specimens were fibroblast growth factor 2 (FGF-basic), platelet-derived growth factor (PDGF-BB), and vascular endothelial growth factor (VEGF). The detection limit for each molecule was determined by the recovery of the corresponding standard, and the lowest values with more than 70% recovery were set as the lowest detection limits. Measurements less than the lower limit of quantification (LLOQ) were assigned a value of half the LLOQ, and measurements more than the upper limit of quantification (ULOQ) were assigned a value of twice the ULOQ for each marker.
Plasma sCD14 was measured according to the manufacturer's protocol for commercial enzyme-linked immunosorbent assay (ELISA) ( Hycult Biotech, Wayne PA, USA).

Other tests
Human hepatitis B virus surface antigen (HBsAg) was tested by ELISA technique (Wan Tai Biomedical Co. Ltd, Beijing, China). CD4 cell counts were assessed by FACSCount (Becton, Dickinson and Co., San Jose, CA, USA).

Statistical analysis
Distributions of categorical variables were compared using chi-square test or Fisher's exact test, and continuous varabiles were compared using t-test or Mann-Whitney U test. Log10-transformation was conducted for plasma cytokines levels for further statistical and regression analyses. A multiple logistic regression analysis with adjustment for age (as a contiuous variable), sex (0-male, 1-female), ethnicity (0-Han, 1-Dai, 2-Jingpo,3-Others, entered as dummy variables), current HIV RNA (0-Undetectable, 1-Detectable), current HCV RNA (0-Undetectable, 1-Detectable), current CD4 cell counts ((0-<200, 1-200-349, 2-≥350, entered as dummy variables), years on cART (as a contiuous variable) and ART regimen type ((0-NVP, 1-TDF, 2-Others, entered as dummy variables) was used to explore the association of liver fibrosis with the plasma level of each of the twenty-seven cytokines and sCD14. The odds ratio (OR) and 95% confidence interval (95%CI) represent the risk of higher FIB-4 with per one log-unit change in plasma cytokine concentration. Spearman correlations were computed to explore correlations between the plasma inflammatory biomarkers. Statistical significance was defined as P <0.05 and Bonferroni P <0.002 (0.05/27≈0.002) for multiple comparison adjustment. All statistical analyses were performed using R software (version 3.3.2).

Demographic and clinical characteristics
The median age of the participants was 35 Table 1).

Prevalence of Advanced Liver fibrosis
Among all participants, 75 (21.9%) had no or mild liver fibrosis (FIB-4), 80 (23.3%) had intermediate liver fibrosis (FIB-4), and 188 (54.8%) had severe or advanced liver fibrosis (FIB-4 >3.25). Compared to patients without advanced liver fibrosis, those with advanced liver fibrosis were older, under different cART regimen, and had longer duration on cART (Table 1).   Data were presented as median and interquartile range (IQR), with μg/mL as the unit for sCD14 and pg/mL for all other cytokines. All the p-values were calculated by Mann-Whitney tests.

Correlations of inflammatory cytokines with advanced liver fibrosis
Multivariable logistic regression adjusting for potential confounders was performed to examine the association of advanced liver fibrosis with each of the 27 cytokines/chemokines and sCD14 levels.

Discussion
Liver complications including liver fibrosis, cirrhosis and ESLD have emerged as an important concern of non-AIDS-defining morbidity and mortality among PLWH in China, especially among those who are coinfected with HCV and/or hepatitis B virus (HBV) [16,20,21] . In the present study, 54.8% of HIV/HCV coinfected patients had advanced liver fibrosis. The prevalence was much higher than studies conducted in western countries which ranged from 22.9% to 40.3% [22][23][24].The discrepancy is possibly attributed to the fact that these the patients in our study are lack of antiviral therapy for viral hepatitis C and have a high rate of heavy alcohol use [25].
HIV and HCV infections are both characterized by systemic inflammation with increased levels of blood inflammatory cytokines. The pattern of cytokine release may have been altered and immunemediated inflammatory responses be exacerbated by HIV infection [26]. Although cART might help reduce plasma inflammatory marker levels in HIV/HCV coinfected patients, the markers may still remain much higher than healthy controls [27]. In the present study, we identified a mixed profile of elevated circulating inflammatory cytokines, chemokines and grow factors including IL-6, IL-10, IFN-γ, GM-CSF and FGF-basic among HIV/HCV coinfected patients with advanced liver fibrosis. This finding further indicates that progressive and advanced liver fibrosis in HIV/HCV coinfected patients is mainly an inflammatory syndrome [12].
In multiple logistic regression analysis adjusting for potential confounders, FGF-basic was shown to be significantly and positively associated with advanced liver fibrosis upon Bonferroni correction for multiple comparisons. FGF-basic or Fibroblast Growth Factor-basic (FGF-basic/FGF-2/FGF-β) is a singlechain polypeptide growth factor that plays a significant role in the process of wound healing, is a potent inducer of angiogenesis, and has potent heparin-binding activity. Recent studies have suggested potential yet controversical associations of FGF-basic with disease status among HIV patients [28,29]. The present finding of the positive association between FGF-basic and advance liver fibrosis may reveal a pathogenic mechanism and a potential clinical intervention target of liver fibrosis in HCV/HIV coinfection.
The role of IFN-γ in liver fibrosis is controversial. In some studies IFN-γ was negatively correlated with liver fibrosis [35], while IFN-γ was also found to have proinflammatory effects that can aggravate disease progression and organ dysfunction [32], and increased production of IFN-γ is observed among cirrhotic patients [36]. Soluable IL-10 was higher in HIV/HCV coinfected patients than in HIV-negative persons [29], but could be reduced following peg-interferon plus ribavirin (PR) treatment for HCV [37].
GM-CSF was higer in HIV patients than in health control subjects [33] ,and may promote liver damage and liver fibrosis [38]. sCD14 is often employed as an indirect biomarker of microbial translocation, given the technical difficulty in measuring lipopolysaccharide (LPS) which is a major marker for microbial translocation [39]. Our study shows that sCD14 level was positively associated with advanced liver fibrosis, consistent with the previous studies [40,41], further suggesting the role of inflammation in liver complications in HIV/HCV patients.
This study has several limitations. First, no causal relationship could be drawn upon the crosssectional design of this study. Second, data are unavailable on participants' alcohol drinking which is known to influence the risk of liver fibrosis and could alter levels of immune/inflammatory markers.
This should be addressed in future research.

Author's contributions
The study was conceived and supervised by NH and SD. RT, SZ, RY, YY, JW and SY conducted field survey, data collection and laboratory testing. XC analyzed the data and drafted the manuscript. XL and NH critically reviewed and revised the manuscript. All authors approved the final manuscript.

Availability of supporting data
Not applicable

Ethics approval and consent to participate
The study was approved by the Institutional Review Board of Fudan University School of Public Health, Shanghai, China. All subjects provided informed consent prior to enrollment.

Consent for publication
Not applicable Figure 1 Comparison of log10 transformed plasma concentration of tested biomarkers between participants with different FIB-4 score. *p<0.05, **p<0.01, and ***p<0.001. All the p-values were calculated by Mann-Whitney tests.

Figure 2
Adjusted odds ratios and 95% confidence intervals for the associations between plasma cytokine levels (log10 transformed) and advanced liver fibrosis among HIV/HCV coinfected patients. Each association was assessed in a separate multivariable logistic regression model adjusting for age, sex, ethnicity, current HIV RNA, current HCV RNA, current CD4 count, years on cART, and ART regimen.

Figure 2
Adjusted odds ratios and 95% confidence intervals for the associations between plasma cytokine levels (log10 transformed) and advanced liver fibrosis among HIV/HCV coinfected patients. Each association was assessed in a separate multivariable logistic regression model adjusting for age, sex, ethnicity, current HIV RNA, current HCV RNA, current CD4 count, years on cART, and ART regimen.

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