APC: Antigen-Presenting Cell; ELISA: Enzyme-Linked Immuno Sorbent Assay; GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor; HAART: Highly Active Antiretroviral Therapy; HCV: Hepatitis C Virus; HIV-1: Human Immunodeficiency Virus; IDU: Intravenous Drug User; IFN: Interferon; IL: Interleukin; MCP-1: Monocyte Chemoattractant Protein; MIP-1α: Macrophage Inflammatory Protein- 1α; PBMC: Peripheral Blood Mononuclear Cell; RANTES: Regulated upon Activation Normal T cell Expressed and Secreted; RPE: R-Phyco Erythrin; TNF-α: Tumor Necrosis Factor-α

Currently the global burden of both human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) is quite significant. HCV currently infects 3% of the world’s population (greater than 170 million people), with approximately 38,000 new infections occurring annually in the United States alone [1]. Currently 33 million people globally are estimated to be living with HIV-1 [2]. Due to similar routes of transmission of these viruses, co-infection with the two is quite common. Intravenous drug use, in particular, has resulted in an increase in rates of HIV-1/HCV co-infection, with incidence reaching or exceeding 90% prevalence [3]. Furthermore, progression to chronic HCV infection following acute infection is increased from 70 - 85% as seen in HIV-1 seronegative individuals to 90% in those individuals co-infected with HIV-1 [3]. Progression of HCV-related disease is further enhanced in HIV-1 positive individuals with advanced immunosuppression [4,5]. Co-infected individuals are known to have higher HCV RNA levels, which may result in greater risks of transmission [3]. Prior to the antiretroviral therapy (ART) era, HCV-related chronic liver disease mortality was masked by extra- hepatic mortality in HIV-1 co-infected individuals [6]. Now that ART has decreased HIV-related morbidity; liver disease constitutes a high proportion of mortalities among HIV-1 patients [7].

The increased levels of immune activation have been demonstrated in HIV-1/HCV co-infected individuals in contrast to HIV-1 monoinfected individuals [8,9], which may account for the high rate of disease progression in these individuals. Immune defects caused by either HIV-1 or HCV can alter the course of secondary infection and deregulated innate immune responses can contribute to more rapid disease progression. In addition, whether HIV-1 or HCV is the primary infection can also lead to different rates of HIV-1 disease progression as observed in various HIV-1/HCV coinfection studies [10,11]. Studies in the past have hinted that pre-existing HIV-1 infection thwarts the ability of the host to clear secondary HCV infection or worsens the individual’s condition as compared to HCV mono-infected individuals [3,12-16 ]. This may possibly be due to the pre-established immunosuppression that occurs subsequent to primary HIV-1 infection. However, is still remains unclear how chronic HCV infection affects secondary HIV 1 infection. Recently, it was found that IL-15 levels were enhanced in HIV-1/HCV co-infected individuals, suggesting a pro-fibrotic role for this cytokine [17]. With respect to inflammation and fibrosis, Blackard et al. [18] demonstrated that suppression of intrahepatic cytokines during HIV-1/HCV co-infection results in an imbalance between profibrogenic and anti-fibrogenic cytokines, favoring HCV replication and fibrosis within the liver. Another study demonstrated impaired IL-2 production by HCV-specific CD4⁺ T cells and impaired IL-2 and IFN-γ production from HCV-specific CD8⁺ T cells in co-infected patients [19]. Barrett et al. [20] examined cytokine profiles within PBMCs from uninfected, HIV-infected, HCV-infected and HIV-1/HCV-co infected individuals in response to HCV proteins. Exposure to HCV antigens was found to increase IL-10 production by PBMCs in uninfected and HIV-monoinfected individuals, but this response was attenuated in chronic HCV infection and HIV-1/HCV-co infection. This selective induction was suggested to play a role in establishing chronic HCV infection, thus contributing to liver pathology [20]. However, a more detailed analysis of steady-state cytokine profile to compare monoinfected individuals to co-infected ones is required. In this regard, the ability to visualize changes in cytokine networks with the onset and progression of disease or treatment is critical to advance our understanding of the immune response to pathogens. The recent Luminex^® technology has revolutionized the simultaneous detection and quantitation of several cytokines and chemokines in clinical samples that are generally available in small quantities, making it difficult to perform individual ELISAs for each cytokine of interest. Here we have utilized Luminex^® technology to compare cytokine and chemokine profiles of mono-infected HIV-1 or HCV individuals and co-infected HIV-1/HCV individuals.

Patients and plasma samples

Plasma samples from a well-characterized cohort of patients were utilized as per the institutional guidelines. The relevant clinical information related to these patient samples is given in Table 1.

Table 1: Patients utilized in the study.

Multiplex-25 bead Luminex^® assay

The human cytokine multiplex-25 bead array assay kit for Luminex was purchased from Invitrogen (Carlsbad, CA) to measure the following cytokines: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IP-10, MCP-1, MIP-1α, RANTES, TNF-α, IFN-α2, IFN-γ, GM-CSF and Eotaxin. The protocol was performed as per the manufacturer’s instructions and as previously described [21,22]. Appropriate dilutions of the human plasma samples in assay diluents were made. The assay was performed in a 96-well filter plate, using all the assay components provided. All incubation steps were performed at room temperature and in the dark to protect the beads from light. All washes were performed using a vacuum manifold. For the detection of cytokines and chemokines, the samples were finally incubated for 30 minutes with streptavidin conjugated to the fluorescent protein, R-phycoerythrin (Streptavidin-RPE, diluted 1:10). After washing to remove the unbound Streptavidin-RPE, the beads (minimum of 50 beads per cytokine) were analyzed in the Luminex 100 instrument, which monitored the spectral properties of the beads while simultaneously measuring the amount of fluorescence associated with R-phycoerythrin. Raw data was analyzed using Bio- Plex Manager software, v4.1 (Bio-Rad) [23]. Each undiluted plasma sample was assayed in duplicate, and cytokine standards supplied by the manufacturer were used to calculate the concentrations of the samples.

Plasma samples from patients who have either HIV-1 or HCV mono-infection or HIV-1/HCV co-infection were analyzed for the presence of 23 cytokines and chemokines by Luminex^® (Table 2). Cytokine concentrations for all analytes across all samples were derived using a 5PL curve fit algorithm. Out of the total 23 cytokines measured, 8 (IFNα2, IL-2, IL-3, IL-6, IL-8, IL12-p70, IL-15 and RANTES) were highest in patients with HIV-1/HCV co-infection (Figures 1 and Figure 2) by comparing the trend of the median value. These included important APC derived antiviral effectors such as IFN-α2 and IL- 12p70, mediators of T-cell expansion and memory differentiation IL-2 and IL-15 (which was previously found to be elevated in HIV-1/ HCV co-infection [17] as well as the key APC chemokine RANTES that is known to be involved in the recruitment of leukocytes to sites of inflammation. Expression of RANTES from one mono-infected individual, HC3, was higher than the two co-infected individuals HIC2 and HIC3 and must be looked at in further detail. The levels of the proinflammatory cytokines IFN-γ and TNF-γ were higher in the HIV-1 mono-infected individuals as compared to the other groups (Figure 1), which correlates with previous findings from Ciuffreda et al. [19] demonstrating that CD8⁺ T cells from HIV-1/HCV coinfected individuals were found to have impaired IFN-γ secretion [19]. However, when comparing each HIV-1 mono-infected individual’s IFN-γ and TNF-α response with the respective proviral loads, no direct relationship was observed. Interestingly, with respect to IP-10, the HIV-1 mono-infected individual with the lowest HIV-1 viral load, HI3, demonstrated the highest IP-10 expression. Nevertheless, this trend did not follow as individual HI1 had the next highest expression of IP-10, but the overall highest HIV-1 viral load of the 3 HIV-1 mono-infected individuals. Additionally, with respect to the HCV mono-infected individuals’ cytokine expression, no correlation with proviral load was observed. Cluster analysis was performed on the derived concentrations of the 19 cytokines (without normalization) to determine a pattern or unique signature (Figure 3A). The cluster analysis was better evaluated by normalizing the derived concentrations for all cytokines to healthy donor plasma and then uploaded to an in-house software suite for complex pattern analysis and visualization of multiplex cytokine and chemokine data (Figure 3B). Standard correlation clustering of the normalized data demonstrated a unique plasma proteomic signature for HIV-1/HCV co infected individuals (Figure 3B, upper dendrogram), demonstrating that in addition to the key cytokines that were highly expressed in these patients, the levels of Eotaxin and MIP-1α were contrastingly decreased in the same individuals when compared to mono-infected individuals (Figure 2). Interestingly, the co-infected individual with the highest HCV viral load (but not the highest HIV-1 viral load), HIC3, demonstrated the greatest expression of 7 of the 8 aforementioned elevated cytokines. With respect to RANTES and IL- 1α, the opposite was observed in which this same individual expressed the lowest amount compared to the other two co infected individuals, suggesting that HCV may have role in with stimulating or suppressing release of these particular cytokines/chemokines. Furthermore, the co-infected individual who had both high HIV-1 and HCV viral loads demonstrated the greatest expression of IL-10, similar to previous findings of HCV-antigen stimulation [20] and MIP-1α (Tables 1 and Table 2). Overall, cytokine expression levels may allow distinguishing co-infected individuals and assisting evaluation of the immune impact of therapeutic strategies in reversing immune activation changes associated with chronic mono or dual infection in such individuals.

Table 2: Absolute plasma concentrations of various cytokines and chemokines from mono HIV-1 and HCV mono- and co-infected individuals.

Figure 1: Plasma concentrations of various cytokines. The individual and median concentrations of various cytokines from HIV-1 mono-infected, HIV-1/ HCV co-infected, and HCV mono-infected individuals (n=3 for each condition and are represented by diamond, circle and square symbols, respectively). The median value is represented by a bar.

Figure 2: Plasma concentrations of various chemokines. The individual and median concentrations of various chemokines from HIV mono, HIV-1/HCV co-infected, and HCV mono-infected individuals (n=3 for each condition and are represented by diamond, circle and square symbols, respectively). The median value is represented by a bar.

Figure 3: Cluster and pattern analysis of plasma cytokine and chemokine expression in infected individuals. (A) Cluster representation of the absolute concentrations of various cytokines and chemokines. (B) The derived concentrations were normalized to healthy donor plasma and a pattern analysis was performed using a core developed software suite specific for Luminex® analysis. Results are presented as the mean plasma cytokine value for the sample (S) over the concentration from normal donor plasma (D) for that cytokine ({S/n)}/(D)) and arranged as an unsupervised distance clustered intensity plot.

These studies were supported in part by the United States Public Health Service/National Institutes of Health Grant R01 AI077414-01 (P. Jain) and also by the departmental start-up funds provided to Dr. Pooja Jain.