Immunomodulatory effects of IFN-[alpha] on dendritic cells: implications for HIV-1/HCV pathogenesis and treatment

Mohit Sehgal

doi:10.17918/etd-6576

HIV-1/HCV co-infection is a significant burden on global economy and public health. Highly active antiretroviral treatment (HAART) against HIV-1 is fairly effective. On the other hand, first generation direct acting antiviral drugs against HCV have improved cure rates but high cost and development of drug resistance are important concerns. Therefore PEGylated interferon (PEG-IFN) and ribavirin (RBV) are still essential components of anti-HCV treatment, and identification of host factors that predict IFN/RBV treatment response is imperative. In my thesis research, we investigated the host genetic and immunological correlates of successful treatment response. We also investigated the mechanisms by which PEG-IFN is able to clear the virus (in responders). Impaired dendritic cell (DC) and T cell responses are associated with HCV persistence. It has been shown that IFN/RBV treatment enhances HCV-specific T cell functions and it is likely that functional restoration of DCs is the underlying cause. To test this hypothesis, we utilized an antibody cocktail (consisting of DC maturation, adhesion and other surface markers) to perform comprehensive phenotypic characterization of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in a cohort of HIV-1/HCV co-infected individuals undergoing IFN/RBV treatment. Our results showed that pretreatment frequencies of mDCs were lower in non-responders (NRs) compared to responders (SVRs) and healthy controls. The treatment was able to restore the frequency of mDCs in NRs, but it downregulated the frequency of CCR7+, CD54+ and CD62L+ mDCs. Pretreatment pDC frequencies were lower in NRs and decreased further upon treatment. NRs exhibited reduced frequency of CCR7+ pDCs and increased pDC PD-L1/CD86 ratio as a result of treatment. These findings demonstrate that functional state of DCs before/during therapy influences the treatment outcome. We also show that before treatment, PBMCs from SVRs secrete higher amounts of IFN-[gamma] compared to controls and NRs. Upon genotyping IFNL3 polymorphisms rs12979860, rs4803217 and ss469415590, we found rs12979860 to be a better predictor of treatment outcome. Collectively, this part of the study led to identification of important correlates of IFN/RBV treatment response in patients. Next, we wanted to investigate how the expression of interferon-stimulated genes (ISGs) affects the treatment outcome. It is already known that high pre-treatment expression of ISGs in liver is a good predictor of poor response to IFN/RBV treatment, but there is no clear consensus regarding the correlation between ISGs expression in PBMCs and treatment outcome. We compared the expression of forty-six ISGs between NRs and SVRs both before and at week 4 of IFN/RBV treatment. We found that the pre-treatment levels of sixteen ISGs were moderately higher in SVRs compared to NRs. At week 4 of IFN/RBV treatment, nineteen ISGs were upregulated and fifteen ISGs were downregulated in SVRs. Next, we identified miRNAs whose expression can be regulated by IFN-[alpha] in PBMCs. miR-155 was one of the many miRNAs upregulated by IFN-[alpha] and its expression varied between NRs and SVRs both before and at week 4 of treatment. Overall, our results indicated that lower miR-155 expression and higher levels of ISGs could be predictive of treatment success. Besides their well-appreciated role in direct antiviral defense, IFN-[alpha] plays a critical role in the regulation of innate and adaptive immune response. Immunomodulatory properties of IFN-[alpha] are facilitated by its action on DCs, professional antigen presenting cells critical to generation of protective immunity. Various microRNAs are known to regulate DC functions but it remains unclear whether IFN-[alpha] exerts its effects on DCs through miRNAs. In this study, we examined miRNA expression patterns in myeloid and plasmacytoid DCs in response to IFN-[alpha] and observed miR-221 downregulation via IFN induced STAT3 inhibition in both. Using in silico approaches followed by experimental validation, BCL2L11, CDKN1C, and SOCS1 were identified as miR-221 targets. Moreover, miR-221 overexpression in mDCs enhanced their secretion of proinflammatory cytokines IL-6 and TNF-[alpha]. In PBMCs isolated from HIV-1/HCV co-infected patients on standard IFN-[alpha] therapy, pre-treatment levels of miR-221 were significantly lower in non-responders compared to responders and healthy controls. Also, in patients PBMCs, miR-221 and CDKN1C, CD54, IL-6 and TNF-[alpha] levels correlated similarly as observed earlier in mDCs. In addition, we isolated total liver cells and kupffer cells (antigen presenting cells in liver) from HCV infected individuals as well as individuals with alcoholic cirrhosis. We found that both total liver cells and kupffer cells from HCV-infected individuals had significantly higher miR-221 levels compared to cirrhotic patients which might indicate HCV-mediated impairment in IFN signaling. Overall, this part of our study demonstrated the role of IFN-[alpha]/miR-221 axis in HCV pathogenesis and response to IFN-based treatments. Collectively, during my thesis research, we were able to demonstrate the correlation between miR-221 and miR-155 expression in PBMCs and response to PEG-IFN/RBV treatment. We were also able to characterize the DC phenotype and ISGs expression (in PBMCs) of NRs and SVRs both before and during the treatment. We are confident that this work will prove to be beneficial for both diagnostic purposes and for understanding of the role of DCs in HCV pathogenesis.

Immunomodulatory effects of IFN-[alpha] on dendritic cells: implications for HIV-1/HCV pathogenesis and treatment

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Immunomodulatory effects of IFN-[alpha] on dendritic cells: implications for HIV-1/HCV pathogenesis and treatment

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