On HIV-1 restriction in human dendritic cells and peripheral blood mononuclear cells

<p>Dendritic cells (DCs) are one of the first cells to encounter HIV-1 during sexual transmission. They may transmit the virus to CD4+ T-cells either locally or in the draining lymph nodes. In the present work, we have focused the studies on monocyte derived DCs. Upon DC maturation, HIV-1 replication is restricted in these DCs. DC maturation can be triggered by pathogens, danger signals and pro-inflammatory mediators such as TNF-α and IFN-α. The maturation signal results in several functional and phenotypic changes in the DCs. In this thesis, we have studied the influence of apoptotic cells and pro-inflammatory cytokines in their capacity to induce DC maturation and inhibit HIV-1 replication. We found that antiviral host APOBEC3 molecules were restricting HIV-1 replication in the DCs. We, furthermore, studied the effect of proteasome inhibitors on HIV-1 replication in primary cells.</p><p>We demonstrated that apoptotic activated CD4+ T-cells (ApoAct) can trigger DC maturation, which was quantified in terms of expression of co-stimulatory molecules. In addition, we detected a reduced frequency of HIV-1 infection in DCs. A prerequisite, for inducing DC maturation and inhibition of HIV-1 replication by the apoptotic cells, was the activation of CD4+ T-cells before inducing apoptosis. Hence, apoptotic resting CD4+ T-cells (ApoRest) did not exert these effects on the DCs. We also found that DCs exposed to ApoAct (either HIV-1 infected or uninfected) secreted MIP-1α, MIP-1β, MCP-1, and TNF-α. Blocking of TNF-α using monoclonal antibodies, partially abrogated induction of co-stimulatory CD86 molecules and reduction of HIV-1 infection in DCs co-cultured with ApoAct. Expression of APOBEC3G in DCs was increased after co-culture with ApoAct, but not ApoRest. Silencing of APOBEC3G in DCs abrogated the HIV-1 inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs/ApoAct co-cultures exposed to HIV-1, which are signs of functional APOBEC3 activities.</p><p>We found that both the cellular and supernatant fractions of apoptotic activated peripheral blood mononuclear cells (PBMC) were involved in triggering DC maturation. More specifically, the TNF-α present in the supernatant was involved and the cell-cell contact dependent signaling engaged beta-2 integrins, DC-SIGN and TLR4. We also found multiple signaling pathways and transcription factors being activated in DCs when they were co-cultured with ApoAct. These molecules include p38, JNK, PI3K-Akt, Src family of tyrosine kinases, NFκB p65 and AP1 transcription factor family members, c-Jun and c-Fos.</p><p>We showed that DCs upon treatment with TNF-α up-regulated co-stimulatory molecules and were able to restrict HIV-1 replication in DCs without inducing the expression of APOBEC3 mRNA (A3G, A3A or A3F). However, when the DCs were treated with low quantities of IFN-α2b they failed to up-regulate co-stimulatory molecules but significantly induced A3G, A3A and A3F mRNA expression and restricted viral replication in DCs. Sequence analyses of the env region from HIV-1 infected DCs treated with low quantities of IFN-α2b, showed an induction of high frequency of G-to-A hypermutations.</p><p>In addition, we also demonstrated that proteasome inhibitors can effectively reduce transcription from the HIV-1 LTR-promoter. Treatment of PBMCs with proteasome inhibitors showed reduced replication of HIV-1 in PBMC. The results were similar when the PBMCs were treated with proteasome inhibitors alone or in combination with other antiretroviral drugs. Futhermore, proteasome inhibitors reduced expression of IL-2 inducible T-cell kinase (Itk), a Tec-family kinase that is involved in HIV-1 replication.</p><p>In conclusion, we have showed that activated apoptotic lymphocytes and pro- inflammatory mediators can induce maturation in DCs and reduce HIV-1 infection, at least in part by inducing APOBEC3 molecules. Low quantities of IFN-α2b restricted HIV-1 replication in DCs while keeping an immature phenotype. We also identified some of the molecules and signaling pathways involved in DC response to ApoAct. Finally, proteasome inhibitors inhibit HIV-1 replication in PBMCs by targeting host factors essential for HIV-1 replication. These finding can be employed in therapeutic and/or prevention strategies.</p><h3>List of scientific papers</h3><p>I. Venkatramanan Mohanram, Ulrika Johansson, Annette E. Sköld, Joshua Fink, Sushil Kumar Pathak, Barbro Mäkitalo, Lilian Walther-Jallow, Anna- Lena Spetz. Exposure to Apoptotic Activated CD4+ T Cells Induces Maturation and APOBEC3G- Mediated Inhibition of HIV-1 Infection in Dendritic Cells. PLoS One. June 2011, 6, e21171. <br><a href="https://doi.org/10.1371/journal.pone.0021171">https://doi.org/10.1371/journal.pone.0021171</a><br><br> </p><p>II. Sushil Kumar Pathak, Annette E. Sköld, Venkatramanan Mohanram, Cathrine Persson, Ulrika Johansson, Anna-Lena Spetz. Activated Apoptotic Cells Induce Dendritic Cell Maturation via Engagement of TLR4, DC-SIGN and Beta-2 integrins. [Manuscript]</p><p>III. Venkatramanan Mohanram, Annette E. Sköld, Sushil Kumar Pathak, Anna- Lena Spetz. Low quantities of interferon-alpha2b induce APOBEC3 family proteins and restrict HIV-1 replication but do not mature dendritic cells. [Manuscript]</p><p>IV. Liang Yu, Venkatramanan Mohanram, Oscar E. Simonson, C.I. Edvard Smith, Anna-Lena Spetz, Abdalla J. Mohamed. Proteasome inhibitors block HIV-1 replication by affecting both cellular and viral targets. Biochemical and Biophysical Research Communications. May 2009, 385, 100-105. <br><a href="https://doi.org/10.1016/j.bbrc.2009.04.156">https://doi.org/10.1016/j.bbrc.2009.04.156</a><br><br> </p>