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

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.

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.

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.

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.

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.

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.

List of scientific papers

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.
https://doi.org/10.1371/journal.pone.0021171

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]

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]

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.
https://doi.org/10.1016/j.bbrc.2009.04.156