Macrophage-Mediated Innate Immunity Against AIDS Virus

Innate immunity plays an important role in the control of viral infections including HIV. Macrophages are the major immune cells in the innate immune system. The importance of macrophage in the pathogenesis of HIV infection is highlighted by its dual roles in HIV infection:to participate in the host anti-HIV immune responses, and to be a target and reservoir for HIV infection. Because HIV can integrate into host cell genome, it is crucial to suppress and eliminate HIV from its target cells and reservoirs. A great deal of research has demonstrated that HIV can counteract antiviral innate immune response of the host, developing chronic and persistent infection. Therefore, the activation of innate immunity suppressed by HIV within its target cells is helpful to control and eliminate HIV. Induction of the antiviral innate immune response depends on a family of innate immune receptors, such as Toll-Like Receptor3(TLR3) and Retinoic Acid-Inducible Gene-I (RIG-I).This project mainly revealed inhibitory effect and mechanism of AIDS virus by activation of TLR3and RIG-I signaling in macrophages. Engagement of TLR3and RIG-I activates signaling cascades that culminate in inflammatory and immune defense to inhibit a number of viral infections including HIV and SIV. The precise mechanisms of IFN-mediated intracellular anti-SIV response induced by TLR3or RIG-I in macaque macrophages were determined in this thesis.1. To establish an efficient technique for in vitro culture of monocyte-derived macrophage (MDM) from rhesus macaquesDue to lack of effective ways to separate and cultivate macaque macrophages, our primary task is to establish a simple, inexpensive and efficient technique for in vitro culture of MDM from rhesus macaques (Macaca mulatto) of Chinese origin. PBMCs were cultured for24h in RPMI1640supplemented with different proportions of autologous serum or fetal bovine serum (FBS). We found that the cells cultured with RPMI1640containing2%autologous serum for7days yielded the best results with regard to macrophage morphology, the response to LPS stimulation and susceptibility to SIV or SHIV infections. The purity of adherent macrophages under2%autologous serum culture condition was more than96%. RPMI1640with2%autologous serum is suitable for in vitro differentiation of peripheral blood monocytes from rhesus macaques. This technique is simple, inexpensive, no need for growth factor and highly effective for macaque monocyte adherence and differentiation. 2. Innate immune responses of SIV-infected rhesus macaques of Chinese OriginA family of innate immune receptors, such as TLR3and RIG-I plays an important role in the control of HIV/SIV infection. The innate intracellular factors such as APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide3G, A3G), A3F, A3B and A3C, also including newly identified anti-SIV miRNAs (miR-29a,-29b,-146a and-9, have the ability to inhibit infection and replication of SIV. We investigated their longitudinal expression and functionality in the SIV/macaque model at acute phase. Four rhesus macaques of Chinese origin were challenged intravenously with SIVmacR71and SIVmacl7E-Br. The four monkeys showed viremia, and the SIV was detected in the Cerebrospinal Fluid (CSF). Innate immunity of SIV-infected monkeys were significantly activated, and induced a variety of antiviral factors. We examined the expression of these antiviral factors in PBMCs of uninfected and SIV-infected Chinese rhesus macaques. Compared with the status before they became infected, we found increased innate immune receptors (TLR3, RIG-I, MDA5), ISGs (MxA, OAS-1), intracellular antiviral factors (A3G, A3F, A3B, A3C), and anti-SIV miRNAs (miR-29a,-29a-146a and miR-9) in PBMCs from SIV-infected macaques. During acute viremia, increased levels of these four miRNAs and APOBEC3were associated with OAS-1, a prototype IFN-stimulated gene, indicating a common regulation by the initial IFN response. These findings suggest a protective effect of TLR3/RIG-I mediated innate immune responses to SIV infection in vivo and indicate regulation of the four anti-SIV miRNAs and APOBEC3by IFN during early infection.3. Activation of TLR3signaling inhibits SIV infection in macaque macrophagesTLR3recognizes double-stranded RNA and induces multiple intracellular events responsible for innate antiviral immunity against viral infections. Here we demonstrated that TLR3signaling of macaque macrophages by poly I:C inhibited SIV infection and replication. Investigation of the mechanisms showed that TLR3activation resulted in the induction of type I and type III IFNs and IFN-inducible antiviral factors, including MxA, A3G, and tetherin. In addition, poly I:C-treated macaque macrophages expressed increased levels of CC chemokines including CCL3, CCL4and CCL5, the ligands for HIV or SIV coreceptor CCR5. Furthermore, TLR3signaling of macaque macrophages induced the expression of cellular microRNAs (miR-29a,-29b and-146a), the newly identified intracellular SIV restriction factors. TLR3activation-mediated anti-SIV effect could be compromised by the knockdown of IRF3and IRF7. These findings indicate that TLR3-mediated induction of multiple viral restriction factors contribute to the inhibition of SIV infection at different stages of viral replication in macaque macrophages.4. RIG-I signaling of macaque macrophage suppresses SIV replicationThe RIG-I signaling pathway is critical in the activation of the type I IFN-dependent antiviral innate-immune response. We thus examined whether RIG-I activation can inhibit SIV/SHIV replication in macrophages. We showed that the stimulation of macaque macrophages with5’ppp-dsRNA, a synthetic ligand for RIG-I, induced the expression of RIG-I, and high-level of type I and III IFNs, and up-regulation of IRF3/7, key regulators of the IFN signaling pathway. In addition, RIG-I activation induced the expression of multiple intracellular SIV-restriction factors, including ISGs, A3G and tetherin. The inductions of these factors were associated with the inhibition of SIV replication in macrophages stimulated by5’ppp-dsRNA. These observations highlight the importance of RIG-I signaling in macrophage innate immunity against SIV, which can be beneficial for the treathent of HIV disease, where intracellular immune defense is compromised by the virus.Collectively, on the basis of the establishment of in vitro culture of macaque macrophages, we focus on the interplay between SIV infection and macaque macrophages. We found that the activation of TLR3/RIG-I mediated innate immune responses by SIV infection plays an important role in the control of viral infection. Our further study revealed the activation of TLR3and RIG-Ⅰ signaling of macaque macrophages inhibits SIV replication. The activation of type Ⅰ and Ⅲ IFNs signaling and induction of multiple cellular antiviral factors against HIV or SIV should account for much of the TLR3/RIG-I-mediated anti-SIV activity at both viral entry and transcriptional levels. At extracellular level of AIDS virus entry into the cells, β chemokines (CCL3, CCL4and CCL5) were secreted by macrophages to block SIV into macrophages, and induction of viral restriction factors, such as A3G, tetherin, MxA, ISGs and anti-SIV miRNAs suppress SIV infection at different steps of viral replication at the intracellular level, which makes HIV/SIV very difficult to produce drug-resistance viruses. Therefore, the activation of TLR3and RIG-Ⅰ signaling pathway is highly effective in control of AIDS virus infection and replication. Our data provide the scientific evidence for supporting the notion of developing new HIV therapy based on the activation of intracellular innate immunity.