Ali A. Ashkar, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N3Z5, Canada. E-mail : firstname.lastname@example.org
Citation Mian MF, Ashkar AA. Induction of innate immune responses in the female genital tract: friend or foe of HIV-1 infection? Am J Reprod Immunol 2011; 65: 344–351
Heterosexual transmission of HIV-1 and HSV-2 across the genital tract epithelial tissue is one of the primary routes for dissemination of these viral infections. Mucosal innate immunity is the first line of defense against invading pathogens. A vast majority of mucosal HIV-1 exposures do not result in productive infections which may indicate that the innate mucosal immune system is highly protective. It has been shown that Toll-like receptors (TLR)-induced innate antiviral immunity in the genital mucosa lead to induction of type I and III interferon and prevention of HSV-2 infection. The innate antiviral function of type I and III interferons and other innate factors at genital mucosa against HIV-1 is not well defined. In this review, we summarize our current understanding and advances of the innate mucosal response to genital viral infections, including HIV-1 and HSV-2, focusing on those factors that may prevent or accelerate initial infection. Understanding how each of these components contributes to mucosal innate antiviral immunity may lead to the development of novel and effective strategies to use microbicides or antiviral agents to control HIV-1 acquisition and/or transmission.
Innate immunity, the first line of defense during both primary and recurrent viral infection, is crucial to limit initial viral replication and subsequently to develop an appropriate adaptive immune response. The innate immune response comprises a complex multilayered system of mechanical interference, secreted defenses, immediate IFN and cytokine/chemokine responses, and rapid recruitment of immune cellular defenses against invading pathogens. Although there is commonality in the mucosal immune response, however, distinct site specificity in immune mechanisms depending upon the function and anatomical location of an organ is also evident. Mucosal surfaces of the female genital tract (FGT) serve as the major portal of entry for sexually transmitted infections (STI) including human immune deficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2). Germ line-encoded hosts pattern recognition receptors of the innate immune system recognize the invading virus, interact immediately with viral pathogen associated molecular patterns (PAMPs) and produce interferons (IFNs), cytokines, and chemokines to prevent viral replication and spread. It is therefore important to understand how innate immune system in the female genital tract interacts with HIV-1.
Despite numerous efforts made during the last decades, HIV-1 remains the most devastated STI which is also on the rise. The annual HIV-1 acquisition was estimated 2–4 million, while the total infected population was about 40 million worldwide.1 There is no effective vaccine or therapy currently available for HIV-1 cure for which AIDS still recognized as a pandemic disease. Heterosexual transmission of HIV-1 through genital mucosal tissue is the primary route of acquisition and dissemination across the globe.2 HIV-1 infections often accompany co-pathogens that can suppress3–6 or in most cases accelerate transmission and clinical progression of HIV-1 infection.7–12 Herpes simplex virus type 2 (HSV-2) is the most common co-pathogen that enhances the susceptibility and transmission of HIV-112 by causing ulcerative lesions on mucosal tissues7 or by inducing inflammation and recruiting HIV-1 targeting cells (macrophages or CD4 T cells).13 The vast majority of the earlier studies on HIV-1 have focused on adaptive immunity to formulate effective vaccine, those however were relatively unsuccessful. Recent studies have focused on exploring innate-mediated antiviral immunity to develop potent microbicides or antiviral agents that can successfully prevent viral acquisition, replication, or dissemination in the FGT. In this review, we summarize the recent advances on the role of innate immune system in sexually transmitted viral infections to the female genital mucosal surfaces in the context of HIV-1 infection.
Innate immune responses in the female genital mucosal surface and HIV-1 infection
The female genital tract (FGT) has evolved with unique innate architecture and physiological activities to provide protection against invading viral, bacterial, or other pathogenic microbes. However, the complex microenvironment of FGT is regulated by multiple immunoregulatory activities such as hormonal regulation, secreted mediators, Toll-like receptors (TLR) stimulations, and regulation by co-infecting pathogens. The epithelial lining of the genital mucosal surface serves as the physical barrier/first line of defense, and a number of secreted factors such as human β-defensins, trappin-2/elafin, secretory leukoprotease inhibitors (SLPI), and lactoferin act to eliminate potential pathogens including HIV-1.14–19 These epithelial-secreted mediators elicit potent antimicrobial, as well as anti-inflammatory responses by inhibiting NF-kB activation with subsequent reduction in pro-inflammatory cytokines or chemokines to prevent pathogen invasions.20–22 Female reproductive hormones often influence the innate immune system in the genital tract.23,24 While estradiol25 prevents, progesterone26, in contrast, increases the susceptibility to HIV-1 acquisition and transmission. Co-infections with other STIs such as Herpes simplex virus type 2 (HSV-2), human cytomegalovirus (CMV), Neisseria gonorrheae, and many others also enhance HIV susceptibility either by breaching the epithelial tissue, recruiting HIV target cells into the site of infection, or by generating a pro-inflammatory local immune milieu. For example, de Jong et al27 have recently demonstrated that TLR agaonists stimulation and genital co-infections by Candida albicans and Neisseria gonorrhea induce production of TNF-α, which in turn enhances replication and transmission of HIV-1 through Langerhans’ cells ex vivo. Several measures targeting genital co-infections, such as vaccines, microbicides, and suppressive therapy, have been found beneficial in the short term and have the potential to curb the HIV-1 pandemic.22,28 Unlike other STIs such as HSV-2, suitable animal models are not yet available to study the host–HIV-1 interactions that led HIV-1 research more complicated and confined only to ex vivo or in vitro models. Therefore, a vast majority of HIV-1 studies rely on either macaques model for SIV or mouse model for HSV-2. Nevertheless, recent advancement in exploiting humanized mouse model to study HSV-229 has opened new windows to explore HIV-1 immunity and pathogenesis studies in the near future.
Induction of mucosal innate antiviral immunity by TLR ligands
Innate immune system has evolved to protect the host from invading pathogens, which relies on strictly conserved pattern recognition receptors (PRRs) that recognize conserved pathogen-associated molecular patterns (PAMPs) synthesized by bacteria or virus. The best characterized receptors that recognize PAMPs are TLRs that are expressed on a wide variety of cells including epithelial cells, macrophages, DCs, NK cells, B, and T cells in the FGT (reviewed in ref. 20). All major TLRs (1–10) are expressed by the human genital tract cells.21,30–34 Of the ten TLRs, TLR3, TLR4, TLR7, TLR8, and TLR9 have been known as antiviral TLRs because they preferentially induce the production of antiviral interferons, IFN-α/β, and IFN-λ. Four of these TLRs including TLRs 3, 7, 8, and 9 reside in the endosome in most cell types and can be stimulated by viral nucleic acid agonists, double-stranded RNA, imidazoquinoline/loxoribine, single-stranded RNAs, and non-methylated CpG ODN, respectively. Exploiting TLR signaling, as well as searching for new PAMPs/ligands possessing potent innate immune capability but lesser side effects is the main focus of today’s sexually transmitted virus research to generate a more comprehensive vaccine or therapeutic interventions. TLR ligand stimulation triggers local inflammation, recruitment of effector cells, and secretion of cytokines/chemokines that modulate both innate and adaptive immune responses.35–37 A few recent important studies utilizing TLR ligands have been successful to provide protection against the devastating STI, HSV-2 in murine model. In this regard, we and others have demonstrated that local delivery of CpG ODN, a TLR9 agonist, through the genital tract provide complete protection of mice from subsequent lethal challenge by vaginal HSV-2.38–41 Mechanistically, local delivery of CpG ODN induces proliferation and thickening of the genital epithelial cells, huge influx of inflammatory cells into the submucosa, and arrests HSV-2 replication but not entry into the genital tract.38,40,42 We further showed that local delivery of TLR3 ligand poly I:C robustly protects mice from intravaginal HSV-2 challenge, which is independent of any local inflammatory reactions43 that is observed with CpG treatment. These two studies indicate distinct mode of responses; while TLR9 ligand inhibits HSV-2 infection through Th1 (pro-inflammatory) inflammation, TLR3 stimulation, in contrast, acts independent of inflammation. Moreover, TLR9 stimulation by CpG results in the expansion of CD8+ T cells and destruction of lymphoid tissues with consequent development of immunosuppression and B cell-related autoimmunity; in contrast, dsRNA-mediated responses are much solid and safer without causing any adverse immunopathological effects. Intravaginal inoculation of poly I:C also found to induce array of innate cytokines/chemokines such as IFN-α, IL-1α, IL-1β, IL-6, IFN-γ/MIP-1α, and RANTES production in the genital tract39,44 and IFN-β release by epithelial cells.45 Therefore, CpG and poly I:C could be potential candidate microbicide for local protection against genital HSV-2 or HIV-1 infections that deserve further studies. As in the case of HIV-1, TLR-3 ligand poly I:C stimulation of genital tract displayed conflicting results; while one study shows poly I:C prevents HIV-1 transmission,46 conversely, the other study found poly I:C as a potent inducer of Langerhans’ cell maturation and HIV-1 transmission.11 More recently, treatments of human primary uterine cell culture ex vivo with TLR-3, 9, and 5 ligands poly I:C, CpG ODN and flagellin, respectively, resulted in 80% inhibition of HSV-2 replication, but only TLR3 stimulation produced significant amounts IFN-β, IL-1β, IL-6, and TNF-α through IRF-3 and NF-kB activation.31 Further evidence that the TLR3 ligand poly I:C induces TH1 response in an HIV-gag protein vaccine depends on MDA-5-mediated recognition in both hematopoietic and stromal cell compartments.47 A more recent study, however, described contrasting roles of TLR5 and TLR9 ligands, flagellin, and CpG ODN, respectively; while flagellin enhances the CCR5- and CCR4-tropic HIV-1 replication, treatment with CpG ODN suppressed both viral variants in lymphoid tissue ex vivo that correlated with CCL3, CCL4 and CCL 5 and CXCL10 and CXCL12 and T-cell activation leading to HIV-1 pathology.48 Similarly, Funderburg et al49 showed differential activation of CD8+ T-cell and CD4+ T-cell responses with HIV-1 infection upon TLR ligands’ (TLR2-9) treatments of human PBMCs ex vivo.
Type1 and type III interferons in HIV-1 infection
Recognition of viruses by pattern recognition receptors of the innate immune system is crucial for rapid production of type I interferon (IFN) and early antiviral defense. Although the role of type 1 IFN in the protection against viral infection has been studied for decades, its distinctive role in the context of HIV-1 infection is not well defined and potentially much more complicated. Type 1 IFN comprises a single IFN-β, and multiple IFN-α members are produced by many cell types in response to stimulation of any transmembrane and cytosolic receptors including TLRs, RLRs (RIG-I-like receptors), RIG-I, and MDA-550,51,52,53 or NOD-like cytoplasmic receptors.54 TLRs 3, 7, 8, and 9 typically reside in the endosome as opposed to at the plasma membrane, where they can gain access to viral nucleic acids.55 A new group of interferon, the type III interferon (IFN-λ or IL-28/29), has recently been emerged as a potent antiviral agent which although structurally distinct but exhibits similar functions as the type 1 IFNs. The expression of IFN-λ receptors is largely restricted to epithelial cells; however, IFN-λ can be induced in many cell type of the innate immune system by TLR stimulation or viral infections and is able to induce type 1 IFNs.56–61 TLR pathways leading to the production of IFN-α/β and IFN-λ have begun to be unraveled in recent years.35,62,63 In most cell types, the common pathway for type 1 IFNs is the activation of TLR3 or RIG-1 or MDA-5 by viral nucleic acids, particularly dsRNA.64 The production of type 1 IFNs can be further augmented in a autocrine manner by activating IRF-7, thereby amplifying type 1 IFN production.65,66 Recently, we and others have demonstrated that activation of TLR3 and TLR9 by poly I:C and CpG ODN, respectively, provided significant protection against HSV-2 genital infections mediated preferentially by IFN-β, not by IFN-α, TN-α, or IFN-γ, production in murine model.67,68 Both genital viral replication and the disease progression were enhanced in HSV-2-infected mice lacking the IFN-α/β receptor. Furthermore, antiviral treatments using agonists to TLR3, 7, and 9, synthetic dsRNA, imiquimod, and CpG ODN, respectively, were strongly dependent on IFN-α/β receptor signaling,68 suggesting requirements of IFN-α/β receptor signaling in the antiviral defense against HSV-2. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent IFN-α/β response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurs independently of viral replication but dependent on viral entry, however, in macrophages and ﬁbroblasts, the HSV-2 is able to replicate but IFN-α/β production depends on both viral entry and replication. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response. We have demonstrated a distinct correlation between IFN-β production and protection from HSV-2 by TLR ligands in vivo. However, it is not clear how IFN-β, following local delivery of TLR ligands, elicits an antiviral activity against HSV-2. It is known that HSV-2 enters the epithelial cells, but at which stage the viral replication is blocked remains unclear. A detail understanding of TLR ligands in innate antiviral responses may provide valuable insights into type 1 IFN-mediated innate defense against HSV-2 and HIV-1 infections.67
Type 1 IFNs are involved in inhibiting HIV-1 replication at multiple steps in the early phase of its life cycle and thereby suppress viral transmission. Failure to induce antiviral interferons in infected macrophages may promote transmission. HIV replication is inefficient in DCs, an indicator of type 1 IFN functional state in DCs. It remains controversial whether type 1 IFN production is increased or reduced during HIV-1 infection.69 A number of studies focused on circulating pDC production of IFN-α during acute and chronic infection upon TLR9 ligand CpG stimulations,70,71 which does not reflect the scenario that occurs in the FGT during HIV-1 infection. However, ex vivo genital epithelial tissue treated with TLR-3 ligands induced IFN-β production and inhibited HSV-2 replication.31,72 Importantly, ssRNA isolated from HIV-1 virion as well as TLR7/8 ligand (R-848) has been shown to inhibit HIV-1 replication by eliciting type 1 IFN production through TLR7/8 signaling.73,74 The innate antiviral defenses are overall largely ineffective at suppressing acute HIV-1 infection in vivo and often progress to a chronic infection. This in part has been attributed to the properties of the virus that inhibit specific host defense factors. Importantly, two recent studies have found valuable clues to how HIV-1 evades type 1 IFN pathway. Yan et al. delineated that a host factor, TREX1 (a cytoplasmic exonuclease), knockdown in CD4+ T cells and macrophages upon HIV-1 infection induces type 1 IFN production through TBK1-STING-IRF-3 pathway and inhibits HIV-1 replication and spread. Another study demonstrates that IRF-3 can direct a robust innate antiviral response to control HIV-1 replication, but that HIV-1 infection potentially disrupts IRF3-dependent signaling pathways and innate antiviral defense during replication cycle in CD4+ T cells that support acute infection in mucosal tissue probably by abrogating type 1 IFN production.75
The newly discovered type III interferon (IFN-λ) retains potent antiviral activity against a broad range of viruses. A number of studies recently have delineated the novel roles of IFN-λ in inhibiting replication of a number of viruses including CMV76 and HSV-2.58 However, a recent in vitro study employing human macrophage cells upon IFN-λ treatment has been shown to inhibit HIV-1 replication.77 The antiHIV response of IFN-λ has been found broad because it prevents the replication of both the laboratory and clinical strains of HIV-1. Mechanistically, IFN-λ although has little effect on CCR5, a receptor for HIV-1 entry, expression, however, induces remarkable upregulation of type 1 IFNs and APOBEC3G/3F, a newly identified anti-HIV-1 cellular factor, expressions. These results provide clear evidence that IFN-λ potentially inhibits HIV-1 replication in macrophages through both extra- and intracellular antiviral mechanisms that make IFN-λ a prospective candidate for therapeutic treatment of HIV-1 infection. Further studies required to evaluate whether local delivery or induction of IFN-λ to the FGT could protect against sexually transmitted viral infections such as HSV-2 or SIV in appropriate animal models.
Concluding remarks and future directions
Advances over the past several years of research on HIV-1 and HSV-2 have provided us important insights into several aspects of these infections, including (i) how these sexually transmitted viruses invade into the genital tissue; (ii) which host factors in the FGT can limit virus replication; and (iii) what are the target cells that the viruses use for initial infection and subsequent replication in the genital mucosa. Additional view comes from the observations that HIV-1 has evolved mechanisms to evade the TLR-IFN pathway.75,78 Although the role of TLRs-mediated innate antiviral immunity or pathogenesis for HIV-1 infection has expanded significantly in recent years, however, many controversies as well as questions regarding virus–host interactions remain to be clarified. It is increasingly evident that TLR ligands provide protective antiviral immunity in some cases, while clearly exacerbate HIV-1-induced disease in others. Therefore, the cellular mechanisms by which TLRs regulate the balance between protective and pathogenic immunity in HIV-1 infections deserve deeper investigations. Nevertheless, a number of efforts have been made to develop TLR agonists-based topical microbicide to prevent sexually transmitted viral infections including HSV-2 and HIV-1 in the genital tract. To this end, intravaginal administration of TLR-3 ligand poly I:C, a unique TLR4 ligand FimH, and TLR9 ligand CpG ODN has been demonstrated to provide complete protection against HSV-2 challenge in mouse model. The major concerns with induction of innate immunity at the genital mucosa are the associated inflammation and other immunoregulatory factors that may favor HIV-1 replication and spread. It may be desirable to induce the high levels of type I and III IFNs to provide potent innate antiviral immunity but to avoid induction of inflammation and recruitment of leukocytes to the site of infection (Fig. 1). Use of Poly I:C and selective targeting of CpG ODN to pDCs and epithelial cells to induce type 1 IFN have been suggested to diminish inflammation and maximize the microbicidal effectiveness against HSV-2. Likewise, TLR 7/8 ligands also could be considered as microbicide against HIV-1 or HSV-2 infection because they induce type 1 IFN-mediated protection. Collectively, TLR-induced type 1 IFNs production at the genital mucosal surface may be a safer way to provide maximum level of protections against HIV-1 and HSV-2. It is very important to understand the role of genital epithelial cells in the production and/or respond to type I and III IFNs in the context of HIV-1 infection. Recently, type III IFN (IFN-λ) has been shown to potentially inhibit HIV-1 and HSV-2 replication in epithelial cells and macrophages by type 1 IFN and chemokine production. Therefore, IFN-λ represents a promising candidate microbicide against HIV-1 and HSV-2 genital infections; however, further studies are required to unveil cellular responses.
This study was supported by a grant from CIHR to Ali A. Ashkar. AAA is a recipient of Career award in Health Sciences from Rx&D/CIHR.