IL-32 and HIV Persistence
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Written by Alain Lafeuillade
Tuesday, 31 May 2011 14:16
IL-32 and HIV Persistence
The proinflammatory cytokine IL-32, was originally identified in 1992, but until recently has received little attention as a moderator of chronic immune activation. Recent experiments support the hypothesis that IL-32 may be a contributing factor in an immunoregulatory axis that collectively acts as a double-edged sword in lentiviral immunodeficiency infections.
In a recent paper published by the A. Haase group (1), evidence is given that IL-32 may have such a role in countering immune activation and inflammation during HIV-1 infection by promoting immune suppression via the induction of immunosuppressive molecules (IDO and Ig-like transcript 4). An increased expression of IL-32 was demonstrated in both gut and lymph nodes during all stages of HIV infection (from acute HIV infection to full blown AIDS) and was inversely correlated with HIV replication in lymph nodes. Compared with uninfected individuals, levels of IL-32 were significantly increased in both gut and LT during all stages of HIV-1 infection, with the highest IL-32 levels in the acute stage of disease for gut (4.8-fold increase) and AIDS stage of disease for lymph node (5.8-fold increase).
This IL-32 production concerned CD4+ T cells, B cells, macrophages and dendritic cells.
IL-32 is viewed as a double-edged sword suppressing both the immune activation but also the antiviral immune response. IL-32, IDO, ILT4, and TReg cells therefore constitute important components of an immunoregulatory axis designed to counter the pathological effects of chronic immune activation in persistent HIV-1 infection. The price the host pays for these moderating effects are impaired host defences.
In conclusion, during HIV infection IL-32 moderates chronic immune activation to avert associated immunopathology but at the same time dampens the antiviral immune response and thus paradoxically supports HIV-1 replication and viral persistence.
Smith AJ, Toledo CM, Wietgrefe SW, Duan L, Schacker TW, Reilly CS, Haase AT. The Immunosuppressive Role of IL-32 in Lymphatic Tissue during HIV-1 Infection. J Immunol. 2011 Jun 1;186(11):6576-84. Epub 2011 Apr 27.
The Immunosuppressive Role of IL-32 in Lymphatic Tissue during HIV-1 Infection - pdf attached
J Immunol 2011;186;6576-6584
Anthony J. Smith, Chad M. Toledo, Stephen W. Wietgrefe, Lijie Duan, Timothy W. Schacker, Cavan S. Reilly and Ashley T. Haase
*Department of Microbiology, Medical School, University of Minnesota, Minneap- olis, MN 55455; Department of Medicine, Medical School, University of Minnesota, Minneapolis, MN 55455; and Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, MN 55455
One pathological hallmark of HIV-1 infection is chronic activation of the immune system, driven, in part, by increased expression of proinflammatory cytokines. The host attempts to counterbalance this prolonged immune activation through compensatory mediators of immune suppression. We recently identified a gene encoding the proinflammatory cytokine IL-32 in microarray studies of HIV-1 infection in lymphatic tissue (LT) and show in this study that increased expression of IL-32 in both gut and LT of HIV-1-infected individuals may have a heretofore unappreciated role as a mediator of immune suppression. We show that: 1) IL-32 expression is increased in CD4+ T cells, B cells, macrophages, dendritic cells, and epithelial cells in vivo; 2) IL-32 induces the expression of immunosuppressive molecules IDO and Ig-like transcript 4 in immune cells in vitro; and 3) in vivo, IL-32-associated IDO/Ig-like transcript 4 expression in LT macrophages and gut epithelial cells decreases immune activation but also may impair host defenses, supporting productive viral replication, thereby accounting for the correlation between IL-32 levels and HIV-1 replication in LT. Thus, during HIV-1 infection, we propose that IL-32 moderates chronic immune activation to avert associated immunopathology but at the same time dampens the antiviral immune response and thus paradoxically supports HIV-1 replication and viral persistence.
Chronic activation of the immune system is a pathological hallmark of HIV-1 infection (1) and now widely accepted as an important negative prognosticator of disease progression in infected individuals (2). This chronic immune activation is thought to be due to the persistent nature of HIV-1 replication and the host's inability to clear the virus (3). As such, the immune response does not contract and fully return to a quiescent-like state, instead remaining in a state of sustained interplay between mediators of immune activation and immune suppression, which ultimately determines the magnitude, pace, and duration of an immune response to the invading pathogen (4). In HIV-1 infection, this indelicate balance between mediators of immune activation and immune suppression is skewed in a way that supports viral persistence, CD4+ T cell depletion, and other pathologies that result in disease progression (5-7).
Immune activation during HIV-1 infection is the result of a robust host response in lymphatic tissue (LT) (6), the primary anatomical site of viral replication, CD4+ T cell depletion, and pathology. This response is characterized by the upregulation of a vast array of genes controlling immune activation and antiviral molecules. Although ongoing immune activation is one important feature throughout HIV-1 infection in LT, there is a compensatory upregulation in expression of genes promoting immune suppression during early HIV-1 infection, likely serving as a counterbalance to moderate the immunopathological consequences of sustained immune activation (6). Maintaining this equilibrium between immune activation and immune suppression is essential, as imbalances can benefit HIV-1 replication-too much immune activation provides permissive target cells for the virus, whereas too much immune suppression can dampen innate and cell-mediated immune responses needed to contain the virus.
We have examined the complex relationship between immune activation and suppression during HIV-1 infection by transcriptionally profiling the global host response to HIV-1 infection in LT (6, 7). Using this experimental approach, we have identified global, stage-specific transcriptional signatures during HIV-1 infection (6), transcriptional correlates of viral load (7), and particular genes that provide tantalizing clues to factors and mechanisms that may be critically affecting HIV-1 replication and the host response in LT.
One such gene, the proinflammatory cytokine IL-32, was originally identified in 1992 as an unknown transcript for which expression increased in activated NK and T cells (8). IL-32 is a multi-isoform cytokine that has received growing attention recently as an important component in numerous autoimmune and inflammatory disorders (9). Thus, when we identified IL-32 in our microarray analysis as a gene increased in expression in LT during HIV-1 infection (6), we investigated its potential functional role in this anatomical niche. In this report, we show that there is a significant increase in IL-32 expression in both lymph node and gut during HIV-1 infection, that this cytokine is a potent inducer of immunosuppressive molecules IDO and Ig-like transcript 4 (ILT4), and that IL-32 expression is associated with a dampening of the antiviral immune response by reducing cell-mediated cytotoxicity, potentially accounting for the correlation between increased IL-32 levels and higher HIV-1 replication in vivo. We thus propose that the nominally proinflammatory cytokine IL-32 actually functions as a double-edged sword during HIV-1 infection, suppressing both immune activation and the antiviral immune response, thereby supporting HIV-1 replication and viral persistence.
IL-32 is thought to play an important role in various inflammatory disorders due to its purported role in fueling inflammation by inducing expression of other proinflammatory mediators (9). However, until recently (29), IL-32 has received little attention as a moderator of chronic immune activation. In this study, we provide evidence that IL-32 may have such a role in countering immune activation and inflammation during HIV-1 infection by promoting immune suppression via the induction of immunosuppressive molecules IDO and ILT4. This observation complements a previous study in which IL-32 was shown to induce the immunosuppressive cytokine IL-10 (17).
The balance between immune activation and immune suppression is critical throughout the immune system, whereby perturbation of this balance can have deleterious, immunopathological consequences for the host (30). Additionally, this balance is crucial in determining the effectiveness and ability of the immune system to initially clear acute infections or partially control persistent infections. Because HIV-1 is usually not cleared in the acute stage of infection (31), the immune system, without antiviral therapy, is confronted over a period of years with the sustained challenge of managing this balance between chronic immune activation, needed to maintain host defenses to partially control persistent viral replication, and moderating the immunopathological consequences of this chronic immune activation.
The transcriptional profiles of the acute, asymptomatic, and AIDS stages of HIV-1 infection (6) have revealed the complexity of managing chronic immune activation in persistent infection. Initially, the host's immune system responds by upregulating expression of large numbers of genes that mediate immune activation and innate and adaptive defenses. With the general failure of these defenses in clearing infection, there is an abrupt decrease in expression of most of these genes to levels indistinguishable from HIV-1-uninfected individuals in the asymptomatic stage of infection, which we have interpreted as immunoregulatory mechanisms mounted by the host to strike a balance between moderating chronic immune activation and maintaining host defenses to partially contain infection.
The mediators of this immunoregulatory transition, however, were not immediately obvious in the lists of genes with altered expression, with the exception of IL-32, IDO, and ILT4 (6), and hence the focus on these genes in the work we now report. From the evidence we present, IL-32 certainly could be one of these early immunoregulatory mediators, with increased expression at the right place and right time-in many immune cells during acute HIV-1 infection in both the gut and lymph node as well as in intestinal epithelial cells (Figs. 1, 2). The induction of IL-32 itself is likely due to other proinflammatory cytokines increased during HIV-1 infection (32), particularly IL-18 (Supplemental Fig. 1), which has been shown to be a potent inducer of IL-32 in vitro (12). The antigenicity of HIV-1 itself is unlikely to stimulate IL-32 production, as Nold et al. (33) demonstrated that infection of PBMCs with various strains of HIV-1 actually inhibited production of this cytokine rather than enhancing it.
We had also observed increased expression of ILT4 and IDO in early HIV-1 infection (6) and conjectured that these immunosuppressors were also partly responsible for the immunoregulatory transition. In this study, we show that IL-32, IDO, and ILT4 are coregulated and associated with decreased immune activation, proliferation, and cytotoxic host factors. In vitro, IL-32 induced both IDO and ILT4 expression in PBMCs, whereas in vivo, the levels of IL-32 in LT and gut correlated with both IDO and ILT4 expression levels and were, in turn, inversely correlated with markers of cell proliferation and cytotoxic NK and T cell markers.
The role of IDO in HIV-1 infection has been well documented (20, 34) in suppressing various arms of the immune system by depleting locally available stocks of the essential amino acid tryptophan. A tryptophan-depleted environment has been repeatedly described in HIV-1 infection (35-38) and thought to be responsible for inhibiting essential cellular functions through its potent antiproliferative and immunosuppressive effects (21, 22, 39). Additionally, high tryptophan catabolism within the environment can promote the local generation of TReg cells (23, 24) and other immunosuppressive molecules (e.g., ILT4) (25), a process that can lead to further IDO induction (34), resulting in a continuous cycle of immunosuppressive amplification.
Like IDO, ILT4 can also serve to further amplify an immunosuppressive environment; ILT4 expression on APCs such as macrophages can inhibit proliferation of immune cells (26, 40), render immune cells anergic and unresponsive to extracellular stimuli (26, 40, 41), and promote the local generation of TReg cells (25, 41, 42). The localization of ILT4 and IDO in vivo, mainly in macrophages, is consistent with these functions.
We think that IL-32, IDO, ILT4, and TReg cells comprise important components of an immunoregulatory axis designed to counter the pathological effects of chronic immune activation in persistent HIV-1 infections and the primate counterparts in pathogenic SIV infections. The price the host pays for these moderating effects are impaired host defenses, ranging from smaller numbers of virus-specific CTLs (27, 43) to the decreased expression of cytotoxic effectors (44-46) associated with increased IL-32 expression. In support of this, a recent study showed that splenocytes isolated from mice injected with IL-32-expressing epithelial cells displayed dampened cell-mediated cytotoxicity compared with splenocytes isolated from control mice (47). Although IL-32 has been shown in vitro to have IFN-mediated antiviral activity (33), we think in vivo that IL-32's immunosuppressive effects contribute, at best, to partial control of untreated HIV-1 infection. In sum, although it remains difficult to quantify the suppressive contributions from IL-32 itself in terms of overall cell-mediated cytotoxicity compromised during HIV-1 infection (48), these data, nevertheless, suggest that IL-32 may be a contributing factor in an immunoregulatory axis that collectively acts as a double-edged sword in lentiviral immunodeficiency infections.
IL-32 expression is increased in lymph node and gut during HIV-1 infection
To examine changes in IL-32 expression during HIV-1 infection, we used Abs recognizing IL-32 to stain gut (ileum and rectum) and lymph node (inguinal) biopsies from uninfected and HIV-1-infected individuals in each clinical stage of disease: acute (defined as individuals sampled within 4 mo of documented seroconversion), asymptomatic (defined as individuals infected for at least 4 mo with a CD4+ T cell count >200 cells/μl), and AIDS (defined as infected individuals with a CD4+ T cell count <200 cells/μl) (Table I). Compared with uninfected individuals, levels of IL-32 were significantly increased in both gut and LT during all stages of HIV-1 infection, with the highest IL-32 levels in the acute stage of disease for gut (4.8-fold increase) and AIDS stage of disease for lymph node (5.8-fold increase) (Fig. 1).
To determine the cellular source(s) of IL-32 within gut and LT, we used Abs to CD4 or CD8 (T cells), CD163 (macrophages), CD20 (B cells), CD11c (dendritic cells), killer cell lectin-like receptor subfamily C, member 1 (NK cells), and cytokeratin (epithelial cells) to colocalize IL-32 with the cell types producing this cytokine. As shown in representative images in Fig. 2, IL-32 was expressed during HIV-1 infection in CD4+ T cells, B cells, macrophages, and dendritic cells; minimal expression was detected in CD8+ T cells or NK cells (data not shown). Additionally, IL-32 was highly expressed in the mucosal epithelium of HIV-1-infected gut. Finally, the majority of IL-32+ cells were also expressing IL-18, a proinflammatory cytokine implicated in initiating IL-32 expression (12) (Supplemental Fig. 1). Thus, IL-32 is significantly increased during HIV-1 infection and broadly expressed in many cell types in both gut and LT.
IL-32 induces IDO expression
IL-32 has been categorized as a proinflammatory cytokine due to its elevated levels in various inflammatory diseases (13-15) as well as its ability in vitro to induce other proinflammatory mediators, such as TNF-α, IL-1ß, and IL-8 (12, 13, 16). Despite IL-32's association with inflammation and induction of proinflammatory cytokines, recent work has also suggested a role for IL-32 in immune suppression through its ability to stimulate IL-10 production (17). To better understand the role of IL-32 during HIV-1 infection, we first examined its effects in vitro. We treated human leukocytes (both polymorphonuclear cells and PBMCs) with rIL-32γ, the most biologically active isoform (18) and then examined expression of markers of immune activation or suppression. We found IL-32γ to be a potent inducer of IDO, a tryptophan-degrading enzyme with far-reaching immunosuppressive effects (19). IL-32γ increased IDO expression ~6-fold (p < 0.001) in both polymorphonuclear cells and PBMCs (Fig. 3). IL-32γ induced IDO production in CD163+ macrophages, CD4+ T cells, Foxp3+ T regulatory (TReg) cells, and CD11c+ dendritic cells (Supplemental Fig. 2). In contrast, IL-32γ had minimal effects on immune activation and proliferation, with no detectable increases in cell activation/proliferation markers such as Ki-67, CD38, or CD69 (data not shown), in agreement with a previous report (14).
We then showed that IL-32 expression correlated with IDO production in vivo. We stained for IDO in a subset of study individuals and found a similar pattern of protein expression as for IL-32 (Fig. 4A), resulting in a significant positive association between IL-32+ cells and IDO expression (r = 0.6850, p = 0.003) (Fig. 4B). Moreover, IDO often was coexpressed with IL-32 (Fig. 4C), predominantly in LT macrophages (Fig. 4D) and gut epithelial cells (Fig. 4A). Thus, increased expression of IL-32 during HIV-1 infection coincides with induction of the tryptophan-degrading enzyme IDO in diverse anatomical compartments such as the gut and lymph node, thereby lowering environmental tryptophan (20), which can inhibit immune cell proliferation/activation (21, 22), promote the generation of TReg cells (23, 24), and stimulate expression of other immunosuppressive molecules such as the ILT receptors (25).
IL-32 induces ILT4 expression
ILT receptors are cell-surface inhibitory molecules that bind MHC class I and render target cells anergic (26). Because we had previously identified ILT4 from our microarray analysis as a gene increased in expression in LT during HIV-1 infection (6), we examined the possibility that IL-32-induced IDO might be creating a local environment in LT that favors ILT4 expression.
We first tested in vitro whether IL-32γ could induce ILT4 expression in PBMCs. As we had shown for IDO induction, IL-32γ also increased ILT4 expression in PBMCs (~2.6-fold; p < 0.001) (Fig. 5). In vivo, we also found a similar pattern of protein expression for IL-32 and ILT4 (Fig. 6A), resulting in a significant positive association between IL-32+ cells and ILT4 expression (r = 0.9246, p < 0.0001) (Fig. 6B). Moreover, ILT4 often was coexpressed with IL-32 (Fig. 6C), predominantly in LT macrophages (Fig. 6D) and gut epithelial cells (Fig. 6A). Not surprisingly, ILT4 and IDO were also significantly associated with one another (r = 0.8790, p < 0.0001) and expressed within the same cell (Supplemental Fig. 3A, 3B). Thus, increased expression of IL-32 during HIV-1 infection likely initiates a cascade of events to moderate immune activation by inducing two potent immunosuppressors, IDO and ILT4, in both gut and lymph node.
IL-32 expression is associated with reduced immune activation, cell proliferation, and cytotoxic factors in vivo
Immunosuppressive molecules such as IDO and ILT4 could play an important role in moderating the immunopathological consequences of chronic immune activation during HIV-1 infection, but these molecules also may be detrimental to the host by dampening the antiviral immune response through inhibition of innate and adaptive cytotoxic functions. Consistent with this concept of a balance between moderating effects on immune activation/proliferation and inhibition of host defenses, we found significant inverse correlations between IL-32 expression and mRNA levels of cell proliferation markers (Ki-67, proliferating cell nuclear Ag), cell activation markers (CD38, HLA-DR), and cytotoxic mediators (perforin, granzyme, NK cell group 7 sequence, and killer-specific secretory protein of 37 kDa) in lymph node (Fig. 7).
IL-32 expression is associated with HIV-1 replication in vivo and inversely correlates with CD4+ T cell survival
The immunosuppressive effects of IDO, other immunoregulatory cytokines, and TReg cells have been implicated in facilitating SIV and HIV-1 replication (22, 27, 28). Because IL-32 can promote IDO and ILT4 expression, we looked at the relationship between HIV-1 replication and IL-32 expression in a small subset of lymph node biopsies and found a qualitative relationship between the number of HIV-1-infected cells and IL-32 expression (Fig. 8). Consistent with a role in immune suppression, we also found an inverse relationship between IL-32 expression and granzyme-producing cells in the lymph node (Fig. 8). Finally, IL-32 expression and augmented HIV-1 replication was inversely correlated with CD4+ T cell numbers in both peripheral blood (r = -0.451, p = 0.035) (Supplemental Fig. 3C) and lymph node (Fig. 8). These data suggest that IL-32-induced IDO and ILT4 expression may suppress cell proliferation/activation and downmodulate cytotoxic mediators required for effective viral clearance, creating an environment more conducive for productive HIV-1 replication, which also contributes to CD4+ T cell depletion.