Proinflammatory IL-18 Increased in HIV+ Despite HAART
"Interleukin (IL)-18 is a multifunctional and pleiotropic cytokine with unique proinflammatory, immune regulatory, proapoptotic, and atherogenic properties.......We and other investigators have shown that concentrations of IL-18 are significantly increased in the circulation of human immunodeficiency virus (HIV) type 1 (HIV-1)-infected individuals....The results presented in this study confirm earlier reports from this and other laboratories ([6-8]; reviewed in ) regarding increased serum levels of IL-18 in HIV-infected subjects, compared with healthy subjects. Of interest, HAART did not result in normalization of these levels, even in patients who responded to the treatment and had undetectable plasma viral loads. Recent studies have shown that certain anti-HIV-1 drugs (eg, protease inhibitors) induce secretion of proinflammatory cytokines like IL-1β, tumor necrosis factor-α, and IL-6.....In this study, we also provide experimental evidence to show that infection of macrophages with HIV-1 contributes to imbalanced production of IL-18 and its antagonist in HIV-infected individuals. It is noteworthy that in vivo infection of macrophages in many tissues of the body has been demonstrated in HIV-infected persons . It is widely believed that macrophages are the first type of cells to be infected via a mucosal route (which is the predominant route of infection in humans). These cells also represent an important reservoir of infection in HAART-treated HIV-infected persons . These observations imply that the observed imbalance in levels of IL-18 and IL-18BP may occur in early stages of the infection. Indeed, increased concentrations of circulating IL-18 have been reported in early stages of the infection .....HAART consisted of different regimens that included >1 nucleoside and/or nonnucleoside reverse-transcriptase inhibitors in single, dual, or triple formulations, in combination with 1 or 2 protease inhibitors or a chemokine (C-C motif) receptor 5 (CCR-5) antagonist (Vicriviroc; Schering-Plough)."
HIV-1 Causes an Imbalance in the Production of Interleukin-18 and Its Natural Antagonist in HIV-Infected Individuals: Implications for Enhanced Viral Replication
The Journal of Infectious Diseases Feb 15 2010;201:608-617
Alexandre Iannello,1,4 Mohamed-Rachid Boulassel,2 Suzanne Samarani,1,4 Cecile Tremblay,3,4 Emil Toma,3,4 Jean-Pierre Routy,2 and Ali Ahmad1,4
1Laboratory of Innate Immunity, Centre Hospitalier Universitaire Sainte-Justine Research Center, 2McGill University Health Center, McGill University, 3Division of Infectious Diseases, Centre Hospitalier de l'Universite de Montreal-Hotel Dieu, and 4Department of Microbiology and Immunology, University of Montreal, Montreal, Quebec, Canada
Background. Concentrations of interleukin (IL)-18 increase in the circulation of human immunodeficiency virus (HIV)-infected persons. However, nothing is known concerning the regulation of IL-18-binding protein (IL-18BP), which neutralizes IL-18 in vivo. This issue is addressed in the present study.
Methods. Serum samples obtained from healthy subjects and HIV-infected patients were analyzed by enzyme-linked immunosorbent assay to determine their IL-18 and IL-18BP contents. Human monocyte-derived macrophages (MDMs) were infected in vitro with HIV type 1 (HIV-1), and the production of these 2 cytokines by these cells was measured. Finally, we determined the effect of IL-18 on HIV-1 replication in human cells.
Results. In contrast to IL-18 levels, IL-18BP levels decreased in the serum of HIV-infected patients. This decrease resulted in enhanced levels of free IL-18 in the serum of such patients. The infection increased production of IL-18 but decreased that of IL-18BP in MDMs. IL-10 and transforming growth factor-β, concentrations of which are increased in HIV-infected persons, also decreased production of IL-18BP by human MDMs. Finally, recombinant human IL-18 enhanced HIV-1 replication in human CD4+ T cells.
Conclusions. Production of IL-18 and its antagonist becomes imbalanced in HIV-1-infected persons. The infection and the cytokine milieu play a role in this decreased production. The increased biological activities of IL-18 may enhance viral replication in human CD4+ T cells.
Interleukin (IL)-18 is a multifunctional and pleiotropic cytokine with unique proinflammatory, immune regulatory, proapoptotic, and atherogenic properties [1, 2]. In concert with IL-12, IL-18 acts as the most powerful inducer of interferon (IFN)-γ in natural killer (NK) and T cells . The cytokine is mainly produced by activated macrophages and several other types of cells in the body. In the circulation, IL-18 is bound specifically with a naturally occurring antagonist, IL-18-binding protein (IL-18BP). The bound IL-18 becomes functionally inactive and cannot transduce signals via IL-18-specific receptors. This protects the body from potentially harmful proinflammatory effects of the cytokine. Like IL-18, IL-18BP is constitutively produced from a wide variety of cells in the body, including macrophages. It has much higher affinity for IL-18, compared with that of IL-18 for IL-18-specific receptor [3-5]. The protein exists in humans in 4 different isoforms, designated as "a-d," which result from alternate splicing of the IL-18BP messenger RNA. Of these isoforms, the "a" isoform accounts for almost all IL-18-neutralizing activity of IL-18BP in the circulation .
We and other investigators have shown that concentrations of IL-18 are significantly increased in the circulation of human immunodeficiency virus (HIV) type 1 (HIV-1)-infected individuals ([6-8]; reviewed in ). However, to the best of our knowledge, no information exists concerning production of IL-18BP in these individuals. This information is important for understanding regulation of the biological activity of IL-18 in HIV-1 infection. We addressed this issue in the present study. We also investigated the effects of HIV-1 infection on the production of these 2 soluble mediators by human monocyte-derived macrophages (MDMs). Increased biological activities of IL-18 in HIV-infected persons may be relevant to the infection, because we observed an enhancing effect of the cytokine on HIV-1 replication in human CD4+ T cells.
The results presented in this study confirm earlier reports from this and other laboratories ([6-8]; reviewed in ) regarding increased serum levels of IL-18 in HIV-infected subjects, compared with healthy subjects. Of interest, HAART did not result in normalization of these levels, even in patients who responded to the treatment and had undetectable plasma viral loads. Recent studies have shown that certain anti-HIV-1 drugs (eg, protease inhibitors) induce secretion of proinflammatory cytokines like IL-1β, tumor necrosis factor-α, and IL-6 . Because IL-1β and IL-18 belong to the same family and share similar mechanisms of production, it is highly likely that these anti-HIV-1 drugs also induce production of IL-18 by human macrophages.
To our knowledge, the present study is the first to show that, concomitant with an increase in IL-18 concentrations, IL-18BP concentrations are significantly reduced in the circulation in HIV-infected individuals. A practical consequence of the decrease in levels of this IL-18 antagonist is that the concentrations of biologically active IL-18 become even more accentuated in the circulation of these individuals. This would have resulted in increased biological activities of IL-18 in the circulation of HIV-infected persons. Indeed, we have previously demonstrated that the biological activity of IL-18 is increased in serum samples obtained from HIV-infected individuals . In this study, we also provide experimental evidence to show that infection of macrophages with HIV-1 contributes to imbalanced production of IL-18 and its antagonist in HIV-infected individuals. It is noteworthy that in vivo infection of macrophages in many tissues of the body has been demonstrated in HIV-infected persons . It is widely believed that macrophages are the first type of cells to be infected via a mucosal route (which is the predominant route of infection in humans). These cells also represent an important reservoir of infection in HAART-treated HIV-infected persons . These observations imply that the observed imbalance in levels of IL-18 and IL-18BP may occur in early stages of the infection. Indeed, increased concentrations of circulating IL-18 have been reported in early stages of the infection .
Studies of IL-18BP have shown that its production is induced in the body as a negative-feedback mechanism in response to IL-18 for controlling proinflammatory biological effects of the cytokine. This induction occurs, at least in part, as a result of IL-18-mediated IFN-γ production by NK and T cells. It is noteworthy that IFN-γ is a powerful stimulus for production of IL-18BP . The HIV-induced decrease in production of this soluble mediator seems to occur independently of IFN-γ, because MDMs are not known to produce this cytokine. Furthermore, our data show that IL-18 can also directly inhibit production of its antagonist in human MDMs. Moreover, decreased production of IFN-γ in HIV-infected persons has been reported elsewhere . Decreased production of TH1 type cytokines, of which IFN-γ is the representative one, has been implicated in the pathogenesis of AIDS . Although IL-18 is a powerful inducer of IFN-γ in NK and T cells, it does so only in combination with other cytokines (eg, IL-12, IL-21, etc). IL-18 alone induces little of this interferon. Because production of IL-12, IL-15, and IL-21 markedly decreases in HIV-infected persons [22-24], it is not surprising that production of IFN-γ also decreases in these patients. Furthermore, immunosuppressive cytokines, such as TGF-β and IL-10, also inhibit production of this interferon in response to IL-18 and other stimuli. The concentrations of these immunosuppressive cytokines are increased in HIV-infected persons [6, 23, 25-27]. We have shown here that treatment of macrophages with these cytokines decreases production of IL-18BP, apparently in an IFN-γ-independent manner. Thus, the cytokine milieu prevalent in the circulation of HIV-infected persons also contributes to decreased production of IL-18BP in these patients.
Conflicting results concerning the effects of IL-18 on HIV-1 replication have been published in the literature . The cytokine stimulates HIV-1 replication in vitro in chronically infected human monocytic and T cell lines but not in primary human cells [28, 29]. However, it was reported to inhibit replication of both M- and T-tropic HIV-1 strains in human PBMCs, and the inhibitory effect was ascribed to IL-18-induced IFN-γ production . In PBMC cultures, the inhibitory effects of IL-18 may prevail because of the cytokine-induced production of IFN-γ by NK cells. However, IL-18 promotes HIV-1 replication in isolated CD4+ T cells, chronically infected T cell lines, and HIV-infected monocytic cells in which IL-18 alone would induce little IFN-γ. As mentioned earlier, IL-18 alone induces little IFN-γ. It induces this interferon only when acting in collaboration with other cytokines (eg, IL-12). Production of the collaborating cytokines becomes compromised in HIV-infected individuals, often in early stages of the infection (as reviewed in ). Furthermore, increased concentrations of immunosuppressive cytokines (ie, TGF-β and IL-10) in the circulation of HIV-infected individuals would also inhibit production of IFN-γ by their PBMCs. Under these circumstances, it should not be surprising if IL-18 promotes HIV-1 replication in the PBMCs (and tissues) of HIV-infected persons. This finding may explain the reported association of AIDS progression with increased serum levels of the cytokine in HIV-infected subjects [7, 31, 32].
In short, the present study provides novel insights concerning the regulation of IL-18 and its antagonist in HIV-1 infection.
Increased IL-18 and decreased IL-18BP serum concentrations in HIV-infected individuals.As shown in Figure 1A, serum samples obtained from all groups of HIV-infected individuals had significantly (p<.001) increased levels of IL-18, compared with serum samples obtained from healthy subjects. The average IL-18 concentrations in the serum samples from infected individuals were about 2.5- to 3-fold higher than those in the serum samples from control individuals. Of interest, no significant differences (p>.05) were observed between the serum IL-18 concentrations in viremic and aviremic individuals receiving HAART. Results of analysis of the IL-18BP contents of these serum samples are shown in Figure 1B. Unlike IL-18, IL-18BP was present in significantly lower (p<.001) median concentrations in the serum samples obtained from HIV-infected persons than in the serum samples obtained from control individuals, with the exception of the CIH individuals. No statistically significant (p>.05) differences were observed within the infected groups, for these 2 parameters. However, the IL-18 concentration tended to increase in aviremic patients, and the IL-18BP concentration tended to increase in chronically infected patients (both viremic and aviremic) who were receiving HAART, compared with chronically infected patients who were not receiving HAART. No statistically significant correlations were found (p>.05) between IL-18 or IL-18BP levels and viral loads, CD4+ T cell counts, or CD8+ T cell counts in all groups of infected individuals (data not shown).
Correlation between serum IL-18 and IL-18BP concentrations in healthy individuals but not in HIV-infected individuals.
Because IL-18BP neutralizes IL-18 in vivo and protects body cells from its tissue-destructive effects, we reasoned that the concentrations of the 2 soluble mediators might be correlated with each other. As shown in Figure 1C, a significant positive correlation (p<.001; r=0.64) was found between the 2 parameters in healthy persons. Of interest, no significant correlations were found between these 2 cytokines in the serum samples of HIV-infected individuals in all groups (p=.32 and r=-0.18 for patients with PI [Figure 1D], p=.47 and r= -0.12 for patients with CI [Figure 1E], p=.58 and r= -0.1 for CIH-V patients [Figure 1F], and p=.41 r= -0.23 and for CIH-A patients [Figure 1G]). These data suggest that coordinated production of these 2 soluble mediators, as observed in control subjects, is lost in HIV-infected individuals. Coordination was not restored even in those patients who responded to HAART with undetectable viral loads.
Increase in serum levels of free IL-18 in HIV-infected individuals.
Decreased levels of IL-18BP with concomitant increased levels of IL-18 resulted in a 3- to 4-fold increase in ratios of IL-18 and IL-18BP concentrations in serum samples obtained from the infected subjects, compared with those in samples obtained from control subjects (p<.001) (Figure 1H). We calculated free IL-1 concentrations in the serum samples obtained from our study participants, using the law of mass action as described in Materials and Methods. As shown in Figure 1I, we found, as was expected, an increase in serum concentrations of free IL-18 in HIV-infected subjects, compared with those in healthy subjects (p<.01). Consequently, the difference in free IL-18 concentrations between HIV-infected and healthy donors is more pronounced, compared with the difference in total IL-18 concentrations.
Effects of HIV infection on production of IL-18 and IL-18BP by human MDMs.Macrophages are the main producers of IL-18 and IL-18BP in the body.
They are also infected with HIV and act as a viral reservoir in HIV-infected individuals . Therefore, we were interested in determining the effects of HIV-1 infection on the production of IL-18 and its antagonist by these cells. For this purpose, we infected MDMs with a dual-tropic HIV-1 strain (89.6) that is known to productively infect MDMs , and we analyzed the cell culture supernatants for IL-18 and IL-18BP contents. As shown in Figure 2A, at each tested time point until 24 days after infection, the virus-infected MDMs produced significantly more (p<.01) IL-18, compared with mock-infected MDMs. However, they produced significantly less IL-18BP (p<.01) at these time points (Figure 2B). The ratios between the IL-18 and IL-18BP concentrations present in the supernatants of the mock- and HIV-infected MDMs are shown in Figure 2C. As expected, the average ratio was increased for HIV-infected MDMs, compared with mock-infected cells, at each time point.
We further calculated the concentrations of free IL-18 present in these cell culture supernatants and observed an increase in concentrations of free IL-18 in HIV-infected cells, compared with those in mock-infected cells, at each time point (Figure 2D). These data suggest that the viral infection itself induces imbalanced production of IL-18 and IL-18BP by MDMs. No significant effects (p>.05) on these 2 soluble mediators were observed when the cells were infected with the viral preparation that had been heated at 56°C for 1 h (data not shown). Essentially, similar results were observed when MDMs were infected with an M-tropic viral strain YU-2 (Figure 3). In our hands, both 89.6 and YU-2 replicate in MDMs with similar kinetics, and they reach peak production of progeny viruses by day 8 after infection (data not shown).
Next, we sought to investigate whether HIV-1 replication was required for the observed alterations in IL-18 and IL-18BP production. For this purpose, we infected MDMs with HIV (89.6) for different lengths of time with or without 3TC, a nucleoside reverse-transcriptase inhibitor that blocks viral replication . As shown in Figure 4A, treatment with 3TC decreased IL-18 production in cell culture supernatants to levels that were almost equal to those seen in mock-infected cells. The treatment also restored IL-18BP production by MDMs (Figure 4B). Taken together, these data suggest that HIV-1 replication-not binding of the virus to MDMs-results in imbalanced production of IL-18 and IL-18BP by these cells. Essentially similar results were observed when human MDMs were infected with the M-tropic HIV-1 strain YU-2 (data not shown).
Effect of IL-10 and TGF-β on IL-18BP production by MDMs.
IL-10 and TGF-β are 2 cytokines whose concentrations are often increased in the circulation of HIV-infected patients with AIDS [15, 16]. Therefore, we sought to determine their effects on the production of IL-18BP by human MDMs. For this purpose, we infected MDMs with the dual-tropic virus and added equimolar concentrations of the 2 cytokines for different lengths of time, and we then measured, by use of ELISA, the levels of IL-18BP secreted in the cell culture supernatants. As shown in Figure 5, the infection decreased IL-18BP in the culture supernatants when they were analyzed on days 1, 3, 5, and 8 after infection. Furthermore, the addition of IL-10, IL-18, or TGF-β to the MDM cultures also decreased production of this mediator on days 3, 5 and 8 after infection. Of interest, at each of these time points, the effects of the infection and the cytokines on the production of IL-18BP from MDMs were additive (Figure 5). Furthermore, the cytokines also acted in an additive manner in decreasing production of IL-18BP. Taken together, these data suggest that decreased concentrations of circulating IL-18BP from macrophages may occur in HIV-infected individuals because of their cytokine milieu, even in aviremic conditions. In contrast to their effects on IL-18BP production, cytokine treatments of the MDMs did not significantly ( ) affect IL-18 production by these cells (data not shown).
In separate experiments, we generated MDMs from 3 viremic patients and 3 HIV-seronegative control subjects. After 8 days of culture at 37°C, no significant difference was observed concerning the concentrations of IL-18 and IL-18BP in their culture supernatants (data not shown).
Enhancement of HIV-1 replication by IL-18 in human CD4+ T cells.
In accordance with our earlier results , no significant correlation existed between serum IL-18 concentrations and viral loads in HIV-infected persons (data not shown). Nevertheless, we were interested in determining the effect of IL-18 on HIV replication, because any replication-enhancing effects of the cytokine may have been suppressed by HAART in the HIV-infected individuals. For this purpose, we infected purified human CD4+ T cells with HIV-1 (strain NL4.3) and added equimolar concentrations of different cytokines (IL-18, IL-12, IL-10, or TGF-β) alone as well as in different combinations. The supernatants from these cultures were quantified for p24 contents on days 3 and 5 after infection. As shown in Figure 6, we observed a significant (p<.01) enhancing effect of recombinant human IL-10, IL-12, and IL-18 on HIV-1 replication. However, no significant (p>.05) effect of TGF-β on viral replication was observed. Furthermore, the cytokines did not show any additive effect when they were added together. We also performed experiments to determine the effect of IL-18 and other cytokines on viral replication in MDMs after infecting them with the dual-tropic HIV-1 strain (89.6). IL-18 alone or in combination with other cytokines caused no significant effect on HIV-1 replication in MDMs (data not shown).
Materials and Methods
Generation of human MDMs.
The MDMs were generated from the peripheral blood mononuclear cells (PBMCs), which were prepared from venous blood samples obtained from HIV-infected individuals and HIV-seronegative healthy subjects, as described elsewhere . The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 media that contained 10% fetal calf serum, 2 mmol/L l-glutamate, 100 µg/mL penicillin, and 100 µg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO2. To generate MDMs, the monocytes present in the PBMCs were isolated by adherence to plastic dishes and were allowed to differentiate into macrophages for 5 days via culture in culture medium with 5% human AB-positive serum and 2 ng/mL recombinant human (rh) granulocyte-macrophage colony-stimulating factor.
Antibodies and recombinant cytokines.
The cytokines used in the present study included rhIL-2 from Roche; rhIL-18, rhIL-10, and rh granulocyte-macrophage colony-stimulating factor from Biosource; and rh transforming growth factor (TGF)-β and rhIL-12 from eBioscience. Phytohemagglutinin was purchased from Sigma-Aldrich, and lamivudine (3TC) and p24 enzyme-linked immunosorbent assay (ELISA) kits were obtained from the AIDS Vaccine Program (National Cancer Institute-Frederick).
Patients and collection of serum samples.
Serum samples were obtained from 114 HIV-infected individuals and 32 age-matched healthy HIV-seronegative individuals. The characteristics of the infected individuals are presented in Table 1. The HIV infection was defined as a primary infection (PI), if <6 months had passed since the date of infection, and as chronic infection (CI), if >6 months had passed since this date. Individuals with CI who were receiving highly active antiretroviral therapy (HAART) were categorized as CIH patients. CIH patients who responded poorly to HAART and had detectable plasma viral loads were characterized as CIH viremic (CIH-V) patients. CIH patients who responded to HAART and had undetectable viral RNA in their plasma (<50 HIV-1 RNA copies/mL) were categorized as CIH aviremic (CIH-A) patients. All individuals in the CIH categories and 15 individuals in the CI category had >1 acquired immunodeficiency syndrome (AIDS)-defining condition. HAART consisted of different regimens that included 1 nucleoside and/or nonnucleoside reverse-transcriptase inhibitors in single, dual, or triple formulations, in combination with 1 or 2 protease inhibitors or a chemokine (C-C motif) receptor 5 (CCR-5) antagonist (Vicriviroc; Schering-Plough). The date of infection of each HIV-infected individual was estimated using his/her clinical and laboratory data as well as his/her medical history, as described elsewhere . We followed the guidelines proposed by the Acute HIV Infection and Early Disease Research Program sponsored by the Division of AIDS of the National Institutes of Allergy and Infectious Disease.
Measurement of IL-18 and IL-18BP concentrations.
Concentrations of IL-18 and IL-18BP in the serum samples and culture supernatants were determined using commercial ELISA kits from Bender Medsystems and R&D Systems, respectively. The detection limits for these kits are 12 pg and 60 pg/mL, respectively. The IL-18 kit measures total IL-18 (both free as well as IL-18BP-bound forms). The IL-18BP kit is specific for measurement of the "a" isoform, which accounts for almost all the IL-18-binding activity in the human circulation . For each individual, free IL-18 was calculated from the individual's data on serum IL-18 and IL-18BP concentrations, in accordance with the law of mass action, as described elsewhere . The calculation was based on the fact that IL-18BP binds IL-18 with a 1:1 stoichiometry and with a dissociation constant (Kd) of 0.4 nmol/L.
HIV-1 infection of MDMs.
In separate experiments, MDMs were infected (multiplicity of infection, 1) with a dual-tropic strain (89.6) or an M-tropic HIV-1 strain (YU-2) for 2 h at 37°C, and they were extensively washed. The cells were incubated at 37°C, and their culture supernatants were collected after different lengths of time (detailed in individual experiments), clarified by centrifugation, aliquoted, and stored at -20°C until they were analyzed. In some experiments, the effects of different cytokines on the production of IL-18 and IL-18BP or on replication of HIV-1 in MDMs were determined. In these experiments, the cytokines were added to the cell cultures at equimolar concentrations (15 µmol/L) at the time of infection.
Isolation and HIV-1 infection of CD4+ T cells.
To investigate the effect of IL-18 (either alone or in combination with other cytokines) on HIV-1 replication in CD4+ T cells, we isolated these cells from the PBMCs of healthy HIV-seronegative donors. The PBMCs were activated with rhIL-2 (100 U/mL) and phytohemagglutinin (10 µg/mL) for 3 days at 37°C. CD4+ T cells were isolated by negative selection performed using a kit (Stem Cell Technology). The purified CD4+ T cells were >95% pure, as verified by flow cytometry (data not shown). The cells were infected (multiplicity of infection, 1) in vitro with HIV-1 (NL4.3) for 2 h at 37°C, and they were extensively washed with the culture medium to remove the residual virus. The cytokines (indicated in individual experiments) were added to the infected cell cultures immediately after the infection. The culture supernatants were collected after different lengths of time (as detailed in individual experiments), clarified by centrifugation, aliquoted, and stored at -20°C until they were analyzed to determine their HIV-1 p24 contents.
Statistical analysis. The IL-18 and IL-18BP data from serum samples were analyzed using nonparametric one-way analysis of variance (Kruskal-Wallis test) and Dunn's multiple comparison test. The data from in vitro experiments were analyzed using classical one-way analysis of variance and the Tukey multiple comparison test. Pearson's correlation was used to find correlation between 2 variables. The software used was GraphPad software (version 4; Prism).