Interleukin-18 enhances HIV-1 production in a human chronically-infected T cell line (H9-V)

Interleukin-18 (IL-18) is a recently identified immunoregulatory cytokine expressed by activated macrophages, that induces production of interferon-gamma (IFN-gamma) and Th-1 development. Recently some investigators reported controversial in vitro data on IL-18 stimulation of HIV-1 replication in several cell lines. In the present study the effect of IL-18 on HIV replication in a human chronically HIV-1-infected lymphocytic T cell line (H9-V) was investigated. HIV-1 replication was determined by an immunoassay method in order to evaluate the content of p24 antigen in the cell culture supernatants. Stimulation of H9-V cells with IL-18 resulted in increased production of p24, especially at concentrations of 0.01 microg ml(-1) and 0.10 microg ml(-1). Moreover a significant and persistent IL-18 stimulation of HIV-1 replication was observed at a concentration of 0.01 microg ml(-1) during a 7-day period. Pre-treatment of IL-18 with a specific neutralizing monoclonal antibody significantly reduced HIV-1 replication. These experiments show that IL-18 promotes the increase of HIV-1 replication in human chronically-infected lymphocytic T cells and confirm the role of IL-18 as a proimflammatory cytokine in stimulating and maintaining HIV-1 replication during the course of the disease. In a successive set of experiments, since one of the main activities of IL-18 is the induction of IFN-gamma, we evaluated the effect of this biological modifier on H9-V cells. In particular, IFN-gamma shows a significant effect on cell replication and on reduction of CD4 and CD71 surface expression.     

Elimination of HIV-1-Infected Primary T Cell Reservoirs in an In Vitro Model of Latency

Establishment of long-lived cellular reservoirs of HIV-1 represents a major therapeutic challenge to virus eradication. In this study, we utilized a human primary cell model of HIV-1 latency to evaluate the requirements for efficient virus reactivation from, and the selective elimination of, latently infected human T cells. Ectopic expression of BCL2 supported the replication and spread of R5-tropic HIV-1 in activated CD4⁺ T cells. After IL-2 withdrawal, the HIV-1-infected T cells survived as resting cells for several months. Unexpectedly, these resting T cells continue to produce detectable levels of infectious virus, albeit at a lower frequency than cells maintained in IL-2. In the presence of HIV-1 inhibitors, reactivation of the resting T cells with γc-cytokines and allogeneic dendritic cells completely extinguished HIV-1 infectivity. We also evaluated the ability of the bacterial LukED cytotoxin to target and kill CCR5-expressing cells. After γc-cytokine stimulation, LukED treatment eliminated both HIV-1-infected resting cells and the non-infected CCR5⁺ cells. Importantly, complete clearance of in vitro HIV-1 reservoirs by LukED required a lower threshold of cytokine signals relative to HIV-1 inhibitors. Thus, the primary T cell-based HIV-1 latency model could facilitate the development of novel agents and therapeutic strategies that could effectively eradicate HIV-1.     

Interleukin-18 stimulates HIV-1 replication in a T-cell line

Interleukin-18 (IL-18) is a recently identified proinflammatory cytokine. Its ability to induce interferon-g suggests a potential virustatic effect. On the other hand, it stimulates NFkB - an activator of HIV replication. Recently, stimulation of HIV-1 in monocytic cells has been demonstrated. In the present study, the influence of IL-18 on HIV-1 replication in lymphatic cells was investigated. Hut78 cells were infected with HIV-1 in the presence of recombinant human IL-18 expressed either in E. coli or eucaryotically by baculovirus in Sf9 cells. HIV-1 replication was monitored by p24 ELISA and endpoint titration of culture supernatants on C8166 cells. The addition of IL-18 led to a 3- to 15-fold enhancement of HIV replication in Hut78 cells. By addition of neutralising monoclonal anti-IL-18 antibodies, this effect of IL-18 was reduced by 75%. Exposure of Hut78 to IL-18 prior to HIV infection could exclude the possibility that IL-18 promotes infection of cells. Taken together, these data provide direct evidence for an IL-18-mediated enhancement of HIV-1 replication in lymphatic cells.     

Regulation of interleukin-18 by THP-1 monocytoid cells stimulated with HIV-1 and Nef viral protein

Interleukin (IL)-18 is a proinflammatory cytokine that plays an important role in both innate and adaptive immune responses against several infectious pathogens. Relatively little is known about its production in HIV-1 infection, and there are controversial data on the influence of IL-18 on HIV-1 replication in vitro. In this study, we investigated the effect of HIV-1 infection, and challenge with recombinant HIV-1 proteins, on IL-18 production by THP-1 cells. This is a monocytoid cell line spontaneously producing IL-18, and consequently is particularly suitable for the study of HIV-1 effects on this type of cytokine regulation. The results reported here demonstrate a significant reduction in IL-18 secretion during HIV-infection. In fact, low levels of IL-18 were released until 120 h from viral challenge (15 +/- 11 pg/mL at 24 h and 17 +/- 13 at 96 h and < 12.5 at 120 h), whereas IL-18 production by uninfected control cells was 193 +/- 104 pg/mL and 214 +/- 114 pg/mL at 24 h and 120 h respectively. At 168 h of incubation, IL-18 production by infected and uninfected cells was found to be 164 +/- 88 pg/mL and 325 +/- 101 pg/mL respectively (p = 0.001). Of the following viral proteins: gp 120, p24 and Nef, only the last one induced decreased IL-18 secretion in the supernatants of THP-1 cells. This effect is more evident with the concentrations of 5 -1.25 microg/mL of Nef protein (p < 0.0001). In conclusion, our data show that HIV-1 and its regulatory protein, Nef, are able to down-regulate the release of IL-18, in vitro. These results confirm that a variety of modulating effects on the immune response, induced by HIV-infection, may facilitate progression of HIV-1 infection.     

Cytokine Regulation of Human Immunodeficiency Virus Type 1 Entry and Replication in Human Monocytes/Macrophages through Modulation of CCR5 Expression

Human macrophages express chemokine receptors that act as coreceptors for human immunodeficiency virus type 1 (HIV-1) and are major targets for HIV-1 infection in vivo. The effects of cytokines on HIV-1 infection of macrophages and on the expression of CCR5, the principal coreceptor for macrophage-tropic viruses, have now been investigated. Expression of CCR5 on the surface of freshly isolated human monocytes was virtually undetectable by flow cytometry with the monoclonal antibody 5C7. However, after culture of monocytes for 48 h in serum-free medium, approximately 30% of the resulting macrophages expressed CCR5 and the cells were susceptible to infection by macrophage-tropic HIV-1. Addition of either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) to the cultures markedly increased both the extent of HIV-1 entry and replication as well as surface expression of CCR5. In contrast, addition of the T-helper 2 (Th2) cell-derived cytokine interleukin-4 (IL-4) or IL-13 prevented the expression of CCR5 induced by culture in medium alone, and IL-4 inhibited virus entry, replication, and cytopathicity under these conditions. IL-4 or IL-13 also prevented the stimulatory effects of M-CSF or GM-CSF on CCR5 expression as well as HIV-1 entry and replication. In addition, IL-4 reversed the increase in CCR5 expression induced by pretreatment of cells with M-CSF. Although IL-10 also inhibits HIV-1 replication in macrophages, it did not suppress surface CCR5 expression induced by colony-stimulating factors. These results indicate that the cytokine environment determines the susceptibility of macrophages to HIV-1 infection by various mechanisms, one of which is the regulation of HIV-1 coreceptor expression.     

Increased Escherichia coli-Induced Interleukin-23 Production by CD16+ Monocytes Correlates with Systemic Immune Activation in Untreated HIV-1-Infected Individuals

The level of microbial translocation from the intestine is increased in HIV-1 infection. Proinflammatory cytokine production by peripheral antigen-presenting cells in response to translocated microbes or microbial products may contribute to systemic immune activation, a hallmark of HIV-1 infection. We investigated the cytokine responses of peripheral blood myeloid dendritic cells (mDCs) and monocytes to in vitro stimulation with commensal enteric Escherichia coli in peripheral blood mononuclear cells (PBMC) from untreated HIV-1-infected subjects and from uninfected controls. Levels of interleukin 23 (IL-23) produced by PBMC from HIV-1-infected subjects in response to E. coli stimulation were significantly higher than those produced by PBMC from uninfected subjects. IL-23 was produced primarily by CD16⁺ monocytes. This subset of monocytes was increased in frequency and expressed higher levels of Toll-like receptor 4 (TLR4) in HIV-1-infected individuals than in controls. Blocking TLR4 on total CD14⁺ monocytes reduced IL-23 production in response to E. coli stimulation. Levels of soluble CD27, an indicator of systemic immune activation, were elevated in HIV-1-infected subjects and were associated with the percentage of CD16⁺ monocytes and the induction of IL-23 by E. coli, providing a link between these parameters and systemic inflammation. Taken together, these results suggest that IL-23 produced by CD16⁺ monocytes in response to microbial stimulation may contribute to systemic immune activation in HIV-1-infected individuals.     

Interleukin-2 Inhibits HIV-1 Replication in Some Human T Cell Lymphotrophic Virus-1-infected Cell Lines via the Induction and Incorporation of APOBEC3G into the Virion

IL-2 has been used in culture of primary T cells to maintain cell proliferation. We have previously reported that IL-27 inhibits HIV-1 replication in primary T cells in the presence of IL-2. To gain a better understanding of the mechanisms involved in this inhibitory effect, we attempted to investigate in detail the effects of IL-27 and IL-2 using several cell lines. Unexpectedly, IL-27 did not inhibit HIV-1 in T cell lines, whereas IL-2 inhibited HIV-1 replication in the human T cell lymphotrophic virus (HTLV)-1-transformed T cell lines, MT-2, MT-4, SLB-1, and ATL-2. No effects were seen in HTLV-1-negative cell lines. Utilizing MT-2 cells, we demonstrated that IL-2 treatment inhibited HIV-1 syncytia-inducing ability and dose-dependently decreased supernatant p24 antigen levels by >90%. Using real time PCR and Western blot analysis, we observed that IL-2 treatment induced the host restriction factor, APOBEC3G with accumulation into the lower molecular mass active form as characterized by FPLC. Further analysis revealed that the virus recovered from IL-2-treated MT-2 cells had impaired replication competency. This was found to be due to incorporation of APOBEC3G into the virion despite the presence of Vif. These findings demonstrate a novel role for IL-2 in regulating production of infectious HIV-1 virions in HTLV-1-infected cells through the induction of APOBEC3G.     

Interleukin-15 but not interleukin-7 abrogates vaccine-induced decrease in virus level in simian immunodeficiency virus mac251-infected macaques

The loss of CD4(+) T cells and the impairment of CD8(+) T cell function in HIV infection suggest that pharmacological treatment with IL-7 and IL-15, cytokines that increase the homeostatic proliferation of T cells and improve effector function, may be beneficial. However, these cytokines could also have a detrimental effect in HIV-1-infected individuals, because both cytokines increase HIV replication in vitro. We assessed the impact of IL-7 and IL-15 treatment on viral replication and the immunogenicity of live poxvirus vaccines in SIV(mac251)-infected macaques (Macaca mulatta). Neither cytokine augmented the frequency of vaccine-expanded CD4(+) or CD8(+) memory T cells, clonal recruitment to the SIV-specific CD8(+) T cell pool, or CD8(+) T cell function. Vaccination alone transiently decreased the viral set point following antiretroviral therapy suspension. IL-15 induced massive proliferation of CD4(+) effector T cells and abrogated the ability of vaccination to decrease set point viremia. In contrast, IL-7 neither augmented nor decreased the vaccine effect and was associated with a decrease in TGF-beta expression. These results underscore the importance of testing immunomodulatory approaches in vivo to assess potential risks and benefits for HIV-1-infected individuals.     

Macrophage Resistance to HIV-1 Infection Is Enhanced by the Neuropeptides VIP and PACAP

It is well established that host factors can modulate HIV-1 replication in macrophages, critical cells in the pathogenesis of HIV-1 infection due to their ability to continuously produce virus. The neuropeptides VIP and PACAP induce well-characterized effects on macrophages through binding to the G protein-coupled receptors VPAC1, VPAC2 and PAC1, but their influence on HIV-1 production by these cells has not been established. Here, we describe that VIP and PACAP reduce macrophage production of HIV-1, acting in a synergistic or additive manner to decrease viral growth. Using receptor antagonists, we detected that the HIV-1 inhibition promoted by VIP is dependent on its ligation to VPAC1/2, whereas PACAP decreases HIV-1 growth via activation of the VPAC1/2 and PAC1 receptors. Specific agonists of VPAC2 or PAC1 decrease macrophage production of HIV-1, whereas sole activation of VPAC1 enhances viral growth. However, the combination of specific agonists mimicking the receptor preference of the natural neuropeptides reproduces the ability of VIP and PACAP to increase macrophage resistance to HIV-1 replication. VIP and PACAP up-regulated macrophage secretion of the β-chemokines CCL3 and CCL5 and the cytokine IL-10, whose neutralization reversed the neuropeptide-induced inhibition of HIV-1 replication. Our results suggest that VIP and PACAP and the receptors VPAC2 and PAC1 could be used as targets for developing alternative therapeutic strategies for HIV-1 infection.     

HIV-1 inhibits phagocytosis and inflammatory cytokine responses of human monocyte-derived macrophages to P. falciparum infected erythrocytes

HIV-1 infection increases the risk and severity of malaria by poorly defined mechanisms. We investigated the effect of HIV-1_Ba-L infection of monocyte-derived macrophages (MDM) on phagocytosis of opsonised P. falciparum infected erythrocytes (IE) and subsequent proinflammatory cytokine secretion. Compared to mock-infected MDM, HIV-1 infection significantly inhibited phagocytosis of IE (median (IQR) (10 (0–28) versus (34 (27–108); IE internalised/100 MDM; p = 0.001) and decreased secretion of IL-6 (1,116 (352–3,387) versus 1,552 (889–6,331); pg/mL; p = 0.0078) and IL-1β (16 (7–21) versus 33 (27–65); pg/mL; p = 0.0078). Thus inadequate phagocytosis and cytokine production may contribute to impaired control of malaria in HIV-1 infected individuals.     

Differential Induction of Interleukin-10 in Monocytes by HIV-1 Clade B and Clade C Tat Proteins

The clade B human immunodeficiency virus, type 1 (HIV-1) Tat (trans-acting regulatory protein) induces interleukin-10 (IL-10) production in monocytes. IL-10, an anti-inflammatory cytokine, down-regulates proinflammatory cytokines and suppresses the immune response, leading to a rapid progression from HIV-1 infection to AIDS. Nine clades of HIV-1 are responsible for the majority of infections worldwide. Recent studies demonstrate that different HIV-1 clades have biological differences in relation to transmission, replication, and disease progression. In this study, we show that the cysteine to serine mutation at position 31, found in >90% of HIV-1 clade C Tat proteins, results in a marked decrease in IL-10 production in monocytes compared with clade B Tat. Additionally, the C31S mutation found in C Tat is responsible for the inability of these Tat proteins to produce high IL-10 levels in monocytes due to its inability to induce intracellular calcium flux through L-type calcium channels. Moreover, we show that p38α/p38β and phosphoinositide 3-kinase are crucial to Tat-induced IL-10 production. These findings provide further evidence that HIV-1 clades differ in their biological properties that may impact HIV-1 pathogenesis and disease progression.     

Interleukin-27 Is Differentially Associated with HIV Viral Load and CD4+ T Cell Counts in Therapy-Na?ve HIV-Mono-Infected and HIV/HCV-Co-Infected Chinese

Human Immunodeficiency Virus (HIV) infection and the resultant Acquired Immunodeficiency Syndrome (AIDS) epidemic are major global health challenges; hepatitis C virus (HCV) co-infection has made the HIV/AIDS epidemic even worse. Interleukin-27 (IL-27), a cytokine which inhibits HIV and HCV replication in vitro, associates with HIV infection and HIV/HCV co-infection in clinical settings. However, the impact of HIV and HCV viral loads on plasma IL-27 expression levels has not been well characterized. In this study, 155 antiretroviral therapy-naïve Chinese were recruited. Among them 80 were HIV- and HCV-negative healthy controls, 45 were HIV-mono-infected and 30 were HIV/HCV-co-infected. Plasma level HIV, HCV, IL-27 and CD4+ number were counted and their correlation, regression relationships were explored. We show that: plasma IL-27 level was significantly upregulated in HIV-mono-infected and HIV/HCV-co-infected Chinese; HIV viral load was negatively correlated with IL-27 titer in HIV-mono-infected subjects whereas the relationship was opposite in HIV/HCV-co-infected subjects; and the relationships between HIV viral loads, IL-27 titers and CD4⁺ T cell counts in the HIV mono-infection and HIV/HCV co-infection groups were dramatically different. Overall, our results suggest that IL-27 differs in treatment-naïve groups with HIV mono-infections and HIV/HCV co-infections, thereby providing critical information to be considered when caring and treating those with HIV mono-infection and HIV/HCV co-infection.     

Selective Loss of Early Differentiated,Highly Functional PD1high CD4 T Cells with HIV Progression

The role of PD-1 expression on CD4 T cells during HIV infection is not well understood. Here, we describe the differential expression of PD-1 in CD127^high CD4 T cells within the early/intermediate differentiated (EI) (CD27^highCD45RA^low) T cell population among uninfected and HIV-infected subjects, with higher expression associated with decreased viral replication (HIV-1 viral load). A significant loss of circulating PD-1^highCTLA-4^low CD4 T cells was found specifically in the CD127^highCD27^highCD45RA^low compartment, while initiation of antiretroviral treatment, particularly in subjects with advanced disease, reversed these dynamics. Increased HIV-1 Gag DNA was also found in PD-1^high compared to PD-1^low ED CD4 T cells. In line with an increased susceptibility to HIV infection, PD-1 expression in this CD4 T cell subset was associated with increased activation and expression of the HIV co-receptor, CCR5. Rather than exhaustion, this population produced more IFN-g, MIP1-a, IL-4, IL-10, and IL-17a compared to PD-1^low EI CD4 T cells. In line with our previous findings, PD-1^high EI CD4 T cells were also characterized by a high expression of CCR7, CXCR5 and CCR6, a phenotype associated with increased in vitro B cell help. Our data show that expression of PD-1 on early-differentiated CD4 T cells may represent a population that is highly functional, more susceptible to HIV infection and selectively lost in chronic HIV infection.