Increased Plasma Levels of the TH2 chemokine CCL18 associated with low CD4+ T cell counts in HIV-1-infected Patients with a Suppressed Viral Load.

The chemokine (C-C motif) chemokine ligand 18 (CCL18) is a structural homolog of CCL3 primarily produced by monocyte-derived cells with an M2 phenotype. Elevated levels of CCL18 have been observed in several diseases associated with malignancies and chronic inflammation. The role of CCL18 in Human Immunodeficiency Virus (HIV-1) infection remains unknown. We analyzed expression levels of T helper cell-mediated (TH2) chemokines CCL18, CCL17, and CCL22 by ELISA in plasma collected from HIV-1-infected and healthy donors. In HIV-1-infected individuals, plasma viral loads were monitored by NucliSense HIV-1 QT assay and T cell counts and expression of the activation marker CD38 were determined by flow cytometry. Our data showed a significant increase in plasma levels of CCL18 in HIV-1-infected individuals compared to uninfected controls (p < 0.001) and a significant correlation between CCL18 levels and viral load in untreated patients. No significant difference of CCL18 levels was detected among the HIV-1-infected patients treated with combined antiretroviral therapy (cART) and HIV-1-untreated patients.CCL18 values are negatively correlated with CD4+CD38+ cell numbers and total CD4+ T cell counts in patients with a suppressed viral load. Notably, plasma levels of the TH2 chemokines CCL17 and CCL22 are also elevated during HIV-1 infection. However, no correlation of CCL17 and CCL22 production with CD4+ T cell counts was detected. Presented data shows that the chemokines, CCL17, CCL18, and CCL22 are increased during HIV-1 infection. However, only increased levels of CCL18, a marker of M2 macrophages, correlate with low CD4+ T cell counts in patients with suppressed viral load, raising the possibility that CCL18 and/or CCL18-producing cells may interfere with their reconstitution in HIV-1-infected patients on cART.

Tubular cell HIV-entry through apoptosed CD4 T cells: a novel pathway.

We hypothesized that HIV-1 may enter tubular cells by phagocytosis of apoptotic fragments of HIV-1-infected T cells infiltrating tubular interstitium. The study was designed to evaluate the interaction of programmed death-1 (PD-1) receptors on CD4 T cells and programmed death ligand-1 (PD-L1) on tubular cells (HK2 and HRPTEC, primary tubular cells). Co-cultivation of HIV-1 infected lymphocytes (HIV-LY) with HK2s/HRPTECs resulted in T cell apoptosis, uptake of the apoptosed HIV-LY by HK2s/HRPTECs, tubular cell activation and HIV expression. Cytochalasin-B inhibited tubular cell HIV-LY uptake and anti-PD-L1 antibody inhibited HIV-LY apoptosis and tubular cell HIV-LY uptake, activation and HIV expression. These observations do indicate induction of apoptosis of T cells due to interaction of PD-1 and PD-L1 upon co-cultivation and subsequent phygocytosis of HIV-laden apoptotic bodies by tubular cells and thus the transfer of HIV-1 into tubular cells. These findings identify a novel pathway that facilitates HIV-1 entry into tubular cell.

EBV LMP1, a viral mimic of CD40, activates dendritic cells and functions as a molecular adjuvant when incorporated into an HIV vaccine.

HIV-1 does not significantly activate cellular immunity, which has made it difficult to use attenuated forms of HIV-1 as a vaccine. In contrast, EBV induces robust T cell responses in most infected individuals, perhaps as this virus contains LMP1, a viral mimic of CD40, which is a key activating molecule for DCs and macrophages. Consequently, studies were conducted using LMP1 and LMP1-CD40, a related construct formed by replacing the intracellular signaling domain of LMP1 with that of CD40. Upon electroporation into DCs, LMP1 and LMP1-CD40 mRNAs were sufficient to up-regulate costimulatory molecules and proinflammatory cytokines, indicating that these molecules can function in isolation as adjuvant-like molecules. As a first step toward an improved HIV vaccine, LMP1 and LMP1-CD40 were introduced into a HIV-1 construct to produce virions encoding these proteins. Transduction of DCs and macrophages with these viruses induced morphological changes and up-regulated costimulatory molecules and cytokine production by these cells. HIV-LMP1 enhanced the antigen-presenting function of DCs, as measured in an in vitro immunization assay. Taken together, these data show that LMP1 and LMP1-CD40 are portable gene cassettes with strong adjuvant properties that can be introduced into viruses such as HIV, which by themselves, are insufficient to induce protective cellular immunity.

Interleukin-21 and cellular activation concurrently induce potent cytotoxic function and promote antiviral activity in human CD8 T cells.

Infection with human immunodeficiency virus (HIV)-1 induces a progressive deterioration of the immune system that ultimately leads to acquired immune deficiency syndrome (AIDS). Murine models indicate that the common γ-chain (γ(c))-sharing cytokine interleukin (IL)-21 and its receptor (IL-21R) play a crucial role in maintaining polyfunctional T cell responses during chronic viral infections. Therefore, we analyzed the ability of this cytokine to modulate the properties of human CD8 T cells in comparison with other γ(c)-sharing cytokines (IL-2, IL-7, and IL-15). CD8 T cells from healthy volunteers were stimulated in vitro via T cell receptor signals to mimic the heightened status of immune activation of HIV-infected patients. The administration of IL-21 upregulated cytotoxic effector function and the expression of the costimulatory molecule CD28. Notably, this outcome was not accompanied by increased cellular proliferation or activation. Moreover, IL-21 promoted antiviral activity while not inducing HIV-1 replication in vitro. Thus, IL-21 may be a favorable molecule for immunotherapy and a suitable vaccine adjuvant in HIV-infected individuals.

HIV-1 harboring renal tubular epithelial cell interaction with T cells results in T cell trans-infection.

Renal biopsy data suggest that renal tubular cells may serve as a reservoir for HIV-1, however the mechanism underlying this finding has not been studied. Here we show that primary human renal proximal tubular epithelial cells (HRPTECs) have the potential to harbor HIV-1 through the DEC-205 receptor. The interaction of HIV-1 with DEC-205 results in the rapid internalization of the virus for lysosomal degradation, without establishing a productive infection. However, a small fraction of incoming virus escapes degradation and can be rescued by T cells. Since pH-modulating agents and an inhibitor of endosomal transport increased HIV-1 accumulation and trans-infection to T cells, it appears that HRPTECs endocytic compartments may be the site of viral persistence and transmission to target cells. The ability of T cells to rescue the virus from HRPTECs further supports the hypothesis that these cells have the potential to serve as a reservoir for HIV-1.

DEC-205-mediated internalization of HIV-1 results in the establishment of silent infection in renal tubular cells.

HIV-1 infection of renal cells has been proposed to play a role in HIV-1-associated nephropathy. Renal biopsy data further suggest that renal tubular cells may serve as reservoir for HIV-1. The mechanism by which HIV-1 enters these cells has not been identified. Renal tubular cells do not express any of the known HIV-1 receptors, and our results confirmed lack of the expression of CD4, CCR5, CXCR4, DC-SIGN, or mannose receptors in tubular cells. The aim of this study, therefore, was to determine the mechanism that enables viral entry into renal tubular cells. An in vitro model was used to study the HIV-1 infection of human kidney tubular (HK2) cells and to identify the receptor that enables the virus to enter these cells. Results of these studies demonstrate that the C-type lectin DEC-205 acts as an HIV-1 receptor in HK2 cells. Interaction of HIV-1 with DEC-205 results in the internalization of the virus and establishment of a nonproductive infection. HIV-1-specific strong-stop DNA is detected in the infected HK2 cells for at least 7 d, and the virus can be transmitted in trans to sensitive target cells. HIV-1 entry is blocked by pretreatment with specific anti-DEC-205 antibody. Moreover, expression of DEC-205 in cells that lack the DEC-205 receptors renders them susceptible to HIV-1 infection. These findings suggest that DEC-205 acts as an HIV-1 receptor that mediates internalization of the virus into renal tubular cells, from which the virus can be rescued and disseminated by encountering immune cells.

Macrophage inflammatory protein 1alpha inhibits postentry steps of human immunodeficiency virus type 1 infection via suppression of intracellular cyclic AMP.

Primary isolates of human immunodeficiency virus type 1 (HIV-1) predominantly use chemokine receptor CCR5 to enter target cells. The natural ligands of CCR5, the beta-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES, interfere with HIV-1 binding to CCR5 receptors and decrease the amount of virions entering cells. Although the inhibition of HIV-1 entry by beta-chemokines is well documented, their effects on postentry steps of the viral life cycle and on host cell components that control the outcome of infection after viral entry are not well defined. Here, we show that all three beta-chemokines, and MIP-1alpha in particular, inhibit postentry steps of the HIV-1 life cycle in primary lymphocytes, presumably via suppression of intracellular levels of cyclic AMP (cAMP). Productive HIV-1 infection of primary lymphocytes requires cellular activation. Cell activation increases intracellular cAMP, which is required for efficient synthesis of proviral DNA during early steps of viral infection. Binding of MIP-1alpha to cognate receptors decreases activation-induced intracellular cAMP levels through the activation of inhibitory G proteins. Furthermore, inhibition of one of the downstream targets of cAMP, cAMP-dependent PKA, significantly inhibits synthesis of HIV-1-specific DNA without affecting virus entry. These data reveal that beta-chemokine-mediated inhibition of virus replication in primary lymphocytes combines inhibitory effects at the entry and postentry levels and imply the involvement of beta-chemokine-induced signaling in postentry inhibition of HIV-1 infection.

Macrophages and lymphocytes differentially modulate the ability of RANTES to inhibit HIV-1 infection.

The beta-chemokines MIP-1alpha, MIP-1beta, and RANTES inhibit HIV-1 infection of CD4+ T cells by inhibiting interactions between the virus and CCR5 receptors. However, while beta-chemokine-mediated inhibition of HIV-1 infection of primary lymphocytes is well documented, conflicting results have been obtained using primary macrophages as the virus target. Here, we show that the beta-chemokine RANTES inhibits virus entry into both cellular targets of the virus, lymphocytes and macrophages. However, while virus entry is inhibited at the moment of infection in both cell types, the amount of virus progeny is lowered only in lymphocytes. In macrophages, early-entry restriction is lost during long-term cultivation, and the amount of virus produced by RANTES-treated macrophages is similar to the untreated cultures, suggesting an enhanced virus replication. We further show that at least two distinct cellular responses to RANTES treatment in primary lymphocytes and macrophages contribute to this phenomenon. In lymphocytes, exposure to RANTES significantly increases the pool of inhibitory beta-chemokines through intracellular signals that result in increased production of MIP-1alpha and MIP-1beta, thereby amplifying the antiviral effects of RANTES. In macrophages this amplification step does not occur. In fact, RANTES added to the macrophages is efficiently cleared from the culture, without inducing synthesis of beta-chemokines. Our results demonstrate dichotomous effects of RANTES on HIV-1 entry at the moment of infection, and on production and spread of virus progeny in primary macrophages. Since macrophages serve as a reservoir of HIV-1, this may contribute to the failure of endogenous chemokines to successfully eradicate the virus.

beta-Chemokine production in CD40L-stimulated monocyte-derived macrophages requires activation of MAPK signaling pathways.

CD40 ligand is a cell surface molecule on CD4(+) T cells that interacts with its receptor, CD40, on antigen presenting cells to mediate humoral and cellular immune responses. Our previous studies demonstrated that a trimeric soluble form of CD40L (CD40LT) activates macrophages to produce beta-chemokines and decrease CCR5 and CD4 cell surface expression, thus inducing resistance to HIV-1 infection. However, the mechanism(s) by which CD40LT mediates these effects in primary macrophages remains unclear. In this report, we demonstrate that CD40LT induces synthesis of beta-chemokines through the activation of MAPK signaling pathways. Treatment of macrophages with CD40LT results in a rapid activation of p38 and ERK1/2 mitogen-activated protein kinases. Inhibitors of these MAPKs blocked beta-chemokine production, while protein kinase A and C inhibitors had little or no effect. We also provide evidence that CD40LT stimulates beta-chemokine production directly, as well as indirectly via a TNF-alpha-dependent mechanism. At the early time points, CD40LT directly stimulated beta-chemokine production, whereas at later time points the effect was mediated to some extent by TNF-alpha. In conclusion, our results suggest that CD40-CD40L interactions are important for the activation of monocyte-derived macrophage antiviral response affecting both viral replication and the recruitment of immune cells.