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Chloroquine To Block Immune Activation - pdf attached
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"Chronic immune activation is a hallmark of HIV-1 pathogenesis....Our results suggests that a safe well tolerated drug such as chloroquine can be proposed as an adjuvant therapeutic candidate along with HAART to control immune activation in HIV-1 infection.....Chloroquine is widely accessible, inexpensive and is well tolerated when administered over periods of several years making it a good candidate for adjuvant therapy along with HAART to control immune activation in HIV-1 infection. The relevance of the findings presented in this study are particularly important as it is now known that non AIDS defining illnesses such as atherosclerosis, liver disease, renal diseases that occur despite effective HAART contribute to mortality. Immune activation and inflammation are the key contributing factors linked to these co-morbidities (49). The use of chloroquine as an adjuvant with HAART could be an effective and inexpensive approach to control immune activation and reduce the risk of comorbidities in HAART treated HIV infected individuals. The use of chloroquine in HIV-1 infected individuals in resource rich and resource poor countries need to be further investigated.....HIV-1 infection hijacks the innate immune response which directly contributes to disease pathogenesis. Chronic stimulation of pDC with non-infectious and infectious virions leads to their enhanced activation, state, production of the cytokine IFNα and an up-regulation of IDO and PDL-1, all of which are known to directly contribute to decreased T cell survival, proliferation, and function. In this study, we have shown that chloroquine blocks TLR signaling in pDC, a critical step in its activation pathway. By blocking pDC activation we observed a decrease in the immunoregulatory cytokine IFNα which in turn could reduce IFNα mediated immune activation and improve T cell survival by blocking negative modulators like IDO and PDL-1."
Chloroquine Modulates HIV-1 Induced Plasmacytoid Dendritic Cell IFN{alpha}: Implication for T cell Activation - pdf attached
Antimicrob. Agents Chemother. doi:10.1128/AAC.01246-09
published online ahead of print on 30 November 2009
Jeffrey A. Martinson, Carlos J. Montoya, Xiomara Usuga, Rollie Ronquillo, Alan L. Landay, and Seema N. Desai*
Department of Immunology and Microbiology, Rush University Medical Center, Chicago, IL-USA; Group of Immunovirology-Biogenesis, University of Antioquia, Medellin-Colombia
* To whom correspondence should be addressed. Email: Seema_N_Desai@rush.edu.
ABSTRACT
Plasmacytoid dendritic cell (pDC) mainly contribute to antiviral immunity through recognition of microbial products and viruses via intracellular toll-like receptors (TLR) 7 or 9 that results in the production of type I interferons. Though interferons reduce viral burden in acute phase of infection their role in chronic phase is unclear. Presence of elevated plasma IFN{alpha} levels in advanced HIV disease and its association with microbial translocation in chronic HIV infection lead us to hypothesize that IFN{alpha} could contribute to immune activation. Blocking IFN{alpha} production using chloroquine an endosomal inhibitor was tested in a novel in vitro model system with the aim to characterize the effects of chloroquine on HIV-1 mediated TLR signaling, IFN{alpha} production and T cell activation. Our results indicate that chloroquine blocks TLR mediated activation of pDC and MyD88 signaling as shown by decrease in the downstream signaling molecules IRAK-4, IRF-7 and inhibition of IFN{alpha} synthesis. Chloroquine decreased CD8 T cell activation induced by AT-2 HIV-1 in PBMC cultures. In addition to blocking pDC activation, chloroquine also blocked negative modulators of T cell response like IDO (indoleamine 2, 3-dioxygenase) and PDL-1 (programmed death ligand-1). Our results indicate that TLR stimulation and production of IFN-{alpha} by pDC contribute to immune activation, and blocking these pathways using chloroquine may interfere with events contributing to HIV pathogenesis. Our results suggests that a safe well tolerated drug such as chloroquine can be proposed as an adjuvant therapeutic candidate along with HAART to control immune activation in HIV-1 infection.
Introduction
Plasmacytoid dendritic cells (pDC) are an integral part of the innate and adaptive immune systems that recognize pathogens through toll-like receptor (TLR) 7 and 9 (2, 34, 42, 43, 45, 73). Since these TLRs are intracellular, their ligands require cellular uptake and endosomal maturation to trigger NF-κB and MAP kinase-mediated signals through the MyD88-dependent pathway. These TLR signals result in pDC activation/maturation as well as the production of pro-inflammatory cytokines and a large amount of type 1 interferon (IFN-α/τ) (17, 43, 46). PDC also provide negative regulatory signals that modulate and establish immune tolerance (62). Several candidate molecules expressed by pDC are implicated in negative modulation of T cell responses including the production of indoleamine 2, 3 dioxygenase (IDO) and the expression of PDL-1. IDO, the rate limiting enzyme involved in tryptophan catabolism, inhibits CD4 T-cell proliferation (8, 10, 53) and enhances T regulatory cell generation and suppressor cell function (16). The PD-1/PDL-1 interaction results in decreased T cell proliferation, cytokine production and cell mediated cytotoxicity (72). TLR mediated induction of PDL-1 expression on pDC may contribute to T cell exhaustion and dysfunction through the PD-1/PDL-1 pathway. Despite a decrease in the number of circulating pDC in HIV-1 infected individuals (3, 24, 26, 73, 74) their stimulation by microbial products via TLR7, TLR9 (48) or by HIV itself (5) results in activation of PDC and interferon-alpha (IFNα) production that contributes to pathogenesis in chronic HIV-1 disease(14). IFNα is known to be associated with elevated LPS levels in chronic HIV infection that may contribute to persistent immune activation (14).
Chloroquine, an anti-malarial drug, has immunomodulatory properties and is used in the treatment of autoimmune disorders (28, 69, 81). It is also commonly used in vitro to study the role of endosomal acidification in cellular processes, such as the TLR activation pathways in pDC induced by HIV-1 (5, 9, 25, 35, 71). It is a weak base that accumulates within the endosome of cells leading to an elevated vacuolar pH thereby inhibiting endosomal maturation and nucleic acid binding to TLR7 and TLR9 (68). HIV-1 activates pDC through TLR7 (5) which makes chloroquine a candidate for preventing HIV-1 induced activation and subsequent downstream effects on T cell activation and function.
The goal of the present study was to characterize the effects of chloroquine on HIV-1 mediated stimulation of pDC and its potential effect on modulating regulators of T cell function including IFNα induced T cell activation. Our results show that chloroquine inhibits pDC activation/maturation, up-regulation of MyD88 pathway signaling molecules IRF-7 and IRAK-4, IFN-α production, IDO synthesis, and PDL-1 expression. We also observed that CD8+ T cell activation induced by IFN-α can be modulated by chloroquine through its ability to decrease IFNα production by pDC. The central role that TLRs play in innate and adaptive immunity makes them an ideal target for therapeutic intervention using chloroquine. We propose that in HIV infection, chronic TLR stimulation and production of IFNα by pDC contributes to immune activation and immune cell dysfunction, and that blocking or modulation of these pathways with chloroquine may interfere with the events of HIV pathogenesis.
Discussion
HIV infection is associated with the impairment of immune function with deficiencies observed in nearly every type of immune cell. A better understanding of the process by which HIV hijacks innate immune cell function could be explored for alternative therapeutic approaches. Among potential mechanisms, chronic activation of pDC by direct interaction with infectious and non-infectious HIV-1 particles contribute to the maintenance of immune cell dysfunction and HIV-associated pathogenesis (8, 11, 21, 36). In this study, we used an in vitro model to demonstrate the potential immunoregulatory function of chloroquine to modulate HIV-1 induced pDC activation/maturation, IFNα production, IDO expression/activity, and PDL-1 expression.
We also show that IFNα, an outcome of pDC activation, directly contributes to CD8+ T cell activation. Using chloroquine, an inhibitor of endosomal fusion and acidification, we were able to suppress HIV-1 mediated TLR signaling in pDC and inhibit the potential deleterious effects of chronic pDC activation. Our data shows that interfering with TLR signaling reduces HIV-1 induced pDC activation products that are known to contribute to immune dysregulation in HIV-1 infection.
More than 99% of HIV-1 particles detected in the circulation are not productively infectious (35, 64). These non-infectious particles contribute to HIV-induced immunopathogenesis (21, 36). We and others have shown that AT-2 HIV-1 at concentrations between 300-1000ng/mL induces pDC activation/maturation and production of IFNα (27, 35, 36, 54, 82). The concentration of HIV (500ng/mL) used in this study is within the range found in the plasma of infected individuals, children and adults which is known to vary depending on stage of HIV disease (44, 66). Using 500ng/mL of noninfectious AT-2 inactivated HIV-1 Ada and MN virus we could activate pDC IFNα pathway. Activation of pDC by HIV-1 requires endocytosis of the virion leading to endosomal TLR7 recognition of viral RNA (5, 58) and initiation of the MyD88 pathway for the production of IFNα by pDC. Chloroquine, an inhibitor of endocytosis and endosomal acidification/maturation, prevented HIV-1 mediated TLR7 signaling in pDC as shown by the failure of pDC to up-regulate the down-stream IRAK-4 and IRF-7 signaling molecules within the MyD88 pathway. By showing that chloroquine decreases IFNα production it can be inferred that AT-2 HIV-1 utilizes TLR7 signaling in pDC and not the cytosolic anti-viral pathway mediated by RIG-1 recognition of ssRNA (41, 65). These data highlight the importance of the endolysosomal pathway in HIV-1 mediated signaling of pDC.
The production of IFNα by pDC in HIV-1 infection may also have important consequences for T cell activation and survival (67). During acute infection HIV-1 high plasma titers of IFNα provide a protective antiviral effect and help trigger the adaptive immune response (73). However, in late-stage disease elevated levels of serum IFNα is an indicator of poor clinical prognosis (30, 80). We demonstrate that 100µM chloroquine has an inhibitory effect on HIV-1 mediated production of IFNα. It has been shown that 100µM chloroquine used in treatment of leucocytes in vitro results in intracellular concentrations comparable to those obtained in vivo during chloroquine therapy (29). Our findings are similar to those of other investigators using chloroquine to modulate IFNα production in HSV infected systems (25, 50). However, in our study at the declining concentrations of chloroquine (0.5-5 µM) we observed a dose-dependent decrease in HIV-1 induced IFNα. At our lowest tested concentration of chloroquine( 0.5 µM), we observed a partial blockage of IFNα which is in contrast to a published report (71) of complete blockage of IFNα with similar concentration of chloroquine (0.5 µM) used to induce pDC activation through TLR9. These differences in concentration of chloroquine required for blocking IFNα could be attributed to their activation pathways. As previously reported, a higher concentration of chloroquine is necessary to block TLR7 endosomal maturation and acidification compared to TLR9 (51). Therefore, our results provide evidence that HIV-1 induced IFNα can be modulated with chloroquine at concentrations that are obtainable for therapeutic application.
Chronic immune activation is a hallmark of HIV-1 pathogenesis. Previous reports of elevated IFNα levels in the plasma and lymphoid tissues along with increased expression of interferon-inducible genes in HIV-infected patients has been shown to correlate with elevated T cell activation characteristic of HIV progression (14, 36, 37). In this study, CD38 activation marker, commonly used to measure the T cell activation state in HIV-infected patients (8, 20, 31, 75), was down-modulated in PBMC cultures when utilizing 5µM chloroquine . As cell separation procedures affect the uptake of chloroquine (29) we used lower dose of chloroquine (5µM) to study its effect on T cell activation in enriched T cells and compared with a similar dose of chloroquine in PBMC cultures. Chloroquine downmodulates T cell activation in AT-2 HIV-1 stimulated PBMC cultures and not in AT-2 HIV-1 stimulated enriched T cells suggesting that inhibition of pDC IFNα decreases T cell activation. Using enriched CD8+ cells we demonstrate that increased CD38 expression is induced by exogenous rIFNα2a and not by direct interaction with HIV-1 suggesting that pDC IFNα certainly contributes to CD8+ T cell activation. As previously reported(67), we did not find IFNα induced activation of CD4+ T cell (data not shown). The ability to block IFNα mediated CD8+ activation with the anti-IFNαR monoclonal antibody (64G12) and not chloroquine highlights the importance of modulating IFNα induced CD8+ T cell activation. This is the first direct evidence of chloroquine blocking pDC IFN-α mediated activation and is supported by a previous study demonstrating the ability of chloroquine to control TLR9 (CpG) or LPS induced pro-inflammatory cytokine production in mice (40).
HIV-1 also modulates immune function through the induction of negative regulatory signals which directly affect the development of the adaptive immune response. One such mechanism evaluated in our studies was the induction of the immunosuppressive enzyme, IDO in pDC following HIV-1 stimulation. We also observed an increase in kynurenine metabolites in the culture supernatants indicative of IDO regulated tryptophan catabolism. As shown by other in vitro studies HIV-1 induces IDO in pDC (10, 22, 57) which directly inhibits T cell proliferation and function through tryptophan depletion and through the activity of kynurenine metabolites (4, 12, 61). Another key regulatory function of IDO is the induction and stimulation of T regulatory cells during HIV infection (18, 23, 33) which dampen HIV specific T cell responses. Thus, development of new treatment modalities (i.e. chloroquine or hydroxychloroquine) to reverse the induction of IDO levels found in both blood cells and lymphoid tissues (4, 10, 61) may prove beneficial for the improvement of T cell proliferation and function in addition to controlling aberrant immune activation in HIV infected individuals. A second mechanism induced by HIV-1 infection to dampen anti-viral responses is the PD-1/PDL-1 pathway (9, 19, 63, 79). Our data suggests that Ònon-infectiousÓ HIV-1 (AT-2 HIV-1) exposure up-regulates PDL-1 ligand expression on pDC and is consistent with reports of other investigators showing TLR induced PDL-1 on pDC and IFNα induced PDL-1 on mDC (1, 15, 78). Manipulation of the PD-1/PDL-1 pathway in HIV-1 infection may prove beneficial in the restoration of virus specific CD8+ T cell responses by decreasing programmed death pathway induced apoptosis.
In addition to the immunomodulatory effects observed in these studies chloroquine has been shown in vitro to have direct inhibitory activity on newly produced HIV virions by altering the glycosylation of the 2G12 epitope which is located on the gp120 envelope surface protein required for virus infectivity (69, 70). Several clinical trials have been performed where chloroquine or hydroxychloroquine was given to HIV infected individuals along with antiretroviral therapy. In one study a decrease in HIV viral load measurements was reported (77). Whilst in another a decrease in plasma p24 capsid antigen levels was observed in chloroquine treated individuals while no alterations in CD4+ T lymphocyte counts were identified compared to control group (76).
HIV-1 infection hijacks the innate immune response which directly contributes to disease pathogenesis. Chronic stimulation of pDC with non-infectious and infectious virions leads to their enhanced activation, state, production of the cytokine IFNα and an up-regulation of IDO and PDL-1, all of which are known to directly contribute to decreased T cell survival, proliferation, and function. In this study, we have shown that chloroquine blocks TLR signaling in pDC, a critical step in its activation pathway. By blocking pDC activation we observed a decrease in the immunoregulatory cytokine IFNα which in turn could reduce IFNα mediated immune activation and improve T cell survival by blocking negative modulators like IDO and PDL-1. Chloroquine is widely accessible, inexpensive and is well tolerated when administered over periods of several years making it a good candidate for adjuvant therapy along with HAART to control immune activation in HIV-1 infection. The relevance of the findings presented in this study are particularly important as it is now known that non AIDS defining illnesses such as atherosclerosis, liver disease, renal diseases that occur despite effective HAART contribute to mortality. Immune activation and inflammation are the key contributing factors linked to these co-morbidities (49). The use of chloroquine as an adjuvant with HAART could be an effective and inexpensive approach to control immune activation and reduce the risk of comorbidities in HAART treated HIV infected individuals. The use of chloroquine in HIV-1 infected individuals in resource rich and resource poor countries need to be further investigated.
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