The effects of heightened microbial translocation on B cells during HIV infection are unknown. We examined the in vitro effects of HIV and lipopolysaccharide (LPS) on apoptosis of CD27+ IgD- memory B (mB) cells from healthy controls. In vivo analysis was conducted on a cohort of 82 HIV+ donors and 60 healthy controls. In vitro exposure of peripheral blood mononuclear cells (PBMCs) to LPS and HIV led to mB cell death via the Fas/Fas ligand (FasL) pathway. Plasmacytoid dendritic cells (pDCs) produced FasL in response to HIV via binding to CD4 and chemokine coreceptors. HIV and LPS increased Fas expression on mB cells in PBMCs, which was dependent on the presence of pDCs and monocytes. Furthermore, mB cells purified from PBMCs and pretreated with both HIV and LPS were more sensitive to apoptosis when cocultured with HIV-treated pDCs. Blocking the interferon receptor (IFNR) prevented HIV-stimulated FasL production in pDCs, HIV-plus-LPS-induced Fas expression, and apoptosis of mB cells. In vivo or ex vivo, HIV+ donors have higher levels of plasma LPS, Fas expression on mB cells, and mB cell apoptosis than controls. Correspondingly, in HIV+ donors, but not in controls, a positive correlation was found between plasma FasL and HIV RNA levels and between Fas expression on mB cells and plasma LPS levels. This work reveals a novel mechanism of mB cell apoptosis mediated by LPS and HIV through the Fas/FasL pathway, with key involvement of pDCs and type I IFN, suggesting a role for microbial translocation in HIV pathogenesis.
Despite the strong correlation of T-cell CD38 expression with HIV disease progression, evidence linking CD38 expression and dysfunction at the single cell level is scant. Since CD38 memory CD4 T cells, especially those from HIV-infected persons, fail to induce CD154 (CD40L) while responding to a superantigen with interferon (IFN)-? or interleukin (IL)-2, we aimed to determine if recall responses to cytomegalovirus (CMV) were similarly affected in the CD38 memory CD4 T-cell subpopulation.
Systemic inflammation has been linked to a failure to normalize CD4(+) T-cell numbers in treated human immunodeficiency virus (HIV) infection. Although inflammatory cytokines such as interleukin 6 (IL-6) are predictors of disease progression in treated HIV infection, it is not clear how or whether inflammatory mediators contribute to immune restoration failure.
The mechanisms of increased memory CD4+ T cell cycling in HIV disease are incompletely understood but have been linked to antigen stimulation, homeostatic signals, or exposure to microbial products and the inflammatory cytokines that they induce. We examined the phenotype and V? family distribution in cycling memory CD4+ T cells among 52 healthy and 59 HIV-positive (HIV+) donors. Cycling memory CD4+ T cells were proportionally more frequent in subjects with HIV infection than in controls, more often expressed CD38 and PD-1, and less frequently expressed OX40 and intracellular CD40L. OX40 expression on memory CD4+ T cells was induced in vitro by anti-CD3, interleukin-2 (IL-2), IL-7, or IL-15 but not by Toll-like receptor ligands. In HIV+ donors, memory CD4+ T cell cycling was directly related to plasma lipopolysaccharide (LPS) levels, to plasma HIV RNA levels, and to memory CD8+ T cell cycling and was inversely related to peripheral blood CD4+ T cell counts but not to the levels of IL-2, IL-7, or IL-15, while in HIV-negative donors, memory CD4+ T cell cycling was related to IL-7 levels and negatively related to the plasma levels of LPS. In both controls and HIV+ donors, cycling memory CD4+ T cells had a broad distribution of V? families comparable to that of noncycling cells. Increased memory CD4+ T cell cycling in HIV disease is reflective of generalized immune activation and not driven primarily by cognate peptide stimulation or exposure to common gamma-chain cytokines. This cycling may be a consequence of exposure to microbial products, to plasma viremia, or, otherwise, to proinflammatory cytokines.
5P12-RANTES is a recently developed chemokine analogue that has shown high level protection from SHIV infection in macaques. However, the feasibility of using 5P12-RANTES as a long-term HIV prevention agent has not been explored partially due to the lack of available delivery devices that can easily be modified for long-term release profiles. Glycosaminoglycans (GAGs) have been known for their affinity for various cytokines and chemokines, including native RANTES, or CCL5. In this work, we investigated used of GAGs in generating a chemokine drug delivery device. Initial studies used surface plasmon resonance analysis to characterize and compare the affinities of different GAGs to 5P12-RANTES. These different GAGs were then incorporated into drug delivery polymeric hydrogels to engineer sustained release of the chemokines. In vitro release studies of 5P12-RANTES from the resulting polymers were performed, and we found that 5P12-RANTES release from these polymers can be controlled by the amount and type of GAG incorporated. Polymer disks containing GAGs with stronger affinity to 5P12-RANTES resulted in more sustained and longer term release than did polymer disks containing GAGs with weaker 5P12-RANTES affinity. Similar trends were observed by varying the amount of GAGs incorporated into the delivery system. 5P12-RANTES released from these polymers demonstrated good levels of CCR5 blocking, retaining activity even after 30 days of incubation.
Human beta defensin-3 (hBD-3) is an antimicrobial peptide with diverse functionality. We investigated the capacity of hBD-3 and for comparison, Pam3CSK4 and LL-37, to induce co-stimulatory molecules and chemokine expression in monocytes. These stimuli differentially induced CD80 and CD86 on the surface of monocytes and each stimulant induced a variety of chemokines including MCP-1, Gro-alpha, MDC and Mip1beta, while only hBD-3 and Pam3CSK4 significantly induced the angiogenesis factor, Vascular endothelial growth factor. HBD-3 induced similar chemokines in monocyte-derived macrophages and additionally induced expression of RANTES in these cells. Comparison of monocytes from HIV+ and HIV- donors indicated that monocytes from HIV+ donors were more likely to spontaneously express certain chemokines (Mip1alpha, Mip1beta and MCP-1) and less able to increase expression of other molecules in response to hBD-3 (MDC, Gro-alpha and VEGF). Chemokine receptor expression (CCR5, CCR2 and CXCR2) was relatively normal in monocytes from HIV+ donors compared to cells from HIV- donors with the exception of diminished expression of the receptor for MDC, CCR4, which was reduced in the patrolling monocyte subset (CD14+CD16++) of HIV+ donors. These observations implicate chemokine induction by hBD-3 as a potentially important mechanism for orchestrating cell migration into inflamed tissues. Alterations in chemokine production or their receptors in monocytes of HIV-infected persons could influence cell migration and modify the effects of hBD-3 at sites of inflammation. This article is protected by copyright. All rights reserved.
HIV infection contributes to accelerated rates of progression of liver fibrosis during hepatitis C virus (HCV) infection, and HCV liver disease contributes to mortality during HIV infection. Although mechanisms underlying these interactions are not well known, soluble and cellular markers of immune activation associate with disease progression during both infections.
The determinants of HIV-1-associated lymphadenopathy are poorly understood. We hypothesized that lymphocytes could be sequestered in the HIV-1+ lymph node (LN) through impairments in sphingosine-1-phosphate (S1P) responsiveness. To test this hypothesis, we developed novel assays for S1P-induced Akt phosphorylation and actin polymerization. In the HIV-1+ LN, naïve CD4 T cells and central memory CD4 and CD8 T cells had impaired Akt phosphorylation in response to S1P, whereas actin polymerization responses to S1P were impaired dramatically in all LN maturation subsets. These defects were improved with antiretroviral therapy. LN T cells expressing CD69 were unable to respond to S1P in either assay, yet impaired S1P responses were also seen in HIV-1+ LN T cells lacking CD69 expression. Microbial elements, HIV-1, and interferon ? - putative drivers of HIV-1 associated immune activation all tended to increase CD69 expression and reduce T-cell responses to S1P in vitro. Impairment in T-cell egress from lymph nodes through decreased S1P responsiveness may contribute to HIV-1-associated LN enlargement and to immune dysregulation in a key organ of immune homeostasis.
HIV disease results in decreased IL-7 receptor expression and IL-7 responsiveness in T cells. To explore mechanisms of these deficiencies, we compared CD127 expression and IL-7 induction of P-STAT5 in T cells from HIV-infected persons with serum concentrations of cytokines (IL-7, IL-6 and IL-15), markers of microbial translocation (sCD14 and LPS), and with an indicator of oxidative stress (malondialdehyde (MDA) adducts). CD127 expression was directly related to IL-7 responsiveness in most CD8+ T cell subsets but not in CD4+ T cells from HIV-infected persons. MDA adducts were increased in serum of HIV-infected patients and were inversely related to IL-7 responsiveness in CD8+ T cells and in central memory CD4+ T cells. Incubation of T cells from healthy controls with hydrogen peroxide resulted in impairments in IL-7 induction of P-STAT5. These findings suggest that oxidative stress that is characteristic of HIV disease could contribute to impairments in IL-7 responsiveness and disrupt T cell homeostasis.
Type-I interferon (IFN-I) has been increasingly implicated in HIV-1 pathogenesis. Various studies have shown elevated IFN-I and an IFN-I-induced gene and protein expression signature in HIV-1 infection, yet the elevated IFN-I species has not been conclusively identified, its source remains obscure and its role in driving HIV-1 pathogenesis is controversial. We assessed IFN-I species in plasma by ELISAs and bioassay, and we investigated potential sources of IFN-I in blood and lymph node tissue by qRT-PCR. Furthermore, we measured the effect of therapeutic administration of IFN? in HCV-infected subjects to model the effect of IFN? on chronic immune activation. IFN-I bioactivity was significantly increased in plasma of untreated HIV-1-infected subjects relative to uninfected subjects (p = 0.012), and IFN? was the predominant IFN-I subtype correlating with IFN-I bioactivity (r = 0.658, p<0.001). IFN? was not detectable in plasma of subjects receiving anti-retroviral therapy. Elevated expression of IFN? mRNA was limited to lymph node tissue cells, suggesting that peripheral blood leukocytes are not a major source of IFN? in untreated chronic HIV-1 infection. Plasma IFN-I levels correlated inversely with CD4 T cell count (p = 0.003) and positively with levels of plasma HIV-1 RNA and CD38 expression on CD8 T cells (p = 0.009). In hepatitis C virus-infected subjects, treatment with IFN-I and ribavirin increased expression of CD38 on CD8 T cells (p = 0.003). These studies identify IFN? derived from lymph nodes, rather than blood leukocytes, as a possible source of the IFN-I signature that contributes to immune activation in HIV-1 infection.
Inflammation and infection are associated with premature birth and with activation of the fetal immune system. We hypothesized that exposure to microbial Toll-like receptor (TLR) ligands plays an important role in neonatal T-cell maturation and that early exposure to microbial products may result in early T-cell maturation and a tendency for these matured effector cells to change their homing receptor patterns.
Human ?-defensin 3 (hBD-3) activates antigen-presenting cells through Toll-like receptors (TLRs) 1/2. Several TLR1/2 agonists have been identified but little is known about how they might differentially affect cellular activation. We compared the effects of hBD-3 with those of another TLR1/2 agonist, Pam(3) CSK(4) , in human monocytes. Monocytes incubated with hBD-3 or Pam(3) CSK(4) produced interleukin-6 (IL-6), IL-8 and IL-1?, but only Pam(3) CSK(4) induced IL-10. The IL-10 induction by Pam(3) CSK(4) caused down-modulation of the co-stimulatory molecule, CD86, whereas CD86 expression was increased in monocytes exposed to hBD-3. Assessment of signalling pathways linked to IL-10 induction indicated that mitogen-activated protein kinases were activated similarly by hBD-3 or Pam(3) CSK(4) , whereas the non-canonical nuclear factor-?B pathway was only induced by Pam(3) CSK(4) . Our data suggest that the lack of non-canonical nuclear factor-?B signalling by hBD-3 could contribute to the failure of this TLR agonist to induce production of the anti-inflammatory cytokine, IL-10, in human monocytes.
HIV infection results in depletion and dysfunction of naïve CD4(+) T cells. The mechanisms underlying these deficiencies are not understood. We investigated the frequencies of CD4(+) naïve subsets in HIV disease as defined by expression of CD25 and/or FoxP3 and the relationship of these frequencies to naïve T cell proliferation function. We observed increased proportions of CD25(+)FoxP3(+) and CD25(+)FoxP3(-) cells and decreased proportions of CD25(-)FoxP3(-) cells within the naïve CD4(+) cell compartment from HIV-infected persons compared with findings in healthy donors. These perturbations were related to higher plasma HIV RNA levels but not with higher immune activation, as measured by the proportions of CD38(+) memory CD4(+) T cells. Naïve T cell proliferation responses to mitogen stimulation were inversely related to the frequencies and absolute numbers of FoxP3(+) naïve T cells. MDA, a marker of oxidative stress, and sCD14, a marker of monocyte activation and a surrogate for microbial translocation, were increased in serum samples from HIV(+) donors; however, neither marker was related to naïve T cell function in HIV(+) donors. These observations suggest that alterations in naïve T cell subset frequencies could contribute to naïve T cell dysfunction in HIV disease, but these alterations are not necessarily the result of chronic immune activation.
Naive B lymphocytes are generally thought to be poor APCs, and there is limited knowledge of their role in activation of CD8(+) T cells. In this article, we demonstrate that class I MHC Ag presentation by human naive B cells is enhanced by TLR9 agonists. Purified naive B cells were cultured with or without a TLR9 agonist (CpG oligodeoxynucleotide [ODN] 2006) for 2 d and then assessed for phenotype, endocytic activity, and their ability to induce CD8(+) T cell responses to soluble Ags. CpG ODN enhanced expression of class I MHC and the costimulatory molecule CD86 and increased endocytic activity as determined by uptake of dextran beads. Pretreatment of naive B cells with CpG ODN also enabled presentation of tetanus toxoid to CD8(+) T cells, resulting in CD8(+) T cell cytokine production and granzyme B secretion and proliferation. Likewise, CpG-activated naive B cells showed enhanced ability to cross-present CMV Ag to autologous CD8(+) T cells, resulting in proliferation of CMV-specific CD8(+) T cells. Although resting naive B cells are poor APCs, they can be activated by TLR9 agonists to serve as potent APCs for class I MHC-restricted T cell responses. This novel activity of naive B cells could be exploited for vaccine design.
Chronic hepatitis C virus (HCV) infection is characterized by reduced numbers of functional HCV-specific T cells. In addition, chronically HCV-infected individuals have reduced response to vaccine. Alterations in naive CD4 T cell phenotype or function may contribute to these immune impairments.
CC Chemokine Receptor 5 (CCR5) is an important mediator of chemotaxis and the primary coreceptor for HIV-1. A recent report by other researchers suggested that primary T cells harbor pools of intracellular CCR5. With the use of a series of complementary techniques to measure CCR5 expression (antibody labeling, Western blot, quantitative reverse transcription polymerase chain reaction), we established that intracellular pools of CCR5 do not exist and that the results obtained by the other researchers were false-positives that arose because of the generation of irrelevant binding sites for anti-CCR5 antibodies during fixation and permeabilization of cells.
Expression of the activation antigen CD38 on T cells is a strong predictor of the risk of HIV disease progression, but it is not known whether CD38 is a marker or mediator of dysfunction. We examined the relationship between CD38 expression and responses to T-cell receptor stimulation in central memory and effector memory CD4 T cells in HIV-infected persons and in healthy controls. Basal CD38 expression was preserved by blocking golgi transport with brefeldin A. Intracellular expression of interleukin 2, interferon ?, and CD154 was measured after stimulating peripheral blood mononuclear cells with the superantigen staphylococcal enterotoxin B with or without anti-CD28 costimulation. Interferon-? responses were comparable or increased in stimulated CD38 memory cells, and the interleukin 2 responses of costimulated CD38 central memory cells were decreased in HIV infection. In CD38 cells and especially in CD38 cells of HIV-infected persons, stimulated memory cells more often failed to express CD154 (CD40 ligand) when induced to express cytokine. A dissociated cytokine and CD154 expression by memory CD4 T cells may impair interactions between T cells and antigen-presenting cells, contribute to impaired immunity and help explain the relationship between CD38 expression and disease progression in chronic HIV infection.
Both clinical experience and a growing medical literature indicate that some persons who have been exposed to human immunodeficiency virus (HIV) infection remain uninfected. Although in some instances this may represent good fortune, cohorts of uninfected persons have been reported who are considered at high risk for infection. In these cohorts a variety of characteristics have been proposed as mediating protection, but to date only the 32–base pair deletion in the chemokine (C‐C motif) receptor 5 gene, which results in complete failure of cell surface expression of this coreceptor, has been associated with high‐level protection from HIV infection. With this in mind, there are probably many other factors that may individually or in combination provide some level of protection from acquisition of HIV infection. Because some of these factors are probably incompletely protective or inconsistently active, identifying them with confidence will be difficult. Nonetheless, clarifying the determinants of protection against HIV infection is a high priority that will require careful selection of high‐risk uninfected cohorts, who should undergo targeted studies of plausible mediators and broad screening for unexpected determinants of protection.
Many immune correlates of CD8(+) T-cell-mediated control of HIV replication, including polyfunctionality, proliferative ability, and inhibitory receptor expression, have been discovered. However, no functional correlates using ex vivo cells have been identified with the known ability to cause the direct elimination of HIV-infected cells. We have recently discovered the ability of human CD8(+) T-cells to rapidly upregulate perforin--an essential molecule for cell-mediated cytotoxicity--following antigen-specific stimulation. Here, we examined perforin expression capability in a large cross-sectional cohort of chronically HIV-infected individuals with varying levels of viral load: elite controllers (n = 35), viremic controllers (n = 29), chronic progressors (n = 27), and viremic nonprogressors (n = 6). Using polychromatic flow cytometry and standard intracellular cytokine staining assays, we measured perforin upregulation, cytokine production, and degranulation following stimulation with overlapping peptide pools encompassing all proteins of HIV. We observed that HIV-specific CD8(+) T-cells from elite controllers consistently display an enhanced ability to express perforin directly ex vivo compared to all other groups. This ability is not restricted to protective HLA-B haplotypes, does not require proliferation or the addition of exogenous factors, is not restored by HAART, and primarily originates from effector CD8(+) T-cells with otherwise limited functional capability. Notably, we found an inverse relationship between HIV-specific perforin expression and viral load. Thus, the capability of HIV-specific CD8(+) T-cells to rapidly express perforin defines a novel correlate of control in HIV infection.
Immune reconstitution after HAART is incomplete, but no widely accepted method to quantify subclinical immune deficiency is available. We immunized 9 HIV-negative subjects and 29 HIV-infected patients with CD4>/=450 cells/microL and undetectable HIV RNA levels with 2 doses of diphtheria/tetanus toxoid (TT) and KLH, a presumed neoantigen. We quantified the response by lymphoproliferative assay, delayed-type hypersensitivity (DTH), and antibody titers up to 59days after enrollment. We assessed T cell proliferative capacity using anti-Vbeta3 and anti-Vbeta5 antibody stimulation, which we herein show induced predominant proliferation of naïve T cells. Subjects with detectable responses to KLH tended to exhibit greater proliferative responses to anti-Vbeta3/Vbeta5 stimulation; no such pattern was seen with response to TT. Several measures of in vitro T cell proliferative capacity correlated significantly with DTH and antibody responses to KLH, but not with TT responses; this association was independent of naïve T cell numbers. Our results indicate that naïve T cell proliferation predicts response to neo-, but not recall antigens, and suggest that it may be a meaningful reflection of in vivo immune competence in HIV-infected persons.
HIV infection is associated with an increased risk of thrombosis; and as antiretroviral therapy has increased the lifespan of HIV-infected patients, their risk for cardiovascular events is expected to increase. A large clinical study found recently that all-cause mortality for HIV(+) patients was related to plasma levels of interleukin-6 and to D-dimer products of fibrinolysis. We provide evidence that this elevated risk for coagulation may be related to increased proportions of monocytes expressing cell surface tissue factor (TF, thromboplastin) in persons with HIV infection. Monocyte TF expression could be induced in vitro by lipopolysaccharide and flagellin, but not by interleukin-6. Monocyte expression of TF was correlated with HIV levels in plasma, with indices of immune activation, and with plasma levels of soluble CD14, a marker of in vivo lipopolysaccharide exposure. TF levels also correlated with plasma levels of D-dimers, reflective of in vivo clot formation and fibrinolysis. Thus, drivers of immune activation in HIV disease, such as HIV replication, and potentially, microbial translocation, may activate clotting cascades and contribute to thrombus formation and cardiovascular morbidities in HIV infection.
Type I interferon (IFNalpha/beta) plays a complex role in HIV-1 infection and has been proposed alternately to have roles in either disease protection or progression. Although IFNalpha/beta plays crucial roles in regulating monocytes and dendritic cells, responsiveness of these cells to IFNalpha/beta in HIV-1 infection is poorly understood. We report significant defects in IFNalpha/beta receptor (IFNalpha/betaR) expression, IFNalpha signaling, and IFNalpha-induced gene expression in monocytes from HIV-1-infected subjects. IFNalpha/betaR expression correlated directly with CD4+ T-cell count and inversely with HIV-1 RNA level and expression of CD38 by memory (CD45RO+) CD8+ T cells, a measure of pathologic immune activation in HIV-1 infection associated with disease progression. In addition, monocytes from HIV-1-infected persons showed diminished responses to IFNalpha, including decreased induction of phosphorylated STAT1 and the classical interferon-stimulated gene produces MxA and OAS. These IFNalpha responses were decreased regardless of IFNalpha/betaR expression, suggesting that regulation of intracellular signaling may contribute to unresponsiveness to IFNalpha/beta in HIV-1 disease. Defective monocyte responses to IFNalpha/beta may play an important role in the pathogenesis of HIV-1 infection, and decreased IFNalpha/betaR expression may serve as a novel marker of disease progression.
The significance of elevated plasma levels of bacterial lipopolysaccharide (LPS) in persons with chronic HIV infection remains undefined. We measured LPS levels by use of limulus lysate assay, and DNA sequences encoding bacterial ribosomal 16S RNA (16S rDNA) were assessed by quantitative polymerase chain reactions in plasma samples obtained from 242 donors. Plasma levels of 16S rDNA were significantly higher in human immunodeficiency virus (HIV)-infected subjects than in uninfected subjects, and they correlated with LPS levels. Higher levels of 16S rDNA were associated with higher levels of T cell activation and with lower levels of CD4 T cell restoration during antiretroviral therapy. Antiretroviral therapy reduces but does not fully normalize plasma levels of bacterial 16S rDNA, an index of microbial translocation from the gastrointestinal tract. High levels of 16S rDNA during therapy are strongly associated with reduced increases in the CD4(+) T lymphocyte count, irrespective of plasma HIV RNA levels. These findings are consistent with the importance of microbial translocation in immunodeficiency and T cell homeostasis in chronic HIV infection.
Loss of interleukin-7 (IL-2) receptor expression has been described in T lymphocytes from persons with human immunodeficiency virus (HIV) infection, potentially contributing to perturbations in T cell homeostasis. We investigated IL-7 receptor signaling by measuring signal transducer and activator of transcription 5 (STAT5) phosphorylation in CD4+ T cell subsets from HIV-infected persons. We determined that CD45RA- memory cell subsets (both CD27+ and CD27-) displayed the most robust immediate responses to IL-7, whereas naive CD4+ T cells sustained the signal most efficiently. Memory CD4+ T cells with a terminal phenotype (CD45RA+CD27-) responded poorly to IL-7 stimulation. Defects in signaling were observed in cells from viremic HIV-infected persons and were especially pronounced in CD45RA-CD27- memory subset. Although CD127 expression was diminished for T cells from HIV-infected persons, it was not directly related to IL-7 receptor signaling function. Instead, age was inversely related to IL-7 signaling in cells from both HIV-infected viremic subjects and healthy control subjects. Thus, HIV infection results in impaired IL-7 responsiveness, especially in memory CD4+ T cells, and this defect is likely compounded by aging.
The mechanisms responsible for increased cardiovascular risk associated with HIV-1 infection are incompletely defined. Using flow cytometry, in the present study, we examined activation phenotypes of monocyte subpopulations in patients with HIV-1 infection or acute coronary syndrome to find common cellular profiles. Nonclassic (CD14(+)CD16(++)) and intermediate (CD14(++)CD16(+)) monocytes are proportionally increased and express high levels of tissue factor and CD62P in HIV-1 infection. These proportions are related to viremia, T-cell activation, and plasma levels of IL-6. In vitro exposure of whole blood samples from uninfected control donors to lipopolysaccharide increased surface tissue factor expression on all monocyte subsets, but exposure to HIV-1 resulted in activation only of nonclassic monocytes. Remarkably, the profile of monocyte activation in uncontrolled HIV-1 disease mirrors that of acute coronary syndrome in uninfected persons. Therefore, drivers of immune activation and inflammation in HIV-1 disease may alter monocyte subpopulations and activation phenotype, contributing to a pro-atherothrombotic state that may drive cardiovascular risk in HIV-1 infection.
Rapidly evolving research examining the extended role of human beta-defensins (hBDs) in chemoattraction, innate immune-mediated response, and promotion of angiogenesis suggest that the collective effects of hBDs extend well beyond their antimicrobial mechanism(s). Indeed, the numerous basic cellular functions associated with hBDs demonstrate that these peptides have dual impact on health, as they may be advantageous under certain conditions, but potentially detrimental in others. The consequences of these functions are reflected in the overexpression of hBDs in diseases, such as psoriasis, and recently the association of hBDs with pro-tumoral signaling. The mechanisms regulating hBD response in health and disease are still being elucidated. Clearly the spectrum of function now attributed to hBD regulation identifies these molecules as important cellular regulators, whose appropriate expression is critical for proper immune surveillance; i.e., expression of hBDs in proximity to areas of cellular dysregulation may inadvertently exacerbate disease progression. Understanding the mechanism(s) that regulate contextual signaling of hBDs is an important area of concentration in our laboratories. Using a combination of immunologic, biochemical, and molecular biologic approaches, we have identified signaling pathways associated with hBD promotion of immune homeostasis and have begun to dissect the inappropriate role that beta-defensins may assume in disease.
Interactions of AMPs with plasma membranes of primary human immune cells are poorly characterized. Analysis of PI exclusion as a measure of membrane integrity indicated that hBD-3 caused membrane perturbations in monocytes but not T or B cells at concentrations typically used to kill bacteria or to induce activation of APCs. Bleb-like structures were observed in monocytes exposed to hBD-3. These cells also increased surface expression of LAMP1, a membrane repair marker after exposure to hBD-3. Furthermore, cell death was enhanced by adding an inhibitor of membrane repair. Removal of cholesterol from membranes resulted in greater susceptibility of cells to hBD-3, but cholesterol content was not different between the cell types, as assessed by filipin staining. Freshly isolated monocytes expressed higher levels of the negatively charged phospholipid, PS, on their outer leaflet compared with B or T cells. Preincubation of monocytes with molecules that bind PS protected these cells from hBD-3-induced membrane damage, suggesting that outer-membrane PS expression can at least partially explain monocyte susceptibility to hBD-3. The potential for membrane disruption caused by AMPs should be evaluated in various cell types when considering these molecules for therapeutic applications in humans.
hBD-3 is an antimicrobial peptide that may contribute to adaptive immune responses by activating professional APCs via a TLR1/2-dependent mechanism. Patients with HIV disease experience increased susceptibility to mucosal infections, which may, in part, stem from diminished APC function. Our current studies demonstrate a reduced capacity of hBD-3 to induce the expression of a costimulatory molecule, CD80, on monocytes and mDCs from HIV-infected persons compared with cells from healthy controls. Although the expression of TLR1 and TLR2 on monocytes was not a strong predictor of hBD-3 responsiveness in bivariate analyses, monocytes and mDCs from HIV-infected persons expressed significantly lower levels of TLR1. Monocyte expression of the activation marker CD69, in cells from HIV-infected persons with therapeutically controlled viremia, was correlated directly with TLR2 and TLR4 expression but not with TLR1 expression. Overall, these studies suggest that immune activation may affect TLR2 and TLR4 expression but may not fully account for reduced TLR1 expression in monocytes from HIV-infected persons. Impairments in hBD-3 responsiveness and TLR1 expression are likely to contribute to increased risk of mucosal infection in HIV disease.
Increased plasma IL-6 levels have been associated with HIV-1 disease progression risk, yet the drivers of IL-6 production in HIV-1 infection are not known. This study was designed to explore the relationship between HIV-1 replication and IL-6 induction.
Interleukin-7 (IL-7) is a cytokine that plays a critical role in T cell homeostasis by promoting proliferation and survival of mature T cells and also by enhancing thymic output for the generation of new T cells. IL-7 receptor expression and signaling function is perturbed in HIV infection and could contribute to disease pathogenesis. Even though highly active anti-retroviral therapy has markedly reduced morbidity and mortality in HIV-infected persons, there remains concern that a significant proportion of treated patients may experience relatively poor CD4+ T cell recovery despite sustained viral suppression. Recent human trials and animal studies suggest that IL-7 administration may provide a powerful tool to enhance T cell reconstitution in HIV-infected persons. The role of IL-7/IL-7 receptor perturbations in HIV pathogenesis and the potential to reconstitute immunity with IL-7 administration in the setting of HIV infection are important areas of investigation.
Type I interferons play important roles in innate immune defense. In HIV infection, type I interferons may delay disease progression by inhibiting viral replication while at the same time accelerating disease progression by contributing to chronic immune activation.
In HIV infections, homoeostasis of T cells is dysregulated such that there is a depletion of CD4(+) T cells and a progressive loss of naïve CD4(+) and CD8(+) T cells. Methodologies that can improve the function of some or all of these cells will likely enhance immune responsiveness in HIV infection. Interleukin-7 (IL-7) is a cytokine that has been shown to be critical in homeostatic expansion of naïve CD8(+) and CD4(+) cells in lymphopenic hosts, as well as regulating effector T cell to memory T-cell transition and memory T-cell homeostasis. In animal studies and clinical trials, repeated injections of IL-7 are used to boost both CD4(+) and CD8(+) cell counts. Daily injections, however, are painful, inconvenient, and provide a frequent route for pathogen entry. We developed a poly (D,L-lactide-co-glycolide; PLGA) microparticle controlled release system to administer IL-7 in which a single injection of microparticles can provide therapeutic delivery of IL-7. IL-7 encapsulated PLGA microparticles were first synthesized using a water/organic/water double emulsion method, release from the particles was then optimized using in vitro release studies and therapeutic effectiveness was finally studied in animal studies. These PLGA microparticles showed effective delivery of IL-7 for 1 week in vitro. These results were translated to in vivo delivery as well, which was followed for 9 days. Controlled release of IL-7 in mice demonstrated biological activity in both CD4(+) and CD8(+) T cells in mice, which was consistent with previously reported results using daily injections.
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