Upregulated IL-32 Expression And Reduced Gut Short Chain Fatty Acid Caproic Acid in People Living With HIV With Subclinical Atherosclerosis.

Despite the success of antiretroviral therapy (ART), people living with HIV (PLWH) are still at higher risk for cardiovascular diseases (CVDs) that are mediated by chronic inflammation. Identification of novel inflammatory mediators with the inherent potential to be used as CVD biomarkers and also as therapeutic targets is critically needed for better risk stratification and disease management in PLWH. Here, we investigated the expression and potential role of the multi-isoform proinflammatory cytokine IL-32 in subclinical atherosclerosis in PLWH (n=49 with subclinical atherosclerosis and n=30 without) and HIV- controls (n=25 with subclinical atherosclerosis and n=24 without). While expression of all tested IL-32 isoforms (α, ß, γ, D, ϵ, and θ) was significantly higher in peripheral blood from PLWH compared to HIV- controls, IL-32D and IL-32θ isoforms were further upregulated in HIV+ individuals with coronary artery atherosclerosis compared to their counterparts without. Upregulation of these two isoforms was associated with increased plasma levels of IL-18 and IL-1ß and downregulation of the atheroprotective protein TRAIL, which together composed a unique atherosclerotic inflammatory signature specific for PLWH compared to HIV- controls. Logistic regression analysis demonstrated that modulation of these inflammatory variables was independent of age, smoking, and statin treatment. Furthermore, our in vitro functional data linked IL-32 to macrophage activation and production of IL-18 and downregulation of TRAIL, a mechanism previously shown to be associated with impaired cholesterol metabolism and atherosclerosis. Finally, increased expression of IL-32 isoforms in PLWH with subclinical atherosclerosis was associated with altered gut microbiome (increased pathogenic bacteria; Rothia and Eggerthella species) and lower abundance of the gut metabolite short-chain fatty acid (SCFA) caproic acid, measured in fecal samples from the study participants. Importantly, caproic acid diminished the production of IL-32, IL-18, and IL-1ß in human PBMCs in response to bacterial LPS stimulation. In conclusion, our studies identified an HIV-specific atherosclerotic inflammatory signature including specific IL-32 isoforms, which is regulated by the SCFA caproic acid and that may lead to new potential therapies to prevent CVD in ART-treated PLWH.

Association of chronic hepatitis C infection with T-cell phenotypes in HIV-negative and HIV-positive women.

BACKGROUND: Hepatitis C virus (HCV) viremia is thought to have broad systemic effects on the cellular immune system that go beyond its impact on just those T cells that are HCV specific. However, previous studies of chronic HCV and circulating T-cell subsets (activation and differentiation phenotypes) in HIV negatives used general population controls, rather than a risk-appropriate comparison group. Studies in HIV positives did not address overall immune status (total CD4⁺ count). METHODS: We used fresh blood from HIV-positive and at-risk HIV-negative women, with and without chronic HCV, to measure percentages of activated CD4⁺ and CD8⁺ T cells, Tregs, and T-cell differentiation phenotypes (naive, central memory, effector memory (EM), and terminally differentiated effector). This included 158 HIV negatives and 464 HIV positives, of whom 18 and 63, respectively, were HCV viremic. RESULTS: In multivariate models of HIV negatives, HCV viremia was associated with 25% fewer naive CD4⁺ (P = 0.03), 33% more EM CD4⁺ (P = 0.0002), and 37% fewer central memory CD8⁺ (P = 0.02) T cells. Among HIV positives, we observed only 1 of these 3 relationships: higher percentage of EM CD4⁺ among HCV viremic women. Furthermore, the association with EM CD4⁺ among HIV positives was limited to individuals with diminished immune status (total CD4⁺ count ≤500 cells/µL), as were associations of HCV viremia with higher percentages of activated CD4⁺ and Tregs. Among HIV positives with high CD4⁺ count, no significant associations were observed. CONCLUSIONS: These data suggest that HCV viremia in HIV negatives is associated with accelerated T-cell differentiation, but among HIV positives, the impact of HCV viremia is less straightforward and varies by total CD4v count.

Optimizing viable leukocyte sampling from the female genital tract for clinical trials: an international multi-site study.

BACKGROUND: Functional analysis of mononuclear leukocytes in the female genital mucosa is essential for understanding the immunologic effects of HIV vaccines and microbicides at the site of HIV exposure. However, the best female genital tract sampling technique is unclear. METHODS AND FINDINGS: We enrolled women from four sites in Africa and the US to compare three genital leukocyte sampling methods: cervicovaginal lavages (CVL), endocervical cytobrushes, and ectocervical biopsies. Absolute yields of mononuclear leukocyte subpopulations were determined by flow cytometric bead-based cell counting. Of the non-invasive sampling types, two combined sequential cytobrushes yielded significantly more viable mononuclear leukocytes than a CVL (p<0.0001). In a subsequent comparison, two cytobrushes yielded as many leukocytes (∼ 10,000) as one biopsy, with macrophages/monocytes being more prominent in cytobrushes and T lymphocytes in biopsies. Sample yields were consistent between sites. In a subgroup analysis, we observed significant reproducibility between replicate same-day biopsies (r = 0.89, p = 0.0123). Visible red blood cells in cytobrushes increased leukocyte yields more than three-fold (p = 0.0078), but did not change their subpopulation profile, indicating that these leukocytes were still largely derived from the mucosa and not peripheral blood. We also confirmed that many CD4(+) T cells in the female genital tract express the α4ß7 integrin, an HIV envelope-binding mucosal homing receptor. CONCLUSIONS: CVL sampling recovered the lowest number of viable mononuclear leukocytes. Two cervical cytobrushes yielded comparable total numbers of viable leukocytes to one biopsy, but cytobrushes and biopsies were biased toward macrophages and T lymphocytes, respectively. Our study also established the feasibility of obtaining consistent flow cytometric analyses of isolated genital cells from four study sites in the US and Africa. These data represent an important step towards implementing mucosal cell sampling in international clinical trials of HIV prevention.

GB virus C infection and B-cell, natural killer cell, and monocyte activation markers in HIV-infected individuals.

GB virus C (GBV-C), a pan-lymphotropic flavivirus capable of persistent infection, is associated with prolonged survival and reduced T-cell activation in HIV-infected patients. GBV-C was associated with reduced CD56brt/CD16- natural killer cell and monocyte activation, and a trend toward reduced B-cell activation by measuring cell surface activation markers or HIV entry coreceptors. The GBV-C association was independent of HIV viral load. Thus, GBV-C may influence non-T-cell immune activation in individuals with HIV infection.

Protection of Tregs, suppression of Th1 and Th17 cells, and amelioration of experimental allergic encephalomyelitis by a physically-modified saline.

In multiple sclerosis (MS) and other autoimmune diseases, the autoreactive T cells overcome the resistance provided by the regulatory T cells (Tregs) due to a decrease in the number of Foxp3-expressing Tregs. Therefore, upregulation and/or maintenance of Tregs during an autoimmune insult may have therapeutic efficacy in autoimmune diseases. Although several immunomodulatory drugs and molecules are available, most present significant side effects over long-term use. Here we have undertaken an innovative approach to upregulate Tregs and achieve immunomodulation. RNS60 is a 0.9% saline solution generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), RNS10.3 (TCP-modified saline without excess oxygen) and PNS60 (saline containing excess oxygen without TCP modification), was found to upregulate Foxp3 and enrich Tregs in MBP-primed T cells. Moreover, RNS60, but not NS, RNS10.3 and PNS60, inhibited the production of nitric oxide (NO) and the expression of iNOS in MBP-primed splenocytes. Incubation of the cells with an NO donor abrogated the RNS60-mediated upregulation of Foxp3. These results suggest that RNS60 boosts Tregs via suppression of NO production. Consistent to the suppressive activity of Tregs towards autoreactive T cells, RNS60, but not NS, RNS10.3, or PNS60, suppressed the differentiation of Th17 and Th1 cells and shifted the balance towards a Th2 response. Finally, RNS60 treatment exhibited immunomodulation and ameliorated adoptive transfer of experimental allergic encephalomyelitis, an animal model of MS, via Tregs. These results describe a novel immunomodulatory property of RNS60 and suggest its exploration for therapeutic intervention in MS and other autoimmune disorders.

A low T regulatory cell response may contribute to both viral control and generalized immune activation in HIV controllers.

HIV-infected individuals maintaining undetectable viremia in the absence of therapy (HIV controllers) often maintain high HIV-specific T cell responses, which has spurred the development of vaccines eliciting HIV-specific T cell responses. However, controllers also often have abnormally high T cell activation levels, potentially contributing to T cell dysfunction, CD4+ T cell depletion, and non-AIDS morbidity. We hypothesized that a weak T regulatory cell (Treg) response might contribute to the control of viral replication in HIV controllers, but might also contribute to generalized immune activation, contributing to CD4+ T cell loss. To address these hypotheses, we measured frequencies of activated (CD38+ HLA-DR+), regulatory (CD4+CD25+CD127(dim)), HIV-specific, and CMV-specific T cells among HIV controllers and 3 control populations: HIV-infected individuals with treatment-mediated viral suppression (ART-suppressed), untreated HIV-infected "non-controllers" with high levels of viremia, and HIV-uninfected individuals. Despite abnormally high T cell activation levels, controllers had lower Treg frequencies than HIV-uninfected controls (P = 0.014). Supporting the propensity for an unusually low Treg response to viral infection in HIV controllers, we observed unusually high CMV-specific CD4+ T cell frequencies and a strong correlation between HIV-specific CD4+ T cell responses and generalized CD8+ T cell activation levels in HIV controllers (P ≤ 0.001). These data support a model in which low frequencies of Tregs in HIV controllers may contribute to an effective adaptive immune response, but may also contribute to generalized immune activation, potentially contributing to CD4 depletion.

Chloroquine modulates HIV-1-induced plasmacytoid dendritic cell alpha interferon: implication for T-cell activation.

Plasmacytoid dendritic cells (pDC) contribute to antiviral immunity mainly through recognition of microbial products and viruses via intracellular Toll-like receptor 7 (TLR7) or TLR9, resulting in the production of type I interferons (IFNs). Although interferons reduce the viral burden in the acute phase of infection, their role in the chronic phase is unclear. The 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 of IFN-alpha production using chloroquine, an endosomal inhibitor, was tested in a novel in vitro model system with the aim of characterizing 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 decreases in the levels of the downstream signaling molecules IRAK-4 and IRF-7 and by inhibition of IFN-alpha synthesis. Chloroquine decreased CD8 T-cell activation induced by aldrithiol-2-treated HIV-1 in peripheral blood mononuclear cell cultures. In addition to blocking pDC activation, chloroquine also blocked negative modulators of the T-cell response, such as indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PDL-1). Our results indicate that TLR stimulation and production of IFN-alpha by pDC contribute to immune activation and that blocking of 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 highly active antiretroviral therapy to control immune activation in HIV-1 infection.

Potent HIV-specific responses are enriched in a unique subset of CD8+ T cells that coexpresses CD4 on its surface.

In humans, approximately 3% of peripheral CD8+ T cells coexpress CD4 dimly on their surface and hence are designated as CD4(dim)CD8(bright) T cells. We evaluated the contribution of this CD4(dim)CD8(bright) T-cell population to anti-HIV immunity. We demonstrate that CD4(dim)CD8(bright) T cells generate greater than 55% of CD8+ T-cell antigen recognition and effector response to HIV, as evaluated by multiple parameters for assessing T-cell antiviral immunity, including HIV tetramer recognition, cytokine production, and cytolytic potential. Inhibition of major histocompatibility class II (MHC-II) on target cells or CD4 on CD4(dim)CD8(bright) T cells diminishes their anti-HIV responses, suggesting that CD4 on effector cells and MHC-II on target cells provides an additional arm of contact between effector and target cells which is critical to CD4(dim)CD8(bright) T-cell function. CD4(dim)CD8(bright) T cells also exhibit features that are indicative of central memory T cells. Finally, CD4(dim)CD8(bright) T cells are elevated in blood of HIV+ long-term nonprogressors in comparison to HIV- donors. Collectively, our findings show that CD4(dim)CD8(bright) T cells designate an enriched antiviral subpopulation of CD8+ T cells that should be targeted for therapeutic intervention or evaluation of vaccine efficacy.

Effect of mucosal fluid from women with bacterial vaginosis on HIV trans-infection mediated by dendritic cells.

Women with bacterial vaginosis (BV) have a higher risk of HIV transmission but the cause of risk is unknown. Dendritic cells (DC) are implicated in transmission of HIV and we previously observed that DC mature when exposed to mucosal fluid from women with BV. We hypothesized that maturation of DC by BV mucosal fluid would enhance DC-mediated trans-infection of HIV. Monocyte-derived DC (MDDC) were treated with mucosal fluid, incubated with HIV(Bal), and HIV trans-infection was evaluated. While LPS-treated MDDC increased HIV(Bal)trans-infection, BV fluid reduced trans-infection. HIV(Bal) DNA levels in MDDC were not affected by BV fluid or LPS but productive infection of MDDC was decreased by LPS and BV fluid. Mucosal fluid from women with BV does not increase MDDC-mediated trans-infection suggesting that BV does not increase HIV susceptibility by increasing DC-mediated trans-infection. However, indirect effects of DC maturation on HIV transmission cannot be ruled out.

Reactive oxygen species up-regulate CD11b in microglia via nitric oxide: Implications for neurodegenerative diseases.

Microglial activation is considered as a hallmark of several neurodegenerative disorders. During microglial activation, the expression of CD11b, the beta-integrin marker of microglia, is increased. However, the molecular mechanism behind increased microglial CD11b expression is poorly understood. The present study was undertaken to explore the role of reactive oxygen species (ROS) in the expression of CD11b in microglial cells. Bacterial lipopolysaccharide (LPS) stimulated the expression of CD11b in mouse BV-2 microglial cells and primary microglia, the effect that was blocked by antioxidants such as N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). Furthermore, comicroinjection of either NAC or PDTC with LPS was also able to suppress LPS-stimulated expression of CD11b in striatum in vivo. Similarly, other neurotoxic molecules, such as interleukin-1beta (IL-1beta), IL-12 p40(2), fibrillar amyloid-beta (Abeta) peptides, HIV-1 gp120, and double-stranded RNA (poly(IC)), also stimulated the expression of CD11b in microglia through the involvement of ROS. Complete inhibition of LPS-stimulated expression of CD11b by catalase, induction of CD11b expression by H2O2 alone, and inhibition of superoxide-stimulated CD11b expression by catalase suggest that H2O2, but not superoxide, is in fact involved in the expression of CD11b. Interestingly, we also demonstrate that ROS stimulated the expression of CD11b after the induction of nitric oxide (NO) production and failed to stimulate CD11b when NO production was inhibited by either 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) or L-N6-(1-iminoethyl)-L-lysine (L-NIL). Taken together, these studies suggest that the up-regulation of CD11b in microglia is redox sensitive and that ROS up-regulates CD11b via NO.

Impact of class A, B and C CpG-oligodeoxynucleotides on in vitro activation of innate immune cells in human immunodeficiency virus-1 infected individuals.

Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl-deoxyguanosine dinucleotides (CpG-ODNs) stimulate Toll-like receptor 9 (TLR9) in plasmacytoid dendritic cells (pDC) and B cells and activate innate and adaptive immunity. Three classes of synthetic CpG-ODNs, class A, B and C, activate cells through TLR9; our goal was to evaluate their effect on cells from human immunodeficiency virus (HIV)-1(+) individuals. We compared the frequencies and the unstimulated activation status of immune effector cells in HIV-1(+) and HIV-1(-) individuals. Fewer pDC, myeloid dendritic cells (mDC), B cells, natural killer (NK) cells and invariant natural killer T cells (iNKT) were present in HIV-1(+) peripheral blood mononuclear cells (PBMC) and their baseline activation status was higher than HIV-1(-) PBMC. Exposure of HIV-1(+) PBMC to all classes of CpG-ODNs led to activation and maturation of pDC based on CD86, CD80, and CD83 expression similar to that of cells from HIV-1(-) individuals. The percentage of CpG-ODN stimulated pDC that express CD40 was dramatically higher when cells were obtained from HIV-1(+) than from HIV-1(-) individuals. B-lymphocytes were activated similarly in HIV-1(+) and HIV-1(-) individuals. mDC, NK and iNKT cell, which lack TLR9, were indirectly activated. Interferon-alpha (IFN-alpha) and interferon inducible protein 10 (IP-10) secretion was induced by class A or C but not class B CpG-ODN, but the concentrations were less than those produced by HIV-1(-) PBMC. HIV-1 infected individuals have fewer innate effector cells that are chronically activated, but these cells can be further activated by CpG-ODN, which suggests that synthetic CpG-ODNs could be used to enhance the immune system in HIV-1 infected individuals.

Dendritic cells from HIV-1 infected individuals are less responsive to toll-like receptor (TLR) ligands.

We compared TLR responsiveness in PBMC from HIV-1-infected and uninfected individuals using the TLR agonists: TLR7 (3M-001), TLR8 (3M-002), and TLR7/8 (3M-011). Activation and maturation of plasmacytoid dendritic cells (pDC) were measured by evaluating CD86, CD40, and CD83 expression and myeloid dendritic cell (mDC) activation was measured by evaluating CD40 expression. All agonists tested induced activation and maturation of pDC in PBMC cultures of cells from HIV+ and HIV- individuals. The TLR7 agonist induced significantly less pDC maturation in cells from HIV+ individuals. Quantitative assessment of secreted IFN-alpha and pro-inflammatory cytokines at the single cell level showed that pDC from HIV+ individuals stimulated with TLR7 and TLR7/8 induced IFN-alpha. TLR8 and TLR7/8 agonists induced IL-12 and COX-2 expression in mDC from HIV+ and HIV- individuals. Understanding pDC and mDC activation and maturation in HIV-1 infection could lead to more rational development of immunotherapeutic strategies to stimulate the adaptive immune response to HIV-1.

Identification of CD19 and CD20 peptides for induction of antigen-specific CTLs against B-cell malignancies.

The purpose of these studies was to develop immunogenic peptides derived from the CD19 and CD20 self-antigens for the induction of antigen-specific CTLs against B-cell malignancies. A total of seven peptides were designed and examined for their HLA-A2.1 affinity and immunogenicity. Of these peptides, we identified two highly immunogenic HLA-A2.1-specific peptides, CD19(150-158) (KLMSPKLYV) and CD20(188-196) (SLFLGILSV), which were capable of inducing peptide-specific CTLs. The CTLs displayed HLA-A2.1-restricted and antigen-specific cytotoxicity against Burkitt's lymphoma, chronic B cell leukemia, and multiple myeloma cell lines. The CD19 or CD20 peptide-specific CTL cytotoxicity was confirmed using HLA-A2.1(+) T2 cells presenting the appropriate peptide. No cytotoxic activity was observed against T2 cells presenting the irrelevant MAGE-3 peptide or T2 cells alone. In addition, the CTLs displayed a significant (P < 0.05) increase in cell proliferation and IFN-gamma secretion (>830 ng/mL) following restimulation with HLA-A2.1(+)/CD19(+)/CD20(+) tumor cells. The CTLs also displayed a distinct phenotype consisting of a high percentage of CD69(+)/CD45RO(+) and a low percentage of CD45RA(+)/CCR7(+) CD4(+) or CD8(+) T cells characteristic of effector memory cell population. Cyclic guanosine 3',5'-monophosphate culture conditions using serum-free AIM-V medium containing human AB serum, recombinant human interleukin 2 (Proleukin) and CD3/CD28 Dynabeads were developed resulting in a 35-fold expansion of CD20 peptide-specific CTLs. The expanded CD20-CTLs retained their cytotoxic activity (28-49%) against the Burkitt's lymphoma cell line. In conclusion, we report here on the identification of novel immunogenic CD19(150-158) (KLMSPKLYV) and CD20(188-196) (SLFLGILSV) peptides that have immunotherapeutic potentials as peptide vaccines or targeted T-cell therapies for treating B-cell malignancies.

Heteroclitic CD33 peptide with enhanced anti-acute myeloid leukemic immunogenicity.

The goal of these studies was to engineer a synthetic CD33 peptide with enhanced immunogenicity for the induction of acute myeloid leukemia (AML)-specific CTLs. Eight modified CD33 peptides YLISGDSPV, YIGSGDSPV, YIIIGDSPV, YIILGDSPV, YIISGISPV, YIISGDLPV, YIISGDSWV and YIISGDSPL were designed for increased HLA-A2.1 or T cell receptor affinity and compared with the native CD33(65-73) peptide, AIISGDSPV, for enhanced immunogenicity. The YLISGDSPV peptide was found to be the most immunogenic epitope producing highly cytolytic CTLs against AML target cells. The CTLs generated withYLISGDSPV peptide showed CD33 peptide-specificity through targeting of both native (AIISGDSPV) and modified (YLISGDSPV) peptide presenting EBV-BLCL. The CTL cultures displayed a distinct phenotype consisting of a high percentage of activated memory (CD69(+)/CD45RO(+))-CD8(+)and a low percentage of naive (CD45RA(+)/CCR7(+))-CD8(+)cells. In addition, T-cell clones specific to the YLISGDSPV peptide were isolated and characterized to target AML cells. The clones exhibited both HLA-A2.1-restricted and AML cell-specific cytotoxicity that was mediated through a granule-dependent pathway. More importantly, the CTL clones did not lyse or inhibit the proliferation of normal CD34(+) progenitor cells. In conclusion, we report on the identification of a highly immunogenic heteroclitic YLISGDSPV CD33 epitope that is a promising candidate for immunotherapy targeting AML.

Identification of novel CD33 antigen-specific peptides for the generation of cytotoxic T lymphocytes against acute myeloid leukemia.

Identification of immunogenic peptides for the generation of cytotoxic T lymphocytes (CTLs) may lead to the development of novel cellular therapies to treat disease relapse in acute myeloid leukemia (AML) patients. The objective of these studies was to evaluate the ability of unique HLA-A2.1-specific nonameric peptides derived from CD33 antigen to generate AML-specific CTLs ex vivo. We present data here on the identification of an immunogeneic HLA-A2.1-specific CD33(65-73) peptide (AIISGDSPV) that was capable of inducing CTLs targeted to AML cells. The CD33-CTLs displayed HLA-A2.1-restricted cytotoxicity against both mononuclear cells from AML patients and the AML cell line. The peptide- as well as AML cell-specificity of CD33-CTLs was demonstrated and the secretion of IFN-gamma by the CTLs was detected in response to CD33(65-73) peptide stimulation. The cultures contained a distinct CD33(65-73) peptide-tetramer(+)/CD8(+) population. Alteration of the native CD33(65-73) peptide at the first amino acid residue from alanine (A) to tyrosine (Y) enhanced the HLA-A2.1 affinity/stability of the modified CD33 peptide (YIISGDSPV) and induced CTLs with increased cytotoxicity against AML cells. These data therefore demonstrate the potential of using immunogenic HLA-A2.1-specific CD33 peptides in developing a cellular immunotherapy for the treatment of AML patients.