Viraga Haridas

T Cell-Specific siRNA Delivery Suppresses HIV-1 Infection in Humanized Mice

Evaluation of the therapeutic potential of RNAi for HIV infection has been hampered by the challenges of siRNA delivery and lack of suitable animal models. Using a delivery method for T cells, we show that siRNA treatment can dramatically suppress HIV infection. A CD7-specific single-chain antibody was conjugated to oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2rgamma-/- mice reconstituted with human lymphocytes (Hu-PBL) or CD34+ hematopoietic stem cells (Hu-HSC). In HIV-infected Hu-PBL mice, treatment with anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R controlled viral replication and prevented the disease-associated CD4 T cell loss. This treatment also suppressed endogenous virus and restored CD4 T cell counts in mice reconstituted with HIV+ peripheral blood mononuclear cells. Moreover, scFvCD7-9R could deliver antiviral siRNAs to naive T cells in Hu-HSC mice and effectively suppress viremia in infected mice. Thus, siRNA therapy for HIV infection appears to be feasible in a preclinical animal model.

CD70 + antigen-presenting cells control the proliferation and differentiation of T cells in the intestinal mucosa

One unresolved issue in gut immunity is how mucosal T lymphocytes are activated and which antigen-presenting cell (APC) is critical for the regulation of this process. We have identified a unique population of APCs that is exclusively localized in the lamina propria. These APCs constitutively expressed the costimulatory molecule CD70 and had antigen-presenting functions. After oral infection of mice with Listeria monocytogenes, proliferation and differentiation of antigen-specific T cells occurred in the gut mucosa in situ and blockade of CD70 costimulation abrogated the mucosal T cell proliferation and effector functions. Thus, a potent CD70-dependent stimulation via specialized tissue-specific APCs is required for the proliferation and differentiation of gut mucosal T cells after oral infection.

Targeted Delivery of Small Interfering RNA to Human Dendritic Cells To Suppress Dengue Virus Infection and Associated Proinflammatory Cytokine Production

ABSTRACT Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-d-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34+ hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvED) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-α), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rγ−/− mouse model engrafted with human CD34+ hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-α effectively suppressed poly(I:C)-induced TNF-α production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.

Cytokine response in children undergoing surgery for congenital heart disease.

Pediatric cardiac surgery with cardiopulmonary bypass (CPB) induces a complex inflammatory response that may cause multiorgan dysfunction. The objective of this study was to measure postoperative cytokine production and correlate the magnitude of this response with intraoperative variables and postoperative outcomes. Serum samples from 20 children (median age, 15 months) undergoing cardiac surgery with CPB were obtained preoperatively and on postoperative days (POD) 1–3. Serum levels of interleukin (IL)-6, IL-8, and IL-10 increased significantly on POD 1 (p < 0.01) vs pre-op values to 271 ± 68, 44 ± 9, 7.5 ± 0.8 pg/ml, respectively, whereas serum IL-1β, IL-12, and tumor neurosis factor -α were not significantly changed. The serum IL-6 and IL-8 levels correlated positively (p < 0.01) with the degree of postoperative medical intervention as measured by the Therapeutic Interventional Scoring System and indicated a greater need for inotropic support (p = 0.057). A negative correlation (p < 0.01) between IL-6, IL-8, and mixed venous oxygen saturation suggested compromised cardiopulmonary function. Patients with single ventricle anatomy had the highest levels of IL-6 and IL-8 (629 ± 131 and 70 ± 17 pg/ml, respectively), with a mean CPB time of 106 ± 23 minutes. Thus, the proinflammatory response after surgery with CPB was associated with postoperative morbidity with increased need for medical intervention.

Concurrent generation of effector and central memory CD8 T cells during vaccinia virus infection.

It is generally thought that during the contraction phase of an acute anti-viral T cell reponse, the effector T cells that escape activation-induced cell death eventually differentiate into central memory T cells over the next several weeks. Here we report that antigen-specific CD8T cells with the phenotype and function of central memory cells develop concomitantly with effector T cells during vaccinia virus (vv) infection. As soon as 5 days after an intraperitoneal infection with vv, we could identify a subset of CD44hi and CD62L+ vv-specific CD8 T cells in the peritoneal exudate lymphocytes. This population constituted approximately 10% of all antigen-specific T cells and like central memory T cells, they also expressed high levels of CCR7 and IL-7R but expressed little granzyme B. Importantly, upon adoptive transfer into naïve congenic hosts, CD62L+, but not CD62L− CD8 T cells were able to expand and mediate a rapid recall response to a new vv challenge initiated 6 weeks after transfer, confirming that the CD62L+ vv-specific CD8 T cells are bonafide memory cells. Our results are thus consistent with the branched differentiation model, where effector and memory cells develop simultaneously. These results are likely to have implications in the context of vaccine design, particularly those based on vaccinia virus recombinants.

Enhanced induction of HIV-specific cytotoxic T lymphocytes by dendritic cell-targeted delivery of SOCS-1 siRNA.

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8(+) T cells from healthy donors. Finally, stimulation of CD8(+) T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8+ T cells from healthy donors. Finally, stimulation of CD8+ T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.

A Role for IFITM Proteins in Restriction of Mycobacterium tuberculosis Infection

SUMMARY The interferon (IFN)-induced transmembrane (IFITM) proteins are critical mediators of the host antiviral response. Here, we expand the role of IFITM proteins to host defense against intracellular bacterial infection by demonstrating that they restrict Mycobacterium tuberculosis (MTb) intracellular growth. Simultaneous knockdown of IFITM1, IFITM2, and IFITM3 by RNAi significantly enhances MTb growth in human monocytic and alveolar/epithelial cells, whereas individual overexpression of each IFITM impairs MTb growth in these cell types. Furthermore, MTb infection, Toll-like receptor 2 and 4 ligands, and several proinflammatory cytokines induce IFITM1–3 gene expression in human myeloid cells. We find that IFITM3 co-localizes with early and, in particular, late MTb phagosomes, and overexpression of IFITM3 enhances endosomal acidification in MTb-infected monocytic cells. These findings provide evidence that the antiviral IFITMs participate in the restriction of mycobacterial growth, and they implicate IFITM-mediated endosomal maturation in its antimycobacterial activity.

TB-IRIS, T-cell activation, and remodeling of the T-cell compartment in highly immunosuppressed HIV-infected patients with TB

Objective:To investigate the impact of tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) upon immunological recovery and the T-cell compartment after initiation of TB and antiretroviral therapy (ART). Design and methods:We prospectively evaluated T-cell immunophenotypes by flow cytometry and cytokines by Luminex assays in a subset (n = 154) of highly immunosuppressed HIV-infected patients with TB from the Cambodian Early versus Late Introduction of Antiretrovirals randomized clinical trial. We compared findings from patients who developed TB-IRIS with findings from patients who did not develop TB-IRIS. Data were evaluated with mixed-effect linear regression, Kaplan–Meier estimates, and Wilcoxon rank-sum tests, and q-values were calculated to control for multiple comparisons. Results:Development of TB-IRIS was associated with significantly greater pre-ART frequencies of HLA-DR+CD45RO+CD4+, CCR5+CD4+, OX40+CD4+, and Fas+ effector memory CD8+ T cells, and significantly elevated levels of plasma interleukin (IL)-6, IL-1&bgr;, IL-8, and IL-10, and viral load. Post-ART initiation, effector memory CD4+ and Fas+ effector memory CD4+ T-cell frequencies significantly expanded, and central memory CD4+ T-cell frequencies significantly contracted in patients who experienced TB-IRIS. By week 34 post-TB treatment initiation, effector memory/central memory CD4+ T-cell ratios were markedly higher in TB-IRIS versus non-TB-IRIS patients. Conclusions:A distinct pattern of pre-ART T-cell and cytokine markers appear to poise the immune response of certain patients to develop TB-IRIS. Experience of TB-IRIS is then associated with long-term remodeling of the CD4+ T-cell memory compartment towards an effector memory-dominated phenotype. We speculate that these pre and post-ART TB-IRIS-associated immune parameters may contribute to superior immune control of TB/HIV co-infection and better clinical outcome.

Imaging flow cytometry analysis of intracellular pathogens

Imaging flow cytometry has been applied to address questions in infection biology, in particular, infections induced by intracellular pathogens. This methodology, which utilizes specialized analytic software makes it possible to analyze hundreds of quantified features for hundreds of thousands of individual cellular or subcellular events in a single experiment. Imaging flow cytometry analysis of host cell-pathogen interaction can thus quantitatively addresses a variety of biological questions related to intracellular infection, including cell counting, internalization score, and subcellular patterns of co-localization. Here, we provide an overview of recent achievements in the use of fluorescently labeled prokaryotic or eukaryotic pathogens in human cellular infections in analysis of host-pathogen interactions. Specifically, we give examples of Imagestream-based analysis of cell lines infected with Toxoplasma gondii or Mycobacterium tuberculosis. Furthermore, we illustrate the capabilities of imaging flow cytometry using a combination of standard IDEAS™ software and the more recently developed Feature Finder algorithm, which is capable of identifying statistically significant differences between researcher-defined image galleries. We argue that the combination of imaging flow cytometry with these software platforms provides a powerful new approach to understanding host control of intracellular pathogens.