Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.

Altered Toll-like Receptor 2-mediated Endotoxin Tolerance Is Related to Diminished Interferon β Production

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-alpha and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-alpha after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-alpha but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-kappa B activation, whereas TNF-alpha expression is blocked at the gene transcription level. Interferon beta plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon beta. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon beta and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

IMMUNOLOGICAL AND CYTOLOGICAL INVESTIGATION OF GAMMA INTERFERON PRODUCTION IN HUMAN T-LYMPHOCYTES INDUCED BY PMA AND PHA (IMMUNOGOLD, CELL CYCLE ANALYSIS)

The present study examined cellular mechanisms involved in the production and secretion of human (gamma)IFN. The hypothesis of this investigation was that (gamma)IFN is an export glycoprotein whose synthesis in human T lymphocytes is dependent on membrane stimulation, polypeptide synthesis in the rough endoplasmic reticulum, packaging in the Golgi complex, and release from the cell by exocytosis.^ The model system for this examination utilized T lymphocytes from normal donors and patients with chronic lymphocytic leukemia (CLL) induced in vitro with the tumor promoter, phorbol 12-myristate 13-acetate (PMA) and the lectin, phytohemagglutinin (PHA) to produce (gamma)IFN. This study reconfirmed the ability of PMA and PHA to synergistically induce (gamma)IFN production in normal T lymphocytes, as measured by viral inhibition assays and radio-immunoassays for (gamma)IFN. The leukemic T cells were demonstrated to produce (gamma)IFN in response to treatment with PHA. PMA treatment also induced (gamma)IFN production in the leukemic T cells, which was much greater than that observed in similarly treated normal T cells. In these same cells, however, combined treatment of the agents was shown to be ineffective at inducing (gamma)IFN production beyond the levels stimulated by the individual agents. In addition, the present study reiterated the synergistic effect of PMA/PHA on the stimulation of growth kinetics in normal T cells. The cell cycle of the leukemic T cells was also responsive to treatment with the agents, particularly with PMA treatment. A number of morphological alterations were attributed to PMA treatment including the acquisition of an elongated configuration, nuclear folds, and large cytoplasmic vacuoles. Many of the effects were observed to be reversible with dilution of the agents, and reversion to this state occurred more rapidly in the leukemic T cells. Most importantly, utilization of a thin section immuno-colloidal gold labelling technique for electron microscopy provided, for the first time, direct evidence of the cellular mechanism of (gamma)IFN production and secretion. The results of this latter study support the idea that (gamma)IFN is produced in the rough endoplasmic reticulum, transferred to the Golgi complex for accumulation and packaging, and released from the T cells by exocytosis. ^

Distinct patterns of peripheral HIV-1-specific interferon- gamma responses in exposed HIV-1-seronegative individuals.

It is unclear how human immunodeficiency virus (HIV) type 1–specific immune responses in exposed seronegative (ESN) individuals differ from those in HIV-1–infected subjects. By use of overlapping peptides spanning Gag, Tat, Nef, Vif, Vpr, and Vpu, peripheral blood mononuclear cells from ESN individuals, their seropositive (SP) partners, and unexposed seronegative control subjects were screened for interferon-? production. Responses were more frequent (95.7% vs. 20%), of a higher magnitude (9-fold), and of wider breadth (median number of peptides recognized, 18 vs. 2.5) in SP than in ESN individuals. Peptides recognized by ESN individuals were less frequently recognized by their SP partners. SP subjects infrequently recognized peptides from Vif, and such responses were subdominant; among ESN individuals, this HIV-1 protein was most frequently recognized. Immunodominant peptides recognized by SP subjects tended to be from relatively conserved regions, whereas peptides recognized by ESN individuals were associated with slow disease progression.

Regulation of human CD4(+) alphabeta T-cell-receptor-positive (TCR(+)) and gammadelta TCR(+) T-cell responses to Mycobacterium tuberculosis by interleukin-10 and transforming growth factor beta.

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor beta [TGF-beta]) cytokines. IL-10 and TGF-beta are produced by M. tuberculosis-infected macrophages. The effect of IL-10 and TGF-beta on M. tuberculosis-reactive human CD4(+) and gammadelta T cells, the two major human T-cell subsets activated by M. tuberculosis, was investigated. Both IL-10 and TGF-beta inhibited proliferation and gamma interferon production by CD4(+) and gammadelta T cells. IL-10 was a more potent inhibitor than TGF-beta for both T-cell subsets. Combinations of IL-10 and TGF-beta did not result in additive or synergistic inhibition. IL-10 inhibited gammadelta and CD4(+) T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4(+) T cells and, to a lesser extent, for gammadelta T cells. TGF-beta inhibited both CD4(+) and gammadelta T cells directly and had little effect on APC function for gammadelta and CD4(+) T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis-infected monocytes to a greater extent than TGF-beta. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-beta both inhibited CD4(+) and gammadelta T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis.

ROLE OF MULTIPLE TOLL-LIKE RECEPTOR ACTIVATION IN REGULATING CYTOTOXIC T CELL PRIMING TO LYMPOCYTIC CHORIOMENINGITIS VIRUS INFECTIONS

Foreign pathogens are recognized by toll-like receptors (TLR), present on various immune cells such as professional antigen-presenting cells (pAPCs). On recognition of its ligand, these receptors activate pAPCs, which may in turn influence naïve CD8+ T cell activation and affect their abilities to clear viral infection. However, how TLR ligands (TLR-L) can regulate CD8+ T cell responses have not been fully elucidated. This thesis will focus on examining how the presence of components from foreign pathogens, e.g. viral or bacterial infection, can contribute to shaping host immunity during concurrent viral infections. Since nitric oxide (NO), an innate effector immune molecule, was recently suggested to regulate proteasome activity; we sought to examine if NO can influence MHC-I antigen presentation during viral infections. The data in this section of the thesis provides evidence that combined TLR engagement can alter the presentation of certain CD8+ epitopes due to NO-induced inhibition in proteasome activity. Taken together, the data demonstrate that TLR ligation can influence the adaptive immune response due to induction of specific innate effector molecules such as NO. Next, the influence of combined TLR engagement on CD8+ T cell immunodominance hierarchies during viral infections was examined. In this section, we established that dual TLR2 and TLR3 stimulation alters immunodominance hierarchies of LCMV epitopes as a result of reduced uptake of cell-associated antigens and reduced cross-presentation of NP396 consequently suppressing NP396-specific CD8+ T cell responses. These findings are significant as they highlight a new role for TLR ligands in regulating anti-viral CD8+ T cell responses through impairing cross-presentation of cell-associated antigens depending on the type of TLR present in the environment during infections. Finally, we addressed TLR ligand induced type I interferon production and the signalling pathways that regulate them in two different mouse macrophage populations – those derived from the spleen or bone marrow. In this study, we observed that concomitant TLR2 stimulation blocked the induction of type I IFN induced by TLR4 in bone marrow-derived macrophages, but not spleen-derived macrophages in SOCS3-dependent manner. Taken together, the data presented in this thesis have defined new facets of how anti-viral responses are regulated by TLR activation, especially if multiple receptors are engaged simultaneously.

Caractérisation de la voie d'activation des interférons de type I

Codirecteur de recherche: Dr Sylvain Meloche

Produção deficiente de citocinas Th1 em camundongos BALB/c jovens

O objetivo do presente estudo foi comparar a produção de IFN-γ, IL-12 e IL-4 entre camundongos jovens (5, 12 e 19 dias de idade) e adultos (30 dias de idade). As avaliações foram feitas por estimulação, in vitro, de células esplênicas com Concanavalina A (ConA) , Staphylococcus aureus (S. aureus) e lipopolissacarídeo (LPS). Diferentes concentrações de cada estímulo foram testadas e os sobrenadantes das culturas foram coletados após 48 horas de incubação e as concentrações de IFN-γ, IL-12 e IL-4 determinadas por ELISA. Células de camundongos jovens e adultos produziram níveis igualmente elevados de IFN-γ após estímulo com ConA. Somente animais adultos produziram IFN-γ em resposta ao estímulo com S. aureus. Em culturas estimuladas com LPS, a produção desta citocina foi baixa e similar nos animais jovens e significativamente elevada nos animais adultos. Somente células de animais adultos estimuladas com S. aureus foram capazes de produzir IL-12. O único estímulo capaz de induzir níveis detectáveis de IL-4 foi ConA, sendo que estes níveis foram mais elevados nos animais com 12 e 19 dias de idade em comparação com animais neonatos e adultos. A diminuição das doses ótimas dos estímulos não mudou o perfil de produção de cada citocina nos animais jovens. Estes resultados permitem concluir que a idade afeta a produção de citocinas: ocorre maior produção de IL-4 em camundongos jovens e maior produção de IL-12 e IFN-γ em animais adultos. Estas informações são importantes devido ao papel destas citocinas na polarização das respostas imunes nos sentidos Th1 e Th2. Palavras-chave: camundongo; citocina; interferon-gama; interleucina-4; interleucina-12.

Avaliação da carga viral do virus da hepatite C em diferentes compartimentos biológicos: influência na predição da recidiva virológica

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

DNA immunization: Induction of higher avidity antibody and effect of route on T cell cytotoxicity

Immunizations of mice with plasmid DNAs encoding ovalbumin (OVA), human Ig, and hen egg lysozyme were compared with doses of soluble protein (without adjuvant) that induced similar IgG responses. The route of immunization influenced the magnitude of the antibody (Ab) response in that intradermal (i.d.) injection elicited higher IgG Ab levels than i.m. injection in both DNA- and protein-immunized mice. Although total IgG levels were similar to soluble protein controls, the avidity of the anti-OVA Abs generated by DNA immunization were 100- and 1,000-fold higher via the i.m. or i.d. route, respectively. However, despite the generation of high-avidity Ab in DNA-immunized mice, germinal centers could not be detected in either DNA- or protein-immunized mice. Examination of the IgG subclass response showed that IgG2a was induced by i.m. DNA immunization, coinciding with elevated interferon γ production, whereas a dominant and elevated IgG1 response, coinciding with detectable interleukin 4 production, was generated after i.d. immunization with DNA or soluble OVA and hen egg lysozyme but not human Ig protein. As expected, cytotoxic T cell (CTL) responses could be detected only after DNA immunization. I.d. immunization produced the strongest CTL responses early (2 weeks) but was similar to i.m. later. Therefore, DNA immunization can differ from protein immunization by its ability to induce rapid CTL responses and higher avidity Ab, both of which are advantageous for vaccination.

Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines

We have reported previously that murine bone marrow-derived dendritic cells (DC) pulsed with whole tumor lysates can mediate potent antitumor immune responses both in vitro and in vivo. Because successful therapy was dependent on host immune T cells, we have now evaluated whether the systemic administration of the T cell stimulatory/growth promoting cytokine interleukin-2 (IL-2) could enhance tumor lysate-pulsed DC-based immunizations to further promote protective immunity toward, and therapeutic rejection of, syngeneic murine tumors. In three separate approaches using a weakly immunogenic sarcoma (MCA-207), the systemic administration of nontoxic doses of recombinant IL-2 (20,000 and 40,000 IU/dose) was capable of mediating significant increases in the potency of DC-based immunizations. IL-2 could augment the efficacy of tumor lysate-pulsed DC to induce protective immunity to lethal tumor challenge as well as enhance splenic cytotoxic T lymphocyte activity and interferon-γ production in these treated mice. Moreover, treatment with the combination of tumor lysate-pulsed DC and IL-2 could also mediate regressions of established pulmonary 3-day micrometastases and 7-day macrometastases as well as established 14- and 28-day s.c. tumors, leading to either significant cure rates or prolongation in overall survival. Collectively, these findings show that nontoxic doses of recombinant IL-2 can potentiate the antitumor effects of tumor lysate-pulsed DC in vivo and provide preclinical rationale for the use of IL-2 in DC-based vaccine strategies in patients with advanced cancer.

Heterogeneity in the granulomatous response to mycobacterial infection in patients with defined genetic mutations in the interleukin 12-dependent interferon-gamma production pathway

Granulomatous Diseases Review Heterogeneity in the granulomatous response to mycobacterial infection in patients with defined genetic mutations in the interleukin 12-dependent interferon-gamma production pathway

Up-regulation of NADPH oxidase components and increased production of interferon-gamma by leukocytes from sickle cell disease patients

We have previously demonstrated that mononuclear leukocytes from patients with sickle cell disease (SCD) release higher amounts of superoxide compared with normal controls. The aim of this study was to further study the NADPH oxidase system in these patients by investigating gene expression of NADPH oxidase components, phosphorylation of p47(phox) component, and the release of cytokines related to NADPH oxidase activation in mononuclear leukocytes from patients with SCD. gp91(phox) gene expression was significantly higher in monocytes from SCD patients compared with normal controls (P = 0.036). Monocytes from SCD patients showed higher levels of p47 phox phosphorylation compared with normal controls. INF-gamma release by lymphocytes from SCD patients was significantly higher compared with normal controls, after 48 h culture with phytohemagglutinin (P = 0.02). The release of TNF-alpha by monocytes from SCD patients and normal controls was similar after 24 and 48 h culture with lipopolysaccharide (P > 0.05). We conclude that monocytes from SCD patients show higher levels of gp91(phox) gene expression and p47(phox) phosphorylation, along with increased IFN-gamma release by SCD lymphocytes. These findings help to explain our previous observation showing the increased respiratory burst activity of mononuclear leukocytes from SCD patients and may contribute to inflammation and tissue damage in these patients.

Inhibition of interferon-gamma-induced nitric oxide production in endotoxin-activated macrophages by cytolethal distending toxin

Introduction: Cytolethal distending toxin (CDT) is a DNA-targeting agent produced by certain pathogenic gram-negative bacteria such as the periodontopathogenic organism Aggregatibacter actinomycetemcomitans. CDT targets lymphocytes and other cells causing cell cycle arrest and apoptosis, impairing the host immune response and contributing to the persistence of infections caused by this microorganism. In this study we explored the effects of CDT on the innate immune response, by investigating how it affects production of nitric oxide (NO) by macrophages. Methods: Murine peritoneal macrophages were stimulated with Escherichia coli sonicates and NO production was measured in the presence or not of active CDT. Results: We observed that CDT promptly and significantly inhibited NO production by inducible nitric oxide synthase (iNOS) in a dose-dependent manner. This inhibition is directed towards interferon-gamma-dependent pathways and is not mediated by either interleukin-4 or interleukin-10. Conclusion: This mechanism may constitute an important aspect of the immunosuppression mediated by CDT and may have potential clinical implications in A. actinomycetemcomitans infections.