Interleukins, cytokines secreted by leukocytes, are predominant messengers modulating immune responses. Interleukin-27 (IL-27), a key immunomodulatory cytokine, functions to induce both pro- and anti-inflammatory effects in various immune cells. IL-27 is a heterodimeric cytokine, composed of IL-27p28 and Epstein-Bar virus induced gene 3 (EBI3) subunits, and binds to a receptor composed of IL-27Rα (WSX-1) and gp130. Initial studies focused on describing IL-27 functions in skewing T helper cell development to a Th1 response, with few reports on functions in monocytes. Thus, in this thesis, I aimed to characterize novel functions of IL-27 in innate immune responses of monocytes. I initially established that IL-27 induced a pro-inflammatory cytokine profile (IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α) mediated via STAT1/3 and NF-κB signaling pathways. Further investigation led to the discovery that IL-27 could enhance LPS responses via upregulation of TLR4 expression and NF-κB signaling. Together, these studies described novel signaling mechanisms (NF-κB and JAK/STAT crosstalk) and gene targets (cytokines and TLR4) of IL-27 that drive inflammatory responses. In continuing the quest for novel IL-27 functions in innate immunity, I reported IL-27 can upregulate expression of the IFN-responsive, antiviral protein called BST-2. My results showing IL-27-induced expression of BST-2 mRNA and cell surface protein were supported by previous studies reporting IL-27-induced expression of other antiviral molecules. Furthermore, previous studies showed IL-27 could inhibit HIV replication via antiviral gene induction, pointing to potential for IL-27 immunotherapies. In light of the posited role for IL-27 in therapeutics, it became inherently critical to describe how IL-27 functions in the setting of HIV infection. Thus, in my final thesis chapters, I described the effect of HIV infection on IL-27 expression and functions, addressing a substantial void in literature. Interestingly, a trend of decreased IL-27 expression and significant impairment of IL-27-induced gene expression was observed in HIV infection. Therefore, decreased circulating IL-27 and decreased IL-27 responsiveness may collectively dysregulate IL-27 function in HIV. This thesis describes novel, IL-27-driven, proinflammatory responses, and highlights impairment of IL-27 function in HIV infection. This work bridged a gap in knowledge of IL-27 functions in monocytes and highlighted multifaceted mechanisms underlying immunoregulation by IL-27. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2012-04-12 13:07:50.588

Interleukin-27 (p28/EBI3) is an immunomodulatory cytokine expressed by activated antigen presenting cells. Although first discovered to be involved in Th1 cell differentiation, further studies demonstrated the immunosuppressive functions of IL-27 including inhibition of Th2 and Th17 differentiation, development of a tolerogenic phenotype in dendritic cells (DC), and promoting type 1 regulatory T cells (Tr1). The anti-inflammatory effects of IL-27 have been demonstrated in vivo in murine models of parasitic infections and autoimmune diseases. Despite the prevalence of studies detailing the induction of IL-27 expression and the role of IL-27 in Tr1 differentiation, little is known about factors that negatively regulate IL-27 expression and Tr1 differentiation. Prostaglandin E2 (PGE2), a lipid mediator abundant at inflammatory sites, was shown to act as a proinflammatory agent in models of inflammatory/autoimmune diseases primarily by promoting CD4 Th1/Th17 differentiation. Here we describe a novel proinflammatory mechanism for PGE2 through the inhibition of IL-27 production in conventional dendritic cells (cDC) and the inhibition of Tr1 differentiation. PGE2 inhibits IL-27 production in bone marrow-derived DC and macrophages, as well as in splenic cDC, through EP2/EP4 receptors, induction of cAMP, and downregulation of IRF1 expression and binding to the p28 IL-27 ISRE site. The inhibitory effect of PGE2 on p28 and irf1 expression does not involve endogenous IFN-β, STAT1 or STAT2, and inhibition of IL-27 does not appear to be mediated through PKA, EPAC, PI3K, or MAPKs. We observed similar inhibition of p28 expression in vivo in splenic DC following administration of dimethyl PGE2 in conjunction with LPS. In addition to the inhibition of IL-27 production in APCs, PGE2 also directly affects Tr1 differentiation by reducing IL-27-induced CD4+CD49b+LAG-3+Foxp3- Tr1 cells and IL-10 production. The inhibitory effect is mediated by EP4 and induction of cAMP in differentiating CD4 T cells. IL-27-induced Tr1 differentiation and function depends primarily on the sustained expression of c-Maf in addition to AhR and Blimp-1. PGE2 significantly reduced expression of c-Maf without affecting AhR and only marginally reducing Egr-2/Blimp-1 expression. The effects of PGE2 on Tr1 cells are independent of STAT1/STAT3 signaling and of IL-21 signaling. In addition, the effect of PGE2 on CD4+CD49b+LAG-3+ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3+ Treg or effector Th17 cells. The effects of PGE2 on both IL-27 production and IL-27-induced Tr1 differentiation represent novel proinflammatory mechanisms of PGE2. / Microbiology and Immunology

Produzida por macrófagos e células dendríticas, a IL-27 é uma citocina heterodimérica capaz de induzir células Tr1 produtoras de IL-10 e consequentemente regular linfócitos Th1, Th2 e Th17, dependendo da doença envolvida. Partindo-se do pressuposto de que a infecção causada por Trypanosoma cruzi normalmente induz miocardite refletida pela migração intensa de linfócitos Th1 para o tecido cardíaco, nós analisamos o papel regulador da IL-27 nesta condição inflamatória. Nós inicialmente verificamos que a IL-27 foi prontamente induzida in vitro em células infectadas com T. cruzi. Para gerar miocardite intensa coordenada por linfócitos Th1, nós polarizamos linfócitos T naïves para o padrão Th1 na ausência de moléculas relacionadas ao perfil Th17 (camundongos IL-17R-/-, IL-23-/- e IL-6-/-). Como esperado, a inflamação cardíaca intensa e o dano tecidual foram observados na ausência das moléculas do padrão Th17, o que contribuiu para a morte prematura dos animais IL-17R-/-, IL-23-/- e IL-6-/-, precisa e notoriamente pela indução da migração excessiva de linfócitos Th1 para o tecido cardíaco via CXCL-9 e CXCL-10. Para explorar os mecanismos pelos quais a IL-27 controla a miocardite induzida pelo T. cruzi, nós encontramos um recrutamento substancial de macrófagos produtores de IL-27 para o tecido cardíaco, o qual foi mediado pelas quimiocinas CCL3 e CCL4 na ausência de moléculas do padrão Th17. Para determinar quais os receptores necessários para a produção de IL-27, nós observamos que macrófagos derivados da medula óssea de camundongos deficientes de TLR4-/-, TLR9-/- e NLRP3-/- aboliram completamente a produção desta citocina após a infecção in vitro com T. cruzi, enquanto o receptor TLR2 foi dispensável. Nós também verificamos que macrófagos produtores de IL-27 suprimiram linfócitos Th1 através da indução de células Tr1 produtoras de IL-10 após a infecção com T. cruzi. Em seguida, nós avaliamos se a IL-27 foi correlacionada com a proteção cardíaca durante a doença de Chagas. Nós observamos níveis séricos elevados de IL-27 tanto em pacientes com a forma clínica indeterminada ou cardíaca leve, enquanto pacientes com cardiomiopatia moderada ou grave produziram níveis reduzidos de IL-27. Neste estudo, nós descrevemos um novo mecanismo regulador desempenhado por macrófagos produtores de IL-27 no controle da miocardite induzida por T. cruzi. Macrófagos produtores de IL-27 podem suprimir processos inflamatórios desencadeados por linfócitos Th1, os principais vilões na doença de Chagas. / IL-27 is a heterodimeric cytokine produced by macrophages and dendritic cells known to induce IL-10-producing Tr1 cells and to regulate Th1, Th2, and Th17 lymphocytes, depending on the underlying disease. Because the infection caused by Trypanosoma cruzi normally induces myocarditis mirrored by an outstanding migration of Th1 cells to the heart tissue, we analyzed the regulatory role of IL-27 in this inflammatory condition. We firstly verified that IL-27 was promptly induced by in vitro T. cruzi-infected spleen cells. To generate a robust myocarditis coordinated by Th1 lymphocytes, we polarized lymphocytes to a Th1 pattern by infecting mice in the absence of Th17-related molecules (IL-17R-/-, IL-23-/-, and IL-6-/- mice). As expected, an impressive cardiac inflammation and damage was observed in the absence of Th17-related molecules, leading IL-17R-/-, IL-23-/-, and IL-6-/- mice to the premature death, precisely and notably by inducing an exuberant Th1 migration to the heart tissue via CXCL9 and CXCL10 chemokines. To explore the mechanisms by which IL-27 controls T. cruzi-induced myocarditis, we found a striking recruitment of IL-27-producing macrophages to the heart tissue mediated by increased levels of CCL3 and CCL4 chemokines in the absence of Th17-associated molecules. To gain further insights into the receptors required to IL-27 production, we observed that bone marrow-derived macrophages from TLR4-/-, TLR9-/-, and NLRP3-/- mice completely abolished IL-27 production after in vitro T. cruzi infection, while TLR2 was dispensable. We also verified that IL-27-producing macrophages supressed Th1 lymphocytes by inducing IL-10-producing Tr1 cells after T. cruzi infection. We next assessed whether IL-27 was correlated to cardiac protection during Chagas Disease. We observed augmented serum levels of IL-27 in either patients with indeterminate (asymptomatic) form or mild cardiac form, whereas patients with moderate or severe cardiomyopathy were poor producers of IL-27. Here, we described a novel regulatory mechanism developed by IL-27-producing macrophages in the control of T. cruzi-induced myocarditis. IL-27-producing macrophages can suppress inflammatory processes caused by Th1 lymphocytes, the bona fide culprits of Chagas Disease.

Produzida por macrófagos e células dendríticas, a IL-27 é uma citocina heterodimérica capaz de induzir células Tr1 produtoras de IL-10 e consequentemente regular linfócitos Th1, Th2 e Th17, dependendo da doença envolvida. Partindo-se do pressuposto de que a infecção causada por Trypanosoma cruzi normalmente induz miocardite refletida pela migração intensa de linfócitos Th1 para o tecido cardíaco, nós analisamos o papel regulador da IL-27 nesta condição inflamatória. Nós inicialmente verificamos que a IL-27 foi prontamente induzida in vitro em células infectadas com T. cruzi. Para gerar miocardite intensa coordenada por linfócitos Th1, nós polarizamos linfócitos T naïves para o padrão Th1 na ausência de moléculas relacionadas ao perfil Th17 (camundongos IL-17R-/-, IL-23-/- e IL-6-/-). Como esperado, a inflamação cardíaca intensa e o dano tecidual foram observados na ausência das moléculas do padrão Th17, o que contribuiu para a morte prematura dos animais IL-17R-/-, IL-23-/- e IL-6-/-, precisa e notoriamente pela indução da migração excessiva de linfócitos Th1 para o tecido cardíaco via CXCL-9 e CXCL-10. Para explorar os mecanismos pelos quais a IL-27 controla a miocardite induzida pelo T. cruzi, nós encontramos um recrutamento substancial de macrófagos produtores de IL-27 para o tecido cardíaco, o qual foi mediado pelas quimiocinas CCL3 e CCL4 na ausência de moléculas do padrão Th17. Para determinar quais os receptores necessários para a produção de IL-27, nós observamos que macrófagos derivados da medula óssea de camundongos deficientes de TLR4-/-, TLR9-/- e NLRP3-/- aboliram completamente a produção desta citocina após a infecção in vitro com T. cruzi, enquanto o receptor TLR2 foi dispensável. Nós também verificamos que macrófagos produtores de IL-27 suprimiram linfócitos Th1 através da indução de células Tr1 produtoras de IL-10 após a infecção com T. cruzi. Em seguida, nós avaliamos se a IL-27 foi correlacionada com a proteção cardíaca durante a doença de Chagas. Nós observamos níveis séricos elevados de IL-27 tanto em pacientes com a forma clínica indeterminada ou cardíaca leve, enquanto pacientes com cardiomiopatia moderada ou grave produziram níveis reduzidos de IL-27. Neste estudo, nós descrevemos um novo mecanismo regulador desempenhado por macrófagos produtores de IL-27 no controle da miocardite induzida por T. cruzi. Macrófagos produtores de IL-27 podem suprimir processos inflamatórios desencadeados por linfócitos Th1, os principais vilões na doença de Chagas. / IL-27 is a heterodimeric cytokine produced by macrophages and dendritic cells known to induce IL-10-producing Tr1 cells and to regulate Th1, Th2, and Th17 lymphocytes, depending on the underlying disease. Because the infection caused by Trypanosoma cruzi normally induces myocarditis mirrored by an outstanding migration of Th1 cells to the heart tissue, we analyzed the regulatory role of IL-27 in this inflammatory condition. We firstly verified that IL-27 was promptly induced by in vitro T. cruzi-infected spleen cells. To generate a robust myocarditis coordinated by Th1 lymphocytes, we polarized lymphocytes to a Th1 pattern by infecting mice in the absence of Th17-related molecules (IL-17R-/-, IL-23-/-, and IL-6-/- mice). As expected, an impressive cardiac inflammation and damage was observed in the absence of Th17-related molecules, leading IL-17R-/-, IL-23-/-, and IL-6-/- mice to the premature death, precisely and notably by inducing an exuberant Th1 migration to the heart tissue via CXCL9 and CXCL10 chemokines. To explore the mechanisms by which IL-27 controls T. cruzi-induced myocarditis, we found a striking recruitment of IL-27-producing macrophages to the heart tissue mediated by increased levels of CCL3 and CCL4 chemokines in the absence of Th17-associated molecules. To gain further insights into the receptors required to IL-27 production, we observed that bone marrow-derived macrophages from TLR4-/-, TLR9-/-, and NLRP3-/- mice completely abolished IL-27 production after in vitro T. cruzi infection, while TLR2 was dispensable. We also verified that IL-27-producing macrophages supressed Th1 lymphocytes by inducing IL-10-producing Tr1 cells after T. cruzi infection. We next assessed whether IL-27 was correlated to cardiac protection during Chagas Disease. We observed augmented serum levels of IL-27 in either patients with indeterminate (asymptomatic) form or mild cardiac form, whereas patients with moderate or severe cardiomyopathy were poor producers of IL-27. Here, we described a novel regulatory mechanism developed by IL-27-producing macrophages in the control of T. cruzi-induced myocarditis. IL-27-producing macrophages can suppress inflammatory processes caused by Th1 lymphocytes, the bona fide culprits of Chagas Disease.

Les cytokines jouent un rôle fondamental dans la régulation des processus biologiques via la cascade de signalisation JAK-STAT. Les « Suppressors of Cytokine Signalling » (SOCS), protéines intracellulaires, inhibent la voie JAK-STAT. Plusieurs études supportent leur implication dans des maladies immunitaires, mais peu d’informations sont disponibles sur leur expression par les lymphocytes T humains. Nous postulons que les cytokines Interféron-β(IFN-β) et Interleukine-27 (IL-27), dotées d’un potentiel immuno-régulateur, ont des rôles bénéfiques via l’induction des SOCS. L’impact de l’IFN-β et l’IL-27 sur l’expression des SOCS-1 et SOCS-3 par des cellules T CD8 et CD4 humaines a été étudié en utilisant des cellules sanguines de donneurs sains. L’expression de ces régulateurs a été évaluée aux niveaux de l’ARNm par qRT-PCR et protéique par immunocytochimie. Les SOCS-1 et SOCS-3 ont été rapidement induits en ARNm dans les deux types cellulaires en réponse à l’IFN-β ou l’IL-27 et une augmentation de l’expression a été confirmée au niveau protéique. Afin de mimer les thérapies à base d’IFN-β, les cellules T ont été exposées chroniquement à l’IFN-β. Après chaque ajout de cytokine les cellules T ont augmenté l’expression du SOCS-1, sans moduler le SOCS-3. L’IL-27 a induit les SOCS-1 et SOCS-3 préférentiellement dans les cellules T CD8 ; ceci corrèle avec des résultats du laboratoire démontrant une plus petite expression des récepteurs à l’IL-27 par les lymphocytes T CD4 que les CD8. Notre projet a permis d’élucider l’expression des SOCS dans deux populations de cellules T et de clarifier les mécanismes d’actions de l’IFN-β et l’IL-27. / Cytokines regulate fundamental biological processes via the JAK-STAT signaling pathway. Suppressors of Cytokine Signaling proteins (SOCS), intracellular proteins, inhibit the JAK-STAT pathway. Emerging evidence supports the involvement of SOCS in diseases of the immune system but no data is available regarding their expression in human T cells. We postulate that the cytokines Interferon-β (IFN-β) and Interleukin-27 (IL-27), both potential immuno-regulators, have beneficial roles through the induction of SOCS proteins. The impact of IFN-β and IL-27 on the SOCS-1 and SOCS-3 expression by human CD4 and CD8 T cells was assessed using peripheral blood mononuclear cells from healthy donors. We evaluated the expression of SOCS-1 and SOCS-3 at the mRNA level by qRTPCR and at the protein level by immunocytochemistry. A rapid increase of SOCS-1 and SOCS-3 mRNA levels was observed upon cytokine addition, and such upregulation was confirmed at the protein level. To mimic patients under IFN-β treatment, both T cell subsets were chronically exposed to IFN-β. We observed an increase of SOCS-1 after each stimulation but not for SOCS-3. IL-27 stimulation increased SOCS-1 and SOCS-3 mRNA levels in CD8 T cells but only slightly in CD4 T cells; these observations correlate with previous observations in our laboratory showing less IL-27 receptors on CD4 T cells than CD8 counterparts. Our project determined the distinct expression of SOCS proteins in different human T cells subsets. This study could highlight the mechanism of action of cytokines such as IFN-β and IL-27.

Les cytokines jouent un rôle fondamental dans la régulation des processus biologiques via la cascade de signalisation JAK-STAT. Les « Suppressors of Cytokine Signalling » (SOCS), protéines intracellulaires, inhibent la voie JAK-STAT. Plusieurs études supportent leur implication dans des maladies immunitaires, mais peu d’informations sont disponibles sur leur expression par les lymphocytes T humains. Nous postulons que les cytokines Interféron-β(IFN-β) et Interleukine-27 (IL-27), dotées d’un potentiel immuno-régulateur, ont des rôles bénéfiques via l’induction des SOCS. L’impact de l’IFN-β et l’IL-27 sur l’expression des SOCS-1 et SOCS-3 par des cellules T CD8 et CD4 humaines a été étudié en utilisant des cellules sanguines de donneurs sains. L’expression de ces régulateurs a été évaluée aux niveaux de l’ARNm par qRT-PCR et protéique par immunocytochimie. Les SOCS-1 et SOCS-3 ont été rapidement induits en ARNm dans les deux types cellulaires en réponse à l’IFN-β ou l’IL-27 et une augmentation de l’expression a été confirmée au niveau protéique. Afin de mimer les thérapies à base d’IFN-β, les cellules T ont été exposées chroniquement à l’IFN-β. Après chaque ajout de cytokine les cellules T ont augmenté l’expression du SOCS-1, sans moduler le SOCS-3. L’IL-27 a induit les SOCS-1 et SOCS-3 préférentiellement dans les cellules T CD8 ; ceci corrèle avec des résultats du laboratoire démontrant une plus petite expression des récepteurs à l’IL-27 par les lymphocytes T CD4 que les CD8. Notre projet a permis d’élucider l’expression des SOCS dans deux populations de cellules T et de clarifier les mécanismes d’actions de l’IFN-β et l’IL-27. / Cytokines regulate fundamental biological processes via the JAK-STAT signaling pathway. Suppressors of Cytokine Signaling proteins (SOCS), intracellular proteins, inhibit the JAK-STAT pathway. Emerging evidence supports the involvement of SOCS in diseases of the immune system but no data is available regarding their expression in human T cells. We postulate that the cytokines Interferon-β (IFN-β) and Interleukin-27 (IL-27), both potential immuno-regulators, have beneficial roles through the induction of SOCS proteins. The impact of IFN-β and IL-27 on the SOCS-1 and SOCS-3 expression by human CD4 and CD8 T cells was assessed using peripheral blood mononuclear cells from healthy donors. We evaluated the expression of SOCS-1 and SOCS-3 at the mRNA level by qRTPCR and at the protein level by immunocytochemistry. A rapid increase of SOCS-1 and SOCS-3 mRNA levels was observed upon cytokine addition, and such upregulation was confirmed at the protein level. To mimic patients under IFN-β treatment, both T cell subsets were chronically exposed to IFN-β. We observed an increase of SOCS-1 after each stimulation but not for SOCS-3. IL-27 stimulation increased SOCS-1 and SOCS-3 mRNA levels in CD8 T cells but only slightly in CD4 T cells; these observations correlate with previous observations in our laboratory showing less IL-27 receptors on CD4 T cells than CD8 counterparts. Our project determined the distinct expression of SOCS proteins in different human T cells subsets. This study could highlight the mechanism of action of cytokines such as IFN-β and IL-27.