IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice.

Immunity to infection with intracellular pathogens is regulated by interleukin 12 (IL-12), which mediates protective T helper type 1 (TH1) responses, or IL-4, which induces TH2 cells and susceptibility. Paradoxically, we show here that when present during the initial activation of dendritic cells (DCs) by infectious agents, IL-4 instructed DCs to produce IL-12 and promote TH1 development. This TH1 response established resistance to Leishmania major in susceptible BALB/c mice. When present later, during the period of T cell priming, IL-4 induced TH2 differentiation and progressive leishmaniasis in resistant mice. Because immune responses developed via the consecutive activation of DCs and then T cells, the contrasting effects of IL-4 on DC development and T cell differentiation led to immune responses that had opposing functional phenotypes.

NY-ESO-1-specific circulating CD4+ T cells in ovarian cancer patients are prevalently T(H)1 type cells undetectable in the CD25+ FOXP3+ Treg compartment.

Spontaneous CD4(+) T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESO-specific CD4(+) T cells in EOC patients with spontaneous immune responses to the antigen are prevalently T(H)1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer(+) cells ex vivo, at an average frequency of 1:25,000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer(+) cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25(+)FOXP3(+)Treg. Thus, spontaneous CD4(+) T-cell responses to ESO in cancer patients are prevalently of T(H)1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines.

Endogenous angiotensin II induces atherosclerotic plaque vulnerability and elicits a Th1 response in ApoE-/- mice.

Rupture of vulnerable plaques is the main cause of acute cardiovascular events. However, mechanisms responsible for transforming a stable into a vulnerable plaque remain elusive. Angiotensin II, a key regulator of blood pressure homeostasis, has a potential role in atherosclerosis. To study the contribution of angiotensin II in plaque vulnerability, we generated hypertensive hypercholesterolemic ApoE-/- mice with either normal or endogenously increased angiotensin II production (renovascular hypertension models). Hypertensive high angiotensin II ApoE-/- mice developed unstable plaques, whereas in hypertensive normal angiotensin II ApoE-/- mice plaques showed a stable phenotype. Vulnerable plaques from high angiotensin II ApoE-/- mice had thinner fibrous cap (P<0.01), larger lipid core (P<0.01), and increased macrophage content (P<0.01) than even more hypertensive but normal angiotensin II ApoE-/- mice. Moreover, in mice with high angiotensin II, a skewed T helper type 1-like phenotype was observed. Splenocytes from high angiotensin II ApoE-/- mice produced significantly higher amounts of interferon (IFN)-gamma than those from ApoE-/- mice with normal angiotensin II; secretion of IL4 and IL10 was not different. In addition, we provide evidence for a direct stimulating effect of angiotensin II on lymphocyte IFN-gamma production. These findings suggest a new mechanism in plaque vulnerability demonstrating that angiotensin II, within the context of hypertension and hypercholesterolemia, independently from its hemodynamic effect behaves as a local modulator promoting the induction of vulnerable plaques probably via a T helper switch.

Th2 activities induced during virgin T cell priming in the absence of IL-4, IL-13, and B cells.

Virgin T cells being primed to Th2-inducing or Th1-inducing Ags, respectively, start to synthesize IL-4 or IFN-gamma as they begin to proliferate. Parallel respective induction of B cells to produce gamma1 or gamma2a switch transcripts provides additional evidence of early divergent Th activity. This report concerns the roles of IL-4, IL-13, and B cells in these early events in vivo. Th2 responses were induced in lymph nodes against hapten-protein given s.c. with killed Bordetella pertussis adjuvant. In T cell proliferation in wild-type mice, IL-4 message up-regulation and gamma1 and epsilon switch transcript production were underway 48-72 h after immunization. The absence of IL-4, IL-13, or B cells did not alter the early T cell proliferative response. The gamma1 and epsilon switch transcript production was still induced in the absence of IL-4, IL-13, or both, but at a reduced level, while the dominance of switching to IgG1 in the extrafollicular hapten-specific plasma cell response was retained. The up-regulation of IL-4 message was not reduced or delayed in the absence of B cells and was only marginally reduced by the absence of IL-13. It is concluded that signals delivered by dendritic cells, which are not dependent on the presence of IL-4, IL-13, or B cells, can prime virgin T cells and induce the early Th2 activities studied. These early events that direct virgin T cells toward Th2 differentiation contrast with the critical later role of Th2 cytokines in selectively expanding Th2 clones and driving further IL-4 synthesis.

Expansion of gamma delta+ T cells in BALB/c mice infected with Leishmania major is dependent upon Th2-type CD4+ T cells.

T cells belong to either the alpha beta+ or gamma delta+ lineage as defined by their antigen receptor. Although both T-cell subsets have been shown to be involved in the immune response to the parasite Leishmania major, very little is known about possible interactions between these two populations. In this study, using a mouse model of infection with L. major, we showed that expansion of a subset of gamma delta+ T cells in vivo is dependent upon the presence of alpha beta+ CD4+ T cells. Moreover, this effect appears to be mediated via the secretion of lymphokines by CD4+ cells with a T-helper 2 (Th2) functional phenotype. Results showing that activation of Th2-type cells in mice treated with anti-immunoglobulin D antibodies or infected with Nippostrongylus brasiliensis also results in gamma delta+ T-cell expansion suggest that this effect of the Th2-type CD4+ cells is a general phenomenon not restricted to infection with L. major.

Propionibacterium acnes Promotes Th17 and Th17/Th1 Responses in Acne Patients.

Propionibacterium acnes is a Gram-positive commensal bacterium thought to be involved in the pathogenesis of acne vulgaris. Although the ability of P. acnes in the initiation of pro-inflammatory responses is well documented, little is known about adaptive immune responses to this bacterium. The observation that infiltrating immune cells consist mainly of CD4(+) T cells in the perifollicular space of early acne lesions suggests that helper T cells may be involved in immune responses caused by the intra-follicular colonization of P. acnes. A recent report showing that P. acnes can induce IL-17 production by T cells suggests that acne might be a T helper type 17 (Th17)-mediated disease. In line with this, we show in this work that, in addition to IL-17A, both Th1 and Th17 effector cytokines, transcription factors, and chemokine receptors are strongly upregulated in acne lesions. Furthermore, we found that, in addition to Th17, P. acnes can promote mixed Th17/Th1 responses by inducing the concomitant secretion of IL-17A and IFN-γ from specific CD4(+) T cells in vitro. Finally, we show that both P. acnes-specific Th17 and Th17/Th1 cells can be found in the peripheral blood of patients suffering from acne and, at lower frequencies, in healthy individuals. We therefore identified P. acnes-responding Th17/Th1 cells as, to our knowledge, a previously unreported CD4(+) subpopulation involved in inflammatory acne.

New mechanisms regulating human Th1 and Th2 cell differentiation

Selective development of human T helper (Th) cells into functionally distinct Th1 and Th2 subtypes plays an essential role in the host immune response towards pathogens. However, abnormal function or differentiation of these cells can lead to development of various autoimmune diseases as well as asthma and allergy. Therefore, identification of key factors and the molecular mechanisms mediating Th1 and Th2 cell differentiation is important for understanding the molecular mechanisms of these diseases. The goal of this study was to identify novel factors involved in the regulation of Th1 and Th2 differentiation processes. A new method was optimized for enrichment of transiently transfected resting human primary T lymphocytes, that allowed the study of the influence of genes of interest in human Th1/Th2 cell differentiation and other primary Th cell functions. Functional characterization of PRELI, a novel activation-induced protein in human Th cells, identified it as a mitochondrial protein involved in the regulation of Th cell differentiation and apoptosis. By influencing the intracellular redox state, PRELI induces mitochondrial apoptosis pathway and downregulates STAT6 and Th2 differentiation. The data suggested that Calpain, an oxidative stress induced cysteine protease, is involved as a mediator in PRELI-induced downregulation of STAT6. PIM serine/threonine-specific kinases were identified as new regulators of human Th1 cell differentiation. PIM1 and PIM2 kinases were shown to be preferentially expressed in Th1 cells as compared to Th2 cells. RNA interference studies showed that PIM kinases enhance the production of IFN, the hallmark cytokine produced by Th1 cells. They also induce the expression of the key Th1-driving factor T-bet and the IL-12 signaling pathway during early phases of Th1 cell differentiation. Taken together, new regulators of human T helper cell differentiation were identified in this study, which provides new insights into the signaling mechanisms controlling the selective activation of human Th cell subsets.

Immune cells and oxidative stress in the endotoxin tolerance mouse model

Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.

The increased but non-predominant expression of Th17- and Th1-specific cytokines in Hashimoto’s thyroiditis but not in Graves’ disease

Hashimoto’s thyroiditis (HT) is considered to be mediated mainly by Th1 cells, but it is not known whether Graves’ disease (GD) is associated with Th1 or Th2 predominance. Th17 cells, a novel subset of Th cells, play a crucial role in the pathogenesis of various autoimmune disorders. In the present study, the expression of IL-17A and IFN-γ was investigated in patients with HT or GD. mRNA expression of IL-17A and IFN-γ in peripheral blood mononuclear cells (PBMC) from 43 patients with autoimmune thyroid disease (AITD) and in thyroid tissues from 40 AITD patients were measured by real-time qRT-PCR. The protein expression of IL-17A and IL-23p19 was examined by immunohistochemistry in thyroid tissues from 28 AITD patients. The mRNA levels of IL-17A and IFN-γ were higher in both PBMC and thyroid tissues of HT patients than in controls (mRNA levels are reported as the cytokine/β-actin ratio: IL-17 = 13.58- and 2.88-fold change and IFN-γ = 16.54- and 2.74-fold change, respectively, P < 0.05). Also, the mRNA levels of IL-17A and IFN-γ did not differ significantly in GD patients (P > 0.05). The high protein expression of IL-17A (IOD = 15.17 ± 4.8) and IL-23p19 (IOD = 16.84 ± 7.87) in HT was confirmed by immunohistochemistry (P < 0.05). The similar high levels of IL-17A and IFN-γ suggest a mixed response of Th17 and Th1 in HT, where both cells may play important roles in the destruction procedure by cell-mediated cytotoxicity.

Mécanismes moléculaires de la réplication préférentielle du VIH-1 dans les cellules à polarisation Th1Th17 versus Th1 : rôle de PPARG dans la régulation négative de la réplication virale

Les cellules T CD4+ humaines sont hétérogènes du point de vue de la permissivité à l’infection par le virus de l’immunodéficience humaine de type 1 (VIH-1). Notre laboratoire a préalablement démontré que les cellules Th1 à phénotype CXCR3+CCR6- sont relativement résistantes à l’infection par le VIH-1 alors que les cellules Th1Th17 à phénotype CXCR3+CCR6+ y sont hautement permissives. La réplication du VIH dépend de plusieurs facteurs cellulaires de restriction ou de permissivité agissant à différentes étapes du cycle viral. Toutefois, malgré plusieurs avancées, la compréhension des voies de signalisation cellulaire impliquées dans la régulation de la réplication du VIH est encore limitée. L’objectif majeur de ce projet de maîtrise est de caractériser les mécanismes moléculaires de la permissivité et de la résistance au VIH respectivement dans les cellules Th1Th17 et Th1. Ce mémoire est divisé en quatre parties qui visent: (i) l’identification des voies canoniques et des fonctions biologiques différemment régulées dans les cellules Th1Th17 versus Th1 par l’analyse de leur transcriptome au niveau du génome entier; (ii) la validation de l’expression différentielle des gènes d’intérêt identifiés par biopuces au niveau des transcrits et des protéines; (iii) la caractérisation du rôle fonctionnel de certains de ces facteurs (i.e., PPARG, AhR) sur la réplication du VIH dans les cellules Th1Th17 versus Th1; et (iv) l’identification du niveau auquel ces facteurs interfèrent avec le cycle de réplication du VIH. Nos résultats d’analyse du transcriptome du génome entier par Gene Set Enrichment Analysis et Ingenuity Pathway Analysis indiquent que les cellules à profil Th1Th17 sont plus susceptibles à l’activation cellulaire et à l’apoptose, favorisent plus l’inflammation et expriment moins fortement les gènes liés à la dégradation protéosomale comparé aux cellules à profil Th1. Ces différences dans la régulation de diverses voies et fonctions biologiques permettent en partie d’expliquer la susceptibilité à l’infection par le VIH dans ces cellules. Nous avons ensuite confirmé l’expression différentielle de certains gènes d’intérêt dans les cellules Th1Th17 (CXCR6, PPARG, ARNTL, CTSH, PTPN13, MAP3K4) versus Th1 (SERPINB6, PTK2) au niveau de l’ARNm et des protéines. Finalement, nous avons démontré le rôle des facteurs de transcription PPARG et AhR dans la régulation de la réplication du VIH. L’activation de la voie PPARG par la rosiglitazone induit la diminution importante de la réplication du VIH dans les cellules T CD4+, alors que l’activation de la voie AhR par les ligands exogènes TCDD et FICZ augmente de façon significative la réplication virale. Nous proposons que la voie PPARG agit comme un régulateur négatif de la réplication du VIH dans ces cellules, en interférant avec la polarisation Th17 et probablement en inhibant l’activité transcriptionnelle du facteur NF-kB. Les rôles des formes nucléaires versus cytoplasmiques du récepteur Ahr semblent être diamétralement opposés, dans la mesure où l’interférence ARN contre AhR s’associe également à l’augmentation de la réplication virale. Il est ainsi possible que la forme cytoplasmique d’AhR, connue par son activité E3 ligase, participe à la dégradation protéosomale des particules virales. Le mécanisme par lequel le AhR nucléaire versus cytoplasmique interfère avec la réplication virale est en cours d’étude au laboratoire. Cette étude représente la première caractérisation de l’expression différentielle de gènes au niveau du génome entier de sous-populations T CD4+ permissives versus résistantes à l’infection par le VIH. Nos résultats identifient de nouvelles cibles moléculaires pour de nouvelles stratégies thérapeutiques visant à limiter la réplication du VIH dans les lymphocytes T CD4+ primaires.

Influence of Th1/Th2 cytokines and nitric oxide in murine systemic infection induced by Sporothrix schenckii

In an attempt to elucidate the effects of Sporothrix schenckii infection on the immune response, our laboratory has developed a murine model of disseminated sporotrichosis. Helper T cells can be further subdivided into Th1 and Th2 phenotypes. The differentiation of two subsets of T lymphocytes is driven by IL-12 and IL-4 cytokines, respectively. Th1 cells produce IFN-gamma that activate macrophages and promote cell-mediated immunity. In addition, we found low levels of iNOS and NO production in the initial (1st and 2nd weeks) and final (9th and 10th weeks) periods of the infection, in contrast with the period of week 4 to 7 of elevated values. The determination of IFN-gamma and IL-12 are in agreement with NO/iNOS detection, showing the presence of cellular immune response throughout the infectious process. However, the production of IL-4 shows an increase in levels after the 5th and 6th weeks suggesting a participation of Th2 response in this period as well. Regarding these results, the study demonstrated that in experimental sporotrichosis infection the cellular immune response participated throughout the period analyzed as a nitric oxide dependent mechanism. In contrast, the presence of Th2 response began in the 5th week, suggesting the participation of humoral immune response in advanced stages of sporotrichosis.

Decreased production of TNF-alpha by lymph node cells indicates experimental autoimmune encephalomyelitis remission in Lewis rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Are the severe injuries of cutaneous leishmaniasis caused by an exacerbated Th1 response?

American tegumentary leishmaniasis (ATL) is a disease whose clinical features are strongly related to the type of immune response it induces. Herein we report an atypical presentation of cutaneous leishmaniasis in a woman with a severe and extensive sore located in her leg, and we describe the differences between the usual local immune response in ATL and the local immune response in this patient. We observed an intense inflammatory response characterized by Th1 cells and cytokines with conspicuous expression of Toll-like receptor 3 (TLR-3). Few parasites were present, but there was an extensive tissue damage. We also discuss the immunological factors that could be related to the atypical presentation.

Intrauterine Undernourishment Alters TH1/TH2 Cytokine Balance and Attenuates Lung Allergic Inflammation in Wistar Rats

IL-4 produced by Th2 cells can block cytokine production by Th1 cells, and Th1 IFN-gamma is known to counterregulate Th2 immune response, inhibiting allergic eosinophilia. As intrauterine undernutrition can attenuate lung inflammation, we investigated the influence of intrauterine undernourishment on the Th1/Th2 cytokine balance and allergic lung inflammation. Intrauterine undernourished offspring were obtained from dams fed 50% of the nourished diet of their counterparts and were immunized at 9 weeks of age. We evaluated the cell counts and cytokine protein expression in the bronchoalveolar lavage, mucus production and collagen deposition, and cytokine gene expression and transcription factors in lung tissue 21 days after ovalbumin immunization. Intrauterine undernourishment significantly reduced inflammatory cell airway infiltration, mucus secretion and collagen deposition, in rats immunized and challenged. Intrauterine undernourished rats also exhibited an altered cytokine expression profile, including higher TNF-alpha and IL-1 beta expression and lower IL-6 expression than well-nourished rats following immunization and challenge. Furthermore, the intrauterine undernourished group showed reduced ratios of the IL-4/IFN-gamma and the transcription factors GATA-3/T-Bet after immunization and challenge. We suggest that the attenuated allergic lung inflammation observed in intrauterine undernourished rats is related to an altered Th1/Th2 cytokine balance resulting from a reduced GATA-3/T-bet ratio. Copyright (C) 2012 S. Karger AG, Basel

Deficient Regulatory T Cell Activity and Low Frequency of IL-17-Producing T Cells Correlate with the Extent of Cardiomyopathy in Human Chagas' Disease

Background: Myocardium damage during Chagas' disease results from the immunological imbalance between pro-and production of anti-inflammatory cytokines and has been explained based on the Th1-Th2 dichotomy and regulatory T cell activity. Recently, we demonstrated that IL-17 produced during experimental T. cruzi infection regulates Th1 cells differentiation and parasite induced myocarditis. Here, we investigated the role of IL-17 and regulatory T cell during human Chagas' disease. Methodology/Principal Findings: First, we observed CD4(+)IL-17(+) T cells in culture of peripheral blood mononuclear cells (PBMC) from Chagas' disease patients and we evaluated Th1, Th2, Th17 cytokine profile production in the PBMC cells from Chagas' disease patients (cardiomyopathy-free, and with mild, moderate or severe cardiomyopathy) cultured with T. cruzi antigen. Cultures of PBMC from patients with moderate and severe cardiomyopathy produced high levels of TNF-alpha, IFN-gamma and low levels of IL-10, when compared to mild cardiomyopathy or cardiomyopathy-free patients. Flow cytometry analysis showed higher CD4(+)IL-17(+) cells in PBMC cultured from patients without or with mild cardiomyopathy, in comparison to patients with moderate or severe cardiomyopathy. We then analyzed the presence and function of regulatory T cells in all patients. All groups of Chagas' disease patients presented the same frequency of CD4(+)CD25(+) regulatory T cells. However, CD4(+)CD25(+) T cells from patients with mild cardiomyopathy or cardiomyopathy-free showed higher suppressive activity than those with moderate and severe cardiomyopathy. IFN-gamma levels during chronic Chagas' disease are inversely correlated to the LVEF (P = 0.007, r = -0.614), while regulatory T cell activity is directly correlated with LVEF (P = 0.022, r = 0.500). Conclusion/Significance: These results indicate that reduced production of the cytokines IL-10 and IL-17 in association with high levels of IFN-gamma and TNF-alpha is correlated with the severity of the Chagas' disease cardiomyopathy, and the immunological imbalance observed may be causally related with deficient suppressor activity of regulatory T cells that controls myocardial inflammation.