T1/ST2 is preferentially expressed on murine Th2 cells, independent of interleukin 4, interleukin 5, and interleukin 10, and important for Th2 effector function

T helper (Th) cells can be categorized according to their cytokine expression. The differential induction of Th cells expressing Th1 and/or Th2 cytokines is key to the regulation of both protective and pathological immune responses. Cytokines are expressed transiently and there is a lack of stably expressed surface molecules, significant for functionally different types of Th cells. Such molecules are of utmost importance for the analysis and selective functional modulation of Th subsets and will provide new therapeutic strategies for the treatment of allergic or autoimmune diseases. To this end, we have identified potential target genes preferentially expressed in Th2 cells, expressing interleukin (IL)-4, IL-5, and/or IL-10, but not interferon-γ. One such gene, T1/ST2, is expressed stably on both Th2 clones and Th2-polarized cells activated in vivo or in vitro. T1/ST2 expression is independent of induction by IL-4, IL-5, or IL-10. T1/ST2 plays a critical role in Th2 effector function. Administration of either a mAb against T1/ST2 or recombinant T1/ST2 fusion protein attenuates eosinophilic inflammation of the airways and suppresses IL-4 and IL-5 production in vivo following adoptive transfer of Th2 cells.

Bordetella pertussis infection in 2-month-old infants promotes type 1 T cell responses.

Neonatal immaturity of the immune system is currently believed to generally limit the induction of immune responses to vaccine Ags and to skew them toward type 2 responses. We demonstrated here that Bordetella pertussis infection in very young infants (median, 2 mo old) as well as the first administration of whole-cell pertussis vaccine induces B. pertussis Ag-specific IFN-gamma secretion by the PBMC of these infants. IFN-gamma was secreted by both CD4(+) and CD8(+) T lymphocytes, and the levels of Ag-induced IFN-gamma secretion did not correlate with the age of the infants. Appearance of the specific Th-1 cell-mediated immunity was accompanied by a general shift of the cytokine secretion profile of these infants toward a stronger Th1 profile, as evidenced by the response to a polyclonal stimulation. We conclude that the immune system of 2-mo-old infants is developmentally mature enough to develop Th1 responses in vivo upon infection by B. pertussis or vaccination with whole-cell pertussis vaccines.

Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response.

Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4(+) T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-kappaB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.

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

The Effects Of Farnesyltransferase Inhibitor Abt-100 On Murine Helper T-Cell Differentiation And Cytokine Secretion

Farnesyltransferase Inhibitors (FTIs) are a class of drugs known to prevent the farnesylation and subsequent membrane attachment of a number of intracellular proteins. In various studies, the administration of FTIs has been found to play a role in the activation and development of T-cells in the immune system. FTIs have also been found to act as immunomodulators in delaying MHC-II mismatched skin allografts in mice. This study focuses on the effect of the FTI, ABT-100, on the differentiation and cytokine secretion of Th1 and Th2 helper T-cells in BALB/C mice to better understand which immune responses are targeted by FTIs. Splenocytes were isolated from BALB/C mice, skewed towards either a Th1 or a Th2 phenotype with the addition of cytokines, and treated with various concentrations of ABT-100. Splenocytes were also isolated and immediately cultured in the presence of ABT-100 to observe differentiation trends of helper T-cells. Cytokine production was measured using intracytoplasmic flow cytometry analysis. I found that ABT-100 treatment does not block Th1 or Th2 cell differentiation. Instead, ABT-100 treatment appears to affect cytokine production from effector T-cells. I found that ABT-100 causes a decrease in IFN-¿ production in mature Th1 cells yet does not affect IL-4 production in mature Th2 cells. This decrease in cytokine production as a result of ABT-100 treatments provides a potential mechanism for how ABT-100 works to delay MHC-II mismatched allograft rejection.

Regulatory T Cells Accumulate in the Lung Allergic Inflammation and Efficiently Suppress T-Cell Proliferation but Not Th2 Cytokine Production

Foxp3(+)CD25(+)CD4(+) regulatory T cells are vital for peripheral tolerance and control of tissue inflammation. In this study, we characterized the phenotype and monitored the migration and activity of regulatory T cells present in the airways of allergic or tolerant mice after allergen challenge. To induce lung allergic inflammation, mice were sensitized twice with ovalbumin/aluminum hydroxide gel and challenged twice with intranasal ovalbumin. Tolerance was induced by oral administration of ovalbumin for 5 consecutive days prior to OVA sensitization and challenge. We detected regulatory T cells (Foxp3(+)CD25(+)CD4(+) T cells) in the airways of allergic and tolerant mice; however, the number of regulatory T cells was more than 40-fold higher in allergic mice than in tolerant mice. Lung regulatory T cells expressed an effector/memory phenotype (CCR4(high)CD62L(low)CD44(high)CD54(high)CD69(+)) that distinguished them from naive regulatory T cells (CCR4(int)CD62L(high)CD44(int)CD54(int)CD69(-)). These regulatory T cells efficiently suppressed pulmonary T-cell proliferation but not Th2 cytokine production.

Modulation of the infant immune responses by the first pertussis vaccine administrations.

Many efforts are currently made to prepare combined vaccines against most infectious pathogens, that may be administered early in life to protect infants against infectious diseases as early as possible. However, little is known about the general immune modulation induced by early vaccination. Here, we have analyzed the cytokine secretion profiles of two groups of 6-month-old infants having received as primary immunization either a whole-cell (Pw) or an acellular (Pa) pertussis vaccine in a tetravalent formulation of pertussis-tetanus-diphtheria-poliomyelitis vaccines. Both groups of infants secreted IFN-gamma in response to the Bordetella pertussis antigens filamentous haemagglutinin and pertussis toxin, and this response was correlated with antigen-specific IL-12p70 secretion, indicating that both pertussis vaccines induced Th1 cytokines. However, Pa recipients also developed a strong Th2-type cytokine response to the B. pertussis antigens, as noted previously. In addition, they induced Th2-type cytokines to the co-administrated antigen tetanus toxoïd, as well as to the food antigen beta-lactoglobulin. Furthermore, the general cytokine profile of the Pa recipients was strongly Th2-skewed at 6 months, as indicated by the cytokines induced by the mitogen phytohaemagglutinin. These data demonstrate that the cytokine profile of 6-month-old infants is influenced by the type of formulation of the pertussis vaccine they received at 2, 3 and 4 months of life. Large prospective studies would be warranted to evaluate the possible long-term consequences of this early modulation of the cytokine responses in infants.

SLAT regulates Th1 and Th2 inflammatory responses by controlling Ca2+/NFAT signaling

SWAP-70-like adapter of T cells (SLAT) is a novel guanine nucleotide exchange factor for Rho GTPases that is upregulated in Th2 cells, but whose physiological function is unclear. We show that SLAT-/- mice displayed a developmental defect at one of the earliest stages of thymocyte differentiation, the double-negative 1 (DN1) stage, leading to decreased peripheral T cell numbers. SLAT-/- peripheral CD4+ T cells demonstrated impaired TCR/CD28-induced proliferation and IL-2 production, which was rescued by the addition of exogenous IL-2. Importantly, SLAT-/- mice were grossly impaired in their ability to mount not only Th2, but also Th1-mediated lung inflammatory responses, as evidenced by reduced airway neutrophilia and eosinophilia, respectively. Levels of Th1 and Th2 cytokine in the lungs were also markedly reduced, paralleling the reduction in pulmonary inflammation. This defect in mounting Th1/Th2 responses, which was also evident in vitro, was traced to a severe reduction in Ca2+ mobilization from ER stores, which consequently led to defective TCR/CD28-induced translocation of nuclear factor of activated T cells 1/2 (NFATc1/2). Thus, SLAT is required for thymic DN1 cell expansion, T cell activation, and Th1 and Th2 inflammatory responses.

SOCS3 regulates onset and maintenance of TH2- mediated allergic responses

Members of the suppressor of cytokine signaling (SOCS) family are involved in the pathogenesis of many inflammatory diseases. SOCS-3 is predominantly expressed in T-helper type 2 (TH2) cells, but its role in TH2-related allergic diseases remains to be investigated. In this study we provide a strong correlation between SOCS-3 expression and the pathology of asthma and atopic dermatitis, as well as serum IgE levels in allergic human patients. SOCS-3 transgenic mice showed increased TH2 responses and multiple pathological features characteristic of asthma in an airway hypersensitivity model system. In contrast, dominant-negative mutant SOCS-3 transgenic mice, as well as mice with a heterozygous deletion of Socs3, had decreased TH2 development. These data indicate that SOCS-3 has an important role in regulating the onset and maintenance of TH2-mediated allergic immune disease, and suggest that SOCS-3 may be a new therapeutic target for the development of antiallergic drugs.

Regulation of Foxp3+ Inducible Regulatory T Cell Stability by SOCS2

Suppressor of cytokine signaling (SOCS) proteins are key regulators of CD4+ T cell differentiation, and in particular, we have recently shown that SOCS2 inhibits the development of Th2 cells and allergic immune responses. Interestingly, transcriptome analyses have identified SOCS2 as being preferentially expressed in both natural regulatory T cells (Tregs) and inducible Tregs (iTregs); however, the role of SOCS2 in Foxp3+ Treg function or development has not been fully elucidated. In this study, we show that despite having no effect on natural Treg development or function, SOCS2 is highly expressed in iTregs and required for the stable expression of Foxp3 in iTregs in vitro and in vivo. Indeed, SOCS2-deficient CD4+ T cells upregulated Foxp3 following in vitro TGF-ß stimulation, but failed to maintain stable expression of Foxp3. Moreover, in vivo generation of iTregs following OVA feeding was impaired in the absence of SOCS2 and could be rescued in the presence of IL-4 neutralizing Ab. Following IL-4 stimulation, SOCS2-deficient Foxp3+ iTregs secreted elevated IFN-? and IL-13 levels and displayed enhanced STAT6 phosphorylation. Therefore, we propose that SOCS2 regulates iTreg stability by downregulating IL-4 signaling. Moreover, SOCS2 is essential to maintain the anti-inflammatory phenotype of iTregs by preventing the secretion of proinflammatory cytokines. Collectively, these results suggest that SOCS2 may prevent IL-4-induced Foxp3+ iTreg instability. Foxp3+ iTregs are key regulators of immune responses at mucosal surfaces; therefore, this dual role of SOCS2 in both Th2 and Foxp3+ iTregs reinforces SOCS2 as a potential therapeutic target for Th2-biased diseases.

Effect of vitamin D on Epstein-Barr (EBV)-specific CD8+T cells in patients with early multiple sclerosis (MS)

Background: Infection with EBV and a lack in vitamin D may be important environmental triggers of MS. 1,25-(OH)2D3 mediates a shift of antigen presenting cells (APC) and CD4+ T cells to a less inflammatory profile. Although CD8+ T cells do express the vitamin D receptor, a direct effect of 1,25(OH)2D3 on these cells has not been demonstrated until now. Since CD8+ T cells are important immune mediators of the inflammatory response in MS, we examined whether vitamin D directly affects the CD8+ T cell response, and more specifically if it modulates the EBV-specific CD8+ T cell response. Material and Methods: To explore whether the vitamin D status may influence the pattern of the EBV-specific CD8+ T cell response, PBMC of 10 patients with early MS and 10 healthy controls (HC) were stimulated with a pool of immunodominant 8-10 mer peptide epitopes known to elicit CD8+ T cell responses. PBMC were stimulated with this EBV CD8 peptide pool, medium (negative control) or anti- CD3/anti-CD28 beads (positive control). The following assays were performed: ELISPOT to assess the secretion of IFN-gamma by T cells in general; cytometric beads array (CBA) and ELISA to determine whichcytokines were released by EBV-specific CD8+ T cells after six days of culture; and intracellular cytokine staining assay to determine by which subtype of T cells secreted given cytokines. To examine whether vitamin D could directly modulate CD8+ T cell immune responses, we depleted CD4+ T cells using negative selection. Results: We found that pre-treatment of vitamin D had an antiinflammatory action on both EBV-specific CD8+ T cells and on CD3/ CD28-stimulated T cells: secretion of pro-inflammatory cytokines (IFNgamma and TNF-alpha) was decreased, whereas secretion of antiinflammatory cytokines (IL-5 and TGF-beta) was increased. At baseline, CD8+ T cells of early MS patients showed a higher secretion of TNFalpha and lower secretion of IL-5. Addition of vitamin D did not restore the same levels of both cytokines as compared to HC. Vitamin D-pretreated CD8+T cells exhibited a decreased secretion of IFN-gamma and TNF-alpha, even after depletion of CD4+ T cells from culture. Conclusion: Vitamin D has a direct anti-inflammatory effect on CD8+ T cells independently from CD4+ T cells. CD8+ T cells of patients with earlyMS are less responsive to the inflammatory effect of vitamin D than HC, pointing toward an intrinsic dysregulation of CD8+ T cells. The modulation of EBV-specific CD8+T cells by vitaminDsuggests that there may be interplay between these twomajor environmental factors of MS. This study was supported by a grant from the Swiss National Foundation (PP00P3-124893), and by an unrestricted research grant from Bayer to RDP.

Changes in histone acetylation and methylation that are important for persistent but not transient expression of CCR4 in human CD4(+) T cells

Although regulation of CXCR3 and CCR4 is related to Th1 and Th2 differentiation, respectively, many CXCR3(+) and CCR4(+) cells do not express IFN-gamma and/or IL-4, suggesting that the chemokine receptor genes might be inducible by mechanisms that are lineage-independent. We investigated the regulation of CXCR3 versus IFNG, and CCR4 versus IL4 in human CD4(+) T cells by analyzing modifications of histone H3. In naive cord-blood cells, under nonpolarizing conditions not inducing IL4, CCR4 was induced to high levels without many of the activation-associated changes in promoter histone H3 found for both IL4 and CCR4 in Th2 cells. Importantly, CCR4 expression was stable in Th2 cells, but fell in nonpolarized cells after the cells were rested; this decline could be reversed by increasing histone acetylation using sodium butyrate. Patterns of histone H3 modifications in CXCR3(+) CCR4(-) and CXCR3(-) CCR4(+) CD4(+) T-cell subsets from adult blood matched those in cells cultured under polarizing conditions in vitro. Our data show that high-level lineage-independent induction of CCR4 can occur following T-cell activation without accessibility-associated changes in histone H3, but that without such changes expression is transient rather than persistent.

Resposta celular Th1 /Th2 na doença periodontal experimental, em ratos: um estudo imuno-histoquímico

Host response plays a major role in the pathogenesis of periodontal disease. Mediators such as inflammatory cytokines which are secreted during the immune response to bacterial challenges have ambiguous functions that may or may not lead to protection of the attacked tissue. In this context, experimental evidence suggests that T-helper 1 (Th-1) and T-helper 2 (Th-2) mediated responses are potentially important during the disease process. The aims of this study therefore were to further clarify the role played by Th2 cells during different time points of the active phase of periodontal disease, as well as, to investigate whether there was any evidence of a Th1 response in the periodontal disease microenvironment. Experimental periodontitis was induced in 30 Wistar male rats by placing cotton ligatures around the mandibular first molars. The rats were then randomly divided into two groups. Group1 (G1=15) and Group 2 (G2=15). In G1 the ligatures were maintained for 2 days, whereas in G2 the ligatures were left for 15 days, a time point that corresponds to the advanced stage of periodontal disease The contra-lateral teeth served as controls (no ligatures). Immunohistochemical investigation for the presence in gingival tissue of Th2 specific transcription factor (GATA3) and the subunit of the IFN-γ receptor was carried out after the disease induction period. Light microscopy analysis revealed a decrease in the expression of GATA-3 as bone loss progressed. On the other hand, although IFN-γ R1 was detected at an early stage of the active phase of disease its expression remained unaltered during the remaining period of the study. These results indicate that the Th2 response have a protective role during the pathogenesis of periodontal disease and that the progression of the periodontal disease is related with the unbalance of the responses Th1/Th2

Recovery from Strongyloides venezuelensis infection in Lewis rats is associated with a strong Th2 response

P>In this study, we investigated the characteristics of the infection and subsequent immunity induced by Strongyloides venezuelensis in Lewis rats. Animals were infected with 4000 L3 of S. venezuelensis and number of eggs per gram of faeces indicated an acute phase around day 8 and a recovery phase around day 32 after infection. A strong Th2 polarization during recovery phase was ascertained by a significant increase in IgG1 and IgE compared with that in the acute period. A shift in the cytokine profile confirmed these findings. A predominant production of IFN-gamma during the acute phase was followed by IL-10 production during recovery. Together these findings show that experimental infection of Lewis rats with S. venezuelensis presents a kinetics of parasite establishment and immunity similar to that described in other models of helminthic infection.

Molekulare Reaktionsmuster in dendritischen Zellen nach Allergenexposition

Allergische Erkrankungen, wie zum Beispiel die allergische Rhinitis oder das allergische Asthma haben im Verlauf der letzten vier Jahrzehnte stark zugenommen. So leidet heute jeder vierte bis fünfte Mensch an einer Allergie. Ausgelöst wird diese IgE-vermittelte Hypersensibilitätsreaktion des Typs I (Allergie vom Soforttyp) von Allergenen und beruht auf der Aktivierung von Mastzellen durch die Interaktion eines Antigens mit dem an eine Mastzelle über die Fc-Rezeptoren gebundenen IgE-Moleküls. Die degranulierende Mastzelle sezerniert Mediatoren, was zu einem Auftreten von allergischen Symptomen führt. Die Bildung von IgE wird durch das von TH2-Zellen produzierte Zytokin IL-4 induziert. Das von TH1-Zellen produzierte Zytokin IFN- ist in der Lage die Sekretion von IL-4 zu inhibieren, wie auch IL-4 hemmend auf die Produktion von IFN- wirkt. Dieses TH1-/ TH2-Gleichgewicht ist bei allergischen Erkrankungen in Richtung TH2 verschoben. Allergene werden von antigenpräsentierenden Zellen aufgenommen, prozessiert und auf der Zelloberfläche präsentiert. Die potentesten antigenpräsentierenden Zellen sind die dendritischen Zellen, die nach Kontakt mit einem Allergen in die benachbarten Lymphknoten wandern, ausreifen und kostimulatorische Moleküle exprimieren. Sie sind so in der Lage T-Zellen zu aktivieren und entweder in TH1- oder in TH2-Zellen differenzieren zu lassen. Die zytokinabhängige TH1- beziehungsweise TH2-Differenzierung führt zur Aktivierung der Januskinasen. Im aktiven Zustand phosphorylieren sie STAT-Moleküle, die dimerisieren und in den Zellkern translozieren, wo sie unter anderem als Transkriptionsfaktoren für Zytokingene dienen. Unreife humane dendritische Zellen von Allergikern zeigen nach Stimulation mit Proteinallergenen eine schnelle Phosphorylierung des mit der TH2-Entwicklung assoziierten STAT6. Dahingegen sind TH1-Antwort hervorrufende Kontaktallergene nicht in der Lage STAT6 oder andere STAT-Moleküle in dendritischen Zellen zu induzieren. Die Transkriptionsfaktoren T-bet und GATA3 sind ebenfalls von Bedeutung für die TH1-/TH2-Entwicklung, da T-bet ausschließlich in TH1-Zellen, GATA3 nur in TH2-Zellen exprimiert wird. Die Regulation des JAK/STAT-Weg unterliegt den Molekülen der intrazellulär vorkommenden Familie der SOCS-Proteine. SOCS3 ist in TH2-Zellen höher exprimiert als SOCS1, wohingegen SOCS1 in TH1-Zellen eine erhöhte Expression gegenüber SOCS3 aufweist. In der vorliegenden Arbeit wurde der Einfluss von Proteinallergenen auf humane dendritische Zellen untersucht. Zunächst konnte eine morphologische Veränderung der unreifen dendritischen Zellen nach Kontakt mit dem Allergenextrakt beobachtet werden. Die beginnende Ausreifung der Zellen konnte mittels Durchflußzytometrie anhand der kostimulatorischen Moleküle CD80 und CD86, insbesondere aber über den Marker für reife dendritische Zellen CD83, nachgewiesen werden. Die zu beobachtende beginnende Ausreifung scheint ein Effekt des bakteriellen Lipopolysaccharids (LPS) zu sein, das in dem Allergenextrakt vorkommt, da sich durch Zugabe des kationischen Antibiotikums Polymyxin B die beginnende Reifung verhindern ließ. Auf RNA-Ebene war es im Rahmen dieser Arbeit möglich, den Einfluss verschiedener Allergene auf unreifen humanen dendritischen Zellen näher zu charakterisieren. So weisen unreife humane dendritische Zellen nach Kontakt mit Proteinallergenextrakt ein TH2-assoziiertes Genexpressionprofil auf, was sich durch eine erhöhte relative Expression der Gene SOCS3 und GATA3 auszeichnet. Im Gegensatz hierzu zeigen unreife humane dendritische Zellen nach Inkubation mit dem Kontaktallergen MCI/MI eine erhöhte relative Expression des Gens T-bet, was mit einer TH1-Antwort assoziiert ist. Nach Zugabe des „TH1-/ TH2-neutralen“ Tetanustoxoids konnten erhöhte relative Expressionen der Gene GATA3, T-bet und SOCS3 gemessen werden. Die Ergebnisse in dem in dieser Arbeit benutzten humanen in vitro System geben Anlass zur Hypothese, dass die Art der Immunantwort (TH1 versus TH2) sich bereits auf Ebene der dendritischen Zellen anbahnt. GeneChip-Analysen mittels High Density Micro Arrays von unreifen humanen dendritischen Zellen, die entweder mit Proteinallergenextrakt oder mit LPS in Berührung kamen, zeigten statistisch signifikant regulierte Gene, die allerdings keine Gemeinsamkeiten aufwiesen. Es konnten für die mit Alllergenextrakt gepulsten dendritischen Zellen insgesamt 10 Gene identifiziert werden, jedoch gelang es nicht, diese näher zu deuten oder in einen Zusammenhang mit der allergischen Erkrankung oder der dendritischen Zelle zu bringen. Für die mit LPS, dem stärkeren Stimulus, gepulsten dendritischen Zellen konnten 40 Gene identifiziert werden, die unter anderem für die Maturierung der dendritischen Zelle verantwortlich sind. Zudem war es möglich, die Daten der Arrays auf Proteinebene exemplarisch anhand des Chemokins CXCL2 (Gro-β) zu verifizieren.