Overexpression of IL-12 and IL-23 has been implicated in the pathogenesis of Crohn’s disease. Targeting these cytokines with monoclonal antibodies has emerged as an effective therapy, but one with adverse reactions. In this study, we sought to develop peptide-based virus-like particle vaccines specific to p40 unit (shared by IL-12 and IL-23) or IL-12 (p35) or IL-23 (p19) and evaluate the effects of the vaccine in 2,4,6-trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulfate (DSS)-induced acute and chronic murine colitis. Three vaccines against p40 induced high-titered and long-lasting antibodies to IL-12, IL-23 and p40 without the use of adjuvants. Vaccine-induced antibodies could block IL-12- and IL-23-induced biological functions in vitro dose-dependently. One of the three p40 vaccines was selected for further evaluation in acute and chronic colitis. Administration of the vaccine before or after the commencement of TNBS or DSS delivery, significantly improved body weight loss and decreased inflammatory scores, collagen deposition, and the expression of p40, IL-12, IL-23, IL-17 and TNF in colon tissues, compared with mice receiving carrier protein (HBcAg) or saline. Moreover, in mesenteric lymph nodes, vaccinated mice exhibited a trend to lower percentages of Th1 cells in acute colitis and of Th17 cells in chronic colitis compared to carrier and saline controls. Vaccinated mice also had higher ratios of Treg/Th1 and Treg/Th17 and higher percentages of apoptosis in Th1 and Th17 cells than controls. Vaccine treatment decreased the infiltration of CD11c+ cells into the gut, but promoted the production of IL-10 from these cells. Safety evaluation indicated that vaccine immunization did not increase the susceptibility to the infection of chlamydia muridarum. Two vaccines specific to IL-12 (against p35) and one vaccine to IL-23 (against p19) were also developed. They induced specific antibodies against IL-12 and IL-23, respectively. IL-23p19 vaccine immunization, not IL-12p23 vaccine, ameliorated TNBS-induced chronic colitis. In summary, IL-12/IL-23p40 vaccine treatment ameliorated murine colitis through rebalancing Th1/Th17/Treg responses, promoting Th1 and Th17 apoptosis, and promoting IL-10 production, and did not increase the severity of chlamydia muridarum infection. This vaccine strategy may provide a novel long-term treatment for Crohn’s disease.

Coccidiosis vaccine associated side effects, oocyst shedding patterns, intestinal lesions, and mucosal gene expression in the turkey were studied. The first study examined vaccine associated side effects and oocyst shedding patterns under experimental conditions. Peak oocyst shedding occurred on days 5-6, 13-17, and 19-20 days post vaccination. Throughout the course of the study, several poults exhibited clinical coccidiosis. Based on body weights, growth was correlated with vaccine cycling. The second study examined coccidiosis vaccine induced lesions and changes in mucosal gene expression on day 5, 10, 13, 17, and 20 days post vaccination. Poults were gavaged the equivalent of 0x, 1/2x, 1x, and 2x the available vaccine dose. Intestinal sections adjacent to the Meckel's diverticulum, ileocecal junction, and middle of the ceca were collected for histological analysis and gene expression. Measurements from the tip of the villus to the base of the lamina propria, villus width, and the muscularis mucosae thickness were acquired from the histological sections. Interleukin-10, IL-1beta, and GAPDH gene expression were measured by extracting mRNA in the tissues and quantified using real-time RT-qPCR. Starting on day five post vaccination, the control group weighed significantly more than the group that received the 2x dose. Body weight and oocyst dose were inversely related through day 17. Intestinal measurements did not necessarily correlate with the vaccine dose, although there appears to be some correlation on day five. There were no significant changes in the mucosal gene expression of IL-10 and IL-1beta in the intestinal tissue adjacent to the Meckel's diverticulum throughout the course of the study. On day five post vaccination, IL-10 and IL-1beta were significantly upregulted in the ileocecal junction. Interleukin-10 was significantly upregulated on day 17 and IL-1beta was significanlty down regulated on day 20 in the ileocecal junction. Both IL-10 and IL-1beta were significantly upregulated in the ceca days 5, 10, and 13 post vaccination. Interleukin-10 was significnalty upregulated in the ceca on day 17 and significantly down regulated on day 20. Individual variation among poults in the same group merits further attention.

Univ., Diss.--München, 1998. / Literaturverz. S. 309 - 337.

A asma acomete cerca de trezentos milhões de pessoas em todo o mundo. A doença é caracterizada por falta de ar, chiado e compressão no peito, tosse como consequência da hiperresponsividade brônquica e limitação de fluxo aéreo, causadas pela inflamação pulmonar Th2 e eosinofílica. Porém, existem evidências de que a IL-22 e a IL-17 participam da patogênese da asma alérgica. Com intuito de compreender melhor o papel da IL-22 na asma alérgica, utilizamos camundongos deficientes em IL-22 (IL-22KO) comparando-os aos animais Wild Type (WT) expostos ao alérgeno. Animais WT e IL-22KO foram sensibilizados e desafiados com ovalbumina (OVA) e avaliados quanto à inflamação eosinofílica, produção de citocinas, produção de muco, e populações celulares no pulmão. Nossos resultados mostram que a ausência de IL-22 resultou em diminuição da eosinofilia, IL-5 e IL-13 no lavado broncoalveolar, de células CD4+IL-4+ nos linfonodos e diminuição na produção de muco nas vias aéreas. Além disso, os camundongos IL-22KO alérgicos apresentam diminuição na porcentagem e número de células dendríticas CD11c+CD11b+CD103- nos pulmões quando comparados aos respectivo grupo WT. A transferência de células Th17 geradas a partir de animais IL-22KO causou diminuição na eosinofilia em camundongos expostos ao alérgeno quando os mesmos foram comparados aos animais que receberam células Th17 geradas a partir de animais WT. Esse resultado atribui mais à IL-22 do que à IL- 17 papel patogênico na asma alérgica. Outro indício da participação patogênica da IL-22 na asma alérgica é o fato de que o tratamento alérgeno específico, combinado ou não com a terapia livre de alérgeno, induziu redução da eosinofilia, do infiltrado de células dendríticas e diminuição de IL-22 no lavado broncoalveolar. A provável ação da IL-22 é a manutenção da viabilidade e sobrevivência de eosinófilos nos pulmões, fazendo que estes leucócitos continuem auxiliando no recrutamento de células dendríticas responsáveis pela captura e apresentação do alérgeno nos linfonodos, onde haverá a diferenciação de linfócitos de padrão Th2. Essas células podem migrar para os pulmões, gerando aumento na inflamação no local. Em síntese, nosso estudo corrobora o papel proinflamatório da IL-22 na asma alérgica e mostra, de forma inédita, que a transferência de células Th17 produtoras de ambas as citocinas, IL-22 e IL-17, mas não a transferência de células produtoras apenas de IL-17, causa exacerbação da inflamação pulmonar, possivelmente relacionada com o papel da IL-22 em prevenir indiretamente a indução de apoptose nos eosinófilos. / Asthma affects approximately three hundred million people worldwide. The disease is characterized by shortness of breath, wheezing and chest compression, coughing as a result of bronchial hyperresponsiveness and airflow limitation caused by Th2 lung inflammation and eosinophilia. However, there is evidence that IL-22 and IL-17 participate in the pathogenesis of allergic asthma. With the intention to better understand the role of IL-22 in allergic asthma, we used IL-22 deficient mice (IL-22KO) comparing them to wild type animals (WT) exposed to the allergen. Animals WT and IL-22KO were sensitized and challenged with ovalbumin (OVA), and assessed for eosinophilic airway inflammation, cytokine production, mucus production, and cell populations in the lungs. Our results show that the absence of IL-22 resulted in decreased eosinophilia, IL-5 and IL-13 in the bronchoalveolar lavage, CD4 + IL-4 + cells in the lymph nodes and decrease in mucus production in the airways. In addition, allergic IL-22KO mice have decreased percentage and number of dendritic cells CD11c + CD11b + CD103- in lungs when compared to their corresponding WT group. The transfer of Th17 cells generated from IL-22KO animals caused a reduction in eosinophilia in mice exposed to the allergen when they were compared to animals that received Th17 cells generated from WT mice. This result assigns more IL-22 than IL-17 pathogenic role in allergic asthma. Another indication of the pathogenic involvement of IL-22 in allergic asthma is the fact that the specific allergen treatment, combined or not with allergen-free therapy induced a reduction of eosinophilia, the dendritic cell infiltration and decreased IL-22 in bronchoalveolar lavage. The possible action of IL-22 is maintaining the viability and survival of eosinophils in the lungs, making these leukocytes remain helping in the recruitment of dendritic cells responsible for capture and allergen presentation in lymph nodes, causing differentiation in lymphocytes Th2. These cells can migrate to the lungs, resulting in increased inflammation at the site. In summary, our study confirms the proinflammatory role of IL-22 in allergic asthma and shows, in an unprecedented manner, the transfer of producing Th17 cells of both cytokines IL-22 and IL-17, but not the transfer of cells producing only IL-17, cause exacerbation of pulmonary inflammation, possibly related to the role of IL-22 on indirectly prevent induction of apoptosis in eosinophils.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-10-14T12:30:34Z No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-10-22T13:05:05Z (GMT) No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) / Made available in DSpace on 2016-10-22T13:05:05Z (GMT). No. of bitstreams: 1 vanessadealmeidabelo.pdf: 412659 bytes, checksum: 2be0607436379c3a9ca0f2415972f9be (MD5) Previous issue date: 2008-02-15 / Nos países tropicais, as perdas causadas pela infestação de carrapatos em bovinos acarretam um grande impacto no sistema de produção animal. Recentes estudos têm mostrado a importância de fatores genéticos ligados a resistência a carrapato em Bos taurus indicus e Bos taurus taurus e que as citocinas têm um papel crítico na prevenção ou progressão de doenças. O objetivo desse trabalho foi avaliar os níveis de expressão dos genes IL-10 e IL-4 relacionados ao perfil imunológico Th2 associado à susceptibilidade ao carrapato e os genes IL-2 e IFN- relacionados ao perfil imunológico Th1 associado à resistência ao parasito. Além destes genes, analisou-se o perfil de expressão do gene TLR-2, importante no processo de reconhecimento de patógenos e os genes IL-8 e TNF-α importantes no processo inflamatório inicial. Seis animais mais resistentes e seis animais mais susceptíveis de uma população F2 de 332 animais, originária do cruzamento de animais F1(½ Holandês: ½ Gir), foram selecionados baseado na contagem de carrapatos e valor genético. Amostras de tecido foram coletadas de pele no 5° e 12° dias após a infestação para extração de RNA total. As PCRs em tempo real foram realizadas usando o gene GAPDH como controle endógeno. Os animais resistentes e susceptíveis apresentaram aumento de expressão do gene IL-10 no 5° (p<0,01) e 12 ° dias após a infestação (p<0,05). O gene IL-2, nos animais resistentes e susceptíveis, no 5° dia após a infestação não apresentou alteração da expressão sendo que 12° dia, em ambos os grupos de animais, este gene passou a ser mais expresso em relação ao animal controle sugerindo um perfil de resposta imunológica do tipo de Th2 nos animais resistentes e susceptíveis nos primeiros dias após a infestação. O gene IL-4 apresentou uma tendência ao aumento de expressão nos animais resistentes e susceptíveis em relação ao controle, sendo o perfil Th2 sugerido atribuído a IL-10 produzida por linfócitos T regulatórios (p>0,05). O gene TNF- apresentou aumento de expressão nos animais susceptíveis no 5° dia após a infestação com posterior diminuição no 12° dia após a infestação (p<0,05). Nos animais resistentes não foi observada alteração da expressão deste gene, isto sugere que ele possa estar mais atuante no início do processo inflamatório, logo após a fixação do carrapato. A mesma observação estende-se para o gene IL-8, em que não foi verificada alteração de expressão nos animais resistentes, embora nos animais susceptíveis este gene apresentou diminuição da expressão no 12° dia após a infestação (p<0,05). Quanto ao gene IFN-, não houve diferença de expressão entre os animais resistentes e susceptíveis, sendo que este gene parece não estar relacionado ao mecanismo de resistência. O gene TLR-2 apresentou diminuição da expressão em ambos os grupos de animais. Estes resultados sugerem que a resposta imune adquirida avaliada neste trabalho não apresenta papel preponderante no mecanismo de resistência e que resposta imune inata poderia está envolvida no mecanismo de resistência ao carrapato. Portanto, avaliação da resposta imunológica horas após a fixação do carrapato poderia nos fornecer resultados mais conclusivos. / In tropical countries losses caused by tick infestation in cattle lead to a major impact on animal production systems. Recent studies have shown the importance of genetic factors linked to tick resistance in Bos indicus and Bos taurus as well as the critical role in the prevention or progression of diseases mediated by cytokines. The aim of this work was to evaluate gene expression of IL-10 and IL-4 in relation to tick susceptibility associated with the Th2 profile and gene expression of IL-2 and IFN- in relation to tick resistance associated with the Th1 profile. In addition, the expression of TLR-2, important in the process the recognition of pathogens, and TNF-α and IL-8 genes, important in the initial inflammatory process, were evaluated. Six tick-resistant and six tick-susceptible animals from a F2 population of 332 animals, originated from the cross of F1 animals (½ Holstein: ½ Gir), were selected based on tick count and breeding value for tick resistance. Skin biopsies were collected in the 5th and 12th days after tick infestation. The GAPDH was used as endogenous control to normalize the amount of starting cDNA target in the real-time PCR assay. Both resistant and susceptible animals showed increased gene expression of IL-10 in the 5th and 12th days after infestation in relation to control animal (p<0.05). The IL-2 gene showed no change of expression in the 5th day after infestation for the resistant and susceptible animals. In the 12th post infestation, both resistant and susceptible animals showed increased gene expression in relation to control animal. These results suggest an enhancement of Th2 profile through the increase of IL-10 mRNA levels and a possible inhibition of the Th1 pattern in both groups (resistant and susceptible) starting 5 days after infestation and return to normal by day 12. Despite our results suggest the occurrence of the Th2 profile, the susceptible and resistant animals did not show variation on gene expression for IL-4 in relation to control animal. The susceptible animals showed increased expression of TNF-α in the 5th day after infestation. However, in the 12th day post infestation it was noted a decrease in the gene expression level. The resistant animals showed no change in the expression of this gene in relation to control animals suggesting that TNF-α could be more actively expressed in the early steps of the inflammatory process. Similarly, the resistant animals showed no variation in the expression of IL-8 while the susceptible animals showed increased expression in the 12th day post infestation. There were no differences of expression between resistant and susceptible animals in relation to IFN-γ what suggests that this gene might not be involved in the resistance mechanism. The TLR-2 gene showed decreased expression in both resistant and susceptible animals (p<0.05). Finally, there was no difference in expression between susceptible and resistant animals in relation to all selected genes in the 5th and 12th days after infestation. These results suggest that the acquired immunity evaluated in this work might not have preponderant role in the resistance mechanism. The innate immunity might be playing a major role in the bovine tick resistance/susceptibility mechanism in early hours after infestation.

In recent years, the subsets of mammalian CD4+ T cells and their repertoire of effector cytokines has expanded beyond the original Th1/Th2 paradigm, to include natural (n) and inducible (i) regulatory T cells (Treg), Th17, Th9, Th22 and follicular T helper (Tfh) cells. Whilst Th1, Th2 and nTreg immune responses have been described in the chicken, the existence of other Th cell subsets is yet to be determined. To investigate Th17 immune responses in the chicken, the mammalian components of these responses currently unannotated in the chicken genome, IL-23 p19 and the IL-23R, were identified and cDNAs cloned. A chicken IL-23 flexiconstruct, containing IL-23 p19 and p40 joined by a linker, was designed. Recombinant chicken IL-23 protein (rchIL-23) was expressed and purified. Bioactivity of rchIL-23 was demonstrated by increased mRNA expression of chIL- 17F and chIL-22 in rchIL-23-stimulated splenocytes. Monoclonal antibodies which identify chIL-12/chIL-23 p40 also recognised purified rchIL-23. Further, chIL-23 p19 mRNA levels were measured and detected in a wide range of tissues but was not up-regulated in stimulated splenocytes, thymocytes or bursal cells. Messenger RNA (mRNA) expression levels of Th17 cytokines (chIL-17A, chIL-17F, chIL-21, chIL-22 and chIL-23) were measured in a chicken tissue panel, in stimulated splenocytes, thymocytes and bursal cells, as well as during infections previously described as initiating typical Th1 or Th2 adaptive immune responses in the chicken. Chicken IL-17A mRNA expression levels were up-regulated in susceptible chickens during infection with Marek’s disease virus (a disease which typically drives a Th1 immune response), but were down-regulated in resistant birds. Chicken CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS) and recombinant Th17-associated cytokines used to attempt to drive the cells towards a Th17 phenotype, as measured by expression of mRNA for chIL-17A and chIL-23R. The sorted chicken CD4+ cells failed to proliferate or respond to Th17 cytokine stimulation. ChIL-23R was also correctly identified and cloned as cDNA, and its mRNA expression measured in a panel of unstimulated and stimulated tissues and cells. The chIL-23R mRNA levels were detected in a wide range of tissues as well as stimulated splenocytes, thymocytes and bursal cells. Future work would seek to positively identify Th17 cells in the chicken and determine the role of Th17 immune responses against avian diseases.

Les lymphocytes T Vgamma9Vdelta2 humains constituent la sous-population majoritaire de lymphocytes Tgammadelta dans le sang chez l'adulte sain et représentent 1% à 4% des cellules mononucléées du sang périphérique (PBMC). Ce sont des lymphocytes T non conventionnels activés par des antigènes non peptidiques, les phosphoantigènes (PAgs) sans nécessité de restriction par les molécules du CMH. Ils jouent un rôle essentiel dans l'immunité anti-infectieuse et antitumorale, notamment en sécrétant des cytokines pro-inflammatoires et des molécules lytiques au contact de leurs cellules cibles. L'efficacité des lymphocytes T Vgamma9Vdelta2 en immunothérapie anticancéreuse est aujourd'hui démontrée mais reste fortement limitée en raison de la grande toxicité de l'IL-2 requise pour leur expansion. Toutes les cytokines de la famille de l'IL-2 possèdent la même toxicité intrinsèque, en raison de la chaîne gamma commune à tous les récepteurs de cette famille. Il est donc nécessaire de trouver une molécule alternative à l'IL 2, moins toxique mais tout aussi efficace, dont la transduction du signal ne dépend pas de la chaîne gamma. L'IL-33 est une cytokine de la famille de l'IL-1 appartenant au groupe des alarmines, dont le récepteur ST2/IL-1 RAcP est gamma chain-indépendant. Elle est naturellement présente dans le microenvironnement tumoral et son récepteur ST2 est exprimé sur de nombreuses cellules de l'immunité innée et adaptative. L'IL-33 est une cytokine pluripotente, pouvant induire à la fois des réponses immunitaires de type Th2 ou Th1. Néanmoins, aucune donnée n'était disponible quant à la bioactivité de l'IL-33 sur les lymphocytes T Vgamma9Vdelta2. Mes travaux de thèse ont donc consisté à déterminer si l'IL-33 pouvait potentialiser les fonctions anticancéreuses des lymphocytes T Vgamma9Vdelta2. Au cours de cette étude, nous avons montré que l'IL-33 associée à un PAg induit la prolifération et l'amplification des lymphocytes T Vgamma9Vdelta2 au sein des PBMC. Après amplification, les lymphocytes T Vgamma9Vdelta2 induits avec de l'IL-33 sécrètent des cytokines de type Th1 INF-gamma et TNF-alpha et ont une activité cytotoxique semblable à ceux induits avec de l'IL-2. De plus, nous avons mis en évidence que la prolifération des lymphocytes T Vgamma9Vdelta2 induite par l'IL-33 est issue d'un mécanisme complexe dépendant d'une interaction avec les lymphocytes T CD4. L'ensemble de ces résultats suggère que l'IL-33 pourrait représenter une bonne alternative à l'IL-2 dans les protocoles d'immunothérapie anticancéreuse impliquant des lymphocytes T Vgamma9Vdelta2. / Human Vgamma9Vdelta2 T cells represent the most prominent subset of gammadelta T cells in the blood of healthy adults, representing 1 to 4% of peripheral blood mononuclear cells (PBMC). These cells are non-conventional lymphocytes activated by non peptidic antigens, the phosphoantigens (PAgs), without restriction by MHC molecules. They are very important actors of anti-infectious and antitumor immune responses. Indeed, their activation upon a contact with their target cells lead them to secrete pro-inflammatory cytokines and lytic granules mediating cell cytotoxicity. Their efficacy in cancer immunotherapy is now demonstrated but appears strongly limited by the toxicity of IL-2 which is essential for their expansion. All the cytokines of the IL-2 family have the same toxicity owing to the gamma chain shared by all the receptors of the family. Therefore, it appears crucial to find as effective but safer alternative to IL-2 which signaling does not depend on the gamma chain. IL-33 is a member of IL-1 family belonging to the alarmins which its receptor ST2/IL-1RAcP does not require the gamma chain. It is naturally present in the tumor microenvironment and ST2 is expressed on numerous innate and adaptive immune cells. IL 33 is a multipotent cytokine able to sustain both Th2 and Th1 immune responses. However, its bioactivity on Vgamma9Vdelta2 T cells has never been studied. The aim of my PhD thesis consisted in determining if IL-33 could promote Vgamma9Vdelta2 T cell anticancer functions. We found that IL-33 enhances the proliferation and the amplification of PAg-activated Vgamma9Vdelta2 T cells. Moreover, we found that IL-33-induced Vgamma9Vdelta2 T cells display the same anticancer functions than those induced by IL-2, through their secretion of Th1 type cytokines and to their cytotoxic activity towards cancer cells. Interestingly, we found that Vgamma9Vdelta2 T cell proliferation induced by IL-33 is due to a complex mechanism requiring interaction with CD4 T cells. Altogether, these results suggest that IL-33 represents a potential alternative to IL-2 in Vgamma9Vdelta2 T cell-based immunotherapies.

Monocyte-derived macrophages (MDMs) from HIV-infected patients and MDMs infected in vitro with HIV manifest inhibition of various cytokines including IL 12. Recently, IL-27 was shown to inhibit HIV replication in macrophages. Whether HIV infection or HIV regulatory proteins such as tat, impact IL-23 or IL-27 production in macrophages remains unknown. I have demonstrated that intracellular HIV-tat expression as well as HIV-tat basic domain peptides inhibited LPS-induced IL-23 and IL-27 proteins and their subunits in MDMs. First I investigated the signalling pathways involved in the regulation of LPS-induced IL-23 and IL-27 production in MDMs infected with control pLXIN retrovirus-infected MDMs. The p38 MAPK, SHP-1 and PI3K signalling molecules positively regulated LPS-induced IL-23 expression. In contrast, Src kinases and JNK MAPK negatively regulated LPS-induced IL-23 production. On the other hand, LPS-induced IL-27 production was positively regulated by the PI3K, p38 MAPKs and SHP-1 and Src kinases. Src kinases positively regulated LPS-induced IL-27 production whereas Src kinases and JNK negatively regulated LPS-induced IL-23 production. HIV-Tat significantly inhibited p38 MAPK and PI3K which were implicated in HIV-Tat-mediated inhibition of LPS-induced IL-23 and IL-27 production. Even though HIV-Tat inhibited ERK and JNK MAPK activation, these kinases were not involved in HIV-Tat-mediated inhibition of LPS-induced IL-23 and IL-27 production. While SHP-1 regulated LPS-induced IL-23 and IL-27 production, HIV-Tat did not inhibit SHP-1 and therefore were not involved in HIV-Tat-mediated inhibition of LPS-induced IL-23 and IL-27 production. HIV-Tat did not inhibit Src kinases and hence were not involved in HIV-Tat-mediated inhibition of LPS-induced IL-27 production. Furthermore, HIV-Tat did not inhibit the expression of upstream TLR4-activated signaling molecules including TRAF3, TRIF, MyD88, IRAK1, IRAK3, IRAK4, TRAF-1, TRAF-2, cIAP-1, cIAP-2 and, xIAP. These results suggest association of IL-23 and IL-27 inhibition by HIV with decreased HIV-specific immune responses, and increased viral replication. These results further suggest novel strategies to improve cellular immune responses and inhibition of HIV replication.

Background: IL-4 and IL-13 are related cytokines with similar functional properties. Differential regulation of IL-4 and IL-13 has not been described. Objective: We have examined the effects of IFN-α on antigen-driven proliferation, IL-4 generation, and IL-13 generation from human PBMCs and T-cell clones. Methods: Proliferation was assessed by 3H-thymidine incorporation. Cytokine generation was assessed by reverse transcription PCR and ELISA. Messenger RNA stability was assessed in the presence of actinomycin D. Results: IFN-α induced a concentration-dependent inhibition of antigen-driven proliferation of TH1 and TH2 clones (median effective concentration, 150 to 200 U/mL); the sensitivity of TH1 and TH2 clones to IFN-α was not significantly different (P = .6). IFN-α induced an analogous concentration-dependent inhibition of antigen-driven IL-13 generation from TH1 and TH2 clones (median effective concentration, 100 U/mL); this effect was evident by 12 hours of culture and persisted beyond 48 hours. However, IL-4 generation from TH2 clones was insensitive to IFN-α at all concentrations and times tested (1 to 10,000 U/mL). A similar inhibitory effect of IFN-α on mitogen-driven proliferation and IL-13 generation from PBMCs was demonstrated; once again, IL-4 generation from PBMCs was insensitive to IFN-α. IL-13 mRNA stability was unaffected by IFN-α, suggesting transcriptional regulation. Conclusion: IFN-α differentially regulates antigen-stimulated IL-4 and IL-13 generation.

Indiana University-Purdue University Indianapolis (IUPUI) / The immune system provides resistance to the myriad of pathogens in the environment, but can also respond inappropriately causing allergic inflammation and autoimmune disease. CD4+ T cells, which play a crucial role in adaptive immune system, can be divided into several subsets based on their effector functions. T helper 9 (Th9) cells, derived by the IL-4/STAT6 and TGF-β signaling pathways, produce IL-9 as a hallmark cytokine, as well as IL-10. Through IL-9 production, Th9 cells protect against parasite infection but are also involved in allergic inflammation and autoimmune diseases. Transcription factors that promote Th9 development include STATs, PU.1, BATF, and IRF4. In this study, we identify ETV5 as a factor that promotes IL-9 and IL-10 production by binding to cis-acting regulatory elements in the respective genes. At the Il9 gene, ETV5 cooperates with PU.1 in regulating gene expression. At the Il10 gene, ETV5 facilitates binding of other transcription factors to the locus. These studies and others suggested that there may be additional cis-acting regulatory elements in the Il9 gene. We demonstrate that a conserved noncoding sequence (CNS) located 25 kb upstream of the Il9 transcription start site, termed Il9 CNS-25, is critical for regulating Il9 expression in Th cell subsets. Th9 cells derived from Il9 CNS-25 mutant (Il9 ΔCNS-25) mice produce significantly less IL-9. Il9 CNS-25 promoted chromatin modifications at the promoter and accessibility of the locus. Il9 ΔCNS-25 mice showed attenuated airway inflammation compared to control mice. The Il9 CNS-25 region in mice is conserved with an IL9 CNS-18 region in the human genome. We deleted CNS-18 in primary human Th9 cells and observed diminished IL-9 production. Thus, we have identified transcription factors that regulate multiple cytokines in Th cell lineages and have demonstrated that the Il9 CNS-25/IL9 CNS-18 elements are respectively critical for Il9/IL9 gene expression.