Interluekin-23 (IL-23) is a pro-inflammatory cytokine critical to the regulation of innate and adaptive immune responses. The main role for this cytokine is in the proliferation and differentiation of the IL-17 producing CD4 T helper cell, Th17. Virus infection deregulates IL-23 expression and function, but little is known about the mechanism behind this phenomena. Here, I demonstrate a reduction of Toll like receptor (TLR) ligand-induced IL-23 expression in lymphocytic choriomeningitis virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs), indicating that a function of these cells is disrupted during virus infection. I propose a mechanism of TLR ligand-induced IL-23 expression inhibition upon LCMV infection via the deactivation of p38, AP-1, and NF-κB. Further analysis revealed a direct relationship between LCMV infection with the IL-10 and SOCS3 expression. To understand IL-23 function, I characterized IL-23-induced JAK/STAT signalling pathway and IL-23 receptor expression on human CD4 T cells. My results demonstrate that IL-23 induces activation of p-JAK2, p-Tyk2, p-STAT1, p-STAT3, and p-STAT4 in CD4 T cells. For the first time I show that IL-23 alone induces the expression of its own receptor components, IL-12Rβ1 and IL-23Rα, in CD4 T cells. Blocking JAK2, STAT1, and STAT3 activation with specific inhibitors detrimentally effected expression of IL-23 receptor demonstrating that activation of JAK/STAT signalling is important for IL-23 receptor expression. I also addressed the effect of viral infection on IL-23 function and receptor expression in CD4 T cells using cells isolated from HIV positive individuals. These studies were based on earlier reports that the expression of IL-23 and the IL-23 receptor are impaired during HIV infection. I demonstrate that the phosphorylation of JAK2, STAT1, and STAT3 induced by IL-23, as well as IL-23 receptor expression are deregulated in CD4 T cells isolated from HIV positive individuals. This study has furthered the understanding of how the expression and function of IL-23 is regulated during viral infections. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2011-08-24 07:52:24.898

Inflammation plays an important role in responding to injury and combating infections. In this thesis, I examine how inflammation is regulated by cytokines responsible for driving initial immune responses to combat infections. Toll-Like receptor (TLR)-mediated activation of monocytes, macrophages and dendritic cells can lead to the co-expression of proinflammatory cytokines including IL-1β, IL-23, and IL-27. IL-23 and IL-27 belong to the IL-12 cytokine family yet have distinct functions; IL-23, along with IL-1β, regulates TH17 cell differentiation, while IL-27 supports TH1 proliferation and inhibits TH17 differentiation. Our lab has previously demonstrated that IL-27 can modulate inflammasome activation, the multi-protein regulatory complex that produces bioactive IL-1β; however, the mechanism behind this is poorly understood. Similarly, the effect of IL-27 on IL-23 expression has not been well described. Using the CD14+ THP-1 monocytic cell line as a model system, I investigated the role of IL-27 on LPS-mediated inflammasome activation and IL-23 expression. To induce inflammasome activation, CD14+ THP-1 cells were treated with LPS and/or IL-27, followed by treatment with ATP. I demonstrated that IL-27-enhanced inflammasome activation, which is associated with increased surface expression of LPS and ATP receptors: TLR4 and P2X7 respectively. Furthermore, costimulation resulted in increased secretion of ATP from CD14+ THP-1 cells. Inhibition of ATP signaling and inflammasome activation significantly decreased secreted IL-1β, suggesting that an ATP autocrine feedback loop is driving IL-1β secretion. Moreover, LPS and IL-27 costimulation increased IL-23 expression concurrent with that of IL-1β and ATP secretion. Furthermore I showed that IL-23 secretion is dependent on inflammasome activation and IL-1β, and ATP signaling following IL-27 and LPS priming. My data point to a novel mechanism of IL-27 enhanced LPS-induced IL-1β and IL-23 secretion from CD14+ THP-1 cells through an ATP autocrine feedback loop. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2014-06-26 15:18:20.124

BACKGROUND: Spondyloarthritis is a family of related inflammatory diseases including psoriatic arthritis and ankylosing spondylitis. The cytokine IL-23 is known to play an important role in spondyloarthritis development. Overexpression of IL-23 using IL-23 minicircle DNA in B10.RIII mice results in the development of a spondyloarthritis-like disease. B10.RIII is a major histocompatibility complex (MHC) congenic mouse strain that is susceptible to a number of autoimmune and autoinflammatory diseases. Contaminating regions outside the congenic interval from the donor strain, RIII, have been previously detected. While B10.RIII mice develop collagen-induced arthritis (CIA) and collagen antibody induced arthritis (CAIA), the background strain, C57BL/10 (B10) does not. The MHC region is known to play a role in the susceptibility of B10.RIII mice to CIA, but not in CAIA development, so other regions must be involved in arthritis development as well. These contaminating RIII-derived regions may control susceptibility to IL-23 minicircle induced arthritis in B10.RIII mice. OBJECTIVE: This study aimed to identify RIII-derived regions in the genome of B10.RIII mice and begin to interrogate their effects on IL-23 minicircle induced arthritis. METHODS: A systematic literature review was conducted using Pubmed and Web of Science. Articles using B10.RIII mice to study inflammatory disease models were included and data about year of publication, inbred mouse strains used, disease model, and inbred strain notation were extracted. Genome sequences of B10.RIII(71NS)/Sn, C57BL/10SnJ, and C57BL/10J were compared to identify RIII-derived clusters of variation in the B10.RIII genome. B10.RIII mice were crossed with B10 mice and subsequently backcrossed to B10.RIII mice to introduce the B10 allele at chromosome 15. Chr15b/b, Chr15b/r, and Chr15r/r B10.RIII mice were hydrodynamically injected with IL-23 minicircles, and arthritis development was monitored every other day for two weeks. RESULTS: The systematic literature review yielded 8 studies that compared arthritis development in B10.RIII to RIII or B10. These studies identified three arthritis susceptibility regions: Cia5/Eae3 on chromosome 3, Eae2 on chromosome 15, and Eae39 on chromosome 5. Eae2 is further split into four sub-regions: Cia30, Cia31, Cia32, and Cia26. Genome sequence comparison identified RIII-derived clusters in B10.RIII mice on chromosomes 10, 14, 15, and 17. The chromosome 15 region overlaps with the Eae2 susceptibility region for approximately 17 Mbp and includes the arthritis susceptibility loci Cia26 and Cia32. When this region was interrogated in vivo, Chr15r/r and Chr15b/r B10.RIII mice developed IL-23 minicircle arthritis, while Chr15b/b mice did not. CONCLUSION: The B10.RIII(71NS)/Sn strain contains several large RIII/WySn-derived regions outside the congenic MHC region. One of these clusters on chromosome 15 includes the arthritis susceptibility loci Cia26 and Cia32 and appears to determine susceptibility to IL-23 minicircle induced arthritis. Future studies will interrogate the role of the chromosome 15 RIII-derived region in arthritis development in more detail and aim to identify the specific gene variants that control arthritis susceptibility. / 2023-01-28T00:00:00Z

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.

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.

Submitted by Jaqueline Silva (jtas29@gmail.com) on 2016-08-30T20:48:41Z No. of bitstreams: 2 Dissertação - Pollyana Guimarães de Oliveira - 2014.pdf: 1550661 bytes, checksum: 2c614da0d23de4cac81d9260ef077071 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-08-30T20:48:58Z (GMT) No. of bitstreams: 2 Dissertação - Pollyana Guimarães de Oliveira - 2014.pdf: 1550661 bytes, checksum: 2c614da0d23de4cac81d9260ef077071 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-30T20:48:58Z (GMT). No. of bitstreams: 2 Dissertação - Pollyana Guimarães de Oliveira - 2014.pdf: 1550661 bytes, checksum: 2c614da0d23de4cac81d9260ef077071 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-10-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Dendritic cells (DCs) are a heterogeneous group of cells and the major activators of naive T lymphocytes. During antigen presentation, different types of cytokines are produced and they will interfere with the immune response profile generated. IL-12p70 promotes the differentiation of Th1 phenotype and IL-23 p enhances the Th17 response. Given the major role of IL-12 and IL-23 on the stabilization of the acquired immune response, the production of these cytokines is highly controlled and it is an issue for several studies. The objective of this work was to characterize a new lineage of DC, described as AP284, based on their surface markers and evaluate the ability of these cells to produce IL-12p40, IL-12p70 and IL-23 after different stimulus in vitro. Additionally it will be evaluate their phagocytic capacity and their ability to induce a Th1 and Th17 response in vivo. The expression of the markers was analyzed by flow cytometry and the quantitation of IL-12 and IL23 was performed by ELISA after stimulation of cells AP284 in vitro with lipopolysaccharide (LPS) and Escherichia coli. AP284 cells express MHC class II, CD11c and 33D1 on its surface, which are characteristic markers of DCs. After stimulus with LPS or E.coli, AP284 cells produce large amounts of IL-12p40 and IL-23 but do not produce IL-12p70. The production of IL-12p70 was not detected even when IFN-γ is added to prime cultures, moreover, priming the cells with IFN-γ promoted inhibition of IL-12p40 and IL-23. Our data sugest that the AP284 is a DC17 lineage. Thus, AP284 can be an important tool to study the mechanisms of induction or regulation of IL-23 production and a possible tool to study generation and maintenance of a Th17 profile. / As células dendríticas (DCs) constituem um grupo heterogêneo de células e são as principais ativadoras de linfócitos T virgens. Durante a apresentação de antígenos são produzidos diferentes tipos de citocinas que interferem com o perfil de resposta imune que será gerado. A produção de IL-12p70 favorece a diferenciação do perfil Th1 e a produção de IL-23 potencializa o perfil de resposta Th17. Devido à importância da IL-12 e IL-23 na estabilização de uma resposta imune adquirida, a produção destas citocinas é altamente controlada e alvo de diversas pesquisas. O objetivo deste projeto foi de caracterizar uma nova linhagem de DC AP284 a partir dos seus marcadores de superfícies e avaliar a capacidade destas células em produzir IL-12p40 e IL-23 após diferentes estímulos in vitro, além de avaliar a sua capacidade fagocítica e indução de um perfil de resposta Th1 ou Th17 in vivo. A expressão dos marcadores foi analisada por citometria de fluxo e a dosagem de IL-12 e IL23 por ELISA após o estímulo de células AP284 in vitro com lipopolisssacáride (LPS) e Escherichia coli. Células AP284 expressam MHC classe II, CD11c e 33D1 em sua superfície, sendo estes marcadores característicos de populações de DCs. Após estímulos com LPS e E.coli, células AP284 produzem grande quantidade de IL-12p40 e IL-23, mas não produzem IL-12p70. A produção de IL-12p70 não foi detectada mesmo após a adição de IFN-γ às culturas, além disso, a primagem das células com IFN-γ promoveu a inibição de IL-12p40 e IL-23. Neste trabalho foi demonstrado que células AP284 são DCs, pois além de expressarem MHC classe II expressam marcadores que são característicos deste grupo celular. Nossos dados ainda suportam a ideia de que a linhagem AP284 seja uma linhagem de DC17, pois produziram grandes quantidades de IL-12p40 e IL-23, mas nenhuma quantidade de Il-12p70. Assim, AP284 pode ser uma ferramenta importante para o estudo dos mecanismos de indução e de regulação da produção da IL-23 e uma possível ferramenta para o estudo da geração e manutenção de um perfil de resposta Th17.

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.

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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21634号 / 医博第4440号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 三森 経世, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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