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

La spondyloarthrite (SpA) est un rhumatisme inflammatoire chronique fréquent avec une prévalence de 0,43% en France, fortement associée à HLA-B27. À l’heure actuelle, il n’existe aucun traitement curatif et les mécanismes physiopathologiques impliqués restent méconnus. Afin de mieux comprendre les mécanismes immunologiques impliqués dans le développement de la SpA, nous avons étudié deux populations cellulaires clé, les cellules dendritiques (DC) et les lymphocytes T (LT) CD4+, chez le rat transgénique pour le HLA-B27 et la β2 microglobuline humaine (rat B27) qui développe spontanément tous les symptômes de la SpA. Il a été démontré que l’accumulation de lymphocytes T helper producteurs d’interleukine 17 (IL-17) pathogénique (lymphocyte Th17), et plusieurs défauts fonctionnels des cellules dendritiques (DCs) sont corrélés avec le développement de la SpA chez les rats B27.Nous nous sommes tout d’abord intéressés aux lymphocytes T régulateurs (Treg), dont le rôle est d’empêcher l’établissement d’une réponse immune pathogène pour l’hôte, chez le rat B27. Nous avons découvert que les Treg de rats B27 présentent un phénotype pro-inflammatoire (surexpression d’IL-17 et sous-expression d’IL-10 anti-inflammatoire), lié à la surexpression de la molécule ICOS. De plus, la sévérité des signes cliniques chez les rats B27 n’exprimant pas ICOS (rats B27 ICOS KO) est diminuée comparé aux animaux HLA-B27 sauvages. Cette protection partielle est corrélée à une réduction de la proportion de lymphocytes Th17. Ces résultats mettent en lumière le rôle majeur d’ICOS dans la physiopathologie de la SpA du rat.La deuxième partie de ce travail s’est concentrée sur les conséquences de la sous-expression d’IL-27 par les DC de rats B27, cytokine connue pour inhiber le développement des Th17. Nous avons observé que l’addition d’IL-27 exogène permet de diminuer la production d’IL-17 et d’augmenter la synthèse d’IL-10 anti-inflammatoire par les LT différenciés (T effecteurs et Treg) et les LT naïfs de rats B27 différenciés in vitro. De façon intéressante, l’IL-27 réduit également la synthèse d’IL-17 par les LT CD4+ circulants de patients atteints de SpA.Ces travaux démontrent pour la première fois le rôle clé de l’IL-27 et d’ICOS dans le contrôle de l’inflammation chez le rat B27 et suggèrent fortement que ces deux molécules sont de nouvelles cibles thérapeutiques prometteuses dans la SpA. / Spondyloarthritis (SpA) is a frequent chronic rheumatic inflammatory disorder with a prevalence of 0.43% in France and closely associated to HLA-B27. To date, there is no curative treatment and pathophysiological mechanisms involved in this pathology remain elusive. To better understand these mechanisms, we studied two crucial cell populations, dendritic cells (DC) and CD4+ T cells in rats transgenic for HLA-B27 and human β2 microglobulin (B27 rats) which spontaneously develop a phenotype closely resembling human spndyloarthritis. Previous studies demonstrated that accumulation of pathogenic IL-17 producing T cells (Th17 cells) and several function defects of DCs are correlated with SpA development in B27 rats.First, we focused on regulatory T cells, whose role is to prevent the establishment of pathogenic immune responses. We discovered that Treg from B27 rats have a pro-inflammatory phenotype. They overexpress IL-17 and underexpress anti-inflammatory IL-10, linked to ICOS overexpression. Furthermore, B27 rats knock-out for ICOS (B27 ICOS KO rats) have reduced severity of clinical symptoms compared to B27 ICOS WT rats. This protective effect is correlated with a reduced proportion of Th17 cells. These results highlight the crucial role of ICOS in rat SpA physiopathology.In the second part of this work we studied the consequences of IL-27 underexpression by B27 DC, a cytokine known to inhibit Th17 development. Addition of exogenous IL-27 reduces IL-17 and increases IL-10 productions by differentiated T cells (Teff and Treg) and by naive T cells polarized in vitro. Interestingly, IL-27 also reduces IL-17 production by circulating CD4+ T cells isolated from blood of SpA patients.This work demonstrate for the first time the key role of IL-27 and ICOS in the control of inflammation in B27 rats and highly suggest that these molecules may be new promising therapeutic targets in SpA.

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.

Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types. My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli. In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules. Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.

Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types. My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli. In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules. Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.

IL-12, an immunoregulatory cytokine, plays a key role in the development of cell-mediated immune responses. However, very little is known about the regulation and induction of the other members of this family, particularly IL-23 and IL-27. The regulation of these cytokines was studied in the human primary monocytes and monocyte-derived macrophages (MDMs) as they play a key role in innate and adaptive immune responses. THP-1 promonocytic cells were employed as a model system to confirm the results obtained with monocytes and MDMs. Two stimuli IFN-γ and LPS were used as both are strong inducers of IL-12 family cytokines. My results show that IFN-γ induced the production of IL-12/23p40 and IL-23p19 mRNA as well as IL-12p40 and IL-23 proteins in primary human monocytes isolated by positive selection. IFN-γ-induced IL-23 and IL-12/23p40 expression was positively regulated by the p38 mitogen-activated protein kinases (MAPK), independent of the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) signaling. In contrast, IL-12 and IL-23 were negatively regulated by the Jak/STAT, phosphoinositide-3 kinase (PI3K) and the c-Jun-N-terminal kinase (JNK) MAPKs in IFN-γ-stimulated monocytes. LPS significantly stimulated IL-23p19 and IL-12/23p40 mRNA expression as well as IL-12/23p40 and IL-23 protein production in THP-1 cells, while IFN-γ stimulation alone did not affect IL-23 mRNA or protein levels. THP-1 cells were pre-treated with ERK, JNK or p38 MAPK inhibitors and then stimulated with LPS. LPS-induced IL-12p40 and IL-23 proteins were positively regulated by the p38 and JNK MAPKs and PI3K, whereas LPS-induced IL-23p19 mRNA expression was negatively regulated by these kinases. These results were confirmed using siRNA in LPS-stimulated THP-1 cells. My results also show that IFN-γ/LPS-induced IL-23 expression is not regulated through MAPK or PI3K signaling pathways in human MDMs. My results also show for the first time that IFN-γ alone without any second stimulus induced IL-27p28 gene expression and IL-27 protein production in human monocytic cells. I investigated the signalling pathways governing the regulation of IL-27 protein and its subunit IL-27p28 following stimulation with IFN-γ in primary human monocytic cells. IFN-γ-mediated IL-27 protein, but not IL-27p28 gene expression was positively regulated by JNK MAPK and PI3K, independent of JAK/STAT signaling in primary human monocytes. I also investigated the signalling pathways governing the regulation of IL-27 and its α subunit, IL-27p28 following stimulation with IFN-γ alone or IFN-γ-primed LPS-stimulated macrophages (IFN-γ/LPS) and THP-1 cells. A differential regulation of IL-27p28 and IL-27 in response to stimulation by either IFN-γ or IFN-γ/LPS was observed. IFN-γ- and IFN-γ/LPS induced IL-27 expression was positively regulated by the JNK, p38 MAPK and PI3K, independent of Jak/STAT signaling in human MDMs and THP-1 cells. Taken together, my results show that IL-23 induction is differentially regulated by different pathways in response to different stimuli, whereas IL-27 expression is regulated by JNK, p38 MAPK and PI3K regardless in the stimulus in human myeloid cells. These results may provide additional strategies aimed at targeting disease, autoimmune disorders and cancer.

Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types. My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli. In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules. Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.

IL-12, an immunoregulatory cytokine, plays a key role in the development of cell-mediated immune responses. However, very little is known about the regulation and induction of the other members of this family, particularly IL-23 and IL-27. The regulation of these cytokines was studied in the human primary monocytes and monocyte-derived macrophages (MDMs) as they play a key role in innate and adaptive immune responses. THP-1 promonocytic cells were employed as a model system to confirm the results obtained with monocytes and MDMs. Two stimuli IFN-γ and LPS were used as both are strong inducers of IL-12 family cytokines. My results show that IFN-γ induced the production of IL-12/23p40 and IL-23p19 mRNA as well as IL-12p40 and IL-23 proteins in primary human monocytes isolated by positive selection. IFN-γ-induced IL-23 and IL-12/23p40 expression was positively regulated by the p38 mitogen-activated protein kinases (MAPK), independent of the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) signaling. In contrast, IL-12 and IL-23 were negatively regulated by the Jak/STAT, phosphoinositide-3 kinase (PI3K) and the c-Jun-N-terminal kinase (JNK) MAPKs in IFN-γ-stimulated monocytes. LPS significantly stimulated IL-23p19 and IL-12/23p40 mRNA expression as well as IL-12/23p40 and IL-23 protein production in THP-1 cells, while IFN-γ stimulation alone did not affect IL-23 mRNA or protein levels. THP-1 cells were pre-treated with ERK, JNK or p38 MAPK inhibitors and then stimulated with LPS. LPS-induced IL-12p40 and IL-23 proteins were positively regulated by the p38 and JNK MAPKs and PI3K, whereas LPS-induced IL-23p19 mRNA expression was negatively regulated by these kinases. These results were confirmed using siRNA in LPS-stimulated THP-1 cells. My results also show that IFN-γ/LPS-induced IL-23 expression is not regulated through MAPK or PI3K signaling pathways in human MDMs. My results also show for the first time that IFN-γ alone without any second stimulus induced IL-27p28 gene expression and IL-27 protein production in human monocytic cells. I investigated the signalling pathways governing the regulation of IL-27 protein and its subunit IL-27p28 following stimulation with IFN-γ in primary human monocytic cells. IFN-γ-mediated IL-27 protein, but not IL-27p28 gene expression was positively regulated by JNK MAPK and PI3K, independent of JAK/STAT signaling in primary human monocytes. I also investigated the signalling pathways governing the regulation of IL-27 and its α subunit, IL-27p28 following stimulation with IFN-γ alone or IFN-γ-primed LPS-stimulated macrophages (IFN-γ/LPS) and THP-1 cells. A differential regulation of IL-27p28 and IL-27 in response to stimulation by either IFN-γ or IFN-γ/LPS was observed. IFN-γ- and IFN-γ/LPS induced IL-27 expression was positively regulated by the JNK, p38 MAPK and PI3K, independent of Jak/STAT signaling in human MDMs and THP-1 cells. Taken together, my results show that IL-23 induction is differentially regulated by different pathways in response to different stimuli, whereas IL-27 expression is regulated by JNK, p38 MAPK and PI3K regardless in the stimulus in human myeloid cells. These results may provide additional strategies aimed at targeting disease, autoimmune disorders and cancer.

Multiple myeloma is an aggressive plasma cell cancer that consistently acquires multi-drug resistance and relapses despite initial treatment successes. Patients may go through greater than 10-lines of therapy, highlighting the need for more effective treatment options. Immunotherapies are the latest evolution in targeted cancer treatments, and thus far have displayed impressive results in several hematological cancers, including multiple myeloma. T cells possess robust anti-tumor functions which can be harnessed and refined for the treatment of cancers. Genetic engineering of T cells to express a chimeric antigen receptor (CAR) confers antigen-specific tumor-targeting, and adoptive transfer of patient-derived CAR-engineered T (CAR T) cells has been efficacious in relapsed/refractory multiple myeloma. Despite the high efficacy, CAR T cell therapy for myeloma is associated with serious adverse events, which limits dose levels and patient eligibility. We have developed a novel synthetic antigen receptor platform, called the T cell antigen coupler (TAC) receptor, which has shown comparatively higher efficacy with a reduced pro-inflammatory profile compared with CAR T cells in pre-clinical models. The TAC receptor was purpose-built to co-opt the natural T cell activation machinery and lacks the costimulatory signaling typically incorporated in CAR designs. This thesis investigates strategies to augment TAC T cell function against for multiple myeloma through the evaluation of ancillary pharmacological and protein stimuli that would complement the anti-tumor functions of TAC T cells without modifying the TAC receptor design. In chapter 2, I investigated a strategy combining TAC T cells with the SMAC mimetic LCL161 to provide transient costimulatory effects. While LCL161 boosted TAC T cells survival and proliferation, the drug also enhanced susceptibility of TAC T cells to apoptosis and offered no advantage to the TAC T cells when challenged with myeloma. In chapter 3, I engineered TAC T cells to secrete IL-27 in an attempt to modulate the myeloma microenvironment and support T cell cytolytic function. IL-27 did not enhance the anti-tumor activity of TAC T cells but forced expression of IL-27 led to a reduction in the production of pro-inflammatory cytokines without altering cytotoxicity. In appendix I, I describe the process of optimizing CRISPR/Cas9 editing of primary TAC T cells. This methodology was required for much of the work in chapter 2. Ph.D. Thesis – Arya Afsahi McMaster University – Biochemistry and Biomedical Sciences v In appendix II, I describe an assessment of mRNA-engineering as a method to produce TAC T cells. This approach proved to be therapeutically futile and was not pursued beyond the work described herein. The work presented here highlights methods of combining TAC T cells with a clinically relevant SMAC mimetic, or the cytokine IL-27, and provides insights into the biological mechanisms that are affected by these approaches. / Thesis / Doctor of Philosophy (PhD)

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