過敏性疾病（如過敏性哮喘和過敏性皮炎）發病率在香港及世界均呈上升趨勢。過敏性哮喘是一種慢性反復發作的炎症疾病，而過敏性皮炎是一種慢性皮膚炎症。呼吸道細菌及金黃色葡萄球菌可分別加重過敏性哮喘病人氣道炎症及過敏性皮炎患者的炎症反應。人體對細菌的先天免疫反應主要通過模式識別受體(PRR)介導。NOD樣受體(NLR)和Toll樣受體(TLR)是兩種重要的PRR。 NLR 家族成員NOD2幾乎識別所有細菌中結構保守的胞壁醯二肽(MDP)。而LTR2識別範圍廣泛的病原相關分子模式，如革蘭氏陽性菌中的肽聚糖(PGN)和脂磷壁酸(LTA)，以及人工合成的脂蛋白Pam3CSK4。 / 本研究包括：NOD2配體MDP，哮喘相關的腫瘤壞死因子家族成員LIGHT對共培養的人嗜鹼性粒細胞和人支氣管上皮細胞的活化作用；熱滅活的金黃色葡萄球菌（HKSA），MDP，TLR2配體PGN，LTA，以及Pam3CSK4對共培養的人嗜鹼性粒細胞和人皮膚成纖維細胞的活化作用；在體內NLR配體對卵清蛋白(OVA)致敏的哮喘小鼠的作用。 / 研究發現，在共培養體系中，MDP能顯著增強嗜鹼性粒細胞與支氣管上皮細胞表面粘附因子（細胞間粘附因子ICAM-1 及血管細胞粘附因子VCAM-1）的表達。同時，MDP能顯著促進共培養體系中炎症相關細胞因子IL-6，趨化因子CXCL8及抗菌肽β-防禦素2的釋放。在MDP刺激下，支氣管上皮細胞是共培養體系中釋放IL-6，CXCL8及β-防禦素2的主要細胞。在MDP刺激下，嗜鹼性粒細胞中包括胞核因子-kappaB（NF-κB）在內的幾個核轉錄因子的表達上升。ICAM-1，VCAM-1，IL-6，CXCL8，及β-防禦素2的表達被信號分子化學抑制劑所抑制，結果表明，嗜鹼性粒細胞與支氣管上皮細胞的相互作用受不同的信號通路（NF-κB, p38 MAPK 及 JNK）調節。OVA致敏小鼠實驗表明，NLR配體能增加分泌粘蛋白的杯狀細胞在肺氣管中的數量，使小鼠支氣管下皮結締組織纖維化並增厚。NLR配體進而提高過敏性哮喘小鼠支氣管肺泡灌洗液中CCL5與IL-13 的表達水平。 / 研究表明，在嗜鹼性粒細胞和皮膚成纖維細胞的共培養體系中，HKSA，MDP，PGN，LTA，或Pam3CSK4顯著誘導ICAM-1, IL-6, CXCL8, CCL2 和 CCL5 的表達。而嗜鹼性粒細胞與皮膚成纖維細胞的直接相互作用是釋放IL-6, CXCL8, CCL2 與 CCL5 所必需的。嗜鹼性粒細胞與皮膚成纖維細胞的相互作用並釋放細胞因子與趨化因子受p38 MAPK 及 NF-κB信號通路調控。 / 在嗜鹼性粒細胞與支氣管上皮細胞共培養體系中，LIGHT 可能通過受體HVEM 與 LTβR顯著增強支氣管上皮細胞表面粘附因子的表達，提高細胞因子IL-6, CXCL8 與 MMP-9的釋放。 / 研究結果表明，在過敏炎症中，通過與組織細胞（如支氣管上皮細胞，人皮膚成纖維細胞）相互作用，嗜鹼性粒細胞有利於組織細胞對病原相關的分子模式作出反應。因此，研究結果對細菌介導的先天性免疫應答與過敏炎症的加重之間的聯繫作出了新的解釋。以上結果也增強了我們對LIGHT在氣道重塑中的免疫病理作用及其作為氣道重塑治療靶標的認識。 / The incidences of allergic diseases such as allergic asthma and atopic dermatitis (AD) are increasing in Hong Kong and worldwide. Allergic asthma is a chronically relapsing inflammatory pulmonary disease, while AD is a chronic inflammatory skin disorder. Respiratory bacterial and Staphylococcus aureus (S. aureus) infection can provoke allergen sensitization and subsequently amplify and sustain inflammation in allergic asthma and AD, respectively. The innate immune system recognizes bacterial infection through pattern recognition receptors (PRRs), two important PRRs involving in inflammatory and immune responses are nucleotide-binding oligomerization domain-like receptors (NLRs) and Toll-like receptors (TLRs). NOD2 is one member of the NLR family, which senses the conserved structural component muramyl dipeptide (MDP) in almost all bacteria. TLR2 recognizes a wide range of pathogen-associated molecular patterns (PAMPs) including peptidoglycan (PGN) and lipoteichoic acid (LTA) from Gram-positive bacteria and synthetic triacylated lipoprotein N-palmitoyl-S-[2,3-bis (palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S] -lysine (Pam3CSK4). / In the present study, we investigated the effect of NOD2 ligand MDP, asthma-related tumor necrosis factor (TNF) family member LIGHT on human basophils co-cultured with human bronchial epithelial cells and the effect of heat-killed S. aureus, MDP, TLR2 ligands PGN, LTA and Pam3CSK4 on basophils co-cultured with human dermal fibroblasts, and the underlying intracellular mechanisms. The in vivo effect of NOD ligands on ovalbumin (OVA)-sensitized allergic asthmatic mice was also studied. / It was found that MDP could significantly enhance the cell surface expression of adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on basophils and primary human bronchial epithelial cells (HBE) in the co-culture system (all p < 0.05). MDP could further enhance the release of inflammatory cytokine interleukin (IL)-6, chemokine CXCL8, and epithelium derived anti-microbial peptide β-defensin 2 in the co-culture. HBE cells were the major source while basophils were the minor source to release IL-6, CXCL8 and β-defensin 2 in the co-culture upon MDP stimulation. The activities of several nuclear transcription factors, including NF-κB, were up-regulated in human basophils upon MDP stimulation. The cell surface expression of ICAM-1 and VCAM-1 and the release of IL-6, CXCL8 and β-defensin 2 were suppressed by the signaling molecule inhibitors, implying that the interaction between basophils and primary human bronchial epithelial cells could be differentially regulated by the NF-κB, p38 MAPK and JNK pathways. The animal study showed that iE-DAP and MDP could increase the number of mucin-secreting goblet cells, the thickness and fibrosis of the bronchial subepithelial tissue of airways from the OVA-sensitized mice. The iE-DAP and MDP could further promote the levels of CCL5 and IL-13 (all p < 0.05) in bronchoalveolar lavage fluid (BALF) of allergic asthmatic mice. / It was found that the induction of ICAM-1, IL-6, CXCL8, CCL2 and CCL5 was significantly promoted upon the interaction between human basophils and dermal fibroblasts activated by heat-killed S. aureus, MDP, PGN, LTA or Pam3CSK4. The release of IL-6, CXCL8, CCL2 and CCL5 might depend on the direct interaction of basophils and dermal fibroblasts. The p38 MAPK and NF-κB pathways should be involved in the release of the cytokines and chemokines upon the interaction of basophils and human dermal fibroblasts. / LIGHT could significantly promote the cell surface expression of adhesion molecule, the release of IL-6, CXCL8 and MMP-9 from human bronchial epithelial cells upon the interaction with basophils, probably through the receptors HVEM and LTβR. / The results suggest that, through the interaction with tissue-resident cells such as bronchial epithelial cells and dermal fibroblasts, basophils may facilitate the activation of tissue-resident cells in response to the PAMPs in allergic inflammation. The results therefore provide a new insight of the crucial link between the bacterial-mediated innate immune response and the exacerbation of allergic inflammation. The above results also enhance our understanding on the immunopathological roles of LIGHT in airway remodeling, and the potential therapeutic target for airway remodeling. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qiu, Huaina. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 165-196). / Abstract also in Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 摘要 --- p.ix / Publications --- p.xi / Table of Contents --- p.xiii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Asthma and atopic dermatitis (AD) --- p.1 / Chapter 1.2 --- Human basophils in allergic inflammation --- p.3 / Chapter 1.2.1 --- Development and morphology of basophils --- p.3 / Chapter 1.2.2 --- Receptors and products of basophils --- p.4 / Chapter 1.2.3 --- Cell surface markers on basophils --- p.7 / Chapter 1.2.4 --- Basophils in allergic inflammation --- p.7 / Chapter 1.3 --- Human bronchial epithelial cells in airway inflammation --- p.10 / Chapter 1.4 --- Human fibroblasts in AD --- p.11 / Chapter 1.5 --- Staphylococcus aureus (S. aureus) in AD --- p.12 / Chapter 1.6 --- NOD2 and TLR2 in allergic inflammation --- p.14 / Chapter 1.7 --- IL-33 in allergic inflammation --- p.18 / Chapter 1.8 --- IL-6 in allergic inflammation --- p.18 / Chapter 1.9 --- CXCL8 in allergic inflammation --- p.20 / Chapter 1.10 --- CCL2 in allergic inflammation --- p.21 / Chapter 1.11 --- CCL5 in allergic inflammation --- p.22 / Chapter 1.12 --- β-defensin 2 (HBD-2) in allergic inflammation --- p.23 / Chapter 1.13 --- ICAM-1 and VCAM-1 in allergic inflammation --- p.25 / Chapter 1.14 --- LIGHT and airway remodeling in allergic asthma --- p.25 / Chapter 1.15 --- Signal transduction pathways in allergic inflammation and pharmacological inhibitors --- p.26 / Chapter 1.15.1 --- Signal transduction pathways in allergic inflammation --- p.26 / Chapter 1.15.2 --- Signaling molecule inhibitors as new drugs for inflammatory diseases --- p.31 / Chapter 1.16 --- Aims and scope of the study --- p.32 / Chapter Chapter 2: --- Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Reagents and buffers for the purification of human basophils --- p.35 / Chapter 2.1.2 --- Primary cells and cell lines --- p.36 / Chapter 2.1.3 --- Heat-killed Staphyloccocus aureus (HKSA) --- p.38 / Chapter 2.1.4 --- Ligands for NLR and TLR2 --- p.39 / Chapter 2.1.5 --- Recombinant human cytokines --- p.39 / Chapter 2.1.6 --- Reagents and buffer solutions for flow cytometry --- p.40 / Chapter 2.1.7 --- RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR), and real-time quantitative PCR (qPCR) --- p.45 / Chapter 2.1.8 --- Cytometric Bead Array (CBA) Kits --- p.48 / Chapter 2.1.9 --- MILLIPLEX® MAP Human Cytokine/Chemokine Magnetic Bead Panel Kit --- p.49 / Chapter 2.1.10 --- Enzyme-linked immunosorbent assay (ELISA) kits --- p.49 / Chapter 2.1.11 --- Procarta Transcription Factor Assay kit --- p.50 / Chapter 2.1.12 --- Signal transduction inhibitors --- p.50 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Purification of primary human basophils and basophil culture --- p.50 / Chapter 2.2.2 --- Culture of KU812 cells --- p.51 / Chapter 2.2.3 --- Culture of primary human bronchial epithelial cells --- p.51 / Chapter 2.2.4 --- Culture of BEAS-2B cells --- p.52 / Chapter 2.2.5 --- Culture of human dermal fibroblasts --- p.52 / Chapter 2.2.6 --- Co-culture of primary human bronchial epithelial cells/human bronchial epithelial cell line (BEAS-2B) cells and basophils/KU812 cells --- p.52 / Chapter 2.2.7 --- Co-culture of human dermal fibroblasts and basophils/KU812 cells --- p.52 / Chapter 2.2.8 --- Co-culture of fixed primary human bronchial epithelial cells and basophils --- p.53 / Chapter 2.2.9 --- Co-culture of human dermal fibroblasts and basophils in the presence of transwell inserts --- p.53 / Chapter 2.2.10 --- CBA assay --- p.53 / Chapter 2.2.11 --- ELISA --- p.54 / Chapter 2.2.12 --- Human Transcription Factor Plex Assay --- p.54 / Chapter 2.2.13 --- Milliplex Human Cytokine / Chemokine Magnetic Panel assay --- p.54 / Chapter 2.2.14 --- Bio-Plex mouse cytokine assay --- p.55 / Chapter 2.2.15 --- Flow cytometric analysis of cell surface expression of target molecules --- p.55 / Chapter 2.2.16 --- Flow cytometric analysis of intracellular expression of target molecules --- p.55 / Chapter 2.2.17 --- Allergic asthmatic mice model --- p.57 / Chapter 2.2.18 --- Statistical analysis --- p.57 / Chapter Chapter 3: --- Muramyl Dipeptide Mediated Activation of Human Bronchial Epithelial Cells Interacting with Basophils: A Novel Mechanism of Airway Inflammation --- p.59 / Chapter 3.1 --- Introduction --- p.59 / Chapter 3.2 --- Results --- p.61 / Chapter 3.2.1 --- Cell surface expression of CD203c on basophils --- p.61 / Chapter 3.2.2 --- Intracellular expression of NOD2 protein --- p.63 / Chapter 3.2.3 --- Cell surface expression of adhesion molecules on basophils and primary human bronchial epithelial cells activated by MDP --- p.67 / Chapter 3.2.4 --- Induction of cytokine, chemokine and β-defensin 2 upon the interaction of basophils and bronchial epithelial cells stimulated by MDP --- p.71 / Chapter 3.2.5 --- Bronchial epithelial cells were the main source for the release of IL-6, CXCL8 and β-defensin 2 in co-culture --- p.74 / Chapter 3.2.6 --- Effects of signaling inhibitors on MDP-induced cytokines and adhesion molecules --- p.77 / Chapter 3.2.7 --- Differential activation of intracellular signaling pathways involved in the interaction of KU812 and BEAS-2B upon MDP stimulation --- p.84 / Chapter 3.2.8 --- In vivo effect of NOD1,2 ligands on IgE and chemokine production in serum and BALF in allergic asthmatic mice --- p.89 / Chapter 3.3 --- Discussion --- p.93 / Chapter Chapter 4: --- NOD2 and TLR2 Ligands Mediated Activation of Basophils Interacting with Human Dermal Fibroblasts in Atopic Dermatitis --- p.100 / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Results --- p.102 / Chapter 4.2.1 --- Cell surface expression of adhesion molecules ICAM-1 on human dermal fibroblasts activated by heat-killed Staphyloccocus aureus (HKSA) --- p.102 / Chapter 4.2.2 --- Induction of chemokines upon the interaction of basophils and human dermal fibroblasts stimulated by HKSA --- p.104 / Chapter 4.2.3 --- Expression of NOD2 and TLR2 protein --- p.107 / Chapter 4.2.4 --- Cell surface expression of adhesion molecule ICAM-1 on human dermal fibroblasts activated by MDP, PGN, LTA or Pam3CSK4 --- p.110 / Chapter 4.2.5 --- Induction of cytokine and chemokines upon the interaction of basophils (with or without IL-33 priming) and human dermal fibroblasts stimulated by MDP, PGN, LTA or Pam3CSK4 --- p.112 / Chapter 4.2.6 --- Direct interaction between human dermal fibroblasts and basophils was required for the release of IL-6, CXCL8, CCL2 and CCL5 upon the stimulation of MDP, PGN, LTA and Pam3CSK4 --- p.118 / Chapter 4.2.7 --- Effect of signaling molecular inhibitors on the expression of adhesion molecule ICAM-1 --- p.121 / Chapter 4.2.8 --- Effect of signaling molecule inhibitors on the release of cytokine and chemokines upon the stimulation by NOD2 and TLR2 ligands --- p.123 / Chapter 4.2.9 --- Differential activation of intracellular signaling pathways involved in the interaction of human dermal fibroblasts and basophilic KU812 upon stimulation of NOD2 and TLR2 ligands --- p.127 / Chapter 4.3 --- Discussion --- p.131 / Chapter Chapter 5: --- Effect of Tumor Necrosis Factor Family Member LIGHT on the Activation of Basophils Interacting with Bronchial Epithelial Cells: Potential Therapeutic Target for Airway Remodeling --- p.138 / Chapter 5.1 --- Introduction --- p.138 / Chapter 5.2 --- Results --- p.139 / Chapter 5.2.1 --- Cell surface expression of HVEM and LTβR --- p.139 / Chapter 5.2.2 --- Effect of LIGHT on the expression of ICAM-1 on basophil or BEAS-2B alone or co-culture --- p.141 / Chapter 5.2.3 --- Induction of cytokine and chemokine upon the interaction of basophils and BEAS-2B cells stimulated by LIGHT --- p.144 / Chapter 5.2.4 --- Induction of MMP-9 upon the interaction of basophils and BEAS-2B cells stimulated by LIGHT --- p.147 / Chapter 5.2.5 --- Effect of LIGHT on the release of TGFβ-1, histamine and periostin upon the interaction of basophils and BEAS-2B cells --- p.149 / Chapter 5.3 --- Discussion --- p.152 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.156 / Chapter 6.1 --- General conclusions --- p.156 / Chapter 6.2 --- Future perspectives --- p.160 / Appendix --- p.163 / References --- p.165

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. Bibliography: 58-64.

The innate immune system provides the first line of defense against infection. Pathogens are detected though a variety of Pattern Recognition Receptors (PRRs), which activate downstream signaling cascades. Effector molecules such as cytokines and chemokines are released upon activation and aid in cell recruitment, control of pathogen replication, and coordination of the adaptive immune response. Nucleic acids that are released into the cytosol during viral and bacterial infection are recognized through a special class of PRRs, coined cytosolic nucleic acid sensors. Upon recognition, these receptors induce the production of type I interferons and other cytokines to aid in pathogen clearance. Although many cytosolic nucleic acid sensors have been discovered, it is unclear how they work in concert to mediate these responses. The Interferon Gamma Inducible protein (IFI)16 and its proposed mouse orthologue IFI204 are cytosolic DNA sensors that have been linked to the detection of cytosolic DNA during infection with Herpes Simplex Virus (HSV-1). IFI16 binds dsDNA that has been released into the cytosol during viral infection and engages the adaptor molecule Stimulator of Interferon Genes (STING) leading to TANK binding kinase-1 (TBK1) dependent phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of type I interferons and interferon stimulated genes. In addition to its role as a sensor, in chapter two of this thesis we describe a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in anti-viral immunity. In an effort to better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as other inducers of IFN such as cyclic-dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to cytosolic DNA ligands and DNA viruses. In contrast, expression of the NF-κB regulated cytokines such as IL-6 and IL-1β were unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of expression of IFN-α and IFN stimulated genes such as RIG-I in response to cyclic GMP-AMP (cGAMP), a second messenger produced in response to cGAS, as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-α promoter in IFI16 knockdown cells suggesting that transcription of ISGs is dependent on IFI16. Since IFI16 knockdown compromised not only DNA virus driven pathways, we propose additional regulatory roles outside of DNA sensing. Collectively, these results indicate that IFI16 plays a role in the regulation of type I IFN gene transcription and production in response to both RNA and DNA viruses. The role of IFI16/IFI204 has been studied extensively in vitro, however the role of the receptors in vivo has yet to be determined. In chapter three of this thesis, we developed a mouse deficient in IFI204 to explore the role of IFI204 in in vivo immune responses to viruses. We investigated the ability of IFI204 deficient cells to induce type I interferons and other cytokines in response to a panel of DNA and RNA ligands in vitro. IFI204 deficient BMDMs displayed a partial defect in type I interferon induction in response to both DNA and RNA ligands and viruses as compared to WT mice. We also observed that this phenotype is time dependent, since there was no change in type I interferon induction after 12 hours post infection as compared to earlier time points. In contrast to these results, expression of the NF-κB regulated cytokines IL-6 and IL-1β were unaffected in IFI16 knockdown cells. These results suggest that IFI204 plays a partial role in the induction of type I interferons in response to both DNA and RNA ligands. Additionally, IFI204 may work in tandem with other receptors in a sequential manner to amplify the type I interferon response. We also studied the involvement of IFI204 in an in vivo model of HSV-1 infection. IFI204 knockout mice produce less brain and serum IFN-β, IL-6, and IL-1β 72 hours post intraperitoneal infection with HSV-1. Furthermore, IFI204 -/- mice are more susceptible to HSV-1 infection as compared to WT mice. These data indicate that IFI204 mediates the response to HSV-1 in vivo by inducing the production of cytokines that are necessary for the control of viral infection.

The innate immune system is an evolutionarily conserved primary defense system against microbial infections. One of the central components of innate immunity are the pattern recognition receptors which sense infection by detecting various conserved molecular patterns of pathogens and trigger a variety of signaling pathways. In this dissertation, the signaling pathways of several classes of these receptors were dissected. In chapters II and III, the role of two NOD-like receptors, NLRP3 and NLRC4 were investigated in the context of infection with the fungal pathogen, C. albicans. C. albicans is an opportunistic pathogen that causes diseases mainly in immunocompromised humans and innate immunity is critical to control the infection. In chapters II and III, we demonstrate that a multiprotein-inflammasome complex formed by the NLR protein, NLRP3 and its associated partners, ASC and caspase-1 are critical for triggering the production of mature cytokine IL-1β in response to C. albicans. NLRC4, another inflammasome forming NLR that is activated by intracellular bacterial pathogens, was not required for this process in macrophages. Thus, our data indicates that NLRP3 inflammasome responds to fungal infections in addition to its known stimuli such as bacterial and viral infections, toxic, crystalline and metabolic signals. Interestingly, this NLRP3 dependent inflammasome response was maintained even when the pathogen is not viable, and is either formalin fixed or heat-killed (HK). Hence, in chapter III, we examined β-glucans, a structural cell wall component, as the potential immunostimulatory component of C. albicans and dissected the inflammasome responses to β -glucans. We observed that NLRP3-ASC-caspase-1 inflammasome was critical for commercially obtained particulate β-glucans similar to the case of C. albicans. β-glucan sensing C-lectin receptor dectin-1 and the complement receptor CR3 mediated inflammasome activation, IL-1β production in response to the glucan particles. Interestingly, CR3 which recognizes glucans as well as complement opsonized pathogens was strongly required for HK C. albicans induced IL-1β, and partially required for that of live C. albicans, while dectin-1 was not required. Consistent with the receptor studies, blocking of β -glucan receptors by pre-incubating cells with nonstimulatory, soluble glucans led to decreased IL-1β production in response to HK C. albicanswith no effect on IL-1β in response to the live fungus. Dectin-1, CR3 and β-glucan sensing also triggered a moderate dendritic cell death response to β-glucans and HK C. albicans. Live C. albicans induced cell death requires phagocytosis but not the inflammasome, β-glucan sensing, dectin-1 or CR3. The Drosophila caspase-8 like molecule DREDD plays an essential, nonapoptotic role in the Drosophila NF-κB pathway called the ‘IMD’ pathway. Owing to the remarkable evolutionary conservation between Drosophila and mammalian innate immune NF-κB pathways, we explored the potential role of caspase-8 in inflammasomes and in TLR signaling. Using casp8-/- Rip3-/- macrophages and dendritic cells, we observed that caspase-8, specifically augments β-glucan and HK C. albicans induced IL-1β as well as cell death in a caspase-1 independent manner, but not that of live C. albicans, in chapter III. We also found that caspase-8 differentially regulates TLR4 and TLR3 induced cytokine production (chapter IV). Caspase-8 specifically promotes TLR4 induced production of cytokines such as TNF, IL-1β in response to LPS and E. coli. On the other hand, caspase-8 negatively regulates TRIF induced IFNβ production in TLR4 and TLR3 signaling in response to LPS and dsRNA. Caspase-8 executed a similar mode of regulation of the cytokine RANTES in MEFs, in part, by collaborating with RIP3. Strikingly, caspase-8 deficiency alone triggers higher macrophage death and IL-1β production in response to TLR ligands, due to the presence of RIP3. Thus, in addition to its conventional roles in apoptosis, caspase-8 modulates TLR4 and TLR3 induced cytokine production and prevents RIP3 mediated hyper inflammation in response to TLR signals. Together, our findings provide valuable information on fungal pattern recognition and inflammasome pathways and define the contribution of β-glucan sensing to C. albicans induced inflammasome responses. In addition, we demonstrate how caspase-8 adds a layer of specificity to inflammasome as well as TLR signaling. Overall, these results also shed light on the cross talk between death signaling components and innate immune pathways to mount a specific and potentially effective innate immune response against microbial pathogens.

The innate immune system provides an essential first line of defense against infection. Innate immune cells detect pathogens through several classes of Pattern Recognition Receptors (PRR) allowing rapid response to a broad spectrum of infectious agents. Activated receptors initiate signaling cascades that lead to the production of cytokines, chemokines and type I interferons all of which are vital for controlling pathogen load and coordinating the adaptive immune response. Detection of nucleic acids by the innate immune system has emerged as a mechanism by which infection is recognized. Recognition of DNA is complex, influenced by sequence, structure, covalent modification and subcellular localization. Interestingly certain synthetic oligodeoxynucleotides comprised of the TTAGGG motif inhibit proinflammatory responses in a variety of disease models. These suppressive oligodeoxynucleotides (sup ODN) have been shown to directly block TLR9 signaling as well as prevent STAT1 and STAT4 phosphorylation. Recently AIM2 has been shown to engage ASC and assemble an inflammasome complex leading to the caspase-1-dependent maturation of IL-1β and IL-18. The AIM2 inflammasome is activated in response to cytosolic dsDNA and plays an important role in controlling replication of murine cytomegalovirus (MCMV). In the second chapter of this thesis, a novel role for the sup ODN A151 in inhibiting cytosolic nucleic acid sensing pathways is described. Treatment of dendritic cells and macrophages with the A151 abrogated type I IFN, TNF-α and ISG induction in response to cytosolic dsDNA. A151 also reduced INF-β and TNF-α induction in BMDC and BMDM responding to the herpesviruses HSV-1 and MCMV but had no effect on the responses to LPS or Sendai virus. In addition, A151 abrogated caspase-1-dependent IL-1β and IL-18 maturation in dendritic cells stimulated with dsDNA and MCMV. Although inhibition of interferon-inducing pathways and inflammasome assembly was dependent on backbone composition, sequence differentially affected these pathways. While A151 more potently suppressed the AIM2 inflammasome, a related construct C151, proved to be a more potent inhibitor of interferon induction. A151 suppressed inflammasome signaling by binding to AIM2 and competing with immune-stimulatory DNA. The interaction of A151 and AIM2 prevented recruitment of the adapter ASC and assembly of the macromolecular inflammasome complex. Collectively, these findings reveal a new route by which suppressive ODNs modulate the immune system and unveil novel applications for suppressive ODNs in the treatment of infectious and autoimmune diseases. The innate immune response to HSV-1 infection is critical for controlling early viral replication and coordinating the adaptive immune response. The cytokines IL-1β and IL-18 are important effector molecules in the innate response to HSV-1 in vivo. However, the PRRs responsible for the production and maturation of these cytokines have not been fully defined. In the third chapter of this thesis, The TLR2-MyD88 pathway is shown to be essential for the induction of pro-IL-1β transcription in dendritic cells and macrophages responding to HSV-1. The HSV-1 immediate-early protein ICP0 has previously been shown to block TLR2 responses and in keeping with this finding, ICP0 blocked pro-IL-1β expression. Following translation, pro-IL-1β exists as an inactive precursor that must be proteolytically cleaved by a multiprotein complex known as the inflammasome to yield its active form. Inflammasomes are composed of cytoplasmic receptors such as NLRP3 or AIM2, the adapter molecule ASC, and pro-caspase-1. In the present study we found that the NLRP3 inflammasome is important for maturation of IL-1β in macrophages and dendritic cells responding to HSV-1. In contrast the related NLRP12 protein controls IL-1β production in neutrophils. These data indicate that sensing of HSV-1 by TLR2 drives pro-IL-1β transcription and infection activates the inflammasome to mature this cytokine. Moreover, these studies reveal cell type-specific roles for NLRP3 and NLRP12 in inflammasome assembly.