Molecular genetic characterization of nod-like receptor 11 (NLRP11) in the regulation of adaptive immune responses

Virtually all organisms have at least a rudimentary defense mechanism to fight invaders, foreign substances or rivals. Comparatively speaking, the immune system of advanced vertebrates, including humans, is a sophisticated, compartmentalized and well-organized defense mechanism. The distinct effector mechanisms of the innate immunity and the adaptive immunity provide the versatility for the immune system of higher vertebrates. The innate immune system takes action within hours of infection and eliminates pathogens in a non-specific manner. However, in the event that a pathogen evades the innate immune response, an infection develops. When the innate immunity is breached, the adaptive immune system is activated and functions in the eradication of specific pathogens. Even though the immune system is categorized into two distinct components, recent studies suggest that there is no clear line seperating the innate immunity from adaptive immunity. For example, it is now clear that components of the innate immune system have roles in the regulation of the adaptive immune system. Furthermore, Patern Recognition Receptors (PRRs) of the innate immunity such as Toll-like receptors (TLRs) and Nod-like receptors (NLRs) can also be found in the cells of the adaptive immunity. Unlike Toll-like receptors, however, Nod-like receptors have not been widely studied in the context of regulation of adaptive immunity. The NLR family is a relatively newly discovered family of PRRs whose functions have been examined almost exclusively in the innate immunity. There are 22 known members of the NLR family in humans, four of which form a multiprotein complex called the inflammasome complex. Aside from inflammasome complex formation, the NLR family has been shown to have associations with several diseases including artherosclerosis, type II diabetes, obesity, Alzheimer's disease, gout and bacterial, viral and parasite infections. One of the most recent discovered members of the NLR family, NLR family Pyrin Containing 11 /NLRP11), is expressed only in primates: however, its cellular functions as well as the specific stimulant(s) that activate it are unknown. As stated above, the function of NLRP11 remains elusive and the regulatory role of NLRP11 in coordinating the adaptive immune system is uncharted territory. In an effort to remedy this dearth of information, we focused in this thesis on the molecular analysis of NLRP11. Because NLRP11 is expressed in Daudi cells, a human Burkitt's B cell lymphoma cell line, we used Daudi cells as a model throughout our experiments. Since the ligand for NLRP11 has not yet been identified, we selected several stimulants including CD40L, PMA/Ionomycine, LPS and adenosine, and screened them to determine whether or not NLRP11 was inducible in Daudi cells. With the exception of adenosine, all these stimulants are commonly used to induce an immune response and the formation of an inflammasome complex in immune cells. We included adenosine in the experiments because of the relatively high expression of the adenosine receptor A2A on Daudi cells. A1, A2A, A2B and A3 adenosine receptors are G-protein-coupled receptors that mediate the biological actions of adenosine, a purin nucleoside. Upon binding to its purinergic receptors which are found on several types of immune cells, adenosine modulates immune responses. The binding of adenosine to A2A and A2B receptors results in immunosuppression. To further investigate the effects of adenosine on NLRP11 expression, we performed western blot analysis and quantitative reverse transcriptase PCR (RT-qPCR). We first tested the effects of dose and duration of treatment and found 50 µM adenosine for 4 hours of stimulation was the optimum condition. We discovered that both mRNA and protein levels of NLRP11 are increased in Daudi cells at 4 hour post-stimulation with adenosine. Overall, these results strongly support our hypothesis that the NLRP11 expression is increased in Daudi cells in response to adenosine stimulation but not to CD40L, PMA/Ionomycine or LPS. To further validate the effect of adenosine on NLRP11, Daudi cells were stimulated with the adenosine inhibitor caffeine, an antagonist of the adenosine A2A receptor. These results indicated that the expression levels of NLRP11 are downregulated in response to caffeine further evidence that NLRP11 regulation might be mediated by an adenosine receptor. Activation of NLR family members AIM2, NLRP1, NLRC4 and NLRP3 results in the formation of a multi-protein complex, the inflammasome, which contains the ASC (Apoptosis-associated speck-like protein containing a CARD) adaptor protein and the cysteine protease caspase-1. Activation of caspase-1 results in the cleavage and maturation of the pro-inflammatory cytokines IL1β (Interleukin-1β) and IL-18. To date, NLRP11 protein has not been reported to form an inflammasome complex via an interaction with ASC and caspase-1 proteins. We reasoned that because of NLRP11 is highly expressed in Daudi, B cell lymphoma, cell line, it might possibly be involved in a dual or triple interaction with ASC and/or caspase-1. We then tested this possibility using a co-immunoprecipitation (Co-IP) assay. The Co-IP assays demonstrated that NLRP11 indeed binds to the ASC adaptor protein after stimulation with adenosine. However, IL1β and IL18 cytokines were not secreted at the protein level in response to the NLRP11-ASC interaction, although we were able to measure these cytokines at the mRNA level by qPCR. We then proceeded to check the caspase-1 activity after stimulating Daudi cells with adenosine. Results from these particular experiments were inconclusive and necessitate optimization for the accurate measurement of caspase-1 activity. B cells are Antigen Presenting Cells (APC) that produce higher quality antibodies upon interaction with T cells. Because of Daudi cells are a B-cell lymphoma cell line, we used these cells to examine what role, if any, NLRP11 played in the interaction between human primary T cells and B cells; in other words, to investigate the role of this NLR family member in shaping the adaptive immune responses. For this purpose, we first verified that Daudi cells have the characteristics of antigen presenting cells for co-culture experiments; therefore, we examined the expression levels of co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86) on Daudi cells. We found that Daudi cells express these co-stimulatory molecules; however, their expression levels are not dependent on NLRP11. We next induced downregulation of NLRP11 in Daudi cells using siRNA (small interfering RNA) and co-cultured these Daudi cells with blood-derived human primary CD4+ T cells. Cytokine release from the CD4+ T cells in the co-culture condition was measured to understand the effect of NLRP11 on T cell polarization. We found that co-culturing with Daudi cells suppressed both Th1 (T Helper 1) and Th17 (T Helper 17) responses, while downregulation of NLRP11 in Daudi cells led to the recovery of these Th1 and Th17 responses, but did not cause any significant changes in Th2 (T helper 2) and Treg (Regulatory T cell) responses when co-cultured with CD4+ T cells. Our findings raise the possibility of new targets for treatment of immune system related diseases originating from deficient T cell responses and lay the groundwork for further studies to understand the mechanism by which NLRP11 regulates T cell skewing.