Activation of inflammasome - Dissertation Example

?Abberant inflammasome activation in ataxia telangiectasia Ataxia telangiectasia is a multi-organ system autosomal recessive condition characterized in part by disorders of the adaptive immune system. It is an autosomal recessive disorder effecting either the innate or adaptive immune systems leaving the organism vulnerable to opportunistic infections. There is a connection between deficiencies in the molecular underpinnings of the adaptive immune system and vulnerability to infections that this report will explore. This study will investigate the most immediate pathogenic effects resulting from this disorder using cells infected with S.typhimurium and L.Monocytogenes to demonstrate vulnerability to infections. Homozygous mutants suffered the highest mortality rates. Assays were conducted to determine the molecular cofactors most involved during the acute disease state by examining the process of inflammasome formation. Mutations along several stages of the process elucidate the cofactors involved. Ataxia Telangiectasia alters the formation of the inflammasomes complex based on caspase recruitment, and is characterized by deficiencies in interleukins as opposed to tumor necrosis factors. The identification of cofactor levels corresponding with the autosomal recessive genotype could potentially support the development of enhanced screening or diagnostic techniques with an ultimate goal of corrective therapy. INTRODUCTION The Immune system offers protection to the body from internal and external threats. Mammalian Immune system is classified into innate and adaptive immunity. Adaptive immune system appeared later in the evolutionary time line and it is more specific than the innate immunity. Through feature called immunological memory, adaptive immune system evokes a quick immune response upon subsequent exposure to similar pathogenic stimuli. These responses are typically dependent on T-cells, which involve not only the removal of active pathogens; but also the elimination by cell-death (Apoptosis). Innate immune system has no immunological memory. This analysis depends upon the genetic basis underlying failure in the immune system due to molecular changes. Innate immunity is the body’s first line of defense. Innate immune recognition and responsiveness to biological danger is mediated by germ-line encoded innate immune receptors called pattern recognition receptors (PRRs). PRRs recognize conserved motifs on pathogens called Pathogen associated molecular patterns (PAMPs) or endogenous molecules called Damage Associated Molecular Patterns (DAMPs) that are released by stressed of dead cells6. PRRs are classified into trans-membrane receptors: Toll like receptors (TLRs) and C-type lectin receptors (CLRs), the cytoplamic families of PRRs: Nucleotide-binding oligomerization (NOD)-like receptor (NLRs), RIG-I like receptors (RLRs) and cytoplasmic DNA receptors (CDRs)6. Cytoplasmic DNA Receptors (CDRs) consist a diverse and growing number of receptors that recognize DNA in the cytoplasm. Members of this family include DAI, IFI16, LRRFIP15. TLRs are type-I trans-membrane proteins composed of an ectodomain domain rich in leucine-rich repeats that play a role in the recognition of PAMPs, trans-membrane region and cytosolic Toll-IL-1 receptor (TIR) domain which are involved in the activation of downstream signaling pathways. 10 and 12 TLRs have been identified in human and mouse, respectively. TLRs are localized at the cell surface and endosomal compartments where they recognize a broad range of bacterial, viral, fungal, protozoan PAMPs such as lipoproteins (recognized by TLR1, TLR2, and TLR6), double-stranded (dsRNA) (by TLR3), lipopolysaccharide (LPS) (by TLR4), flagellin (by TLR5), single-stranded RNA (ssRNA) (by TLR7 and TLR8), and DNA (by TLR9)1. NLRs consists of a large family of receptors that recognize broad spectrum of PAMPs and DAMPs in the cytoplasm8. Till now 34 murine and 23 human NLR genes have been identified. Plants contain NLR orthologues as R genes. Plants lacking adaptive immune system completely depend on innate immune system, which is mediated by R genes1. NLRs contains three domains, N-terminal protein interaction domain, C-terminal LRR domain and central nucleotide-binding domain. Functionally NLR can be categorized into those like NOD1 and NOD2 that mediate transcriptional activation of inflammatory and anti-microbial genes and those like NLRP3, NLRP1, NLRC4, and NLRC5 that mediate activation of the caspase 1 - inflammasome dependent inflammatory processes namely the maturation IL-1 ? and IL- 18 and induction of pyroptotic cell death2. CLRs belonging to the family of trans-membrane protein receptors, recognize the carbohydrate moiety present on pathogens (viruses, bacteria, fungi) by the carbohydrate binding domain and elicit inflammatory response 2 RLRs family consists of 3 members: Retinoic acid-inducible gene-I (RIG-I), Melanoma Differentiation-Associated gene5 (MDA5), and Laboratory of Genetics and Physiology 2 (LGP2). RLRs contain two N-terminal caspase recruitment domains (CARDs), C-terminal regulatory domain and a central domain DEAD box helicase / ATPase domain. These cytoplamic receptors recognize genomic RNA, dsRNA viruses, dsRNA generated by the replication of ssRNA viruses and bacteria which replicate in cytoplasm. RLRs elicit the type I and inflammatory responses. RIG-I detects short viral RNAs while MD5 recognize the long dsRNAs like poly I: C. LGP2 has been suggested to be both a positive and negative regulator of RIG-I and MDA53. This signaling is important in T-cells, where LGP2 controls survival of CD-8 cells, upregulating them in cases of infection. Evidence indicates the AT mutation includes a disruption of this signaling pathway, preventing increases in these cofactors, and interfering with innate immunity3. Below is a graphic demonstration of the functional mechanism underlying immune reactions. The surface antigens of various pathogens shown below trigger immune cofactors. This is a survival adaptation, for the imune system to exhibit elevated reactivity in response to the distinctive features of potential pathogens. This elevation in sensitivity is a well-established principle used later in this analysis to demonstrate dysfunction in inflammation by removing cofactors through mutation and testing whether the expected reactivity still occurs, and at what rate. The graph below demonstrates the standard mechanisms. Figure1: (Anand, et al. 2011)4 Diagram of the activation and conformation process involved in the assembly of Inflammasomes. Pathogen-Associated molecular patterns can be seen initiating the signaling process by way of membrane-bound antibodies functioning as receptors. Toll-like receptors signal NF-?B activation and transcription9. Although important against infections and tissue repair, unregulated activation of PRRs is known is deleterious to the host. In fact the immunopathological manifestation of many inflammatory diseases including sepsis, rheumatoid arthritis, inflammatory bowel diseases, neurodegenerative diseases and metabolic diseases have been ascribed to dysregulations in activation of PRR signaling pathways. Ataxia-telangiectasia (AT) is an autosomal recessive disease caused by deficiency in the Ataxia Telangiectasia Mutated kinase (ATM). ATM is a member of the phosphatidylinositol 3-kinase (Pi3K) family of protein kinases that plays a central role in DNA Damage Response (DDR). DDR signaling pathways mediate several cellular responses chief of which is repair of DNA lesions and activation of cell cycle arrest or cell death if the DNA damage is too severe5. Activation of DDR signaling pathways can also activate inflammatory responses6. Although usually stereotyped as neurodegenerative disease, AT is a multisystem disease. Its manifestations include genomic instability, immunodeficiency, cancer susceptibility retarded growth, accelerated aging, sterility6. Curiously most of the AT patients (estimated median survival age of 25 years) die due to sepsis caused pulmonary bacterial infections6 . The immunodeficiency in AT has in part been explained in terms of the defects in adaptive immune responses. Whether the immunopathological manifestation in AT are also due to defects in activation of innate immune system and why such patients specifically succumb to bacterial infections is not clear. Furthermore whether such infections contribute to neuropathology and the role of PRRs thereof remains to be established. In response to PAMPs and DAMPs, PRRs activate the downstream intracellular signaling pathways that result in the induction of inflammatory cytokines/chemokines and type I interferons (IFNs). Pro-inflammatory cytokines mediate innate immune responses against a variety of infections including bacteria, fungal and parasites, while type I IFNs are involved in protection against viruses. Both Pro-inflammatory cytokines and type I IFNs are also key modulators of adaptive immunity. Thus apart from the innate immune system PRRs activation is essential for the development of the adaptive immune system. Abnormalities in these cytokines characterize Ataxia telangiectasia6. The experiment in question focuses upon a mutated kinase causing a susceptibility to pulmonary infections. Other mutations of immunological cofactors are also included to further elucidate the process. Results: The assays described below utilize the organism Salmonella typhimurium as an adjuvant mechanism to characterize the activation of innate immunity. Its proteins possess the ability to activate multiple pattern recognition receptors associated with immune responses as listed below: TLR's, RLR's, NLR's, and CDR's, as well as type I interferons. Immune reactions were provoked using bacterial derived lipopolysaccharides in the bone derived macrophages described in the assay below. These bacterial vectors are useful in terms of provoking immune reactions, allowing researchers to elucidate the extent to which different molecular cofactors respond during the inflammation and the inflammasomes formation process. These mutant types allow investigators to map out the process of molecular activity by describing the consequences to the inflammation process when one or more genes are deactivated. These mechanisms allow us to determine which pattern recognition receptors are important in inflammasomes formation by studying the consequences of their deactivation. As described below, this study provides evidence in regards to which pathways are influenced by ataxia telangiectasia, providing more evidence for the role of these genes by studying their dysfunction. The lipopolysaccharide cell wall components of many bacteria serve to enhance immune reactions, most likely as a result of direct selection pressure in the evolution of the immune system to combat bacteria specifically; but more research on the specifics could be conducted. Central to the experiments described in this analysis are the effects of mutations associated with ataxia telangiectasia in the operation of pattern recognition receptors. We are able to determine that the disorder serves as a negative regulator of toll like receptors, as well as certain nod like receptors. The rationale behind Listeria monocytogenes and salmonella typhimurium in this instance are that they should indeed provoke very strong immune reactions, for valid reasons of defense against infection. Seeing when and where this expected reaction breaks down allows investigators to characterize the molecular mechanisms of the disorder – especially pertaining to inflammasomes activation. ATM is a negative regulator of PRR pathways of TBK1, NF-kB and MAPK activation Listeria monocytogenes (L.m.) is a model microoerganisms for studying innate immunity. It activates multiple PRRs including TLRs (e.g. TLR2, TLR9) and RLRs, NLRs and CDRs to activate MAPK, NF-kB and TBK1-IRF3 pathways leading to the production of pro-inflammatory and type I interferons2. To investigate the role of ATM in regulation of PRR signaling pathways for pro-inflammatory responses, WT and ATM -/- BMDMs infected with L.m. for different were evaluated for the activation of NF-kB and MAPK pathways. ATM-/-BMDM exhibited an elevated TBK1 phosphorylation at basal conditions (data not shown) and in response to L.m. ATM deficiency also resulted in - a higher ERK1/2 phosphorylation and a more sustained phosphorylation of p38 MAPK (Figure 1.A). In response to LPS stimulation, ATM-/- BMDMs also exhibited enhanced (-) ERK1/2 activation (Figure 1 B). ATM deficiency is associated with enhanced proinflammatory NF-kB and MAPK pathways play a critical role in transcriptional activation of pro-inflammatory mediators. In view of the above data, next we evaluated the effect of ATM deficiency on pro-inflammatory cytokine production in response to bacterial or TLR stimulation. ATM-/- BMDMs elicited a higher production of the pro-inflammatory cytokines TNF-a, IL-6 and Il-1b in response to L.m. or infection (Figure 2A&B), or stimulation with TLR2 and TLR4 ligands Pam2CSK and LPS respectively (Figure 2C&D). To analyze type I IFN response, we used the IFN-b luciferase (IFN-???-luc/??-luc) reporter mice. These mice were bred with ATM-/- mice to generate ATM-/-IFN-???-luc/??-luc. Compared to the ATM sufficient cells (i.e IFN-???-luc/??-luc), ATM-/-IFN-???-luc/??-luc BMDMs elicited higher IFN-? response in response to L.m. or S.p. infection or LPS stimulation (Figure 3 A-C). Together these data suggest that ATM is a negative regulator of PRR pathways for pro-inflammatory responses and that its deficiency results in enhanced cytokine production. FIGURE 2: Role of ATM in Innate immune signaling pathway Figure 2: Bone marrow derived macrophages WT, ATM-/-, IFNAR-/-, ATM IFNAR DKO cells stimulated with LPS (500ng/ml) (A and B) and infected with Listeria monocytogenes (MOI=50) (C and D) and spun down at 400g for 5 min (C&D). Cells were incubated for different time points (0, 5, 15, 30, 60, 120, 180 min.) Cell lysates were immunoblotted with antibodies against –PhosphoIk?, Ik?, phosphoTBK1, phosphoP38, phosphoERK1 and 2 . ?-actin is used as the loading control. The TBK-1 row provides initial data concerning the molecular differences between the wild type and ATM mutants. Electrophoresis data shows residual bands reflecting a conformation distinction. It is probable that P-TBK-1 reflects a structure incompletely assembled in cases of ATM -/-. Role of ATM in Inflammasome activation The synthesis, processing and release of mature IL-1? require two signals: first, an inflammatory stimulus which via NF-kB and MAPK pathways primes the cells to transcribe and synthesize pro-IL-1? and a second stimulus e.g. ATP which acts via the ligand gated ion channel P2X7R to activate the caspase 1-inflammasome complex2. Activation of inflammasome results in autocatalytic processing of 45kDa caspase-1 to generate the p20 and p10 subunits. These subunits make up the active caspase-1, a (p20/p10)2 tetramer, responsible for the cleavage of precursor IL-1 (pro-IL-1?) as well as for induction of pyroptotic cell death program2. In the absence of the second stimulus, pro-IL-1? is either degraded or remains intracellular and unprocessed. However, bacterial pathogens like Listeria, promote the potassium efflux through pore forming bacterial toxins (listeriolysin O) which subsequently results in the activation of NLRP3 inflammasome2. ATM deficiency is associated with enhanced IL-1b production. Interestingly, we also that in the absence of ATP, ATM-/- cells were could still to secrete significant amount of Il-1b in response LPS. This suggests that the higher Il-1b production in ATM-/- cells was not only due to higher transcription response but most probably also due to enhanced activation of the inflammasome. To investigate, lysates and supernatants of WT, ATM-/-, ASC-/- cells infected with either L.m. S.t or stimulated with LPS and (or not with) ATP. Immunoblotting analysis revealed a diminished secretion of mature IL-1b or cleaved caspase 1 (Figure 4). As expected no secretion of active caspase -1 nor mature IL-1? was observed in ASC-/- cells in response to LPS +ATP stimulation or infection with L.m. Curiously, even at basal conditions, ATM-/- cells exhibited an elevated Il-1b expression and a detectable active caspase 1 (Figure 3). Such spontaneous activation of caspase 1 in ATM-/-might in part account for Il-1b processing and secretion in cells only primed with LPS. In our experiments where we treated BMDM cells with L.m. active form of caspase-1 was detected only in the cell lysates. However, secreted forms of caspase-1 and IL-1? are not detected. This probably due to the combination of a suboptimal MOI of infection and a too early time point of sample collection which did permit detection secreted caspase-1 and IL-1?. The Immunoblots below describe inflammasomes activation through Caspase activity. Specifically caspases 1 and interleukin 1 beta. Listeria monocytogenes provided the antigens necessary to stimulate immune activity. Figure: 3 i) Role of ATM in Activation of procaspase-1 and release of IL-1? BMDM cells of WT, ATM-/-, ASC-/- mice were stimulated with LPS (500ng/ml) for 4hours and then added 5mM pulse of 30min and Infection with listeria monocytogenes (MOI=30) incubated for 30min at 370C. Then gentamycine was added for 30 min and incubated overall for 5 hours. After four hours both stimulated and infected cells supernatants were collected and cells were lysed and supernatants were precipitated by using methanol-chloroform. The experiment in Figure 3 represents a stimulation of the immune response with bacterial lipopolysaccharides to provoke a reaction, by way of caspase recruitment of the inflammasome complex. The most noteworthy reaction is the Apoptosis-associated speck-like protein mutants. The lack of this gene influences the conformation of the protein and electrophoretic migration found in the wild type and ATM mutants for the supernatant assay. 2.) To investigate whether ATM deficiency also impairs the ASC independent inflammasomes we generated mice doubly deficeint in ATM and ASC. Compared to the wild types BMDMs from ATM-/- mice elicited diminished caspase 1 processing and IL-1b secretion while ASC-/- and ATM/ASC-/- cells elicited no such responses. To investigate the non-canonnical ASC independent pathway of IL-1b secretion, WT, ATM-/- ASC, and ATM/ASC-/- cells were challenged with Salmonella typhimuriums then analyzed for IL-1b and caspase 1 processing. Whereas ATM-/- cells exhibited a diminished caspase 1 processing compared to the wild type, no caspase 1 processing was detected in ASC and ATM/ASC. However albeit lower that in WT and ATM-/- cells, ASC and ATM/ASC-/- cells were found to a significant IL-1b processing. There was not difference in IL-1b response between ASC and ATM/ASC-/- cells suggesting that ATM deficiency does not impair the ASC-Caspase 1-independent inflammasome activation. In addition to cytokine secrection, is one of the consequences of inflammasome activation is pyroptosis. However measurement of cell membrane damage showed no significant difference between WT and ATM-/- macrophages upon LPS+ATP stimulation, as shown in figure C. Inflammasome conformation is a multistage process, with a range of cofactors dependent on various pattern recognition receptors. S.typhimurium is the primary agent used to stimulate immune reactions needed to engage the process. A) B) Figure 3: A-B. Electrophoresis results from stimulation with S. typhimurium. This data is most revealing when comparing the wild type with ATM-/- mutants. Few significant differences can be observed in the migration of interleukin and pro-interleukin. Distinction is apparent in the caspase and pro-caspase rows in terms of molecular weight of the products. The caspase rows provide data concerning the importance of this molecule; and its interaction with ASC (DKO). This confirms a chain of causation with ASC in the foundations of inflammation beginning with caspase reqruitment. C) Figure 3: C. Graphic representations of assays. DAPI positive cells increased by a factor of 10 minimum when LPS is combined with ATP. These results are indicative of control over the immune reactions pertaining to inflammasomes when compared with responses from both cell types with CRTL or LPS alone. ATP (Adenosine triphosphate) is clearly integral to the reaction; which is not improbable given the typical function of ATP as energy currency catalyzing a wide range of biochemical reactions. 4. ELISA analysis revealed a lower lower IL-1b levels in supernatants of ATM-/- cells stimulated with LPS+ATP. Such cells however elicited enhanced a higher TNF-a suggesting that impaired IL-1b response was not due to a block in priming. Immunoblot analysis also revealed diminished IL-1b secretion by ATM-/- cells. Such response was not due to impaired IL-1b synthesis but was associated with impaired secretion of mature IL-1b and active caspase 1. In fact, closer analysis revealed a higher basal IL-1b synthesis in the ATM-/- cells. This is consistent with a role of the genes damaged by AT mutations concerning negative regulation of cytokine gene transcription during infection. Fig4. Impaired IL-1b and inflammasome but not TNF-a activation in macrophages ATM-/- mice in response to PAMPs. TNF-a secretion (A) response was not impaired in ATM-/- BMDMs. Impaired IL-1b production by ATM-/- cells in response to LPS+ATP (B). Enhanced priming but impaired IL-1b and caspase 1 processing in ATM-/- BMDMs. These results have limitations for the function of interleukins pertaining to the disorder in question. It suggests the TNF is not implicated in the major symptoms of the disorder, but a deficiency of interleukins represents a significant symptom. This molecular data could be useful in the determination of diagnostic screening with more development. MATERIAL AND METHODS Mice C57BL/J wild type mice (WT), Ataxia telangiectasia mutated knockout mice (ATM-/-), Apoptosis-associated speck -like protein knockout mice (ASC-/-) and ATM ASC double knockout mice (ATM ASC DKO). Bacterial and Mammalian cell cultures Bone marrow derived macrophages (BMDM) from WT and knockout mice (ATM-/-, ASC-/-) were generated by differentiating bone marrows cells in IMDM media consisting of 10% heat inactivated fetal bovine serum (FBS), 10% L929 cell supernatant containing 1% penicillin+streptomycin. L.monocytogenes and S.typhimurium were grown aerobically at 37 0C in BHI media overnight and next day 1/3 overnight culture added in fresh BHI media and grow for 2 hrs to O.D 0.4 – 0.6. After washing in cold PBS, the culture was adjusted using PBS to O:D 0.4 which according to a predetermined growth curve corresponds to 0.03 x10^9 cfu/ml (L. monocytogenes) and 1.14x10^8 cfu/ml (S.typhimurium). Analysis of phosphorylation status of signaling molecules in responses bacterial infection or TLR stimulation. BMDM cells were plated in 6 well cell culture plates at 1.5x106 cells/well. The following day, cells were stimulated (or not) with LPS (500 ng/ml) and incubated at 37C 5% CO2. 5, 15, 30, 60, 120, 180 minutes before lysis with RIPA buffer (150mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 0.1% Triton X, and 50mM Tris pH 8.0) supplemented with complete EDTA free protease inhibitor and phosSTOP phosphatase inhibitor mixture (Roche Applied Science). Alternatively, cells were exposed to L.monocytogenes (MOI 50), centrifuged at 1000 rpm for 5 min then incubated for 5, 15, 30, 60, 120, 180 minutes before lysis. Such cell lysates were then analyzed by immunoblotting for IkBa, phospho-ERK1/ERK2, phospho-TBK1, phospho-TAK1, phospho-JNK1 and ?-actin. Antibodies Anti-P-IK?a (SC-371, santacruz), anti-IK?a (9246S, cell signaling), anti-Phosphop-38 (4631S, cell signaling), anti-phospho ERK1/2 (ab78238, Abcam), anti-phospho TBK1 (ab109272,Abcam ), anti-Phospho TAK1 (ab79583, Abcam), anti-Phospho JNK1 ( ab46821, Abcam), anti caspase 11 (17d9;Sigma), anti-caspase1-p20 (4B4, Genentech) and anti- IL-1? (ab9722, abcam) and for loading control, anti-?- actin (SC-81178, santacruz). Enzyme-Linked Immunosorbent assays allow labeling and identification of specified antigens for visual identification, used in this case to identify interleukins and determine their role in inflammasome formation. Analysis of Inflammasome activation To evaluate caspase 1 and IL-1b processing by immunoblotting, BMDM cells were plated 1.5x106 cells/well in 6 well cell culture plates in antibiotic free IMDM medium overnight. The following day such cells were infected with L. monocytogenes (MOI 20) or S.typhimurium (MOI 30) for 30 min before adding gentamycin (50?g/ml) to the kill extracellular bacteria then incubated for an additional 4 -18 hours. Alternatively, cells were primed by stimulating with LPS (500 ng/ml) for 4 hours followed by a short pulse with 5mM ATP for 30 min. Thereafter cell supernatantas were collected and concentrated 15 fold by speed vacuum concentration while cells were lysed in laemmli buffer. Both the cell lysates and supernatants were then dissolved in 2M SDS sample buffer boiled for 10 min then analyzed by SDS-PAGE immunoblotting for Caspase1, IL-1b. This data permits us to determine not only the pattern recognition receptors most important to wild type immune responses, it permits investigators data from which to build a scenario concerning the signaling pathway for the molecular cofactors involved, including interleukins and related molecules in the signaling cascades responsible for caspases recruitment pertaining to the formation of an inflammasomes complex. In describing the process of recruitment, it is possible to determine the effects of the ATM mutation at all stages, including early recruitment of pro-inflammatory mediators. The deficiency provides evidence of enhanced cytokine production and negative regulation of pattern recognition receptors essential in the beginning of inflammation. Even if genetic disorders such as this are not immediately responsive to curative efforts, the above results with respect to inflammasomes formation and pre-inflammation signaling enhance the understanding of the immune system and provide avenues for a wide range of future research interests concerning inflammasomes, the role of interleukins and signaling pathways essential to innate immunity. DISCUSSION Bacterial vectors were used as mechanisms in the results section as a method of provoking immune responses. It is known that the surface proteins associated with bacteria interact to varying degrees with the cells and receptors responsible for the recruitment of further immune responses. The figures described above utilizing listeria provide data concerning the function of ATM in the regulation of pattern recognition receptors. The data allows an evaluation of the activation of NF-kB and MAPK pathways. Deficiencies in ATM resulted in sustained phosphorylation of p38, MAPK, and TBK1 (data not shown). ERK1/2 activation of BMDM's was also enhanced. This data and supports a hypothesis that deficiencies in the ATM gene enhance pro-inflammatory reactions with implications for the entire immune system. By disabling cofactors through mutation, it is possible to develop a functional map of the underlying cascade leading to recruitment, and construction of inflammasomes, as the results demonstrate. Aberrations based on mutating cofactors is a classic method to determine the functionality of those cofactors. Among these results are interleukin secretion-findings based on ELISA. Initial production of interleukin beta is not specifically impeded in ATM cells, yet it is diminished in the supernatant. It is therefore probably that ATM mutants produce the same amount, but this cofactor cannot be recruited and distributed in the usual fashion as would occur in wild types. This hints at a recruitment/distribution mechanism affecting interleukins that is disabled in these mutants. The assays combined with the interleukin data allow us to portray the genes for ataxia telangiectasia as an important negative regulator of pattern recognition receptors preceding inflammation. And immediate reaction to their efficiency is an increase in cytokine production, thus leading to a cascade affecting multiple organs systems with debilitating implications in terms of inflammation, and autoimmunity. In terms of actual molecular pathways, a priming reaction is necessary involving via NF-kB and MAPK pathways; which trigger the transcription of Pro interleukin 1-beta which itself goes on to activate the ligand gated ion channel P2X7R which triggers the conformation of the inflammasome complex. Formation of all subunits and cleavage of precursor stages occurs through autocatalytic processing of caspase-1, which is able by itself to stimulate apoptosis (pyroptotic). This is indicated by the results shown in figure 4, with ASC knockout mutants displaying differences in electrophoretic migration when the supernatant was examined. Apoptosis speck-like protein can be inferred to have an involvement in caspase recruitment. Bacterial antigens can also influence this process through pore formation that directly triggers inflammasomes activation, and as such they were useful in this assay. In order to characterize diverse circumstances under which inflammasomes could be stimulated. In the case of ataxia telangiectasia mutants, interleukin 1-beta could still be secreted even in the absence of the normal energy currency ATP. The lack of a negative regulator is responsible for a cascade of inappropriate immune reactions forming one of the principal manifestations of the disorder, which was also demonstrated in figure 3, which provided evidence of impairments in interleukin and caspase processing in ATM -/- mutants. The condition itself does not appear susceptible to an obvious treatment modality based upon known facts and the results indicated here, however this research is important because in studying the deficiencies associated with ataxia telangiectasia, it is apparent based upon the results of figure 2 that certain inflammasomes are indeed subject to artificial stimulus, and this data could be used as the groundwork for future therapeutic modalities involving inflammation and autoimmunity. REFERENCES 1.) Rothenfusser, S. et al. The RNA helicase Lgp2 inhibits TLR-independent sensing of viral replication by retinoic acid-inducible gene-I. J. Immunol. 175, 5260–5268 (2005). 2.) Bergsbaken, T., Fink, S. L. & Cookson, B. T. 2009. Pyroptosis: host cell death and inflammation. Nat. Rev. Microbiol. 7, 99–109 (2009). 3.) Suthar, M.S., Ramos,H.J., Brassil, M.M., et al. 2012. The RIG-I-like Receptor LGP2 Controls CD8+ T Cell Survival and Fitness. Copyright © 2012 Elsevier Inc. All rights reserved. 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