A Tale of Two SNPS: Polymorphism Analysis of Toll-like Receptor (TLR) Adapter Proteins: A Dissertation

The innate immune system is the first line of defense against invading pathogens. Recognition of microbial ligands by the innate immune system relies on germ-line encoded, evolutionarily conserved receptors called pattern recognition receptors (PRRs). Toll-like receptors (TLRs) are one such family of PRRs and are involved in innate defenses to a variety of microbes. At the core of TLR signaling pathways are Toll interleukin-1 receptor (TIR) domain containing adapter proteins. Much of the specificity of TLR pathways arise from the differential use of these adapter proteins. The TLR signaling cascade that ensues upon ligand recognition is marked by finely orchestrated molecular interactions between the receptor and the TIR domain containing adapter proteins, as well as various downstream kinases and effector molecules. Conserving the structural integrity of the TLR components is thus essential for maintaining a robust host defense system. Sometimes, changes in a protein can be brought about by single nucleotide polymorphisms (SNPs). Studies carried out in this thesis focus on polymorphisms in MyD88 adapter-like (Mal) and myeloid differentiation protein 88 (MyD88), two TIR domain-containing adapter proteins, which incidentally are also highly polymorphic. Mal is a 235 amino acid protein that is involved in TLR2 and TLR4 signaling. The known polymorphisms in the coding region of Mal were screened with an aim to identify SNPs with altered signaling potential. A TIR domain polymorphism, D96N, was found to be completely defective in TLR2 and TLR4 signaling. Immortalized macrophage-like cell lines expressing D96N have impaired cytokine production as well as NF-κB activation. The reason for this loss-of-function phenotype is the inability of Mal D96N to bind the downstream adapter MyD88, an event necessary for signaling to occur. Genotyping studies reveal a very low frequency of this polymorphism in the population. Similar SNP analysis was carried out in myeloid differentiation protein 88 (MyD88). MyD88 is a key signaling adapter in TLR signaling; critical for all TLR pathways except TLR3. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knockout mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. S34Y mutant has a dramatically different localization pattern as compared to wild type MyD88. Unlike wild type MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. IRAK4, a downstream kinase, colocalizes with MyD88 in these aggregates or “Myddosomes”. S34Y MyD88, however, is unable to assemble into Myddosomes, thus demonstrating that proper cellular localization of MyD88 is a feature required for MyD88 function. This thesis thus describes two loss‐of‐function polymorphisms in TLR adapter proteins Mal and MyD88. It sheds light not only on the structural aspects of signaling by these two proteins, but also has implications for the development of novel pharmaceutical agents

MyD88 adapter-like (Mal)/TIRAP interaction with TRAF6 is critical for TLR2- and TLR4-mediated NF-kappaB proinflammatory responses

Toll/interleukin-1 (TIR)receptor-containing adapters are critical in orchestrating the different signal transduction pathways following Toll-like receptor (TLR) activation. MyD88 adapter-like (Mal), also termed TIRAP, is involved in bridging MyD88 to the receptor complex for TLR-2 and TLR4 signaling in response to bacterial infection. We have previously reported an interaction between Mal and tumor necrosis factor receptor-associated factor 6 (TRAF6) via a TRAF6-binding motif, the disruption of which inhibited TLR-mediated NF-kappaB-luciferase reporter activity. Given the recent report of intracellular TRAM localization promoting sequential signaling in TLR4 responses, we further characterized Mal interaction with TRAF6, the cellular localization, and the outcomes of disrupting this association on TLR inflammatory responses. We found that Mal and TRAF6 directly interact in response to TLR2 and TLR4 stimulation, although membrane localization is not necessary to facilitate interaction. Critically, reconstitution of murine Mal-deficient macrophages with MalE190A, containing a mutation within the TRAF6-binding motif, fails to reconstitute the proinflammatory response to TLR2 and TLR4 ligands compared with wild type Mal. Furthermore, Mal interaction with TRAF6 mediates Ser phosphorylation of the p65 subunit of NF-kappaB and thus controls transcriptional activation but not nuclear translocation of NF-kappaB. This study characterizes the novel role for Mal in facilitating the direct recruitment of TRAF6 to the plasma membrane, which is necessary for TLR2- and TLR4-induced transactivation of NF-kappaB and regulation of the subsequent pro-inflammatory response

TLR3‐mediated IFN‐β gene induction is negatively regulated by the TLR adaptor MyD88 adaptor‐like

There is limited insight into the mechanisms involved in the counterregulation of TLR. Given the important role of TLR3/TIR domain‐containing adaptor‐inducing IFN‐β (TRIF)‐dependent signalling in innate immunity, novel insights into its modulation is of significance in the context of many physiological and pathological processes. Herein, we sought to perform analysis to definitively assign a mechanistic role for MyD88 adaptor‐like (Mal), an activator of TLR2/4 signalling, in the negative regulation of TLR3/TRIF signalling. Biochemical and functional analysis demonstrates that Mal negatively regulates TLR3, but not TLR4, mediated IFN‐β production. Co‐immunoprecipitation experiments demonstrate that Mal associates with IRF7 (IRF, IFN regulatory factor), not IRF3, and Mal specifically blocks IRF7 activation. In doing so, Mal impedes TLR3 ligand‐induced IFN‐β induction. Interestingly, Mal does not affect the induction of IL‐6 and TNF‐α upon TLR3 ligand engagement. Together, these data show that the TLR adaptor Mal interacts with IRF7 and, in doing so, impairs IFN‐β induction through the positive regulatory domains I‐III enhancer element of the IFN‐β gene following poly(I:C) stimulation. Our findings offer a new mechanistic insight into TLR3/TRIF signalling through a hitherto unknown mechanism whereby Mal inhibits poly(I:C)‐induced IRF7 activation and concomitant IFN‐β production. Thus, Mal is essential in restricting TLR3 signalling thereby protecting the host from unwanted immunopathologies associated with excessive IFN‐β production

Correlation of FCERIB gene haplotypes with histamine release from basophils in atopic asthma

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TGFβ1 haplotypes and asthma in Indian populations

Background: Asthma is a complex disorder of the airways of the lungs. TGF-β1 plays a key role in airway remodeling and asthma by having both proinflammatory and anti-inflammatory activities, making TGFβ1 an important candidate gene to study. Objective: To investigate the association of TGFβ1 gene polymorphisms with asthma. Methods: A case-control study was designed for identifying polymorphisms and haplotypes associated with asthma and associated phenotypes. We have verified our results in 2 independent cohorts collected from northern (number of patients, 187; number of controls, 187) and western India (number of patients, 209; number of controls, 190). We measured the serum TGF-β1 levels of selected individuals and correlated them with genotypes and haplotypes. Results: A novel (CT)n(CA)m repeat polymorphism (BV209662) 24.9 kb upstream of TGFβ1 was identified. A significant association was seen at the level of alleles and genotypes with asthma in the 2 cohorts studied independently (P < .05). Interestingly, a novel 3-locus haplotype, 23_G_T, was found to be significantly associated with asthma (P = .00001 in cohorts A and B) as well as with higher serum TGF-β1 level (P = .01). On the other hand, a novel haplotype, 22_G_C, was negatively associated with asthma (P = .00001 for cohorts A and B) and with lower serum TGF-β1 level (P = .0019). Conclusion: This is the first study identifying novel risk and protective haplotypes-23_G_T and 22_G_C, respectively-in the TGFβ1 gene that are associated with asthma. We also demonstrate the functional significance of these haplotypes with serum TGF-β1 levels. These results would be valuable in elucidating the role of TGF-β1 in asthma pathogenesis

Combined action of nucleic acid-sensing Toll-like receptors and TLR11/TLR12 heterodimers imparts resistance to Toxoplasma gondii in mice

Triple-defective (3d) mice carrying a mutation in UNC93B1, a chaperone for the endosomal nucleic acid-sensing (NAS) Toll-like receptors TLR3, TLR7, and TLR9, are highly susceptible to Toxoplasma gondii infection. However, none of the single or even the triple NAS-TLR-deficient animals recapitulated the 3d susceptible phenotype to experimental toxoplasmosis. Investigating this further, we found that while parasite RNA and DNA activate innate immune responses via TLR7 and TLR9, TLR11 and TLR12 working as heterodimers are required for sensing and responding to Toxoplasma profilin. Consequently, the triple TLR7/TLR9/TLR11-deficient mice are highly susceptible to T. gondii infection, recapitulating the phenotype of 3d mice. Humans lack functional TLR11 and TLR12 genes. Consistently, human cells produce high levels of proinflammatory cytokines in response to parasite-derived RNA and DNA, but not to Toxoplasma profilin, supporting a more critical role for NAS-TLRs in human toxoplasmosis

LXR Agonism Upregulates the Macrophage ABCA1/Syntrophin Protein Complex That Can Bind ApoA‑I and Stabilized ABCA1 Protein, but Complex Loss Does Not Inhibit Lipid Efflux

Macrophage ABCA1 effluxes lipid and has anti-inflammatory activity. The syntrophins, which are cytoplasmic PDZ protein scaffolding factors, can bind ABCA1 and modulate its activity. However, many of the data assessing the function of the ABCA1–syntrophin interaction are based on overexpression in nonmacrophage cells. To assess endogenous complex function in macrophages, we derived immortalized macrophages from <i>Abca1</i>^<i>+/+</i> and <i>Abca1</i>^{<i>–/–</i>} mice and show their phenotype recapitulates primary macrophages. <i>Abca1</i>^<i>+/+</i> lines express the CD11B and F4/80 macrophage markers and markedly upregulate cholesterol efflux in response to LXR nuclear hormone agonists. In contrast, immortalized <i>Abca1</i>^{<i>–/–</i>} macrophages show no efflux to apoA-I. In response to LPS, <i>Abca1</i>^{<i>–/–</i>} macrophages display pro-inflammatory changes, including an increased level of expression of cell surface CD14, and 11–26-fold higher levels of IL-6 and IL-12 mRNA. Given recapitulation of phenotype, we show with these lines that the ABCA1–syntrophin protein complex is upregulated by LXR agonists and can bind apoA-I. Moreover, in immortalized macrophages, combined α1/β2-syntrophin loss modulated ABCA1 cell surface levels and induced pro-inflammatory gene expression. However, loss of all three syntrophin isoforms known to bind ABCA1 did not impair lipid efflux in immortalized or primary macrophages. Thus, the ABCA1–syntrophin protein complex is not essential for ABCA1 macrophage lipid efflux but does directly interact with apoA-I and can modulate the pool of cell surface ABCA1 stabilized by apoA-I

LXR Agonism Upregulates the Macrophage ABCA1/Syntrophin Protein Complex That Can Bind ApoA‑I and Stabilized ABCA1 Protein, but Complex Loss Does Not Inhibit Lipid Efflux

Macrophage ABCA1 effluxes lipid and has anti-inflammatory activity. The syntrophins, which are cytoplasmic PDZ protein scaffolding factors, can bind ABCA1 and modulate its activity. However, many of the data assessing the function of the ABCA1–syntrophin interaction are based on overexpression in nonmacrophage cells. To assess endogenous complex function in macrophages, we derived immortalized macrophages from <i>Abca1</i>^<i>+/+</i> and <i>Abca1</i>^{<i>–/–</i>} mice and show their phenotype recapitulates primary macrophages. <i>Abca1</i>^<i>+/+</i> lines express the CD11B and F4/80 macrophage markers and markedly upregulate cholesterol efflux in response to LXR nuclear hormone agonists. In contrast, immortalized <i>Abca1</i>^{<i>–/–</i>} macrophages show no efflux to apoA-I. In response to LPS, <i>Abca1</i>^{<i>–/–</i>} macrophages display pro-inflammatory changes, including an increased level of expression of cell surface CD14, and 11–26-fold higher levels of IL-6 and IL-12 mRNA. Given recapitulation of phenotype, we show with these lines that the ABCA1–syntrophin protein complex is upregulated by LXR agonists and can bind apoA-I. Moreover, in immortalized macrophages, combined α1/β2-syntrophin loss modulated ABCA1 cell surface levels and induced pro-inflammatory gene expression. However, loss of all three syntrophin isoforms known to bind ABCA1 did not impair lipid efflux in immortalized or primary macrophages. Thus, the ABCA1–syntrophin protein complex is not essential for ABCA1 macrophage lipid efflux but does directly interact with apoA-I and can modulate the pool of cell surface ABCA1 stabilized by apoA-I