Expression Regulation and Signaling

Hui Xiao, Xiaoxia Li, and Derek W. Abbott contents

3.1 Introduction 39

3.2 Analysis of TLR Expression 42

3.2.1 Detection of TLR Expression at the Message Level 42

3.2.2 Detection of TLR Expression at the Protein Level 44

3.3 Analysis of Signaling Complexes 45

3.4 Analysis of IRAKI Modification and Degradation 48

3.5 Analysis of IRAK and TAK1 Activation 49

3.6 Analysis of IKK Signalosome Activation 52

3.6.1 Detection of NEMO Ubiquitination 52

3.6.2 Detection of IKK Activation 53

References 55

3.1 introduction

The innate immune system recognizes and responds to pathogenic organisms. In doing so, this system is responsible for initiating a cytokine response designed to tailor the adaptive immune system to eradicate the offending organism. Because this initial cytokine release must be tightly regulated, signal transduction pathways leading to this cytokine release are highly coordinated. This coordination begins at the cell surface with the initial recognition of pathogens by Toll-like receptors (TLRs). TLRs recognize components of bacteria, fungi, or viruses (collectively called pathogen-associated molecular patterns, or PAMPs) and play a major role in host defense against infection.1 The majority of the TLR family members are abundantly expressed in monocytes, macrophages, and dendritic cells.2 These receptors activate a highly conserved signaling network and ultimately lead to the activation of a variety of transcription factors, including NF-kB, ATF2, c-JUN, and IRF3/7. These transcription factors then act in synergy to induce the expression of hundreds of cellular genes.3

This gene induction is cell-type specific and determines the adaptive immune response. For example, monocytes and macrophages produce proinflammatory cytokines such as IL-1, TNFa, and interferons to initiate an acute inflammatory response,4

while TLR engagement in dendritic cells induces the expression of co-stimulatory molecules that facilitate dendritic cell maturation and antigen presentation ability.5 TLRs are also expressed in a subset of B and T cells, and recent work suggests that lymphocytic TLR signaling is essential to regulate lymphocyte activation and proliferation, antibody production, and antigen presentation.6,7 Lastly, a subset of TLRs (i.e., TLR2, TLR4, and TLR9) is expressed by endothelial cells and by certain epithelial cells, where they appear to play a role in maintaining mucosal homeosta-sis. Thus, TLR signaling in endothelial cells or gastrointestinal epithelial cells may contribute to the pathogenesis of atherosclerosis and colitis, respectively.89

Some TLRs within the TLR family also show a cell-type-specific expression pattern. TLR3 is mainly expressed in dendritic cells, and can be induced in macrophages upon LPS stimulation.10 TLR5 is highly expressed on the basolateral surface of the GI epithelium,11 while TLR11 is expressed in the kidneys and liver.12 In addition, TLR7 and TLR9, but not the other TLRs, are present in plasmocytoid dendritic cells (pDCs), and cause robust secretion of type I interferons aimed at inhibiting viral infection.1314 This cell-type-specific expression of TLRs argues for the existence of strict control of TLR expression. Very little is known about the mechanisms underlying this tissue-specific expression; however, it is tempting to speculate that the cytokines that regulate immune development and differentiation may play a role in this process. In this regard, proinflammatory cytokines such as TNFa and IFNy, as well as bacterial or viral infections, have been shown to induce the expression of TLR2 and TLR3, but not the other TLRs.8,15,16 These studies highlight the likely role of transcriptional factors NF-kB, STATs, and IRF3/7 in the regulation of TLR expression.

An additional level of regulation of the TLRs lies in their intracellular location. While TLRs 2, 4, and 6 are plasma membrane-localized receptors, TLRs 3, 7, and 9 are located intracellularly within endosomal compartments,1718 This cellular localization is required for TLRs 3 and 9 to recognize a viral infection, while at the same time not allowing the inadvertent recognition of host nucleic acids. This allows a degree of specificity to the differentiation of host versus pathogen.19 Lastly, TLR9 is localized in the endoplasmic reticulum in resting cells, but translocates to the endosome when CpG or viral DNA is presented.18,20 In fact, pDCs produce much more type I interferons than conventional DCs (cDCs) because CpG is translocated into the endosome in pDCs, while it is translocated mainly into the lysosome in cDCs.21 This type of regulation of ligand and receptor translocation is crucial for appropriate TLR signaling and again illustrates an additional level of regulation by which host and pathogen can be distinguished.

Despite the complexity of both the expression and the localization of the TLRs, a basic mechanism by which the TLRs transmit signaling information has emerged. Upon ligand engagement, the TLRs oligomerize. With the exception of TLR3, these oligomerized TLRs recruit MyD88 to induce a highly conserved signaling pathway leading to NF-kB activation.22 This signaling pathway involves dynamic complex formation and a cascade of kinase activation featuring four key mechanisms by which TLR signaling is achieved22-24 (see Figure 3.1). Thus, immediately after the engagement of TLR, complex I (consisting of TLR-MyD88-IRAK4-IRAK1-TRAF6) is formed. In this complex, the death domain-containing serine/threonine kinases

figure 3.1 TLR-induced NF-kB Activation

Note: Upon TLR ligand stimulation, adapter molecules, such as MyD88 and/or Trif, associate with the engaged receptor and, in turn, recruit IRAK1, IRAK4, and TRAF6, resulting in the formation of the receptor complex (complex I). Upon the formation of complex I, IRAK4 is activated, leading to the hyperphosphorylation of IRAK1, which creates an interface for the interaction of Pellino proteins with the IRAK1-IRAK4-TRAF6 complex (intermediate complex). IRAK1 bound to TRAF6 then leaves the intermediate complex and binds to TAK1-TAB1-TAB2/3 to form complex II on the plasma or endosomal membrane. In complex II, IRAK1 is ubiquitinated and degraded, resulting in the translocation of the TRAF6-TAK1-TAB1-TAB2/3 complex (complex III) from the membrane to the cytosol, where TAK1 is activated. Subsequently, TAK1 activates IKKP, which, in turn, activates NF-kB through the degradation of IKBa. In addition, intermediate complex can bind to MEKK3 to form complex IV in the cytosol. Complex IV can also mediate NF-kB activation through a process not involving IKBa degradation.

figure 3.1 TLR-induced NF-kB Activation

Note: Upon TLR ligand stimulation, adapter molecules, such as MyD88 and/or Trif, associate with the engaged receptor and, in turn, recruit IRAK1, IRAK4, and TRAF6, resulting in the formation of the receptor complex (complex I). Upon the formation of complex I, IRAK4 is activated, leading to the hyperphosphorylation of IRAK1, which creates an interface for the interaction of Pellino proteins with the IRAK1-IRAK4-TRAF6 complex (intermediate complex). IRAK1 bound to TRAF6 then leaves the intermediate complex and binds to TAK1-TAB1-TAB2/3 to form complex II on the plasma or endosomal membrane. In complex II, IRAK1 is ubiquitinated and degraded, resulting in the translocation of the TRAF6-TAK1-TAB1-TAB2/3 complex (complex III) from the membrane to the cytosol, where TAK1 is activated. Subsequently, TAK1 activates IKKP, which, in turn, activates NF-kB through the degradation of IKBa. In addition, intermediate complex can bind to MEKK3 to form complex IV in the cytosol. Complex IV can also mediate NF-kB activation through a process not involving IKBa degradation.

(i.e., IRAK4 and IRAK1) are activated. This activation leads to the phosphorylation of IRAK1, and phosphorylated IRAK1 bound to TRAF6 leaves complex I and binds to TAK1/TAB2/3 to form complex II on the plasma or endosomal membrane. In complex II, autoubiquitinated TRAF6 promotes the phosphorylation of TAK1/ TAB2/TAB3. At this point, hyperphosphorylated IRAK1 is polyubiquitinated, and subsequently degraded in a proteasome-dependent manner. This process facilitates the movement of the TRAF6-TAK1/TAB2/3 complex into the cytosol, where it binds to IKK to form complex III. In complex III, TAK1 phosphorylates the activation loop of IKKp. Activated IKKp then phosphorylates IKBa, ultimately targeting it for polyubiquitination and proteosomal degradation. Degradation of IKBa allows the NF-kB transcription factors to enter the nucleus and activate target genes Within this canonical activation pathway, additional, recently recognized proteins such as SIGIRR, Tollip, or Pellino proteins can also bind to various signaling complexes to either positively or negatively regulate NF-kB activation.2526 Furthermore, phosphorylation, ubiquitination, and sumoylation of the IKK scaffolding protein, NEMO, play a critical role in regulating IKKp activity.27 Lastly, in addition to the TAK1-dependent pathway described above, MEKK3 mediates NF-kB activation in IL-1 and TLR7 signaling.28,29 However, the detailed mechanism by which this TAK1-independent pathway affects TLR signaling is only in its infancy and is in need of further investigation.

Given the precision with which TLRs must recognize their ligands and tailor the adaptive immune system response, it is not unexpected that their expression and activity are regulated at multiple levels. This chapter aims to serve as a synopsis by which this regulation can be studied. A set of methods designed to monitor the strength and duration of the TLR response is presented, and common artifacts associated with these methods as well as ways to circumvent them are discussed.

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