Role of Mal/Tirap in Tlr2- and Tlr4-, but Not Tlr5-Induced Corneal Inflammation

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ROLE OF MAL/TIRAP IN TLR2- AND TLR4-, BUT NOT TLR5-INDUCED CORNEAL INFLAMMATION

SUSAN RENEE WILLIAMS

Submitted in partial fulfillment of the requirements

For the degree Master of Science

Thesis Adviser: Dr. Eric Pearlman

Department of Pathology

CASE WESTERN RESERVE UNIVERSITY

January 2010 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

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TABLE OF CONTENTS

Table of Contents 1 List of Figures 2 List of Abbreviations 3 Abstract 5

Introduction 6 Overview 6 Keratitis Disrupts Corneal Transparency and Impairs Vision 6 The Normal Cornea 6 Keratitis 12 Etiologic Agents of Bacterial Keratitis 14 Gram-Positive Bacterial Keratitis 14 Gram-Negative Bacterial Keratitis 15 Toll-Like Receptors 16 TLR2 18 TLR4 18 TLR5 19 TIR-Adaptor mediated TLR signaling 21 MyD88-dependent signaling 21 Mal/TIRAP 22 TICAM-1/TRIF-dependent signaling 22 TICAM-2/TRAM 23 SARM 23

Materials and Methods 26 Results 29 Discussion and Future Directions 40 Summary of Results and Key Findings 40 TLR-Induced Inflammatory Responses 40 Role of Epithelial Cells and BMDCs in Inflammatory Responses 41 Role of NLRs and IL-1 in Inflammation 42 Role of TIR Adaptor Activity in TLR/IL-1R1-Mediated Keratitis 43

References 45

LIST OF FIGURES

Figure 1 Anatomy of the visual axis 10

Figure 2 Structure of the normal cornea 11

Figure 3 Differential transcription factor activation by TLRs 17

Figure 4 TLR4-induced signaling 17

Figure 5 TIR-domain-containing adaptor domains and motifs 25

Figure 6 TLR2-induced stromal haze is abrogated in TIRAP-deficient 29 mice

Figure 7 TLR2-induced neutrophil infiltration is abrogated in 31 TIRAP-deficient mice

Figure 8 TLR5-induced stromal haze is unaffected by TIRAP 33 deficiency

Figure 9 TLR5-induced neutrophil infiltration is unaffected by TIRAP 35 deficiency

Figure 10 TLR4-induced stromal haze is abrogated in TIRAP-deficient 36 mice

Figure 11 TLR4-induced neutrophil infiltration is abrogated in 38 TIRAP-deficient mice

LIST OF ABBREVIATIONS

AC anterior chamber APC antigen-presenting cell BMDC bone marrow-derived cell Btk Bruton's tyrosine kinase CARD Caspase recruitment domain CD cluster of differentiation DC dendritic cell DD death domain ECM extracellular matrix GPCR G protein-coupled receptor HCEC human corneal epithelial cell ID intermediary domain IFN interferon Ig Immunoglobulin IKK inhibitor of NF-κB kinase IL interleukin IL-1R IL-1 receptor IRAK IL-1R-associated kinase IRF IFN regulatory factor JNK Jun N-terminal kinase LBP LPS-binding protein LC Langerhans cell LPS lipopolysaccharide LRR leucine-rich repeat Mal MyD88 adaptor-like MD-2 myeloid differentiation factor-2 MHCII major histocompatibility complex class II MAPK mitogen-activated protein (MAP) kinase MKK MAPK kinase MyD88 myeloid differentiation primary-response gene 88 NAK NF-κB-activator-binding kinase NF-κB nuclear factor-κB NLRC4 NLR family CARD domain-containing protein P2RX7 P2X purinoreceptor 7 P3C Pam3CysK4, triacylated synthetic lipoprotein

PGE2 prostaglandin E2 PKCε protein kinase Cε RIP1 receptor-interacting protein 1 SARM sterile α- and armadillo-motif-containing protein SIGRR single immunoglobulin IL-1R-related molecule SLRP small leucine-rich proteoglycan Sp. species SNP single-nucleotide polymorphism TAB TAK1-binding protein

TAK1 transforming-growth-factor-β-activated kinase 1 TBK1 TRAF-family-member-associated kinase 1 TGF transforming growth factor TICAM-1/2 TIR domain-containing adapter molecule-1/2 TIR Toll/interleukin-1 receptor TIRAP TIR domain-containing adaptor protein TLR Toll-like receptor TNF tumor necrosis factor TNFR TNF receptor TRAM TRIF-related adaptor molecule TRAF TNFR-associated factor TRIF TIR-domain-containing adaptor protein inducing interferon-β

Role of Mal/TIRAP in TLR2- and TLR4-, but not TLR5-Induced Corneal Inflammation

Abstract

SUSAN RENEE WILLIAMS

Neutrophil recruitment to the cornea and corneal haze produced by cellular

infiltration and tissue damage are characteristic features of infectious bacterial keratitis or sterile keratitis due to bacterial products. In this context, these responses are mediated by

TLRs, particularly TLR2, TLR4 and TLR5. Understanding the regulation of the activity of these receptors in the cornea may be important for dissecting host responses to

pathogens and designing interventions for therapeutic targets. The TIRAP adaptor protein was examined as a regulator of these responses in mouse models of Pam3CysK4/TLR2-,

LPS/TLR4- and flagellin/TLR5- induced sterile keratitis; we demonstrated that TIRAP participates in TLR2- and TLR4- induced neutrophil recruitment and stromal haze but did not influence TLR5-induced responses. Moreover, TIRAP-independent TLR4 responses were not apparent. Based on these findings, we hypothesize that TIRAP mediates TLR2 and TLR4 induced keratitis responses upon bacterial product recognition.

INTRODUCTION

Overview

Bacterial keratitis is a major cause of visual impairment in the developed and

developing worlds. Corneal inflammation is initiated upon recognition of conserved

bacteria components by host TLRs, which induce inflammatory cascades resulting in

edema and neutrophil infiltration of the cornea. Neutrophils are a major component of a rapid first-line host response that reduces bacterial proliferation and dissemination, but they may also cause profound damage to well-organized and delicate corneal tissues.

TLR2, TLR4 and TLR5 are important for the recognition of common bacterial pathogens and have been shown to initiate inflammatory responses in the cornea and other tissues.

Hence, corneal disease induced by a pathogen or its constituents may be influenced by the regulation of the activity of a particular TLR. TLRs recruit TIR-domain containing adaptor proteins that initiate the signature TLR signaling cascades leading to gene transcription and other downstream effects. However, the contribution of the Mal/TIRAP adaptor protein to the responses of corneal TLRs that recognize bacteria has not been examined.

Keratitis Disrupts Corneal Transparency and Impairs Vision

The Normal Cornea

The cornea plays a crucial role in the manipulation of light for clear vision. The

cornea refracts light along with the lens; in humans it accounts for two-thirds of the eye’s refractive power with a fixed focus. Vision begins with the sequential passage of light through the cornea, aqueous humor, pupil, lens and vitreous humor to fall finally onto the retina. In retinal tissue this physical stimulus interacts with photoreceptor pigments to

become the neural impulses that are interpreted as the sense of sight. Inappropriate

curvature of the cornea causes improper light focusing, leading to blurred vision in the form of hyperopia, myopia or astigmatism. Transparency of the cornea is also crucial for clear vision by minimizing light scatter and is produced and maintained by the cornea’s unique structure.

Maintenance of corneal transparency begins at the tear film-epithelial interface, the most significant component of refractive power. Lipid, aqueous and mucinous tear film components moisten and lubricate the lids and epithelial surface and wash away debris with blinking. Lacrimal glands secrete proteins and enzymes into the aqueous phase including lysozyme, lactoferrin, IgA, and beta-lysin that modulate ocular surface flora via bacterial lysis and opsonization (2). Mucins and microplicae of the superficial epithelium increase free surface area and tear film retention and stability. Disruption of the smoothness of this interface by epithelial irregularity, dryness or edema reduces visual acuity. Five to seven layers of nonkeratinized, stratified, squamous and regularly arranged cells constitute the underlying corneal epithelium. Epithelial tight junctions form an impermeable barrier to fluid transport into or out of the cornea and microbial invasion, maintaining its hydration and preventing infection. The epithelium overlays

Bowman’s membrane of collagen and proteoglycans which merges with the stroma posteriorly (3).

The stroma is principally composed of type I and type V collagen fibrils and

SLRPs of four types: decorin, lumican, keratocan, and mimecan. The latter three SLRPs are cornea-selective keratan sulfate proteoglycans which form rings arranged around fibrils and maintain uniform interfibrillar spacing. Decorin is a dermatan sulfate

proteoglycan that forms chains connecting alternate fibrils, regulating interfibrillar spacing as well as organization of parallel fibrils into lamellar sheets (4, 5).

Proteoglycans also bind cations and water to promote stromal hydration. Uniform interfibrillar spacing that is less than half the wavelength of light is maintained by SLRPs and is necessary for transparency. Stromal keratinocytes are found parallel to collagen bundles in cell sheets that alternate with collagen lamellae and maintain the ECM (5, 6).

Orthogonal paired lamellae - cell sheets form a continuous lattice within the circumference of the limbus in an arrangement that eliminates light scatter via destructive interference and limits vascular requirements via facilitated diffusion between lamellae

(5, 7). In contrast, randomly arranged collagen and a rich vascular supply produces opacity at the limbus and beyond (5, 8). Beneath the stroma is Descemet's layer, an endothelium-secreted basement membrane of primarily type IV collagen. The endothelium forms a relatively fluid-permeable monolayer of cells linked by gap junctions at the most posterior aspect of the cornea and actively transports bicarbonate to the aqueous humor. This dehydrating mechanism counteracts proteoglycan-mediated hydration to regulate corneal hydration, with compromise leading to stromal edema and loss of transparency (3).

Transparency may also be maintained by a unique immunologic function. The normal cornea is avascular and lacks lymphatics, in contrast to the limbus and conjunctiva which are well supplied with blood vessels and lymphatics. Immature APCs may be found throughout the cornea, while they recognize antigens relatively poorly.

Additional mechanisms that maintain these APCs in an immature state may be present

but are not well known, although PGE2, TGF-ß and IL-10 may be contributors. Corneal

endothelium may produce endogenous PGE2 and TGF-ß may be supplied by the cornea and aqueous humor (9). Similarly, the uveal tract contains macrophages and DCs that weakly express costimulation markers and do not migrate to draining lymph nodes (10).

Immature APCs have a high capacity for phagocytosis and antigen processing but a low

costimulation capability which are reversed upon maturation. Four populations have been

defined in the cornea. Epithelial LCs of a CD45(+) CD11c(+) CD3(-) CD11b(-) CD40(-)

CD80(-) CD86(-) phenotype have been found with a low density in the central cornea and

a higher density peripherally near the limbus. These also tend to be MHCII(-) centrally and MHCII(+) peripherally. CD8α(-) CD11b(+) CD11c(+) DCs are found in the anterior stroma, which are distinct from the CD45(+) CD11b(+) CD11c(-) cells of the posterior stroma. Undifferentiated CD14(+) monocytic precursor cells are also found throughout the stroma that lack typical monocytic or dendritic cell markers (11-13). Recent studies found CD45(+) CD11c(+) MHC-II(–) DC-LAMP(–) Langerin(–) CD1a(–) DC-SIGN(–) cells in the epithelium, an additional population of cells that may be recently arrived LC progenitors (14). MHCII+ LCs express membrane nanotubule networks thought to function in intercellular communication and traffic to the normal corneal epithelium by a

CX3CR1-mediated process (15-17), while CCR2 mediates bone marrow-derived cell recruitment to the stroma (18).

Figure 1. Schema of the visual axis. (Image taken from the NEI website http://www.nei.nih.gov/health/cornealdisease/) Light from a visual stimulus travels an imaginary path known as the visual axis. The visual axis traverses the tear film, cornea, anterior chamber, pupil, lens and vitreous, and finally rests on the retina. Structures in or near the axis influence visual acuity.

A B

Figure 2. Structure of the normal cornea. A. (Image taken from http://dels.nas.edu/ilar_n/ilarjournal/40_2/40_2BacterialCorneal.shtml) B. (Image taken from http://education.vetmed.vt.edu/Curriculum/VM8054/EYE/CRNSCLRA.HTM) The cornea is a five-layered structure composed of epithelium anteriorly, Bowman’s membrane, stroma, Descemet’s membrane and endothelium posteriorly. The normal cornea has a stroma composed of dense, well organized collagen and proteoglycans with a minimal cellular content.

Keratitis The normal flora of the ocular surface includes nonpathogenic aerobic and anaerobic organisms such as S. epidermidis and P. acnes which minimize the colonization of pathogens. Certain bacterial groups are more commonly found in the cornea such as Staphylococcus sp., Streptococcus sp., Pseudomonas sp., and

Enterobacteriaceae sp. (especially Serratia), whereas others such as Moraxella sp. and

Proteus sp. are uncommon causes of keratitis corneal infections. However, almost any bacteria can infect the cornea if the epithelial barrier has been compromised. Many bacterial keratitis risk factors involve epithelial barrier compromise or immune compromise; they include aqueous tear deficiencies, dacrocystitis, structurally abnormal eyelids, preexisting corneal disease, alcoholism, diabetes, and topical corticosteroid use, among others (3). The most common risk factor in developed countries is contact lens- wear, which may compromise the epithelium via anoxia and trauma or may be associated with contaminated lenses and solutions. Developing countries have fewer contact-lens wearers, but commonly have keratitis cases due to traumatic inoculation of the stroma

(19-23). Typical symptoms include blurred vision, eye pain, photophobia, tearing and injection. The disease process may include ulceration, stromal abscess formation, hypopyon and even corneal perforation and secondary endophthalmitis; corneal leukoma and irregular astigmatism may be sequelae (3). The disease course chiefly depends on the virulence of the infectious agent. Staphylococcus-, Moraxella- and Klebsiella-induced keratitis advances more slowly and with less inflammation than that induced by

Pseudomonas sp. and Streptococcus sp. Contact lens wear may also contribute to less severe “sterile” keratitis due to bacterial products in the absence of active infection, with

symptoms observed as contact lens associated red eye (CLARE) (24), contact lens peripheral ulcers (CLPU) (24-27), and contact lens associated corneal infiltrates (CLACI)

(28).

Introduction of bacteria or bacterial products to the cornea may induce the production of antimicrobial peptides and proinflammatory cytokines and chemokines, leading to recruitment of primarily neutrophilic infiltrates to the site of infection (29, 30).

Corneal epithelial cells, keratocytes and BMDCs have all been demonstrated to contribute to the induction of keratitis responses in vivo and in vitro. ELR+ CXC chemokines mediate neutrophil chemotaxis and activation by binding GPCRs CXCR1 and CXCR2 in humans or CXCR2 in mice (31-39). Neutrophils release numerous antimicrobial enzymes and reactive oxygen species that eliminate pathogens but also damage host tissues. Epithelial and stromal cells at the injured site undergo necrosis and also release substances that lead to stromal destruction, edema, neovascularization and opacity along with bacterial toxins and proteases (40, 41). Keratocyte gene expression shifts from transparency-related crystallins to macrophage-like genes and proinflammatory chemokines (42). Resolution involves epithelial regeneration from basal epithelial cells or limbal stem cells and stromal repair via keratocyte transformation into fibroblasts or myofibroblasts and fibrosis with deposition of type VI and VIII collagens (43). Proteoglycan composition becomes altered with increased hydration, leading to edema. Newly synthesized collagen fibrils are disorganized with variable diameters, increasing light scatter; disruption of collagen organization by scarring leads to corneal opacities (4, 44)). Resident LCs and DCs may be primed by the proinflammatory milieu for more rapid and severe responses to corneal insults via

upregulated MHCII, costimulatory markers and proinflammatory cytokine production.

APCs may also migrate into the central cornea from the limbus and conjunctiva to promote detrimental keratitis responses (45).

Etiologic Agents of Bacterial Keratitis

Gram-Positive Bacterial Keratitis

The majority of keratitis cases involve Gram-positive organisms, particularly

Staphylococcus, which is commonly found on the skin and eyelids. S. epidermidis and S.

aureus may be almost equally common causes of ulceration, but S. epidermidis is associated with more indolent inflammation and ulceration whereas S. aureus infection is

more aggressive and induces more severe inflammation and hypopyon. Both species

typically produce a well-localized, deeply penetrating inflammatory focus with abscess

and perforation (3). Models of S. aureus-induced keratitis demonstrate chemokine production, neutrophil recruitment and edema resulting from infection (46-52). In rabbit models, alpha (dermonecrotic factor), beta (beta-hemolysin or sphingomyelinase C) and

gamma toxins were shown to mediate corneal pathology with live S. aureus infection or

CLPU, but these toxins were not required for pathology (46, 48, 53). Staphylococcal

protein A, which binds Fc-Ig and inhibits opsonization and phagocytosis, did not play a

role in a rabbit model in vivo (46). It mediated proinflammatory responses to live S.

aureus by HCECs in vitro, but failed to induce antimicrobial peptide expression in

contrast to S. aureus-derived peptidoglycan (54-56). Other toxins that may be significant

are delta toxin, leukocidin, exfoliative toxin and coagulase.

Streptococcus sp. also cause acute and severe corneal infections. S. pyogenes is

associated with severe infection, producing cytolytic toxins streptolysin O and

streptolysin S that damage cholesterol-containing cell membranes, lyse cells and inhibit

phagocytosis. S. pneumoniae causes indolent ulcerations and superinfections of

compromised corneas and produces cytolysin, which may activate host collagenases to

produce complete opacification of rabbit corneas (3).

Gram-Negative Bacterial Keratitis

Gram-negative infections proceed with rapid ulcerations and perforation in short

periods; indolent courses are less common. P. aeruginosa is the most common cause of

Gram-negative bacterial keratitis and contact-lens associated keratitis and produces the most virulent disease course, although other Pseudomonas sp. are also found in corneal

ulcers (21). Diffuse epithelial graying due to inflammatory edema may be found distal to

the main focus of inflammation; ring ulcers may progress to corneal melting with

mucopurulent discharge, descemetocele and perforation (3). P. aeruginosa produces various proteases and toxins that contribute to corneal virulence including alkaline phosphatase and elastases (LasA and LasB) although these were found to be unnecessary for initiating or maintaining infection (57). This organism activates NF-κB, JNK and p38

and induces proinflammatory cytokine production in primary and cell line HCECs (58-

60). Cytokines such as IL-10, KC, IL-1β, IL-6 and MIP-2 have been shown to be important mediators of the response to P. aeruginosa infection as well as the chemokine receptor CXCR2 (37, 61-64). ST2, a member of the TIR receptor family that binds IL-33, has been shown to decrease corneal disease associated with P. aeruginosa infection (65).

SIGIRR was also shown to decrease harmful proinflammatory responses (66). Contact

lenses may increase susceptibility to P. aeruginosa-induced corneal disease by blocking

of hBD-2 antimicrobial peptide production by HCECs (22).

Other gram-negative rods such as Klebsiella, E. coli, and Proteus usually are less pathogenic, inducing relatively mild inflammation and localized indolent ulcerations in compromised corneas. Moraxella sp. in particular infect debilitated, diabetic, alcoholic or chronically malnourished patients after trauma. S. marcescens is also a significant ocular pathogen (3).

Toll-Like Receptors

TLRs recognize microbial products and initiate inflammatory responses. They are type I integral membrane proteins with extracellular ligand-binding LRR motifs, a single transmembrane region and a cytoplasmic TIR domain for signaling. To date, thirteen mouse TLRs and eleven human TLRs have been identified which primarily respond to lipids, (TLR2/1, TLR2/6, and TLR4) proteins (TLR5, TLR11) or nucleic acids (TLR3,

TLR7, TLR8, TLR9). Protein and lipid-binding TLRs are primarily located on the cell surface, whereas nucleic acid-binding TLRs are found intracellularly in acidic endosomes

(67-69). Corneal TLR activation has been shown to induce keratitis responses in vivo and in vitro (52, 56, 70-76). TLR1, TLR2, TLR3 TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 mRNA expression in HCECs has been reported, and so has functional TLR2,

TLR3, TLR4 and TLR5 protein expression (33, 55, 70, 74, 77-80).

Figure 3. Differential transcription factor activation by TLRs. (Image taken from (1)) Various TLRs use a common set of TIR-containing adaptor molecules to mediate distinct signaling and gene transcription programs. The mechanisms underlying diverse responses by common proteins are not fully characterized. The only currently known adaptor that does not associate with specific TLRs to initiate signaling is SARM.

Figure 4. TLR4-induced signaling. (Image taken from (1)) TLR4 is unique among TLR by utilizing four adaptor molecules to signal. It is also unique in using an adaptor (TRAM) that is not known to be utilized by any other TLR. SARM also interacts with the TLR4/TRAM/TRIF signaling pathway. MyD88- and TRIF dependent pathways are redundant for Nf-κB activation, with divergent signaling mechanisms and kinetics.

TLR2

TLR2 recognizes various Gram-positive and Gram-negative bacterial cell wall lipopeptides, with additional specificity conferred by cooperation with TLR1 or TLR6.

The TLR1/TLR2 heterodimer binds bacterial triacylated lipoproteins such as the synthetic compound Pam3Cys with the help of nonsignaling accessory protein CD14

(81). TLR2/TLR6 binds diacylated lipoproteins from mycoplasma, such as mycoplasma lipoprotein-2 (MALP-2) and the bacterial glycolipid lipotechoic acid (LTA) (82-84).

TLR2 was reported to be expressed in a HCEC cell line and primary cells in a form that

was retained within the cytoplasm and unresponsive to peptidoglycan stimulation (85).

However, S. aureus-derived lipoproteins prepared by Triton X-114 extraction activated

HCECs and reporter cells in a TLR2-dependent fashion in another report (86). Similarly,

TLR2 was found to be surface-localized in primary and cell-line HCECs and was activated by S. aureus, its exoproducts or Pam3Cys (54). Killed S. aureus and Pam3Cys were also shown to induce TLR2- and MyD88-dependent, but TLR4- and TLR9- independent keratitis responses in murine corneas in vivo (52, 72). Keratitis responses to killed S. aureus and Pam3Cys via TLR2 have also been shown to be medicated by JNK

(87). Group B streptococcus has been shown to activate TLR2 and produce factors that upregulate PGE2 production by interacting with the TLR2/6 heterodimer (88, 89).

TLR4

TLR4 binds outer cell membrane lipopolysaccharide (LPS) of Gram-negative

bacteria. It cooperates with several accessory molecules including LBP, CD14 and MD-2

to enable LPS binding and increase LPS sensitivity. LBP and CD14 have been found in

tear fluid; MD-2 may be a serum component (90, 91). LBP extracts single endotoxin

molecules from aggregates or bacterial membranes to form monomeric endotoxin: CD14 18

complexes. CD14 transfers LPS to MD-2 at an appropriate binding conformation and

LPS:MD-2 activates TLR4 (92-94). However, MD-2 is essential for TLR4 signaling whereas LBP and CD14 are nonessential (95-100). Eritoran is a competitive LPS antagonist derived from R. sphaeroides that was found to inhibit TLR4 activation by occupying a pocket of the MD-2 protein that binds LPS (101-103). It was also shown to reduce LPS-induced keratitis responses in vivo (104).

TLR4 and CD14 were first shown to be expressed on the surface of HCECs and mediate IL-8 and IL-6 production in vitro (79) whereas in another study it was found that

TLR4 was sequestered intracellularly in a HCEC cell line and lacked responsiveness to even cytosol-translocated LPS (85). However, TLR4-dependent keratitis responses to

LPS or Gram-negative bacteria have been produced in several mouse and rabbit models

(16, 30, 71, 72, 104-106). TLR4 is necessary for resolution of live P. aeruginosa keratitis with expression downregulated by SIGIRR (66, 107). Corneal fibroblasts also express

TLR4 and respond to LPS treatment; their responses are enhanced by serum or exogenous CD14 and LBP (108-110). TLR4 activation of the cornea was found to be mediated by BMDCs and to increase nanotubule expression by MHCII(+) LCs (16, 111).

Uveal resident APCs also express TLR4 protein in contrast to ciliary, retinal and scleral tissues (112).

TLR5

TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-

negative bacteria. Flagellin has four domains and is the major constituent of flagella, which promote bacterial motility and invasion of host tissues. Domain D1 contains conserved regions involved in TLR5 ligation which are hidden in intact flagella, and are

accessible to TLR5 only within flagellin monomers which may be shed by bacteria or by host proteases and detergents. Two exposed-surface regions of TLR5 bind these conserved flagellin regions, with similar important sequences recognized by human and murine receptors (113). N- and C-terminals regions responsible for polymerization form domain D0; D2 and D3 domains are surface-exposed hypervariable regions that contribute to induction of adaptive immune responses (114).

Flagellin-induced TLR5-mediated inflammatory responses to P. aeruginosa, B. cenocepacia and L. pneumophila have been demonstrated in airway epithelia, as well as a human TLR5 SNP producing a dominant negative protein that increases carrier susceptibility to L. pneumophila pneumonia (115-118). TLR5 is also a major mediator of inflammatory responses to pathogens by intestinal epithelium (114, 119, 120). TLR5 protein expression is polarized to basal and wing cell regions of the human corneal epithelium and the basolateral portions of conjunctival and intestinal epithelia, which may restrict TLR5 activation to instances of pathogen invasion (74, 119, 121).

Interestingly, purified flagellins of ocular-pathogenic P. aeruginosa and S. typhimurium were shown to activate HCECs in vitro but those of ocular-nonpathogenic S. marcescens and B. subtilis were not (77). This report may indicate that corneal TLR5 distinguishes between flagellins independently of invasion, or simply that these bacteria have flagellin mutations that abolish TLR5. HCECs with pre-exposure to low-dose flagellin had enhanced antimicrobial gene expression and decreased inflammatory responses to a high-dose flagellin or live P. aeruginosa challenge, perhaps indicating that TLR5 contributes to antimicrobial responses with less destructive inflammatory sequelae (74,

122, 123).

TIR Adaptor-Mediated TLR Signaling

Ligand binding leads to dimerization of TLR ectodomains which is required for signaling by both homodimers (TLR3) and heterodimers (TLR2/1, TLR2/6 or IL-1R1 and its accessory protein (IL-1RAcP) (124-128). Dimerization brings conserved TIR domains in the cytoplasmic tails into close proximity to form a stable, bridged TIR-TIR structure (129). This structure recruits TIR domain-containing adaptor molecules which initiate signaling cascades by associating with downstream signaling molecules in turn.

Five adaptor molecules have been discovered to date, and broadly mediate two divergent signaling pathways initiated by the MyD88 or TRIF adaptors. MyD88 initiates signaling by also IL-1R, IL-18R and all TLRs except TLR3 (130-134). The TRIF-dependent pathway is activated by TLR3 and TLR4, and leads to induction of type I IFNs associated with viral infection (135-138). TLR4 is therefore notable for the ability to activate cells through two independent signaling pathways (139, 140). The other adaptor molecules associate with TLRs more specifically and modulate MyD88 or TRIF pathway activation.

TIRAP and TRAM are plasma membrane-bound; TIRAP recruits MyD88 to TLR2 and

TLR4 and TRAM recruits TRIF to TLR4 (140-146). SARM is known to negatively regulate TRIF-dependent signaling (134, 147).

MyD88-Dependent Signaling MyD88 has been shown to play a role in responses to various pathogens, although

the differential function of TLR vs. IL1R activation must be carefully considered.

MyD88 contains an N-terminal DD, an ID intermediate domain and a C-terminal TIR domain. It recruits IRAKs, specifically IRAK-4 which also contains an N-terminal DD to

the receptor-MyD88 complex (148, 149). IRAK-4 is required for IL-1R1 and TLR responses and IRAK-1 phosphorylation (150) and has been shown to be clinically

relevant; humans deficient in IRAK-4 are more susceptible to pyogenic bacterial infections (151). IRAK-4 recruits and activates IRAK-1, which interacts with TRAF6.

TRAF proteins are ubiquitin E3 ligases that lead to activation of NF-κB and MAPK signaling cascades. Activated TRAF6 activates MAPKKs, which lead to JNK and p38

MAPK activation and downstream activation of AP-1 and NF-κB (152). It also activates the IKK complex leading to IκB degradation by proteasomes and release of NF-κB (67,

153).

Mal/TIRAP

TIRAP has been shown to be clinically important in bacterial infection; SNP

S180L heterozygosity produces attenuated TLR2 signaling and protection against

pneumococcal disease, bacteremia, malaria and TB (154). It was identified due to its

sequence similarity to MyD88 (141, 155, 156), but TIRAP-deficient mice and MyD88- deficient mice are similar only upon TLR4 and TLR2 activation (157). Along those lines,

TIRAP-deficiency abrogates inflammatory responses to flagellin-deficient P. aeruginosa

and other unflagellated bacteria in the airways (118, 158). Unlike MyD88, TIRAP

localizes to the plasma membrane via a phosphatidylinositol-4,5-bisphosphate binding

domain, facilitating MyD88 recruitment to the membrane in proximity to TLR4 (142).

TIRAP may associate with TRAF6 to mediate NF-κB and MAPK activation (159). It also

interacts with Btk, which phosphorylates it to enables downstream signaling as well as its

degradation (160, 161). Mal is also processed by caspase-1 cleavage in order to function

(162).

TICAM-1/TRIF-Dependent Signaling

TRIF was discovered as a TIR-containing protein that associated with TLR3 (163,

164). TLR4-induced genes are expressed in a MyD88-independent TRIF-dependent fashion including in murine macrophage microarrays including CCL5, IP-10 and M-CSF

(165-167). TRIF-deficient mice have impaired TLR4-induced inflammatory cytokine production and impaired TLR3- and TLR4-induced IRF3 activation and IFN-β

production (138, 163). TRIF activation results in activation of NF-κB, IRFs and MAPKs

without the participation of IRAKs by forming a complex with TBK1/NAK, IKKε and

IRF-3(168). TBK1 phosphorylates IRF-3, inducing dimerization, nuclear translocation and transcription of IFN-α/β. TRIF interacts directly with TRAF6 and TAK1 to

contribute to NF-κB and MAPK activation and also interacts with RIP1 to indirectly activate TRAF6 and TAK1 (1, 67, 133). In addition, TLR4 may induce delayed NF-κB activation through the TRIF pathway via IRF3-mediated TNF-α production, with

subsequent TNFR binding leading to prolonged NF-κB activation (169).

TICAM-2/TRAM

TLR3 associates with TRIF directly, but TLR4 requires TRAM as a bridging

adaptor that associates with the plasma membrane via N-terminal myristoylation (145).

TRAM must also be phosphorylated by PKCε in order to function, but the roles of this

modification are otherwise unknown (170). TLR4 was recently proposed to recruit

TRAM and TRIF to the plasma membrane with subsequent internalization via dynamin-

mediated endocytosis to initiate TRAM/TRIF-dependent signaling in early endosomes

(144, 171).

SARM

SARM interacts with TRIF to inhibit NF-κB and IRF activation, leading to

decreased TLR3 and TLR4-induced cytokine production. TLR4 activation may negatively regulate TRIF-dependent signaling by increasing SARM activity via

posttranscriptional modifications, a possible mechanism of negative feedback regulation.

However, SARM was shown to play no role in macrophage TLR signaling, but increase

neuronal resistance to oxidative stress and apoptosis in SARM-deficient mice (133, 134,

147).

Figure 5. TIR-domain-containing adaptor domains and motifs. (Image taken from (1) Adaptor molecules all contain a common TIR-domain involved in interactions with TLRs and other adaptors. Other components of adaptor molecule structure enable specific regulation of adaptor function and downstream signaling responses.

MATERIALS AND METHODS

TLR agonists

All TLR agonists were purchased from Invivogen (San Diego, CA) and diluted to

the proper concentrations for use with endotoxin-free sterile water. Pam3CysK4 is a

synthetic tripalmitoylated lipopeptide that mimics the acylated amino terminus of

bacterial lipopeptides; it specifically binds TLR2/1. Ultrapure E. coli K12 LPS activates

only TLR4. Standard flagellin purified from S. typhimurium was used for TLR5

activation.

Mice C57BL/6 mice (6 to 8 weeks old) were obtained from Jackson Laboratories (Bar

Harbor, ME). TIRAP-deficient mice were provided by Dr. S. Akira (Osaka University;

Osaka, Japan). Animals were housed in filter-covered microisolator cages in the animal

facility of Case Western Reserve University (Cleveland, OH).

Animal model of TLR-ligand-induced keratitis

Six- to 10-week-old C57BL/6 and TIRAP-/- mice were anesthetized by

intraperitoneal injection of 0.4 ml 2,2,2-tribromoethanol (1.2%; Sigma-Aldrich), and the central corneal epithelium was abraded without stromal penetration with 3 scratches using a 26-gauge needle. 5 µg of Pam3Cys, 40 µg of LPS, 15 µg of flagellin or sterile water was applied to the surface in a 2 µl drop. Eyes were closed and taped shut to prevent drying until mice awoke from anesthesia.

Confocal microscopy analysis of stromal haze

Cellular infiltrate was analyzed by in vivo confocal microscopy (Nidek

ConfoscanTM; Fremont, CA). Twenty-four hours post-challenge, mice were euthanized

with CO2 and immobilized on a secure platform. A 40x objective was focused on the

central cornea with the assistance of a transparent gel medium (Genteal; Novartis

Ophthalmics; Duluth, GA). Images were captured every 1 – 2 um between basal

epithelium and endothelium with corresponding numerical values for light intensity and

stored as a stack for analysis (NAVIS; Lucent Technologies; Murray Hill, NJ). Stromal

haze was quantified by saving light intensity values to a spreadsheet (Excel; Microsoft;

Redmond, WA) and exporting them into statistical software (Prism; Graph Pad Software;

San Diego, CA) to generate a nonlinear regression curve. The total area under the under

the curve represented the total light intensity of the corneal stroma and was used to

measure corneal haze. Naïve untreated and sterile water-treated corneas were used as

controls.

Immunohistochemistry of corneal sections

Eyes were enucleated and frozen in OCT (Tissue-Tek; Sakura; Torrance, CA). 5-

µm cryosections of the central cornea were fixed in 4% formaldehyde for 30 minutes.

The sections were then washed with PBS and incubated with 8 µg/ml (rat anti-mouse)

NIMP-R14 IgG (Abcam; Cambridge, MA) in PBS with 1% FCS for 2 hours, then 5

µg/ml FITC-conjugated rabbit anti-rat IgG (H+L; Vector Laboratories, Burlingame, CA)

in PBS with 1% FCS for 45 minutes. After excess antibody was removed, sections were

dried and mounted in anti-fade medium containing 4',6'-diamino-2-phenylindole which

stains cell nuclei (Vectashield; Vector Laboratories; Burlingame, CA) and viewed with

fluorescence microscopy (magnification, x40) (Olympus Optical Co. Ltd.; Tokyo, Japan).

Neutrophils were quantified by direct counting from limbus to limbus.

Statistics

Statistical analysis was performed using an unpaired t-test (Prism; Graph Pad

Software; San Diego, CA); statistical significance was defined as a P value of less than

0.05.

RESULTS

P = Experiment 1

8000 7000 6000 5000 4000 3000 2000 1000 0 WT N AIVE T IR AP N AIVE WT H2O TIRAP H2O WT P3C TIRAP P3C

P = Experiment 2

8000 7000 6000 5000 4000 3000 2000 1000 0 WT N AIVE T IR AP N AIVE WT H2O TIRAP H2O WT P3C TIRAP P3C

Figure 6. TLR2-induced stromal haze is abrogated in TIRAP-deficient mice.

WT and TIRAP-/- corneas were abraded and exposed to Pam3CysK4 (5 µg) which activates TLR2/1. Light diffraction of the corneal stroma produced by cellular infiltrates was then measured by confocal microscopy after 24 hours. The results of two separate experiments are shown, with each data point representing a single cornea. In both experiments, stromal haze is increased by TLR2 activation in WT corneas compared to water (trauma) controls. TLR2 activation in TIRAP-/- corneas produced significantly

less haze that appeared similar to that of water controls. Thus, TIRAP appears to mediate

TLR2-induced keratitis responses as measured by stromal haze.

Experiment 1 P =

550 500 450 400 350 300 250 200 150 100 50 0 WT N AIVE T IR AP N AIVE WT H2O TIRAP H2O WT P3C TIRAP P3C

Experiment 2 P = 550 500 450 400 350 300 250 200 150 100 50 0 WT N AIVE T IR AP N AIVE WT H2O TIRAP H2O WT P3C TIRAP P3C

Figure 7. TLR2-induced neutrophil infiltration is abrogated in TIRAP-deficient mice.

WT and TIRAP-/- corneas were abraded and to Pam3CysK4 as described. Eyes were then removed, sectioned and immunostained with NIMP-R14 for neutrophil identification. Labeled cells were quantified by direct counting of the whole section, with each data point representing a single cornea. In Experiment 1, water controls had greater numbers of infiltrating neutrophils than in Experiment 2, although infiltration was similar in for both genotypes. In both experiments TLR2 activation induced greater neutrophil

infiltration in WT corneas relative to TIRAP-/- corneas. Thus, TIRAP appears to mediate

TLR2 keratitis responses as measured by neutrophil infiltration. However, neutrophil infiltration was not ablated to water control levels in either experiment.

Experiment 1 P = 8000 7000 6000 5000 4000 3000 2000 1000 0 WT N AIVE T IR AP N AIVE WT H2O TIRAP H2O WT FLAG T IR AP FLAG

P = Experiment 2

8000 7000 6000 5000 4000 3000 2000 1000 0 WT N AIVE T IR AP N AIVE WT H2O TIRAP H2O WT FLAG T IR AP FLAG

Figure 8. TL5-induced stromal haze is unaffected by TIRAP deficiency.

WT and TIRAP-/- corneas were exposed to S. typhimurium flagellin (15 µg) which activates TLR5. Corneas were analyzed by confocal microscopy after 24 hours and results are shown as described previously. Naïve and water controls are the same as those for Pam3CysK4 treatment (Pam3CysK and flagellin treatments were performed in the same experiment). In Experiment 1, TLR5-induced stromal haze was reproducible and significantly decreased in TIRAP-/- corneas relative to WT corneas. However, WT

corneas had similar levels of stromal haze in response to water or TLR5 activation. In

Experiment 2, TLR5-induced stromal haze was less reproducible but was not

significantly different between genotypes. TIRAP does not appear to mediate TLR5- induced keratitis responses as measured by stromal haze.

Experiment 1

550 P = 500 450 400 350 300 250 200 150 100 50 0 WT N AIVE T IR AP N AIVE WT H2O TIRAP H2O WT FLAG T IR AP FLAG

Experiment 2 P =

550 500 450 400 350 300 250 200 150 100 50 0 WT N AIVE T IR AP N AIVE WT H2O TIRAP H2O WT FLAG T IR AP FLAG

Figure 9. TLR5-induced neutrophil infiltration is unaffected by TIRAP deficiency. WT and TIRAP-/- corneas were exposed to flagellin and neutrophil infiltration

was quantified; results are shown as described, sharing naïve and water controls with

Pam3CysK4 treatment. In Experiment 1, TLR5—induced neutrophil infiltration was

reproducibly similar in WT and TIRAP-/- corneas. In Experiment 2, TLR5-induced

neutrophil infiltration was less reproducible but appeared to be significantly greater than

that of water controls for both genotypes. It also was not significantly different in WT and TIRAP-/- corneas. Thus, TIRAP does not appear to mediate TLR5-induced keratitis responses as measured by neutrophil infiltration. 35

P = Experiment 1 8000 7000 6000 5000 4000 3000 2000 1000 0 WT N AIVE WT H2O TIRAP H2O WT LPS TIRAP LPS

Experiment 2 P =

8000 7000 6000 5000 4000 3000 2000 1000 0 WT N AIVE T IR AP N AIVE WT H2O TIRAP H2O WT LPS TIRAP LPS

Figure 10. TLR4-induced stromal haze is abrogated in TIRAP-deficient mice.

WT and TIRAP-/- corneas were exposed to ultrapure E. coli K12 LPS (40 µg) which activates TLR4 exclusively. Corneas were analyzed by confocal microscopy after

24 hours and results are shown as described. Naïve and water controls are specific to these experiments. TLR4-induced stromal haze was greater than that of water controls in both experiments. TLR4-induced stromal haze was significantly decreased in TIRAP-/- corneas relative to WT corneas in both experiments. TIRAP-/- mice have been shown to be functionally equivalent to MyD88-/- mice upon TLR4 stimulation, which would lead 36

to sole activation of the TLR4-induced TRIF signaling. However, TLR4-induced stromal haze does not appear to be different than water-induced stromal haze in TIRAP-/- corneas. Thus, TLR4-induced keratitis responses are mediated by TIRAP but not TRIF as measured by stromal haze.

Experiment 1 P = 550 500 450 400 350 300 250 200 150 100 50 0 WT N AIVE WT H2O TIRAP H2O WT LPS TIRAP LPS

Experiment 2 P =

550 500 450 400 350 300 250 200 150 100 50 0 WT N AIVE T IR AP N AIVE WT H2O TIRAP H2O WT LPS TIRAP LPS

Figure 11. TLR4-induced neutrophil infiltration is abrogated in TIRAP-deficient mice. WT and TIRAP-/- corneas were exposed to E. coli K12 LPS, neutrophil

infiltration was quantified and results are shown as described. In Experiment 1, water

controls are similar between WT and TIRAP-/- corneas. TLR4 induces an increase in

neutrophil infiltration relative to water controls in WT corneas, which is significantly

decreased to water control levels in TIRAP-/- corneas. In Experiment 2, TLR4-induced

neutrophil infiltration is significantly less in TIRAP-/- corneas relative to WT corneas.

Thus, TLR4-induced keratitis responses as measured by neutrophil infiltration may be

mediated by TIRAP. However, TLR4-induced neutrophil infiltration in TIRAP-/- corneas was not equivalent to that of water controls. This result may indicate a role of TLR4- induced TRIF activity or other factors.

DISCUSSION AND FUTURE DIRECTIONS Summary of Results and Key Findings These results demonstrate that TLR2, TLR4 and TLR5 activation in the cornea

leads to neutrophil recruitment to the stroma and increased stromal haze. Both of these keratitis responses are ablated in the corneas of TIRAP-/- mice during TLR2 and TLR4 activation, but are unaffected during TLR5 activation. Thus, TLR2- and TLR4- but not

TLR5–induced keratitis are TIRAP-dependent processes. TLR4 did not appear to have

TIRAP-independent effects on these inflammatory processes. TRIF has been shown to be

present and active in the cornea in vivo and in vitro, and to mediate TLR3-induced keratitis responses (73). MyD88 has been shown to mediate TLR2- and TLR4-induced keratitis responses and to suppress TLR3-induced TRIF-mediated responses via JNK in

HCECs but not in macrophages, indicating a novel interdependent role for these adaptors in the cornea (72, 73). The role of this mechanism in TLR4-induced TRIF-mediated responses has not been fully addressed, although such effects were not apparent in this study, assuming that TIRAP deficiency and MyD88 deficiency would produce the same findings. This study contributes to our knowledge of TLR-induced keratitis by confirming the role of TIRAP in the cornea as an essential initiator of signaling upstream of MyD88 for TLR2 and TLR4. It was also seen that TLR5 signaling was unaffected by the absence of TIRAP.

TLR-induced inflammatory responses

The cornea is constantly exposed to commensal and pathogenic bacteria and their products. However, inflammatory responses that could lead to corneal opacification and visual loss may be dampened by the corneal epithelium, which forms a physical barrier to pathogens and may constitutively express antimicrobial agents in the absence of

inflammation (172). TLRs may be separated from bacterial products by that barrier, but

once it is penetrated bacterial products and TLRs may combine to initiate a

proinflammatory response. Polarized and intracellular TLR expression and activity have

been investigated in various mucosal epithelial tissues including the corneal epithelium.

Various in vivo studies have shown that absence of a TLR relevant to an infectious

organism decreases protective inflammation and increases tissue load of organism, or

downregulation of a recognized PAMP increases an organism’s ability to proliferate and

disseminate (172, 173, 174). When multiple TLRs may be involved in recognition

different patterns of cooperation may be observed: additive response or redundant served responses with a lack of TLR recognition leading to bacterial dissemination.

Paradoxically, more intense inflammatory responses may be observed when TLRs are

attenuated, perhaps due to loss of non-inflammatory host gene expression that partially

controls infection, increased bacterial dissemination and invasion with activation of

additional inflammatory pathways. Some studies have found that TLRs may modulate the

expression and activity of other TLRs involved in pathogen recognition, further

increasing the complexity of the pathogen-host interaction (31, 117).

Role of NLRs and IL-1 in inflammation

The NLR family is another group of innate immune PRRs that recognize PAMPs

in the cytoplasm, while TLRs are associated with cell surface or endosomal membranes.

It includes the Nod1 and Nod2 receptors which bind PGN synthetic intermediates and

activates MAPKs and NF-κB via the RIP2 adaptor similarly to TLRs; they may play a

major role in inflammatory responses when TLRs are attenuated. It also includes

receptors responsible for inflammasome and caspase-1 activation: NLRC4 binds

cytosolic flagellin and NLRP3 cooperates with P2X7R to activate caspase-1 in response

to various PAMPs. NLRs and TLRs may be activated simultaneously during infection

and synergize via the process of IL-1β production and secretion. TLR-mediated NF-κB activation leads to production of inactive pro-IL1β; NLR-mediated caspase-1 activation cleaves pro-IL-1β to IL-1β, its biologically active form which then mediates continued induction of inflammatory cascades. The relative role of NLRs and TLRs in inflammatory responses may be more significant when considering invasive or intracellular pathogens and those with product translocation mechanisms into or across cells such as P. aeruginosa. For example, NLRC4 activation requires a T3SS found in

bacteria like P aeruginosa (173-176). Noninvasive flagellated enteropathogenic E. coli but not its flagellin-deficient mutant activated Caco-2 immortalized enterocytes in a

TLR5- and flagellin-dependent manner, with absence of a T3SS increasing flagellin- independent responses; flagellin-deficient mutants activated the cells at later timepoints

(177). In this case, flagellin contributed to responses to the extent that it could be recognized by the various PRRs present, a property likely shared by other bacterial products in the cornea. The contribution of the inflammasome and IL-1 to the bacterial keratitis model could be assessed using ASC-/- or caspase-1-/- mice.

Role of epithelial cells and BMDCs in inflammatory responses

Mucosal inflammatory responses are modulated by their constituent resident cell

types and BMDCs. Studies in various models have indicated that these cell types may

cooperate to maximize the induction of inflammation (178, 179). These cell types may

also contribute to inflammatory responses in different ways, with the activity of each

depending on population size, response amplitude and TLR expression or activity. In

lung and corneal models of P. aeruginosa infection, APCs enhanced inflammatory

responses and bacterial clearance (180, 181). It is possible that resident cells may be activated by certain TLR stimuli while corneal APCs downregulate those particular TLRs

and thus antibacterial inflammation or vice-versa: skin LCs have been shown to respond weakly to TLR2, TLR4 and TLR5 stimulation in vitro after TGF-β-mediated development in vitro (182). In the intestine, CD11c+ LCs may downregulate TLR4 and upregulate TLR5, with a potentially greater role of TLR5 in colitis initiation: LPS stimulation prompts anti-inflammatory responses while flagellin induces proinflammatory chemokine production (183, 184). The converse may the case as well; these differences in TLR expression and activity may have important consequences regarding the role of particular TLRs in mucosal inflammation and disease phenotype, depending on the cell type with dominant activity (76, 184).

The relative contribution of corneal resident cell and APC TLR expression to keratitis responses may be assessed using bone marrow chimeric mouse models of TLR agonist- or organism-induced keratitis with TLR2, TLR4 or TLR5-deficient recipient mice reconstituted with WT bone marrow or vice versa. Alternatively, transgenic

“Mafia” mice may be pharmacologically depleted of APCs prior to TLR agonist- or organism-induced keratitis. These approaches have demonstrated that BMDCs play a more significant role than resident cells in TLR4-induced keratitis in vivo and may elucidate the roles of myeloid TLR5 and TLR2 in vivo as well (111).

Role of TIR Adaptor Activity in TLR/IL-1R1-mediated keratitis

Adapter protein expression and activity or novel signaling mechanisms in the different cell types involved produces variations in signaling among the same TLRs.

Similarly, TLR4 has been reported to function differently in macrophage cell lines and nonmyeloid cells compared to primary human or murine macrophages: LPS induced

IFN production by human and mouse primary macrophages but not by endothelial cells, synovial fibroblasts or RAW 264.7 cells (177). In addition, the role of TLR4-induced

TRIF signaling in corneal antibacterial innate immunity is not well characterized, and

IRF-3 activation and IFN-β secretion leading to TNF production and delayed NF-κB activation may be an important contributor to the course and severity of keratitis

(167).TRIF was found to contribute to clearance of E. coli and P. aeruginosa but not H. influenza with reduced clearance, perhaps secondary to CCL5 and alveolar macrophage chemokine production (179-181). On the other hand, MyD88, IRAK, TRAF6, and Tollip but not TIRAP, were found to be necessary for HAEC responses to P. aeruginosa, perhaps indicating that TLR5 but not TLR4 is indispensable for responses (118). In this case, ablation of TLR4 -or TIRAP-dependent responses may reduce inflammatory

morbidity without compromising pathogen clearance, a possible therapeutic target. Some

pathogens circumvent TLR responses this way: Brucella produces a TIR domain- containing protein similar to TIRAP that abrogates TLR2 and TLR4 activation; Y. pseudotuberculosis and S. typhimurium injects proteins which abrogate TLR4 signaling.

The role of the various adaptor molecules in keratitis responses may be assessed in mutant mice and chimeric models of TLR-induced keratitis.

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