The Emerging Role of Toll-Like Receptor Pathways in Surgical Diseases

REVIEW ARTICLE The Emerging Role of Toll-Like Receptor Pathways in Surgical Diseases

Laszlo Romics, Jr, MD, PhD, MRCS; Gyongyi Szabo, MD, PhD; John Calvin Coffey, PhD, AFRCSI; Jiang Huai Wang, PhD; Henry Paul Redmond, MCh, FRCSI

Objective: To outline the emerging significance of Toll- of pathogens renders them a key figure in the activation like receptor (TLR) signaling pathways in surgical dis- of both innate and adaptive immune responses during eases. sepsis. However, emerging evidence points to fundamen- tally important roles in ulcerative colitis, Crohn dis- Data Sources: A systematic review of the literature was ease, and Helicobacter pylori infection in the gastrointes- undertaken by searching the MEDLINE database for the tinal tract and in the development of atherosclerotic period 1966 to 2005 without language restriction. plaques in the cardiovascular system. Furthermore, recent studies suggest that the regulation of the TLR path- Study Selection: Original or review articles that de- way fulfills a central role in anticancer immunotherapy scribed experimental data on the activation of TLR sig- and in organ rejection after transplantation. naling pathways in surgically relevant diseases were selected for inclusion in this review. Conclusion: Given the clinical significance of TLR pathways, the targeting of individual molecular components Data Extraction: Data were obtained from peer- is likely to offer a broad range of future therapeutic mo- reviewed articles and references. dalities.

Data Synthesis: The role of TLRs in the recognition Arch Surg. 2006;141:595-601

OLL-LIKE RECEPTORS (TLRS) TLR5 is responsible for bacterial flagellin belong to the pattern rec- recognition. TLR9 is important in the recognition receptor super- ognition of unmethylated CpG DNA de- family that recognizes dis- rived from bacteria. TLR1, TLR6, and tinct pathogen-associated TLR10 can coassociate with TLR2 and sub- molecularT patterns. They have a broad sequently enhance ligand specificity. range of functions, from being respon- Toll-like receptors represent an impor- sible for dorsoventral polarity in the Dro- tant link between the “innate” and “adap- sophila embryo1 to protecting the latter tive” immune systems. Activation of TLRs from fungal infections.1 Since 1997, 11 triggers nonspecific immune responses in members of the mammalian homologue of monocytes and macrophages. In addi- the Drosophila Toll receptor family have tion, TLRs are also found on immature been described.2 dendritic cells (DCs) and are capable of Structurally, TLRs consist of a leucine- triggering their maturation. In this man- rich repeat in the extracellular domain and ner, DCs matured through TLR activa- a Toll–interleukin 1 (IL-1) receptor ho- tion stimulate specific or adaptive im- mologous region3 (TIR domain) in their mune responses. intracellular portion. TLR4 (the so- In this article, we review recent data that called endotoxin receptor) recognizes li- link various surgical diseases to TLR path- popolysaccharide (LPS) present in the cell ways. We searched the MEDLINE data- Author Affiliations: wall of gram-negative bacteria and also en- base for the period 1966 to 2005 without Department of Surgery, Cork dogenous ligands such as heat shock pro- language restriction. Original or review ar- University Hospital, National teins (HSPs) 60 and 70. TLR2 is respon- ticles that described experimental data University of Ireland, Cork (Drs Romics, Coffey, Wang, and sible for the recognition of peptidoglycans published in original papers or summa- Redmond); and Department of (PGN), lipoteichoic acid (LTA), and bac- rized in review papers on the activation of Medicine, University of terial lipoprotein (BLP), as well as zymo- TLR signaling pathways in surgically rel- Massachusetts Medical School, san from fungi. TLR3 recognizes double- evant diseases were selected for inclu- Worcester (Dr Szabo). stranded RNA (dsRNA) from viruses. sion in this review.

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 TLR3, 5, TLR1 and 6 TLR2 MD-2 TLR4 TLR2 and 4 PGN LPS dsRNA 7, and 9 Polymorphism MD-2 CD14 TLR TLR TLR 2 4 3 Cytoplasm TIRAP TIRAP Crohn Disease Sepsis Sepsis Sepsis Ulcerative Colitis Atherosclerosis SIRS Atherosclerosis Enteritis CAD Ulcerative Colitis Transplantation Immunotherapy Immunotherapy Crohn Disease MyD88 TRIF Wound Healing Wound Healing Atherosclerosis CAD Cytoplasm Sepsis Immunotherapy IRAK Tollip ? Experimental Colitis Wound Healing Transplantation Helicobacter pylori Infection Cell Necrosis TRAF6 ? AMI Helicobacter pylori Infection

Sepsis MAPK NF-κB IRF-3 MyD88 Atherosclerosis Activation of TLR2/4 Immunotherapy Transcription IRAK Transplantation Nucleus Nucleus Pyogenic Infections

DNADNA DNA Figure 2. Surgically relevant Toll-like receptor (TLR) pathways and their associations with various surgical diseases. AMI indicates acute myocardial Figure 1. Toll-like receptor (TLR) 2, TLR3, and TLR4 signaling pathways. infarction; CAD, coronary artery disease; IRAK, interleukin 1 Deficiency both in myeloid differentiation factor 88 (MyD88) and receptor–associated kinase; MyD88, myeloid differentiation factor 88; and Toll-interleukin 1 (IL-1) receptor domain-containing adaptor-inducing SIRS, systemic inflammatory response syndrome. interferon ␤ (TRIF) results in complete loss of nuclear factor ␬B (NF-␬B) activation, a factor that has a central role in inflammatory cytokine production. dsRNA indicates double-stranded RNA; IRAK, IL-1 Activated MyD88 recruits a death domain–containing receptor–associated kinase; IRF-3, interferon regulatory factor 3; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; serine–threonine kinase, the IL-1 receptor–associated ki- PGN, peptidoglycans; TIRAP, Toll-IL-1 receptor domain–containing adaptor nase (IRAK) (Figure 1). The IRAK family consists of IRAK-1, protein; Tollip, Toll-interacting protein; and TRAF6, tumor necrosis factor -2, -4, and -M isoforms. Notably, IRAK-4–deficient mice receptor–associated factor 6. are unresponsive to LPS-induced proinflammatory stimulation,11 whereas IRAK-1–deficient mice exhibit a partial THE SIGNALING PATHWAYS OF TLRs attenuation in LPS-induced inflammatory responses. Phos- phorylated IRAK associates with tumor necrosis factor To better understand the functions of the TLRs, their as- (TNF) receptor–associated factor 6 (TRAF6), which leads sociated signaling pathways should first be described to the activation of NF-␬B and mitogen-activated protein (Figure 1). TLR4 is the most widely studied TLR. Af- kinase (MAPK) (Figure 1). ter LPS ligation, TLR4 forms stable clusters (recepto- A central role has been attributed to TIR domain– somes) that recruit the adaptor molecule, MD-24 containing adaptor proteins (TIRAPs): TIRAP/MyD88- (Figure 1). MD-2–deficient mice are resistant to LPS- adaptor–like (Mal)12,13 and Toll/IL-1 receptor domain- induced shock, as are TLR4-mutant and TLR4-deficient containing adaptor-inducing interferon ␤ (TRIF) (Figure 1). mice.5 During gram-negative septic shock, circulating LPS This suggestion is based on the finding that a dominant is bound by LPS-binding protein (LBP) in the serum. This negative form of TIRAP/Mal prevents TLR4- but not TLR9- complex is subsequently recognized by CD14, which fa- mediated NF-␬B activation. Yamamoto et al14 showed that cilitates LPS-induced signaling.6 MD-2 either resides in mice deficient in both MyD88 and TRIF exhibit a com- a complex with TLR4 or is secreted in the serum when plete loss of TLR4-mediated NF-␬B activation. These data in excess.7 Having ligated with TLR4 at the cell surface, indicate that LPS-triggered NF-␬B activation involves either LPS is internalized rapidly via an unknown mechanism. MyD88 or TRIF. These molecules may represent future TLR activation induces either a core TLR response or therapeutic targets in the regulation of TLR-mediated a specific cellular response via the induction of different signaling. signaling pathways. As part of the core signaling pathway, myeloid differentiation factor 88 (MyD88) plays a TLRs IN SEPSIS AND THE SYSTEMIC central role (Figure 1). MyD88 interacts with TLRs via INFLAMMATORY RESPONSE SYNDROME their TIR domain. MyD88-deficient mice are unresponsive to LPS,8 and MyD88-deficient macrophages do not Figure 2 summarizes surgical diseases that are cur- respond to PGN and lipoproteins (TLR2 ligand) or rently associated with TLR pathways. Much of the evi- CpG DNA (TLR9 ligand). On the other hand, MyD88- dence that supports a role for TLR-mediated signaling deficient mice are highly susceptible to Staphylococcus in sepsis stems from murine studies. TLR4 activation is aureus infection.9 MyD88 deficiency modulates the now known to occur during the systemic inflammatory kinetics of the LPS-induced inflammatory response. The response syndrome (SIRS). The development of SIRS, af- activation of nuclear regulatory factor ␬B (NF-␬B) ter the administration of soluble heparan sulfate or elas- and c-Jun N-terminal kinases is delayed.10 Given the cen- tase, is TLR4 dependent. In keeping with this observa- tral role of this molecule in LPS-mediated signal trans- tion, Johnson et al15 demonstrated that exogenous duction, it represents a potential focal point for future macromolecules acting on TLRs can initiate a process that therapies. culminates in SIRS.

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 In humans, up-regulation of TLR4 and TLR2 levels ing sepsis. The role of the TLR pathway in the generation occurs in monocytes isolated from patients with sep- of tolerance is somewhat controversial. We and others sis.16,17 Takeuchi et al9 showed that in murine models of have demonstrated that the phenomena of homotoler- acute polymicrobial sepsis, TLR2, TLR4 messenger ance and heterotolerance occur for ligands of TLR2, TLR4, (mRNA), and TLR4 protein levels are up-regulated early TLR5, TLR7, and TLR9.33-41 Numerous intracellular during polymicrobial sepsis, which correlates with in- mechanisms have been implicated in tolerance. Our labo- creased mortality. Transgenic mice that overexpress TLR4 ratory has recently demonstrated a down-regulation of are more sensitive to LPS.18 Absence of the TLR4 gene TLR2 surface expression in tolerance induced by expo- protects against LPS-induced lethality but makes the host sure to low-dose bacterial lipoprotein.33 Other studies38 more susceptible to gram-negative bacterial infection.19 demonstrate reduced recruitment of MyD88 to TLR4 in TLR2 and MyD88 knockout mice show increased sus- LPS-induced tolerance. Emerging mechanisms include ceptibility to S aureus infection compared with wild- the down-regulation of IRAK-1 and IRAK-M as well as type mice.20 In addition, MyD88-deficient mice are more reduced colocalization between IRAK isoforms and resistant to polymicrobial sepsis.21 This finding points to MyD88.33,39-41 a common TLR4/TLR2 signaling pathway that follows the Recently it was observed that TLR4 and MD-2 exist activation of both receptors by gram-positive and gram- in a soluble form in the circulation.42-44 Soluble TLR4 at- negative bacteria. Interestingly, TLR4, TLR2, and com- tenuates LPS-induced NF-␬B activation in vitro.42 Pu- bined TLR4/TLR2 deficiency do not improve survival in gin et al43 demonstrated that soluble MD-2 levels are in- experimental polymicrobial sepsis.22 creased in the plasma, urine, and lung edema fluid from Several studies23-29 point to the importance of genetic patients with severe sepsis. We have shown that in- alterations in influencing outcome in sepsis. Polymor- creased hepatic LPS sensitivity is associated with tran- phisms in the TLR4 gene have been implicated in the de- scriptional up-regulation of MD-2 in a mouse model for velopment of septic shock. Arbour et al24 showed that fulminant hepatic failure.45 Similarly, excessive TLR4 re- common, cosegregating missense mutations (Asp299Gly activity, which can occur as a “second hit” after severe and Thr399Ile) in the TLR4 gene lead to a blunted in- injury, might lead to multiple organ failure.46 flammatory response after LPS inhalation in humans. Finally, the high-mobility group box 1 (HMGB1) pro- Gram-negative infection occurs in a significantly higher tein has been shown to bind TLR2 and TLR4.47 Secreted percentage of the patients with allelic mutations.25 The HMGB1 acts a cytokine, and therapeutic administration Asp299Gly mutation was found to be associated with in- of HMGB1 antagonists rescues mice from lethal sepsis. creased mortality in SIRS.26 In contrast, monocytes sepa- Therefore, the interaction of HMGB1 with TLR2 and TLR4 rated from heterozygous TLR4 mutation carriers did not may provide an explanation for the ability of HMGB1 to demonstrate an altered response to LPS.27,28 However, a generate inflammatory responses and accelerate lethal- coding mutation in the MD-2 gene (TLR4 coreceptor) re- ity in sepsis. sults in reduced LPS-induced signaling.29 Polymorphisms in the TLR2 gene have been shown TLRs IN THE GASTROINTESTINAL TRACT to correlate with both the incidence and outcome of septic shock. In vitro, functional studies30 confirmed that cells In normal conditions, intestinal epithelia strongly express that express mutant TLR2 are significantly less respon- TLR3 and TLR5, whereas TLR4 and TLR2 expression is sive to TLR2 ligands than wild-type controls. In accor- weak.48,49 Rakoff-Nahoum et al50 demonstrated that the dance with this, Meng et al31 demonstrated that an anti- interaction between commensal bacteria and TLRs on TLR2 antibody prevents a lethal shocklike syndrome in surface epithelia is crucial to the maintenance of gut epi- mice. thelial homeostasis and protection against gut injury. The clinical significance of genetic alterations in the In ulcerative colitis (UC) and Crohn disease (CD), TLR4 downstream pathways has been illustrated by Picard et expression in primary epithelial cells is increased.51 In con- al,32 who recently described the cases of 3 unrelated chil- trast, TLR3 expression is down-regulated in active UC but dren with recurrent severe pyogenic bacterial infec- not in active CD. Interestingly, TLR2 and TLR5 levels re- tions. They identified a defect in human TLR-IRAK sig- main unchanged during both.51 Recent findings indicate naling in these children that resulted in an increased that in murine models of experimental colitis, TLR4/ susceptibility to pyogenic bacteria. However, this defect MD-2 and CD14 are expressed mainly in distally located was associated with a relative protection against most other colonic epithelial cells, whereas TLR2 is expressed pri- ubiquitous pathogens (Figure 2). marily in the proximal colon. This is referred to as TLR The ability to predict a patient’s individual suscepti- compartmentalization in colitis52 (Figure 2). bility to infection would represent an enormously pow- Nucleotide-binding and oligomerization domain 2 erful therapeutic advance. Given our emerging under- (Nod2), a receptor that is structurally similar to TLRs, standing of single-nucleotide polymorphisms and given is also involved in intracellular LPS recognition. Muta- microarray technology, it is foreseeable that a single test, tions in the Nod2, TLR4 (Asp299Gly), and TLR9 genes performed before patients undergo surgery, would readily are associated with increased susceptibility to CD.53,54 The determine susceptibility to infection and thereby guide Asp299Gly and Thr399Ile TLR4 mutations are associ- prophylactic treatments. ated with a higher incidence of UC.55-57 Tolerance to bacterial cell wall components repre- Lodes et al58 showed that the TLR5 agonist, flagellin, sents an adaptive host response during infection. Toler- is a dominant antigen in CD. Although TLR5 is local- ance may also lead to severe immunosuppression dur- ized mainly on the basolateral surface of colonic cell lines,49

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 it occurs mainly at the apical pole of ileal enterocytes.59 TLRs IN SURGICAL ONCOLOGY TLR5 expression on intestinal endothelial cells (in colonic microvessels) raises the exciting prospect of an over- At present, many forms of immunotherapy exploit a tu- lap between localized innate immune responses and sys- mor antigen–specific immune response that is initiated temic vascular responses.60 by antigen-presenting DCs. Numerous phase 1 and 2 trials Several studies point to a role for TLRs in the recog- are currently under way using autologous DC vaccines nition of Helicobacter pylori, a crucial pathogenic factor against non–small cell lung cancer, melanoma, hepato- in chronic gastritis, peptic ulcer disease, and gastric neo- cellular carcinoma, and gastric, colon, breast, pancreas, plasia. TLR4 mRNA levels are up-regulated by H pylori prostate, ovarian, and cervical cancer.79 Toll-like recep- in gastric epithelial cell lines.61 However, H pylori– tors play a critical role in DC maturation. In many DC- activated cytokine production occurs independently of based vaccines, maturation most likely results from the TLR4 activation.62 In addition, H pylori–associated flagel- activation of TLRs by bacillus Calmette-Gue´rin (BCG) lin evades TLR5, a property that may contribute to its cell wall skeleton, dsRNA, LPS, PGN, lipoprotein, and persistence in the host63 (Figure 2). CpG DNA.80,81 One of the best known adjuvants, BCG cell wall skeleton, is known to induce both TLR2 and TLR4.80 Imiquimod, a synthetic antitumor agent, is a TLR7 TLRs IN VASCULAR AND CARDIAC SURGERY ligand. Synergistic activation of TLR3 and TLR9 resulted in an enhanced antimetastatic effect, as demon- Chlamydia pneumoniae infection is associated with the de- strated by Whitmore et al82 (Figure 2). Toll-like recep- velopment of atherosclerotic plaques.64 Elevated C pneu- tor signaling also critically overlaps with apoptotic moniae–derived HSP-60 levels are found in patients with cascades. The regulation of apoptosis by TLR ligands rep- peripheral vascular diseases and in experimental mod- resents an attractive anticancer treatment modality. TLR2 els of atherosclerosis. Elevated serum HSP-60 levels are activation induces apoptosis in macrophages and endo- found in patients with atherosclerosis and even in those thelial cells by recruiting MyD88, IRAK-1, and the Fas- with borderline hypertension. Numerous studies65,66 have associated death domain (FADD)–caspase 8 (and hence identified the presence of C pneumoniae in the walls of the extrinsic) proapoptotic pathway.83,84 abdominal aortic aneurysms. These findings point to There is increasing evidence, albeit indirect, that im- an association between HSP-60 and early cardiovascu- plicates LPS (and therefore TLR) signaling in the accel- lar disease.67,68 erated growth of minimal residual neoplastic disease af- TLR4 recognizes both the whole bacterium and C pneu- ter excisional surgery for cancer cure. It is well accepted monia–derived HSP-60. Chlamydia pneumoniae induces that after surgical stress, local and systemic neoplastic proliferation of human vascular smooth muscle cells. This disease is potentiated and undergoes acceleration in effect occurs via TLR4-mediated activation of p44/p42 growth.85 Although multiple mechanisms have been im- MAPK.69 Furthermore, TLR4 levels are up-regulated in plicated in this phenomenon, enhanced bacterial trans- atherosclerotic plaques compared with the normal ves- location and systemic endotoxemia are directly corre- sel wall.70 TLR4 polymorphisms have also been associ- lated.86 We have demonstrated that antiendotoxin agents, ated with the development of atherosclerotic lesions. Evi- such as taurolidine and probiotics, exert a protective effect dence is emerging that TLR4 polymorphisms are against accelerated metastatic tumor growth after sur- associated with a protective antiatherosclerotic effect in gery.87,88 Overall, these findings strongly implicate LPS the clinical setting. Kiechl et al71 demonstrated that the signaling, and by definition TLR signaling, in the accel- Asp299Gly TLR4 allele is associated with a lower risk of eration of metastatic tumor growth that follows exci- carotid atherosclerosis and a smaller intima media thick- sional surgery for apparent cancer cure. ness in the common carotid artery, either in the homo- zygous or heterozygous form. Furthermore, TLR4 poly- TLRs IN TRANSPLANTATION morphisms are associated with a reduced risk of acute coronary events.72 However, TLR4 polymorphisms do not Dendritic cells play a role in graft rejection after trans- appear to correlate with a reduced risk of stroke.73 TLR2 plantation. The role of TLR signaling has been investi- has recently been implicated in carotid artery plaques and gated in this regard. Hemmi et al89 showed that minor in particular with the vulnerable plaque phenotype.74 antigen-mismatched acute rejection does not occur in Hence, these findings indicate that the adaptive and/or MyD88-deficient mice. This experimental observation cor- innate immune response is implicated in atherogenesis relates with the presence of decreased numbers of ma- and may therefore be modulated with a view to altering ture DCs in draining lymph nodes. An investigation of the disease (Figure 2). the ischemia-reperfusion injury after liver transplanta- Frantz et al75 showed increased TLR4 expression on tion revealed that TLR2, CD14, and LBP levels were in- the border of necrotic and viable myocardium. In- creased within 6 to 12 hours after liver transplantation90 creased TLR2 and TLR4 levels have been demonstrated and also that TLR4 deficiency attenuated hepatic ischemia- in circulating monocytes 2 days after coronary artery by- reperfusion injury.91 These findings indicate that mul- pass grafting.76 In contrast, a protective role has been tiple components of the LPS signaling pathway are acti- shown for TLR2. TLR2 has been proved to attenuate the vated during ischemia-reperfusion injury after liver effects of oxidative stress–related NF-␬B activation in the transplantation.92 In keeping with this, TLR4 polymor- myocardium.77 In addition, TLR2 plays an important role phisms are associated with decreased acute rejection af- in ventricular remodeling after myocardial infarction.78 ter lung transplantation.93 Regarding heart transplanta-

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/24/2021 tion, allograft endothelial dysfunction is associated with pathogenesis. The suggestion therefore arises that con- elevated TLR4 levels on peripheral blood monocytes.94 ditions formerly associated with different molecular pro- Endothelial dysfunction is an early indicator of chronic cesses may involve common signaling pathways. This is rejection after cardiac transplantation. These data dem- an enticing prospect, since the therapeutic subversion of onstrate that TLR pathways contribute to organ rejec- these pathways may greatly simplify the treatment of sur- tion and ischemia-reperfusion injury after liver trans- gical disease. Such is the current level of understanding plantation (Figure 2). of the TLR signaling processes, and the components thereof, that it may soon be possible to therapeutically COMMENT dissect these pathways by selectively targeting adaptor molecules (Mal/TIRAP, TRIF, TRAM, and MyD88) or co- Toll-like receptors recognize specific patterns present in receptors (MD-2 and CD14). We suggest that the thera- molecules within microbes and then regulate the activa- peutic manipulation of TLR signaling will ultimately find tion of both innate and adaptive immunity. Currently it its place at the bedside and represent a powerful adju- is unclear whether the net effect of TLR activation is ben- vant in the management of surgical disease. eficial or detrimental to the host. Although TLR4 deficiency increases susceptibility to gram-negative bacte- Accepted for Publication: June 1, 2005. rial sepsis, it protects against endotoxin-induced 18,19,22 Correspondence: Henry Paul Redmond, BSc, MCh, sepsis. Similarly, TLR2-deficient mice are more sus- FRCSI, Department of Surgery, Cork University Hospi- ceptible to S aureus infection then are their wild-type coun- 20 tal, Wilton Road, Wilton, Cork, Ireland (redmondhp terparts. In humans, TLR4 polymorphisms are associ- @shb.ie), or Gyongyi Szabo, MD, PhD, Department of ated with an increased incidence of gram-negative Medicine, University of Massachusetts Medical School, sepsis24-26 but inflammatory hyporesponsiveness to se- 24 364 Plantation St, LRB 215, Worcester, MA 01605-2324 lective ligands and inhaled LPS. Similarly, patients with ([email protected]). certain types of TLR2 polymorphisms are at increased risk 95 Funding/Support: This work was supported by the Irish of life-threatening staphylococcal infections. These data Research Council for Science, Engineering and Technol- indicate that defective TLR signaling is detrimental to the ogy Basic Research Grants Scheme (SC/2003/27), the host in bacterial infection. However, when bacterial cell Health Research Board in Ireland, and the Hungarian State wall components are used as ligands, defective TLR sig- Scholarship “Eotvos” (2001). naling appears protective. The whole bacterium theo- retically might activate other, as yet unknown, pattern recognition receptors besides TLRs. These receptors may REFERENCES interfere with the TLR signaling process or trigger the 1. Lemaitre B, Nicolas E, Michaut L, et al. The dorsoventral regulatory gene cas- production of different effector molecules, resulting in sette spatzle/Toll/cactus controls the potent antifungal response in Drosophila an altered cytokine environment. adults. Cell. 1996;86:973-983. The complexity of signaling pathways triggered by 2. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. TLRs has been extensively characterized. Some of these Nat Immunol. 2004;5:987-995. studies have focused on the relationship between inflam- 3. Medzhitov R. 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