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Originally published online as doi:10.1189/jlb.0703321 on November 3, 2003

Published online before print November 3, 2003
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(Journal of Leukocyte Biology. 2004;75:428-433.)
© 2004 by Society for Leukocyte Biology

Shielding the double-edged sword: negative regulation of the innate immune system

Koichi S. Kobayashi* and Richard A. Flavell*,{dagger},1

* Section of Immunobiology
{dagger} Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut

1Correspondence: Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, 300 Cedar Street, CAB S-569, New Haven, CT 06520-8011. E-mail: richard.flavell{at}yale.edu


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ABSTRACT
 
The innate immune system is evolutionarily conserved among all multicellular organisms and is the first line of defense against microorganisms. It enables the host not only to combat pathogenic organisms but also to cohabit with nonpathogenic microorganisms by balancing the host-microorganism interaction. The innate immune response is activated rapidly (within hours) compared with adaptive immunity. Activation of the innate immune system allows the activation of the adaptive immune response by production of proinflammatory cytokines and by providing stimulatory signals via major histocompatibility complex molecules and costimulatory molecules such as CD40, CD80, or CD86; together, these lead to the full activation of both immune systems to fight against pathogenic microorganisms. Activation of the innate immune system, however, can be a double-edged sword for the host. Proinflammatory cytokines mediate a positive feedback loop on the innate immune system, and overproduction of cytokines, if unchecked, is hazardous to the host and may cause severe outcomes such as hyperthermia, organ failure, and even death in extreme cases. Moreover, if the overproduction of proinflammatory cytokines persists, it may cause chronic inflammatory diseases. During evolution, the innate immune system has acquired complicated regulatory systems to control itself so that this "sword" will not kill the host. Various mechanisms including inhibition of Toll-like receptor signaling by interleukin-1 receptor-associated kinase-M have evolved for this purpose and are important not only to fight against pathogenic microorganisms efficiently but also are critical for the peaceful coexistence with commensal bacterial flora.

Key Words: innate immunity • TLR • IRAK-M • signaling • endotoxin


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INTRODUCTION
 
Toll-like receptors (TLRs)
The recent, rapid progress in our knowledge of this type I receptor family has revealed that TLRs play central roles in the process of activation of the innate immune system. TLRs detect microorganisms at the cell surface and in vesicles. The extracellular domain, which consists of leucine-rich repeats, plays a critical role in the specificity of recognition. The microbial ligands recognized by TLRs are called pathogen-associated molecular patterns (PAMPs) [1 ]. For example, TLR1 recognizes bacterial lipoproteins [2 , 3 ]. Likewise, TLR2 also recognizes bacterial lipoproteins and peptidoglycan [4 ]. TLR3 recognizes double-stranded RNA, which mimics viral RNA [5 ]. TLR4 recognizes lipopolysaccharide (LPS), a component of the outer cell wall of gram-negative bacteria [6 7 8 ], and TLR5 recognizes flagellin, a component of flagellar bacterium [9 ]. Finally, TLR9 recognizes unmethylated CpG DNA or bacterial DNA [10 ]. Signaling from TLRs requires an adaptor molecule, MyD88, and serine/threonine kinases, interleukin-1 (IL-1) receptor-associated kinase (IRAKs) [11 ]. These components cause activation of the nuclear factor (NF)-{kappa}B and mitogen-activated protein kinase (MAPK) pathways leading to the up-regulation of surface costimulatory molecules and eventual secretion of cytokines such as IL-1ß, IL-6, IL-8, IL-12, and tumor necrosis factor {alpha} (TNF-{alpha}) by antigen-presenting cells [11 12 13 ].

TLR signaling
The cytoplasmic tail of TLRs is a protein-binding moiety, a TLR/IL-1 receptor homology domain (TIR), which undergoes homophilic interaction with other TIR domain-containing molecules [11 , 14 ]. TLR signaling is mediated by an adaptor molecule, MyD88, and serine/threonine kinases, IRAKs [11 ]. MyD88 has two protein-binding domains: The first is a TIR domain, and the second is a death domain (DD). All TLRs use this adaptor molecule for signal transduction. The studies of MyD88-deficient mice showed that MyD88 is a critical molecule required to produce cytokines upon TLR stimulation [12 ].

IRAKs are serine/threonine kinases, which have an N-terminal DD. Upon the activation of TLRs, MyD88 and IRAKs are recruited to TLRs by TIR–TIR interaction and by DD–DD interaction, respectively (Fig. 1 ). Among IRAK family proteins, IRAK-1 and IRAK-4 have been shown to be important mediators of signal transduction following IL-1 and LPS stimulation, as established using mouse genetic studies [15 16 17 18 ]. IRAK-4 seems to act as an upstream kinase of IRAK-1, as recombinant IRAK-1 is phosphorylated by IRAK-4 and as IRAK-1 is not able to phosphorylate IRAK-4 [19 ]. Recruited IRAKs are phosphorylated by auto- or cross-phosphorylation [19 20 21 ]. Phosphorylated IRAKs lose their affinity for MyD88 and activate downstream molecules such as TRAF6, resulting in the activation of cascades of signaling including NF-{kappa}B, c-jun NH2-terminal kinase (JNK), p38, and extracellular-regulated kinase (ERK)1/2 [11 , 12 , 22 , 23 ].



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  		Figure 1. A proposed model of TLR signaling and its inhibitory molecules. TLR activation by PAMPs induces multimerization of these receptors, which causes recruitment of MyD88 or IRAK proteins. Proximity of IRAKs causes auto- or cross-phosphorylation. The conformational change of IRAKs results in reduced affinity for the TLR signaling complex, and IRAKs are released to make possible the activation of downstream molecules such as TNF receptor-associated factor (TRAF)6. IRAK-M is recruited to the signaling complex together with IRAK-1 and IRAK-4. This association inhibits the release of IRAK-1/IRAK-4 from the TLR signaling complex by inhibiting the phosphorylation of IRAK-1/IRAK-4 or stabilizing the TLR/MyD88/IRAK-1/-4 complex, resulting in the interruption of downstream signaling. Suppressor of cytokine signaling (SOCS)-1 directly inhibits TLR4 signaling, possibly by interacting with IRAK-1. A20 has inhibitory roles in TNF and TLR4 signaling. A20 can associate with NF-{kappa}B essential modulator (NEMO)/I{kappa}B kinase (IKK){gamma}, A20 binding inhibitor of NF-{kappa}B activation (ABIN), or ABIN-2; thus, it may exert its inhibitory effects on NF-{kappa}B activation by binding these molecules. Toll-interacting protein (Tollip) binds to IRAK-1 and may inhibit IL-1R and TLR signaling directly. MyD88s is an alternative, short form of MyD88. The overexpression of MyD88s inhibits TLR and IL-1R signaling. TAK1, transforming growth factor-ß-activated kinase 1; TAB2, TAK1-binding protein 2.


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THE MECHANISM OF NEGATIVE REGULATION OF TLR SIGNALING
 
Endotoxin tolerance
Although the activation of the innate immune response is critical to control infection caused by pathogenic microorganisms, excessive cytokine production without proper regulation is harmful to the host and may lead to microcirculatory dysfunction, tissue damage, shock, or even death in extreme cases [24 , 25 ]. Prior exposure to LPS or endotoxin causes transient unresponsiveness to LPS challenge [26 27 28 ]. This phenomenon is called endotoxin tolerance (also called LPS tolerance, LPS unresponsiveness, or LPS refractoriness) and is observed in vivo and in vitro. Endotoxin tolerance is regarded as a protection mechanism against endotoxin shock during gram-negative bacterial infection to limit tissue damage by excessive immune response [29 30 31 ].

There is a long history of research in endotoxin tolerance. During the last century, it was known to clinicians that in patients being given intravenous injection of typhoid vaccine as a method of fever therapy, the dose of vaccine had to be increased at successive treatments to obtain comparable elevation of body temperature, although the mechanism was unknown [26 ]. Endotoxin was also called pyrogen in those days, as fever was the only reliable method to quantify the results following administration. Studies were performed using animals and extracts from gram-negative bacteria, and pre-exposure to such extracts was shown to cause unresponsiveness to subsequent challenge, resulting in nonhyperthermia [26 ]. In the 1960s, human studies were performed with healthy human volunteers, and endotoxin tolerance was confirmed at the controlled, experimental level in humans [32 ]. Although the phenomenon itself has been known for many decades, it is only recently that we have started to comprehend the underlying mechanisms of endotoxin tolerance. Considering the heavy load of bacterial flora in the gut and the strong immunogenicity of LPS, it is very reasonable to expect that humans and other animals have multiple mechanisms to ensure endotoxin tolerance at various levels of signaling.

Endotoxin tolerance is characterized as a state of unresponsiveness of an organism and its macrophages to LPS, in terms of cytokine production [33 34 35 ]. In macrophages previously exposed to LPS, activation of NF-{kappa}B, p38, MAPK, ERK1/2, and JNK upon LPS stimulation is reduced [36 37 38 39 ]. Prior exposure to LPS also causes reduced IRAK-1 phosphorylation and kinase activity upon LPS stimulation [40 , 41 ]. It has also been reported that recruitment of MyD88 to TLR4 is abrogated in endotoxin-tolerant monocytes after LPS treatment [41 ].

Although LPS is the only PAMP that causes "shock" on its own, recent studies indicate that different PAMPs cause "cross-tolerance" in vitro. In this case, pre-exposure to certain PAMPs causes unresponsiveness to subsequent LPS challenge. Such cross-tolerance was observed between TLR2 and TLR4 [42 , 43 ], TLR7 and TLR4 [44 ], and TLR9 and TLR4 [45 ]. It has been shown that cross-tolerance is an endogenous event and is not caused by paracrine mediators [42 ].


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MISSING TLR
 
TLR4 is a receptor for LPS, which is one of the most potent, known immune stimulators. Humans have a large burden of commensal bacterial flora in the intestinal tract. The epithelial cells of the normal intestinal tract do not express TLR4 [46 , 47 ]. This is probably the consequence of evolution following millennia of cohabitation with gram-negative bacteria in the intestinal tract, which provide a vast source of LPS; the mechanism of "negative regulation" has coevolved with innate immune signaling. In addition to intestinal epithelium, there is no expression of TLR4 on epithelial cells of the cervix and vagina, which contain bacterial flora such as Lactobacillus [48 ]. In contrast, in the trachea and urinary bladder, in which no commensal bacteria reside, epithelial cells have TLR4 and can respond to LPS stimulation, leading to production of cytokines [49 50 51 ].

Intestinal epithelial cells also interact with Gram-positive bacteria in gut flora, which may provide large amounts of TLR2 ligands, particularly peptidoglycan and teichoic acid. It has been shown that intestinal epithelial cells are broadly unresponsive to those TLR2 ligands secondary to deficient expression of TLR2 and TLR6 [52 ].

TLR5 recognizes flagellin, a component of flagellar bacteria [9 ]. Studies using T84, a human colonic epithelial cell line, have shown that the expression of TLR5 is restricted to the basolateral but not apical surface of epithelia, providing a mechanism by which flagellar bacteria invading intestinal epithelium but not commensal bacteria induce inflammatory response [53 ].


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DOWN-REGULATION OF SURFACE TLR4
 
One of the mechanisms that causes endotoxin tolerance is probably down-regulation of surface expression of TLR4. Although the expression levels of TLR4 do not change much at the RNA and protein levels, the surface expression level becomes reduced after exposure to LPS [39 ]. The kinetics and level of TLR4 down-regulation are dependent on the dose of LPS. Cell lines transfected with TLR2 or TLR4 are still able to become endotoxin-tolerant, suggesting that down-regulation of TLRs is not the sole mechanism for tolerance [54 , 55 ]. It would be of interest to know if there is a down-regulatory mechanism specific for TLR4 or if there is a common mechanism for various TLRs.


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NEGATIVE REGULATORS OF TLR SIGNALING
 
Several molecules have been proposed to inhibit TLR signaling. Some of these were discovered by mouse genetic approaches, and some were proposed simply by overexpression studies, which should therefore be confirmed by more physiological experiments.

IRAK-M
One such inhibitory molecule is IRAK-M [56 ]. This was surprising, as IRAK-M is one of several IRAK family proteins, and all IRAKs were originally thought to have redundant functions [21 ]. There are four IRAK proteins: IRAK-1, IRAK-2, IRAK-M, and IRAK-4 [19 20 21 , 57 ]. Whereas IRAK-1 and IRAK-4 have serine/threonine kinase activity, IRAK-M and IRAK-2 are enzymatically inactive, as they lack the catalytically active aspartate residue in the kinase domain [21 , 56 ]. Overexpression of IRAK-M in HEK293T cells by transfection results in NF-{kappa}B activation, although to a lower level than that induced by IRAK-1 and IRAK-4 [21 ]. Studies of IRAK-M-deficient mice, however, demonstrated a negative regulatory role of this molecule in TLR signaling [56 ]. IRAK-M-deficient cells showed greater production of proinflammatory cytokines upon TLR and IL-1R stimulation. Moreover, IRAK-M-deficient cells produced more cytokines when infected by gram-negative and gram-positive bacteria. In vivo, IRAK-M-deficient mice orally infected with Salmonella typhimurium showed a severe inflammatory response in the Peyer’s patch of the intestinal tract. IRAK-M is, therefore, a negative regulator of inflammatory signaling.

How does IRAK-M inhibit TLR responses? Upon stimulation of TLR4 and TLR9, the activation of NF-{kappa}B and MAPKs was increased and accelerated in IRAK-M-deficient cells, indicating a negative regulatory role for IRAK-M in TLR signaling. In transfection studies, IRAK-M did not inhibit the formation of the IRAK-1/TRAF6 complex, but it prevented dissociation of IRAK-1 and IRAK-4 from MyD88. This suggests that IRAK-M makes a complex with IRAK-1 or IRAK-4 together with MyD88 and inhibits the release of IRAK-1/IRAK-4 from the TLR signaling complex by inhibiting the phosphorylation of IRAK-1/IRAK-4 or stabilizing the TLR/MyD88/IRAK-1/-4 complex, resulting in turn in the interruption of downstream signaling [56 ].

IRAK-M expression is inducible upon TLR stimulation in macrophages. This is important, as it indicates that there is a negative feedback loop in the TLR system, which up-regulates this inhibitory molecule, and thus, IRAK-M may contribute to stabilize the homeostasis of the innate immune system (Fig. 1) . Indeed, tolerance to LPS was impaired in IRAK-M-deficient cells, showing the importance of IRAK-M for endotoxin tolerance and innate immune homeostasis [56 ].

SOCS-1
SOCS-1, also called signal transducer and activator of transcription (STAT)-induced STAT inhibitor-1 or Janus tyrosine kinase (JAK)-binding protein-1, is a negative regulatory molecule of the JAK–STAT signal cascade [58 59 60 ]. It inhibits signaling by the receptors for interferon-{gamma} (IFN-{gamma}), IL-4, IL-6, and leukemia inhibitory factor. SOCS-1 interacts with JAK tyrosine kinases and inhibits kinase activity, thereby suppressing cytokine signal transduction. Recent work using SOCS-1-deficient mice indicates a negative regulatory role of this molecule in LPS challenge [61 , 62 ]. SOCS-1 is inducible by LPS stimulation in macrophages. SOCS-1-deficient cells have an increased response to LPS stimuli. SOCS-1 deficiency causes abrogation of endotoxin tolerance in vivo and in vitro, and SOCS-1-deficient mice are susceptible to LPS challenge. This is partly because of the loss of the inhibitory effect of SOCS-1 on IFN-{gamma} signaling, as SOCS-1/STAT1 doubly deficient mice are less susceptible to LPS. However, SOCS-1/STAT1 doubly deficient mice are still more susceptible to LPS challenge than wild-type mice, and SOCS-1/IFN-{gamma} doubly deficient macrophages produce more TNF-{alpha} and nitrite than IFN-{gamma} single-deficient macrophages, suggesting a direct inhibitory role of SOCS-1 in TLR4 signaling [61 , 62 ].

A20
A20 is a cytoplasmic zinc finger protein, which is induced by a wide range of stimuli such as IL-1, TNF, CD40, and latent membrane protein 1 [63 64 65 66 ]. Activation of NF-{kappa}B by these stimuli is important for transcriptional up-regulation [67 ]. The function of A20 seems to be to create a negative feedback loop; A20 inhibits NF-{kappa}B activity and TNF-mediated programmed cell death [68 ]. The recent demonstration that A20-deficient mice develop severe inflammation and are hyper-responsive to LPS suggests that A20 may play a key role in regulating the inflammatory response [69 ]. In addition to an inhibitory role in TNF signaling, overexpression of A20 causes decreased NF-{kappa}B activation through TLR4, suggesting a direct, negative regulatory role of A20 in the TLR signaling [70 ]. Although the mechanism of the inhibitory function of A20 is not fully elucidated, A20 can associate with NEMO/IKK{gamma}, ABIN-1, and ABIN-2; thus, it may exert its inhibitory effects on NF-{kappa}B activation by binding these molecules [70 71 72 73 ].

Tollip
Tollip is another adaptor molecule, originally cloned using the yeast two-hybrid assay using IL-1 receptor-associated protein as bait [74 ]. Tollip can associate with IL-1R and IRAK-1 in vitro, and a negative regulatory role has been proposed in IL-1 receptor signaling by overexpression studies [74 ]. More recently, a negative regulatory role of Tollip in TLR signaling was proposed. Overexpression of Tollip causes interaction with TLR2 and TLR4 and inhibits TLR signaling, although the function of this molecule under physiological conditions remains to be elucidated [75 ].

MyD88s
MyD88s is generated by alternative splicing [76 ]. This form lacks only the short intermediate domain separating the DD and TIR domains [76 ]. MyD88 but not MyD88s strongly binds to IRAK-4. The overexpression of MyD88s suppresses the phosphorylation of IRAK-1 and inhibits NF-{kappa}B activation upon IL-1 and LPS stimulation, suggesting that it may have a negative regulatory role in TLR/IL-1R signaling [76 , 77 ].


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CONCLUSION
 
The recent, rapid progress made in studies of the TLR system has revealed the critical importance of this system in the initiation of immune response upon encounter with microorganisms. Endotoxin shock, one of the most important causes of morbidity in our medical history, is now revealed to be a result of the overactivation of the innate immune system. These recent findings on the negative regulation of the TLR system should pave the way to enable control of this system and eventually to the development of new pharmaceutical strategies to combat endotoxin shock and other inflammatory disorders.

Received July 11, 2003; accepted September 25, 2003.


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