Toll-like receptor signaling in transplantation

Abstract

Purpose of the review

This review summarizes recent advances on the role of endogenous and exogenous Toll-like receptor (TLR) ligands in the activation and inhibition of immune responses in transplantation.

Recent findings

During an alloresponse, TLRs can be engaged by both damage-induced endogenous ligands or microbial-associated molecular patterns. The damage-induced molecule high mobility group box 1 protein (HGMB1) and its binding to TLR4 have been identified as major initiators of anti-tumor and anti-transplant immune responses. Type I interferon (IFN) signaling plays an important role in the pro-rejection effect mediated by TLR agonists and some bacteria. However, similar pathways in neonates can result in inhibition rather than activation of alloimmune responses.

Summary

The consequences of TLR engagement by endogenous and exogenous ligands in transplantation may depend on the relative induction of inflammatory and regulatory pathways and the stage of development of the immune system.

Introduction

TLR-mediated signals play a role in acute allograft rejection and antagonize the induction of transplantation tolerance. However, TLR engagement can also promote tolerance in several experimental models. Recent developments in the biology of TLR signaling in transplantation include a better understanding of endogenous and exogenous ligands that can generate TLR signals in allograft recipients, as well as increased attention to possible negative regulatory loops induced by TLR ligation.

1. TLR signals promote acute allograft rejection

TLRs are pattern-recognition receptors expressed on both hematopoietic and non-hematopoietic cells such as endothelial and epithelial cells. At least 13 TLRs have been identified to date. All of them require adaptor MyD88 for signal transduction except for TLR3, which utilizes the adaptor Trif, and TLR4, which can signal via both MyD88 and Trif pathways. Using mice deficient in MyD88 or MyD88 and Trif, it has been shown that TLR signals by donor and recipient cells are absolutely necessary for the rejection of allografts expressing minor histocompatibility antigen mismatches [1,2], while they promote, but are not required for, acute rejection of allografts with major mismatches [3]. Furthermore, genetic ablation of MyD88 facilitates the induction of transplantation tolerance by costimulation-targeting therapies [4,5] and, conversely, administration of synthetic TLR agonists prevents transplantation tolerance by the same regimens [5,6]. The current model states that antigen-presenting cells stimulated by TLRs at the time of transplantation will incur maturation and enhanced presentation of alloantigen, resulting in augmented responses of alloreactive T cells that may then become resistant to tolerance induction. However, the ligands that trigger these TLR signals after transplantation remain to be ascertained. It is clear that molecular patterns expressed by microorganisms can engage TLRs and we have proposed that translocation of commensal bacteria (or bacterial products) during the surgical procedure, or concurrent infections in donors or recipients, may result in TLR signaling and its consequences on alloresponses [5]. However, several reports have also argued for the existence of endogenous non-microbial ligands generated as a consequence of cellular damage. This concept had initially been met with skepticism given the difficulty to definitively exclude the contribution of potential microbial contaminants [7]. However, recent results lend credence to a role of damage-associated molecular patterns (DAMPs) in vivo and a role for selected endogenous ligands of TLRs in transplant responses is emerging.

2. Non-microbial endogenous ligands of TLRs

Several endogenous ligands have been described that are released during inflammation, cellular stress and damage and can engage TLRs. These include heat shock proteins (HSPs), extracellular matrix-derived products, HMGB1, β-defensin, surfactant protein A, and minimally modified LDL [8]. To date, only HMGB1, HSPs and hyaluronan have been linked to transplant responses and will be discussed herein.

2.1 HMGB1

HMGB1 was described as a nuclear protein with fast migration properties during electrophoresis and was named accordingly [9]. Its sequence has been highly conserved during evolution and HMGB1 proteins from all mammals are virtually identical. Its expression is essential as mice deficient in HMGB1 die shortly after birth from hypoglycemia [10]. HMGB1 mostly resides in the nucleus where it is known to bind without sequence specificity to minor DNA grooves, resulting in DNA bending and promotion of protein assembly [11]. HMGB1 can both be passively released by necrotic cells [12], or be actively secreted by upon cell exposure to tumor necrosis factor (TNF), interleukin-1β(IL-1β), lipopolysaccharide (LPS) or other TLR agonists [13] [14,15]. Thus, it can be present in soluble form during both sterile and non-sterile inflammation.

Soluble HMGB1 has been shown to bind the receptor for advanced glycation end products (RAGE), expressed at low levels on many cells types [11], but also to selected TLRs, namely TLR 2 and TLR4. Ligation of HMGB1 to TLR2 was initially controversial as macrophage cell lines transfected with dominant negative constructs of these TLRs showed reduced HMGB1 signaling [16], but not macrophages from TLR2-deficient mice [17]. These discrepancies may be due to redundancy between RAGE and TLR signaling in response to HMGB1. More recently, HMGB1 has also been shown to associate with TLR9 [14,18]. HMGB1 interacted with CpG-A oligonucleotides resulting in enhanced activation of plasmacytoid dendritic cells (pDCs), B cells and macrophages in a RAGE- and TLR9-dependent manner [14,18]. Thus the association of HMGB1 with TLR9, though indirect, may facilitate TLR9 signaling by DNA-containing immune complexes and perhaps contribute to autoimmunity.

In support of an important role for the HMGB1/TLR pathway in immune responses in vivo, recent evidence shows that the anti-tumor immune response elicited by radiation therapy or chemotherapy is due to the release by dying tumor cells of HMGB1, which acts on TLR4/MyD88 on dendritic cells. This anti-tumor effect is abolished in either anti-HMGB1-treated mice or mice deficient in TLR4 but not in other TLRs [19]. These results strongly support an important role for endogenous HMGB1 via TLR4 engagement in the absence of bacterial contamination. At least part of the inflammatory effect of HMGB1 may be due to its ability to induce IL-1 production by monocytes in a CD14 and TLR4-dependent manner, resulting in production of IFN-γ and IL-17 by T cells [20]. Furthermore, HMGB1 signaling via TLRs activates the transcription factor NF-κB, which itself induces further upregulation of HMGB1 and its receptors thereby amplifying inflammation in a positive feedback loop [15].

The role of HMGB1 in transplantation may be many fold. Ischemia/reperfusion injury (IRI) induces tissue damage and liver IRI has been shown to result in rapid release of HMGB1 [21]. Its production by ischemic hepatocytes was demonstrated to be TLR4-dependent and due to induction of reactive oxygen species and subsequent calcium/calmodulin-dependent kinases signaling [22]. Extracellular HMGB1 in turn serves as a chemoattractant for inflammatory cells and leads to hepatic tissue injury, as damage can be reduced using blocking antibodies to HMGB1. These harmful effects of HMGB1 depend on TLR4 expression on hematopoietic cells [23,24].

Following IRI, damage of a transplanted organ can occur as a consequence of alloresponses or autoreactivity. It was recently shown that cellular targets of cytotoxic T cells can release HMGB1 [25]. In addition, activated immune cells, as found infiltrating transplanted organs undergoing acute rejection, can actively secrete HMGB1. Blockade of HMGB1 using a recombinant A-box that acts as a selective antagonist by reducing activity of full-length HMGB1, resulted in prolonged survival of fully mismatched cardiac allografts in mice, that correlated with reduced intra-graft expression of TNF, IFN-γ and impaired Th1 immune responses [26]. Similar results in another murine cardiac allograft model were obtained upon blocking HMGB1 with soluble RAGE, which may prevent engagement of HMGB1 to all its ligands [27]. Whether these pro-rejection effects of HMGB1 are mediated via TLRs 2, 4 or 9, RAGE or a combination of these receptors remains to be established. Interestingly, endogenous production of anti-HMGB1 antibodies was identified during a tolerance phase after liver transplantation in rats, as well as in a patient following withdrawal of immunosuppressive therapy [28]. Although this study did not demonstrate that these endogenous antibodies could block HMGB1 ligation to its receptors, it further suggests that blockade of HMGB1 (either therapeutic or spontaneous) may facilitate graft acceptance.

2.2 Heat shock proteins

HSPs are important chaperones for protein folding and cytoprotection and are mostly found in the cytoplasm of cells, although some are also encountered in mitochondria or nuclei [29]. HSPs are classified into families according to their approximate molecular weight. Although they are expressed at low levels in all cells, their expression is markedly induced under conditions of cellular damage that can also result in their extracellular release by a mechanism that is not yet fully understood [30]. HSPs have been reported to use several surface receptors, including CD14, CD40, CCR5 and TLRs [29]. Both HSP60 and HSP70 are thought to signal via TLR2 and TLR4 [30].

The role of endogenous HSPs in allograft rejection remains to be firmly established. Expression of HSPs, including HSP60 and HSP70, has been detected in transplanted organs [31] and transgenic expression of HSP60 on donor skin has been shown to accelerate allograft rejection [32]. However, the combined deficiency in HSP70.1 and 70.3, the two forms of HSP70 induced by stress in the mouse, in both donor and recipient animals did not affect allograft rejection, dendritic cell maturation or Th1 alloresponses in a minor mismatched murine skin graft model whose rejection is known to depend on MyD88-mediated signals [33]. This result is in contrast to earlier work in which single deletion of HSP70.1 in only donor skin resulted in a statistically significant prolongation of allograft survival in a fully mismatched mouse model, albeit only by a couple of days [34]. It is possible that various HSPs have redundant roles and would all need to be targeted to observe more biologically significant impact on allograft survival.

Hyaluronan

Hyaluronan is a component of the extracellular matrix that accumulates during tissue injury. Clearance of hyaluronan products is mediated by the main hyaluronan receptor CD44. However, hyaluronan fragments can also bind TLR2 and TLR4 resulting in activation of macrophages [35]. Levels of hyaluronan have been shown to be elevated during acute rejection in rat serum and cardiac allografts [36] and in mouse skin allografts [37] and to be associated with bronchiolitis obliterans syndrome in human lung allograft recipients [37]. Treatment of dendritic cells with hyaluronan fragments induced their maturation and enhanced their capacity to initiate alloimmunity in vitro in a manner dependent on signaling via TIR-associated protein (TIRAP), an adaptor for TLR2 and TLR4, but independent from MyD88 and Trif [37]. Thus, components of the extracellular matrix can act as immune adjuvants and potentiate alloreactivity.

2. Microbial ligands of TLRs

Transplantation, like other major surgical procedures can result in translocation of commensal bacteria from skin incisions and intestinal stress. In particular, bacterial and endotoxin translocation have been identified following clinical liver and intestinal transplantation [38,39]. Because transplanted patients are heavily immunosuppressed to prevent allograft rejection, they are more susceptible to pathogenic viral, bacterial and fungal infections. It is conceivable that exposure to both commensal and pathogenic microorganisms promotes allograft responses in transplanted patients.

2.1 Common pathogens in transplanted patients

The incidence and clinical characteristics of infections after solid organ transplantation was described in a retrospective study of 2702 solid organ transplant recipients from the database of the Spanish Network of Infection in Transplantation (RESITRA) [40]. The incidence of infections in the first month after transplantation was observed to be significantly higher (3.5 episodes per 1000 transplant-days) compared to that in the late period (0.4 episodes per 1000 transplantation days), with higher rates of infections following kidney-pancreas, lung and liver allografts (11.52–14.47 episodes per 1000 transplantation days) and lower rates for kidney and heart allografts (4.91–8.78 episodes per 1000 transplantation days). In the late period of >6 months post-transplantation, infection rates were highest for lung allografts (1.4 episodes per 1000 transplantation days), intermediate (0.76 episodes per 1000 transplantation days) for liver, and low for kidney and heart allografts (0.28–0.34 episodes per 1000 transplantation days). Bacterial infections were the most common, and represented 80% of infections in the first month post-transplantation and 50% of all infections after six months post-transplantation. Viral infections, especially CMV, were the second most frequent, contributing 13% and 46% of all infections in the first and >6 months post-transplantation. The rate of fungal infections was 2.5–5% for the study duration, which had median follow-up of 13 months.

The incidence of blood stream infections after solid organ and hematopoietic stem cell transplantation was examined from the same RISTRA database [41]. The incidence of blood stream infections was 7.3–10% for kidney, heart, lung and liver recipients and 20% for pancreas recipients, and significantly higher for hematopoietic stem cell transplant recipients (42.3% for allogeneic recipients and 26% for autologous recipients). Blood stream infections were largely the result of Gram-negative and Gram-positive bacterial infections, with coagulase-negative staphylococci (37%) being the most frequent, followed by E. coli (17%).

Fungal, bacterial and viral infections can profoundly impact the morbidity and mortality of transplant patients [42–44]. In addition, there is evidence that viral infections, especially CMV, contribute to the development of acute and chronic rejection [45]. The impact of bacterial infections on graft rejection is less appreciated. Nonetheless, there is a significant literature correlating bacterial infections and graft rejection (reviewed in [46]). The evidence for fungal infections precipitating rejection is least well documented. Because of adjustments to immunosuppression made in response to infections, the relationships between immunosuppression, infections and allograft rejection are difficult to definitively appreciate solely from clinical observations.

2.2 Pathogen recognition by TLRs

Pathogens, including bacteria, viruses and fungi, can be recognized by host innate immune cells through an array of pattern recognition receptors (PRRs), of which, the best characterized are the TLRs. TLRs that are located on the cell surface recognize cell wall components of bacteria and fungi or envelop proteins of viruses, while endosomally located TLRs recognize viral dsRNA or ssRNA, or unmethylated CpG motifs in DNA. Table I lists potential ligands of pathogens commonly found in clinical transplantation and the TLRs they can engage. Non-TLR PPRs include retinoic-acid-inducible gene I-like helicases (RLH), nucleotide-oligomerization domain leucine-rich repeat (NOD-LRR), C-type lectin and cytosolic DNA-dependent activator of IFN-regulatory factors (DAI) proteins (reviewed in [47–50]). All PRRs can trigger distinct intracellular signals that induce the production of cytokines and inflammatory mediators, providing the cues for the development of adaptive immune responses that, in most instances, control the infection.

Table I. Common infections in transplant recipients and their potential ligands for TLRs.

CMV (cytomegalovirus) HSV-I (herpes virus simplex type I)

Infections	Microbial Agent or Component	TLR
Bacterial	LPS	TLR4
	Diacyl lipopeptides	TLR2/TLR6
	Triacyl lipopeptides	TLR2/TLR1
	Lipoteichoic acid	TLR2/TLR6
	Phenol-soluble modulin	TLR2
	Atypical LPS	TLR2
	Flagellin	TLR5
	CpG DNA	TLR9
	Uropathogenic bacteria	TLR11
Viral	Envelope Proteins of CMV and HSV-1	TLR2
	F protein of respiratory syncytial virus (RSV)	TLR4
	Double strand RNA virus	TLR3
	Single strand RNA virus	TLR7/TLR8
	DNA virus/CpG DNA	TLR9
Fungal	Apergillus	TLR2/TLR4
	Saccharomyces – Zymozan	TLR2/TLR6
	Saccharomyces – Mannan	TLR4
	Candida – phospholipomanan	TLR2
	Cryptococcus - Glucuronoxymannan	TLR4

The relative frequency of bacterial, viral and fungal infections post-transplantation, the ability of the innate immune system to sense these infections, and the cross-talk between innate and adaptive immunity predicts an impact of infections on alloreactivity and graft rejections. We and others had previously reported that TLR-ligation, with CpG (TLR9), LPS (TLR4), Pam₃CysK₄ (TLR1/2) and polyinosinic:polycytidylic acid (poly I:C, TLR3), at the time of allograft transplantation, prevents the ability of anti-CD154-based therapy to induce long-term graft survival [5,6]. More recently, Garantziotis and colleagues reported that inhaled LPS promoted lymphocytic bronchiolitis, epithelial injury and obliterative bronchiolar lesions in allogeneic bone marrow transplant recipients [51]. The inflammation induced by the inhaled LPS was shown to be dependent on TLR4-expression on donor-derived hematopoietic cells and not on recipient structural lung tissue, and resulted in sustained elevations of the inflammatory chemokine, CCL5, but not the cytokine, TNF. In a model of established bone marrow mixed chimeras, Chakraverty and colleagues reported that the transfer of donor T cells does not trigger GVHD. In contrast, the induction of inflammation by administration a TLR7 agonist, imiquimod to the skin, at the time of T cell transfer, triggers massive infiltration of T cells to the skin and localized GVHD [52]. These observations demonstrate that tissue inflammation, triggered by TLRs, can induce the trafficking and activation of alloreactive T cells, and allude to the existence of a tissue-localized checkpoint for GVHD. More recently, Durakovic and colleagues reported that the co-administration of CpG or imiquimod, intraperitoneally augmented the effector function of adoptively transferred donor lymphocyte infusions into stable mixed bone marrow chimeras and promoted graft-versus-host and graft-versus-leukemia activities [53]. These experimental data complement previous clinical reports of possible associations between TLR4 mutations and the risk for acute GVHD, although potentially increased risk for bacterial infections may complicate the interpretation of data in the clinical studies [54,55].

2.3 Effector pathways: Type I IFNs

Both viral and bacterial infections can elicit the production of type I IFNs, which is controlled by two major classes of PPRs: endosomal or plama membrane-associated TLRs and cytosolic receptors, such as RLHs and DAI [55,56]. The membrane-bound TLR4, and the endosomally-localized TLRs 3, 7, 8 and 9 can signal the production of type I IFNs. pDCs express high levels of TLR7 and TLR9, which stimulate the production of type I IFNs in a MyD88-dependent manner. Conventional DCs (cDCs) preferentially express TLR3, TLR8 and low levels of TLR4. TLR3 stimulate type I IFN production in a MyD88-independent and Trif-dependent pathway, while TLR4 does so in a TRAM/Trif and TIRAP/MyD88-dependent manner. TLR8 bears significant homology to TLR7, which stimulates type I IFN production in a MyD88-dependent manner. Kumaigai et al. reported that systemic infection with Newcastle disease virus resulted in the production of IFN-α by plasmacytoid DCs in a TLR-dependent manner, and by conventional DCs and macrophages in a RIG-I-like helicase-dependent manner [57]. In contrast, lung infections induced IFN-α production by alveolar macrophages and conventional DCs in a RIG-I-like helicase-dependent manner, but no production of IFN-α by plasmacytoid DCs [57]. Thus the cells responsible for, and the signaling pathways controlling, type I IFN production are defined by the infectious agent as well as the location of the infection [48,57].

Mechanistic studies by Thornley and colleagues revealed that both LPS and poly I:C were able to prevent the induction of skin tolerance by anti-CD154 plus donor specific transfusion [6]. LPS was shown to utilize a TLR4- and MyD88-dependent pathway, while the effects of poly I:C were independent of TLR-3 [58]. Nonetheless, the ability of both LPS and poly I:C to antagonize the effects of anti-CD154-based tolerance induction was dependent on IFN-α/β greceptor I (IFN-α/β RI) signaling [58]. The administration of IFN-β recapitulated the effects of LPS and poly I:C, demonstrating the sufficiency of type I IFNs to prevent the induction of allograft tolerance.

While synthetic ligands have proven invaluable for dissecting the impact of TLRs on alloreactivity, an important extension of the observations by Thornley et al. [58] is to investigate if real infections also alter allogeneic immune responses. To this end, we have tested the impact of a Listeria monocytogenes infection in a mouse model of heart allograft tolerance [59]. Listeria is a Gram-positive intracellular bacterium that, upon invasion into the cytosol of resting macrophages, induces the production of IFN-β [60]. We observed that Listeria infection at the time of transplantation antagonizes the ability of anti-CD154 and donor specific transfusion to induce long-term heart or skin allograft survival [59]. The effect of Listeria infection was lost in IFN-α/β RI-deficient mice, and recapitulated in wildtype mice injected with IFN-β. The pro-rejection effect of Listeria and its ability to induce IFN-β production were both largely independent of MyD88, consistent with previous reports that type I IFN production is dependent on the sensing of Listeria by cytosolic receptors in infected macrophages [60,61].

These observations demonstrate that viral and bacterial infections that elicit a strong production of type I IFNs, either through TLRs or non-TLR signaling pathways, may antagonize the induction of tolerance. It is clear that some infections do not elicit type I IFN production, and it would be of interest to determine whether such infections can also prevent the induction of tolerance and if so, define their effector mechanisms.

3. Inhibitory effects of TLR signaling

TLR activation leads to immunity, however excessive TLR signaling would induce inflammation and subsequent disease. TLR induced inflammation is controlled by negative feedback pathways via several inhibitory adaptor proteins. These include IRAK-M, A20, SOCS1 [62–64]. Previous studies have demonstrated that mice deficient in these genes manifest exaggerated responses to TLR activation and subsequent inflammation [62,63]. Additionally, repeated TLR activation in vitro can lead to reduced inflammatory responses, for example the phenomenon of endotoxin tolerance [65]. It was recently shown that altered chromatin modifications lead to gene silencing in endotoxin tolerance [66], although the in vivo significance of this finding is not clear. Furthermore, TLR activation directly on regulatory T cells can diminish immune responses [67–69]. In sum, although TLR activation leads to the initiation of potent inflammatory responses, immune regulatory pathways co-evolved to dampen immune responses and prevent undesirable immune pathology.

3.1 Immunoregulatory pathways in neonates

Prior studies have demonstrated that neonates are more prone to infectious disease and to immune tolerance [70–73]. Indeed, in human cardiac transplantation, neonatal recipients can receive ABO incompatible allografts under the cover of generalized immunosuppression [74]. One possible explanation for these findings is that neonates manifest exaggerated immune regulatory pathways and recent studies support this postulate. The work from the Lo-Man laboratory has elegantly demonstrated that neonatal B cells possess unique immunoregulatory properties, specifically upon TLR9 activation [75]. Their initial work demonstrated that neonatal DCs were capable of promoting Th1 immune responses [75]. However, neonatal CD5⁺ B cells reduced DC inflammatory responses upon CpG activation via an IL-10 dependent process. Work published this year by the same group demonstrated that neonatal CD5⁺ B cells are critical for protecting neonates from lethal CpG challenge [76]. In this study, neonatal B cells produced type I IFNs, and these cytokines down graded inflammatory responses upon TLR activation both in vitro in and in vivo. In contrast, type I IFNs in adult mice were inflammatory. Thus, these data demonstrate that neonatal B cells possess specific immunoregulatory properties that impair potent pro-inflammatory signals. These pathways may have evolved to limit inflammation in the neonatal period during the initial exposure to the environment.

In agreement with the Lo-Man laboratory, we have found that TLR-activated neonatal B cells (in response to activation from either TLR 2/6 via peptidoglycan, TLR4 via LPS or TLR9 via CpG) alter DC maturation responses [77]. Specifically, we determined that TLR activated neonatal B cells impaired the ability of adult myeloid DCs to upregulate costimulatory molecules. Furthermore, we found that TLR activated neonatal B cells impaired the ability of DCs to produce IL-12 upon TLR ligation. The presence of TLR activated neonatal B cells skewed T cells to a Th2 phenotype during in vitro mixed lymphocyte reactions. In vivo, we found that TLR activated neonatal B cells, reduced Th1 immune priming in response to immunization with allogeneic spleen cells or in response to skin allografts. Similar to findings from the Lo-Man laboratory [76], we found that IL-10 produced by TLR-activated neonatal B cells was both necessary and sufficient for the ability of neonatal B cells to alter in vitro alloimmune responses [77]. Thus, TLR activated neonatal B cells, in contrast to adult counterparts, modulate both in vitro and in vivo alloimune responses. Clearly, it will be important in future studies to determine if such pathways contribute to the immune tolerance-prone state of neonates.

3.2 Immunoregulatory pathways of endogenous TLR ligands

Similarly to well known reports of regulatory pathways triggered by exogenous ligands of TLRs, such as endotoxin tolerance [78], emerging evidence suggests that endogenous ligands of TLRs can also inhibit immune responses under certain circumstances. In contrast to the pro-inflammatory effects of HMGB1 described earlier, pre-conditioning with HMGB1 has been shown to protect against hepatic damage induced by liver IRI [79]. This anti-inflammatory effect was dependent on intact TLR4 signaling and was associated with upregulation of IRAK-M, one of the inhibitory proteins in the TLR pathways.

Growing evidence suggests that HSPs can also carry anti-inflammatory properties [29]. HSP gp96 is an endoplasmic reticulum chaperone for TLRs. Its predominant role appears to be pro-inflammatory as macrophages from mice with a macrophage-specific targeted deficiency in gp96 failed to respond to engagement of TLRs 2, 4, 5, 7 and 9, resulting in mice that are resistant to endotoxin shock in vivo [80]. However, HSP gp96 can also inhibit immune responses, as treatment with HSP gp96 resulted in improved survival of mouse skin allografts bearing either minor or major mismatches [81]. In a model of rat cardiac allograft, administration of high doses of HSP gp96 purified from donor but not recipient strain livers also resulted in prolongation of graft survival that was associated with T cell hyporesponsivenes to polyclonal stimuli [82]. Finally, although transgenic expression of membrane-bound HSP gp96 resulted in hyper-responsiveness to LPS and a TLR4-dependent lupus-like syndrome, it was also shown to increase the suppressive function of Tregs in a TLR4-dependent manner, providing a putative mechanism for the regulatory properties of damage-associated TLR ligands [83]. Collectively, these results point to potential therapeutic uses of stress-induced molecules.

Conclusion

In summary, growing evidence points to engagement of TLRs by damage-induced endogenous ligands after transplantation resulting in enhanced alloimmune responses. In addition, ligation of TLRs by exogenous ligands can prevent transplantation tolerance in a mechanism dependent on the production of type I IFNs, which may be shared by pathogenic microorganisms. Finally, despite the well known pro-inflammatory role of TLR signals, it is becoming clear that TLR pathways can also lead to immunosuppression and such mechanisms may be harnessed in the future for novel therapeutic manipulations in transplanted patients.