Toll-like receptor 4 is a member of the cell surface pattern recognition receptors involved in pathogenesis of several infectious and autoimmune diseases. The wide range of Toll-like receptor 4 extrinsic and intrinsic ligands means that it has considerable ability to trigger infectious and sterile inflammation, the latter assumed to be the principal cause of ischemia-reperfusion injury. With the rising number of renal transplant procedures using deceased donors, in addition to prolonged ischemia time due to organ transport and consequently increased risk of ischemia-induced injuries, the prevention of detrimental immune responses and/or overcoming these after they initiate could be beneficial for graft survival. This review aims to summarize past and present studies conducted about the role of Toll-like receptor 4 in early and late phases of transplant, including gene expression and polymorphism evaluations.
Key words : Deceased donors, Graft survival, Immune response, Ischemia-reperfusion injury
Toll-like receptors (TLRs) have been recognized as one of the most important receptors of innate immunity, which are involved in initiation of immune responses and development of inflammation.1 The role of TLRs has been investigated in several immune-based diseases, particularly its role in posttransplant events and autoimmunity. The implications of the numerous ligands of Toll-like receptor 4 (TLR4) and their considerable diversity in disease pathogenesis are of interest in the clinic. Toll-like receptor 4 gene expression has been studied in different phases of transplant, especially in early phases. These efforts are directed toward finding novel methods of manipulating immunologic factors to improve graft survival.2
Toll-like receptor 4 belongs to the leucine-rich receptor family, which consists of a wide variety of receptors involved in pattern recognition, signal transduction, and cell-cycle regulation. The extra- and intracellular components of TLR4 are composed of 608 and 187 residues, respectively. The intracellular portion is called the Toll-interleukin 1 receptor (TIR) domain. The extracellular domain has 24 leucine-rich receptor modules, each one containing variable residues (20-33 amino acids); these modules are arranged in a special form such that the LxxLxLxxN motifs shape a concave surface by generating B sheets and the hydrophilic motifs induce a convex surface that contains several 310 helices, creating a horseshoe shape for this molecule.
There are 3 subdomains in the TLR4 molecule: the N-terminal, the C-terminal, and the central area with different hydrophobicities and curvatures that twist the 2 ends of the horseshoe structure inward and outward.3 Toll-like receptor 4 is often displayed when binding to its coreceptor, myeloid differentiation protein 2, on convex surfaces of the central subdomain. The TLR4-myeloid differentiation protein 2 combination appears in a dimeric form on the cell surface4 (Figure 1).
Ligands and Function
The best known ligand of TLR4 is the bacterial antigen lipopolysaccharide (LPS); others recognized by TLR4 include the pathogen-associated molecular pattern teichuronic acid, mannuronic acid polymers, mannan, F protein of respiratory syncytial virus, flavolipin from Flavobacterium meningosepticum, and plant paclitaxel. Moreover, some endogenous molecules can bind to TLR4 and elicit immunologic responses. These molecules have been categorized into 2 groups of extracellular and intracellular ligands.5 Extracellular ligands include hyaluronan, fibronectin, heparan sulfate, biglycan, and tenascin C.6 These molecules usually must undergo deformation or degradation to bind to TLR4. For example, huge glycosaminoglycan hyaluronan matrix molecules, after breaking into small fragments due to tissue damage, can stimulate tissue antigen-presenting cells. The TLR4 intracellular ligands include heat shock proteins (HSPs),7 high-mobility group box 1 protein (HMGB1), surface protein A, beta-defensin-2, calcineurin B, S100 proteins, resistin, fibrinogen, and amyloid B.8 These ligands are either released because of cell damage or secreted due to unusual stimulation.
Among these ligands, HMGB1 and HSPs are the most studied endogenous activators of TLR4 in transplant. The activator HMGB1 is an intranucleos stabilizing factor that organizes DNA transcript, but it can be secreted actively from immune cells or released passively from necrotic cells during tissue damage such as during ischemia.9 Heat shock proteins are chaperone molecules that help with folding of intracellular proteins; however, in stress conditions, they are released from apoptotic cells and can induce inflammatory responses via TLR4 activation.10
All of these above-mentioned ligands have stimulatory effects on TLR4; nonetheless, there have been 2 molecules described as TLR4 antagonists: LPSs of Rhodobacter species11 and LPS-like (CyP) molecules of Oscillatoria species. These render unresponsiveness or attenuate reactions after binding to TLR4; therefore, there have been some attempts to apply such molecules in ameliorating exaggerated responses to microbial antigens12 (Table 1).
Toll-like receptor 4 arranges signaling by 2 MyD88-dependent and MyD88-independent pathways. In the first pathway, the TIR domain-containing adaptor protein moves toward TIR and activates MyD88. Activated MyD88 induces phosphorylation of interleukin 1 receptor-associated kinase (IRAK-1) and IRAK-4 molecules. Next, IRAK-1 activates tumor necrosis factor (TNF) receptor-associated factor 6, which switches on nuclear factor κB and activator protein 1 (AP-1) transcription factors; these molecules then translocate to the nucleus and initiate proinflammatory cytokine gene transcription.13
In the MyD88-independent pathway, the TIR-domain-containing adapter-inducing interferon-β related adaptor molecule binds to TIR and activates the TRIF molecule. The TRIF molecule as an adaptor protein phosphorylates TNF receptor-associated factor 3 (not shown) and receptor-interacting protein 1, leading to activation of TANK binding kinase 1 and mitogen-activated protein kinase (MAPK), respectively. In consequence, MAPK activates the AP-1 molecule, whereas TANK binding kinase 1 recruits interferon regulatory factor 3 (IRF3) together with other members of this family, including IRF7. As a result, transcription factors AP-1, IRF3, and IRF7 enter the nucleus and trigger type I interferon and production of other proinflammatory cytokines14 (Figure 2).
Tissue and Blood Expression
The main TLR4-expressing cells are antigen-presenting cells like monocytes, Langerhans, dendritic, and B cells; TLR4 is also expressed constitutionally in certain somatic cells such as endothelium, thyroidal mesangium, and endometrium, as well as in myocytes and adipocytes.15 The expression of TLR4 has also been detected in epithelial cells of proximal tubules and Bowman capsules in the kidney.16 The rationale of TLR4’s presence on nonimmune cells has not yet been fully understood, but it is hypothesized that this molecule is involved in developing initial responses to stress conditions in tissue.
Ischemia due to transplant procedures occurs as a result of organ preservation (cold ischemia) and/or surgery processes (warm ischemia). During the ischemic period, some delicate components of tissues are disrupted; therefore, some rarely or nonexposed molecules are generated, spread, and become available to damage-associated molecular pattern (DAMP) receptors such as TLR4.17 The most known molecules in this context as mentioned before are HMGB1, heat shock proteins, hyaluronan, fibronectin, and heparin sulfate.18 Toll-like receptor 4 is constitutionally expressed on proximal tubules of nephrons; however, after reperfusion, it is upregulated in other parts of renal tissue such as distal tubules, glomeruli, vascular endothelium,19 and podocytes20; therefore, during ischemia-induced stress, both TLR4 expression and endogenous ligand release are increased, resulting in proinflammatory responses. The activation of TLR4 leads to 2 events. First is adhesion molecule upregulation. Subsequent to signaling, ICAM-1, VCAM-1, and E-selectin molecule expression is enhanced in vascular endothelium, facilitating leukocyte recruitment and trafficking in allograft tissue. This process is followed by the second event, which is secretion of interleukin (IL)-6, IL-1, TNF-α, MIP-2, and MCP-1 cytokines.21 Afterward, graft damage occurs due to immune cell invasion and activation. Reactive oxygen species and proteolytic enzymes are produced by macrophages and neutrophils, which exacerbate the tissue damage process and result in more DAMP release. These cells recruit lymphocytes to the allograft; however, considering that the invited lymphocytes have originated from the recipient, their encounter with alloantigens and minor antigens like agrin and angiotensin II receptor,22 which were hidden before, may cause sustained inflammation and more injuries.
To find out the direct and indirect influences of early TLR4 activation on transplant prognosis, several studies have been conducted in animal models and in human patients. Shen and colleagues, in their comparison of wild-type and TLR4 knockout mice in a liver transplant model after 24 hours of organ preservation at 4°C, demonstrated less infiltration of CD4-positive T cells and lower serum levels of interferon-γ, CXCL10, TNF-α, IL-1b, and IL-2 and less ICAM-1 endothelial expression in the knockout group. However, IL-4 and IL-10 cytokine production was increased.23 Less myocardial infarction injuries were shown in TLR4-deficient mice after ischemia induction, whereas the wild-type group expressed significantly more IL-12 and interferon-γ in response.24
The role of TLR4 in initiating ischemia-reperfusion injury after liver transplant was examined in wild-type versus TLR knockout mice in 2 separate studies with the same findings, which was that lack of TLR4 in recipients protects the allograft from ischemia-induced injuries.25,26 Chong and associates have also reported reduced injuries after heart ischemia in TLR4-/- mice.27 TLR4-/- and MyD88-/- mice do not show enhanced expression of NKG2D ligands, retinoic acid early inducible 1, murine ULBP-like transcript 1, and histocompatibility 60 molecule on tubular epithelium in the ischemia-reperfusion phase of renal transplant.28 Recently, Chang and associates showed that administration of TAK-242 (a TLR4 antagonist) significantly protects islet cell transplant against ischemia-reperfusion injury in a mouse model.29 Pulskens and associates, who studied TLR4-/-, MyD88-/-, and TRIF-/- mutant mice under ischemia-reperfusion injury conditions, found significantly different grades of tissue damage between TLR4-/- and wild-type mice.30 Surprisingly, other mutations did not show any considerable influence on transplant outcomes.30
On the other hand, TLR4 overexpression could have a negative influence on the allograft. Chen and associates induced TLR4 expression in endothelial cells by hydrogen peroxide in vitro; later, in ischemic conditions, these cells expressed significantly more CD54 and CD62E adhesion molecules on their surface, whereas the knockout group did not show any gene upregulation.19
To define the influence of TLR4 ligands on ischemia-reperfusion injury, Wu and associates demonstrated that anti-HMGB1 antibody administration in mice can promote allograft condition and reduce proinflammatory responses; conversely, recombinant HMGB1 caused negative effects with transplant.31
Regarding the implication of TLR4 in ischemia-related injuries, there have been some efforts to reduce ischemia-reperfusion injury by preventing TLR4 activation through administration of high-dose dexamethasone32 or recombinant HMGB133 before transplant in mouse models. Treatment with dexamethasone reduced IκB-α phosphorylation and nuclear factor κB expression; moreover, preconditioning with recombinant HMGB1 induced Siglec-G molecule expression, which regulates HMGB1-mediated TLR4 activation.32,33 Together, most collected data have suggested considerable involvement of TLR4 in ischemia-reperfusion injury and the association between its overexpression and more ischemia-reperfusion injury. On the other hand, knockout animals have shown better outcomes and tissue survival after the ischemic period.
After initial inflammation due to ischemia-reperfusion injury, a sustained inflammatory process could be induced in the allograft. The tissue damage-immune activation cycle due to allo-/autoantigens appears to be continually released subclinically even after immunosuppressive drug administration. This immune activation can be amplified if additional stimulatory factors like infections are included. A wide range of microbial antigens could be recognized by TLR4 and induce immune responses; viral infections such as Cytomegalovirus (CMV) play considerable roles here.
The role of TLR4 in acute rejection has been investigated in various solid-organ transplant models. In their investigation of a mouse intestinal transplant model, Krams and associates showed upregulated expression of TLR4 and TLR2 in allograft tissue; they also reported prolonged survival in TLR4-/- recipients in fully mismatched models together with less inflammatory cytokines in the circulation.34 Moreover, Goldberg and associates attributed the protective role of carbon monoxide against acute rejection in a mouse islet cell transplant model at least in part to TLR4 inhibition.35 Testro and associates showed increased baseline and stimulation-induced expression of the TLR4 gene, as well as elevated TLR4-dependent production of IL-6 and TNF-α cytokines in peripheral blood monocytes of liver transplant patients with acute rejection.36 In their microarray study using nephrectomy allograft tissues from patients with acute and chronic renal transplant rejection, Nogueira and associates showed TLR2 and TLR4 gene expression in tubular epithelial cells but did not find any enhanced gene expression in rejected tissues.37 We have also investigated TLR4 and TLR2 gene expression in biopsy samples from kidney transplant recipients during acute rejection episodes; however, despite MyD88 gene upregulation, neither TLR2 nor TLR4 genes were overexpressed (unpublished observations from Assadiasl and associates).
In nonfunctional TLR4, deleted TLR4, and wild-type skin transplant mice, Samstein and colleagues could not find any significant differences in graft survival between these groups across minor and major histocompatibility mismatch antigens.38 In another study, Zhai and associates showed that TLR4 stimulation by LPS did not break the anti-CD154 monoclonal antibody-induced tolerance in a murine cardiac transplant model.39 In brief, according to the evidence, TLR4 activation in the acute rejection process is not well-defined. To clarify the direct and indirect association of TLR4 with acute rejection, it is necessary to conduct larger studies and perform meta-analysis of all available data.
Despite numerous molecular studies, causes of chronic rejection or chronic allograft nephropathy (CAN), which are characterized by interstitial fibrosis, tubular atrophy, and arteriopathy, have not been clearly understood. Chronic allograft nephropathy is defined as a continuously developing process with various speeds among recipients, induced by a range of predisposing factors such as sustained alloreactivity, immunosuppressive drug toxicity, infections, and repeated acute rejection episodes.40 Prolonged damage of allograft tissue is accompanied by fibroblast activation and fibrin accumulation in interstitium, leading to tissue deformation and loss of function.41 In addition to a proinflammatory environment in the allograft due to alloimmune response, TLR4 can be involved in CAN in other ways. Similar to ischemia-reperfusion injury, which is mediated by DAMP release, obstructive vasculopathies due to chronic rejection could also activate TLR4 through sustained ischemia, resulting in the same injuries.
There is accumulating evidence supporting the role of TLR4 in chronic alloimmune responses in renal transplant recipients. Braudeau and associates showed that expression of TLR4 and MyD88 genes in tissue samples and peripheral blood monocytes of chronic rejection patients was significantly increased compared with operationally tolerant recipients, with expression in stable recipients resembling that of healthy volunteers.42 Our similar findings showed that expression of TLR2, TLR4, and MyD88 genes was increased in tissue from patients with chronic rejection; however, in peripheral blood monocytes, only TLR4 overexpression was detected (unpublished observations from Assadiasl and associates). In an animal study from Wang and associates, TLR2/4-/-, MyD88-/-, and TRIF-/- recipients from wild-type donors demonstrated significantly better graft function; lower levels of IL-6, IL-10, MCP-1, and IL-12p70; and reduced numbers of T cells, dendritic cells, and macrophages in tissue. Moreover, deficient recipients showed less deposition of collagens I and III and smooth muscle actin-positive cells in allografts.43
The implication of TLR4 in chronic tissue damage and progressive fibrosis has also been studied in other solid-organ transplant patients. Methe and colleagues compared CD14-positive monocyte levels in patients with chronic cardiac rejection with those of stable recipients and found significant expression of TLR4 in circulating monocytes of patients with rejection. These cells were shown to produce more IL-12 and TNF-α cytokines. They also reported similar findings in a mouse cardiac transplant model, with mice with rejection exhibiting elevated TLR4 mRNA levels.44 Palmer and associates detected increased levels of hyaluronan as an intrinsic ligand of TLR4 in lung transplant chronic rejection tissue (BOS syndrome).45 Despite limited numbers of studies, almost all suggested an association between TLR4 and chronic allograft damage. Activation of TLR4 during ischemia could be due to vascular thickening and occlusion followed by DAMP release, resulting in a sustained subclinical inflammatory process and fibrin formation, which induce a defective cycle of ischemic fibrosis.
Up to now, there have been 13 TLR4 gene polymorphisms recognized in White populations.46 Among these polymorphisms, 2 ecto-domain mutations are of clinical significance because, in their presence, TLR4 activity is attenuated, affecting immune response to its ligands. These polymorphisms consist of Asp299Gly (aspartic acid replacement with glycine; rs4986790) and Thr399Ile (threonine substitution with isoleucine; rs4986791), which are relatively common with a frequency of approximately 5% and are usually inherited in cosegregated form.47
Although these variations make organ recipients more susceptible to infectious disease, especially opportunist species like CMV, they could be beneficial in the context of graft survival because hyporeactive TLR4 do not respond properly to DAMPs. Many studies were conducted to find whether significant correlations existed between TLR4 polymorphisms and allograft outcomes.
In 238 renal transplant recipients, Ducloux and associates reported less atherosclerotic events and reduced episodes of acute rejection in TLR4-mutant recipients; however, these patients experienced more bacterial, CMV, and opportunistic infections.48 The association between the Asp299Gly loss-of-function mutation and lower ischemia-reperfusion injury after liver transplant has already been reported.49 Lung transplant recipients with Asp299Gly or Thr399Ile also exhibited decreased acute rejection rates over a 6-month follow-up compared with those who possessed wild-type TLR4.47 Palmer and associates also reported reduced rejection episodes in heterozygote donors with Asp299Gly or Thr399Ile alleles, although recipients possessing these polymorphisms did not show better outcomes.50 Souza and associates demonstrated that TLR4 nonfunctional mutations protected mice against chronic kidney disease progression and fibrosis secondary to inflammation.51 Kwan and associates also found no significant differences in long-term allograft survival (100 days) between wild-type and TLR4-mutant type mice; however, lower creatinine levels, CD11c-positive dendritic cell accumulation, and IL-2 overexpression and increased indoleamine 2,3-dioxygenase were shown in graft tissue during the first 2 weeks after transplant in mutant mice.52
Mutlubas and associates demonstrated an association between Thr399Ile mutations and reduced rates of CAN in pediatric renal transplant recipients, a finding that was not obtained with Asp299Gly mutations.53 In a follow-up study of 216 renal transplant donor-recipient pairs, rs10759932 CC genotype presence in either recipient or donor was accompanied by less episodes of acute rejection, with the authors suggesting that this mutation was a protective factor against rejection that promoted graft survival.54 In 201 renal transplant donor/recipients pairs, Nogueira and colleagues found no significant differences in acute tubular necrosis and acute rejection incidence between recipients from TLR4-mutant and wild-type donors. Mutant recipients also did not exhibit better outcomes versus the wild-type group. Bacterial infection rate also did not show any correlation with TLR4 mutations.55 However, the group later demonstrated that rs4986790 (pAsp299Gly) and rs4986791 (pThr399Ile) polymorphisms are associated with attenuated TNF-α secretion and lower TLR4 expression in peripheral monocytes and neutrophils of renal transplant recipients.56 Kruger and associates earlier showed that mutant TLR4 with lower affinity to HMGB1 contributed to better graft function and was associated with lower TNF-α and MCP1 serum levels in renal transplant patients.18
In a pediatric study, neither acute/chronic rejection nor posttransplant infection incidence (including CMV) in recipients was affected by the TLR4 nonfunctional mutation (Asp299Gly).57 In addition, Abdolvahabi and associates investigated single-nucleotide polymorphisms of the TLR4 gene in 239 renal allograft recipients but could not find any significant differences between stable recipients and those who had experienced acute rejection episodes.58
Several studies have evaluated the influence of TLR4 ligand polymorphisms on posttransplant events. Fekete and associates reported higher frequencies of HSPA1B (1267)AA and TLR4 (299)AG genotypes in successful renal transplant recipients after 15 years of follow-up, whereas HSPA1A G(190)C polymorphism frequency was not different between groups with and without rejection.59
Although most polymorphism studies are in favor of the detrimental role of TLR4 activation after transplant, no consensus has been shown about the beneficial effects of Asp299Gly/Thr399Ile mutations in recipients or donors. Interpretations of recipient and donor-based studies are not clear because some trigger stimulators, like hypoxia, activate TLR4 within a donor’s allograft tissue, whereas the recipient’s TLR4 contributes to the rejection process due to alloimmune responses in peripheral blood monocytes and the infectious stimulators that involve both donors and recipients.
Determining the implications of innate immunity in allograft damage can be helpful in creating new methods to prevent rejection and promote graft survival, with TLRs as initiator factors in inflammation playing a considerable role. Studies on the function of TLR4 during various periods of transplant have suggested that TLR4 plays a considerable role in ischemic allograft injuries at both early and late phases. However, not enough evidence is available regarding its involvement in acute rejection. Non- and low-functional polymorphisms of TLR4 may have a protective role in transplant.
Volume : 16
Issue : 3
Pages : 245 - 252
DOI : 10.6002/ect.2017.0308
From the 1Molecular Immunology Research Center, Tehran University of Medical
Sciences, Tehran, Iran; the 2Department of Immunology, School of Medicine,
Tehran University of Medical Sciences, Tehran, Iran; and the 3Department of
Hematology, Faculty of Allied Medicine, Bushehr University of Medical Sciences,
Acknowledgements: The authors have no sources of funding for this study and have no conflicts of interest to declare.
Corresponding author: Aliakbar Amirzargar, Immunology Building No.7, School of Medicine, TUMS, Tehran, Iran
Figure 1. Toll-Like Receptor 4 Structure
Figure 2. Toll-Like Receptor 4 Signaling
Table 1. Toll-Like Receptor 4 Ligands