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Departments of Medicine, Surgery
Pathology, Duke University Medical Center and the Veterans Administration Medical Center, Durham, North Carolina
ABSTRACT
Rationale: Lung transplantation is characterized by very high rates of acute and chronic allograft rejection. We hypothesize that activation of innate immunity augments adaptive immunity, leading to rejection after lung transplantation. In support of this idea, we have recently demonstrated that lung recipients heterozygous for either of two functional polymorphisms (Asp299Gly or Thr399Ile) in Toll-like receptor 4 (TLR4) associated with endotoxin hyporesponsiveness have decreased acute rejection over the first 6 months after transplant. Objectives: In the current analysis, we sought to extend our initial observations and investigate the effect of these TLR4 polymorphisms on post-transplant acute rejection beyond the first 6 months, bacterial infections, bronchiolitis obliterans syndrome, and survival. Methods: Genotyping was performed on 170 lung transplant recipients. Measurements and main results: Recipients heterozygous for either Asp299Gly or Thr399Ile had significantly reduced frequency (p = 0.02) and incidence of acute rejection (p = 0.04) sustained over 3 years after transplant, but no differences were observed in the overall onset of bronchiolitis obliterans syndrome. A trend, however, toward reduced onset of bronchiolitis obliterans syndrome grade 2 or 3 was observed in TLR4 heterozygotes. Conclusion: Our results demonstrate that activation of recipient innate immune responses through TLR4 has a significant and sustained effect on the development of acute lung rejection. Targeting innate immune signaling represents a promising area for future clinical studies in the prevention of lung allograft rejection.
Key Words: innate immunity lung transplant TLR4
Innate immunity is an essential component of host defense against foreign pathogens. Secreted, cellular, and intracellular components of host innate defense recognize and respond to a wide range of pathogen-associated molecular patterns. The best-described group of innate immune receptors is a family of proteins known as Toll-like receptors (TLRs), each of which recognizes a different spectrum of pathogen-associated molecular patterns (1). Toll was originally described in Drosophila as a receptor directing dorsoventral polarity, and later shown to be critical for antifungal responses (2). The pathogen-associated molecular patterns recognized by innate immune receptors represent highly conserved molecular structures invariant among large classes of microbial pathogens that permit discrimination between self and microbial nonself. Endotoxin, or LPS, for example, is a prototypic trigger of innate immunity commonly found on gram-negative bacteria recognized by TLR4 (3).
Despite its central importance in immunity to foreign pathogens and discrimination of self from nonself, the role of innate immunity in transplant biology is just beginning to be defined. Current mechanistic paradigms of allograft rejection emphasize the role of adaptive immunity in orchestrating organ rejection. A potential role for innate immunity in allograft rejection was recently raised in studies of murine minor histocompatibility antigen mismatch skin-graft rejection. In this model, deficiency of the downstream TLR-signaling molecule, MyD88, in both donor and recipient prevented the development of skin-graft rejection (4). In a follow-up study, immune responses were also diminished in the absence of MyD88 signaling in fully major histocompatibility antigen (MHC) mismatched murine heart or skin transplants, although rejection still occurred in those model systems (5).
Because of constant environmental and inhalational exposures in the lung allograft, we believe that innate immune stimulation occurs frequently in the post-transplant lung environment and hypothesize that activation of innate immunity through TLRs directly contributes to the development of allograft rejection. In support of this hypothesis, we recently demonstrated reduced early acute rejection in lung transplant recipients with either of two loss-of-function polymorphisms in TLR4: Asp299Gly (299) or Thr399Ile (399) (6). These cosegregating single nucleotide polymorphisms of the TLR4 have been shown to produce blunted responses to endotoxin in vitro and in vivo (7).
The association between genetically determined, reduced endotoxin responsiveness and reduced early lung transplant rejection provides the first evidence in humans that activation of innate immunity is linked to the activation of adaptive immunity in the context of transplant rejection. Our initial observations, however, were limited to acute rejection within the first 6 months post-transplant. In the current analysis, we sought to extend our initial observations in a larger cohort of lung transplant recipients with extended follow-up (n = 170) and focus on multiple clinically relevant long-term patient outcomes. Specifically, we sought to investigate the effect of the 299/399 TLR4 polymorphisms on the following: post-transplant acute rejection beyond the first 6 months, bacterial infections, onset of bronchiolitis obliterans syndrome (BOS), onset of BOS grade 2 or higher, survival free from BOS grades 2 or 3, and survival.
METHODS
Transplant Population
Appropriate institutional review board approval was obtained. All recipients who underwent primary cadaveric (i.e., nonlive donor) lung transplant at Duke University from October 1998 to January 2003, had DNA available for genotyping, and survived at least 6 months post-transplant were eligible for inclusion. During this time, 170 patients met study inclusion criteria. Analysis included those patients who survived at least 6 months post-transplant because eligibility for BOS requires a minimum survival of at least 6 months. Further study of donors was not included in this analysis because of negative prior results (6). Standardized surgical techniques and post-transplant management protocols were used, as described elsewhere (8). Surveillance biopsies were done at 1, 3, 6, and 12 months post-transplant, then yearly until at least two negative biopsies were obtained. Biopsies were performed at any time post-transplant if clinical indications developed, according to a predefined protocol (e.g., new infiltrate, fever, or > 20% decline in FEV1 from post-transplant baseline).
Acute Rejection
Acute rejection was defined and graded according to standard histologic criteria (9). An acute rejection episode was defined by the presence of acute rejection ( grade A1) on lung biopsy. First episodes of biopsy-proven acute rejection ( grade A1) were treated with 500 mg/day of methylprednisolone for 3 days followed by a prednisone taper. The acute rejection score reflects the cumulative total of the "A" grades of each separate rejection episode (e.g., a patient with one grade 3 rejection and one grade 1 rejection has an acute rejection score of 4). Episodes of rejection were considered distinct if at least 6 weeks apart. A separate analysis considered acute rejection episodes with inclusion of "B" grade rejection as well.
BOS
BOS severity was defined according to the standard International Society for Heart and Lung Transplant definitions (10). Briefly, in patients who survive at least 6 months after transplant, the best post-transplant FEV1 is used to calculate a baseline and the current FEV1 is expressed as a percentage of the baseline. BOS severity was graded as follows: BOS 1, which occurs when the post-transplant FEV1 drops from 66 to 80% of baseline; BOS 2, which occurs when the post-transplant FEV1 drops to 51 to 65% of baseline; or BOS 3, which occurs when the post-transplant FEV1 drops to or below 50% of baseline.
Bacterial Infections
Infection was defined by (1) the presence of a positive culture for bacteria from urine, blood, or bronchoalveolar lavage fluid, (2) appropriate clinical signs or symptoms for infection, and (3) initiation of antimicrobial treatment. Regarding bronchoalveolar lavage specimens, samples were obtained during either surveillance or diagnostic bronchoscopy. We did not, however, consider an isolated positive culture in the absence of the other indications as an infectious event. Infections were classified as either gram-positive or gram-negative based on the organism isolated. The time to first positive culture, total number of infection episodes, and specific organisms were noted for each patient.
Determination of TLR4 Polymorphisms
Genomic DNA was isolated from blood by using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). TaqMan assays were performed for genotyping the 299/399 polymorphisms of the human TLR4, as described previously (11). The TLR4 TaqMan assay results were performed at least in triplicate, with 100% reproducibility of results, and further validated with direct DNA sequencing of selected samples.
Statistics
Patient demographic characteristics are reported using descriptive statistics. Comparison of dichotomous variables was performed using 2 testing or Fisher's exact test, as appropriate. Comparison between continuous variables was performed using two sample t tests or nonparametric Wilcoxon rank-sum test, as appropriate. All statistical tests were two-tailed, and p values of 0.05 or less were defined as significant. Freedom from acute rejection, BOS 1 or greater, BOS 2 or greater, survival free from BOS 2 or greater, and survival were analyzed using Cox proportional hazards models. Survival analysis reflects time to death or retransplant during the follow-up period (i.e., graft, not patient, survival). All statistical analysis was performed using SAS software version 8.2 (Cary, NC).
RESULTS
Patient Characteristics
Results demonstrated that 18 of 170 lung transplant recipients (11%) were heterozygous for either 299 or 399 polymorphisms, whereas 152 (89%) were wild-type. No homozygotes for either 299 or 399 were observed. Both of these commonly cosegregating mutations were identified in 15 patients, 299 was identified alone in one patient, and 399 was identified alone in two patients. The frequency and linkage disequilibrium of the Asp299Gly or Thr399Ile polymorphisms in this study is similar to previous reports (11, 12).
There were no significant differences among demographic characteristics of the two groups, as illustrated in Table 1. Both groups were similar in terms of native lung disease, recipient age, donor age, sex, and ethnicity. There were slightly more patients with chronic obstructive pulmonary disease among the 299/399 heterozygotes and slightly fewer patients with cystic fibrosis. Consistent with our program's preference for bilateral transplants for most native diseases, a majority of all patients underwent bilateral lung transplantation. The immunologic risk for post-transplant rejection as reflected in the degree of HLA mismatch and in an elevated pretransplant panel reactive antibody of more than 10% was similar, regardless of genotype. The length of follow-up was also similar and more than 3 years in each group, with a mean of 3.9 years post-transplant survival in wild-type recipients and 3.4 years post-transplant survival in 299/399 heterozygotes.
Acute Lung Rejection Analysis
Consistent with our prior report, in this larger population of lung transplant recipients with extended follow-up we continued to observe a significant reduction in the absolute rate of acute rejection in 299/399 heterozygous recipients as compared with wild-type recipients. Only 44% of heterozygous recipients had any episode of biopsy-proven acute rejection, whereas 71% of wild-type recipients experienced at least one episode of biopsy-proven acute rejection (p = 0.02, 2 test) over a mean post-transplant follow-up of more than 3 years in each group. The onset of acute rejection was significantly reduced and sustained over follow-up in 299/399 heterozygotes (p = 0.04, log-rank test), as shown in Figure 1. Differences in rejection rates were not from differences in the number of bronchoscopic procedures performed. In 299/399 versus wild-type recipients the mean (± SD) number of bronchoscopies with transbronchial lung biopsy performed per patient was 5.3 (± 1.5) versus 5.7 (± 1.9), respectively (p = 0.48, two-sample t test).
During the post-transplant follow-up, 299/399 heterozygous recipients experienced significantly fewer episodes of acute rejection. Heterozygotes had a mean of 0.72 episodes of rejection per patient, as compared with a mean of 1.3 per patient in wild-type recipients (p = 0.02, two-sample t test, unequal variance). Furthermore, no patients with 299/399 heterozygotes experienced more than two episodes of acute rejection; in contrast, 25 wild-type recipients experienced three or more episodes of acute rejection (0 vs. 16%, respectively; p = 0.08, Fisher's exact test). There was also a trend toward less severe rejection in heterozygous recipients (mean acute rejection score 1.5 in 299/399 heterozygotes vs. 2.2 in wild-type; p = 0.20, two-sample t test, unequal variances). Finally, we also analyzed our results with inclusion of B-grade rejection as well. This results in an increase in the overall rate of acute rejection among wild-type recipients with no change in rate among TLR4 heterozygotes (74 vs. 44%, respectively; p = 0.01, 2 test).
Infectious Complications
The overall rates and type of bacterial infection were almost identical among patients with 299/399 heterozygotes and wild-type recipients. No significant differences were observed in the rate of gram-negative bacterial infection or mean number of gram-negative infections per patient, or in the time to first gram-negative infection among wild-type recipients and TLR4 heterozygotes (Table 2). Specifically, at least one post-transplant infection with a gram-negative pathogen occurred in 57% of patients with wild-type polymorphisms and 61% of heterozygous recipients (p = 0.75). The most common cause of gram-negative infection was Pseudomonas aeruginosa, which accounted for 45% of all gram-negative infections and occurred with a similar frequency regardless of genotype. Other gram-negative pathogens observed in both groups of patients included Burkholderia cepacia, Stenotrophomonas maltophilia, and Klebsiella pneumonia. Similar rates of gram-negative infection were also observed between heterozygous and wild-type recipients, when patients with cystic fibrosis were excluded from the analysis (56 vs. 48%, respectively; p = 0.51). In a multivariable model, only a native disease of cystic fibrosis (not TLR4 genotype) was predictive of post-transplant infection with a gram-negative pathogen (p = 0.0001). Finally, similar rates of infections with gram-positive pathogens were also observed in both groups (Table 2). Staphylococcus aureus was the most common gram-positive infection in patients, regardless of genotype, and accounted for 90% of isolates.
BOS and Survival Analysis
BOS 1 or higher developed in a similar percentage of patients in both groups (p = 0.56, log-rank test). At 3 years post-transplant, Kaplan-Meier estimates were that BOS 1 or higher developed in 33% of wild-type recipients versus 31% of 299/399 heterozygous recipients. In contrast, there was a trend toward reduced BOS grades 2 or 3 in TLR4 heterozygous recipients. Only 2 of 18 (11%) of patients with 299/399 heterozygotes developed BOS 2 or greater over the period of post-transplant follow-up, whereas this occurred in 43 of 152 (28%) of wild-type recipients (p = 0.08, log-rank test). Furthermore, there was also greater long-term survival among patients with 299/399 heterozygotes, although this value was not statistically different. Only 15% (3 of 18) heterozygous patients died during the follow-up period (including the two who developed BOS) as compared with 26% (39 of 152) of wild-type recipients (p = 0.38). When the composite endpoint of BOS 2 or 3 or death was considered, there was a significant increase in freedom from high-grade BOS or death among 299/399 heterozygotes as compared with wild-type recipients (p = 0.05), as shown in Figure 2. Furthermore, in a multivariable model that considered TLR4 genotype, donor age, recipient age, type of transplant operation, recipient sex, recipient ethnicity, and immunologic risk as reflected by degree of HLA mismatch and panel reactive antibody, only TLR4 heterozygosity at 299/399 was predictive of survival free from BOS grade 2 or greater (p = 0.05).
DISCUSSION
Our results demonstrate a reduced frequency, severity, and onset of acute rejection over extended follow-up in lung transplant recipients heterozygous for either of two TLR4 polymorphisms associated with reduced endotoxin responsiveness. Furthermore, differences were not observed in overall freedom from BOS among the groups, but a trend toward reduced BOS 2 or 3 was observed in heterozygous recipients. In contrast to the differences in acute rejection, a similar and relatively high rate of infectious complications was observed in all patients, regardless of genotype.
Our results suggest activation of innate immune signaling through TLR4 contributes to the development of acute rejection and possibly higher grades of BOS. Innate immunity has been shown to modulate adaptive immune responses through a variety of mechanisms (13). In the lung, in particular, TLR4 is highly expressed on alveolar macrophages and also on airway epithelia. Activation of TLR4 leads to production of proinflammatory cytokines and chemokines, which recruit and activate other immune cells, including lymphocytes. Furthermore, activation of TLR4 on antigen-presenting cells, such as alveolar macrophages, increases expression of MHC molecules and costimulatory molecules, thus facilitating a robust adaptive immune response (14). Chimerism between donor and recipient cells is reported to occur in the airway epithelia, which express TLR4, over time after lung transplant (15). Thus, TLR4 recipient genotype could influence the epithelial response to innate pathogens. Although the effects of innate immune activation in the airway epithelia on the development of chronic allograft rejection are unknown, there is some evidence that impaired innate immune signaling is protective against vascular injury in endothelial cells (16).
Furthermore, recent animal studies also highlight a potential role for TLR signaling in the development of the alloimmune response. Using minor histocompatibility antigen mismatch mice (female to male in B6), in which both donors and recipients had targeted deletions of both copies of MyD88 (MyD88eC/eC), an adapter molecule shared by most TLRs, skin-graft rejection was prevented (4). Decreased numbers of mature dendritic cells were evident in the draining lymph nodes of the MyD88eC/eC mice in response to skin graft, as compared with wild-type mice, implying that the failure to reject the graft in MyD88eC/eC mice is because of impairments in the initiation of the alloimmune response. These findings were recently extended to fully allogeneic murine models where acute allograft rejection of both skin and cardiac allografts occurred without a meaningful delay despite the absence of MyD88 (i.e., transplanting Balb/c-MyD88eCdeficient donors into B6-MyD88eCdeficient recipients) (5). However, MyD88 signaling was important for the ability of allogeneic dendritic cells to prime naive recipient T-cell responses, particularly toward TH1. Thus, these studies demonstrate activation of innate immunity through MyD88 participates in the development of adaptive immune responses to murine skin and cardiac allografts. In these model systems, MyD88 is essential for the rejection of minor incompatible skin, but not major incompatible skin or heart transplants. Innate immunity might play a critical and unique role in pulmonary and skin transplant because of the constant environmental exposures and innate responses initiated through resident alveolar macrophages, dendritic cells, and Langerhans' cells. Although none of these models are directly relevant to lung transplant, they provide further support for our hypothesis that activation of innate immunity occurs in the context of organ transplant and participates in the development of the adaptive alloimmune response.
LPS and other TLR4 ligands likely contribute to the development of immune stimulation in the transplant population. Although LPS is found on gram-negative pathogens and is present in the aerosol of many domestic and work environments, making post-transplant exposure common, other exogenous ligands are now recognized for TLR4 and may contribute to the pathogenesis of rejection post-transplant. For instance, respiratory syncytial virus fusion protein is a recognized ligand of TLR4 (17) and has been reported to be associated with the development of acute rejection and BOS (18, 19). More recently, endogenous ligands have also been described, including fragments of hyaluronic acid, fibrinogen, and fibronectin (17). Transplant surgery and the process of perioperative lung ischemiaeCreperfusion would be expected to cause tissue injury and lead to exposure of many of these potential endogenous ligands.
Our results are consistent with previous reports of the functional and clinical significance of heterozygosity or homozygosity for the TLR4 299/399 alleles. In particular, heterozygosity for either of these single nucleotide polymorphisms is associated with a blunted physiologic response to inhaled endotoxin, reduced systemic responsiveness to inhaled endotoxin, and lower serum levels of proinflammatory cytokines, acute phase reactants, and soluble adhesion molecules (11, 16). Clinical associations have been found between these alleles and a predisposition for gram-negative sepsis, premature birth, and protection from carotid artery atherogenesis (12, 16, 20, 21).
Although prior studies in healthy subjects have suggested an increased risk for bacterial infections in patients with 299/399 polymorphisms, we observed a similar high rate of bacterial infections in our population, regardless of the TLR4 genotype. There are several possible explanations for these findings. First, in the lung transplant population, the underlying type of end-stage lung disease and prior colonization with infectious agents makes it difficult for a single gene change to substantially influence the prevalence of infection. Second, effects of these single nucleotide polymorphisms on infection were likely overshadowed by the effects of the potent post-transplant immunosuppressive agents, which led to a high rate of bacterial infections in all patients. Third, we recognize that several innate pathogen-associated molecular patterns can stimulate via multiple TLRs. This redundancy of innate immunity might help prevent a marked increase in infections in the TLR4 recipients. Finally, we hypothesize that there are differences in the innate immune response to a transplant allograft as compared with a bacterial pathogen. Although exposure to a transplant or bacterial infection might activate innate immunity through TLR4, subtle differences in the nature of signal and effector response are likely to occur. For example, differential effects of these polymorphisms on antigen presentation by alveolar macrophages or on regulatory T-cell responses could lead to differential effects regarding the development of rejection distinct from the response to infection.
Although our results identified these two polymorphisms in TLR4 as an important genetic determinant of allograft rejection after lung transplant, 44% of heterozygous recipients still experienced acute allograft rejection and no significant differences were observed regarding BOS. Our results are consistent with acute rejection and BOS as distinct and complex traits that result from interactions between several different genetic and exogenous stimuli. Many common disorders arise from inheriting combinations of unfavorable alleles that collectively predispose individuals to disease. Prior studies in lung transplant suggest a potential role for both donor and recipient HLA alleles in influencing the adaptive immune response to donor alloantigens after lung transplant (22, 23). In addition, one study of 93 lung recipients has suggested that the combination of high-expression alleles in the interleukin 6 and IFN- genes significantly increases the risk for BOS (24). Polymorphisms in several other cytokine genes (e.g., transforming growth factor , interleukin 10) have also been linked to the development of acute and chronic allograft rejection in other solid organ transplant populations (25).
Furthermore, although the 299/399 polymorphisms in TLR4 are the most extensively studied TLR variants, other less common polymorphisms in TLR genes have recently been associated with human disease. For example, a polymorphism in TLR2 (Arg753Gln) is associated with staphylococcal infections and a common polymorphism in TLR5 abolishes flagellin signaling and is associated with increased risk for Legionnaire's disease (26, 27). Thus, consistent with other complex genetic traits, we anticipate that several variants in innate and adaptive immune response pathways influence the development of pulmonary allograft rejection. We are actively pursuing studies of the clinical and biological significance of other TLR variants and downstream-signaling molecules in lung transplant.
In addition, our results highlight a potential discordance between the development of acute rejection and BOS. One interpretation of our results is that the genetic and environmental influences responsible for BOS differ from those involved with acute rejection. Alternatively, limitations of the BOS nomenclature and the limited power of our sample to detect differences in BOS could also explain our results. One major limitation of the BOS nomenclature is the reliance on a clinical syndrome of post-transplant airflow obstruction as a marker for the histologic process of obliterative bronchiolitis. We have previously demonstrated that single lung transplants have an earlier onset of BOS as compared with bilateral lung transplant recipients, most likely as a result of the definition of the clinical syndrome rather than an immunologic effect of the transplant operation (28). Furthermore, given the much higher rates of acute rejection as compared with BOS in our sample, it is possible that with additional follow-up time or a larger patient population, a similar reduction in the rates of BOS would be observed among heterozygotes as was seen in acute rejection.
Finally, our results suggest that the targeting of innate immunity represents a potentially useful means to prevent or treat lung rejection. Furthermore, prior studies have suggested current calcineurin-based post-transplant immunosuppression has little effect on inflammatory cytokine production by alveolar macrophages (29). Currently, there is intense interest in developing strategies to block TLR signaling for a variety of clinical conditions (30). As clinically relevant strategies are developed, studies in lung transplant are warranted to determine if targeting innate immune signaling effectively reduces allograft rejection and improves long-term transplant outcomes.
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