CYTOKINE GENES AND BOS OR ALLOGRAFT FIBROSIS
A number of studies have investigated the association between genetic polymorphisms in tumor necrosis factor alpha (TNFA), interferon gamma (IFNG), transforming growth factor beta-1 (TGFB1), interleukin (IL)6, and IL10, and the development of BOS or allograft fibrosis after lung transplantation (10, 11, 16, 17). These genetic polymorphisms were chosen on account of the proven inflammatory, profibrotic, or anti-inflammatory properties of their gene products.
In four independent studies, no association was detected between genetic polymorphisms in TNFA and IL10 and the development of BOS or allograft fibrosis (10, 11, 16, 17).
A significant association was detected between homozygosity for the major T allele of IFNG at position +874 A/T and the development and earlier onset of BOS (11). Two other studies did not confirm this association (10, 16), but a fourth study showed that allele 2 of the CA repeat in IFNG was most commonly observed in the group with allograft fibrosis compared with the group without allograft fibrosis (18), but this association was not replicated.
Homozygosity for the major allele of codon 25 of TGFB1 was associated with allograft fibrosis diagnosed by histology in two studies (19, 20). One of these studies showed that a second genetic polymorphism (cytosine deletion at position +72) was also associated with allograft fibrosis and that the G allele at position −800 was associated with lung transplant recipients who developed fibrosis compared with healthy controls, although the frequency was not significantly different between recipients with and without allograft fibrosis (20). Other studies, which used either the BOS criteria according to the International Society of Heart and Lung Transplantation (10, 11) or the term chronic rejection (16, 17), did not confirm this association.
Homozygosity for allele 1 of the 86-bp repeat of the IL-1 receptor antagonist gene was associated with chronic rejection in a cohort of thoracic transplant recipients (17, 21), and an almost twofold increased risk for the major allele at position 8061 C/T in IL-1 receptor antagonist was found. These associations were not replicated in another independent cohort.
In IL6, carriership of the G allele of the IL6 gene (−174 G/C) was associated with the development and an earlier onset of BOS in two studies (10, 11) but could not be validated in three other cohorts (10, 16, 17).
INNATE IMMUNITY GENES AND BOS
Five studies have investigated the associations between genetic polymorphisms in innate immunity genes and BOS, but none of the following positive associations have been replicated in another independent cohort.
Lung transplant recipients carrying the minor allele for either one of the functional single-nucleotide polymorphisms (SNPs) in Toll-like receptor 4 (TLR4), Asp299Gly (rs4986790) or Thr399Ile (rs4986791), showed a trend toward reduced onset of BOS grade 2 or 3 (9). Other genetic polymorphisms in TLR2 (rs1898830), TLR4 (rs1927911), and TLR9 (rs352162 and rs187084) were associated with an increased risk to develop BOS (22). In this study, the BOSpos patients had significantly more risk alleles in TLR2, TLR4, and TLR9 together compared with the BOSneg patients and controls (22).
Homozygotes for the minor allele (T) of CD14 at position −159 C/T had a higher overall incidence and an earlier onset of BOS than patients with other genotypes (8).
Patients who received a graft from a donor homozygous for the Y allele of the mannose-binding lectin (MBL) gene had a worse BOS-free survival compared with patients who received a graft from a donor with a X/X or X/Y genotype. Furthermore, a negative effect of the donor HYPA haplotype on the development of BOS was observed. However, these negative effects disappeared after introduction of a new immunosuppressive regimen because of a dramatic increase in the 1-year BOS-free survival. Recipient MBL genotype was not associated with transplant outcome (23).
Furthermore, the presence of the inhibitory haplotype A of the killer immunoglobulin-like receptors (KIRs) and the absence of KIR2DS5 were reported to be associated with BOS (24).
REPAIR GENE AND BOS
Only one study investigated the association between genetic polymorphisms in repair genes and BOS. Lung transplant recipients homozygous for the major alleles of rs17098318, rs11569919, and rs12285347 and for the minor allele of rs10502001 of the matrix metalloproteinase (MMP)7 gene had an increased risk to develop BOS. Haplotypes constructed with three or four of these risk alleles correlated with lower serum levels of MMP-7 and were more often present in the BOSpos patients (25).
COMMENTS ON THE PUBLISHED GENETIC ASSOCIATIONS
The results of this review show that significant associations have been reported between functional genetic polymorphisms in several cytokine genes and the development of BOS or allograft fibrosis after lung transplantation. In addition, significant associations in innate immunity genes and a repair gene were found in relation to BOS.
In the majority of the cytokine gene association studies, the same subset of cytokine genes was analyzed. The association between the genetic polymorphism in the IL6 gene and BOS was reported by Lu et al. and Snyder et al. (10, 11). Snyder et al. (10) were unable to confirm the association of IL6 and IFNG with BOS. However, they could conclude that SNPs in the IL6 and IFNG genes were associated with an earlier onset of BOS and suggested that the conflicting results might be attributed to small sample size and differences in ethnic backgrounds, immunosuppressive regimens, and follow-up time (10). The existence of an association between IFNG and BOS is supported by genetic linkage of the T allele at position +874 and allele 2 of the CA repeat (26).
IL10 and TNFA have never been associated with BOS or allograft fibrosis, and therefore, in our opinion these two cytokines can be excluded from future gene association studies.
The associations of both IL1 and TGFB1 with BOS or allograft fibrosis need to be interpreted with caution. The study that reported the association between IL1 and chronic rejection used a cohort of different types of thoracic transplant recipients of which the number of lung transplant recipients was too small to analyze separately (17). In the studies of El-Gamel et al. (19) and Awad et al. (20), the cohorts of lung transplant recipients were largely overlapping. Therefore, the association between codon 25 in the TGFB1 gene and allograft fibrosis is not positively replicated in another independent cohort. In addition, these studies found an association between a genetic polymorphism in the TGFB1 gene and allograft fibrosis. Allograft fibrosis and BOS may not be equivalent entities as the presence of fibrotic changes on transbronchial biopsies does not necessarily identify patients with changes of obliterative bronchiolitis. The difference between allograft fibrosis and BOS is recently illustrated by a new concept describing BOS no longer as the only form of chronic lung allograft dysfunction. Another form of chronic lung allograft dysfunction, called restrictive allograft syndrome (RAS), exhibits restrictive functional changes with fibrotic processes in peripheral lung tissue than the classical finding of small airway obliteration seen in BOS (27).
Furthermore, in the past years, two different phenotypes of BOS are distinguished based on the response to the treatment with azithromycin (28, 29). The first phenotype is called neutrophilic reversible allograft dysfunction and showed increased bronchoalveolar lavage levels of neutrophils and different proteins, inflammatory active lesions on histology, and is responding to azithromycin. The second phenotype includes the fibroproliferative BOS that showed no neutrophils and another protein pattern in bronchoalveolar lavage, pure fibrosis on histology, and no response to azithromycin (28, 30). RAS and the two different phenotypes of BOS were described recently and have therefore not been included in the definitions of BOS or allograft fibrosis in the gene association studies in this review. Nevertheless, part of the patients who were diagnosed with BOS or allograft fibrosis in these studies might meet the criteria of these new subtypes, which might influence the present conclusions. For example, as azithromycin seems to reduce inflammation by inhibiting components of the innate immune response (29, 31), treatment of lung transplant recipients with azithromycin might influence the associations found between innate immunity genes and BOS. Before RAS and neutrophilic reversible allograft dysfunction can be used in future association studies, they need to be evaluated and confirmed.
Early after lung transplantation, the transplanted lungs exist of donor cells. Nevertheless, chimerism between donor and recipient cells is reported to occur in the lungs of lung transplant recipients (32). Epithelial structures displaying signs of chronic injury, as present in the development of BOS, showed a higher degree of chimerism (32). From this point of view, Palmer et al. (9) concluded that TLR4 recipient genotype could influence the epithelial response to innate pathogens. Besides chimerism, shown to be present in transplanted lung, the genetic profile of the donor will also be involved in the development of BOS. Munster et al. (23) showed that the genetic profile of the donor, and not of the recipient, is associated with the development of BOS.
FUNCTIONALITY OF THE GENETIC POLYMORPHISMS
The functionality of the genetic polymorphisms in the cytokine genes has been previously investigated. The T allele (+874 A/T) and the CA repeat allele 2 of IFNG are in linkage disequilibrium with each other and are associated with an increased production of IFN-γ (33). Furthermore, the −174 G allele of IL6 is also associated with an increased production of its gene product (14).
Homozygosity for the major allele of codon 25 of the TGFB1 gene, which is in linkage disequilibrium with a cytosine deletion at position +72, is also associated with a higher TGF-β1 production than the other genotypes (20).
The mechanisms by which these genetic variations contribute to the development of BOS are currently not exactly known. It is, however, likely that they influence the immune response toward inflammation and fibrosis. IL-6 and IFN-γ are involved in acute inflammatory responses in general, but both are also known for their profibrotic properties (34, 35). TGF-β plays a pivotal role in the development of fibrosis (35). This suggests that genetically determined variability in cytokine production capacity could play a role in interindividual differences in the intensity of the inflammatory process and in the subsequent fibrogenesis leading to BOS.
Significant associations between genetic polymorphisms in the innate immunity genes and BOS were also found. Especially the association of two genetic variants in TLR4 (Asp299Gly and Thr399Ile) is of great interest because there is evidence that carriers of the minor allele have a reduced production of proinflammatory cytokines and chemokines on stimulation, which might have a protective effect on the pulmonary epithelium (36). The functionality of the other SNPs in the TLR2, TLR4, and TLR9 genes has not been investigated (22); however, the risk alleles of these SNPs might contribute to the development of BOS by an increased secretion of cytokines and chemokines that is followed by injury of the pulmonary epithelium. The functionality of the genetic polymorphisms in the CD14, MBL, and KIR genes is known as well. First, CD14 binds to lipopolysaccharide and promotes signaling through TLR4 (37). Homozygotes for the risk allele of CD14 had higher levels of CD14, TNF-α, and IFN-γ in their peripheral blood implying a heightened state of innate immune activation (8). Second, the Y allele of MBL was found to be associated with high production of the gene product that may result in more inflammation and tissue damage and an increased antigen presentation (38). Third, natural killer (NK) cells are important components of the innate immunity and their activation is influenced by KIRs (39). KIR haplotypes are associated with the number of functional inhibitory and activating KIR genes. Haplotype A contains six inhibitory and one activating KIR gene, and this haplotype is associated with functional down-regulation of the NK-cell activity. Haplotype B contains a mixture of functional activating and inhibiting KIRs (40, 41). The association between haplotype A and BOS is against the expectation, because the presence of haplotype A on NK cells is associated with less reactivity against donor cells recognized on lung allografts and thus the absence of BOS (24).
Finally, a genetic association was found between BOS and MMP7, a repair gene. MMP-7 is involved in the repair of the pulmonary epithelium, and its expression is primarily regulated at the transcriptional level (42). The genetic polymorphisms in the MMP7 gene may contribute to aberrant tissue repair and fibrosis through insufficient levels of MMP-7 (25).
The foregoing evidence supports that genetic polymorphisms in innate immunity genes and in a repair gene might contribute to the development of BOS by influencing the inflammatory response and the process of fibrogenesis. However, the association of genetic polymorphisms in the innate immunity genes and in MMP7 with BOS has never been replicated; therefore, validation in an independent cohort is required.
APPLICATION OF GENETIC RISK PROFILING TO CLINICAL PRACTICE
In the future, genetic risk profiling may become a tool for the clinician to stratify the risk of developing BOS after lung transplantation and to adjust the treatment. Palmer et al. (43) already suggested that TLR4 genotyping before transplant permits assessment of the risk for acute rejection. In addition, genetic risk profiling may allow individualization of the immunosuppressive treatment. For example, if a lung transplant recipient has a genetic profile conferring a greater risk of BOS after lung transplantation, it is not unlikely that he/she may benefit from adaptation of the standard immunosuppressive treatment regime. Furthermore, knowledge of the genetic polymorphisms that contribute to BOS might lead to alternative therapies to prevent or treat BOS, such as prevention of the activation of innate immunity through TLRs or inhibition of IL-6, IFN-γ, and TGF-β, that is, by blocking their receptors, to slow down the inflammation and fibrosis. Lung transplant recipients receive multiple anti-inflammatory medications to prevent acute and chronic rejection. Nowadays, the treatment of BOS consists of augmenting or changing the type of immunosuppressive drug (3). Recently, there is evidence that treatment of lung transplant recipients with azithromycin has promising results. A randomized controlled trial showed that azithromycin prophylaxis after lung transplantation attenuates the inflammatory response, improves the FEV1, and reduced the occurrence of BOS (44). Furthermore, treatment of BOSpos patients with azithromycin led to an increase in FEV1 and to a better survival (45, 46). Azithromycin modulates, in particular, the innate immune response by decreasing the response of several cytokines, such as IL-4, IL-8, and TNF-α, inhibiting the chemotaxis of neutrophils, inducing the apoptosis of neutrophils and lymphocytes, and disturbing the interaction between host and pathogen (31).
With the knowledge that BOS is also a fibrotic disease, the question arises whether the treatment of BOS might profit from antifibrotic agents, next to the anti-inflammatory agents.
Although risk stratification of lung transplant recipients with genetic profiling seems to be a promising approach for the future, which absolutely warrants further research, the results of the present studies discussed in this review are not yet sufficient to implement the use of a genetic profile into clinical practice.
RECOMMENDATIONS FOR THE FUTURE
While comparing and summarizing the literature, several limitations were encountered in the studies on genetic polymorphisms and the development of BOS.
First, in most studies only a few genetic polymorphisms or the same subset of genes were studied, which makes the list of candidate gene studies far from exhaustive. There is evidence that a combination of risk alleles is present in BOSpos patients. For example, in a study on genetic polymorphisms in several TLR genes, BOSpos patients had more risk alleles compared with BOSneg patients and controls (22). Furthermore, concomitant presence of high-expression SNPs in both the IL6 and the IFNG gene was higher in BOSpos patients than in BOSneg patients (11). In the light of genetic profiling, future association studies should investigate a combination of multiple genes. For example, in addition to MMP-7, other MMPs have shown to be involved in the development of BOS by their role in remodeling and degradation of the extracellular matrix and, therefore, might be interesting candidate genes (47–49). For the future, aiming at identifying genes relevant in BOS candidate genes can also be selected on the basis of their assumed involvement in pathways leading to BOS. Genetic polymorphisms in IFNG and its gene product are both associated with the development of BOS (50–52), therefore, receptors of IFN-γ and pathways that are activated by IFN-γ might be promising as well. An alternative way of identifying pathways involved in the development of BOS might benefit from whole genome association studies or SNP chips for specific pathway analysis. However, these approaches require a large group of patients to correct for type 1 errors.
Second, the sizes of most study populations were small, which might influence the results through insufficient statistical power. In addition, other risk factors for BOS, such as human leukocyte antigen mismatches, autoimmune responses, cytomegalovirus infection, and type of transplantation, are difficult to control in a statistical analysis because of the small sample size. In larger cohorts, these different risk factors should be included in a multivariate analysis together with the genetic profile, thus enabling a more accurate prediction of the risk of developing BOS.
Third, the follow-up period between the studies is different. The development of BOS is a time-dependent diagnosis; therefore, studies with a relatively short follow-up do not allow BOS to develop and this may lead to false conclusions.
Fourth, the definition BOS or allograft fibrosis is different between studies. Some studies use the BOS criteria according to the International Society of Heart and Lung Transplantation guidelines, while others use histological criteria to grade fibrosis, and in some studies, the definition of allograft fibrosis or BOS is lacking. In addition, RAS and two different phenotypes of BOS are identified as described earlier (27–29). The existence of these subtypes needs to be taken into consideration in future studies.
Fifth, in the majority of studies, the ethnic composition is not described, which influences the results because ethnicity influences the distribution of genetic polymorphisms, as reported in cytokine genes (53, 54).
Finally, differences in immunosuppressive treatment might lead to discrepancies in the results of the various groups, because immunosuppressive medication might mask a possible effect of the genetic polymorphisms. To promote the implementation of genetic profiling, we underline the proposal of Holweg et al. (7) of starting a database, in which allele and genotype frequencies of both donor and recipient, standardized definitions for complications after transplantation, and characteristics of transplant recipients are collected to improve gene association studies on BOS in the future.
The results of this review show that genetic polymorphisms in cytokine, innate immunity, and repair genes have been linked to the susceptibility to develop BOS after lung transplantation. However, exact causality of many of the associations, for example, by regulating the inflammatory response, cytokine and chemokine production, and facilitation of repair, still needs to be proven. Combining of the relevant genetic associations into a SNP chip for the stratification of the risk to develop BOS might be a promising approach. Genetic profiling could help clinicians to set out individualized treatment regimens for the prevention and treatment of BOS. Further studies are, however, needed to prove this concept.
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Bronchiolitis obliterans syndrome; Genetic polymorphisms; Innate immunity genes; Cytokine genes© 2012 Lippincott Williams & Wilkins, Inc.