Recipient NOD2/CARD15 Variants: A Novel Independent Risk Factor for the Development of Bronchiolitis Obliterans after Allogeneic Stem Cell Transplantation

Article Outline

• Abstract
• Introduction
• Patients and Methods
  • Patients
  • Collection of DNA Samples, Informed Consent, and Allelic Discrimination of the NOD2/CARD15 Gene
  • Statistical Analysis
• Results
• Discussion
• Acknowledgments
• References
• Copyright

Abstract 

Bronchiolitis obliterans (BO) is a serious complication after allogeneic stem cell transplantation. We hypothesized that single nucleotide polymorphisms (SNPs) of the NOD2/CARD15 gene (= NOD2/CARD15 variants) contribute to changes in host defense and subsequent alloreaction, leading to BO. We analyzed 427 donor–recipient pairs for the association of NOD2/CARD15 variants (SNP8 [Arg702Trp], SNP12 [Gly908Arg], and SNP13 [Leu1007fsinsC]) with BO occurrence. Overall, 11 patients (2.6%) developed BO. The cumulative incidence of BO rose from 1.3% in donor–recipient pairs without mutation to 18.7% in pairs with donor or recipient NOD2/CARD15 variants (P < .001). Recipient NOD2/CARD15 variants alone led to BO in 22.3% (P < .001), whereas donor variants alone associated with BO in 13.2% (P = .04). Multivariate analysis proved recipient but not donor NOD2/CARD15 variants to be a novel independent risk factor for BO development, and NOD2/CARD15 typing may help identify patients at increased risk for BO.

Key Words: Bronchiolitis obliterans, Chronic GVHD, NOD2, Stem cell transplantation

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Introduction 

Bronchiolitis obliterans (BO) is a serious pulmonary complication leading to late morbidity and decreased survival after allogeneic stem cell transplantation (allo-SCT). The pathophysiology of BO has yet to be defined, but experimental data from heterotopic trachea transplant models suggest that both loss of bronchial epithelial cell (EC) integrity and an alloantigen-reactive T cell response are involved 1, 2. We recently reported an association of single nucleotide polymorphisms (SNPs) of the nucleotide-binding oligomerization domain containing 2/caspase recruitment domain family, member 15 (NOD2/CARD15) gene, resulting in a diminished nuclear factor-kappa B (NF-κβ) response to bacterial cell wall products, leading to increased graft-versus-host disease (GVHD) severity and treatment-related mortality (TRM) 3, 4. Because NOD2/CARD15 is expressed not only in monocytes/macrophages and intestinal Ecs, but also in bronchial Ecs, as shown by Hippenstiel et al. [5] and our group (unpublished data), we hypothesized that NOD2/CARD15 variants contribute to changes in host defense and thus facilitate alloreaction, leading to BO.

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Patients and Methods 

Patients 

The study group comprised 467 patients of Caucasian ethnic background who underwent allo-SCT at 4 European SCT centers (Regensburg and Rostock, Germany; Vienna, Austria; and Barcelona, Spain). A total of 427 donor–recipient pairs were included in the analysis (Table 1), because no information about donor NOD2/CARD15 mutations could be obtained for 40 patients. Mutations assessed included SNP8 (Arg702Trp), SNP12 (Gly908Arg), and SNP13 (Leu1007fsinsC). Median time of follow-up was 674 days (range, 6-3484 days). BO development was assessed by pulmonary function tests, including forced expiratory volume in the first second (FEV₁), single-breath diffusion lung capacity for carbon monoxide (DLCO), total lung capacity (TLC), vital capacity, residual volume (RV), and functional residual capacity (FRC). BO was defined as an FEV₁ ≤ 80% of predicted value (n = 11/11) plus RV%TLC > 25% or FRC%TLC > 40%. Lung function score categorizing incorporating changes in FEV₁ and DLCO was done as described previously 6, 7. Supportive histopathologic findings of BO or pulmonary GVHD were found in 3 patients, and computed tomography (CT) scans demonstrating BO or BO-organizing pneumonia were found in 7 and 2 patients, respectively.

Table 1. Major characteristics of patients, donors, and transplantation procedures in donor-recipient pairs without and with NOD2/CARD15 variants

	Recipient var(+)	Donor var(+)	Recipient + Donor var(+)	Neither var(+)
Number of patients	49	37	18	323
Patient sex
Female	19 (38.8%)	14 (37.8%)	6 (33.3%)	149 (46.3%)
Male	30 (61.2%)	23 (62.2%)	12 (66.7%)	173 (53.7%)
Patient age, years	39.0 (19-63)	39.8 (18-64)	40.6 (16-58)	40.7 (16-65)
Donor age, years	36.0 (17-57)	37.6 (21-58)	38.9 (12-53)	39.0 (11-66)
Disease entity
Acute leukemia	26 (53.1)	20 (54.1%)	11 (61%)	142 (44.0%)
Chronic leukemia	10 (20.4%)	8 (21.6%)	2 (11%)	81 (25.1%)
Lymphoma	9 (18.4%)	7 (18.9%)	4 (22%)	85 (26.3%)
BMF	2 (4.1%)	2 (5.4%)	1 (6%)	12 (3.7%)
Others	2 (4.0%)	0 (0%)	0 (0%)	3 (0.9%)
Disease stage
early/intermediate	31 (63.3%)	23 (62.2%)	7 (39%)	203 (62.8%)
advanced	18 (36.7%)	14 (37.8%)	11 (61%)∗	120 (37.2%)
Donor type
Sibling	18 (36.7%)	13 (35.1%)	16 (89%)	200 (61.9%)
URD	31 (63.3%)∗	24 (64.9)∗	2 (11%)	122 (37.8%)
MRD	0 (0%)	0 (0%)	0 (0%)	1 (0.3%)
T cell depletion
None	31 (63.3%)	28 (75.7%)	15 (83%)	194 (60.1%)
ATG/campath/CD34+selection	18 (36.7%)	9 (24.3%)	3 (17%)	129 (39.9%)
Conditioning
Standard	36 (73.5%)	31 (83.8%)	14 (78%)	237 (73.4%)
RIC	13 (26.5%)	6 (16.2%)	4 (22%)	86 (26.6%)
TBI yes	34 (81%)	23 (79.3%)	12 (70.6%)	188 (64.8%)
TBI no	8 (19%)	6 (20.7%)	5 (29.4%)	102 (35.2%)
Busulfan yes	0 (0%)	2 (7.1%)	2 (11.8%)	11 (3.9%)
Busulfan no	42 (100%)	26 (92.9%)	15 (88.2%)	270 (96.1%)
Stem cell source
Bone marrow	21 (42.9%)	16 (43.2%)	3 (17%)	97 (30.0%)
PBSC	28 (57.1%)	21 (56.8%)	15 (83%)	226 (70.0%)
Acute GVHD
<2	32 (66.7%)	23 (62.2%)	9 (50%)	209 (65.1%)
≥2	16 (33.3%)	14 (37.8%)	9 (50%)	112 (34.9%)
Chronic GVHD
No	28 (57.1%)	18 (50%)	12 (71%)	199 (61.6%)
Yes	21 (42.9%)	18 (50%)	5 (29%)	124 (38.4%)
BO
Yes/no	4/45	2/35	2/16	3/320

var(+) indicates presence of at least one of the SNP8, SNP12, or SNP13; standard conditioning regimens consisted of 8 Gy to 12 Gy fractionated total body irradiation (TBI) followed by high-dose cyclophosphamide or classic busulfan/cyclophosphamide; reduced-intensity conditioning (RIC) consisted mainly of fludarabin/BCNU and melphalan regimens. For T cell depletion, serotherapy with antithymocyte globulin (ATG) or with alemtuzumab (Campath®) was given in the course of pretransplantation conditioning. Some patients were enrolled on protocol for CD34⁺selection. All criteria were equally distributed between groups expect^∗ increased percentage of URD donors in the recipient var(+) and donor var(+) group and more patients with advanced stage of disease at time of transplantation in the recipient var(+) and donor var(+) group. BMF, bone marrow failure syndrome (severe aplastic anemia, osteomyelofibrosis and sclerosis, paroxysmal nocturnal hemoglobinuria).

Collection of DNA Samples, Informed Consent, and Allelic Discrimination of the NOD2/CARD15 Gene 

DNA from 467 patients and from 427 of their donors was collected and retrospectively analyzed by allelic discrimination of the NOD2/CARD15 gene as described previously [3]. At admission and before stem cell collection, all patients and donors had given informed written consent to genetic analysis of hematopoeitic stem cell transplantation—related complications in their biological samples. The study was approved by each center´s ethics committee.

In addition, the presence of NOD2/CARD15 mutations was determined in an independent group of 4 patients who developed BO after allo-SCT at Hannover Medical University.

Statistical Analysis 

Individuals heterozygous for at least 1 of the 3 NOD2/CARD15 SNPs analyzed (SNP8, SNP12, SNP13) were considered variant, whereas individuals lacking these SNPs were considered wild-type. Genotype frequencies observed in donors and recipients were compared with Hardy-Weinberg expectations using χ² statistics. The development of BO was analyzed in relation to NOD2/CARD15 variants by forming groups according to the occurrence of any variant in (1) recipient and donor, (2) in donors only, (3) in recipients only, (4) in either recipient or donor, or (5) none. Cox regression was used to compare groups and analyze hazard ratios. The following variables were included in the univariate analysis: NOD2/CARD15 variants, acute and chronic GVHD (aGVHD, cGVHD), recipient age, stage of disease, donor type (sibling vs unrelated), stem cell source, T cell depletion (TCD), conditioning regimen (standard conditioning vs reduced-intensity conditioning [RIC]; busulfan; total body irradiation [TBI]). Multivariate analysis by Cox regression included NOD2/CARD15 variants, acute and chronic GVHD, stem cell source, recipient age, conditioning intensity, and stage of disease. For cumulative BO development, actuarial curves were obtained by the Kaplan-Meier method and compared statistically using the log-rank test. Allele frequencies were compared using the χ² test. P values were 2-sided, and a significance level of α = .05 was used.

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Results 

We investigated the association of variants of the NOD2/CARD15 gene with the development of BO in 427 donor–recipient pairs after allo-SCT. First, we determined the frequency of NOD2/CARD15 variants in donors and recipients by allelic discrimination for SNP8 (Arg702Trp), SNP12 (Gly908Arg), and SNP13 (Leu1007fsinsC), which represent 82% of the mutated chromosomes. Genotype frequencies both in recipients and donors were consistent with Hardy-Weinberg equilibrium, comparing the observed with the expected genotype frequencies by χ² statistics for SNP8 (donors, P = .42; recipients, P = .55), SNP12 (donors, P = .98; recipients, P = .94), and SNP13 (donors, P = .93; recipients, P = .93). Allele frequencies for SNP8 were 4.46% for donors and 5.04% for recipients, those for SNP12 were 0.94% for donors and 1.64% for recipients, and those for SNP13 were 1.76% for donors and 1.91% for recipients. Total allele frequencies for these 3 main NOD2/CARD15 mutations were 7.16% for donors and 8.59% for recipients, and both individual and total frequencies were comparable to those reported by other studies 8, 9, 10.

At least 1 NOD2/CARD15 variant was carried by 15.7% of all recipients and 12.9% of all donors. In 323 pairs (75.6%), both donor and recipient carried wild-type alleles. In 49 pairs (11.4%), the recipient only had at least 1 mutation; in 37 pairs (8.7%), the donor only had at least 1 mutation. NOD2/CARD15 variants of both donor and recipient were present in 18 pairs (4.2%). Patient- and donor-specific characteristics, as well as transplantation-specific procedures, were distributed equally between patient–donor pairs with and without mutations (Table 1).

Eleven patients (2.6%) developed BO, with a median time of diagnosis of 581 days (range, 186-1407 days) after allo-SCT (Table 2). As shown in Table 3, NOD2/CARD15 variants were observed at higher frequencies in the donor–recipient pairs with BO than in those donor–recipient pairs that did not develop BO (donor: 36.4% vs 12.2%, P = .04; recipient: 54.5% vs 14.6%, P = .003; Figure 1A). These data were confirmed by analysis of NOD2/CARD15 variants in a second independent cohort of 4 cases with BO after allo-SCT from the University of Hannover that found that 75% of the donors and 50% of the recipients carried a mutation (Table 4).

Table 2. Pulmonary function results in 11 patients developing BO after allo-SCT

LFS indicates lung function score; TLC, total lung capacity; VC, vital capacity; RV, residual volume; FRC, functional residual capacity; n/a, not available.

Table 3. Presence of NOD2/CARD15 variants in recipients (a), donors (b), and donors and recipients (c) of patients with BO after allo-SCT, Regensburg-Rostock-Vioenna-Barcelona cohort

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• Figure 1 

  NOD2/CARD15 variants (var) of the recipient independently increase the risk of developing BO after allo-SCT. A, Frequency of NOD2/CARD15 variants in donors and recipients of patients with (n = 11) and without (n = 416) BO (^∗P < .05). B-D, Cumulative incidence of BO after allo-SCT depending on whether (B) the recipient, the donor, or both carry a NOD2/CARD15 variant (n =104); (C) only the recipient has a NOD2/CARD15 variant (n = 49); or (D) only the donor has a NOD2/CARD15 mutation (n = 37). Wt represents wild-type controls, that is, neither donor nor recipient has a NOD2/CARD15 variant (n = 323) (^∗P < .05; ^∗∗P < .01). E, Chronic GVHD and recipient NOD2/CARD15 variant are independent risk factors for BO development.

Table 4. Presence of NOD2/CARD15 variants in recipients and donors with BO after allo-SCT, Hannover cohort

We next compared the pathophysiologic relevance of donor and recipient NOD2/CARD15 variants for BO development by determining the cumulative incidence in dependence on the absence or presence of a mutated allele in the donor, the recipient, or both. The incidence of BO increased from 1.3% in the wild-type group to 18.7% when the donor, the recipient, or both carried a NOD2/CARD15 variant (P < .001; Figure 1B). The incidence was 22.3% (P < .001; Figure 1C) if a mutation was present in the recipient only, 13.2% if a mutation was present only in the donor (P = .04; Figure 1D), and 13.8% if a mutation was present in both the donor and recipient (P < .001; data not shown). Cox regression univariate analysis and hazard ratio (HR) assessment revealed that recipient variants (P < .0001; HR = 9.0), donor variants (P = .02; HR = 4.3), cGVHD (P = .02; HR = 11.7), and advanced stage of disease (P = .03; HR = 3.9) were significant risk factors for BO development, whereas aGVHD, recipient age, TCD, conditioning regimen (standard vs RIC; busulfan; TBI), stem cell source, and donor type were not.

These data suggest that NOD2/CARD15 variants of both donor and recipient may contribute to the increased risk of developing BO. However, multivariate Cox regression analysis revealed a much stronger impact of recipient NOD2/CARD15 variants than donor variants on BO development, with only cGVHD (P = .007; HR = 22.9) and NOD2/CARD15 mutations of the recipient (P = .004; HR = 9.0) remaining as independent risk factors (Figure 1E). Interestingly, no association was found between cGVHD and NOD2/CARD15 variants of the recipient (P = .6), the donor (P = .2), recipient or donor (P = .4), or recipient and donor (P = .4) in our cohort, supporting a unique contribution of NOD2/CARD15 mutations to BO but not to cGHVD in general.

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Discussion 

BO is a late-onset noninfectious pulmonary complication of allo-SCT that primarily affects the smaller airways, leading to severe, progressive pulmonary obstruction and impaired survival. BO can manifest months to years after allo-SCT, with a reported incidence between 2% and 10% 11, 12, 13, 14. Reported risk factors for BO development include aGVHD and cGVHD, busulfan-based conditioning, peripheral blood stem cell transplantation (PBSC), long duration between diagnosis and transplantation, male recipients receiving transplants from female donors, and prior episode of interstitial pneumonitis 11, 12, 14, 15, 18. Risk factors differed among the studies because of differences in study design, patient characteristics and disease definitions. Santo Tomas et al. [11] performed an analysis on the International Bone Marrow Transplant Registry (IBMTR) database, finding 76 cases of BO in 6275 patients. Consistent with previous studies 15, 16, 17, these authors found, among others, aGVHD as a risk factor for BO, but were unable to obtain an unbiased estimate of the effect of cGVHD because the registry questionnaire incorporated BO as a manifestation of cGVHD [11]. In contrast, in the present study, we classified cGVHD and BO separately, and multivariate analysis revealed a significant association between cGVHD and an increased risk of BO development, confirming previous reports 12, 14, 17, 19, 20, 21, 22. aGVHD, which has been described not only as a potential risk factor for BO, but also as a definite risk factor for subsequent cGVHD, did not influence the development of BO in our study. In addition, no association between the development of chronic GVHD and NOD2/CARD15 variants of donor, recipient, or both was seen. This strongly suggests that BO pathophysiology is not based on alloantigen-specific immune responses of the donor immune system against host lung tissue alone, but rather involves additional distinct and unique features that do not apply to all patients, but rather define patients at higher risk for BO development, helping explain why only a small percentage of patients develop this deleterious complication after allogeneic SCT.

NOD2/CARD15, an intracellular receptor for the bacterial cell wall product muramyldipeptide (MDP), is expressed in epithelial cells of the intestinal tract, in bronchial epithelium, and in macrophages, monocytes, and antigen-presenting cells 5, 23, 24, 25, 26, 27, 28, 29. After infection of pulmonary epithelial cells with Streptococcus pneumoniae, expression of NOD2 increases in these cells in vitro and overall expression is up-regulated in mouse lungs infected with pneumococci [27]. TNFα and IFNγ up-regulate the expression of NOD2/CARD15 in gastrointestinal epithelial cells [29] as well as in bronchial epithelial cells, as has been demonstrated by our group (manuscript in preparation). The antimicrobial peptides α- and β-defensins, which participate in the innate host immune defense, have been described as markers of bronchial inflammation as well as of BO development after lung transplantation 30, 31, 32, and MDP–NOD2/CARD15 interactions lead to the activation of the innate immune system by inducing α- and β-defensin secretion as a first line of defense in response to a bacterial attack and also by increasing the production of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-1β, and IL-8 23, 24, 28, 33, 34, 35, 36, 37, 38, 39.

By demonstrating the important role for NOD2/CARD15 in the development of BO, our study provides the first report directly linking BO with mutation of a gene encoding for an intracellular receptor for bacterial cell wall products as a critical part of the innate immune system. Decreased monocyte/macrophage function and altered epithelial defense mechanisms may increase susceptibility to subclinical pulmonary infections, causing dysregulation and prolongation of inflammation 23, 24, 25, 26 and possibly resulting in bronchial epithelial injury, obliteration of bronchi and bronchioli, and airway obstruction. Various models have shown a loss of airway epithelium associated with the development of BO 40, 41, 42, and the integrity of the airway epithelium seems essential to the prevention of BO [43]. Furthermore, Hill et al. [44] recently demonstrated a preventive role of interferon (IFN)-γ on the development of idiopathic pneumonia syndrome after allo-SCT, mediated through lung parenchymal cells. This leads to the speculation that in the context of BO, increasing NOD2/CARD15 expression in epithelial cells by IFNγ administration may be beneficial in patients carrying a NOD2/CARD15 mutation by compensating for the loss of function implied by the mutation.

The association of BO with activation of the innate immune system is also supported by recent reports on successful treatment of BO using azithromycin [45], although the exact mechanism is not yet understood. Further supporting this theory, an association between the innate immune system and obstructive airway disease after allo-SCT has been recently described, with mutations in the bactericidal/permeability-increasing protein gene leading to an increased risk of decreased airflow [46]. In addition, NOD2/CARD15 signaling has been shown to directly cross-talk with and potentially down-regulate T cell responses [47], thus NOD2/CARD15 variants also may contribute to BO development by preventing protective T cell responses and augmenting cytotoxic T lymphocyte injury to the lung in an alloantigen- specific manner. Whereas the NOD2/CARD15 variants analyzed in this study are located near the C-terminal leucine-rich repeat domain and cause decreased NF-κB activation, mutations affecting the central nucleotide–binding NACHT domain result in increased NF-κB activation and have been shown to play a role in Blau syndrome and in early-onset sarcoidosis 48, 49. Although Blau syndrome has historically been seen as a familial inflammatory granulomatous disease affecting the eyes, skin, and joints, Becker et al. [49] recently reported a case of interstitial pneumonitis in Blau syndrome. Pulmonary involvement and manifestation of these diseases strongly support the suggestion that NOD2/CARD15 mutations leading to NF-κB hyperactivation also can promote certain lung pathologies, and emphasize the contributing role of the innate immune system, although the underlying pathophysiologic mechanisms may be completely different.

In summary, our data strongly suggest that NOD2/CARD15 mutations represent a novel risk factor for the development of BO, and thus that future risk assessment before allo-SCT using NOD2/CARD15 typing may help identify patients at greater risk for BO and improve their clinical outcome after allo-SCT. In addition, our data further strengthen the role of both adaptive and innate immune responses and their interplay in SCT immunology.

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Acknowledgments 

This work was partially supported by European Community grant QLRT-CT-2001-01936 (“Transeurope”).

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