Introduction
Cytomegalovirus (CMV) infection commonly involves the colonic mucosa, and while being generally subclinical, still leaves a permanent latent CMV pool in mucosal monocyte-lineage cells1 and expanded anti-CMV effector-memory populations of CD8 + T cells and natural killer (NK) cells.2 Certain immunosuppressant IBD therapies increase the occurrence of primary or reactivated CMV infection, and the resultant viral colitis exacerbates intestinal inflammation and complicates the management of disease activity in inflammatory bowel disease.3 However, neither resolved CMV infection nor NK receptor genetics has been studied as a contributor to Crohn’s disease (CD) behavior or localization. Here, we assess these potential contributors using data from a large pediatric Crohn’s disease inception cohort.
Methods
We studied 514 CD patients and 201 control subjects from the RISK stratification cohort, a pediatric CD inception study with 3 years of available clinical follow-up.4 Subjects were genotyped for 15 killer inhibitory receptor (KIR) genes, histocompatibility leucocyte antigen (HLA)-B and HLA-C, and CMV exposure assessed by IgG serology.5 Analysis was stratified on ancestry determined by ADMIXTURE.6 Ileal RNASeq was imputed by xCELL7 to determine relative abundance of mucosal cell types.
Results
Cytomegalovirus seropositivity was 20% in both Crohn’s disease patients and controls. The RISK ileal biopsy RNASeq data set was tested for gene expression features associated CMV seropositivity. By gene set variation analysis, no individual genes reached significance (false detection rate [FDR] q < 0.05). We performed a further analysis using xCELL to estimate the relative abundance of 64 cell types in each sample data set. Among these cell types, association with CMV seropositivity reached significance for granulocyte-macrophage precursors (3.65-fold, q = 0.02). This cell type displayed a significant positive correlation with NK and NKCD56lo cells in CMV seropositive (q = 0.013 and 0.046) but not seronegative (q = 0.235 and 0.673) subjects.
Using a single variable analysis of disease risk (625 and 684 patients, European and All) and the study variables (CMV, HLA, and KIR), we only observed a modest association between Crohn’s disease with HLA (HLA-Bw4 risk of 1.23 and 1.26, P = .10 and .06, for European and All subjects, respectively; Table 1A).
Table 1.
Association with disease risk and behavior (single variable analysis).
| Association With Disease Risk | ||||
|---|---|---|---|---|
| Europeans (n = 625) | All (n = 684) | |||
| Variable | Risk | P | Risk | P |
| CMV | 1.169 | .497 | 0.952 | .817 |
| HLA-B | 1.234 | .100 | 1.259 | .060 |
| HLA-C | 1.094 | .495 | 1.074 | .568 |
| KIR3DL1 | 0.914 | .840 | 1.073 | .860 |
| KIR3DS1 | 0.980 | .890 | 0.861 | .380 |
| KIR2DL2/3 | 0.964 | .835 | 0.900 | .541 |
| KIR2DS2 | 1.167 | .386 | 1.025 | .885 |
| KIR2DL1 | 1.270 | .694 | 1.360 | .592 |
| KIR2DS1 | 1.006 | .975 | 0.892 | .518 |
| Association with disease behavior | ||||
| Europeans (n = 349) | All (n = 379) | |||
| Variable | Risk | P | Risk | P |
| CMV | 0.952 | .909 | 1.104 | .808 |
| HLA-B | 1.271 | .320 | 1.209 | .420 |
| HLA-C | 0.954 | .845 | 0.928 | .759 |
| KIR3DL1 | 0.383 | .110 | 0.379 | .110 |
| KIR3DS1 | 0.861 | .660 | 0.819 | .560 |
| KIR2DL2/3 | 1.646 | .146 | 1.808 | .082 |
| KIR2DS2 | 1.302 | .440 | 1.459 | .268 |
| KIR2DL1 | 0.000 | .985 | 0.000 | .985 |
| KIR2DS1 | 1.021 | .953 | 1.071 | .840 |
Disease risk: CMV is presence (1) or absence (0) of seropositivity. HLA-B indicates Bw4 allele frequency (0-2). HLA-C is C1 allele frequency (0-2). Disease behavior: Patients with 3-year follow-up for disease behavior (uncomplicated, Montreal B1; complicated, Montreal B2 or B3) and disease location were analyzed using a model conditioned on disease location.
Patients with 3-year follow-up for disease behavior (uncomplicated, Montreal B1; complicated, Montreal B2 or B3; 349 and 379 for European and all subjects) were analyzed for association with the study variables. Multivariate logistic regression used a model adjusting for disease location inasmuch as disease location is confounded with disease behavior.8 However, as shown in Table 1B, no significant signals were detected for any study variables.
In contrast, for disease location at 3-year follow-up, there was a striking association of colonic-only disease with HLA-Bw6 (ie, the reciprocal of HLA-Bw4; risk 2.19 and 2.48, P = .048 and .040, for European and All subjects) and also with CMV (risk 9.09 and 2.71, P = .029 and .116; Table 2A). Consistent with this finding, ileal disease (Montreal L1 + 3 vs L2) was conversely associated with HLA-Bw4 (risk 1.44 and 1.59, P = .12 and .04; Table 2B).
Table 2.
Association with disease location.
| Colonic-only disease | ||||
|---|---|---|---|---|
| Europeans (n = 77) | All (n = 89) | |||
| Variablea | Risk | P | Risk | P |
| HLA-B | 0.456 | .048 | 0.404 | .014 |
| HLA-C | 1.133 | .734 | 0.986 | .968 |
| CMV | 9.097 | .029 | 2.714 | .116 |
| KIR3DL1 | — | .991 | 1.965 | .658 |
| KIR3DS1 | 1.818 | .251 | 1.370 | .522 |
| KIR2DL2/3 | 1.677 | .313 | 1.885 | .192 |
| KIR2DS2 | 0.974 | .959 | 1.111 | .830 |
| KIR2DL1 | — | .991 | — | .991 |
| KIR2DS1 | 1.265 | .652 | 1.118 | .821 |
| Ileal disease | ||||
| Europeans (n = 390) | All (n = 425) | |||
| Variable | Risk | P | Risk | P |
| HLA-B | 1.437 | .122 | 1.594 | .037 |
| HLA-C | 0.974 | .908 | 1.048 | .833 |
| CMV | 0.661 | .267 | 0.602 | .156 |
| KIR3DL1 | 0.000 | .987 | 0.355 | .333 |
| KIR3DS1 | 0.715 | .296 | 0.853 | .614 |
| KIR2DL2/3 | 0.932 | .825 | 0.928 | .809 |
| KIR2DS2 | 1.027 | .933 | 1.135 | .687 |
| KIR2DL1 | 0.000 | .986 | 0.000 | .985 |
| KIR2DS1 | 0.925 | .810 | 1.115 | .736 |
Colonic-only disease location: single variable analysis of disease location in patients with disease location documented at year 3 follow-up (Montreal L1 = 0, Montreal L2 = 1). Association with ileal disease location: single variable analysis of ileal disease location in patients with disease location documented at year 3 follow-up (Montreal L2 = 0, Montreal L1/L3 = 1).
aCMV represents presence or absence of seropositivity. HLA B indicates Bw4 allele frequency (0-2). HLA C indicates C2 allele frequency (0-2).
Discussion
The observed CMV infection rate was somewhat lower than reported for population-level data from the same decade for this age group within the US population (~30%).9 The reason for this lower rate in the RISK cohort is uncertain but may relate to evolving regional, ethnic, and racial factors affecting CMV incidence. Past CMV infection was associated with changes in mucosal NK cell and monocyte subpopulations. These findings are novel observations regarding the human post-CMV mucosal compartment and consistent with the biologic mucosal imprint of prior CMV infection on cell types known to serve as a latent virus pool and the anti-CMV host response.1,2
The disease association parameters were consistent with previous studies that reported an association of Crohn’s disease risk with HLA-B genotypes and not with CMV seropositivity.3,5 However, these observations were distinct from a number of studies reporting relationships of HLA-C and KIR genes with Crohn’s disease.10,11 The reason for this discordance is uncertain. The present cohort was large compared with most other reported studies, so the lack of replication is not likely due to statistical power. The strongest HLA-C and KIR associations were reported for Japanese and Korean cohorts, and other studies involved potentially diverse cosmopolitan without admixture adjustment used in the present study. The present study was also distinguished from prior studies as a pediatric vs adult age cohort. Thus, it is possible that our observations may relate to epidemiologic and biologic differences between pediatric- vs adult-onset Crohn’s disease. No associations were observed for disease behavior, consistent with the landmark study of Cleynen et al, which uncovered no genetic associations (HLA and others) for disease behavior8; here we extend this negative observation to KIR genes.
The main and novel finding of this report was the strong association of CMV and HLA-B with disease location. In prior studies, the strongest reported genetic associations to disease location included nucleotide oligomerization domain (NOD)2, macrophage stimulating (MST)1, chromosome region 3p21, and HLA, the latter particularly related to class 2 loci.5,8 However, the latter study also reported individual HLA-B alleles associated with colonic vs ileal localization, including ones associated with HLA-Bw6 and Bw4. We are not aware of studies testing the relationship of disease localization to CMV seropositivity. Although the potential mechanism of this association is uncertain, we note that the colon is a significant intestinal reservoir for latent CMV, which is associated with low-level anti-CMV host inflammatory activity.1,2 The cellular imputation results in this study affirm and extend this association. Thus, colonic CMV latency and the accompanying mucosal inflammation might prime the colonic intestinal region for Crohn’s disease susceptibility, perhaps via a selective HLA-C2 antiviral response.
We note several limitations of this study, including observational design and the lack of a validation cohort. Strengths of the study are the novelty of assessing CMV experience and KIR genetics for disease phenotype, the molecular stratification of population structure, and the relatively large size of this pediatric study. These observations relating the environmental factor of CMV with genetic contributions to Crohn’s disease phenotype merit further study for validation and mechanistic refinement.
Acknowledgments
The authors are grateful to the Crohn’s disease and healthy volunteer subjects whose selfless participation in this project made this study possible and Ted Hughes for guidance.
Glossary
Abbreviations
- CD
Crohn’s disease
- CMV
cytomegalovirus
- HLA
human leucocyte antigen
- NK
natural killer
Contributor Information
Terri Shih, David Geffen UCLA School of Medicine, Los Angeles, CA, 90095, USA.
Susy Yusung, David Geffen UCLA School of Medicine, Los Angeles, CA, 90095, USA.
Rivkah Gonsky, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.
Rhiannon Dutra-Clarke, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.
David Ziring, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.
Shervin Rabizadeh, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.
Subra Kugathasan, Emory University School of Medicine, Atlanta, GA, 30322, USA.
Lee A Denson, Cincinnati Children’s Medical Center, Cincinnati, OH, 45229, USA.
Dalin Li, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.
Jonathan Braun, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.
Author Contribution
J.B., S.Y., L.A.D., S.K. and D.L. designed the study. L.A.D., S.K., S.R., and D.Z. recruited subjects. T.S., S.Y., R.G., D.L., D.C., and R.D.P. performed bioinformatics analysis. J.B., T.S., and R.D.P. prepared the manuscript.
Funding
Supported by NIH PO1-DK046763 (J.B., D.L.), T32-DK007180 (S.Y.), U01-DK06214 (D.L.), the National Center for Advancing Translational Science (NCATS) UL1TR000124, and the Crohn’s and Colitis Foundation (S.K., L.A.D).
Conflicts of interest
No authors declare any conflicts of interest related to this study.
Data Availability
Ileal biopsy gene expression data has been previously archived under GEO repository accession number GSE101794. Other data cannot be shared publicly due to privacy of individuals that participated in the study; these data will be shared on reasonable request to the corresponding author.
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Associated Data
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Data Availability Statement
Ileal biopsy gene expression data has been previously archived under GEO repository accession number GSE101794. Other data cannot be shared publicly due to privacy of individuals that participated in the study; these data will be shared on reasonable request to the corresponding author.