This study represents the largest pediatric IBD population reported to date in which serologic responses to microbial antigens in children with Crohn’s disease have been examined. Moreover, we have demonstrated that these responses are affected by age at diagnosis with possible implications for understanding the pathogenesis of this complex disease.
In adult populations with known CD, the frequency of ASCA (either IgG or IgA) has been reported to range from 28–59% (11
), while that of anti-ompC ranges from 37–56% (15
). Published investigations in children with CD have revealed somewhat higher rates of detectable ASCA (44–76%) (5
) but a lower rate for anti-ompC (24%) (5
). In the present study, the overall rates of anti-ompC and ASCA are lower than previous pediatric reports. However, the results are likely affected by the relatively large number of very young children in our study cohort, as the rate of anti-ompC rises to almost 20% and ASCA to 40% in the 8–15 year old subjects. pANCA, an ulcerative colitis associated marker, has been reported to be present in 10–20% of adults with “UC-like” CD (19
). Our findings reveal a similar, if somewhat greater frequency, with nearly 30% of children of all ages with isolated colonic CD having detectable pANCA (data not shown).
In adult populations, anti-CBir1 antibodies appear to be a specific serologic marker for CD, having been identified in approximately 50% of adult CD patients but very few UC patients (7
). In our overall population, 56% of children less than 16 years of age at diagnosis had detectable anti-CBir1. At all age groups, anti-CBir1 appears to identify a significant subpopulation of children who otherwise would be characterized as serologically negative. It therefore appears that the addition of the anti-CBir1 assay to a serologic panel should enhance the sensitivity of testing when serology is used as a diagnostic test to identify children likely to have IBD. This speculation must be explored in additional studies, however, as our study population only includes children with documented CD.
For ASCA and anti-CBir1, the age at diagnosis appears to be an important factor affecting the likelihood of identifying positive serologic responses in children with CD. ASCA is rare in the younger children, while anti-CBir1 is particularly common. These observations may be particularly important clues toward understanding the pathogenesis and early immunologic dysfunction that leads to the development of CD.
CD appears to arise from a dysregulated immune response to the endogenous enteric microflora in a genetically susceptible host (22
). Normal innate immune responses recognize microbial antigens such as muramyl dipeptide (a component of lipopolysaccharide), resulting in controlled responses by gut associated immunoreactive cells. Defective innate immune responses, such as those resulting from allelic variants of NOD2/CARD15 or various toll-like receptor (TLR) genes, cause a cascade of events that ultimately are expressed as chronic intestinal inflammation (22
The CBir1 antigen has been shown to have a high degree of homology to bacterial flagellins, resembling known flagellins from enteric microbiota including Butyrivibrio
, and Clostridium
). Antibodies against the CBir1 antigen have been identified in a number of different mouse models of IBD, including one whose underlying genetic predisposition for IBD causes defective epithelial barrier function and a number of others characterized by different defective regulatory T-cell functions (7
The serologic patterns identified in the different age groups reported in the present investigation strongly suggest that the host microbial interactions underlying the development of CD at varying ages are different. As a group, children less than 8 years of age at diagnosis are predominantly anti-CBir1 positive, ASCA and anti-ompC negative, while those older than 8 years of age are more commonly both ASCA and anti-CBir1 positive. These observations suggest that there may be different underlying genetic defects in children who present at different ages. Which specific genes might be most important in the youngest children requires additional study. Our data suggest that the Toll-like receptor (TLR) gene family may be critically important to investigate, as TLR-5 has been reported to mediate the normal innate immune response to bacterial flagellin (24
). In addition, a recently described linkage between a specific haplotype of the NFkB1 gene and expression of anti-CBir1 identifies another potential gene of particular interest in this population (25
Another potentially fertile area for additional investigation arises from the alternative hypothesis that the enteric microbes central to the development of CD differ in younger compared to older children. The high frequency of anti-CBir1 antibodies in the children less than 8 years of age at diagnosis suggests that microbes such as Clostridia may play an important role in precipitating CD in the youngest children, whereas yeast and E. coli, the organisms whose antigens are associated with ASCA and anti-ompC, may play a more important role in children who present at an older age.
In summary, our data demonstrate that the anti-CBir1 assay identifies a subgroup of children with CD who would otherwise not be characterized serologically. This appears especially important in the youngest children with CD, in whom anti-CBir1 appears to be particularly common. These data suggest, but do not prove, that the addition of the anti-CBir1 assay to a panel of markers including ASCA, anti-ompC and pANCA may increase the sensitivity of serology as a screening tool for the diagnosis of CD in children. The age-associated differences in the patterns of antimicrobial seropositivity demonstrated in the present study also suggest that there may be different, and as yet unrecognized, genetic, immunologic and/or microbial factors leading to CD in the youngest children.