While several studies have examined the pathophysiology of growth failure in children with CD, many gaps in our knowledge remain. Previous studies have suggested a genetic predisposition to growth retardation in a subset. A longitudinal population-based study showed that children who underwent surgery were smaller throughout childhood, prior to the clinical diagnosis of CD, compared to those who did not require surgery (12
). A recent paper showed that the parents of patients with growth impairment had significantly decreased HTz compared to parents of children without growth impairment (20
). Studies have consistently shown that small bowel disease location was associated with reduced linear growth (1
). We have previously reported that elevated GM-CSF Ab in both pediatric and adult CD are strongly associated with ileal location and stricturing and penetrating disease requiring surgery (15
). Our current studies have now shown that pediatric CD patients with both a CARD15
SNP and elevated GM-CSF Ab have reduced HTz at diagnosis compared to children with CD with one or neither factor.
In our univariate analysis, we observed an additive effect of elevated GM-CSF Ab and a CARD15
risk allele; the combination reached significance for association with reduced HTz. Small bowel location and WTz were also strongly associated with HTz. Consistent with our prior report, the C15+
state was strongly associated with small bowel location. The C15+
state was not associated with WTz, demonstrating an effect on linear growth independent of variation in weight. In our stepwise multivariate analysis, the inclusion of small bowel location reduced the effect of the C15+
state upon HTz, while the inclusion of WTz reduced the effect of small bowel location upon HTz. We did not observe an effect of age or gender on HTz once they were included in the multivariate analysis. This differs from previous studies that found that age and male gender were associated with reduced linear growth (1
). These differences may have been due to the inclusion of the specific genetic and immune factors in the current analysis, the pubertal status of the cohorts, or the definitions used to classify linear growth. Collectively, our analysis suggests that the C15+
state suppresses linear growth in CD via a mechanism which involves small bowel location, independent of differences in weight.
Remarkably, we did not observe a difference in serum LBP, TNFα, or IL-6 between the C15+
group and disease controls. This suggested that reduced growth was not due simply to global differences in inflammation. We have recently reported that children with CD and elevated GM-CSF Ab exhibit a specific increase in intestinal permeability and anti-endotoxin antibodies, in the absence of differences in systemic or intestinal inflammation (21
). We have previously shown in animal models that endotoxin can directly reduce liver Ghr abundance, via both transcriptional and post-transcriptional mechanisms (6
). Serum GHBP may be used as a surrogate marker for tissue GHR abundance and is associated with BMI and linear growth in healthy children (22
). We identified a significant reduction in serum GHBP in C15+
patients compared to disease controls. Our results suggest that the C15+
state may lead to a specific reduction in tissue GHR as measured by serum GHBP, in the absence of a difference in overall weight or inflammatory status. Ongoing studies will determine whether this is due to increased permeability and chronic endotoxemia.
The patient-based studies could not establish a causal relationship between the C15+
state and reduced tissue GHR abundance. We therefore asked whether induction of ileitis via gm-csf neutralization in the Card15
deficient host would lead to down-regulation of the hepatic Ghr and GH resistance. We confirmed that the hepatic GH:Ghr:Igf-1 axis was profoundly suppressed in this setting, with an intriguing sexual dymorphism. We found that serum LBP concentration was elevated in the murine model of ileitis, with no gender differences, and that this was comparable to the degree of elevation observed in the patients. However, the hepatic Ghr and GH activation of STAT5 were significantly reduced only in male mice with ileitis. However, serum Igf-1 was reduced in both male and female mice with ileitis. We speculate that the gender differences we observed are secondary to differences in sensitivity to endotoxin dependent suppression of Ghr
gene transcription and protein abundance. The reduction in serum Igf-1 observed in both sexes may be due to post-Ghr effects of IL-6 upon Igf-1 stability (24
). Collectively, the murine studies demonstrated that liver Ghr abundance and GH activation of the anabolic transcription factor Stat5 were suppressed in male mice with ileitis due to gm-csf neutralization and Card15
deficiency, while circulating Igf-1 was reduced in both males and females. These changes occurred despite normal weight gain relative to controls, demonstrating an effect of the C15+
state independent of variation in weight. The reduction in circulating Igf-1 was strongly associated with induction of the hepatic APR as measured by serum LBP, while the reduction in liver Ghr and pSTAT5 protein abundance were not. This was consistent with our observation in patients that suppression of circulating GHBP in the C15+
group was not associated with a difference in systemic inflammation as measured by serum LBP or cytokines compared to disease controls.
Given the retrospective nature of our study, there are some limitations. The blood samples for measurement of GM-CSF auto-antibodies, cytokines, and growth factors were obtained on average three years after diagnosis (). While our studies to date have shown that GM-CSF auto-antibodies are stable over time, it is possible that in some patients these may have been different at diagnosis (15
). The results for the cytokines and growth factors are also limited by the cross-sectional nature of the analysis, and may not reflect differences that were present at diagnosis. We did not have access to Tanner staging of the patients, or bone age measurements, and so could not control for pubertal status. Mid-parental heights were not available, which would have been useful in estimating height deficits relative to genetic potential. We were not able to assess protein and calorie intake, or malabsorption, which may differ between CD patients with colon-only disease and those with small bowel involvement. We were not able to obtain a history for NSAID exposure, which may have influenced the serum cytokines or growth factors. Finally, we were not able to measure mucosal disease activity. An ongoing prospective study will address these potential weaknesses.
In summary, we have shown that defects in innate immunity due to GM-CSF auto-antibodies in a CARD15
deficient host are associated with linear growth failure in children with CD, and induce liver GH resistance in a murine model of ileitis. Growth failure in the patients and GH resistance in the animal model occurred in the absence of differences in inflammation or weight, suggesting a specific effect of the C15+
state. Based upon our recent study showing increased intestinal permeability and evidence of endotoxin exposure in children with CD and elevated GM-CSF Ab, we speculate that defective barrier function may directly induce GH resistance and growth failure in this setting (21
). This would imply that therapies designed to augment barrier function might also restore GH action and growth in children with CD.