Crohn’s disease (CD) presents as chronic and often progressive intestinal inflammation, but the contributing pathogenic mechanisms are unclear. Our goal was to identify underlying alterations in intestinal host cells that could contribute to the chronic and progressive course of CD. We performed RNA-seq with RNA extracted from formalin-fixed, paraffin-embedded (FFPE) ileal tissue sections obtained from CD subjects (n=32) and controls without inflammatory bowel disease (n=36). This method allowed us to select relatively uninflamed samples prior to molecular profiling and to perform histological validation with serial tissue sections. Accurate sampling of microvillar length was achieved by measuring 5 cells per villi and 10 villi per sample. We developed new methods to visualize an overlapping modular network of dysregulated genes in CD. With partitional clustering, we identified multiple down-regulated gene clusters present in nearly all of the CD samples. One cluster of 1,056 down-regulated genes was functionally associated with formation and stabilization of microvilli, leading us to quantify microvillar length. CD samples had a significant reduction in microvillar length relative to controls and this directly correlated to average microvillar gene cluster expression, validating our results. Network analysis supported a common regulatory mechanism for several of the common down-regulated gene clusters associated with altered metabolism in the intestinal tissue of CD subjects and this super-cluster was linked to the microvillar cluster. The FFPE transcriptomic approach can identify and link transcriptional alterations to histological alterations in CD tissue, such as a previously unrecognized alteration in microvillar length. We propose that decreased enterocyte microvillar length and gene expression represents an ongoing epithelial malfunction that is independent of active inflammation or local tissue damage.
A sizeable body of data demonstrates that membrane ICAM-1 (mICAM-1) plays a significant role in host defense in a site-specific fashion. On the pulmonary vascular endothelium, mICAM-1 is necessary for normal leukocyte recruitment during acute inflammation. On alveolar epithelial cells (AECs), we have shown previously that the presence of normal mICAM-1 is essential for optimal alveolar macrophage (AM) function. We have also shown that ICAM-1 is present in the alveolar space as a soluble protein that is likely produced through cleavage of mICAM-1. Soluble intercellular adhesion molecule-1 (sICAM-1) is abundantly present in the alveolar lining fluid of the normal lung and could be generated by proteolytic cleavage of mICAM-1, which is highly expressed on type I AECs. Although a growing body of data suggesting that intravascular sICAM-1 has functional effects, little is known about sICAM-1 in the alveolus. We hypothesized that sICAM-1 in the alveolar space modulates the innate immune response and alters the response to pulmonary infection. Using the surfactant protein C (SPC) promoter, we developed a transgenic mouse (SPC-sICAM-1) that constitutively overexpresses sICAM-1 in the distal lung, and compared the responses of wild-type and SPC-sICAM-1 mice following intranasal inoculation with K. pneumoniae. SPC-sICAM-1 mice demonstrated increased mortality and increased systemic dissemination of organisms compared with wild-type mice. We also found that inflammatory responses were significantly increased in SPC-sICAM-1 mice compared with wild-type mice but there were no difference in lung CFU between groups. We conclude that alveolar sICAM-1 modulates pulmonary inflammation. Manipulating ICAM-1 interactions therapeutically may modulate the host response to Gram negative pulmonary infections.
Abstract Lung epithelial progenitors use a complex network of known and predicted transcriptional regulators to influence early lung development. Here, we evaluate the function of one predicted regulator, Cux1, that we identified from transcriptional regulatory analysis of the SOX9 + distal lung progenitor network. We generated a new Cux1-floxed mouse model and created an epithelial-specific knockout of Cux1 using Shh-Cre (Cux1 ShhCre-LOF ). Postnatal Cux1 ShhCre-LOF animals recapitulate key skin phenotypic features found in prior constitutive Cux1 knockout animals, confirming functionality of the new floxed model. Postnatal Cux1 ShhCre-LOF mice displayed subtle alveolar simplification and a transient delay in alveologenesis without persistent lung phenotypes or alterations in lung epithelial cell allocation. Cux1 ShhCre-LOF mice developed failure to thrive in their second and third weeks of life due to delayed ileal maturation, which similarly resolves by postnatal day 35. Finally, we challenged Cux1 ShhCre-LOF with influenza-mediated lung injury to demonstrate that Cux1 ShhCre-LOF mice undergo productive alveolar regeneration that is indistinguishable from WT animals. Together, these findings indicate that epithelial-specific loss of Cux1 leads to transient developmental delays in the skin, lung, and intestine without defects in definitive organogenesis. One-Sentence Summary Deletion of key DNA binding domains leads to loss of Cux1 function in the lung, intestine, and skin characterized by transient failure to thrive without significant adult disease.
Abstract Objective Therapeutic efficacy of biologics has remained at about 50% for 2 decades. In Crohn’s disease (CD) patients, we examined the predictive value of an epithelial cell biomarker, ileal microvillar length (MVL), for clinical response to ustekinumab (UST) and vedolizumab (VDZ), and its relationship to another biomarker, intestinal epithelial cell (IEC) pyroptosis with respect to response to VDZ. Design Ileal biopsies from the UNITI-2 randomized controlled trial were analyzed for MVL as a predictor of clinical response to UST. In a 5-center academic retrospective cohort of CD patients, ileal MVL was analyzed to determine its predictive value for response to VDZ. Correlation between ileal MVL and IEC pyroptosis was determined, and the discriminant ability of the combination of two biomarkers to VDZ was examined. Results Clinical response in UST was significantly higher than placebo (65% vs. 39%, p=0.03), with patients with normal MVL (>1.7 µm) having the greatest therapeutic effect: 85% vs. 20% (p=0.02). For VDZ, clinical response with MVL of 1.35-1.55 µm was 82% vs. 44% (<1.35 µm) and 40% (>1.55 µm) (p=0.038). There was no correlation between ileal MVL and IEC pyroptosis. The combination criteria of ileal pyroptosis < 14 positive cells/1000 IECs or MVL of 1.35-1.55 µm could identify 84% of responders and 67% of non-responders (p=0.001). Conclusions Ileal MVL was predictive of response to UST and VDZ in prospective and retrospective CD cohorts. It was independent of ileal IEC pyroptosis, combination of the two biomarkers enhanced the discriminate ability of responders from non-responders to VDZ.
Therapeutic efficacy of biologics has remained at about 50% for 2 decades. In Crohn's disease (CD) patients, we examined the predictive value of an epithelial cell biomarker, ileal microvillar length (MVL), for clinical response to ustekinumab (UST) and vedolizumab (VDZ) and its relationship to another biomarker, intestinal epithelial cell (IEC) pyroptosis, with respect to response to VDZ.Ileal biopsies from the UNITI-2 randomized controlled trial were analyzed for MVL as a predictor of clinical response to UST. In a 5-center academic retrospective cohort of CD patients, ileal MVL was analyzed to determine its predictive value for response to VDZ. Correlation between ileal MVL and IEC pyroptosis was determined, and the discriminant ability of the combination of 2 biomarkers to VDZ was examined.Clinical response in UST was significantly higher than placebo (65% vs 39%; P = 0.03), with patients with normal MVL (>1.7 µm) having the greatest therapeutic effect: 85% vs 20% (P = 0.02). For VDZ, clinical response with MVL of 1.35 to 1.55 µm was 82% vs 44% (<1.35 µm) and 40% (>1.55 µm; P = 0.038). There was no correlation between ileal MVL and IEC pyroptosis. The combination criteria of ileal pyroptosis <14 positive cells/1000 IECs or MVL of 1.35 to 1.55 µm could identify 84% of responders and 67% of nonresponders (P = 0.001).Ileal MVL was predictive of response to UST and VDZ in prospective and retrospective CD cohorts. It was independent of ileal IEC pyroptosis, and combination of the 2 biomarkers enhanced the discriminate ability of responders from nonresponders to VDZ.
