Exabis Library

Innate Lymphoid Cells (ILC) develop from Common Lymphoid Precursors in the bone marrow, and ILC precursors (ILCP) migrate to mucosa where they mature, promote homeostasis, and provide a potent, antigen-non-specific sources of cytokines. Deciphering what local stimuli drive the final stages of ILCP maturation in these tissues remains a pressing question, as ILC frequencies can become dysregulated during chronic infection and inflammatory diseases. For example, Type-1 innate lymphoid cells (ILC1) are enriched in the mucosa of patients with active inflammatory bowel disease (IBD) and the impact of this accumulation remains elusive.

Here, we develop and use co-cultures of both murine and human iPSC-derived gut and lung organoids with ILCP and with mature ILC isolated from IBD patients’ intestinal biopsies.

Harnessing these versatile models, we demonstrate that epithelial cells provide a complex niche capable of supporting the final maturation of all helper-like ILC1, ILC2, and ILC3. Notably, organoid identity was sufficient to robustly recapitulate tissue-specific ILC imprints and frequencies, even in the absence of microbial stimuli, other cell types, or cytokine supplementation.

In addition, we show that that ILC1 drive expansion of the epithelial stem cell crypt through p38γ phosphorylation, driving a potentially pathological proliferative feedback loop between β-catenin and Cd44v6. We harnessed this model to elucidate that this phenotype was unexpectedly regulated by ILC1-derived TGFβ1. We further show that human gut ILC1 also secrete TGFβ1, and drive CD44v6 expression in both HIO epithelium and mesenchyme. As TGFβ1 is a master regulator of fibrosis, the leading indicator for surgery in IBD, we next characterised the ability of ILC1 to regulate matrix remodelling using a functionalized, synthetic hydrogel system. We show that ILC1 drive both matrix stiffening and degradation, which we posit occurs through a balance of MMP9 degradation and TGFβ1-induced fibronectin deposition.

Taken together, our work provides unprecedented insight into in situ ILC maturation, which we show to be driven by epithelial signals, and into ILC function. We also report that intestinal ILC1 modulate epithelial and matrix remodelling, which may drive either wound healing in homeostasis, but may tip toward pathology when enriched in IBD.
Moreover, our work introduces a modular organoid platform, which provides exquisite control over both environmental stimuli and host genetics, making it a powerful tool for dissecting the interactions between complex mucosal tissues and rare cell subtypes in development and disease.

Extracellular RNAs (exRNAs) are RNA species present outside of the cells in which they were transcribed. They are found in human serum, though the exact role of circulating exRNAs remains to be established, especially in inflammatory bowel diseases (IBD). Besides their potential help in our pathophysiological understanding of disease, they might serve as liquid biopsies or non-invasive biomarkers.  We characterised exRNAs in serum from IBD patients, and questioned their potential in separating ulcerative colitis (UC) from Crohn’s disease (CD).

We carried out SILVER-seq (Small Input Liquid Volume Extracellular RNA-sequencing) on serum droplets (5-7ml) from a cross-sectional cohort of 26 IBD patients (15 UC, 11 CD) with active endoscopic disease (Mayo endoscopic sub score  or Simple Endoscopic Score for Crohn’s disease ) (Table 1). Normalization and differential expression were done using DESeq2 R package, co-expression network analyses performed using WGCNA (FDR adjusted p ≤0.05). Using randomized generalized linear modelling (RGLM), a diagnostic exRNA marker was designed to separate UC from CD samples (15 UC, 11 CD). 

We detected 60,675 exRNAs in serum from IBD patients, capturing 76.1% of all genes expressed in intestinal tissue, and including highly abundant intestinal genes (e.g MUC2) and intestinal barrier genes (e.g claudin 8, occludin and RETNLB). Co-expression network analysis identified 69 clusters of which 1 significantly correlated with the distinction between CD and UC (FDR p=0.003, r=-0.70). One of the hub genes within this module (consisting of 148 genes, upregulated in UC) was GNA12 (p=2.3E-4, r=0.66 for correlation with the module eigengene), encoding for a membrane bound GTPase that plays a key role in tight junction assembly and has previously been identified as UC-specific SNP in GWAS (Figure 1). Serum GNA12 expression was not associated with faecal calprotectin (p=0.55, r=0.12), disease duration (p=0.24, r=0.25), age (p=0.43, r=0.16) or gender (p=1.0), but did correlate with other UC-specific genes including TNFRSF14 (p=0.04, r=0.4), HNF4A (p=0.04, r=-0.4) and CAMK2A (p=0.004, r=0.54). Through machine learning within the UC-specific module (containing 148 genes), we identified an 8-gene exRNA panel, including GNA12, that could accurately discriminate between UC and CD patients (accuracy 96.2%).

Figure 1: Visualisation of the identified exRNA network including GNA12

Liquid biopsies are a novel non-invasive tool in IBD biomarker development. Although larger in-depth studies are required to further validate, explore and characterise the potential of serum exRNAs in the field of IBD, the current pilot project identified a new non-invasive tool to accurately distinguish CD from UC patients.

To address the question if pediatric CD patients responding to nutritional induction therapy can be maintained in remission on dietary therapy without the use of immunosuppressive drugs, we designed a prospective randomized trial (CD-HOPE) comparing cyclic exclusive enteral nutrition (EEN) to daily supplement over a 12 month period.

CD patients (6-17 years) who successfully completed at least 6 weeks of EEN with clinical remission (wPCDAI ≤12.5) were recruited in 21 sites of the French GETAID pédiatrique between 12.2014 and 09.2018. All drug therapy had to be stopped at least 4 weeks prior to inclusion. A total of 112 patients were screened with 100 patients randomized to group A cyclic EEN (100% of caloric requirement) every 8 weeks for 2 weeks or group B daily supplementary nutrition (25% of caloric requirement). Patient stratification according to age (< 10 years or older) and previous drug exposure or not. EEN and the nutritional supplement were in form of MODULEN IBD®. Except for the two weeks of EEN in group A food access was not restricted. Primary objective was the comparison of relapse rates at 12 months (defined as a wPCDAI >12.5 at two consecutive visits) between the two groups (log-rank test per protocol). Additional analyses were performed using a multivariate regression analysis and cox model.

49 CD patients were randomized to group A (cyclic EEN) and 51 to group B (daily supplement) with 43/49 and 44/51 newly diagnosed patients without any previous drug exposure. Baseline characteristics were comparable between the two groups. Median age was 12 and 13 years, group A and B respectively. At the final 12 months visits a total of 25/49 patients (group A) remained in remission without disease activation compared to 12/51 patients (group B) (p=0.004) with a hazard ratio of 0.48 (0.29-0.80) (p= 0.0051). Kaplan Maier survival remission rates are shown in figure 1. Mean fecal calprotectine levels showed no significant difference between the two groups (297, 399 and 469 at month 0, 3, and 12 visits in group A and 480, 606, and 283 at month 0,3, and 12 visits in group B). Mucosal healing at M12 months was achieved in 25/49 patients (group A) and 18/51 patients (group B), with a mucosal healing rate of 52%  (group A) and 33% (group B). Both treatment arms showed a significant catch-up growth.

This is first trial indicating that children/adolescents with CD responding to EEN as induction therapy can be maintained on remission with a nutritional therapy without immunosuppressors/biologics. However, daily nutritional supplement with normal access to food was not successful with a relapse rate of 76%.
This study was supported by an unrestricted grant from Nestlé Health Science and sponsored by APHP.

Isolated colonic (L2) Crohn’s disease (CD) in adults is thought to have unique clinical and genetic features compared with ileal (L1) CD and ulcerative colitis (UC). Similar studies in paediatrics are scarce. Our goal was to characterize the clinical features of paediatric patients with isolated colonic CD and compare them to patients with ileo-cecal CD and those with UC.

This was a multi-center retrospective study including 21 sites affiliated with the Porto IBD group and IBD interest group of ESPGHAN. Data of paediatric patients diagnosed between 2014-2017 with L1 or L2 CD, or with UC, was collected, including information on demographic, clinical and laboratory parameters at diagnosis, end of induction, 1 year and 3 years after diagnosis (or at last follow-up).

Data was collected on 300 children (102 L1, 94 L2, 104 UC) with similar demographic features. At diagnosis, bloody stools were identified in 45% of L2 patients, compared with 15% and 95% of L1 and UC patients, respectively (P<0.001), while fever was documented in 27% of L2 patients, compared to 13% and 3% of L1 and UC patients, respectively (P<0.001). At the time of diagnosis, the median pediatric Crohn’s disease activity index for patients with L1 and L2 was 25 (IQR 17.5-37) and 27.5 (20-40), respectively, while the median pediatric ulcerative colitis activity index was 40 (30-55) for patients with UC. C-reactive protein levels were significantly higher among CD patients (both L1 and L2), compared to patients with UC, and calprotectin values were comparable. ASCA was positive in 55%, 25% and 2% (P<0.001) and pANCA in 2%, 17% and 53% (P<0.001) in L1, L2 and UC patients, respectively. Granulomas were identified in 36% of L2 patients, similar to patients with L1 (33%). For induction therapy, exclusive enteral nutrition, oral steroids and mesalazine were used in 50%, 45% and 38% of patients with L2 CD, compared with 72%, 28% and 9%, and 0%, 52% and 75% of L1 and UC patients, respectively (P<0.001). Steroid-free clinical remission at the end of induction was overall similar between groups, around 55%. At 1-year post-diagnosis, 62%, 68% and 40% were on an immunomodulator (P=0.03) and 41%, 26% and 22% were receiving anti-TNFα agent (P=0.01), of patients with L1, L2 and UC, respectively. While time to initiation of an anti-TNFα agent was significantly shorter in L1 patients compared with L2 and UC (P=0.03), time to admission and time to surgery were similar.

Paediatric patients with isolated colonic CD exhibit several clinical features which differentiate them from ileo-cecal CD and UC. Prospective studies are required to understand the pathogenesis of this unique entity and define short- and long-term outcomes.

Patients with Crohn’s disease (CD) can develop complications including stricturing and penetrating disease [1, 2]. Although reliable baseline predictors of disease progression are urgently needed to inform management strategies, few studies have comprehensively explored the phenotypic and genetic determinants of disease progression in a sufficiently powered cohort.

We used data from 13,926 patients with CD in the UK IBD BioResource to investigate the effects of clinical phenotypes and genetics on CD progression. Median follow-up was 10.6 years and total follow-up was 193,033 patient-years. We applied the Montreal classification system to define disease as B1 (inflammatory), B2 (stricturing) and B3 (penetrating). Patients with B2 or B3 disease (N = 5,185) were compared to patients with B1 disease (N = 8,471) in a multivariate model fitted with both phenotype data and a polygenic score that we developed. Associations with q-values (false discovery rate adjusted p-values) less than 0.05 were defined as statistically significant.

CD progression occurred over time from diagnosis (Figure 1). Consistent with previous findings, we confirmed factors including smoking, disease location and perianal disease were associated with disease progression [3] (Table 1). The impact of a genetic influence on disease progression was confirmed and shown to be independent of genetic effects on disease location [4]. Early prescription of medications showed a protective effect on disease progression: Infliximab, adalimumab and thiopurines significantly reduced the chance of B2/B3 progression when prescribed within two years of diagnosis. Additionally, we observed a decreased progression to B2/B3 disease in patients diagnosed recently (between 2012-2020) compared to those diagnosed before 2012. This finding persisted after conditioning on exposure to biologics and correcting for follow-up time and interval to first thiopurine prescription, and thus may be indicative of other improvements in standards of care in recent years.

Using a large, well-characterised cohort we confirm the importance of disease location, smoking status and genetics on disease progression. We highlight the positive impact of early medication prescription on disease progression and discover an independent signal relating to potential improvements in the standard of care in CD over time. These results create the framework for reliable predictors of CD progression that may better guide future CD management strategies.

Reference:
1. Cosnes, J., et al., Inflamm Bowel Dis, 2002. 8(4): 244-50
2. Lo, B., et al.,J Crohns Colitis, 2018. 12(3): 265-272
3. Torres, J., et al., J Crohns Colitis, 2016. 10(12): 1385-1394
4. Cleynen, I., et al., Lancet, 2016. 387(10014): 156-67