Introduction

Transcriptomics is a fast-evolving field with translational applications and is broadly used in research. However, gene expression (GE) can be affected by many environmental and biological factors. The effect of commonly prescribed medications on GE is still understudied, especially in the context of a large cohort of chronic inflammatory disease (CIDs) patients. The unravelling of transcriptomic changes induced by therapies used by CIDs patients can further aid in the minimization of disease-unspecific signals and contribute to insights into medications’ mechanisms of action and repurposing.

Aims & Methods

The study aimed at the identification of prednisolone (PRED), azathioprine (AZA), and anti-TNF biologics signatures on the transcriptome of patients with inflammatory bowel disease (IBD) and other CIDs. RNA-seq was performed in peripheral blood samples of 625 CIDs patients included in a cross-sectional cohort. CIDs comprised IBD, rheumatoid arthritis, psoriasis, psoriatic arthritis, systemic lupus erythematosus, and arthrosis. Patients using the systemic therapies PRED and AZA, and anti-TNF biologics were compared against patients using no systemic therapies or no biologics, respectively. Linear mixed models were used with sex, age, body mass index (BMI), diagnosis, and C-reactive protein (CRP) as covariates. Differentially expressed genes (DEGs) (q-value < 0.05) were compared to the overall top 2000 varying genes (TVGs) to investigate how much the medication DEGs contribute to the general variation in gene expression.

Results

PRED investigation with low clinical disease activity patients revealed 224 down and 602 upregulated genes that were among the TVGs. Significantly enriched gene ontology (GO) terms of downregulated genes included adaptive immune response, immunoglobulin production, and B cell proliferation, whereas upregulated genes were related to acute inflammation, innate immune response, and positive NF-κB regulation. The AZA investigation comprised 24 IBD patients using AZA and 103 IBD patients using no systemic therapies, all with low clinical disease activity. Significant GO terms from the 144 downregulated genes part of the TVGs were related to cytokine production, cell proliferation, and T, B, and NK cell activity, whereas GO terms from the 301 upregulated genes included platelet aggregation and erythrocyte development. IBD, T-helper cell differentiation and NK cell-mediated toxicity were among suppressed enriched pathways. Variance partition analysis revealed that 151 genes have AZA as the driver of at least 25% of variation and 70 of those were part of the TVGs. GO terms of the upregulated DEGs in common with TVGs from the anti-TNF analysis were related to B cell and innate immune response activities, whereas the downregulated DEGs terms showed no direct relation to immune processes.

Conclusion

Transcriptomics analysis revealed that AZA and PRED, two commonly prescribed therapies to treat CID, modulate the expression of genes that contribute to the overall variation in gene expression and are potentially involved in other gene expression analyses. We suggest that the use of these medications should be considered when performing other transcriptomic analysis on blood samples from CID patients.

Introduction

Lymphocytic colitis (LC) is a debilitating, non-destructive inflammatory bowel disease (IBD) that causes intense watery, non-bloody diarrhoea. LC is diagnosed by an increased lymphocyte infiltration in the mucosa that has been correlated with increased Th1/Th17 cytokine secretion and subsequent sodium malabsorption. Still, LC pathomechanisms have not been fully characterized and its relation to other forms of IBD is unclear.

Aims & Methods

Our aim was to define a LC-specific transcriptome to gain insight into LC pathology, identify genetic signatures uniquely linked to LC, and uncover potentially druggable disease pathways. For that, we performed whole mucosa bulk RNA-sequencing of LC and collagenous colitis (CC) samples from patients with active disease, and healthy controls (n=4-10 per cohort). Differential gene expression was analyzed by gene-set enrichment and deconvolution analyses to identify pathologically relevant pathways and cells, respectively, altered in LC. Key findings were validated using reverse transcription quantitative PCR and/or immunohistochemistry. Finally, we compared our sequencing data to a previous cohort of ulcerative colitis and Crohn’s disease patients (n=4 per group) to distinguish non-destructive from classic IBD.

Results

The LC-specific transcriptome was defined by a limited mucosal immune response against microbiota compared to CC and classic IBD samples. In contrast, we noted a distinct induction of regulatory non-coding RNA species in LC samples. Moreover, compared to CC, we observed decreased water channel and cell adhesion molecule gene expression, which was associated with reduced intestinal epithelial cell proliferation.

Conclusion

We conclude that LC is a pathomechanistically distinct disease that is characterized by a dampened immune response despite massive mucosal immune cell infiltration. Our results point to regulatory micro-RNAs as a potential disease-specific feature that may be amenable to therapeutic intervention.

References

1. Carrasco A. et al. Immunological Differences between Lymphocytic and Collagenous Colitis. J Crohns Colitis. 2016 Sep;10(9):1055-66.
2. Kumawat AK. et al. Microscopic colitis patients demonstrate a mixed Th17/Tc17 and Th1/Tc1 mucosal cytokine profile. Mol Immunol. 2013 Oct;55(3-4):355-64.
3. Barmeyer C. et al. ENaC Dysregulation Through Activation of MEK1/2 Contributes to Impaired Na+ Absorption in Lymphocytic Colitis. Inflamm Bowel Dis. 2016 Mar;22(3):539-47.

Disclosure

CEH, SK and AM received financial support from Ferring Pharmaceuticals (Switzerland). AM has received salary for consultancies from Tillots Pharma AG, Ferring, Vifor, and Dr Falk Pharma, speaker's honoraria from Tillots Pharma AG and Vifor. The remaining authors declare no conflicts of interest.

Introduction

Xanthine oxidase (XO) catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid (UA) with the production of superoxide anion. The accumulation of UA has been shown to initiate the inflammatory process through NLRP3 inflammasome and the production of reactive oxygen species (ROS), which contributes to inflammation-related tissue damage observed in inflammatory bowel disease (IBD). In murine models of colitis, XO inhibitors, allopurinol or febuxostat, reduced the expression of proinflammatory cytokines supporting the involvement of XO in the intestinal inflammatory process.

Aims & Methods

The aim of this study was to evaluate the expression level and enzymatic activity of XO in the intestinal mucosa of IBD patients (pts), both ulcerative colitis (UC) and Crohn’s disease (CD), compared to healthy controls (HC).
Sigmoid-colon biopsies from consecutive UC and CD pts, with a confirmed diagnosis (by ECCO guidelines) in regular follow-up at University Hospital of Cagliari with an indication to endoscopy according to standard clinical practice, were collected. Site-matched biopsies from individuals undergoing screening colonoscopy for colorectal cancer were used as controls.
Demographic data and endoscopic grade for disease activity scored by endoscopic Mayo and SES-CD score were collected. Real-Time PCR was used to evaluate XO relative expression levels (RQ) and data was confirmed by analyzing GEO database (study code GSE11223). XO activity was measured in whole protein extracts from biopsies by spectrophotometric quantification of UA production and expressed as nmol of UA/min/mg of total protein. The relative abundance of XO was further investigated by Western Blot. Finally, XO expression was assessed in sections of paraffin-embedded intestinal biopsies by immunohistochemistry.

Results

Biopsies of 15 HC, 20 UC, and 15 CD pts were collected. Mean age was 50 for UC, 49 for CD and 54 for HC. Mayo endoscopic subscore was 0 in 25% of UC pts, 1 in 18,75%, 2 in 21,2% and 3 in 25%. SES-CD was between 0-2 in 28,6% of CD pts, 3-6 in 21,4%, 7-14 in 42,8% and >14 in 7,1%. Real-time PCR showed that XO expression values were increased in the sigmoid mucosa of pts with UC compared with HC individuals (RQ 1,93 vs 0,99. p=0,004). This data was confirmed by analysis of XO expression in the GEO database study code GSE11223. The difference in XO expression between HC and CD was not statistically significant (p=0,13). Western blot analysis of XO protein expression in CD and UC pts compared with HC showed a significant increase in IBD pts (p<0,001).
XO activity was significantly higher in UC and CD pts as compared to HC. XO activity mean was 3,75 in UC pts and 5,10 in CD pts vs 1,11 nmol of UA/min/mg of protein in HC individuals (p<0.001). Moreover, in UC pts XO activity was correlated with Mayo endoscopic sub-score (r= 0.346; p=0.05). Immunohistochemistry confirmed higher expression of XO in IBD pts than HC with a prevalent expression observed in epithelial cells.

Conclusion

XO expression was higher in UC pts than in HC individuals. XO activity was higher in both CD and UC pts than HC and in UC pts XO activity correlated with endoscopic disease suggesting its potential role as therapeutic target.

Introduction

For the precision medicine, patient-derived organoid (PDO) is regarded as one of the promising models. However, current PDOs were mainly derived from surgical specimen.

Aims & Methods

We aimed to analyze the single cell RNA-sequencing (scRNA-seq) of PDO from various sampling methods with different pathologic outcomes.
In 2021, three pancreatic cancer and one gallbladder cancer samples were collected from surgical specimen, endoscopic ultrasound guided-fine needle biopsy (EUS-FNB), and endoscopic retrograde cholangiopancreatography (ERCP)-guided biopsy. The samples were mechanically dissociated with dissociation buffer and incubated using shaking incubator. Dissociated cells were seeded into growth factor reduced Matrigel. From these PDOs, to evaluate the histological similarity between the PDOs and cancer tissue samples, a pathologist evaluate the PDOs and cancer samples using H&E staining. And we generated scRNA-seq data in each experimental design, and corrected batch effects that could occur when multiple samples are considered simultaneously in downstream analysis. Marker genes representing each cell subcluster were identified, and statistical hypothesis tests were performed to identify genes differentially expressed according to differences in biopsy methods and/or pathological results.

Results

Pathologically, when comparing the PDOs and cancer tissues, the PDOs showed well-visualized malignant glandular epithelium and similar architecture forming glandular structures, just like cancer tissue samples. We found the optimal 13 types of sub cell-type clusters with a resolution parameter of 0.3 in clustering analysis based on the Uniform Manifold Approximation and Projection (UMAP) algorithm in terms of silhouette score. As a result of examining the expression levels of KRT19, a marker gene for malignant ductal cells, and RGS5, a stellate marker gene, which showed the same pattern as the existing PDAC transcriptome studies, it was confirmed that most of the cells derived from PDOs were malignant ductal cells. Finally, we identified a heterogeneous effect on gene expression patterns in ductal cells depending on not only pathological positivity, but also biopsy methods for organoid.

Conclusion

According to our scRNA-seq, most cells were malignant cells and EUS-FNB could be a good sampling method for PDOs. However, further analysis about a heterogeneous effect on gene expression patterns is necessary.

Introduction

Granulocyte–monocyte apheresis (GMA) is a non-pharmacological therapy approved for the treatment of ulcerative colitis (UC), mainly in steroid-dependent cases. Its mechanism of action is based on the removal of activated leukocytes, but its exact immunological changes have not been fully described yet.

Aims & Methods

Our aim was to characterize the response in the transcriptome at the single-cell resolution level and cell population effects of GMA device. We analysed scRNASeq data from peripheral blood mononuclear cells (PBMC) of two UC patients undergoing their first GMA session. Their gene expression profile was compared before (PRE) and one month after (POST) the GMA treatment, from the inflow and outflow lines, respectively. The analytical pipeline included quality control and filtering, cell-type annotation, differential gene expression and pathway enrichment analysis.

Results

Two patients with UC (mean age 59 years; both E2) were included. Overall, the cell populations that appear to have been affected by the GMA treatment were natural killer cells and monocytes with both populations being reduced after the treatment. The distribution of the annotated cell-types was 2,369 B cells (PRE:50%, POST:49%), 11,462 CD4+ (PRE:49%, POST:50%) and 8,845 CD8+ T-cells (PRE:51%, POST:48%), 47 dendritic cells (PRE:40%, POST:59%), 3,773 natural killer cells (PRE:54%, POST:45%) and 2,352 monocytes (PRE:59%, POST:40%). Annotation at higher resolution identified 27 cell-types with monocytes being subannotated as 1,972 CD14 (PRE:62%, POST:37%) and 380 CD16 (PRE:48%, POST:51%) monocytes. Differential gene expression analysis identified in total 86 significant genes across six cell-types (CD4+ naive and central memory T-cells, natural killer cells, B intermediate cells, double-negative T-cells and CD14 monocytes) with 63% of these genes being downregulated after the GMA treatment. Pathway enrichment analysis identified higher contribution of the double-negative T-cells to the enriching genes. More specifically, after the treatment the double-negative T-cells exhibited upregulation of the NEFL that is associated with the MAPK cascade and downregulation of genes related to immune response and signalling pathways. Regarding the CD14 monocytes, after the GMA treatment we observed significant downregulation of the genes LINC02315, IGHEP1 and IGHE, with the latter being linked to innate immune response pathways.

Conclusion

GMA induces a range of modifications in the gene expression profile across different cell types that change the immunological environment of UC patients.

Disclosure

Financial support from an unrestricted grant from Adacyte, who had no role in the study design, interpretation of data or drafting the abstract.

Introduction

Ulcerative colitis (UC) is an inflammatory disorder of the colon of unknown aetiology characterised by intestinal barrier dysfunction associated with colonic epithelial goblet cell depletion and a shift in intestinal stem cell retention and function^1,2. Pancreastatin (PST), an intestinal epithelial cells-derived peptide highly expressed in colonic tissues of patients with active UC, positively correlates with UC early onset, disease severity, and inflammatory markers³. However, the effect of PST inhibition on colonic epithelial barrier function remains undetermined.

Aims & Methods

This study aimed to investigate the impact of pancreastatin inhibitor 8 (PSTI8) on colonic epithelial barrier function in a preclinical mouse model of UC. Male and female C57BL/6 mice were treated intrarectally with PSTi8 (2.5mg/mL/kg) or PBS for six days and received 5% dextran sulfate sodium (DSS) to induce colitis or water (control) for five days. The disease activity index (DAI) was assessed daily. ELISA quantified inflammatory cytokines, interleukin (IL)-6, IL-18 and IL-22. Markers to characterise differentiated goblet cells (Mucin, MUC2), transcription factors for goblets’ lineage commitment (growth factor independent, GFI1 and Kruppel-like factor, KLF1), and colonic stem cells plasticity (fast-cycling Lgr5, fetal-like Ly6a, and reserve Hopx cells) were quantified via RT-PCR and immunofluorescence.

Results

PSTi8 treatment increased stool consistency (p<0.01) and delayed the onset of bleeding in colitic male mice compared to PBS-treated mice (2-4 days). Conversely, no differences in the DAI were noted between colitic PSTi8 and PBS-treated female mice. In non-colitic conditions, PSTi8 treatment increased (p<0.03) IL-22 in males and decreased (p<0.01) its level in female mice with no effect of IL-6. In colitic conditions, although IL-22 levels were decreased (p<0.01) in female mice and IL-6 levels were increased (p<0.001) in male mice, PSTi8 treatment did not show any effect. Conversely, in colitic females, IL-18 was significantly increased (p<0.02), and PSTi8 abolished that increase. The mRNA expression level of MUC2 remains unaltered in male and female non-colitic and colitic mice treated or not with PSTi8. Yet, immunofluorescence staining in non-colitic and colitic conditions demonstrated a decrease of MUC2 in PSTi8-treated female mice. In concordance, transcription factors KLF1 and GFI1 were significantly increased (p<0.001 and p<0.02, respectively) in colitic female mice, and PSTi8 treatment abolished that increase. No differences were seen in male mice except for a significant increase of KLF1 in PSTi8-treated colitic mice. In colitic conditions, fast-cycling cells marker Lgr5 expression in both sexes was significantly decreased (p<0.0001), and treatment with PSTi8 did not show any effect. However, in colitic male mice, fetal-like stem cells makers Ly6a expression was significantly increased (p<0.007), and PSTi8 treatment significantly decreased (p<0.02) that expression. Although a significant decrease (p<0.0001) of reserve cells marker Hopx was only seen in colitic female mice, PSTi8 treatment did not regulate that marker in male and female mice.

Conclusion

PST inhibition protects male mice from DSS-mediated colitis while exacerbating susceptibility to colitis in female mice. PST inhibition regulates the expansion of goblet and intestinal stem cells in a sex-dependent manner. Further investigation is needed to delineate the signalling pathways involved in PST action on the mucosa in both sexes. Targeting PST may lead to novel therapeutic strategies in UC.

References

1. Singh V, Johnson K, Yin J, et al. Chronic Inflammation in Ulcerative Colitis Causes Long-Term Changes in Goblet Cell Function. Cmgh. 2022;13(1):219-232.
2. Yui S, Azzolin L, Maimets M, et al. YAP/TAZ-dependent reprogramming of colonic epithelium links ECM remodeling to tissue regeneration. Cell Stem Cell. 2018;22(1):35-49.e7.
3. Eissa N, Elgazzar O, Hussein H, Hendy GN, Bernstein CN, Ghia JE. Pancreastatin reduces alternatively activated macrophages, disrupts the epithelial homeostasis and aggravates colonic inflammation. A descriptive analysis. Biomedicines. 2021;9(2):1-18.

Introduction

The melanocortin 1 receptor (MC1R)–specific agonist PL8177 and its main metabolite PL8435 have demonstrated MC1R binding affinity and functional activity that mirrors that of α-melanocyte–stimulating hormone, which has been shown to be effective in reducing inflammation in numerous experimental models. This study investigates the effects of PL8177 on inflammation, cell population composition, and gene and protein expression in colons from a dextran sulfate sodium (DSS)–induced rat model of colitis. The objective is to determine the effectiveness of PL8177 in this model and to characterize its underlying mechanism of action.

Aims & Methods

Male Wistar rats received 5% DSS in drinking water for 3 days to induce colitis (each group n=6). Rats in the sham group received drinking water only. The other groups received vehicle control (placebo)–filled capsules; PL8177-filled capsules at 20, 50, and 100 μg per animal (by oral gavage); or oral mesalazine (positive control). At termination on day 8, 24 hours after the last dose, colon tissues were harvested, dissected, and snap frozen with liquid nitrogen. Colon samples were analyzed for cytokine levels, single nuclei RNA-seq, and data-independent acquisition tandem mass spectrometry. Colitis was assessed by a disease activity index (diarrhea and rectal bleeding), colon length shortening, colon weight gain, and histopathological assessment. The total colitis index was used to assess inflammatory damage.

Results

Colitis was induced in rats treated with DSS. Treatment with oral PL8177 50 μg showed a significant (P<0.05) improvement in colon weight (53% reduction), stool consistency, and fecal occult blood score compared to vehicle. There was a significant (P<0.05) improvement in the total colitis index for the PL8177 100-μg group vs vehicle control group. All PL8177 cohorts showed greater improvement in the total colitis index compared to the mesalazine-treated cohort. PL8177 50 μg showed a significant (P<0.05) improvement in colon weight (53% reduction), whereas mesalazine treatment was associated with a marked reduction in colon length, but only moderate improvement (35%) in colon weight. Single nuclei RNA-seq analysis showed that after oral PL8177 100 µg treatment, relative cell populations and key gene expression levels were closer to those of healthy controls. A subclustering analysis revealed significant differences between the PL8177 100-µg and vehicle populations. Although both showed the presence of macrophages, those present in vehicle-treated colons were primarily M1 macrophages that are involved in inflammation. In the PL8177 100 µg–treated colons, macrophages were primarily M2, which are pro-resolution. Proteomic analysis showed that the colons of PL8177-treated rats are more similar to sham colons than to vehicle colons. Additionally, after treatment with PL8177, a protein known to promote intestinal homeostasis and a blood-based biomarker protein showed expression similar to that seen in the sham group, a result not seen in vehicle-treated colons.

Conclusion

In a rat model, oral PL8177 treatment of colonic inflammation showed significant improvement in anatomical markers of colitis vs the vehicle and mesalazine control groups, supporting the aim of treating inflammatory bowel disease (IBD) in humans. Transcriptomics and proteomics data show that oral PL8177 treatment causes diseased colons to move toward the healthy state and to resolve inflammation. Resolving inflammation—rather than blocking it—provides the possibility of efficacy coupled with safety in treating IBD.

Disclosure

Priyanka Dhingra, Alison Obr, Carl Spana, John H. Dodd, and Paul S. Kayne are employees of Palatin Technologies, Inc.