Introduction

The NLRP3 inflammasome is a multiprotein complex consisting of the receptor protein NLRP3, the linker protein ASC, and the effector protein caspase-1. Activation of NLRP3 can lead to increased production of proinflammatory cytokines, which disrupt intestinal barrier function and induce the development of ulcerative colitis.

Aims & Methods

Whether RNF31 regulates the expression of the NLRP3 inflammasome is not known. In this study, we examined the expression of RNF31, and NLRP3 in ulcerative colitis tissues by GEO database analysis and immunohistochemical assays, and subsequently determined that RNF31 can regulate NLRP3 inflammasome expression by constructing an RNF31-deficient cell model, and elucidated the role of RNF31 in the development of ulcerative colitis.

Results

We found that the expression of NLRP3 decreased after the addition of MG132 inhibited the level of ubiquitination. a cycloheximide (CHX)-chase analysis showed that RNF31 was able to make NLRP3 more stable, which was most likely due to the fact that RNF31 changed the level of ubiquitination on NLRP3. Multiple co-immunoprecipitation (CO-IP) assays showed that there was an interaction between RNF31 and NLRP3 and that this interaction increased the level of ubiquitination on NLRP3. And CO-IP experiments using truncated RNF31 plasmids showed that the RBR structural domain interacted with NLRP3, increasing the level of ubiquitination on NLRP3 and making NLRP3 more stable. By constructing a cell model with RNF31 deletion as well as overexpression and using CO-IP experiments we found that the K63 ubiquitination chain plays an important role in stabilizing NLRP3. Finally, we constructed RNF31-deficient mouse models using adenovirus and showed that the expression of NLRP3 and inflammatory factors IL-18 and IL-1β were reduced in the absence of RNF31.

Conclusion

Our study shows that RNF31 can interact with NLRP3 through the RBR structural domain and increase the level of the K63 ubiquitin chain on NLRP3 thereby making NLRP3 more stable and activating the assembly of the inflammasome, elucidating how RNF31 is involved in the mechanism of colitis development.

References

1. D. Oikawa, et al., Linear Ubiquitin Code: Its Writer, Erasers, Decoders, Inhibitors, and Implications in Disorders, Int. J. Mol. Sci. 21 (9) (2020) 3381.
2. F. Humphries, et al., The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome, Nat. Commun. 9 (1) (2018) 1560.
3.X. Wu, et al., MyD88-Dependent Signaling Is Required for HOIP Deficiency Induced Autoinflammation, J. Immunol. 207 (2) (2021) 542–554.
4.K. Bulek, et al., Epithelial-derived gasdermin D mediates nonlytic IL-1β release during experimental colitis, J. Clin. Investig. 130 (8) (2020) 4218–4234.
5.P. Wan, et al., Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation, FASEB J 33 (4) (2019) 5793–5807.

Introduction

Ulcerative colitis (UC) is a chronic, non-specific inflammatory disease of the bowel of unknown etiology. This disease is characterised by high difficulty in cure, high recurrence and cancer rates, which seriously endangers the lives and health of our population. Taraxasterol (TA), a major constituent of the traditional Chinese medicine dandelion, has been reported in several studies to have good anti-inflammatory and antioxidant activities as well as anti-tumour effects.

Aims & Methods

However, the molecular targets and mechanisms of action of TA against UC have not been fully elucidated.The Swiss Target Prediction, PharmMapper databases were used to obtain relevant targets for the drug TA. Targets for UC were obtained from GeneCards, OMIM, PharmGkb, DrugBank databases. Targets of TA anti-UC identified by intersection of drug target and disease target. The relevant targets of TA anti-UC were uploaded to the String database and the resulting data were imported into Cytoscape 3.9.1 software to construct a protein-protein interaction (PPI) network of drug-disease targets. At the same time, the top 10 hub targets were screened using CytoHubba software. Gene ontology (GO) and Kyoto Encyclopedia of Genomes (KEGG) pathway enrichment analyses were performed on TA anti-UC targets using R software, and molecular docking was performed by Autodock Vina 1.1.2. The GEO dataset was used to analyze the differential mRNA expression of the TA anti-UC top 10 hub gene in UC patients and the normal population. Lipopolysaccharide (LPS)-induced intestinal epithelial cells (NCM460) were used to construct an in vitro cellular inflammation model and a Dextran Sulfate Sodium Salt (DSS) induced acute mouse colitis model. The pivotal targets and key signaling pathways were also validated by RT-qPCR and Western blotting.

Results

A total of 129 relevant targets of TA against UC were obtained, of which the top 10 hub targets were GRB2, PTPN11, SRC, PIK3R1, LCK, EGFR, MAPK1, IGF1, ESR1 and JAK2. The PI3K-Akt pathway was significantly associated with TA treatment of UC as identified by KEGG enrichment analysis. GEO dataset analysis demonstrated that EGFR and GRB2 were upregulated in UC patients, while SRC, PIK3R1 and JAK2 were down-regulated. In vitro experiments revealed that TA treatment reversed the expression of EGFR, GRB2, PIK3R1 and JAK2 differential genes. Furthermore, TA was able to up-regulate the mRNA expression of pro-inflammatory factor IL-10 and decrease the mRNA levels of anti-inflammatory factors IL-6, IL-1β and TNF-α in LPS-induced NCM460 cells. In vivo assay confirmed that TA significantly alleviated DSS-induced colonic inflammation in mice, including reversal of weight loss and shortening of colon length, reduction of DAI score in mice, and improvement of intestinal barrier disruption. In vivo and in vitro experiments have confirmed that TA also inhibits the activation of the PI3K/Akt pathway, thus exerting an anti-inflammatory effect.

Conclusion

TA may play a therapeutic role in UC by inhibiting the PI3K-Akt pathway. EGFR, GRB2, PIK3R1 and JAK2 may be the most relevant potential therapeutic targets for TA in the treatment of UC.

Introduction

Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), are non-infectious chronic inflammatory disorders of the gastro-intestinal tract. The continuous presence of inflammatory stimuli, such as increased circulation of cytokines and danger-associated molecular patterns (DAMPs), leads to an enhanced immune response in these patients that could explain some types of disease relapse. An important receptor for DAMPS and pathogen-associated molecular patterns (PAMPs) is NLRP3, a cytosolic pattern recognition receptor. The NLRP3 inflammasome is exclusively activated by inflammatory stimuli, like DAMPs and PAMPs, and therefore not expressed without inflammation. It is thought that NLRP3 associates with ASC to recruit pro-Caspase 1 to the NLRP3 inflammasome and cleave it to its active form. In turn, Caspase 1 cleaves pro-interleukin 1 beta (IL-1β) and pro-interleukin 18 (IL-18) to their mature forms. IL-1β is a known elevated pro-inflammatory cytokine in IBD, promoting intestinal inflammation and immune cell recruitment. We hypothesized that the NLRP3 inflammasome pathway is therefore upregulated in IBD patients.

Aims & Methods

We first set out to systematically analyze gene expression of NLRP3 and IL1B in IBD patients by performing a systematic literature review and subsequent meta-analysis according to the PRISMA guidelines and Cochrane Collaboration methods. Subsequently, an observational cohort of 70 active and quiescent patients with CD or UC was sampled for whole blood, to study NLRP3 inflammasome gene regulation, protein production, assembly and secretion. We performed ex vivo LPS and ATP challenges of whole blood, with and without NLRP3 inflammasome formation inhibitor MCC950.

Results

Across thirty studies with publicly available mRNA expression data, we found NLRP3 and IL1B expression to be significantly higher in IBD patients than in non-IBD controls. In fresh IBD patient-derived whole blood, LPS and ATP stimulation activated the NLRP3-related genes, protein production and subsequent cytokine secretion. No IL-1β and IL-18 were secreted upon incubation with MCC950, suggesting a crucial role for NLRP3 inflammasome assembly in IL-1β and IL-18 production in immune cells. In patients with active IBD, the inflammasome was transcribed at a higher level than in patients in remission. However, this effect was lost on the protein level. We found a large spread in IL-1β and IL-18 production, suggesting a variation in activation of the NLRP3 inflammasome between individuals which was not related to disease activity.

Conclusion

NLRP3 and IL1B mRNA transcription is significantly increased in IBD patients compared to non-IBD controls, and significantly higher in patients with active disease than in patients in remission. While this fits the inflammatory state of active IBD, we found that the level of IL-1β and IL-18 production by the NLRP3 inflammasome is not significantly different between disease states. This suggests a variance in individual immune response to challenges, that could help identify a subset of IBD patients that would benefit from additional therapies directed at NLRP3 and IL-1β.

Introduction

Ulcerative colitis (UC) is a refractory inflammatory bowel disease of the colon with repeated relapses and remissions that requires lifelong treatment. Previous studies indicated that proinflammatory cytokines involved the onsets and developments of UC. New treatments for targeting these cytokines, such as TNF-α, IL-12 and IL-23 have been developed in a recent decade, underlying that immune abnormalities are the core for the etiology of UC.
Platelets play a role not only in hemostatic coagulation but in inflammatory mediator by interacting with various cells in the innate and adaptive immunity. Chronic inflammatory response confers platelets activation leading to elevated platelet count and hypercoagulability. Moreover, platelets aggregates with leukocyte in active inflammatory milieu. In fact, thrombocytosis, thromboembolism and upregulation of platelet-monocyte complex (PMC) in UC patients have been reported, implying that platelets could be novel biomarker, therapeutic target and clues for elucidating the pathology of UC. However, the clinical significance of platelet activation and PMC formation in UC is not fully elucidated and few studies were conducted to assess how platelet activation contributes to disease aggravations of UC. Therefore, we comprehensively analyzed platelet activation and platelet-monocyte complex by focusing on a correlation with clinical severity of UC.

Aims & Methods

Patients with UC (n=70) including MES0-2 group (n=34) and MES3 group (n=27), diagnosed according to a combination of symptoms, endoscopic findings, histologic findings, and the absence of alternative diagnoses, and age and sex-matched healthy controls (HCs) (n =19) were enrolled for this study. We analyzed the surface markers such as CD62P, CD40L, CD63, PAC-1 and Annexin V on CD61+ platelets by flowcytometry (FCM) in patients with UC in comparison with those in healthy adults. We also analyzed the proportion of CD61+CD14+ platelet-monocyte complexes and quantified the activation of PMCs by FCM. Moreover, we also investigated the correlation among surface markers, proportion of platelet-monocyte aggregates and Mayo endoscopic score (MES) in UC patients.

Results

The expression of CD62P but not other surface markers on platelets was significantly upregulated in UC when compared to HCs (p<0.01), and was significantly upregulated in in MES3 group when compared to the MES0-2 group (P<0.001). The proportion of PMC was also higher in MES3 groups when compared to MES0-2 group (P<0.05), and was markedly reduced by treatment (P<0.05). In addition, CD16+ monocytes, phenotypically mature and pro-inflammatory cell population, were increased in patients with UC when compared to HCs (P<0.01). The expression of CD16 on monocytes aggregated with platelets was significantly higher when compered to that without platelet (P<0.001).

Conclusion

Our study suggests that platelet activation accelerates mucosal inflammation via adhesion with monocytes in UC and contributes to the aggravation of disease.

References

Semple JW, et al. Platelets and the immune continuum.
Nat Rev Immunol. 2011 Apr;11(4):264-74.
Collins CE, et al. Platelets circulate in an activated state in inflammatory bowel disease.
Gastroenterology. 1994 Apr;106(4):840-5.

Introduction

Rarely is it mentioned how the synergistic disruption of the gut vascular barrier (GVB) and intestinal epithelial barrier (IEB) contributes to the pathophysiology of ulcerative colitis (UC). Enteric glial cells (EGCs) play a vital role in intestinal homeostasis maintenance, but their possible interactions with dual intestinal barrier function are unknown.

Aims & Methods

We aimed to investigate the role of EGCs in regulating dual intestinal barrier function in the pathogenesis of UC and the underlying mechanisms. This study was performed on the mouse models of DSS-induced colitis and biopsies from UC patients. Impaired dual intestinal barrier function was examined by permeability assays and electron microscopy. The phenotype and function of EGCs were assessed by Western Blot, Immunofluorescence, and Flow Cytometry. Dihydroartemisinin (DHA) was used to reduce inflammation in vivo and in vitro.

Results

Mice with chronic colitis had impaired IEB and GVB such as increased double intestinal barrier permeability, reduced intestinal tight junction protein expression, and increased blood circulating endotoxin levels have been observed in the chronic colitis mice model. EGCs, mainly the subgroup of GFAP⁺/S100⁺-EGCs, have been activated and proliferated when the dual intestinal barrier function is compromised. And the content of GFAP⁺/S100⁺-EGCs corresponds to the damage severity of the barrier. Both proliferation and activation of GFAP⁺/S100⁺-EGCs were markedly reduced by the DHA treatment. In addition to alleviating weight loss and colonic shortening in the model mice, DHA treatment considerably ameliorates intestinal epithelial and vascular endothelial barrier function, thus significantly reducing intestinal permeability. Moreover, DHA treatment reduced the proliferative activity of GFAP⁺/S100⁺-EGCs in vitro, and restricted the recruitment of monocytes to EGCs by inhibiting the production of CCL2, thereby alleviating intestinal inflammatory response.

Conclusion

EGCs are essential for coordinated regulation of the double intestinal barrier efficient operation, and the proliferation and activation of GFAP⁺/S100⁺-EGCs speed up UC development. Those findings will contribute to identifying novel UC treatment targets and practical neurological markers.

Disclosure

The authors declare no competing interests.

Introduction

GLPG3970, a potent and selective small molecule salt-inducible kinase (SIK) 2/3 inhibitor, exhibits a dual mechanism modulating both pro-inflammatory and immunoregulatory pathways that holds potential as a novel therapeutic option for inflammatory bowel disease.^1,2 Oral administration of GLPG3970 in colitis mouse models reduced disease activity scores, inhibited pro-inflammatory cytokine release and improved histological endpoints including protection of goblet cell health and MUC2 expression.^2,3 Mechanistic investigations demonstrated a broad immunomodulatory activity on colonic immune cell types. It remains unclear whether SIK2/SIK3 inhibition exerts a direct therapeutic activity on intestinal epithelial cell types or if these effects result indirectly from modulating pro-inflammatory and immunoregulatory mechanisms.³

Aims & Methods

We aimed to unravel the activity of SIK2/SIK3 inhibition using GLPG3970 in ulcerative colitis (UC) patient-derived epithelial cells focusing on SIK target engagement markers, inflammation and mucus production. Differentiated intestinal organoids from patients with UC were exposed to vehicle control, GLPG3970 (3 µM and 20 µM) or PGE2 (20 µM; positive control) for 24 hours (gene expression) or 48 hours (immunohistochemistry). Gene expression was evaluated in organoid cultures derived from five patients with UC, with or without 24 hours of pro-inflammatory stimulation (using a mix of 100 ng/mL TNFα, 20 ng/mL IL-1β and 1 µg/mL flagellin)⁴ prior to 24 hours of exposure in combination with the compound, and measured by RT-qPCR compared with vehicle control (Friedman, Dunn’s multiple comparison). Immunohistochemistry was performed for MUC2, DAPI, CK14 (n = 3 patients, minimum 10 images/condition, 3 replicates). Randomized images were analysed by Imaris Software by sectioning regions of interest (two-way ANOVA, Dunnett’s multiple comparison).

Results

Upon GLPG3970 exposure, UC patient-derived organoids showed transcriptional upregulation of SIK target engagement markers such as the CREB¹ activation genes NR4A1, NR4A2 and NR4A3. There was no effect with GLPG3970 on gene expression of inflammatory markers in colon epithelial cells, in the presence or absence of inflammatory stimulation, except for IL-1β and IL-8 upregulation after 24 hours of GLPG3970 exposure. Genes related to mucus production were upregulated in both noninflamed (MUC5AC, 20 µM, p < 0.01; AGR2, 20 µM, p < 0.05) and inflamed conditions (MUC2, 20 µM, p < 0.01; MUC5AC, 3 µM and 20 µM, p < 0.01) at the highest GLPG3970 concentration tested. Immunohistochemistry confirmed increased MUC2 secretion (secreted MUC2 surface × secreted MUC2 intensity) after 48 hours of GLPG3970 stimulation (3 µM, p < 0.05; 20 µM, p < 0.01); however, no significant increase was measured in absolute goblet cell number or goblet surface intensity compared with vehicle control.

Conclusion

SIK2/SIK3 inhibition by GLPG3970 showed a significant upregulation of target engagement markers as measured by the CREB activation genes NR4A1/2/3 in UC patient-derived colon epithelial cells, whereas no direct downregulation of inflammatory markers was observed. Exposure of UC patient-derived colonic organoids to GLPG3970 20 µM resulted in a signature of enhanced mucus secretion, both in the presence and absence of inflammation. Overall, these data indicate a direct impact of SIK2/SIK3 inhibition in intestinal epithelial cells with potential benefit in promoting barrier reinforcement via goblet cell mucus production in patients with active UC.

References

1. Peixoto C et al. J Med Chem 2024;67:5233–58.
2. Pereira-Fernandes A et al. [abstract] United European Gastroenterol J 2021;9:S8.
3. Lavazais S et al. [abstract] United European Gastroenterol J 2021;9:S8.
4. Arnauts K et al. Gastroenterology 2020;159:1564–67.

Disclosure

This study was funded by Galapagos NV (Mechelen, Belgium).
Editorial and publications management support was provided by PharmaGenesis London, London, UK, and was funded by Galapagos NV.
LG and KA are joint first authors.
LG and KA report no conflict of interest.
DM was an employee of Galapagos at the time of the work, and is now an employee of NovAliX.
BV reports research support from AbbVie, Biora Therapeutics (formerly Progenity), Landos Biopharma, Pfizer, Sosei Heptares and Takeda; speaker fees from Abbvie, Biogen, Bristol Myers Squibb, Celltrion, Chiesi, Falk, Ferring Pharmaceuticals, Galapagos, Janssen Pharmaceuticals, Lily, MSD, Pfizer, R-Biopharm, Sandoz, Takeda, Tillots Pharma, Truvion Healthcare and Viatris; consultancy fees from Abbvie, Alfasigma, Alimentiv, Applied Strategic, AstraZeneca, Atheneum, BenevolentAI, Biora Therapeutics (formerly Progenity), Boxer Capital, Bristol Myers Squibb, Galapagos, Guidepont, Landos, Lily, Mylan, Inotrem, Ipsos, Janssen Pharmaceuticals, Pfizer, Sandoz, Sanofi, Santa Ana Bio, Sapphire Therapeutics, Sosei Heptares, Takeda, Tillots Pharma and Viatris; and stock options from Vagustim.
SV reports speaker and/or consultancy fees from AbbVie, Abivax, AbolerIS Pharma, AgomAb Therapeutics, Alimentiv, Arena Pharmaceuticals, AstraZeneca, Biora Therapeutics (formerly Progenity), Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Cytoki Pharma, Dr Falk Pharma, Ferring Pharmaceuticals, Galapagos, Genentech/Roche, Gilead Sciences, GSK, Hospira, IMIDomics, Janssen Pharmaceuticals, Johnson & Johnson, Lilly, Materia Prima, Mestag Therapeutics, MiroBio, Morphic Therapeutic, MRM Health, MSD, Mundipharma, Pfizer, ProDigest, Prometheus Biosciences, Robarts Clinical Trials, Surrozen, Takeda, Theravance Biopharma, Tillotts Pharma, VectivBio, Ventyx Biosciences and Zealand Pharma; grants from AbbVie, Galapagos, Johnson & Johnson, Pfizer and Takeda; and participation on a data safety monitoring board for Sanofi.
SDV is an employee of Galapagos.