Introduction

Abdominal surgery frequently results in postoperative ileus (POI), a neuroinflammation-based disorder of the gastrointestinal tract, characterized by motility disturbances leading to prolonged hospital stay and increased health care costs. The underlying neuroinflammation involves activation of resident macrophages and monocyte-derived macrophages (MDM) infiltration into the intestine's muscularis externa (ME). Immune checkpoint proteins, including PD-1 and its ligands PD-L1 and PD-L2, play a prominent role in cancer immunology, while less is known about their role in non-oncological inflammatory diseases.

Aims & Methods

The aim of this study was to investigate the role of PD-1 signaling in POI development. POI was induced by a surgical intestinal manipulation in wildtype (wt), PD-1^-/-, PD-L1^-/- and PD-L2^-/- mice. We performed immunohistochemistry and flow cytometry to identify the cellular sources of PD-1 and PD-L1 and used a blood cell transfer model to determine the role of a leukocyte-restricted PD-1 deficiency on immune cell infiltration. By qPCR, bulk 3´mRNA sequencing and use of the SMART-Seq2 technology in sorted CX3CR1^GFP/+ resident and monocyte-derived macrophages, we analysed transcriptional and functional differences between wt and PD-1^-/- macrophage populations. Furthermore, we quantified neuronal cell loss, in vitro and in vivo macrophage phagocytosis, GI transit measurements and metabolic activity by Seahorse XF Real-Time ATP rate assays to further characterize functional changes linked to PD-1 deficiency.

Results

PD-1, PD-L1 and PD-L2 mRNA levels were induced in the postoperative ME upon intestinal manipulation. PD-1^-/- and PD-L1^-/-, but not PD-L2^-/- mice, were protected from POI. PD-1^-/- mice showed a 50% reduction of MDM infiltration and reduced inflammation-mediated neuronal loss. A cell transfer experiment confirmed a 60% reduction of postoperative ME infiltration of PD-1 deficient leukocytes compared to wt leukocytes. Ly6C^-CX3CR1⁺ resident macrophages and Ly6C⁺CX3CR1⁺ MDM were identified as the primary cellular source of PD-1 in POI. PD-1 deficient Ly6C⁺CX3CR1⁺ MDM showed reduced in vivo phagocytosis, which was confirmed in bone-marrow derived macrophage cultures. Gene ontology analysis of postoperative ME samples of PD-1 and wt mice revealed lower enrichment levels of genes regulating metabolic respiratory chain processes, oxidative stress, cell death and immune functions. Moreover, GSEA analyses of sorted PD-1^-/-CX3CR1⁺Ly6C⁺ MDM showed reduced enrichment of genes associated with various immune functions, host defense and regulation of immune and neuronal cell death as well as an alteration of metabolic genes in POI. Seahorse XF Real-Time ATP rate measurement showed no difference between naïve monocytes from wt and PD-1^-/- mice. However, PD-1^-/- monocytes identified an increased oxidative chain reaction-dependent ATP generation during LPS stimulation.

Conclusion

Our data provide new evidence on the role of PD-1 signaling in MDM during postoperative intestinal neuroinflammation following abdominal surgery. PD-1 deficiency prevents POI, results in a metabolic switch and reduces inflammation-mediated neuronal loss as well as infiltration capacity and phagocytosis of MDM. Interaction in PD-1 signaling might be a potential target in prevention of POI.

Introduction

Recently, artificial intelligence (AI)-based diagnostic support tools for early gastric cancer have been developed and reported to be superior to endoscopists (accuracy: 70% and sensitivity: 85%).¹ However, These tools could not guarantee reliable diagnostic accuracy in clinical practice. In previous reports, magnifying endoscopy has been recognized as superior to white-light imaging endoscopy for endoscopic diagnosis of early gastric cancers. Therefore, we developed an AI-based diagnostic support system using magnifying endoscopy with narrow band imaging (M-NBI) for early gastric cancers² and improved its application in clinical practice using nondegraded images obtained from selective depth inspection (SDI) output for real-time diagnosis. This study is the first to create an AI-based diagnostic support system using non-degraded M-NBI images obtained from SDI output, as several previous papers have analyzed damaged M-NBI images that were converted from various recording devices.^3,4

Aims & Methods

We built a new AI system (M-NBI AI) which was assembled using four different image recognition models with non-degraded M-NBI images (normal mucosa:1776 images, gastric cancers: 1947 images, and non-neoplastic lesions: 2451 images). We evaluated the performance of this M-NBI AI for diagnosis of early gastric cancer. The diagnostic performances of M-NBI AI and endoscopists (five specialists and five non-specialists) were compared using non-degraded M-NBI images (three images per lesion) of 50 early gastric cancer and 50 non-cancerous lesions. Early gastric cancers included histopathological types of well, moderately, and poorly differentiated adenocarcinomas and signet-ring cell adenocarcinomas. The primary outcome was the superiority of the M-NBI AI (based on accuracy) over the endoscopists. The secondary outcome was the superiority of the M-NBI AI (based on sensitivity) over the endoscopists.

Results

	M-NBI AI	Endoscopists
		Specialists	Non-specialists
Accuracy, % (95% CI)	87.0 (0.80–0.94)	80.0 (0.72–0.88)	71.0 (0.62–0.79)
Sensitivity, % (95% CI)	86.3 (0.77–0.96)	74.2 (0.63–0.85)	70.6 (0.58–0.83)
Specificity, % (95% CI)	88.0 (0.79–0.97)	89.0 (0.80–0.99)	71.0 (0.59–0.84)
PPV, % (95% CI)	88.0 (0.79–0.97)	92.0 (0.84–0.99)	72.0 (0.60–0.84)
NPV, % (95% CI)	86.0 (0.76–0.96)	68.0 (0.55–0.81)	70.0 (0.57–0.83)
PPV, positive predictive value; NPV, negative predictive value

The M-NBI AI was superior to the non-specialists based on accuracy (87.0%, 95%CI: 0.80–0.94 versus 71.0 %, 95%CI: 0.62–0.79; p<0.01), without being superior to the specialists (87.0%, 95%CI: 0.80–0.94 versus 80.0%, 95%CI: 0.72–0.88; p=0.18). The M-NBI AI was superior to the specialists and non-specialists based on sensitivity (86.3%, 95%CI: 0.77–0.96 versus 74.2%, 95%CI: 0.63–0.85 and 70.6%, 95%CI: 0.58–0.83; p<0.05, respectively).

Conclusion

The M-NBI AI was created as a new AI system that could be implemented for current endoscopy systems as a real-time diagnostic support tool for early gastric cancers. It demonstrated a robust performance and may indicate equalization of diagnostic accuracy and superadded effect over the white-light endoscopy for diagnosis of early gastric cancers in clinical practice.

References

1. Ishioka M et al. Dig Endosc. 2022. doi: 10.1111/den.14455.
2. Ueyama H et al. J Gastroenterol Hepatol. 2021;36(2):482-489.
3. Horiuchi Y et al. Dig. Dis. Sci. 2020;65:1355–1363.
4. He X et al. Gastrointest Endosc. 2022;95(4):671-678.

Introduction

Motorized spiral enteroscopy (MSE) has shown to be safe and effective for deep small bowel enteroscopy in patients with and without prior abdominal surgery, including patients with Crohn’s disease.
This case series reports our first results regarding the feasibility and safety of the MSE with endoscopic balloon dilatation in patients with small bowel strictures due to Crohn’s disease.

Aims & Methods

Data were collected retrospectively on patients who underwent MSE for endoscopic balloon dilatation of small bowel strictures due to Crohn’s disease. Strictures were either postoperative (P.O.) or post-inflammatory (P.I.).

Results

A total of ten consecutive patients with Crohn’s disease underwent 13 procedures. Propofol sedation without endotracheal intubation was used in all procedures. Demographic and procedure related data are shown in the table. One patient underwent three procedures with 3–4 month intervals. During the third procedure triamcinolone acetonide was injected at four quadrants of the stricture. Fluorescence imaging was used during six procedures. The procedure was technically successful in 12/13 of procedures. In one case, dilatation was not performed because of a longer than anticipated and a sharply angulated stricture.
The number of treated strictures ranged from one to five per procedure. Dilatation of multiple strictures in two patients (4 and 5) was achieved within a relatively short procedure duration of 55 and 70 minutes. Two retained video capsules were retrieved.
Besides the anticipated mild abdominal discomfort and/or sore throat in three patients, no adverse events occurred.

	Patients (age/F/M)	Type stenosis	Procedure route	No. of strictures/ Location (cm)		Maximal dilatation (mm)	Procedure duration (min)	Fluorescence imaging (+/-)
1	44/M	P.O	Antegrade	1/ 200 cm from Treitz		15/18/18 *	50/45/40*	+/+/-
2	54/F	P.I. and VCE retention	Ante-/retrograde	4/ 300 cm from Treitz/ 150 cm from ICV		15	70	+
3	56/F	Mixed	Antegrade	5/ 150-180 cm from Treitz		18	55	-
4	25/M	P.I. and VCE retention	Antegrade	1/ 100 cm from Treitz		18	35	+
5	26/M	P.O.	Ante-/retrograde	1/ 120 cm from Treitz/280 cm from ICV		18 mm via retrograde route	45	-
6	23/F	P.O.	Ante-/retrograde	1/ 450 cm from Treitz/ 50 cm from ICV		15 mm of ileo-ileal anastomosis (pinpoint)	80	+
7	33/M	P.O.	Ante-/retrograde	1/ 350 cm from Treitz /100 cm from ICV		18 mm via retrograde	60	-
8	52/M	P.O.	Antegrade	1/ 200		18	30	-
9	50/F	P.I.	Retrograde	1/ 100		18	30	+

*= 3 Procedures; P.O= Postoperative; P.I= Post-inflammatory; VCE= Video capsule endoscope; ICV= Ileocecal valve
NB: data on patient number 10 is not shown in the table because of the limited number of rows (10) allowed. No dilatation was done.

Conclusion

These retrospective data show that MSE with CRE dilatation is feasible and safe in a cohort of patients with strictures due to Crohn’s disease, postoperatively as well as post-inflammatory. The fact that the distal 16 cm of the endoscope are not covered by the spiral overtube makes cautious introduction of the endoscope beyond a recently dilated stricture feasible and safe. Therefore, dilatation of multiple strictures in one session is possible, provided that they are at a short distance from each other. Fluorescence imaging guidance may be helpful in severe stenosis, multiple strictures, or sharp angulations.

Introduction

Filgotinib (FIL) is a once-daily, oral, Janus kinase 1 preferential inhibitor approved for the treatment of ulcerative colitis (UC). Previous analyses of data from the phase 2b/3 SELECTION trial (NCT02914522) demonstrated that FIL 200 mg (FIL200) improved health-related quality of life (HRQoL) and led to comprehensive disease control (comprising clinical, biomarker, HRQoL and endoscopic improvements) in patients with UC.¹

Aims & Methods

We evaluated individual Inflammatory Bowel Disease Questionnaire (IBDQ) item data from SELECTION to assess which items drive the benefits of FIL treatment. In SELECTION, adults with moderately to severely active UC were randomized 2:2:1 to receive FIL200, FIL 100 mg or PBO once daily for 11 weeks in Induction Studies A and B (biologic-naive and -experienced patients, respectively). Patients who responded to FIL were rerandomized 2:1 to continue their induction FIL dose in the 47-week Maintenance Study. In this post hoc analysis, standardized effect sizes (SES) were calculated to evaluate the relative magnitude of the difference in mean scores of individual IBDQ items at week 10 (W10) between FIL200 versus PBO-treated patients, and at week 58 (W58) between patients who continued FIL200 treatment (FIL200→FIL200) versus those who were rerandomized to PBO (FIL200→PBO). SES were also calculated for the mean change in score of individual IBDQ items from baseline to W10, and from maintenance baseline to W58. SES were categorized by Cohen’s thresholds (‘trivial’ [<0.2]; ‘small’ [≥0.2–<0.5]; ‘moderate’ [≥0.5–<0.8]; and ‘large’ [≥0.8]). A last observation carried forward imputation approach was used for missing data.

Results

At W10, patients treated with FIL200 overall (n=506) had improvements in all individual IBDQ items versus those treated with PBO (n=277) and had moderate to large increases from baseline (SES ≥0.5) in all individual IBDQ items. The IBDQ items with the five largest SES for FIL200 versus PBO at W10 (analysed by induction study), and for FIL200→FIL200 (n=199) versus FIL200→PBO (n=98) at W58 are summarized in the Table. FIL200 treatment consistently resulted in the largest benefits in items related to pain (‘abdominal pain’ and ‘abdominal cramps’), ability to engage socially (‘satisfied with life’, ‘depressed or discouraged’, ‘social engagement’ and ‘problems with activities’) and bowel urgency (‘loose bowel movements’ and ‘finding a washroom’) versus PBO at W10, and versus FIL200→PBO at W58

Table. Top five individual IBDQ item scores ranked by SES for FIL200 versus PBO groups at W10 and FIL200→FIL200 versus FIL200→PBO groups at W58.

FIL200 group – end of induction (W10)

Biologic-naive (n=245)

Biologic-experienced (n=261)

IBDQ items

W10 score, mean (SD)

SES vs PBO

Change in mean score from baseline, mean (SD)

SES vs baseline

IBDQ item

W10 score, mean (SD)

SES vs PBO

Change in mean score from baseline, mean (SD)

SES vs baseline

Social engagement Satisfied with life Depressed or discouraged Abdominal cramps Felt angrya

5.8 (1.4) 4.6 (1.2) 5.4 (1.3) 5.5 (1.4) 5.6 (1.3)

0.62 0.62 0.58 0.56 0.55

1.8 (1.9) 1.3 (1.5) 1.6 (1.7) 1.8 (1.6) 1.6 (1.7)

0.82 0.73 0.78 0.99 0.81

Loose bowel movements Problems with activities Abdominal pain Bowel movement frequency Rectal bleeding

4.1 (1.9) 4.7 (1.8) 5.1 (1.6) 5.2 (1.9) 5.0 (2.0)

0.72 0.64 0.63 0.62 0.62

1.9 (1.9) 1.9 (1.9) 1.6 (1.6) 2.2 (2.1) 2.6 (2.1)

0.88 0.89 1.02 0.83 1.01

FIL200→FIL200 group (n=199) – end of maintenance (W58)

IBDQ item

W58 score, mean (SD)

SES vs FIL200→PBO

Change in mean score from maintenance baseline, mean (SD)

SES vs maintenance baseline

Bowel movement frequency Rectal bleeding Abdominal cramps Problems with activities Finding a washroom

6.3 (1.4) 6.3 (1.4) 6.2 (1.1) 6.0 (1.3) 6.1 (1.4)

0.57 0.55 0.47 0.44 0.41

0.4 (1.6) 0.2 (1.6) 0.5 (1.4) 0.6 (1.4) 0.4 (1.4)

0.19 0.10 0.28 0.42 0.27

aThe items ‘felt angry’ and ‘irritable’ had the same SES versus PBO; however, ‘felt angry’ had the larger change in mean score from baseline. SES for comparison with PBO/FIL200→PBO was calculated as follows: mean differences/pooled SD. SES for comparison with baseline was calculated as follows: mean change from baseline/(SD/√(2*(1−r))). SES were categorized by Cohen’s thresholds (‘trivial’ [<0.2], ‘small’ [≥0.2–<0.5], ‘moderate’ [≥0.5–<0.8] and ‘large’ [≥0.8]). FIL200→FIL200 treatment group = patients who responded to FIL200 during induction and were rerandomized to continue FIL200 during maintenance. FIL200→PBO treatment group = patients who responded to FIL200 during induction and were rerandomized to PBO during maintenance. FIL200, filgotinib 200 mg; IBDQ, Inflammatory Bowel Disease Questionnaire; PBO, placebo; r, Pearson correlation coefficient; SD, standard deviation; SES, standardized effect size; W10, week 10; W58, week 58.

Conclusion

FIL200 treatment provides improvements in HRQoL versus PBO, with large SES seen for most IBDQ items, and the greatest improvements in items related to pain, social engagement and bowel urgency over 58 weeks. This analysis reveals the specific IBDQ items that drive the benefits to HRQoL of FIL and adds further evidence to that showing that FIL contributes to restoration of health through comprehensive disease control for patients with UC.¹

References

1. Schreiber S et al. J Crohns Colitis 2023; https://doi.org/10.1093/ecco-jcc/jjad018

Disclosure

BF reports grants and personal fees from AbbVie, Amgen, AstraZeneca, Bristol Myers Squibb, Janssen Biotech/Centocor, Johnson & Johnson/Janssen, Pfizer, Receptos and Takeda; and personal fees from Ablynx, ActoGeniX, AdMIRx, Inc., Akebia Therapeutics, Inc., Allergan, Atlantic Pharmaceuticals, Avaxia Biologics, Inc., Avir Pharma, Baxter Healthcare Corporation, Biogen Idec, BiomX Israel, Boehringer Ingelheim, Boston Pharmaceuticals, Calypso Biotech, Celgene, Elan/Biogen, Eli Lilly, enGene, Ferring Pharmaceuticals, Galapagos, Genentech/Roche, gIcare Pharma, Gilead, Given Imaging, Gossamer Bio, GSK, Inception IBD, Inc., Ironwood Pharmaceuticals, Japan Tobacco Company, Kyowa Hakko Kirin Co. Ltd, Lexicon Pharmaceuticals, Lycera, Mesoblast, Millennium Pharmaceuticals, MSD, Nestlé, Nextbiotix, Novartis, Novo Nordisk, Par’Immune, Progenity, Prometheus Therapeutics and Diagnostics, Protagonist, Qu Biologics, Salix Pharmaceuticals, Shire, Sienna Biopharmaceuticals, Sigmoid Pharma, Synergy Pharma, Teva Pharmaceuticals, TiGenix, Tillotts Pharma, UCB, Vertex Pharmaceuticals, VHsquared, Vivelix Pharmaceuticals, Wyeth, Zealand Pharma and Zyngenia.
SV reports grants from AbbVie, Galapagos, J&J, Pfizer and Takeda; consulting and/or speaker fees from AbbVie, AbolerIS Pharma, AgomAb, Alimentiv, Arena Pharmaceuticals, AstraZeneca, Avaxia Biologics, Bristol Myers Squibb, Boehringer Ingelheim, Celgene, CVasThera, Cytoki Pharma, Dr Falk Pharma, Ferring Pharmaceuticals, Galapagos, Genentech/Roche, Gilead, GSK, Hospira, Imidomics, Janssen, J&J, Lilly, Materia Prima, MiroBio, Morphic, MrMHealth, Mundipharma, MSD, Pfizer, Prodigest, Progenity, Prometheus, Robarts Clinical Trials, Second Genome, Shire, Surrozen, Takeda, Theravance Biopharma, Tillotts Pharma and Zealand Pharma.
LP-B reports speaking fees from AbbVie, Amgen, Arena Pharmaceuticals, Biogen, Celltrion, Eli Lilly, Ferring Pharmaceuticals, Galapagos, Genentech, Gilead, Janssen Pharmaceuticals, Medac Pharma, MSD, Pfizer, Sandoz, Takeda, Tillotts Pharma, Viatris and Vifor Pharma;grants from Celltrion, Fresenius Kabi and Takeda; and consulting fees from AbbVie, Abivax, Alimentiv, Alma Bio Therapeutics, Amgen, Applied Molecular Transport, Arena Pharmaceuticals, Biogen, Bristol Myers Squibb, Celltrion, CONNECT Biopharma, Cytoki Pharma, Eli Lilly, Enthera, Ferring Pharmaceuticals, Fresenius Kabi, Galapagos, Genentech, Gilead, Gossamer Bio, GSK, H.A.C. Pharma, IAG Image Analysis, Index Pharmaceuticals, Inotrem, Janssen Pharmaceuticals, medac Pharma, Mopac, Morphic, MSD, Norgine, Novartis, OM Pharma, ONO Pharmaceutical, OSE

Introduction

Crohn's disease is characterised by accumulation of immune cells in the intestinal mucosa. This includes effector T cells, thought to play key roles in disease through cytotoxic activity and pro-inflammatory cytokine production. The CCR9-CCL25 axis is the dominant chemokine system that mediates recruitment of mononuclear cells into small bowel mucosa. We sought to develop a novel humanized anti–CCR9 mAb with enhanced effector function (AZD7798) that potently depletes circulating and tissue resident CCR9 expressing cells.

Aims & Methods

Mice were immunized with CCR9 overexpressing cells and CCR9 hybridomas binders were selected. A lead clone was then humanised and optimised via CDR mutagenesis. Cell binding assays was used to identify specific binders to human and cynomolgus CCR9, and antibody-dependent cell-mediated cytotoxicity (ADCC) was confirmed in Molt4 as well as primary human PBMCs. CCR9 expressing cells in human peripheral blood as well a human gut tissue were characterised by flow cytometry. Finally, CCR9 cell depletion flow cytometric assays were established to investigate circulating CCR9 cell depletion in non-human primates (Macaca fascicularis) at a dose of 10 mg/kg, and ileal depletion was assessed by immunohistochemistry.

Results

8,448 hybridoma were screened and 10 unique hits were identified. The AZD7798 parent antibody was humanised and optimised leading to 40 fold potency improvement. AZD7798 demonstrated specific binding to human and cynomolgus, but not murine, CCR9. In the absence of NK cells, AZD7798 prevented binding of CCL25 to CCR9, and in the presence of NK cells induced potent ADCC in an in vitro human PBMC assay. We showed that CCR9 is highly expressed in human intestinal tissue, with highest levels in the ileum (small intestine). Memory T cells, B cells and T regs accounted for the majority of intestinal CCR9 expression, with minimal expression on other immune populations. CCR9-expressing CD4+ and CD8+ T cells showed increased pro-inflammatory cytokine production compared to CCR9-negative T cells, demonstrating their pathogenic potential. Finally, in cynomolgus monkey, AZD7798 depleted circulating and ileal CCR9 expressing T cells, achieving complete depletion in blood within 2 days.

Conclusion

AZD7798 delivers potent blood and tissue depletion of CCR9 expressing mononuclear cells in-vivo, in non-human primates. Safety of AZD7798 is currently being assessed in Ph1, clin trial registry number NCT05452304. This might provide a novel treatment approach for small bowel inflammatory diseases, including Crohn’s disease.

Disclosure

All authors are employees/shareholders of AstraZeneca

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

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.