Introduction

Ulcerative colitis (UC) involves inappropriate apoptosis of intestinal epithelial cells and an imbalance between pro-apoptotic and pro-survival molecules, leading to intestinal damage (1). Vagus nerve stimulation (VNS) is of interest for UC treatment due to its anti-inflammatory and protective effects (2). Noninvasive transcutaneous auricular VNS (NitaVNS) can exert protective effects without invasive VNS-associated side effects (3), making it a promising option for UC treatment. Recently, we showed a beneficial correlation between NitaVNS and inflammatory markers in a preclinical model of UC (4).

Aims & Methods

We assessed the effectiveness of NitaVNS in regulating apoptosis in acute UC colitis in mice. Colitis was induced in C57BL/6 male mice (11–12 weeks old) by administering 5% dextran sodium sulfate (DSS) for five days, while the control mice received regular water. NitaVNS (10 V voltage, 10 min stimulation, 500 µS pulse width, and 20 Hz frequency) or sham stimulation started one day before colitis induction and lasted six days. The disease activity index (DAI) was evaluated daily, and macro- and histological scores were evaluated at sacrifice. Colonic and splenic tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, tumor growth factor (TGF)-β, IL-10, pro-apoptotic markers Bcl-2-associated X protein (BAX), Bcl-2 homologous antagonist killer (BAK1), caspase-8, and pro-survival BCL2 associated agonist of cell death (BAD) were assessed using qRT-PCR and ELISA.

Results

NitaVNS significantly reduced DAI, the severity of colitis associated with rectal bleeding, stool consistency, weight loss, the decrease of colon length, and the macroscopic score. In addition, NitaVNS decreased histological scores, as indicated by mucosal damage, crypt loss, and immune cell infiltration. NitaVNS colitic mice demonstrated unaltered IL-10 protein levels in the colon. However, NitaVNS increased the mRNA expression of IL-10 in both colitic and non-colitic mice and the expression of TGF-β in non-colitic mice. NitaVNS significantly downregulated the mRNA expression of BAX, BAK1, and caspase-8 in colitic mice. Conversely, NitaVNS upregulated BAD mRNA expression in non-colitic conditions. Although the splenic protein levels of IL-1β and IL-6 were not modified under all conditions, NitaVNS decreased TNF-α levels in non-colitic mice. NitaVNS did not alter splenic protein levels of IL-10 but significantly increased TGF-β levels in colitic and non-colitic mice.

Conclusion

These findings suggest that NitaVNS can attenuate the severity of experimental colitis and the inflammatory process by modulating pro-apoptotic, pro-survival molecules and anti-inflammatory cytokines. The use of NitaVNS may lead to novel therapeutic strategies in ulcerative colitis.

References

1. Negroni A, Cucchiara S, Stronati L. Apoptosis, Necrosis, and Necroptosis in the Gut and Intestinal Homeostasis. Mediators Inflamm. 2015;2015:250762.
2. Ghia JE, Blennerhassett P, Kumar-Ondiveeran H, Verdu EF, Collins SM. The vagus nerve: a tonic inhibitory influence associated with inflammatory bowel disease in a murine model. Gastroenterology. 2006;131(4):1122-30.
3. Verma N, Mudge JD, Kasole M, Chen RC, Blanz SL, Trevathan JK, Lovett EG, Williams JC, Ludwig KA. Auricular Vagus Neuromodulation-A Systematic Review on Quality of Evidence and Clinical Effects. Front Neurosci. 2021 Apr 30;15:664740.
4. Hesampour F, Tshikudi D, Veysel Özden A, Bernstein CN, Ghia JE. A54 The impact of noninvasive transcutaneous auricular nerve stimulation (NitaVNS) on the development of experimental colitis in mice. J Can Assoc Gastroenterol. 2023 Mar 7;6(Suppl 1):30.

Disclosure

F. Hesampour: No conflict of interest; A. Veysel Özden: Employee of the company providing the stimulator; C. N. Bernstein: No conflict of interest; J.-E. Ghia: No conflict of interest.

Introduction

Decreased vagus nerve activity and increased inflammatory immune cells are observed in ulcerative colitis (UC) (1, 2). Due to its anti-inflammatory and protective properties, vagus nerve stimulation (VNS) has garnered attention for UC therapy (3). Non-invasive transcutaneous auricular vagus nerve stimulation (NitaVNS), devoid of the side effects associated with invasive techniques, demonstrates protective anti-inflammatory effects in our mouse UC-like model of colitis (4), although the specific cellular mechanisms remain unclear.

Aims & Methods

To evaluate the effects of NitaVNS on the distribution of granulocytes, macrophages, natural killer (NK) cells, and CD4⁺ and CD8⁺ T cells in the colon tissue, spleen, and mesenteric lymph nodes of a UC-like colitis mouse model, NitaVNS was administered to C57BL/6 male mice daily for 10 min, beginning one day before colitis induction with 5% dextran sulfate sodium for 5 days in water (the control group received regular water). This continued until the end of the study, with no stimulation in sham mice. The daily disease activity index (DAI) was evaluated along with distal macroscopic scores at the time of sacrifice. The frequencies of granulocytes (Ly6G), macrophages (F4/80), NK cells (NK1.1), CD4⁺ T cells (CD3, CD4), and CD8⁺ T cells (CD3, CD8) were analyzed in the cecum, proximal, mid and distal colon, spleen, and mesenteric lymph nodes (MLNs) using flow cytometry.

Results

NitaVNS in colitic mice significantly improved the DAI and macroscopic scores related to rectal bleeding, colon bleeding, fecal consistency loss, and shortened colon length compared to sham-stimulated colitic mice. In contrast, no changes were observed in the non-colitic mice. While NitaVNS did not alter the frequency of Ly6G⁺ cells in the cecum, mid/distal colon, and MLNs, it significantly decreased the positivity of these cells in the proximal colon and spleen of mice with colitis without affecting non-colitic mice. NitaVNS significantly decreased the frequency of F4/80⁺ cells in the mid-colon but did not influence these cells in the cecum, proximal and distal colon, spleen, or MLNs. In colitic conditions, NitaVNS significantly increased NK cell frequency in the spleen without affecting non-colitic mice. Moreover, NitaVNS did not alter the frequency of NK cells in the colon or MLNs in colitic or non-colitic mice. Additionally, NitaVNS did not induce changes in CD4⁺ and CD8⁺ T cell distribution in the colon tissue, spleen, or mesenteric lymph nodes of colitic and non-colitic mice.

Conclusion

These results suggest that NitaVNS could potentially reduce inflammation in colitic mice, locally and systemically, by decreasing the frequency of granulocytes in the colon and spleen and colonic macrophages. Additionally, NitaVNS increased the number of splenic NK cells, potentially contributing to its systemic effects. These findings indicate promising therapeutic potential for NitaVNS.

References

1. Ghia JE, Blennerhassett P, Kumar-Ondiveeran H, Verdu EF, Collins SM. The vagus nerve: a tonic inhibitory influence associated with inflammatory bowel disease in a murine model. Gastroenterology. 2006;131(4):1122-30.
2. Wiese JJ, Manna S, Kühl AA, Fascì A, Elezkurtaj S, Sonnenberg E, Bubeck M, Atreya R, Becker C, Weixler B, Siegmund B, Patankar JV, Prüß MS, Schumann M. Myenteric Plexus Immune Cell Infiltrations and Neurotransmitter Expression in Crohn's Disease and Ulcerative Colitis. J Crohns Colitis. 2024 Jan 27;18(1):121-133.
3. Verma N, Mudge JD, Kasole M, Chen RC, Blanz SL, Trevathan JK, Lovett EG, Williams JC, Ludwig KA. Auricular Vagus Neuromodulation-A Systematic Review on Quality of Evidence and Clinical Effects. Front Neurosci. 2021 Apr 30;15:664740.
4. Hesampour F, Tshikudi D, Veysel Özden A, Bernstein CN, Ghia JE. A54 The impact of non-invasive transcutaneous auricular nerve stimulation (NitaVNS) on the development of experimental colitis in mice. J Can Assoc Gastroenterol. 2023 Mar 7;6(Suppl 1):30.

Disclosure

F. Hesampour: None Declared, D. Tshikudi: None Declared, A. Veysel Özden: The employee of the company providing the stimulator, C. N. Bernstein: None Declared, J-E. Ghia: None Declared.

Introduction

Ulcerative colitis (UC) treatment aims for mucosal healing as a therapeutic goal. However, in some cases, especially in the rectal mucosa, healing cannot be achieved. Moreover, the reason why the rectum is the predilection site of UC remains unknown. In this study, we hypothesized that differences in intestinal epithelial cells (IECs) in the rectum and ascending colon play an important role in the pathogenesis of UC, which may lead to the different treatment responses in the rectum and colon. Therefore, we focused on the differences in gene expression in IECs between the rectum (as the lesion site) and the ascending colon (as the non-lesion site) in UC patients.

Aims & Methods

Colorectal organoids were established from lesional and non-lesional sections of total colectomy specimens from UC patients and cultured under non-inflammatory conditions. We then performed microarray analysis on these organoids and identified genes potentially associated with UC pathology. The expression of candidate genes was validated by q-PCR in lesional and non-lesional areas of surgical specimens from UC patients. To further investigate the role of the selected gene, we knocked down (KD) PITX2 in organoids derived from the ascending colon of a healthy individual using a lentiviral system. These organoids were exposed to TNF-α (100ng/ml), IL-1β (10ng/ml), Flagellin (100ng/ml) for one week, following a previously established human IBD in vitro model(J Crohns Colitis. 2021,2022). We assessed the inflammatory response by measuring the expression of IL-8 and DUOXA2, downstream genes of NF-κB, using qPCR. In addition, fluorescent immunostaining for NF-kB p65 was performed, and the nuclear-to-cytoplasmic fluorescence intensity ratio of p65 was analyzed using ImageJ software. Cell proliferation was evaluated using the CellTiter-Glo^🄬 assay. Stem cell function was assessed by sphere formation assay, and apoptosis was analyzed by Western blot for caspase-3 and caspase-7.

Results

We identified PITX2 as a gene specifically upregulated in both organoids derived from the ascending colon and non-lesional tissue of UC patients. Inflammatory response assessment, based on IL-8 and DUOXA2 expression, showed no significant differences between WT and PITX2-KD organoids. Similarly, fluorescent immunostaining for NF-κB p65 revealed no significant difference in nuclear translocation between WT and PITX2-KD organoids, suggesting that PITX2 does not influence inflammatory signaling pathways. PITX2-KD organoids from the ascending colon exhibited similar cell proliferation to WT under normal conditions. However, under inflammatory stimulation for one week, PITX2-KD organoids showed reduced proliferation compared to WT. The sphere-forming ability of PITX2-KD organoids was comparable to WT under both normal and inflammatory conditions, suggesting that PITX2 plays a role in promoting proliferative cells in an inflammatory environment. Western blot analysis revealed increased expression of cleaved caspase-3 and 7 in PITX2-KD organoids following inflammatory stimulation compared to WT. These findings suggest that PITX2 may regulate apoptosis via effector caspases.

Conclusion

PITX2 was identified as a gene specifically upregulated in the ascending colon and non-lesional tissue of UC patients, playing a role in promoting cell proliferation under inflammatory conditions. These findings suggest that PITX2 may contribute to UC pathology by regulating epithelial cell survival and apoptosis in an inflammatory environment.

References

1. Watanabe S, et al. Schlafen 11 Is a Novel Target for Mucosal Regeneration in Ulcerative Colitis. J Crohns Colitis. 2021 Sep 25;15(9):1558-1572.
2. Watanabe S, et al. Importance of Telomere Shortening in the Pathogenesis of Ulcerative Colitis: A New Treatment From the Aspect of Telomeres in Intestinal Epithelial Cells. J Crohns Colitis. 2022 Jan 28;16(1):109-121.

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

Previous research indicate the contact system (CAS) is associated with ulcerative colitis (UC). The activation of coagulation factor XII leads to the activation of plasma kallikrein. Plasma kallikrein generates bradykinin and cleaved H-kininogen (cHK) by cleaving H-kininogen. Bradykinin induces inflammation. C1 inhibitor (C1-INH) is the most important regulator of CAS, while activated factor XII and plasma kallikrein are the accelerators. Interestingly, experimental colitis can be induced and alleviated in UC animal models by respectively activating and inhibiting CAS^1-3. Increased activity of CAS in plasma and colonic tissue was detected in patients with active disease^{4, 5}. Human studies are still limited. We, therefore, aimed to characterise CAS in patients with active UC by employing our unique panel of CAS assays.

Aims & Methods

We included adults with active UC at the Section of Gastroenterology, Esbjerg and Grindsted Hospital, Denmark from May 2021 to June 2023. Inclusion criteria were: Patient reported outcome 2 (PRO2) score ≥1, Mayo endoscopic subscore (MES) ≥1 and negative stool culture. The PRO2 considers stool frequency and passage of blood. Pregnancy and conditions influencing CAS or inflammation was prohibited. Patient were not allowed to use oral contraceptives, non-steroid anti-inflammatory drugs, systemic corticosteroids or anticoagulants. Controls were gender-matched blood donors recruited at the Unit for Thrombosis Research, Esbjerg and Grindsted Hospital, Denmark from 2019 to 2021. In- and exclusion criteria were the Danish blood donation criteria. We used the Montreal classification, MES, Nancy histological index, PRO2, CRP, and fecal calprotectin to describe the UC activity. Biochemical assays determined the kallikrein generation and plasma concentrations of coagulation factor XII, prekallikrein, H-kininogen, C1-INH and cHK (a surrogate marker of bradykinin generated by plasma kallikrein).

Results

We included 94 patients and 100 controls. Patients were primarily female (n=53) non-smokers (n=91) with a median age of 43 years and limited comorbidity (n=26). Half of the patients had proctitis. Patients displayed mild to moderate disease with medians: MES 2, Nancy histologic index 2, PRO2 4, CRP 2.5 mg/L and fecal calprotectin 515 mg/kg. Although, patients and controls generated similar amounts of plasma kallikrein, patients still had higher cHK levels in plasma than controls (1.38 [0.21-3.98] µg/mL vs 0.83 [0.02-2.88] µg/mL, p <0.0001). The same applied for C1-INH levels (0.22 [0.14-0.33] g/L vs 0.21 [0.16-0.29] g/L, p =0.0340). The remaining CAS components were similar in patients and controls. Nor, did CAS correlate to UC extension or severity. Interestingly, cHK remained higher even in patients displaying mild disease (see Table 1).

Conclusion

This is currently the largest study to profile CAS in patients with UC. By employing unique and novel assays, we can conclude that CAS contributes to active UC as verified by increased plasma levels of cHK and C1-INH in patients. Further, cHK might be useful as a serological marker for distinguishing patients with UC from healthy subjects.

References

1. Wang B, Yang A, Zhao Z, et al. The Plasma Kallikrein-Kininogen Pathway Is Critical in the Pathogenesis of Colitis in Mice. Front Immunol 2018;9:21.
2. Arai Y, Takanashi H, Kitagawa H, et al. Effect of icatibant, a bradykinin B2 receptor antagonist, on the development of experimental ulcerative colitis in mice. Dig Dis Sci 1999;44:845-51.
3. Lu F, Fernandes SM, Davis AE, 3rd. The role of the complement and contact systems in the dextran sulfate sodium-induced colitis model: the effect of C1 inhibitor in inflammatory bowel disease. Am J Physiol Gastrointest Liver Physiol 2010;298:G878-83.
4. Stadnicki A, Gonciarz M, Niewiarowski TJ, et al. Activation of plasma contact and coagulation systems and neutrophils in the active phase of ulcerative colitis. Dig Dis Sci 1997;42:2356-66.
5. Stadnicki A, Pastucha E, Nowaczyk G, et al. Immunolocalization and expression of kinin B1R and B2R receptors in human inflammatory bowel disease. Am J Physiol Gastrointest Liver Physiol 2005;289:G361-6.

Disclosure

The authors have nothing to declare