Introduction

Ulcerative colitis (UC) is a difficult-to-treat gastrointestinal disease, but its etiology and pathogenesis have not been clarified. An increasing number of studies have shown that the development and recurrence of UC are closely related to immune memory dysfunction. Our team's previous clinical and animal experiments have suggested that the treatment of UC with the Qingchang Wenzhong Decoction (QCWZD) is effective and can regulate intestinal mucosal immunity, but the exact mechanism is not yet clear. In this study, tissue-resident memory CD4⁺T cells (CD4⁺T_RM cells) were used as the entry point to further investigate the immunological mechanism of the QCWZD for the treatment of UC.

Aims & Methods

To explore the regulatory effect of QCWZD on CD4⁺T_RM cells in mice with DSS-induced colitis. This study aims to provide a new theoretical basis for the treatment of UC. Female C57BL/6 mice were randomly divided into control group, model group, QCWZD low-dose group, QCWZD medium-dose group, QCWZD high-dose group. Mice in control group were fed and watered freely throughout the whole process. Model group and doses group of QCWZD were used to replicate the UC model by freely drinking 2.5 % dextran sulfate sodium (DSS) (w/v) solution for 7 days. At the same time, model group was given deionized water gavage, and each dose group of QCWZD were given the corresponding concentration of QCWZD intervention for 1 week (Fig1 A). During this period, the mice were observed daily for general condition, weight measurement, detection of fecal occult blood, recording of fecal properties. The proportion of CD4⁺T_RM cells was performed with flow cytometry and the expression levels of proteins in related signaling pathways was assessed.

Results

Our findings demonstrated that DSS intervention resulted in colon inflammation with clinical changes, while QCWZD appreciably reversed these phenomenon, as evidenced by retarded body weight loss, recovery of rectal bleeding and diarrhea, decreased disease activity index scores (Fig1 B-C). Moreover, the proportion of CD4⁺T_RM cells in the lamina propria of mice in the DSS group were significantly higher than those in the Control group, while the CD4⁺T_RM cells proportion was significantly reduced after the intervention with the QCWZD (Fig1 D-E). Mechanistically, the results from RT-qPCR analysis of the colonic tissue showed that QCWZD could significantly reduce the expression of glucose transporter protein Glut1/2/4 mRNA (Fig2 F-H), as well as the levels of key enzyme genes of glycolytic pathway (HK2, PKM2, G6PC, LDHA mRNA) (Fig2 I-J), suggesting that QCWZD had an inhibitory effect on glycolytic metabolism in colitis mice. We further found that the mRNA expressions of colon LepR and HIF-1α mRNA were significantly downregulated in QCWZD-treated mice (Fig2 M-N). These results demonstrated that QCWZD regulated the glycolytic metabolism of CD4⁺T_RM cells by inhibiting the activity of LepR/HIF-1α signaling axis.

Conclusion

The results from our current study demonstrated that orally administrated QCWZD regulates CD4⁺T_RM cells immune homeostasis through the modulation of glycolytic metabolism, and hold the therapeutic potential for immunology-targeted strategy in the treatment of UC.

References

[1] Zaiatz Bittencourt V, Jones F, Doherty G, et al. Targeting Immune Cell Metabolism in the Treatment of Inflammatory Bowel Disease. Inflamm Bowel Dis. 2021, 27(10):1684-1693.
[2] Gonzalez-Vivo M, Lund Tiirikainen MK, Andreu M, et al. Memory T Cell Subpopulations as Early Predictors of Remission to Vedolizumab in Ulcerative Colitis. Front Med (Lausanne). 2022, 9:837294.

Introduction

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease mainly involving the mucosa and submucosa. The pathogenesis of UC has not been clearly elucidated and there are limitations of existing treatment measures. Dietary factors have been implicated closely related to the pathogenesis and clinical outcome of UC. Our team in the early clinical trials observed that UC patients had aggravation or recurrence of their disease after taking in lamb. However, the relationship between them is unclear. Does lamb aggravate the intestinal inflammation of UC patients? What is the mechanism if lamb has a negative impact on intestinal inflammation? It is urgent for us to study further.

Aims & Methods

We aimed to clarify the effect of lamb on intestinal inflammation in UC mice and to reveal the mechanism, so as to provide scientific basis for clinical dietary guidance for UC patients.
Mice were given 2.5% DSS (w/v) solution for 5 days and deionized water for 7 days for 1 cycle, repeated 3 cycles to establish the chronic UC model. Take the start of giving DSS solution as day1, corresponding measures of lamb suspensions at a dose of 0.25, 0.50, 1.00 g/mL or sterile water were administered for 31 days. Then the general condition and the severity of colitis were monitored. Colon tissues were obtained to assess the degree of pathological damage by HE staining and detect the levels of key factors of intestinal barrier by RT-qPCR. Spleen tissues were collected to detect the levels of M1/M2 macrophages and related factors by flow cytometry and RT-qPCR.

Results

Our results showed that lamb aggravated intestinal inflammation of UC mice, as indicated by higher weight loss and DAI, shorter colon and more severe pathological damage. Also, the colonic mechanical barrier-associated factors including ZO-1, Claudin-1, Occludin, E-cadherin mRNA and colonic mucosal barrier associated-factors such as Muc1, Muc2, Muc3 mRNA showed significant decrease in lamb-treated colitis mice. Moreover, colitis mice with lamb treatment showed significant increased M1 macrophages and reduced M2 macrophages in the spleen, leading to an increased the ratio of M1/M2 macrophage. Meanwhile, the M1 macrophage-associated inflammatory factors gene TNF-α, IL-6 mRNA increased and M2 macrophage-associated inflammatory factors gene IL-10, TGF-β mRNA decreased significantly in lamb-treated colitis mice.

Conclusion

Our results show that lamb aggravates the intestinal inflammation and damages the mucosal barrier of colitis mice, whose mechanism is related to the promotion of macrophages to M1 cells and inhibition M2 cells differentiation, thereby aggravating the imbalance of M1/M2 macrophages.

Introduction

Inflammatory bowel disease (IBD) is a chronic recurrent inflammatory bowel disease, whose cause involves the interaction between genetic and environmental factors. At present, the pathogenesis of IBD has not been fully understood. Although there is a recognized link between immune response and IBD, the causal relationship between circulating immune cell counts and IBD remains controversial.

Aims & Methods

This study aimed to elucidate the causal relationship between genetically predicted circulating immune cell counts and IBD. We conducted a bidirectional two-sample Mendelian randomization (MR) study using aggregated statistics from genome-wide association studies. The causal relationship between five circulating leukocytes cells (monocytes, lymphocytes, eosinophils, basophils and neutrophils) counts and IBD, including Ulcerative colitis (UC) and Crohn's disease (CD) was analyzed. Inverse variance weighting (IVW) was used as the primary MR analysis method, and horizontal pleiotropy test and heterogeneity test were used to ensure the stability of the results.

Results

Our findings indicated that monocytes, lymphocytes, eosinophils, and basophils count were not significantly associated with IBD, however, elevated circulating neutrophils count was significantly associated with higher risk of IBD [odds ratio (OR): 1.002; 95% confidence interval (CI): 1.001–1.003; p = 0.009] and UC [OR: 2.465; 95%CI: 1.236–4.916; p = 0.01]. In addition, we also found that IBD [ OR: 12.07; 95% CI: 1.909-76.316; P = 0.008] and UC [OR: 2.465; 95% CI: 1.236 - 4.916; P = 0.01] were significantly associated with higher circulating neutrophils count in reverse MR.

Conclusion

This MR study provides genetic evidence for the causal relationship between the genetically predicted increase in circulating neutrophils count and the risk of IBD (UC and CD). This finding stresses the need for further exploring physiological functions of neutrophils in order to develop effective strategies against IBD.

Disclosure

The authors state that the study was conducted in the absence of any business or financial relationships that could be interpreted as potential conflicts of interest.

Introduction

Tissue factor (TF) on cell membranes initiates blood clotting upon vessel trauma and exposure of subendothelial cells to blood flow. The risk of thromboembolic events is increased in patients with ulcerative colitis (UC). Tofacitinib, a JAK1 & 3 inhibitor, recently introduced in UC therapeutics, may further increase the risk¹.

Aims & Methods

To delineate thrombosis pathophysiology in this context, TF expression in primary human colonic mesenchymal cells (PHCMC) of patients with active UC was correlated with clinical, biochemical and endoscopic data. We then treated those PHCMC with tofacitinib, with or without cytokines to better mimic the in vivo milieu they are exposed to.
PHCMC were isolated and set to ex vivo culture from endoscopic biopsies of the inflamed mucosa of 10 UC patients with an endoscopic Mayo score ≥ 2 were treated with each one of all major T helper (Th)1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13) or T regulatory (Treg; TGF-β, IL-10) cytokines with or without tofacitinib. Cells were lysed, RNA was isolated and reverse-transcribed to cDNA. TF and RPL4 (housekeeping) cDNAs were quantified as C_T cycles with real-time, reverse-transcription PCR. Wilcoxon and Mann-Whitney U tests were used to compare TF ΔΔC_T for paired or unpaired values, respectively, and Spearman’s rho to correlate TF ΔΔC_T with scale clinical variables and continuous laboratory values.

Results

Increased TF mRNA abundance in PHCMC was associated with increased partial Mayo score (Spearman's rho 0.661, p < 0.044), reduced serum albumin (Spearman's rho -0.723, p < 0.05) and more severe endoscopic lesions (endoscopic Mayo score 3). No association with CRP was found (p > 0.999). Cells originating from UC patients of an endoscopic Mayo score 3 expressed 20 times more TF than those from an endoscopic Mayo score 2 (p < 0.017). Furthermore, PHCMC from difficult-to-treat patients, defined as requiring > 1 biologics, expressed 9 times more TF than those from patients requiring ≤ 1 biologics.
Interestingly, treatment of PHMC with tofacitinib did not upregulate TF. PHCMC expressed receptors and responded to treatment with all major Th1 (TNF-α, IFN-γ), Th2 (IL-4, IL-13) or Treg (TGF-β, IL-10) cytokines by further upregulating TF by 5.5-7.5 times (p 0.001 - 0.035). IL-13 had the maximal effect. Even when tofacitinib was added together with the aforementioned cytokines, it did not further upregulate TF. Instead, it tended to partially inhibit their effects; for example, it decreased upregulation of TF by IL-13 by 40%.

Conclusion

The expression of TF mRNA in active UC is significantly associated with clinically and endoscopically severe disease and resistance to treatment. Tofacitinib per se does not increase TF. Instead, it may limit the upregulating effect of pro-inflammatory cytokines.

References

1. Sandborn WJ, Panés J, Sands BE, Reinisch W, Su C, Lawendy N, Koram N, Fan H, Jones TV, Modesto I, Quirk D, Danese S. Venous thromboembolic events in the tofacitinib ulcerative colitis clinical development programme. Aliment Pharmacol Ther. 2019 Nov;50(10):1068-1076.

Disclosure

Procurement of reagents and consumables for this project was partially supported by Pfizer Inc. with competitive grant # 64132481.

Introduction

Inflammatory bowel disease (IBD), a chronic inflammatory disease of the gastrointestinal tract, is caused by various factors, including genetic factors and environmental factors, as well as dysregulated immune responses. LMT503, a novel organic small-molecule compound, has the potential to suppress pro-inflammatory cells and induce anti-inflammatory cells by modulating their cell metabolisms. We evaluated the anti-inflammatory effects of LMT503 in a murine adoptive T cell transfer-induced colitis model.

Aims & Methods

This study was conducted to evaluate the anti-inflammatory effect of LMT503 in the murine chronic colitis model. Naïve CD4⁺CD25^-CD45RB^high CD62L^high T cells that were isolated from wild-type mice by flow cytometry based-method and transferred to Rag1 knock-out mice by intraperitoneal injection to induce murine adoptive T cell transfer-induced colitis. LMT503 was orally administrated (50mg/kg, 100mg/kg) for 2 weeks starting at 5 weeks after naïve T cell transfer. The disease activity index (DAI) was checked daily, and histopathological score, macroscopic injury score, and colon length were evaluated upon sacrifice. Macrophages and T cells from the spleen were analyzed by flow cytometry. Gene expression in colon tissue was measured by qRT-PCR and cytokine profiles in plasma was measured by cytometric bead array. Myeloperoxidase (MPO) activity was measured using the MPO activity kit.

Results

Oral administration of LMT503 ameliorated adoptive T cell transfer-induced colitis in mice. The DAI, histopathological score, and macroscopic injury score were significantly decreased after the administration of LMT503 in a dose-dependent manner. The LMT503-treated group showed significantly increased mucin production in the colon. Th1, Th17 cells, and M1 macrophages in the spleen were suppressed, and M2 macrophages were induced by the administration of LMT503. MPO activity and gene expressions of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α, in colon tissue were suppressed by the administration of LMT503. And the level of inflammatory cytokines, such as IL-2, TNF-α, and IFN-γ, in plasma were significantly suppressed by the administration of LMT503. Especially, LMT503 treatment significantly increased the gene expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a), a master regulator of mitochondrial biogenesis, suggesting that LMT503 modulates cellular metabolism towards an anti-inflammatory metabolism.

Conclusion

Our data suggest that the anti-inflammatory effect of LMT503 ameliorates murine adoptive T cell transfer-induced colitis by modulating cellular metabolism and immune cell modulation. Therefore, LMT503 may be a novel therapeutic drug that opens new avenues for the treatment of IBD.

References

1. Erben U, Loddenkemper C, Doerfel K, et al. A guide to histomorphological evaluation of intestinal inflammation in mouse models. Int J Clin Exp Pathol. 2014;7(8):4557-4576.
2. Ostanin DV, Bao J, Koboziev I, et al. T cell transfer model of chronic colitis: concepts, considerations, and tricks of the trade. Am J Physiol Gastrointest Liver Physiol. 2009;296(2):G135-G146.
3. Ghoreschi K, Jesson MI, Li X, et al. Modulation of innate and adaptive immune responses by tofacitinib (CP-690,550). J Immunol. 2011;186(7):4234-4243.
4. Burke JR, Cheng L, Gillooly KM, et al. Autoimmune pathways in mice and humans are blocked by pharmacological stabilization of the TYK2 pseudokinase domain. Sci Transl Med. 2019;11(502):eaaw1736.
5. Kiesler P, Fuss IJ, Strober W. Experimental Models of Inflammatory Bowel Diseases. Cell Mol Gastroenterol Hepatol. 2015;1(2):154-170.