Involvement of the NLRP3/IL-1β pathway in activation and effector functions of γδT17 cells in patients with ulcerative colitis

Abstract

BACKGROUND

The interleukin-17 (IL-17) mediated aberrant immune-inflammatory response plays a paramount role in ulcerative colitis (UC). γδT17 cells are one of the critical sources of IL-17, but the role they play in UC remains under debate.

AIM

To clarify the role of γδT17 cells in patients with mild-to-moderate UC.

METHODS

A single-centre observational pragmatic study was conducted on patients with UC who attended the outpatient and inpatient departments of Xiyuan Hospital of the China Academy of Traditional Chinese Medicine from September 2020 to December 2022. The research population consisted of two groups of adult patients. The first group consisted of healthy volunteers with no significant abnormalities on colonoscopy, and the other group consisted of patients with mild-to-moderate ulcerative colitis. Serum samples from healthy volunteers and patients with UC were collected for the detection of relevant inflammatory factors. Moreover, five colon mucosa samples were randomly selected from each group for testing and analyses.

RESULTS

An increased number of γδT17 cells and hyperactivation of the NLR family pyrin domain containing 3/IL-1β signaling pathway were observed in colonic mucosal tissues from patients with UC.

CONCLUSION

Hyperactivation of the NLR family pyrin domain containing 3/IL-1β signaling pathway promotes the activation of γδT17 cells in colonic mucosal tissues of patients with UC.

Key Words: Ulcerative colitis; Abnormal immune response; γδT17 cells; NLR family pyrin domain containing 3/interleukin-1β pathway; Observational study

Core Tip: The contribution of γδT17 cells in patients with ulcerative colitis (UC) is controversial. In this study, serum samples from healthy volunteers and UC patients were analysed for associated inflammatory factors, and five randomly selected colonic mucosal tissue biopsy samples from each group were analysed by immunohistochemistry and immunofluorescence to clarify the role of γδT17 cells in UC patients. The serum levels of inflammatory factors secreted after γδT17 cell activation were significantly elevated in patients with UC, the expression level of γδT17 cells in colonic mucosal tissues was significantly elevated, and the NLR family pyrin domain containing 3/interleukin-1β signaling pathway was hyperactivated.

INTRODUCTION

Inflammatory bowel disease (IBD) encompasses a series of chronic conditions impacting the gastrointestinal tract and extraintestinal organs[1]. As a global disorder, with more than 6 million cases diagnosed per year worldwide and a prevalence in developed countries approaching 5%, IBD may become a critical health and policy issues around the world[2]. Ulcerative colitis (UC) and Crohn’s disease are the two clinical-histological variants of IBD, the former presents as continuous-type of inflammation of the colorectal or total colonic mucosa and submucosa, and the latter can involve the entire gastrointestinal tract in a discontinuous fashion, most often involving the terminal ileum, colon and perianal area. In addition to protracted course and recurrent attacks[3], up to 24% of IBD patients experience extraintestinal manifestations, such as peripheral arthritis, ankylosing spondylitis and anterior uveitis[4], which can severely impact their quality of life. Furthermore, the risk of colorectal cancer in patients with UC is 2.4 times greater than that in the general population[5]. Despite the pathogenesis of UC is still incompletely understood, abnormal innate and adaptive immune responses are pivotal factors in recurrences and exacerbation of the disease[6].

γδT17 cells are a highly heterogeneous subpopulation of T lymphocytes with surface receptors consisting of γ and δ chains and are mainly distributed in the skin, intestine and respiratory tract[7]. As one of the most abundant T-cell subsets in the intestine, γδT17 cells can interact with a variety of intestinal immune cells, and can participate in abnormal immune-inflammatory responses in the intestinal epithelium[8,9]. Relevant studies have shown elevated levels of γδ T cells in the peripheral blood and intestinal mucosa of patients with IBD[10]. However, other studies have shown a decrease in the number of circulating γδ T cells in patients with Crohn’s disease compared with the normal patients[11,12]. In spontaneous colitis in mice, a special class of γδ T cells with pro-colitis effects has been identified[13], which express two key intestinal homing integrin molecules, CD103 and α4β7, and produce interleukin-17 (IL-17) in response to tretinoin. The mechanism of action of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in intestinal homeostasis has attracted much attention, and as an important part of innate defence, the NLRP3 inflammasome is involved in mediating the pathological process of UC[14]. IL-1β produced by aberrant activation of the NLRP3 inflammasome can induce γδT17 cell activation and thus promote IL-17 secretion. However, in different inflammatory microenvironments, γδT17 cells are plastic and have different functions[15]. Thus, their exact role in IBD remains controversial[16-18].

MATERIALS AND METHODS

All participants were enrolled from Xiyuan Hospital, China Academy of Chinese Medical Sciences (Beijing, China) from September 2020 to December 2022. The diagnostic criteria for UC were in accordance with the 2019 American College of Gastroenterology Clinical Guidelines[19]. This clinical protocol was approved by the ethics committee of Xiyuan Hospital (No. 2019XLC003-2), and all the subjects voluntarily participated and signed an informed consent form. The quantities of cytokines such as IL-1β, IL-23, IL-21, tumor necrosis factor α (TNF-α), granulocyte-macrophage colony stimulating factor (GM-CSF), IL-22, IL-17, IL-10 and transforming growth factor β (TGF-β), were analysed with the appropriate ELISA kits. Type-17 immune response mediated by IL-17 plays a crucial role in the pathology of UC and γδT17 cells are one of the important sources of IL-17. Therefore, we measured the expression levels of IL-17 and γδT17 cells in the colonic mucosal tissues of UC patients by immunohistochemistry and immunofluorescence staining.

Inclusion/exclusion criteria

The study population consisted of an UC group and a normal group, and the ages of all the participants were between 18 and 70 years. Patients with mild-to-moderate UC in the active phase of the disease were eligible for inclusion (active phase: Total modified Mayo index score ≥ 2; mild to moderate defined by modified Truelove and Witts disease severity staging: Stool frequency < 4, light or no blood in the stool, normal pulse, temperature and hemoglobin, and ESR < 20 mm/hour) and rectal biopsy was necessary. Adults with no abnormal clinical manifestations, no significant organic changes on colonoscopy and who were willing to undergo colon biopsy were included in the normal group. Patients with severe heart, liver, kidney or other major organ disease; haematopoietic, neurological, psychiatric disease or combined with other organic lesions of the digestive system (e.g., peptic ulcer), or with systemic diseases affecting the dynamics of the digestive tract (e.g., hyperthyroidism or diabetes mellitus) were excluded.

Clinical samples

This study is a clinical observational study. Blood serum and biopsy tissue samples were obtained from eligible participants. The process of this study strictly adhered to the Declaration of Helsinki.

Haematoxylin and eosin staining

During each colonoscopy, at least two biopsies were obtained from different lesion sites. All biopsies were fixed in 10% buffered formalin and subsequently paraffin embedded, cut into 4-μm-thick sections and then stained with haematoxylin and eosin (HE).

Enzyme-linked immunosorbent assay

Absolute concentrations of cytokines in blood serum were quantified with ELISA kits, including IL-21 (Lot number: 10354), IL-22 (Lot number: 10356), IL-23 (Lot number: 10357), TNF-α (Lot number: 11776), GM-CSF (Lot number: 11005), IL-17 (Lot number: 10344), IL-18 (Lot number: 10349), IL-1β (Lot number: 10351), TGF-β (Lot number: 11800), and IL-10 (Lot number: 13626), according to the manufacturer’s instructions.

Immunohistochemistry and Immunofluorescence

Four-micrometer-thick slices of colonic tissue were deparaffinized in xylene and stained with anti-NLRP3 antibody (1:500, Lot number: 19771-1-AP), anti-apoptosis-associated speck-like protein (ASC) antibody (1:100, Lot number: ab180799), anti-caspase-1 antibody (1:100, Lot number: 22915-1-AP), anti-IL-β antibody (1:500, Lot number: ab283818), and anti-IL-17A antibody (1:200, Lot number: ab189377). For immunofluorescence, sections were incubated with in anti-T cell receptor γ/δ antibody (1:50, Lot number: TCR-1061) and anti-IL-17A antibody (1:100, Lot number: 17-7177-81).

Outcomes

The primary aim of this study was to measure the expression levels of γδT17 cells in intestinal mucosal tissues from UC patients. The secondary aim was to evaluate the status of the NLRP3/IL-1β pathway in intestinal mucosal tissues from UC patients.

Statistical analysis

Statistical data were analysed with GraphPad Prism 8.0 software. The calculation data were tested by χ²-test. The data are expressed as mean ± SDs. Data with normal distribution and homogeneity of variances were analysed by t-test; Mann-Whitney test was used for data without normal distribution or homogeneity of variances. P < 0.05 (two-tailed) was considered statistically significant.

RESULTS

From September 2020 to December 2022, 32 patients with UC and 46 volunteers were screened. Four patients with severe UC and 10 patients who declined to participate were excluded, and volunteers who did not meet the inclusion criteria and declined to participate were also excluded, ultimately 18 patients with UC and 20 healthy volunteers completed our observational study. Figure 1 shows the screening process for the participants in this study. The clinical characteristics such as age, genders and ethnological of the participants in both groups are displayed in Table 1.

Figure 1 The screening process for participants in this study. 32 patients who met the diagnosis of ulcerative colitis were included, of which 4 patients belonged to severe ulcerative colitis and 10 patients refused to participate. Finally, total of 18 patients completed this study: 46 healthy volunteers were screened, of which 15 did not meet the inclusion criteria, 11 refused to participate, and 20 volunteers completed the experiment. UC: Ulcerative colitis.

Table 1 Baseline characteristics of ulcerative colitis group and normal group.

Characteristics	UC group	Normal group	P value
Number	18	20
Age	42.17 ± 10.04	44.45 ± 10.13	0.49
Male/female, %	55.6/44.4	40/60	0.34
Race
Han, n (%)	17 (94.4)	19 (95)	0.94
Other, n (%)	1 (5.6)	1 (5)

UC: Ulcerative colitis.

HE staining of colonic mucosal tissue from patients with UC

Microscopic images of HE stained sections of colonic mucosal tissues are shown in Figure 2, compared with healthy controls, patients with UC showed a decreased number of glands, deformation, distortion, branching of crypt structures, and an increase in population of inflammatory cells in the lamina propria of the colonic mucosal tissues.

Figure 2 Histopathological manifestations of healthy controls and ulcerative colitis patients. The number of glands in the colonic mucosal tissue of ulcerative colitis patients was reduced, crypt structures were distorted, twisted and branched, and the population of inflammatory cells in the lamina propria were increased compared with healthy control (magnification: 200 ×). UC: Ulcerative colitis.

Cytokines induced γδT17 cells activation and cytokines secreted by γδT17 cells were both increased, and anti-inflammatory factors were decreased in patients with UC

The quantities of cytokines such as IL-1β, IL-23, IL-21, TNF-α, GM-CSF, IL-22, IL-17, IL-10 and TGF-β were analysed with the appropriate ELISA kits. As shown in Figure 3, the levels of cytokines that can induce γδT17 cell activation, such as IL-1β and IL-23, were significantly elevated, and cytokines secreted by γδT17 cells such as IL-21, TNF-α, GM-CSF, IL-22 and IL-17 were elevated to different degrees, all of which indicated abnormal activation of γδT17 cells in patients with UC, while concomitant anti-inflammatory cytokines IL-10 and TGF-β were decreased.

Figure 3 Levels of serum inflammatory factors in healthy controls and ulcerative colitis patients. A: Interleukin-21 (IL-21); B: IL-22; C: IL-23 were significantly elevated; D: tumor necrosis factor α; E: Granulocyte-macrophage colony stimulating factor; F: IL-17; G: IL-18; H: IL-1β were significantly elevated; I: Anti-inflammatory cytokines transforming growth factor β were decreased; J: Anti-inflammatory cytokines IL-10 were decreased. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs healthy control. UC: Ulcerative colitis; IL: Interleukin; TNF-α: Tumor necrosis factor α; GM-CSF: Granulocyte-macrophage colony stimulating factor; TGF-β: Transforming growth factor β.

The expression level of γδT17 cells in colonic mucosal tissues of UC patients was increased

As shown in Figure 4, compared with expression levels measured in the normal group, the expression levels of IL-17 and γδT17 cells were significantly elevated in UC patients, and our results also showed γδT17 cells scattered in the epithelium and lamina propria of the colon, which indicated that the elevated IL-17 in UC patients was significantly correlated with abnormal activation of γδT17 cells.

Figure 4 The expression levels of interleukin-17 and γδT17 cells in healthy controls and ulcerative colitis patients. A: The expression level of interleukin-17 in colonic mucosal tissues of ulcerative colitis patients was markedly elevated. vs healthy control (magnification: 200 ×); B: The expression level of γδT17 cells in colonic mucosal tissues of ulcerative colitis patients was markedly elevated. ^bP < 0.01, ^cP < 0.001 vs healthy control (magnification: 40 ×, 200 ×, 400 ×). UC: Ulcerative colitis; IL: Interleukin; TCR: T cell receptor.

The expression levels of NLRP3, ASC, caspase-1, and IL-1β were differentially elevated in colonic mucosal tissues from UC patients

As shown in Figure 5, the expression levels of NLRP3, ASC, caspase-1 and IL-1β in colon mucosal tissue were all elevated in different degrees in UC patients compared with healthy control. The above results indicated that hyperactivation of the NLRP3 inflammasome in colonic tissues of UC patients leads to aberrant activation of γδT17 cells, which can be one of the important pathological mechanisms of UC.

Figure 5 The expression levels of NLR family pyrin domain containing 3, apoptosis-associated speck-like protein, caspase-1 and interleukin-1β in colonic mucosal tissues. NLR family pyrin domain containing 3, apoptosis-associated speck-like protein, caspase-1, and IL-1β are widely expressed in cytoplasm and their expression levels were elevated to different extents in colonic mucosal tissues of ulcerative colitis patients. ^aP < 0.05, ^bP < 0.01 vs healthy control (magnification: 200 ×). NLRP3: NLR family pyrin domain containing 3; ASC: Apoptosis-associated speck-like protein; IL: Interleukin; UC: Ulcerative colitis; IOD: Integrated optical density.

DISCUSSION

UC is a lifelong inflammatory disease characterized by chronic inflammation of the colorectal mucosa and ulcer formation, with the lesions mainly involving the mucosa or submucosa[7]. IL-17 is a T-cell-derived cytokine with multiple functions such as defence against pathogenic microorganisms, recruitment of inflammatory cells, and induction of proinflammatory cytokine secretion[20]. Some studies have shown that elevated levels of IL-17 gene expression are a prominent feature in the pathogenesis of UC[21]. Our findings also showed that the expression level of IL-17 was dramatically elevated in both serum and colonic mucosal tissues from UC patients. However, what are the sources of IL-17 in UC patients? It is widely known that αβ T cells, γδ T cells, and innate lymphoid cells all have the ability to secrete IL-17, among which Th17 cells are one of the main sources of IL-17 and play a pivotal role in the pathological process of UC[22]. At present, a growing body of clinical evidence has demonstrated that the content of γδT17 cells are significantly elevated in colonic tissues form patients with IBD and colorectal tumours[23,24]. Our results showed that expression levels of γδT17 cells were significantly elevated in colonic mucosal tissues from UC patients compared to expression levels in colonic mucosal tissues from healthy volunteers, which is consistent with existing clinical research. Based on the properties of the δ chain, human γδ T cells can be classified into two main subtypes: Vδ1 cells are mainly expressed in mucosal tissues, whereas infiltrating Vδ2 T cells are mainly expressed in peripheral blood and secondary lymphoid organs, have a distinct memory effect and terminal differentiation phenotype, and produce high levels of TNF-α and IL-17, which correlate with inflammatory responses and the severity of IBD[20], and could be another point of in-depth study for our group.

The inflammatory response triggered by innate immunity is a primary host response against foreign invasion, removing pathogens and attracting adaptive immune cells to the site of injury. NLRP3 is an NLR-family protein that responds to extensive stimulation by forming inflammasomes. The role of inflammasomes is to activate caspases to produce inflammatory mediators such as IL-1β and IL-18. Studies have shown that NLRP3, IL-1β, caspase-1, and ASC expression levels are significantly elevated in colonic mucosal biopsy tissues from patients with in-remission or active IBD[25], and recent investigations have shown that nucleotide polymorphisms in the IL-18 and IL-1β genes of patients with IBD are correlated with poor response to treatment with anti-TNF-α[26]. The regulatory role of the NLRP3 inflammasome in intestinal immune-inflammatory homeostasis is receiving increasing attention, and its continuous activation leads to overproduction of IL-1β, which can bind to IL-1 receptors on the surface of γδT17 cells and promote abnormal activation, resulting in production of large amounts of IL-17 and exacerbating intestinal inflammatory responses. Therefore, we further measured the expression levels of NLRP3, ASC, caspase-1 and IL-1β in the colonic mucosal tissues of UC patients. The results of our study revealed that the expression levels of NLRP3, ASC, caspase-1, and IL-1β in patients with UC were significantly elevated, and that overexpressed IL-1β leads to abnormal activation of γδT17 cells, which exacerbates the abnormal immune-inflammatory response. The results of this simple, observational study will be used as the basis for a multicenter, randomized, double-blind, placebo-controlled study to provide high-level, evidence-based clinical findings, which can address the limitations, such as small sample sizes, regional limitations and variability in patient populations of this current study.

CONCLUSION

In conclusion, our observational study provides new ideas for clinical research by demonstrating that overactivation of the NLRP3/IL-1β signaling pathway mediated abnormal activation of γδT17 cells, which is an important pathological mechanism in UC patients (Video 1).