Inhibition of Piezo1 Ameliorates Intestinal Inflammation and Limits the Activation of Group 3 Innate Lymphoid Cells in Experimental Colitis

Inflammatory bowel disease (IBD) is considered to arise from inappropriate activation of the gut mucosal immune system, as a result of the complex interactions between genetics, environment, gut microbiota, and other factors [1]. One of the earliest events of IBD is when innate immune cells, such as neutrophils, macrophages, and dendritic cells (DCs), infiltrate into the intestinal lamina propria and produce elevated levels of pro-inflammatory cytokines. Furthermore, naïve T cells are primed to differentiate into T helper (Th) cells, including Th1, Th2, and Th17 cells, resulting in excessive adaptive immune activation [2, 3]. More recent studies have shown that group 3 innate lymphoid cells (ILC3s) play a dual role in colitis through the generation of cytokines such as interleukin-22 (IL-22) and IL-17A [4‒7]. Current IBD treatments, especially the application of immunomodulators and biologicals, have greatly improved the outcome of the disease over the past decades. Unfortunately, there is still a significant proportion of patients who fail to respond [8, 9]. Therefore, novel therapeutic targets to regulate the immune responses for the treatment of IBD are in urgent need.

Immune cells are constantly exposed to various mechanical cues in vivo, which collaborate with other microenvironmental factors to regulate immune responses [10]. Mechanosensory ion channels (MSICs) located on the cell membrane mediate the effects of mechanical cues by converting them into electrochemical signals [11]. Piezo1 is one of the most well-studied MSICs. It responds to a broad range of mechanical stimuli, such as shear stress, hydrostatic pressure, compression, and matrix stiffness, by allowing cation influx with a special three-blade propeller conformation, which subsequently affects signaling and cell behaviors [12]. The crucial roles of Piezo1 have been delineated in numerous physiological processes [13‒15]. A series of recent studies have highlighted the important role of Piezo1 in regulating immune cell functions and the pathogenesis of immune-related diseases. In monocytes, Piezo1-mediated mechanical stimuli promote pro-inflammatory gene expression. Loss of Piezo1 causes a defective anti-infection response in the model of Pseudomonas infection but protects mice from bleomycin-induced pulmonary fibrosis [16]. In DCs, Piezo1 is involved in proliferation, activation, and cytokine production and alters DC-mediated CD8⁺ T cell activation in models of autoimmune diabetes and tumor immunotherapy [17]. In T cells, it is reported that Piezo1 deletion selectively restrains regulatory T (Treg) cells and attenuates experimental autoimmune encephalomyelitis [18]. These results suggest that Piezo1 is prospective to be a new target for the treatment of immune-related diseases. However, considering the disparate effects of Piezo1 among different organs and diseases, future studies designed to explore the role of Piezo1 in the context of other immune-related diseases are clearly warranted.

Orchestrated dynamics is fundamental for the intestine in the maintenance of normal digestion. Alterations in the motility and mechanical microenvironment occur in disease states like IBD. For instance, decreased colonic motility and increased intraluminal pressure are observed in IBD patients [19, 20]. Further, mucosal edema is typically seen in IBD, which indicates the alteration of interstitial pressure [21]. In addition, various degrees of fibrosis are recognized in both ulcerative colitis (UC) and Crohn’s disease (CD), in which case excessive extracellular matrix proteins deposit and increase the stiffness of the bowel wall. These changes in mechanical properties have been associated with the progression of IBD [22‒25]. Previous studies of IBD mostly focused on interactions between cells and molecules, overlooking the effect of mechanical perturbations on gut inflammation. In spite of its role in transducing mechanical stress and modulating the function of various immune cells, Piezo1 has not been intensively studied in gut immunity. Considering these Piezo1-regulated innate and adaptive immune cells are the central link in the pathogenesis of IBD, we were therefore spurred to investigate the role of Piezo1 in intestinal immunity and colitis. The spider venom-derived peptide GsMTx4 specifically targets cation MSICs [26‒28]. Given its immunomodulatory effect via Piezo1 inhibition under several inflammatory conditions [16, 29, 30], a protective role of GsMTx4 in colitis is thus postulated.

In our present study, the role of Piezo1 in colitis and gut immunity was investigated. We report for the first time that GsMTx4 alleviates experimental colitis in mice. Inhibition of Piezo1 by GsMTx4 eased the innate immune responses during colitis. Specifically, we found that Piezo1 regulated the activation of ILC3s with the participation of the PI3K-Akt-mTOR signaling pathway. The present study provides a theoretical basis for the development of Piezo1 as a novel therapeutic target to treat IBD.

Male and female C57BL/6 mice were purchased from GemPharmatech. All the mice were maintained in the specific pathogen-free animal facility at Shandong University, with a 12-h light/dark cycle at the temperature of 20–26°C. The mouse sample size was calculated using power analysis on G*Power 3.1. All animal procedures were carried out with the approval of the Ethical and Institutional Animal Care and Use Committee of Qilu Hospital of Shandong University (DWLL-2021-007).

Dextran sulfate sodium (DSS)-induced colitis model was established according to the protocol described by Wirtz et al. [31]. In brief, 8-week-old mice were administered 2.5% DSS (MP Biologicals) in drinking water for 7 consecutive days, with intravenous injection of GsMTx4 (DSS+GsMTx4 group, 10 mg/kg [32, 33], Alomone Labs) or solvent (DSS group) on days 2, 4, and 6. Mice given normal drinking water were used as the Control group. The weight loss, rectal bleeding, and stool consistency were monitored daily. Disease activity index (DAI) scores were calculated as previously described [31]. Mice were euthanized on day 7 post-DSS. The colon length was measured, and the colon tissues were collected for subsequent experiments.

Total RNA from colon tissues or ILC3s was extracted using TRIzol (Invitrogen) and reversely transcribed into cDNA using ReverTra Ace^® qPCR RT Kit (Toyobo). Quantitative real-time PCR (qRT-PCR) was done by using SYBR^® Green Realtime PCR Master Mix (Toyobo). All the gene-specific primers were synthesized by Sangon. Primer sequences are listed in online supplementary Table S1 (for all online suppl. material, see https://doi.org/10.1159/000533525). Relative expression levels were normalized to Gapdh mRNA expression and calculated using the 2^−ΔΔCt method.

Colon tissues were homogenized and lysed on ice for 30 min in RIPA lysis buffer (Solarbio). Protein concentrations were determined by using a bicinchoninic acid protein assay kit (ABP Biosciences). Protein extracts were mixed with 5× SDS-PAGE Sample Loading Buffer (Beyotime), boiled at 95°C for 10 min, loaded into the 4–8% SDS-PAGE gel for electrophoresis, and transferred to the polyvinylidene difluoride membrane (Millipore), successively. After blocking with 5% non-fat milk, the membranes were incubated with Piezo1 Polyclonal antibody (1:400, 15939-1-AP; proteintech) and Beta Actin Polyclonal antibody (1:5,000, 20536-1-AP; proteintech) overnight at 4°C. Next, the membranes were washed and incubated with HRP-conjugated anti-rabbit secondary antibody (1:5,000, ZB-2301; ZSGB-BIO) for 1 h at room temperature. The membranes were visualized in an enhanced chemiluminescence (Millipore). The chemiluminescent density of protein bands was quantified using Quantity One (Bio-Rad).

The distal colon sections were fixed in 4% paraformaldehyde solution over 24 h. After being embedded in paraffin, the colon tissues were sliced into 4-μm sections and stained with Hematoxylin and Eosin (HE; Solarbio) following the manufacturer’s protocol. Histology scores were assessed based on the degree of epithelial damage, inflammatory infiltration, and ulcerations according to the criteria described previously [31]. For immunofluorescence staining, colon sections were subjected to heat-induced epitope retrieval in citrate buffer. After being blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature, the sections were incubated with CD45 monoclonal antibody (1:100, 60287-1-lg; proteintech) and PIEZO1 antibody (1:400, NBP1-78446; Novus biologicals) at 4°C overnight. Then, sections were washed and incubated in goat anti-mouse lgG H&L (Alexa Fluor^® 488; 1:1,000, ab150113; Abcam) and goat anti-rabbit lgG H&L (Alexa Fluor^® 647; 1:1,000, ab150079; Abcam). The 4′,6-diamidino-2-phenylindole (DAPI) was added for nucleus staining, and images were captured under an Olympus fluorescence microscope.

Total RNA from colon tissues was extracted. RNA Integrity Number (RIN) was evaluated. RNA sequencing (RNA-seq) was conducted by LC-Bio. Differential expression analysis was carried out using the R package “DESeq2” (https://bioconductor.org/packages/release/bioc/html/DESeq2.html) with a p-adjust threshold of 0.05 and an absolute log 2 (fold change) threshold of 1. Heatmaps were generated with the R package “pheatmap” (https://CRAN.R-project.org/package=pheatmap). Volcano plot was generated with GraphPad Prism 8.0.2. Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the R package “clusterProfiler” (http://www.bioconductor.org/pack-ages/release/bioc/html/clusterProfiler.html).

For colon tissues, samples were first weighed, and the supernatants of the tissue homogenate were collected. The concentrations of IL-6 were measured by the Mouse IL-6 ELISA Kit (Lianke Biotechnology) according to the manufacturer’s instructions. For ILC3s, cell-free supernatants were harvested after treatment. The IL-17A concentrations were determined by the Mouse IL-17A High Sensitivity ELISA Kit (Lianke Biotechnology).

Colonic lamina propria lymphocyte (LPLs) were obtained following a previously established method [34]. In brief, colons were opened longitudinally, cut into 0.5-cm pieces, and washed in ice-cold phosphate buffer solution. Hank’s balanced salt solution (HBSS) containing 10 mm HEPES (Gibco) and 2 mm EDTA (Invitrogen) was used to remove intestinal epithelial cells. The remaining pieces were then digested in RPMI-1640 medium (Gibco) supplemented with 5% fetal bovine serum (Gibco), 1 mg/mL collagenase VIII, and 0.1 mg/mL DNase (Sigma-Aldrich) at 37°C for 1.5 h. After being sifted through 70-μm cell strainers, the cell suspension was centrifuged with 40–80% Percoll (Sigma-Aldrich) to collect the LPL-enriched population. For flow cytometry, cells were incubated with the LIVE/DEAD^™ Violet Viability/Vitality Kit (Invitrogen) at room temperature for 25 min. After being blocked with CD16/32 antibody (Invitrogen), cells were stained with the following antibodies: F4/80 (BM8), Foxp3 (MF-14), CD4 (GK1.5), CD3 (17A2), CD19 (1D3/CD19), Ly-6C (HK1.4), CD64 (X54-5/7.1), RORγt (AFKJS-9), MHCII (M5/114.15.2), CD11b (M1/70), IFN-γ (XMG1.2), IL-6 (MP5-20F3), IL-17A (TC11-18H10.1), CD45 (I3/2.3), CD11c (N418), and CD3ε (145-2C11). Lin comprised CD11c (N418), CD11b (M1/70), CD3ε (145-2C11), CD19 (1D3/CD19), CD45R/B220 (RA3-6B2), TER-119 (TER-119), FcεRIα (MAR-1), CD5 (53-7.3), Ly-6G (1A8), and CD16/32 (93). All these antibodies were purchased from Bio-Legend, except RORγt (Invitrogen). Samples were detected using a Gallios flow cytometer (Beckman Coulter), and flow cytometry data were analyzed with FlowJo 10 software (FlowJo LLC).

Isolated colonic LPLs were incubated with the LIVE/DEAD^™ Violet Viability/Vitality Kit and CD16/32 antibody as above. Then, cells were incubated with CD127 (A7R34), KLRG1 (2F1/KLRG1), CD45 (I3/2.3), and Lin (same as above). All these antibodies were purchased from BioLegend. The cell isolation was conducted in a MoFlo Astrios EQ (Beckman Coulter). Sorted ILC3s were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco) containing 10% fetal bovine serum, IL-2 (10 μg/mL, Peprotech), IL-7 (10 μg/mL, Peprotech), and IL-23 (20 μg/mL, BioLegend). For Yoda1 (Selleck) or GsMTx4 treatment, 5 μm GsMTx4 was administered, and 30 min later, 25 μm Yoda1 was added [16, 17]. In the signaling pathway studies, isolated ILC3s were pretreated with 30 μm LY294002 (Beyotime) or 10 nm rapamycin (Selleck) 30 min before Yoda1 administration [35, 36]. Following overnight treatment, the culture supernate was collected for ELISA. Cells were pelleted and collected for subsequent experiments.

All the statistical analysis was performed by GraphPad Prism 8.0.2 software (GraphPad Prism). Data were expressed as mean ± SD. The Shapiro-Wilk test was used for normality tests. Statistical significance was determined using unpaired Student’s t test, one-way ANOVA, or two-way ANOVA when data were normally distributed. Otherwise, Kruskal-Wallis test was used for data that were not normally distributed. p < 0.05 was considered statistically significant.

To evaluate the expression of Piezo1 in colitis, we resorted to the publicly available datasets from the Gene Expression Omnibus (GEO) database. The analyzed data showed that PIEZO1 expressions were consistently increased in active UC patients in three independent UC cohort microarray analyses (Fig. 1a–c). Meanwhile, we also found the Piezo1 mRNA in murine experimental colitis increased with disease progression (Fig. 1d). Moreover, we confirmed the gradually elevated expression of Piezo1 in DSS-induced colitis at both transcript (Fig. 1e) and protein levels (Fig. 1f). Together, we conclude that Piezo1 expression was enhanced in activated colitis.

Genetic inactivation of Piezo1 leads to embryonic lethality in mice [37]. GsMTx4 has been verified to be effective at inhibiting the activation of MSICs, including Piezo1, both in vivo and in vitro [38‒40]. Both the GEO dataset (GSE196220) and our RNA-seq data showed that Piezo1 was the most abundantly expressed MSICs among all the major mammalian MSICs analyzed in the murine colon (Fig. 2a; online suppl. Fig. S1). We therefore applied GsMTx4 to the established model of murine colitis induced by DSS to investigate the effect of global Piezo1 inhibition on colitis. Mice were administered with 2.5% DSS for 7 days, and GsMTx4 (10 mg/mL) was intravenously injected at days 2, 4, and 6 after colitis induction (Fig. 2b). Interestingly, GsMTx4 significantly ameliorated the major parameters of the severity of colitis, including weight loss, shortened colon length, and DAI score of DSS-induced colitis mice (Fig. 2c–f). Histological analysis showed less epithelial damage and inflammatory infiltration in GsMTx4-treated mice compared to that with DSS-induced colitis (Fig. 2g, h). These observations indicate that GsMTx4 was obviously effective in mitigating DSS-induced colitis.

To further evaluate the effect of GsMTx4 on colitis, we carried out bulk RNA-seq in the colon tissue of GsMTx4-treated mice versus that of the model group. Hierarchical clustering analysis indicated distinguishable transcription patterns between the DSS and DSS+GsMTx4 groups (Fig. 3a). Treatment with GsMTx4 resulted in 1,685 differentially expressed genes (DEGs, p-adjust <0.05, absolute log 2 [fold change] > 1), including 657 up-regulated and 1,028 down-regulated genes, compared to the DSS group. Among these DEGs, various chemokines (Cxcl1, Cxcl3, Cxcl5, and Cxcl13), cytokines and their receptors (Il6, Il16, Il33, and Il7r), as well as inflammation-related enzymes Ptgs2, were downregulated upon GsMTx4 treatment (Fig. 3b). Expression of Il1b was also decreased in response to GsMTx4 but not to a significant degree. Of note, no significant up-regulation of intestinal barrier-related genes (Cldn1, Ocln, and Tjp1) was detected in GsMTx4-treated mice as compared to the DSS group. We further compared the down-regulated DEGs with the list of “inflammatory response” genes in the GSEA database, and 31 genes overlapped (Fig. 3c). Moreover, GSEA revealed a decrease in IBD gene set in colitis tissue after GsMTx4 treatment (Fig. 3d). We then validated the decrease of the major inflammation-related genes in colitis, Il1b, Il6, Ptgs2, and Il7r, by qRT-PCR (Fig. 3e–h). These RNA-seq data uncover that GsMTx4 regulates the pro-inflammatory transcriptional profile in DSS-induced colitis.

Given the fact that GsMTx4 might also interfere with the activation of other MSICs [41], we next co-stimulated murine colonic LPLs with Yoda1, the Piezo1-specific activator, and GsMTx4 to examine if the immunoregulatory activity of GsMTx4 was due to its inhibition of Piezo1. LPLs isolated from the colon of DSS-induced colitis mice highly express Piezo1 (online suppl. Fig. S2a). Furthermore, immunofluorescence confirmed that mouse colonic leukocytes robustly expressed Piezo1 (Fig. 3i), and flow cytometry revealed the expression of Piezo1 in macrophages, DCs, Th cells, and ILCs (online suppl. Fig. S2f, g). Exposure to Yoda1 caused an increase in the expression of Il1b, Il6, Ptgs2, and Il7r in LPLs from mice with colitis, which genes were previously shown to be suppressed by GsMTx4 in the colon of DSS-induced colitis mice. As expected, GsMTx4 could restore the expression of these genes induced by Yoda1 (online suppl. Fig. S2b–e). These results suggest that GsMTx4 regulated the expression of pro-inflammatory mediators in colitis at least partially through Piezo1.

We next investigated the specific effect of GsMTx4 on intestinal immune cells in colitis. IL-6 is a typical pro-inflammatory cytokine released from innate immune cells, including macrophages and DCs. Excessive production of IL-6 plays a central role in the progression of colitis [42, 43]. Macrophages in the colonic lamina propria (cLP) of GsMTx4-treated mice exhibited reduced intracellular levels of IL-6 compared to those of DSS-induced colitis mice (Fig. 4a). Similarly, IL-6 production by cLP DCs was also diminished by GsMTx4 (Fig. 4b). Consistently, the levels of IL-6 in the colon tissue were decreased in GsMTx4-treated mice than mice with colitis (Fig. 4c). However, we detected no significant alteration in either Th1 or Th17 cell proportion after GsMTx4 administration (Fig. 4d–g) and an even lower frequency of Treg cells (Fig. 4h, i) as compared to acute colitis mice. The latter might be attributed to the mitigation of disease severity by GsMTx4. These results imply that the immunomodulatory role of GsMTx4 might mainly benefit from its effects on innate immune cells.

Our RNA-seq and qRT-PCR data showed a reduced expression of Il7r in colitis treated by GsMTx4 (Fig. 3b, c, h). The IL-7-IL-7R signaling is crucial for the functions of Th cells and ILCs [4, 44]. Since GsMTx4 showed no significant effect on Th cells in DSS-induced colitis, we therefore focused on the role of Piezo1 on ILCs. Previous studies showed ILC3s were the predominant IL7-R⁺ ILCs, and blockade of IL-7R reduced intestinal ILCs and attenuated colitis [4]. Using the publicly available database ImmGen, we noted that Piezo1 was expressed in ILC3s (Fig. 5a). The expression of Piezo1 in ILC3s was further verified by qRT-PCR (Fig. 5b) and flow cytometry (online suppl. Fig. S2f, g). Interestingly, neither Yoda1-induced nor GsMTx4-suppressed Piezo1 activation influenced Il7r expression in ILC3s sorted from cLP of DSS-induced colitis mice (Fig. 5c), which indicated that GsMTx4-caused reduction of Il7r level in colitis was mainly due to the decrease of Il7r-expressing cell number. We next found that Piezo1 activation by Yoda1 increased both the cell counts and the ratio of Ki67⁺ cells of ILC3s, which effect can be diminished by GsMTx4 (Fig. 5d, e). Moreover, ILC3s stimulated by Yoda1 or GsMTx4 showed no significant alteration in cell viability (Fig. 5f). These results suggested that Piezo1 changed ILC3’s number mainly by affecting cell proliferation. Furthermore, we demonstrated that Yoda1 increased the expression and release of IL-17A in ILC3s, which can be suppressed by GsMTx4 (Fig. 5g, h). Consistently, the frequency and IL-17A expression of ILC3s were decreased in GsMTx4-treated mice compared to colitis mice (Fig. 5i–l). All the above data reveal a regulatory role for Piezo1 in ILC3 activation in colitis.

To gain insight into the mechanism involved in Piezo1-regulated ILC3 activation, we further performed enrichment analysis based on our RNA-seq data. Interestingly, as one pathway associated with both Piezo1 function and ILC3 activation [45‒48], the PI3K-Akt pathway was suggested to play a significant role (Fig. 6a). Moreover, GSEA implied that the down-regulated DEGs were significantly enriched in the same pathway (Fig. 6b). To determine the function of PI3K-Akt signaling and its down-stream target mTOR in Piezo1-regulated ILC3 activation, we treated ILC3s with Yoda1, PI3K inhibitor LY294002, and mTOR inhibitor rapamycin. The Yoda1-enhanced increase in the cell counts and the frequency of Ki67⁺ ILC3s were blocked by LY294002 or rapamycin (Fig. 6c–e). Increased IL-17A expression and production in Yoda1-treated ILC3s were also suppressed by LY294002 or rapamycin (Fig. 6f, g). These results suggest that Piezo1-related ILC3 activation was at least partially mediated by the PI3K-Akt signaling pathway.

Alteration in colonic structural and physical properties is a typical signature of colitis and has been proved to correlate with the severity and prognosis of IBD [22‒25]. However, studies considering the effect of biomechanical changes on colitis remain rare, which may be partially due to the limitations of effective interventions on the mechanical features of the intestine. Piezo1 has been delineated to be crucial in mediating mechanosensory and thus affecting numerous physiological and pathological processes [13‒16, 18]. In the present study, we demonstrated an enhanced expression of Piezo1 in DSS-induced colitis, which was consistent with a recent study showing that Piezo1 expression was positively correlated with the disease activity of CD [49]. Several inflammatory stimulating factors have been identified to be associated with the expression of Piezo1, including interferon-gamma (IFNγ), lipopolysaccharide (LPS), and IL-1α [50, 51], the upregulation of which might be the potential inducer of Piezo1 expression during colitis.

Increasing evidence suggests an immunomodulatory role for Piezo1 in a variety of immune-related diseases [16, 18, 29]. A recent study indicated that Yoda1, the Piezo1-specific activator, aggravated DSS-induced colitis, while Piezo1-deficiency in myeloid cells decreased the severity of colitis [52]. However, the effect of global inhibition of Piezo1 on intestinal inflammation has not been systematically investigated. GsMTx4 acts as a specific inhibitor of cation MSICs [26‒28], and a large number of studies have verified its efficiency in suppressing Piezo1 both in vivo and in vitro [38‒40]. The present study showed that GsMTx4 alleviated the disease severity and restrained the pro-inflammatory gene expression in colitis. In addition, we found that Piezo1 was the highest expressed MSICs in both murine colon tissue and LPLs, and GsMTx4 directly inhibited the Yoda1-induced increase of the inflammatory cytokine levels in LPLs. These results suggest that Piezo1 mediates, at least part of, the immunomodulatory effects of GsMTx4 on colitis. However, it is still possible that GsMTx4 concurrently acts on other MSICs to regulate immune responses, which remains to be further elucidated via genetic methods.

During the onset of colitis, innate immune cells, such as macrophages and DCs, infiltrate the intestinal mucosa and produce excessive inflammatory mediators, such as IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α). In turn, these cells present antigens to CD4⁺ T cells, which leads to the impaired balance between pro-inflammatory Th1/17 cells and anti-inflammatory Treg cells [2, 3]. GsMTx4 suppressed IL-6 production in macrophages as well as DCs in the cLP of DSS-treated mice. These results are in agreement with the reports showing that Piezo1-mediated mechanosensory is crucial for the immune responses in macrophages and DCs [16, 17, 52]. We observed no significant alteration in the percentage of Th1 and Th17 cells in colitis after GsMTx4 treatment under the conditions of our study, which is consistent with the previous study showing that Piezo1 does not influence either proliferation or polarization of Th1 and Th17 cells [18]. The fraction of Treg cells during DSS-induced colitis was decreased in GsMTx4-treated mice, which might be due to the milder severity of colitis. However, it was demonstrated that Piezo1 deletion favored Treg cell expansion in a murine model of experimental autoimmune encephalomyelitis [18]. This discrepancy may be explained by the differences between these two organs or diseases. Previous studies showed that Piezo1 activation suppressed the expression of tight junction protein in intestinal epithelial cells [53], but positively regulated Mucin2 expression in intestinal goblet cells [54]. Moreover, Piezo1 has been identified as an RNA sensor of enterochromaffin cells that governs 5-HT production [15]. But in our RNA-seq data, we found no significant alteration in the expression of the corresponding genes. All the above indicate that the beneficial potency of Piezo1 inhibition by GsMTx4 may be mainly attributed to its regulation of innate immune responses.

ILCs are innate counterparts of T cells. Although lacking antigen-specific receptors, ILCs are able to respond rapidly to external stimuli with their excellent perception of tissue microenvironment disturbance [55]. While ILCs are continuously exposed to the dynamically changing mechanical microenvironment of the intestine, how ILC functions are regulated by mechanical cues remains elusive. ILC3s are the dominant ILC population in the intestine [5]. The present study, for the first time to our knowledge, demonstrated the expression of Piezo1 in ILC3s. We found that Piezo1 positively regulated the proliferation and production of the pro-inflammatory cytokine IL-17A in ILC3s. These results suggest that Piezo1 may be a potential regulator of ILC3 activation. ILC3s emerge as a double-edged sword in IBD. On the one hand, ILC3s maintain gut mucosal homeostasis with IL-22 and granulocyte macrophage colony-stimulating factor in the physiological state [6, 7]. On the other hand, accumulating evidence indicates that ILC3s produce IL-17A and IFNγ and contribute to the pathogenesis of colitis [4, 5]. We found that Piezo1 inhibition via GsMTx4 decreased the number and proportion of ILC3s and suppressed their IL-17A production in DSS-induced colitis mice. Our results suggest that Piezo1 can directly regulate the activation of ILC3s. Given the complex environment that ILC3s encounter in vivo [55], further exploration is required to fully understand the effect of Piezo1 in combination with other microenvironmental stimuli on ILC3 functions during colitis.

Our RNA-seq data revealed alterations of several signaling pathways in colitis after GsMTx4 treatment, among which PI3K-Akt is one of the most significant. PI3K-Akt and their downstream target mTOR play an essential role in sensing and integrating microenvironmental cues to instruct subsequent immune responses [56, 57]. It has been confirmed that the PI3K-Akt-mTOR pathway sustained the proliferation and function of ILC3s, whereas inhibition of this signaling pathway in ILC3s suppressed their expansion and cytokine secretion [6, 48]. Increasing evidence supports the PI3K-Akt-mTOR pathway as one of the downstream mechanisms of Piezo1 activation [45‒47]. In line with these studies, we found that inhibition of PI3K or mTOR suppressed Yoda1-induced proliferation and IL-17A production in ILC3s. These results suggest that Piezo1 regulates ILC3 proliferation and its effector function at least partially through the PI3K-Akt-mTOR signaling pathway. As a cationic ion channel, Piezo1 mainly allows the entry of Ca2⁺, which is involved in multiple steps of the PI3K-Akt signaling pathway, such as phosphorylation of both PI3K and Akt [45, 58‒60]. Future research is required to determine whether the Piezo1 activation of PI3K-Akt-mTOR depends on the Ca2⁺ signal in ILC3s.

The present study is subject to certain limitations. First, although few adverse effects of GsMTx4 have been reported [61], its selectivity remains to be further verified due to its various roles in different biological processes. Second, besides direct effects, GsMTx4 may regulate intestinal immunity by influencing other cells. For instance, since Piezo1 has been reported to regulate cytokine release from astrocytes [30, 39, 62], it may also affect enteric glia, which are known to interact with immune cells in colitis [63, 64]. Therefore, future gene manipulation experiments are still warranted to elucidate the exact contribution of Piezo1 in specific cell types to the pathogenesis of colitis. Nevertheless, the present study provides a global view of intestinal immunity changes after GsMTx4 intervention in colitis and identifies a novel role of Piezo1 in regulating ILC3 activation (Fig. 7). More specific approaches targeting Piezo1 may become promising strategies to modulate intestinal immunity in IBD.

The animal study protocol was reviewed and approved by the Ethical and Institutional Animal Care and Use Committee of Qilu Hospital of Shandong University (DWLL-2021-007).

The authors have no conflicts of interest to declare.

This work was supported by the National Natural Science Foundation of China [Grant No. 82070551, 82270570]; the Taishan Scholars Program of Shandong Province; and the Natural Science Youth Foundation of Shandong Province [Grant No. ZR2020QH030]. The funding sources had no role in the study design; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to submit it for publication.

C.L. designed and performed the research, analyzed data, and wrote the manuscript; Y.X. made important contributions to the performance of experiments; S.F., B.F., and L.X. provided technical advice during experiments; F.M. assisted in the analysis of RNA-seq data; L.L. supervised the experiment and revised the manuscript; and X.Z. supervised the project and provided financial support.

The RNA-seq dataset generated in this study has been deposited in the GEO database under accession number GSE215772. All relevant data supporting the key findings of this study are available within the article and its supplementary material, or from the corresponding author upon reasonable request.