From scRNA-seq to therapeutic targets: Unveiling the impact of activated mast cells on intestinal dysfunction in acute pancreatitis

Abstract

Wei et al reported a comprehensive single-cell transcriptomic analysis of the small intestine during early acute pancreatitis (AP) and identified activated mast cells and their secretion of CCL5 as pivotal factors driving gut barrier dysfunction. By integrating scRNA-seq with in vitro and in vivo functional assays, this study advances our understanding of the cellular and molecular events underlying AP-associated intestinal injury. In this commentary, I highlight the methodological innovations employed in the study, contextualize its findings in the literature, and propose directions for future research. As an avid researcher in single-cell sequencing, I approached this letter with a spirit of academic inquiry and welcome any further discussion or corrections that may enhance my understanding.

Key Words: Acute pancreatitis; Single-cell RNA sequencing; Mast cells; CCL5; Intestinal barrier

Core Tip: Applying advanced single-cell transcriptomics, Wei et al’s study elucidates the cellular heterogeneity of the intestine during early acute pancreatitis (AP) and identifies activated mast cells and their CCL5 secretion which are key contributors to gut barrier disruption. These findings provide novel insights into AP pathophysiology and highlight potential avenues for targeted therapeutic interventions. Moreover, the study underscores the value of integrative, multidimensional approaches in deciphering complex inflammatory responses.

TO THE EDITOR

I read with great interest the article by Wei et al[1] entitled “scRNA-seq of the intestine reveals the key role of mast cells in early gut dysfunction associated with acute pancreatitis” published in the World Journal of Gastroenterology. The authors deserve commendation for their innovative application of single-cell RNA sequencing (scRNA-seq) integrated with rigorous functional validations to elucidate the complex cellular dynamics of the small intestine during early acute pancreatitis (AP). AP has a worldwide incidence of approximately 34 cases per 100000 person-years. It can lead to severe systemic complications, including sepsis and multiorgan failure, primarily through the occurrence of intestinal barrier dysfunction[2-4]. Recent single-cell transcriptomic studies have transformed our understanding of the cellular heterogeneity of the gut in both healthy and diseased individuals[5,6] and of the active involvement of mast cells (MCs)—traditionally linked to allergic responses[7,8]—in causing inflammatory damage in the intestine. Their findings, particularly the activation of MCs and their secretion of CCL5, offer critical insights into the mechanisms underlying gut barrier dysfunction in AP. As a researcher with a strong interest in single-cell sequencing, I wrote this letter aiming to deepen my understanding of the methodologies and interpretations presented in the study. I hope that my comments will contribute constructively to the ongoing discourse, and I welcome any further discussion or suggestions.

AP is a prevalent and potentially severe abdominal condition that can result in life-threatening complications[9,10]. Disruption of the intestinal barrier in AP markedly increases the risk of bacterial translocation, systemic inflammation, sepsis, and necrotizing pancreatitis[2-4]. Despite extensive research on epithelial damage and tight junction alterations, the early molecular events driving gut barrier dysfunction in AP remain poorly characterized. Recent advancements in single-cell transcriptomics have transformed our understanding of tissue heterogeneity[11]. Landmark studies have elucidated the complex cellular landscape of the intestine in both homeostatic and diseased states[5,6]. However, a critical knowledge gap persists regarding the role of immune cells, particularly MCs, in the early stage of AP. Traditionally associated with allergic responses, MCs are increasingly recognized for their essential functions in innate immunity and tissue homeostasis[12]. Their capacity to secrete inflammatory mediators, such as CCL5 (flow cytometry results in the study by Wei et al[1] showed that the proportion of CCL5⁺ MCs increased from 12.6% in the controls to 24.9% and 28.8% in the AP1 and AP2 test samples, respectively, as shown in figure 9C of the published article), suggests a pivotal role in recruiting neutrophils and macrophages, potentially exacerbating gut barrier dysfunction (figure 8 in the published article). Within this context, the study by Wei et al. is both timely and highly significant.

Strengths and methodological innovations

Wei et al[1] exhibited exceptional methodological precision by integrating advanced scRNA-seq with rigorous functional validation. Their study not only maps the intricate cellular landscape but also elucidates key inflammatory mechanisms underpinning AP. The principal findings are summarized in Table 1, with the key aspects detailed as follows.

Table 1 Key outcome summary.

Key outcome	Observation in AP small intestine	Implication
Intestinal cellular heterogeneity	scRNA-seq of the small intestine identified 17 distinct cell clusters from > 33000 cells across the control, AP1, and AP2 samples	Provides a high-resolution atlas of the small-intestinal cell composition and reveals dynamic shifts during early AP
Intestinal MC activation	scRNA-seq showed an apparent reduction of MC recovery but immunofluorescence staining (c-Kit and toluidine blue) confirmed stable MC counts with degranulation	Highlights activation-induced MC fragility during dissociation and underscores MC mediator release as a driver of barrier dysfunction
CCL5 upregulation in intestinal MCs	scRNA-seq and flow cytometry demonstrated significant CCL5 overexpression in MCs in the AP1 and AP2 groups	Identifies CCL5 as a key chemokine, mediating neutrophil and macrophage recruitment to the gut barrier
Tight junction disruption in small intestine	IHC revealed decreased expression of ZO-1 and occludin in AP1 and AP2 intestinal epithelia	Confirms compromised epithelial barrier integrity at early stages of AP
Programmed cell death in enterocytes	IHC showed increase in cleaved caspase-3 (apoptosis) and p-MLKL (necroptosis) levels in intestinal enterocytes from the control to AP1 to AP2 group	Demonstrates enterocyte loss via multiple cell-death pathways contributing to barrier breakdown

AP: Acute pancreatitis; MC: Mast cell; IHC: Immunohistochemistry.

Methodological precision: Wei et al[1] employed a robust droplet-based scRNA-seq strategy to profile 33232 cells from the small intestine. The high-resolution cellular atlas so-obtained revealed a notable drop in the proportion of MCs from 9.07% of the total cell cluster in the control group to 2.2% and 2.37% in the AP1 and AP2 groups, respectively (figure 2B in the original article). However, a relatively stable MC density alongside clear morphological signs of MC activation and degranulation were observed in the AP groups upon immunofluorescence staining of c-Kit using toluidine blue (figure 7A in the original article). In light of this, the authors reasonably hypothesized that MCs, upon activation, exhibit increased fragility and are less likely to survive enzymatic dissociation, thereby leading to their reduced representation in the scRNA-seq data. Flow cytometric analysis further quantified this phenomenon by showing that CCL5⁺ MCs increased in number from 12.6% in the control group to 24.9% and 28.8% in the AP1 and AP2 groups, respectively (figure 9C in the original article), thereby substantiating the hypothesis of a functional alteration of MCs due to activation rather than a quantitative loss.

Temporal dynamics and functional validation: Examining two early-AP murine models (AP1: 4 caerulein injections, sacrifice carried out 2 hours after the last injection; AP2: 8 caerulein injections, sacrifice carried out 4 hours after the last injection), the study delineated dynamic cellular responses. Immunofluorescence staining for ZO-1 showed a 45%-60% reduction in junctional staining intensity in the AP1 group, with further decline in the AP2 group (figure 3A in the published article), while immunohistochemistry (IHC) for cleaved caspase-3 and p-MLKL revealed an increase in apoptotic/necroptotic enterocytes in the AP groups compared with the control group (figure 3F in the original article). These results thus provide compelling evidence of barrier disruption and programmed cell death, further strengthening the inferences of the transcriptomic studies and validating the proposed mechanisms.

Clinical and pathophysiological implications: The identification of CCL5 secreted by activated MCs as a driver of neutrophil and macrophage infiltration marks a significant advancement in our understanding of AP-associated intestinal injury. More importantly, this mechanistic insight reveals a novel therapeutic target, CCL5, and underscores intestinal barrier integrity preservation as an important strategic intervention for mitigating AP complications.

Therapeutic context (targeting the CCL5–CCR5 axis): In addition to its mechanistic importance, CCL5 is a clinically actionable target: Maraviroc, an Food and Drug Administration-approved CCR5 antagonist used for human immunodeficiency virus treatment[13], has shown anti-inflammatory effects in rheumatoid arthritis and graft-versus-host disease patients[14-16]; Cenicriviroc, a dual CCR2/CCR5 inhibitor in phase II trials for nonalcoholic steatohepatitis, has been found to significantly reduce hepatic inflammation[17]; and preclinical studies of Met-RANTES (a modified CCL5 analog) and anti-CCL5 monoclonal antibodies have demonstrated their therapeutic benefit in experimental colitis and atherosclerosis models[18,19]. Collectively, these examples highlight the feasibility of pharmacologically modulating the CCL5–CCR5 axis and underscore its translatability to intestinal barrier integrity preservation in AP.

Prior single-cell transcriptomic studies of the intestine have primarily focused on steady-state conditions[20,21] or chronic inflammatory diseases such as ulcerative colitis[22], while prior investigations into intestinal barrier dysfunction in AP have relied on bulk RNA-seq or histological analyses with limited cell-type resolution[23,24]. Wei et al[1] are the first to apply high-throughput droplet-based scRNA-seq to an early-AP model, profiling over 33000 cells and integrating in vitro and in vivo functional assays. This multi-dimensional approach provides an unprecedented, cellular-level characterization of early gut injury in AP, which highlights its unique contribution to both basic and translational gastroenterology.

Recommendations and future directions

Although the study presents a thorough investigation, several aspects warrant further exploration. I propose the following key directions for future research, as summarized in Table 2. This table delineates pivotal areas requiring further inquiry, addressing existing gaps while aligning with advancing research in inflammation and gut barrier integrity. These recommendations provide a strategic framework for translating current findings into clinical applications.

Table 2 Recommendations for future research.

Recommendation	Specific approach (key points)
Longitudinal single-cell profiling	(1) Time points (post AP induction): Early (2 hours, 6 hours, and 24 hours), mid (3 days and 7 days) and late (14 days and 28 days); and (2) Assays: FITC-dextran permeability and serum LPS (barrier); histological score and IHC/FACS for neutrophil/macrophage infiltration along with IL-6/TGF-β (inflammation); Masson’s staining and Col1a1/Col3a1 mRNA (fibrosis); and 16S rRNA sequencing (microbiome recovery)
MC activation mechanisms	(1) Techniques: scATAC-seq + phospho-proteomics on sorted MCs; and (2) Targets: PAR2/tryptase, the FcεRI-independent pathway, and NF-κB/AP-1
Human spatial validation	(1) Samples: Ileal/jejunal biopsies (Ranson < 3 vs ≥ 3); (2) Platform: 10 × Visium or GeoMX DSP; and (3) Readouts: MC density, CCL5, and immune infiltrates
Preclinical intervention studies	(1) Drugs: Cromolyn sodium and Maraviroc; and (2) Endpoints: Gut histology, 16S rRNA, cytokines, and 72 hours survival

AP: Acute pancreatitis; MC: Mast cell; LPS: Lipopolysaccharide; IHC: Immunohistochemistry; FITC: Fluorescein isothiocyanate; FACS: Fluorescence-activated cell sorting; IL-6: Interleukin 6; TGF-β: Tumor growth factor β.

Longitudinal single-cell and barrier function profiling: To define both the acute and long-term trajectories of intestinal injury and recovery in AP, we propose serial ScRNA-seq at early (2 hours, 6 hours, and 24 hours), middle (3 days and 7 days), and late (14 days and 28 days) time points after caerulein induction, each paired with functional assays: fluorescein isothiocyanate-dextran permeability and serum lipopolysaccharide (LPS) assays to assess barrier integrity; histopathological scoring, IHC/fluorescence-activated cell sorting quantification of neutrophil and macrophage infiltration, and measurements of interleukin 6 and tumor growth factor β to evaluate inflammation; Masson’s trichrome staining and quantitative analysis of Col1a1/Col3a1 mRNA to evaluate the extent of fibrosis; and 16S rRNA sequencing to track microbiome restoration. This integrated approach will help map cellular composition, barrier function, inflammatory persistence, fibrotic remodeling, and microbial community dynamics over time.

Mechanistic elucidation of MC activation: To understand how MCs become activated in early AP, we recommend isolating intestinal MCs at key time points and applying single-cell ATAC-seq alongside phospho-proteomic profiling. Focusing on PAR2/tryptase-mediated signaling, FcεRI-independent activation, and downstream transcription factors such as NF-κB and AP-1 will help identify the upstream regulators driving CCL5 expression and MC degranulation, thereby illuminating precise molecular targets for intervention.

Human cohort validation with spatial transcriptomics: Although, as demonstrated in the study by Wei et al[1], it is possible to gain mechanistic insights into early intestinal responses during AP using murine caerulein-induced AP models, species-specific differences in MC frequency, activation thresholds, and CCL5 receptor expression may limit their direct extrapolation to humans pathophysiology, thus necessitating spatially resolved validation. We suggest obtaining ileal or jejunal biopsies from AP patients along with severity stratification (Ranson score < 3 vs ≥ 3) within 48 hours of hospital admission. Using platforms such as 10 × Visium or NanoString GeoMX DSP, investigators can map mast-cell density, CCL5 expression, and immune cell infiltration such as plasma zonulin or LPS.

Preclinical evaluation of targeted interventions: Finally, we recommend preclinical trials to assess therapeutic strategies. Administering Cromolyn sodium and/or the CCR5 antagonist Maraviroc in murine AP models and using histological scoring of gut injury, bacterial 16S rRNA translocation, cytokine profiling, and survival over 72 hours as the endpoints will help determine whether MC stabilization and CCL5/CCR5 axis blockade can effectively preserve intestinal barrier integrity and improve outcomes, laying the groundwork for future clinical translation.

Conclusion and broader implications

Wei et al[1] have constructed a high-resolution single-cell atlas of the early-AP intestine, defining 17 cell clusters, and identified a novel MC-CCL5 axis that drives neutrophil and macrophage infiltration, bringing about barrier breakdown. These results align with those from mammalian colitis models in which MC-derived CCL5 was found to exacerbate mucosal injury[25] and with results of sepsis studies showing that CCL5 blockade preserves tight junction integrity[26,27]. Collectively, these data highlight MCs as central sentinels in acute intestinal inflammation.

Scientific contribution: This study generates a high-resolution cellular atlas of the intestinal microenvironment in AP and identifies MC-derived CCL5 as a critical driver of gut barrier dysfunction. These findings not only refine our understanding of AP-related inflammatory cascades but also resonate with recent advances in single-cell transcriptomics across diverse tissue systems.

Clinical implications: Maintaining intestinal barrier integrity is essential for reducing severe AP-associated complications. The identification of CCL5 as a viable therapeutic target presents promising intervention strategies. Targeting MCs stabilization and blocking CCL5-mediated pathways may attenuate inflammatory cell infiltration and preserve gut homeostasis.

Scholarly engagement and future directions: My deep interest in single-cell sequencing research inspired this commentary, which I approached as both an academic exercise and a contributor to the field. I welcome constructive criticism and scholarly dialogue, as such interactions are invaluable for refining my research perspectives.

In conclusion, Wei et al[1] applied high-throughput droplet-based scRNA-seq of early-AP intestinal samples, detailing the cellular atlas of > 33000 cells and uncovering 17 distinct cell types. Crucially, they showed that MCs are promptly activated and they markedly upregulate CCL5, which in turn brings about targeted neutrophil and macrophage infiltration and drives barrier breakdown. This discovery of an MC–CCL5 axis bridges a key gap in AP pathophysiology and positions both CCL5 and MC stabilization as promising therapeutic targets. We advocate for further research to expand these findings and facilitate their translation into targeted therapeutic strategies. I look forward to engaging in ongoing discussions and fostering collaborative scientific exchanges. We advocate for further research to expand these findings and facilitate their translation into targeted therapeutic strategies. I look forward to engaging in ongoing discussions and fostering collaborative scientific exchanges.