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Comprehensive single-cell RNA-sequencing study of Tollip deficiency effect in IL-13-stimulated human airway epithelial cells

BMC Research Notes volume 18, Article number: 194 (2025) Cite this article

Abstract

Objective

Toll-interacting protein (Tollip) suppresses excessive pro-inflammatory signaling, but its function in airway epithelial responses to IL-13, a key mediator in allergic diseases, remains unclear. This study investigates Tollip knockdown (TKD) effects in primary human airway epithelial cells using single-cell RNA sequencing, providing the first single-cell analysis of TKD and the first exploring its interaction with IL-13.

Results

IL-13 treatment upregulated key genes, including SPDEF, MUC5AC, POSTN, ALOX15, and CCL26, confirming IL-13’s effects and validating our methods. IL-13 reduced TNF-α signaling and epithelial-mesenchymal transition in certain cell types, suggesting a dual role in promoting type 2 inflammation while suppressing Th1-driven inflammation. Tollip deficiency alone significantly amplified TNF-α signaling and inflammatory pathways in goblet, club, and suprabasal cells. Comparisons between TKDIL13 vs IL13 and TKD vs CTR revealed that IL-13 does not substantially alter Tollip deficiency response in most cell types, reinforcing findings in TKD vs CTR. Tollip deficiency alters the response to IL-13 in a cell-type-specific manner, strongly downregulating TNF-α signaling in goblet cells but only weakly in basal and club cells. Tollip deficiency enhances IL-13’s suppression of Th1 inflammatory responses in goblet cells. These novel insights in Tollip-IL-13 interactions offer potential therapeutic targets for asthma and related diseases.

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Introduction

Toll-interacting protein (Tollip) is a ubiquitously expressed adaptor protein crucial to innate immunity [1, 2]. Tollip is a negative regulator of toll-like receptor signaling to prevent excessive inflammation [1, 3,4,5]. Reduced Tollip expression is linked to various diseases, including asthma, tuberculosis, idiopathic pulmonary fibrosis, atopic dermatitis, and Alzheimer’s [6,7,8,9,10]. Our prior work demonstrated that the Tollip AG/GG SNP rs5743899 is associated with poor airflow in asthma patients [11]. However, cell type-specific effects of Tollip are yet unclear.

Interleukin 13 (IL-13) is a type 2 cytokine [12], regulating eosinophilic inflammation and enhancing airway hyperresponsiveness [13,14,15]. IL-13 is central to chronic respiratory diseases including asthma and chronic obstructive pulmonary disease (COPD) [16, 17].

This report aimed to determine the individual and combined effects of Tollip knockdown and IL-13 on human airway epithelial cells at the single-cell level. This is the first comprehensive single-cell RNA sequencing (scRNA-seq) study of Tollip-deficient airway epithelial cells and cell type-specific response, and the first exploring the co-influence of Tollip deficiency and IL-13.

Main text

Methods

Primary human tracheobronchial epithelial cells were obtained from a healthy adult donor (National Jewish Health IRB approval #HS-3209). Tollip knockdown was achieved in submerged culture using CRISPR/Cas9 [6]. Verified Tollip knockdown cells were seeded onto collagen-coated Transwell inserts (Corning) in PneumaCult-ALI Medium (STEMCELL Technologies) at air–liquid interface for 21 days to induce mucociliary differentiation. Cells were treated with the following conditions for 3 days:

  1. 1.

    CTR (Control, Tollip-sufficient)

  2. 2.

    IL13 (10 ng/mL, Tollip-sufficient + IL-13)

  3. 3.

    TKD (Tollip-knockdown, no IL-13)

  4. 4.

    TKDIL13 (Tollip-knockdown + IL-13)

Cells were dissociated, loaded into a Chromium Controller (10× Genomics), and processed with Next GEM 3ʹ v3.1 reagents following manufacturer’s protocols. Libraries were sequenced on an Illumina platform (~ 350 million read pairs per sample).

Reads were aligned to GRCh38-2024-A using Cell Ranger (v8.0.0). Quality filters included > 500 genes/cell, < 20% mitochondrial reads, and log10 (UMI counts) within median ± 5 × MAD [18]. CellBender removed ambient RNA [19], and Scrublet filtered doublets [20]. We retained 3114 (CTR), 2148 (IL13), 2447 (TKD), and 1898 (TKDIL13) high-quality cells.

The dataset was aggregated using Cell Ranger’s aggr function to normalize sequencing depth across samples, then processed with the reanalyze function, focusing on barcodes that passed filtering.

Cell types were assigned using Azimuth label transfer against a Human Lung Cell Atlas reference [21,22,23], comprising 584,944 cells from 107 individuals [24], ensuring robust epithelial annotation. Cell-type markers were displayed in Additional file 1 and our previous publication [25].

Harmony was used to integrate single-cell data [26], aligning similar cell types across conditions. Samples were normalized using sctransform v2 [27], merged, and analyzed with 20 principal components. UMAP (reduction = “harmony,” dims = 1:20) and Louvain clustering (resolution 0.5) were performed in Seurat 5 [23] for visualization.

Differential expression for each cell type between conditions was calculated using Loupe Browser 8.0.0, which applies the sSeq test or edgeR for large UMI counts [28]. Pathway analysis used gene set enrichment analysis (GSEA) with FGSEA [29, 30] and hallmark gene sets from MSigDB [31, 32].

Results and discussion

UMAP-based clustering (Fig. 1) identified key airway epithelial cell types: basal, suprabasal, club, goblet, multiciliated, and SMG duct cells. Tollip knockdown (TKD, TKDIL13) consistently reduced multiciliated cells and increased goblet cells compared to CTR and IL13. This finding suggests that Tollip plays a critical role in multiciliated cell development or maintenance, with their loss potentially impairing mucociliary clearance and increasing infection risk [33, 34]. Coupled with heightened inflammatory signaling, this underscores Tollip’s multifaceted role in maintaining airway homeostasis. Cell counts are in Additional file 2, while genes contributing to the significant enrichment of each pathway between conditions are in Additional file 3.

Fig. 1
figure 1

UMAP illustrating the distribution of individual cells across 8 major cell types in human airway epithelial cells cultured at air–liquid interface under each of the 4 treatment conditions

Full size image

IL-13 vs CTR: validation of IL-13-mediated responses

Comparing IL13 vs CTR revealed how IL-13 reshapes the Tollip-sufficient epithelium. SPDEF and MUC5AC were significantly upregulated in goblet and club cells (Additional file 4). Activated by IL-13 via STAT6, SPDEF induces mucus production and interacts with TTF-1 to exacerbate goblet cell hyperplasia, contributing to airflow obstruction [35]. Targeting the IL-13–SPDEF pathway with IL-13/IL-4Rα blockers can effectively reduce goblet cell hyperplasia and mucus hypersecretion [36]. Elevated MUC5AC, a key mucin, impairs mucociliary clearance and fosters airway obstruction, hallmarks of chronic respiratory diseases like asthma and COPD [37]. Therapeutic monoclonal antibodies against IL-4/IL-13 have been shown to decrease mucus production, highlighting MUC5AC as a principal mucus-obstructive component of asthma and chronic bronchitis [38].

IL-13 elevates POSTN in basal, club, and suprabasal cells (Additional file 5), supporting its involvement in extracellular matrix deposition and airway remodeling such as fibrosis and stiffening in asthma [39, 40]. Across all cell types, IL-13, via JAK–STAT pathways, upregulates ALOX15, which through converting arachidonic acid into 15-HETE, promotes eosinophilic inflammation and exacerbates airway hyperresponsiveness in asthma [41]. ALOX15 represents another potential target for therapies to reduce airway eosinophilic inflammation. We observed that basal cells also increased POSTN and ALOX15 under IL-13 stimulation, implicating the role of differentiating cells in tissue remodeling. While secretory cells respond to IL-13 by producing mucus and chemokines, basal cells respond by secreting factors that prime the airway for structural changes, suggesting IL-13 drives mucus hypersecretion and airway remodeling through different cell populations.

Similarly, IL13-induced CCL26 drives eosinophil recruitment [42], which correlates with more frequent asthma exacerbations. Given the effectiveness of IL-4/13 blockers [38], targeting CCL26 and its receptor CCR3 offers a promising therapeutic strategy [42].

Together, induction of these genes not only confirms IL-13’s known regulatory effects and validates our methods and results but also provides cell-type-specific insights.

TNF-α signaling

GSEA (Fig. 2a) revealed that IL-13 suppressed TNF-α–NF-κB signaling in all cell types, most notably club cells. Key downregulated genes include TLR2, TNFAIP3, CCL20, and IL-18, which supports previous studies indicating IL-13’s inhibition of Th1 cytokine and TNF-α transcription [43,44,45]. Mechanistically, via the phosphoinositide 3-kinase pathway, IL-13 increases IRAK-M, subsequently suppressing TLR2 signaling, NF-κB activation, and innate immunity [46]. Overall, this suggests a dual role of IL-13 in promoting type 2 inflammation (SPDEF, MUC5AC, etc.), while suppressing type 1 inflammation, a shift that promotes Th2-dominant conditions, characteristics of asthma.

Fig. 2
figure 2

A Hallmark (H) pathways significantly downregulated by IL-13 treatment (IL13) compared to control (CTR). The size of the dot represents the Normalized Enrichment Score (NES), indicating the strength of pathway enrichment in a given cell type, with larger dots reflecting stronger enrichment. The color of the dot represents p-adjusted value, indicating the statistical significance of the pathway. B Pathways significantly downregulated in Tollip knockout + IL-13-treated (TKDIL13) compared to Tollip knockout-only (TKD)

Full size image
Inflammatory response

IL-13 also reduced the overall inflammatory response in suprabasal, basal, club, and multiciliated cells. While many previous studies on IL-13 have focused on its activation of Th2 allergic inflammation [47,48,49]. our research supports IL-13’s emerging role in suppressing Th1 inflammation [45]. Among our significant inflammatory response genes, most are more closely associated with a Th1 immune response than a Th2 response, including CCL20, CXCL8, IL-18, IL-6, and TLR2, all downregulated.

Epithelial-mesenchymal transition (EMT)

IL-13 suppressed the EMT pathway in club and multiciliated cells. This may be attributed to IL-13’s known role in inducing goblet cell metaplasia from club and multiciliated cells [50, 51]. EMT is typically associated with the transition of epithelial cells to more migratory phenotypes [52], contrary to the stationary phenotype of goblet cells. Notable downregulated genes related to the EMT pathway include IL-6, CXCL6, CXCL8, IGFBP3, LAMC1, and LAMC2.

The suppression of EMT suggests a potential protective effect against airway remodeling processes typically associated with fibrosis. This finding, contrasted with the increased expression of mucins and Th2 inflammation-related genes, adds to the growing evidence that IL-13 has both anti-inflammatory and pro-inflammatory effects.

Effect of Tollip deficiency in the absence of IL-13

TNF-α signaling

In TKD cells, the TNF-α signaling pathway is upregulated across all cell types except multiciliated cells (Fig. 3a). Goblet and club cells exhibited the greatest change in TNF-α-signaling-related expression, with significant upregulation of TNF, TNF-αIP2, NFKB1, TRAF1, and RIPK2. This upregulation corroborates Tollip’s known role in inhibiting TNF-α signaling, as its knockdown reduces this suppression [2, 53].

Fig. 3
figure 3

A Pathways significantly upregulated due to Tollip knockout (TKD) compared to control (CTR). B Pathways significantly upregulated in Tollip knockout + IL-13-treated (TKDIL13) compared to IL-13 treatment-only (IL13)

Full size image
Inflammatory response

The inflammatory response pathway in TKD cells was upregulated across all cell types except multiciliated cells. Once again, goblet and club cells exhibited the greatest change in inflammatory response gene expression. Our previous work found that Tollip inhibits neutrophilic inflammation in response to rhinovirus infection [6]. IL1R1, IRAK2, NFKB1, TNFRSF1B, TLR3, and TLR1, all associated with neutrophilic inflammation, were significantly upregulated in TKD cells, corroborating our previous findings. Multiciliated cells, however, showed no significant change.

IL6-JAK-STAT3 signaling

In TKD cells, the IL6-JAK-STAT3 signaling pathway is upregulated across all cell types, excluding multiciliated cells. Goblet, club, suprabasal, basal, and SMG duct cells showed significant upregulation of related genes, including IL1B, HMOX1, IL6ST, STAT1, and IRF1. This corroborates our earlier research that Tollip deficiency promotes STAT3 activation [54] and that lower Tollip expression is associated with higher IL-6 protein levels [11].

Effect of Tollip deficiency in the presence of IL-13

The TKDIL13 vs TKD results compared to those of IL13 vs CTR show how Tollip deficiency modifies response to IL-13 treatment (Fig. 2b).

TNF-α signaling

Similar to the IL13 vs CTR comparison results, TNF-α signaling via NF-κB is downregulated in TKDIL13 vs TKD across goblet, suprabasal, and multiciliated cells, highlighting IL-13’s role in suppressing pro-inflammatory signaling, even in the absence of Tollip. However, some differences were observed. Goblet cells exhibited a notably stronger downregulation. SMG duct cells, omitted from the IL13 vs CTR comparison due to low cell count, also displayed downregulation of TNF-α signaling in TKDIL13 vs TKD. In contrast, basal and club cells no longer showed any significant enrichment of TNF-α signaling, suggesting that Tollip’s interaction with IL-13 is cell-type-specific.

Inflammatory response

The expression of the inflammatory response pathway differs significantly between IL13 vs CTR and TKDIL13 vs TKD, suggesting that the addition of TKD modifies the response to IL-13 across various cell types. Suprabasal, basal, club, and multiciliated cells all no longer showed significant expression changes in the inflammatory response pathway in the latter comparison. Goblet cells, meanwhile, had many more downregulated genes related to Th1 inflammatory response in TKDIL13 vs TKD compared to IL13 vs CTR, including CSF3, TNFSF15, IL1B, TLR2, CXCL10, and IRF1.

Epithelial–mesenchymal transition

Changes in EMT in multiciliated cells were largely similar between the two comparisons. Notable downregulated genes included SNAI1, TWIST1, SERPINE1, CDH2, and CCL20.

Effect of IL-13 treatment on Tollip-deficient cells

TKDIL13 vs IL13 and TKD vs CTR results were surprisingly similar, suggesting IL-13 does not induce significant changes in response to Tollip deficiency. This similarity validates findings from TKD vs. CTR and elucidates IL-13’s role in modulating the response to Tollip deficiency. Pathway differences were analyzed between TKDIL13 vs. IL13 (Fig. 3b) and TKD vs CTR. In TKDIL13 vs. IL13, suprabasal cells showed the same upregulation in all pathways discussed in TKD vs. CTR. Basal cells also exhibited similar results. However, some differences were observed. Due to low club cell count in TKDIL13, their strong pathway upregulation in TKD vs. CTR was absent in TKDIL13 vs. IL13. Additionally, multiciliated cells experienced upregulation of inflammatory response and TNF-α signaling in TKDIL13 vs IL13, though not in TKD vs CTR. Meanwhile, goblet cells showed slightly weaker upregulation for both pathways in TKDIL13 vs IL13.

Integrated view

Our study provides a detailed analysis of the effects of Tollip deficiency and IL-13 treatment on human airway epithelial cells at the single-cell level. The findings highlight Tollip’s critical role in regulating pro-inflammatory pathways and maintaining multiciliated cell populations while also revealing IL-13’s dual function in activating Th2 inflammation and concurrently suppressing neutrophilic inflammation and EMT-associated remodeling. The complex interactions between Tollip deficiency and IL-13 suggest that therapeutic strategies targeting these pathways will need to be tailored to specific cell types and disease contexts to achieve optimal outcomes.

Future research should focus on elucidating the molecular mechanisms by which Tollip deficiency leads to the upregulation of TNF-α signaling and other key pro-inflammatory pathways, as well as exploring the potential therapeutic benefits of targeting these pathways in asthmatic individuals with reduced Tollip function. Additionally, further studies are needed to investigate broader physiological implications of the observed cell-type-specific responses to Tollip deficiency and IL-13 treatment, particularly in the context of airway remodeling and chronic inflammation.

Limitations

While comparisons between TKDIL13 vs IL13 and TKD vs CTR validate the effect of Tollip knockdown, the relatively low cell count (2000–3000 cells/condition), single-patient data, and in-vitro design limit statistical power and generalizability. Future mechanistic studies with larger sample sizes and in-vivo models could further confirm and expand these findings.

Availability of data and materials

The dataset supporting the conclusions of this article is available in the Gene Expression Omnibus database under accession number GSE279044.

Abbreviations

CTR:

Control sample

TKD:

Tollip knockout-only sample

IL13:

IL-13 treatment-only sample

TKDIL13:

Tollip knockout and IL-13 treated sample

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Funding

This research was funded by the National Institutes of Health (NIH) U19AI125357, R01AI150082, R01AI152504 and R01AI161296. The NIH does not have a role in any of the stages of this work.

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Authors and Affiliations

  1. Singulomics Corporation, Bronx, NY, USA

    Grace Yihua Lee

  2. Department of Medicine, National Jewish Health, Denver, CO, USA

    Niccolette Schaunaman, Hamid Reza Nouri & Hong Wei Chu

  3. Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Monica Kraft

Authors
  1. Grace Yihua Lee
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  2. Niccolette Schaunaman
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  3. Hamid Reza Nouri
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  4. Monica Kraft
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  5. Hong Wei Chu
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Contributions

G.Y.L. conducted the data analysis and interpretation and wrote the manuscript. N.S. and H.R.N. performed the cell culture experiment. M.K. and H.W.C. contributed to the experimental design and manuscript editing.

Corresponding author

Correspondence to Hong Wei Chu.

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Ethics approval and consent to participate

This study, protocol #HS-3209, was determined to be not human research by “the Institutional Review Board (IRB) at National Jewish Health”. Our research project used only banked and de-identified donor lung cells. The need for consent to participate was waived by the IRB at National Jewish Health since this was considered not human research.

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The authors declare no competing interests.

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Supplementary Information

Additional file 1. Marker genes used for cell typing of human airway epithelial cells.

Additional file 2. Cell count and percentage composition for each cell type and treatment condition.

13104_2025_7255_MOESM3_ESM.xlsx

Additional file 3. The raw data from the Gene Set Enrichment Analysis (GSEA) are presented in this table. The three left-most columns indicate the comparison groups and cell types in which each pathway is significantly enriched, along with the direction of expression change (upregulated or downregulated). The right-most column lists the leading-edge genes, which are the significantly differentially expressed genes that contribute to the pathway’s significance. We discussed some of these genes in our results section. The pathways themselves are illustrated in Figures 2, 3.

Additional file 4. Violin plots for SPDEF (A) and MUC5AC (B) in each cell type across conditions.

Additional file 5. Violin plots for POSTN (A), ALOX15 (B), and CCL26 (C) in each cell type across conditions.

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Lee, G.Y., Schaunaman, N., Nouri, H.R. et al. Comprehensive single-cell RNA-sequencing study of Tollip deficiency effect in IL-13-stimulated human airway epithelial cells. BMC Res Notes 18, 194 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13104-025-07255-7

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BMC Research Notes

ISSN: 1756-0500

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