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Cited by 25T-cell and B-cell ImmunologyIL-33, ST2, and ILC PathwaysInflammatory Bowel Disease

PU.1-driven Th9 Cells Promote Colorectal Cancer in Experimental Colitis Models Through Il-6 Effects in Intestinal Epithelial Cells

Open access
Gerlach, Katharina, Popp, Vanessa, Wirtz, Stefan, Al-Saifi, Ragheed, Gonzalez Acera, Miguel, Atreya, Raja, Dregelies, Theresa, Vieth, Michael +5 more·2022·Journal of Crohn's and Colitis
DOI 10.1093/ecco-jcc/jjac097PMC PMC10197880
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Abstract

Background and Aims: Colorectal cancer [CRC] is one of the most frequent malignancies, but the molecular mechanisms driving cancer growth are incompletely understood. We characterised the roles of the cytokine IL-9 and Th9 cells in regulating CRC development. Methods: CRC patient samples and samples from AOM/DSS treated mice were analysed for expression of IL-9, CD3, and PU.1 by FACS analysis and immunohistochemistry. IL-9 citrine reporter mice, IL-9 knockout mice, and PU.1 and GATA3 CD4-Cre conditional knockout mice were studied in the AOM/DSS model. DNA minicircles or hyper-IL-6 were used for overexpression of cytokines in vivo. Effects of IL-6 and IL-9 were determined in organoid and T cell cultures. Claudin2/3 expression was studied by western blotting and bacterial translocation by FISH. Results: We uncovered a significant expansion of IL-9- and PU.1-expressing mucosal Th9 cells in CRC patients, with particularly high levels in patients with colitis-associated neoplasias. PU.1+ Th9 cells accumulated in experimental colorectal neoplasias. Deficiency of IL-9 or inactivation of PU.1 in T cells led to impaired tumour growth in vivo, suggesting a protumoral role of Th9 cells. In contrast, GATA3 inactivation did not affect Th9-mediated tumour growth. Mechanistically, IL-9 controls claudin2/3 expression and T cell-derived IL-6 production in colorectal tumours. IL-6 abrogated the anti-proliferative effects of IL-9 in epithelial organoids in vivo. IL-9-producing Th9 cells expand in CRC and control IL-6 production by T cells. Conclusions: IL-9 is a crucial regulator of tumour growth in colitis-associated neoplasias and emerges as potential target for therapy.

AI snapshot

Written from the paper’s full text.

TL;DR

In human colorectal cancer and colitis-associated cancer, IL-9- and PU.1-expressing Th9 cells expand, and in AOM/DSS mouse models IL-9 or T-cell PU.1 deficiency reduces tumour growth while GATA3 deficiency does not. Mechanistically, IL-9 promotes T-cell IL-6 production and, together with IL-6, drives epithelial STAT3 signalling, proliferation, and barrier defects.

Key findings

  • Human CRC and colitis-associated cancer [CAC] showed elevated IL-9 mRNA and Th9-associated factors, with more PU.1+CD4+ T cells and CD3+IL-9+ cells in tumour tissue than controls; CAC showed particularly high levels (Figure 1; n = 5–7 per high-power field for PU.1/CD4, five patients for CD3/IL-9).
  • In AOM/DSS tumours, IL-9 mRNA was significantly induced versus untreated controls (n = 7), IL-9 was produced mainly by CD4+ T cells rather than CD19+ B cells or CD8+ T cells (n = 3), and CD4+PU.1+ T cells increased; Spi1 expression was analysed in n = 10.
  • IL-9 deficiency reduced inflammation after the third DSS cycle and significantly reduced tumour numbers, size, and score (three independent experiments, n = 18); anti-IL-9 antibody reduced tumours and tumour score (n = 3); IL-9 overexpression via minicircle augmented tumour growth in IL-9-deficient mice (WT n = 15, IL-9 KO n = 16) but had no significant effect in IL-9-proficient WT mice.
  • T-cell PU.1 deficiency suppressed DSS colitis after the third cycle and decreased tumour burden (Spi1flflCd4cre; three independent experiments, five mice per group), whereas T-cell GATA3 deletion augmented colitis activity during the third DSS cycle and increased tumour burden (seven mice per group).
  • IL-9 controlled IL-6: PU.1+ Th9 cells expressed more IL-9 receptor, produced IL-6, and IL-9 stimulation increased IL-6 and IL-9-producing cells; Il-6 and Il-9 mRNA positively correlated in tumours, and hyperIL-6 treatment of IL-9 knockout mice restored tumour development comparable to WT.
  • In epithelial cells, IL-9 downregulated proliferation while IL-6 upregulated it; the combination abrogated IL-9’s anti-proliferative effect. IL-9 upregulated SOCS1/SOCS3 and reduced pSTAT3, IL-6 upregulated pSTAT3, and the combination suppressed SOCS induction and activated STAT3. IL-9 deficiency lowered claudin-2, induced claudin-3, and reduced bacterial translocation.

Methods

  • Human colonic specimens from patients with colorectal cancer, colitis-associated cancer, and controls were analysed by FACS and immunohistochemistry for IL-9, CD3, and PU.1; Table 1 reports n = 36 total (9 controls, 11 CRC, 16 CAC), mean age 57 years, range 25–82.
  • Colitis-associated neoplasias were induced in mice by intraperitoneal AOM [10 mg/kg] followed by three cycles of 2% DSS in drinking water for 1 week and normal water for 2 weeks. IL-9 citrine reporter, IL-9 knockout, PU.1 and GATA3 CD4-Cre conditional knockout, and wild-type mice were studied; IL-9 minicircle DNA, hyper-IL-6 [2 µg weekly i.p.], and anti-IL-9 antibody [80 μg i.p.] were used in vivo.
  • Analyses included high-resolution mini-endoscopy and histopathology, FACS, immunofluorescence, western blotting, FISH for bacterial translocation, intestinal organoid and T cell cultures, and real-time PCR; statistics used Student’s t test with p < 0.05 and Spearman’s rho for correlations.

Limitations

  • The paper has no dedicated limitations section. In the Discussion, it states that reasons for differences from prior studies in melanoma and colitis “are not entirely clear” and may relate to colitis models, microbial factors, cytokine patterns, the local gut microenvironment, or IL-9R expression on tumour cells.
  • The text notes that some CD4+ and CD8+ T cell number comparisons between IL-9 knockout and wild-type mice were “data not shown,” limiting assessment of those negative findings.
  • Evident from the text: human data are descriptive and based on a modest sample (n = 36 total), while causal mechanistic claims rely largely on the AOM/DSS mouse model and organoid/T cell cultures; several experiments use small group sizes (e.g., n = 3).

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1093/ecco-jcc/jjac097

Cites

55 of 55 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

22 of 40 cited sources checked · 7 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    PU.1, rather than IRF4, GATA3, or STAT6, is the main driver for Th9 cell differentiation.

    “Moreover, the transcription factor PU.1, rather than IRF4, GATA3, or STAT6, is the main driver for Th9 cell differentiation.20, 21”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [20] Staudt, 2010, Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells, Immunity source
    • cites [21] Goswami, 2012, STAT6-dependent regulation of Th9 development, J Immunol source

      The Introduction identifies both PU.1 and IRF4 as required for Th9 development. The Results say STAT6 is required for Th9 development, and the Discussion describes a network of factors rather than PU.1 as the main driver instead of the others. Our copy does not provide page numbers for these passages. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation

    IL-6 production may enhance mucosal inflammation via T cell activation.

    “Moreover, IL-6 production may enhance mucosal inflammation via T cell activation,6,35,36 providing a link between inflammation and tumour growth.”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [6] Neurath, 2014, Cytokines in inflammatory bowel disease, Nat Rev Immunol source
    • cites [35] Atreya, 2005, Involvement of IL–6 in the pathogenesis of inflammatory bowel disease and colon cancer, Clin Rev Allergy Immunol source
    • cites [36] Yamamoto, 2000, IL-6 is required for the development of Th1 cell-mediated murine colitis, J Immunol source
  • Premisecitation

    IL-6 has been previously identified as a key cytokine that directly induces tumour growth in CRC via STAT3 activation in IECs.

    “IL-6 has been previously identified as a key cytokine that directly induces tumuor growth in CRC via STAT3 activation in IECs.12,15,46,47.”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [12] Grivennikov, 2009, IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer, Cancer Cell source

      The Introduction and Discussion report that IL-6 promotes tumor growth through STAT3 activation in intestinal epithelial cells. The experiments establish this in a mouse model of colitis-associated cancer (CAC), not colorectal cancer (CRC) generally, so the claim extends the finding beyond the studied setting. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [15] Becker, 2004, TGF–beta suppresses tumor progression in colon cancer by inhibition of IL–6 trans–signaling, Immunity source
    • cites [46] Bollrath, 2009, gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis, Cancer Cell source

      The Summary and Discussion (page numbers unavailable) report that IEC Stat3 activation promotes tumor growth in a mouse model of colitis-associated cancer and that IL-6-dependent Stat3 activation contributes to this process. The paper also identifies IL-11-dependent activation and describes IL-6 and IL-11 as having redundant effects; it does not establish IL-6 alone as the key driver of CRC growth generally. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [47] Rokavec, 2014, IL-6R/STAT3/miR-34a feedback loop promotes EMT-mediated colorectal cancer invasion and metastasis, J Clin Invest source

      The Results (p. 2) show that IL-6 activates STAT3 in a colorectal cancer cell line and promotes EMT and invasion. The paper does not establish the more specific claim that IL-6 directly induces tumour growth through STAT3 activation in intestinal epithelial cells; its reported outcomes here are invasion and metastasis, not tumour growth. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    IL-6 plays an important regulatory role for tumour initiation in CRC.

    “Thus, IL-6 plays an important regulatory role for tumour initiation in CRC.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [14] Landskron, 2014, Chronic inflammation and cytokines in the tumor microenvironment, J Immunol Res source

      Section 5, “Colorectal Cancer and Inflammatory Bowel Disease” (page unavailable), reports that murine models demonstrate a crucial role for IL-6 in the initiation and progression of colitis-associated cancer. This supports a role in tumour initiation, but the claim extends that finding to CRC generally without the colitis-associated and murine-model qualifiers. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    This observation is in contrast to previous findings demonstrating a role of Th9 cells in controlling anti-tumour immune responses in solid tumours such as melanomas.

    “This observation is in contrast to previous findings demonstrating a role of Th9 cells in controlling anti-tumour immune responses in solid tumours such as melanomas.26,27”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [26] Purwar, 2012, Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells, Nat Med source
    • cites [27] Lu, 2012, Th9 cells promote antitumor immune responses in vivo, J Clin Invest source
  • Classified as probably not a substantial claim. 12
  • May combine two claims. 27

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In Th9 cells, IL-9 gene expression is regulated by TGFβ, IL-4, and IL-33 and transcription factors like PU.1, STAT6, GATA3, and IRF4.

    “In these cells, IL-9 gene expression is regulated by the cytokines TGFβ, IL-4, and IL-33 and transcription factors like PU.1, STAT6, GATA3, and IRF4.19–22”
    Quote in the paperNot a substantial claim3 of 4 sources checked
    • cites [19] Chang, 2010, The transcription factor PU.1 is required for the development of IL–9–producing T cells and allergic inflammation, Nat Immunol source

      The Introduction and Discussion describe TGF-β1 and IL-4 in TH9 development, identify PU.1 as a regulator of IL-9, and say STAT6 and GATA3 are required for TH9 development. The Discussion says IRF4 represses Il9 in TH2 cells, not that it regulates IL-9 expression in TH9 cells; no passage in our copy identifies a role for IL-33. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [20] Staudt, 2010, Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells, Immunity source
    • cites [21] Goswami, 2012, STAT6-dependent regulation of Th9 development, J Immunol source

      The Introduction and Discussion describe TGFβ and IL-4 signals and roles for PU.1, STAT6, GATA3, and IRF4 in Th9-cell IL-9 regulation. The Results also show that excess GATA3 decreases IL-9 production, so its role is not simply promotional. No passage in our copy supports the attributed role for IL-33 (searched: IL-33). (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [22] Kaplan, 2015, The development and in vivo function of T helper 9 cells, Nat Rev Immunol source

      The Regulation of TH9 cell development section describes TGFβ and IL-4 signalling and roles for PU.1, STAT6, GATA3 and IRF4 in TH9-cell development or IL-9 production. The copy does not establish IL-33 as a regulator of IL-9 gene expression in TH9 cells; IL-33 appears in the References as the subject of a study on IL-9 production in human CD4+ T cells and basophils. Page numbers are unavailable in the indexed copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    IL-6 binds to gp130 on target cells to activate STAT3 in tumour cells and tumour-associated inflammatory cells.

    “IL-6 binds to gp130 on target cells to activate the signal transducer and activator of transcription 3 [STAT3] in tumour cells and tumour-associated inflammatory cells 12, 15.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [12] Grivennikov, 2009, IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer, Cancer Cell source

      The Introduction (page not available) says IL-6 binds IL-6R, which interacts with gp130 to activate downstream STAT3; it does not say IL-6 binds gp130 directly. The Significance section (page not available) identifies STAT3 as mediating IL-6’s effects on pre-malignant intestinal epithelial cells, but describes inflammatory myeloid cells as IL-6 producers, not as cells in which IL-6 activates STAT3. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [15] Becker, 2004, TGF–beta suppresses tumor progression in colon cancer by inhibition of IL–6 trans–signaling, Immunity source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Chronic inflammation and flares of disease are risk factors for CAC.

    “In agreement with this link, chronic inflammation and flares of disease have been identified as risk factors for CAC.37–39”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [37] Jess, 2012, Risk of colorectal cancer in patients with ulcerative colitis: a meta-analysis of population-based cohort studies, Clin Gastroenterol Hepatol source

      The Discussion (page not provided) identifies severity of colonic inflammation as a determinant of colorectal neoplasia risk in ulcerative colitis and says ongoing, extensive inflammation might increase colorectal cancer risk. It does not identify disease flares as a separate risk factor, so that part of the claim is unsupported by this copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [38] Beaugerie, 2013, Risk of colorectal high-grade dysplasia and cancer in a prospective observational cohort of patients with inflammatory bowel disease, Gastroenterology source

      The Background & Aims passage says chronic intestinal inflammation promotes carcinogenesis and identifies active intestinal inflammation as an established risk factor for colorectal cancer in IBD. It does not specifically identify disease flares as a risk factor; its reported cohort findings focus on long-standing extensive colitis. No page number is provided for the passage. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [39] Broström, 1987, The risk of colorectal cancer in ulcerative colitis. An epidemiologic study, Scand J Gastroenterol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The IL-6/JAK/STAT3 signalling pathway plays an important role in human malignancies such as CRC.

    “The IL-6/JAK/STAT3 signalling pathway plays an important role in human malignancies such as CRC.12,15,46,48,49”
    Quote in the paperNot a substantial claim3 of 5 sources checked
    • cites [12] Grivennikov, 2009, IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer, Cancer Cell source
    • cites [15] Becker, 2004, TGF–beta suppresses tumor progression in colon cancer by inhibition of IL–6 trans–signaling, Immunity source
    • cites [46] Bollrath, 2009, gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis, Cancer Cell source

      The Introduction describes STAT3 activation as a feature of many human malignancies, and the Discussion reports a role for gp130/STAT3 signaling in inflammation-associated tumors. However, the paper’s direct evidence concerns a mouse model of colitis-associated cancer; it does not establish the role of the full IL-6/JAK/STAT3 pathway in human colorectal cancer specifically. The supplied copy gives no page numbers for these sections. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [48] Liu, 2015, IL-6 enriched lung cancer stem-like cell population by inhibition of cell cycle regulators via DNMT1 upregulation, Int J Cancer source

      The Discussion reports that IL-6/JAK2/STAT3 signalling promotes lung cancer stem-like cell proliferation and describes its activity in breast cancer cells, supporting a role in human malignancies. The passage mentioning colorectal cancer links it to chronic inflammation, but does not establish a role for this specific pathway in colorectal cancer. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [49] Kroon, 2013, JAK-STAT blockade inhibits tumor initiation and clonogenic recovery of prostate cancer stem-like cells, Cancer Res source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    During inflammation, IL-6 induces free radicals causing DNA damage, favouring tumour initiation.

    “During inflammation, IL-6 induces free radicals causing a DNA damage, favouring tumour initiation 14.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [14] Landskron, 2014, Chronic inflammation and cytokines in the tumor microenvironment, J Immunol Res source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Claudin-3 has been shown to control malignancy and metastasis in CRC.

    “Whereas claudin-3 has been shown to control malignancy and metastasis in CRC,52,53 claudin-2 led to a leaky epithelium in tumour tissue.54”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [52] Tokuhara, 2018, Nuclear expression of claudin-3 in human colorectal adenocarcinoma cell lines and tissues, Oncol Lett source

      The passage accompanying Table IV (page not supplied) reports that nuclear claudin-3 expression is higher in mucinous colorectal adenocarcinomas and advanced T4 tumors, supporting an association with tumor behavior. It explicitly says further studies are needed to establish whether claudin-3 regulates proliferation or metastasis, so it does not show that claudin-3 controls malignancy and metastasis. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [53] de Souza, 2013, Claudin-3 overexpression increases the malignant potential of colorectal cancer cells: roles of ERK1/2 and PI3K-Akt as modulators of EGFR signaling, PLoS One source

      The Discussion reports that claudin-3 overexpression increased the malignant potential of HT-29 colorectal cancer cells. The Abstract reports increased colony formation, but says claudin-3 overexpression did not alter migration. Our copy does not show that claudin-3 controls metastasis; the Discussion’s mention of lymph-node metastases concerns claudin-1. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Gut bacteria favour protumoral mucosal Th17 signatures and local inflammation in colorectal neoplasias.

    “This impairment of barrier function during tumorigenesis may contribute to IL-9 effects in tumour development, as gut bacteria favour protumoral mucosal Th17 signatures and local inflammation in colorectal neoplasias.45”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [45] Grivennikov, 2012, Adenoma–linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth, Nature source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Mucosal inflammation may further induce the expansion of microorganisms with genotoxic capabilities which favour tumour progression in CRC.

    “Mucosal inflammation in turn may further induce the expansion of microorganisms with genotoxic capabilities which favour tumour progression in CRC.4”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [4] Arthur, 2012, Intestinal inflammation targets cancer-inducing activity of the microbiota, Science source

  • May combine two claims.

    Findingown data

    Mucosal Th9 cells produce both IL-9 and IL-6.

    “Additionally, we saw that mucosal Th9 cells not only produce IL-9 but also the pro-inflammatory cytokine IL-6 [Figure 5B].”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IL-9 stimulation of PU.1+ Th9 cells causes increased production of IL-6 and an increased percentage of IL-9-producing cells.

    “Finally, we searched out that IL-9 stimulation of PU.1+ Th9 cell caused increased production of IL-6 and an increased percentage of IL-9-producing cells [Figure 5D].”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Tumour numbers, tumour size, and tumour score are significantly reduced in IL-9 knockout mice in the AOM/DSS model.

    “Tumour numbers, tumour size, and tumour score in IL-9 knockout mice were significantly reduced [Figure 3A].”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Th9-related transcription factors GATA3 and SPI1 are elevated in CRC and more elevated in CAC.

    “Elevated levels of the Th9-related transcription factors GATA3 and SPI1 were found in CRC and even more in CAC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Colitis-associated tumours had even higher levels of Th9-associated factors and marked correlations between GATA3, SPI.1, and IL-9 mRNA levels.

    “Furthermore, we analysed colitis-associated tumours and detected even higher levels of Th9-associated factors and marked correlations between GATA3, SPI.1, and IL-9 mRNA levels, suggesting the relevance of Th9 cells in these patients.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The combination of IL-9 and IL-6 abrogated the induction of SOCS1 and SOCS3 levels by IL-9 and induced pSTAT3.

    “The combination of IL-9 and IL-6 abrogated the induction of SOCS1 and SOCS3 levels by IL-9 and induced pSTAT3.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    Treatment of IL-9 knockout mice with hyperIL-6 abrogated the protective effects of IL-9 deficiency and led to a marked tumour growth.

    “Indeed, treatment of IL-9 knockout mice with hyperIL-6 abrogated the protective effects of IL-9 deficiency and led to a marked tumour growth, thereby demonstrating that IL-9 is an important regulator of IL-6 trans-signalling effects in colorectal neoplasias.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CRC patients showed elevated IL-9 and PU.1 levels in mucosal T cells, indicating an enrichment of Th9 cells in the tumour tissue.

    “CRC patients showed elevated IL-9 and PU.1 levels in mucosal T cells, indicating an enrichment of Th9 cells in the tumour tissue.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IL-9 treatment induced claudin-2 but reduced claudin-3 levels in IECs.

    “Conversely, IL-9 treatment induced claudin-2 but reduced claudin-3 levels in IECs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Th9 cells express IL-9R and respond to IL-9 stimulation with the production of the pro-tumorigenic cytokine IL-6.

    “Indeed, mechanistic studies revealed that Th9 cells express IL-9R and respond to IL-9 stimulation with the production of the pro-tumorigenic cytokine IL-6.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    In the presence of both IL-9 and IL-6, IL-9-dependent effects on SOCS3 were suppressed and STAT3 was activated.

    “In the presence of both IL-9 and IL-6, however, IL-9-dependent effects on SOCS3 were suppressed and STAT3 was activated.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Tumour cells in experimental colorectal neoplasias exhibited a higher IL-9R expression that controls IECs, suggesting that tumour cells are sensitive to IL-9-mediated signal transduction.

    “Similarly, our studies revealed that tumour cells in experimental colorectal neoplasias exhibited a higher IL-9R expression that controls IECs, suggesting that tumuor cells are sensitive to IL-9-mediated signal transduction.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    There is an auto-regulatory loop in which mucosal Th9 cells produce IL-9, express IL-9R, and generate increased levels of cytokines such as IL-6 upon IL-9 exposure.

    “These findings suggested the presence of an auto-regulatory loop in which mucosal Th9 cells produce IL-9, express IL-9R, and generate increased levels of cytokines such as IL-6 upon IL-9 exposure.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    IL-9 and Th9 cells are present in experimental colorectal tumours.

    “indicating the presence of IL-9 and Th9 cells [Figure 2A, left panel].”
    • Also stated in: § 3.2. Mucosal PU.1+ T cells producing IL-9 are induced in experimental colorectal cancer · hedged
      “thereby suggesting the presence of Th9 cells [Figure 2D].”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Anti-IL-9 antibodies restrained colitis.

    “Anti-IL-9 antibodies also restrained colitis, suggesting that IL-9 could be used efficiently for colitis therapy 24.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [24] Gerlach, 2014, TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells, Nat Immunol source

  • May combine two claims.

    Premisecitation

    In murine colitis models, IL-9 deficiency or PU.1 inactivation in T cells suppressed colitis activity.

    “In murine colitis models, IL-9 deficiency or PU.1 inactivation in T cells suppressed colitis activity.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [24] Gerlach, 2014, TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells, Nat Immunol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Signal specificity of FISH was demonstrated using Escherichia coli as a positive control with the EUB-338 probe.

    “Signal specificity was demonstrated by using Escherichia coli as positive control with the EUB-338 probe.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Colitis severity was assessed using the established Murine Endoscopic Index of Colitis Severity (MEICS) scoring system based on five parameters.

    “Murine Endoscopic Index of Colitis Severity [MEICS] scores for colitis severity were assigned at the end of the experiment on the basis of five parameters [translucency, granularity, fibrin, vascularity, and stool] according to an established scoring system 29–31.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [29] Becker, 2006, High resolution colonoscopy in live mice, Nat Protoc source
    • cites [30] Wirtz, 2007, Chemically induced mouse models of intestinal inflammation, Nat Protoc source
    • cites [31] Wirtz, 2017, Chemically induced mouse models of acute and chronic intestinal inflammation, Nat Protoc source

  • May combine two claims.

    Interpretationown data

    PU.1-expressing Th9 cells are induced in patients with CRC and play a dominant protumoral role in the experimental CRC model.

    “Here, we showed that PU.1-expressing Th9 cells are induced in patients with CRC and play a dominant protumoral role in the experimental CRC model.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Detection of β-Actin served as a loading control in western blot analysis.

    “Detection of ß-Actin [Santa Cruz, 1:5000] served as loading control.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    IL-9 signalling controls IL-6 production by tumour-infiltrating T cells, leading to IL-6-dependent pSTAT3 activation and proliferation of intestinal epithelial cells.

    “Mechanistic experiments demonstrated that IL-9 signalling controls IL-6 production by tumour-infiltrating T cells, leading to IL-6-dependent pSTAT3 activation and proliferation of intestinal epithelial cells [IEC].”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    IL-9 and Th9 cells play an important role in driving colitis activity and tumour growth in vivo in murine DSS and AOM/DSS models.

    “Functional studies in the murine DSS and AOM/DSS models identified an important role of IL-9 and Th9 cells in driving colitis activity and tumour growth in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The pro-inflammatory cytokines IL-6 and IL-9 together are able to stimulate proliferation of IECs, thus explaining the pro-tumoral role of these cytokines in CRC.

    “Thus, the pro-inflammatory cytokines IL-6 and IL-9 together were able to stimulate proliferation of IECs, thus explaining the pro-tumoral role of these cytokines in CRC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    IL-9 controls the balance between the sealing protein claudin-3 and the pore-forming claudin-2 protein, indicating that IL-9 impairs barrier function.

    “Additional studies in IL-9 knockout mice revealed that IL-9 controls the balance between the sealing protein claudin-3 and the pore-forming claudin-2 protein, indicating that IL-9 impairs barrier function.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    IL-9 deficiency has limited effects on tumour growth by regulating mucosal inflammation but may directly modulate tumour growth via a decreased pro-inflammatory cytokine milieu.

    “However, this observation suggested that IL-9 deficiency has limited effects on tumour growth by regulating the mucosal inflammation but may directly modulate tumour growth via a decreased pro-inflammatory cytokine millieu.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    SOCS3 is a downstream target gene of the IL-6/JAK/STAT3 signalling cascade and inhibits STAT3 activation in CRC.

    “One of the downstream target genes of this signalling cascade is the negative feedback regulator suppressor of cytokine signalling 3 [SOCS3] that inhibits STAT3 activation in CRC.50,51”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [50] Yin, 2015, SOCS3 and its role in associated diseases, Hum Immunol source
    • cites [51] Li, 2010, Disease-related expression of the IL6/STAT3/SOCS3 signalling pathway in ulcerative colitis and ulcerative colitis-related carcinogenesis, Gut source

  • May combine two claims.

    Interpretationown data

    The induction of IL-6 function reversed the protective phenotype of IL-9 knockout mice and restored tumour induction to levels indistinguishable from wild-type mice.

    “Thus, the induction of IL-6 function reversed the protective phenotype of IL-9 knockout mice and restored tumour induction to levels indistinguishable from wild-type mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    PU.1-driven Th9 cells, rather than GATA3-expressing T cells, are essential for tumorigenesis.

    “Thus, PU.1-driven Th9 cells, rather than GATA3-expressing T cells, are essential for tumorigenesis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    IL-9 controls tumour cell proliferation in colorectal neoplasias via IL-6 and impairs barrier function markedly.

    “Thus, IL-9 controls tumour cell proliferation in colorectal neoplasias via IL-6 and impairs barrier function markedly.”
    Quote in the paperSubstantial claim
37 of 37

  • The authors state this as an assumption.

    The molecular mechanisms controlling IL-6 production in colorectal cancer were poorly understood.

    “However, the molecular mechanisms controlling IL-6 production remained poorly understood.”

  • The authors state this as an assumption.

    Little was known about the functional role of IL-9 in colorectal cancer.

    “Opposite to the function of IL-9 in chronic intestinal inflammation, little is known about the functional role of IL-9 in CRC.”
2 of 2

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • IL-6 dose for organoid cultures differs between methods and Figure 6

    Major

    The organoid-culture method specifies 100 ng hIL-6, while the Figure 6C legend specifies 50 ng hIL6 for organoids.

  • GATA3 inactivation is described as having no effect despite increased tumour burden

    Major

    The abstract says GATA3 inactivation did not affect tumour growth, whereas the results report higher tumour burden when GATA3 was absent in T cells.

  • Mean patient age does not match Table 1

    Minor

    The 36 listed ages average 58.69 years, which rounds to 59 rather than the reported 57.

    Reported 57 · recomputed 58.694444

3 of 3

Cites

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Related

Authors
Gerlach, K., Popp, V., Wirtz, S., Al-Saifi, R., Gonzalez Acera, M., Atreya, R., Dregelies, T., Vieth, M., Fichtner-Feigl, S., McKenzie, A. N. J., Rosenbauer, F., Weigmann, B., & Neurath, M. F.
Affiliations
  • Friedrich-Alexander-Universität Erlangen-Nürnberg (DE)
  • Department of Medicine 1, Kussmaul Campus for Medical Research, University of Erlangen-Nuremberg , Erlangen , Germany
  • Department of General and Visceral Surgery, Medical Center-University of Freiburg , Freiburg , Germany
  • MRC Laboratory of Molecular Biology (GB)
  • University of Münster (DE)
Corresponding author
Weigmann, Benno
ORCID
Venue
Journal of Crohn's and Colitis
Publisher
Oxford University Press (OUP)
Published
2022-07-06
Volume
16
Issue
12
Pages
1893-1910
ISSN
  • 1873-9946 (ISSN-L)
  • 1876-4479
DOI
10.1093/ecco-jcc/jjac097
PMC
PMC10197880
OpenAlex
W4284890438
Semantic Scholar
16e375bb79d1f7475793525feb89a9ace5872b8a
Cited by
25
References
55
Citations by year
2026: 4 · 2025: 8 · 2024: 7 · 2023: 5 · 2022: 1
FWCI
1.62
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Crohn s and Colitis (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
Funders
  • Interdisciplinary Center for Clinical Research
  • Collaborative Research Centers
  • German Research Council
  • Emerging Fields Initiative and the Ludwig Demling Center
  • DFG (GE3043/3-1 [434401404], WE4656/2-2, CRC 1181/2-B02)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
  • Life Sciences › Immunology and Microbiology › Immunology › IL-33, ST2, and ILC Pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Inflammatory Bowel Disease
Keywords
colorectal cancer, Th9 cells
MeSH terms
Animals, Colitis, Epithelial Cells, T-Lymphocytes, Helper-Inducer, Mice, Inbred C57BL, Colorectal Neoplasms, Interleukin-6, Cytokines, Interleukin-9, Mice, Knockout, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-21.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-21.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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