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. 2018 Apr 17;13(4):e0196034.
doi: 10.1371/journal.pone.0196034. eCollection 2018.

Role of IL-23 Signaling in the Progression of Premalignant Oral Lesions to Cancer

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Free PMC article

Role of IL-23 Signaling in the Progression of Premalignant Oral Lesions to Cancer

Blaine Caughron et al. PLoS One. .
Free PMC article

Abstract

Mice bearing carcinogen-induced premalignant oral lesions were previously shown to have a pro-inflammatory phenotype, which is replaced with an immune inhibitory phenotype as lesions progress to cancer. Since Th17 cells are prominent at the premalignant lesion state and their levels are supported by IL-23, studies used mice that were IL-23 receptor deficient (IL-23R KO) to determine the requirement for IL-23 signaling in the immunological and clinical status of mice with premalignant oral lesions. The results showed a dependence on IL-23 signaling for the pro-inflammatory state of mice with oral lesions as levels of IL-2, IFN-γ, IL-6, IL-17 and TNF-α were elevated in wildtype mice with premalignant oral lesions, but not in IL-23R KO mice. In contrast, as lesions progressed to cancer, the pro-inflammatory phenotype subsided and was replaced with the inhibitory mediator IL-10 and with Treg cells in wildtype mice, although not in IL-23R KO mice. Clinically, early progression of premalignant oral lesions to cancer was enhanced in IL-23R KO mice compared to progression in wildtype mice. These results show the importance of IL-23 signaling in both the pro-inflammatory phenotype characteristic of premalignant oral lesions and the inhibitory phenotype as lesions progress to cancer.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Development of IL-23R conditional knockout mice.
Schematic representation of the gene targeting strategy to generate Il23r conditional knockout mice. A STOP cassette, consisting of splicing acceptor (SA) and three repeats of poly A signal (3xpA), was inserted into the second intron of the endogenous Il23r locus, thus abolishing the expression of IL-23R. The neomycin selection cassette was then removed by crossing with a Flp-expressing mouse line, leaving a single Frt site on the genome. Stu: Stu I restriction site; Bm: BamHI restriction site; open triangle: loxP site; closed triangle: Frt site.
Fig 2
Fig 2. FACS analysis demonstrating the absence of IL-23R expression in the IL-23R KO mice.
Spleen cells from wildtype and IL-23R knockout mice were skewed toward activated Th17 cells and then immunostained for IL-23R. Positive-staining cells were identified by flow cytometry.
Fig 3
Fig 3. Timelines for 4NQO treatment, endoscopic examination and immunological assessments.
Mice were treated with 4NQO in their drinking water for 7 weeks. Once treatment was initiated, they were endoscopically examined at weekly intervals. Subgroups of mice were euthanized for immunological analyses once lesions appeared and at 4 week intervals thereafter.
Fig 4
Fig 4. The absence of IL-23 signaling accelerates progression of premalignant oral lesions toward cancer.
4NQO-treated mice were monitored weekly by endoscopy for the appearance of premalignant oral lesions on the tongue. Once lesions appeared, endoscopic images were taken of the tongue and the severity of the lesions was given a score between 1 and 4 by a blinded scorer. Statistically significant differences (p<0.05) in the clinical score between wildtype and IL-23R KO mice at each time point is indicated (✶).
Fig 5
Fig 5. Endoscopic images showing examples of more advanced premalignant oral lesions in IL-23R KO mice.
Wildtype and IL-23 KO mice that were treated with 4NQO were monitored endoscopically for the development and progression of premalignant oral lesions on the tongue. Shown are endoscopic images of tongues from 4 representative mice of each group taken at 11 and 16 weeks after onset of 4NQO treatment.
Fig 6
Fig 6. Dependence on IL-23 signaling for the increased spleen cell production of pro-inflammatory cytokines and reduced production of inhibitory cytokines during early stages of premalignant lesion development.
Spleen cells of untreated- or 4NQO-treated wildtype or IL-23R KO mice were cultured for 3 days on anti-CD3 antibodies. Culture supernatants were then collected for cytokine quantitation. Significant differences (p<0.05) between cytokine levels secreted by spleen cells from wildtype versus IL-23R KO mice are indicated (✶).
Fig 7
Fig 7. Wildtype, but not IL-23R KO mice, have an increase in splenic levels of CD4+ cells and CD4+ cells expressing IFN-γ during lesion development, which wanes as lesions advance to cancer.
Spleen cells of untreated- and 4NQO-treated wildtype and IL-23R KO mice were immunostained for CD4 and IFN-γ. Cells were analyzed by flow cytometry and significant differences (p<0.05) between the percentages of positive-staining spleen cells from wildtype versus IL-23R KO mice are indicated (✶).
Fig 8
Fig 8. Dot blots showing increased levels of CD4+ cells and CD4+/IFN-γ+ cells in spleens of wildtype mice, but not IL-23R KO mice during lesion development.
Spleen cells of untreated- and 4NQO-treated wildtype and IL-23R KO mice were immunostained for CD4 and IFN-γ. Cells were analyzed by flow cytometry. Representative dot blots of analyses of spleen cells from wildtype or IL-23R KO mice are shown.
Fig 9
Fig 9. Increased splenic levels of Treg in wildtype mice bearing advanced lesions but not in lesion-bearing IL-23R KO mice.
Spleen of untreated- and 4NQO-treated wildtype and IL-23R KO mice were immunostained for CD4+ cells expressing Foxp3. Cells were analyzed by flow cytometry and significant differences (p<0.05) between the percentages of positive-staining spleen cells from wildtype versus IL-23R KO mice are indicated (✶).
Fig 10
Fig 10. Dot blots showing increased levels of Treg cells in spleens of wildtype mice bearing advance premalignant lesions but not in lesion-bearing IL-23R KO mice.
Representative dot blots are shown of spleen cells from untreated- or 4NQO-treated wildtype or IL-23R KO mice that were immunostained for CD4+ cells expressing Foxp3.

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Grant support

This study was supported by the Clinical Sciences Research Program of the Department of Veterans Affairs; Grant number I01 CX000851. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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