Fig 2: Dectin‐3−/− mice have increased tumor load upon AOM‐DSS treatment than WT miceSingle‐housed WT mice and Dectin‐3−/− mice (n = 8 for each group) were injected intraperitoneally with one dose of AOM (10 mg/kg), followed by three cycles of feeding water with 2% DSS. After induction of tumorigenesis, mice were euthanized on day 100. ARepresentative images of colon tumors were shown. Scale bars, 10 mm.BTumor number, tumor size, and tumor load in each mouse were measured.C, DHistological analysis of colon tumors was shown by hematoxylin and eosin (HE) staining. Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist. Scale bars, 25 µm.E, FTumor tissues were stained for Ki67 and p‐STAT3. The percentage of Ki67‐positive and p‐STAT3‐positive cells was quantified. Scale bars, 25 µm.GProportion of CD11b+F4/80+ and RORγt+/CD45+lin− cells was analyzed in colonic LP cells and mLNs among tumor‐bearing WT and Dectin‐3−/− mice by flow cytometry.HRelative expression of Il‐6, Il‐22, Cxcl1, and Il‐17a in LP cells from tumor‐bearing WT and Dectin‐3−/− mice were detected using qPCR. Data information: Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by unpaired Student’s t‐test. See also Fig EV1.

Fig 3: Up‐regulation of IL‐22 in Dectin‐3 −/− mice contributes to CAC development A, BMice were treated as described in Fig 2A. LP cells were isolated from each mouse and were culture for 48 h. Cytokine and chemokine production of LP cells were detected using multiplex cytokine assay. Color from blue to red indicates enrichment of gene expression.CmRNA expressions of IL‐22, β‐defensin, Reg3g, and Cxcl1 in LP cells were detected by qPCR.DLP cells and mLNs were isolated from each mouse and were culture for 48 h. Production of IL‐22 in LP cells and mLNs were detected by ELISA.ELP cells were isolated from each mouse and were culture for 48 h. Expression of Il‐17 in LP cells were detected by qPCR. Production of IL‐17 in LP cells were detected by ELISA.FProtein level of p‐STAT3 and STAT3 in colonic epithelial cells was detected by using Western blot (one mouse per lane). The relative expression of p‐STAT3 was calculated.GWT and Dectin‐3−/− mice were intraperitoneally treated with anti‐IL22 antibody or anti‐IgG antibody as control during AOM‐DSS administration (n = 5, each group). Mice were euthanized on Day 100, tumor number, tumor size, and tumor load in colons were measured. Data information: Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by Student’s t‐test. See also Fig EV3.

Fig 4: Single-cell transcriptomics reveals diabetes-induced alterations in lymphocyte populations.A. Initial clustering analysis identified three distinct T lymphocyte populations (clusters 8, 14, and 18) characterized by positive Cd3g expression, validating their T-cell lineage identity. B. Violin plots demonstrating differential expression of immunoregulatory genes (Furin, Ikzf2, Il2ra, Tgfb1) and pro-inflammatory mediators (Il17a, Nfkbia, Xcl1, Nkg7) between normoglycemic (NG) and hyperglycemic (HG) conditions. C. UMAP demonstrates co-expression patterns of Il17a with Cd3g, Foxp3 with Ikzf2, and Cd8a with Nkg7 within respective cellular clusters. D. Quantification of IL-17A+CD3+ cells by flow cytometry, expressed as the absolute number of IL-17A+CD3+ cells at baseline and six weeks after oral inoculation with Pg and Fn to induce periodontitis. Data shown are the mean ± SEM from three independent experiments. P < 0.05, Student’s t-test. (Right) Representative flow cytometry plots showing IL-17A+CD3+ cell frequencies from the gingiva of normoglycemic and diabetic mice. E. Quantification of IL-17A+CD3+ cells by flow cytometry, expressed as the absolute number of IL-17A+CD3+ cells in human gingival tissue. Data shown are the mean ± SEM from normoglycemic (N = 5) and hyperglycemic (N = 3) individuals. P < 0.05, Student’s t-test. (Right) Representative flow cytometry plots showing IL-17A+CD3+ cell frequencies. F. Quantification of CD8+CD3+ cells by flow cytometry in murine gingival tissues, expressed as the absolute number of CD8+CD3+ cells at baseline and two weeks after oral inoculation with Pg and Fn to induce periodontitis. Data shown are the mean ± SEM from three independent experiments. P < 0.05, Student’s t-test. (Right) Representative flow cytometry plots showing CD8+CD3+ cell frequencies. G. Quantification of CD8+CD3+ cells by flow cytometry in human gingival tissue, expressed as the absolute number of CD8+CD3+ cells. Data shown are the mean ± SEM from normoglycemic (N = 5) and hyperglycemic (N = 3) individuals. P < 0.05, Student’s t-test. (Right) Representative flow cytometry plots showing CD8+CD3+ cell frequencies. H. A new object was created from the lymphocyte subcluster identified in Fig. 4A and re-clustered by R/Seraut, highlighting Il17a+ (cluster 0), Ikzf2+ (cluster 2), and Nkg7+ (cluster 5) populations. I. Cell populations consisting of 10 distinct clusters are identified in the table. J. Bar graph depicting percentage lymphocyte counts per cluster, emphasizing clusters 0, 2, and 5 changes between normoglycemic and hyperglycemic phenotypes. K. Dot plot visualization of differential gene expression across T lymphocyte subclusters. L. Quantification of CD3+Foxp3+cells by flow cytometry, expressed as a percentage of CD3+ cells at baseline, two weeks, and six weeks after oral inoculation with Pg and Fn to induce periodontitis and from human gingiva in subjects with periodontitis with obtained in normoglycemic and hyperglycemia. Data for mice shown are the mean ± SEM from three independent experiments and for human subjects n = 5. *P < 0.05, Student’s t-test. M. Quantification of Foxp3+ cells by flow cytometry, expressed as a percentage of total CD3+ cells in human gingival tissue. The mean ± SEM is shown from normalglycemic (N = 5) or abnormally high glycemic (N = 3) individuals. *P < 0.05, Student’s t-test. N. Quantification of CD3+Tcr γδ + cells by flow cytometry, expressed as a percentage of CD3+ cells at baseline, two weeks, and six weeks after oral inoculation with Pg and Fn to induce periodontitis. Data shown are the mean ± SEM from three independent experiments. *P < 0.05, Student’s t-test.

Fig 5: Glycemic-Dependent Pathway Analysis of Tregs and γδ T-cells with γδ T-cell IL-17A Production StudiesA. Canonical IPA analysis of differentially expressed genes between normalglycemic and hyperglycemic phenotypes within cluster 0 (identified as γδ T-cell) B. Canonical IPA analysis of differentially expressed genes between normalglycemic and hyperglycemic phenotypes within cluster 2 (identified as Tregs). C. Relative mRNA expression of IL17A quantified by RT-qPCR using mouse splenic γδ T-cells incubated in normal glucose (NG, 5 mM), osmotic mannitol control (M, 25 mM), or high glucose condition (HG, 25 mM). D-F. Relative IL17A mRNA expression in γδ T-cells incubated in NG or HG with varying concentrations of (B) lipopolysaccharide (LPS; 0.2, 1 and 5ug/ml), (C) advanced glycation end products (AGE; 50, 100, 200ug/ml) or (D) tumor necrosis factor (TNF; 10, 50, 250 ng/mL). G. Quantification of IL17A/F protein in the supernatant by ELISA using mouse splenic γδ T-cells under NG, M, or HG conditions. H. Quantification of IL17A/F in γδ T-cells under NG or HG conditions that were treated with LPS (left), AGE (middle), or TNF (right) at given concentrations. Fig. 6C–H: Data is shown as mean ± SEM from three independent experiments. *P < 0.05, Student’s t-test. Abbreviations: ns: not significant, *P < 0.05 by one-way ANOVA with Tukey’s post-hoc test.