Additional file 1: of Prevalence of Candida albicans and non-albicans on the tongue dorsa of elderly people living in a post-disaster area: a cross-sectional survey

Relationships of demographic character, oral conditions, systemic conditions, lifestyle, medications and relocation from home with colonization of C. albicans and non-albicans (N = 264). Results of multinomial logistic regression analysis for colonization of C. albicans and non-albicans. Relationships of C. albicans and non-albicans colonization with all independent variables examined in this study are presented in this table. (DOCX 22 kb

IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

<div><p>Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^−/−) mice and RAG-2 and RORγt double deficient (RAG-2^−/− × RORγt^−/−) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected <i>RORC</i> expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^−/− mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^−/− × RORγt^−/− mice that lacked T and NKT cells. Surprisingly, RAG-2^−/− × RORγt^−/− mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^−/− mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^−/− mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.</p></div

Hepatic ILCs express RORγt and are decreased in RAG-2^−/− × RORγt^−/− mice.

<p>(A) RORγt expression in hepatic MNCs. RORγt mRNA expression in the indicated hepatic MNCs subsets was measured by RT-qPCR and normalized relative to β-actin expression. Error bars represent SEM of triplicate samples. (B) SCA-1/Thy1 staining of the Lin⁻ fraction of hepatic MNCs from RAG-2^−/− or RAG-2^−/− × RORγt^−/− mice. (C) Ratio of SCA-1⁺Thy1^high ILCs in Lin⁻ hepatic MNCs from RAG-2^−/− or RAG-2^−/− × RORγt^−/− mice. Data show the mean ± SEM (n = 4/group). (D) Expression of NK1.1, NKp46, IL-7R, c-kit, CD25, CD44, CCR6 and RORγt on hepatic ILCs from RAG-2^−/− mice. Data are representative of three independent experiments.</p

Protective role of RORγt-dependent hepatic ILCs in acute hepatitis.

<p>RORγt-dependent hepatic ILCs attenuate acute liver injury through the production of IL-22.</p

RAG-2^−/− × RORγt^−/− mice develop more severe CCl₄-induced hepatitis than RAG-2^−/− mice.

<p>(A) IL-22 expression in hepatic MNCs of CCl₄-treated RAG-2^−/− mice. IL-22 mRNA expression in the indicated hepatic MNCs subsets was measured by RT-qPCR and normalized relative to β-actin expression. Error bars represent SEM of triplicate samples. (B) Intracellular staining of IL-22, IL-17A and IFN-γ of hepatic ILCs. Hepatic MNCs were collected from RAG-2^−/− or RAG-2^−/− × RORγt^−/− mice 12 h after CCl₄ injection, and stimulated in the presence or absence of IL-23 for 3 h. Data are representative of eight mice in each group. Mean percentages of cytokine producing cells in the ILC subset. Data show the mean ± SEM (n = 8/group). Data are representative of two independent experiments.</p

Depletion of ILCs with anti Thy-1 mAb exacerbates CCl₄-induced hepatitis in RAG-2^−/− mice.

<p>(A) Serum ALT levels of olive oil or CCl₄-injected RAG-2^−/− mice, or anti-Thy1 mAb and CCl₄ (Thy1 CCl₄) or anti-NK1.1 mAb and CCl₄ (NK1.1 CCl₄)-treated RAG-2^−/− mice 12 h after CCl₄ or olive oil injection. Data show the mean ± SEM (n = 9/group). (B) Representative photomicrographs of H&E-stained liver from each group and pathological score. (C) CD11b/CD11c staining of hepatic MNCs from each group 12 h after CCl₄ or olive oil treatment. Ratio and absolute number of CD11b⁺ macrophage in the hepatic MNCs. Data show the mean ± SEM (n = 5/group). (D) TCRβ/NK1.1 staining of hepatic MNCs. Ratio and absolute number of NK cells in the hepatic MNCs. Data show the mean ± SEM. (E) SCA-1/Thy1 staining of Lin⁻ hepatic MNC. Ratio of ILCs in the Lin⁻ fraction and absolute number of ILCs in each group. Data show the mean ± SEM. Data are representative of three independent experiments.</p

RAG-2^−/− × RORγt^−/− mice develop more severe CCl₄-induced hepatitis than RAG-2^−/− mice.

<p>(A) Serum ALT levels of RAG-2^−/− or RAG-2^−/− × RORγt^−/− mice 12 h after CCl₄ (RAG-2^−/− n = 9, RAG-2^−/− × RORγt^−/− n = 8) or olive oil injection (n = 8/group). Data show the mean ± SEM. (B) Representative photomicrographs of H&E-stained liver from each group and pathological score. (C) CD11b/CD11c staining of hepatic MNCs from RAG-2^−/− or RAG-2^−/− × RORγt^−/− mice 12 h after CCl₄ (RAG-2^−/− n = 5, RAG-2^−/− × RORγt^−/− n = 4) or olive oil treatment (n = 5/group). Ratio and absolute number of CD11b⁺ macrophages in the hepatic MNCs. Data show the mean ± SEM. (D) TCRβ/NK1.1 staining of hepatic MNCs. Ratio and absolute number of NK cells in the hepatic MNCs. Data show the mean ± SEM. (E) SCA-1/Thy1 staining of Lin⁻ hepatic MNCs. Ratio of ILCs in Lin⁻ fraction and absolute number of ILCs. (F) Cytokine mRNA expression in the hepatic MNCs from CCl₄-injected RAG-2^−/− or RAG-2^−/− × RORγt^−/− mice 12 h after CCl₄ injection was determined by RT-qPCR. Data show the mean ± SEM Cytokine mRNA expression in the hepatic MNCs from CCl₄-injected RAG-2^−/− or RAG-2^−/− × RORγt^−/− mice 12 h after CCl₄ injection was determined by RT-qPCR. Data show the mean ± SEM (n = 4/group). Data are representative of three independent experiments.</p

RORγt^−/− mice develop CCl₄-induced hepatitis.

<p>(A) Serum ALT levels of WT or RORγt^−/− mice 12 h after CCl₄ (WT n = 5, RORγt^−/− n = 4) or olive oil injection (n = 5/group). Data show the mean ± SEM. (B) Representative photomicrographs of H&E-stained liver from each group and pathological score. (C) CD11b/CD11c staining of hepatic MNCs from WT or RORγt^−/− mice 12 h after CCl₄ or olive oil treatment. Ratio and absolute number of CD11b⁺ macrophages in the hepatic MNCs. Data show the mean ± SEM. (D) TCRβ/NK1.1 staining of hepatic MNCs. Ratio and absolute number of NKT cells in the hepatic MNCs. Data show the mean ± SEM. Data are representative of two independent experiments.</p

Anti-asialo GM1 Ab treatment does not affect the degree of CCl₄-induced hepatitis or number of hepatic ILCs.

<p>(A) Serum ALT levels of olive oil- or CCl₄-injected RAG-2^−/− mice, or anti-asialo GM1 Ab and CCl₄ (asialo GM1 CCl₄) treated RAG-2^−/− mice 12 h after CCl₄ or olive oil injection. Data show the mean ± SEM (n = 5/group). (B) Representative photomicrographs of H&E-stained liver from each group and pathological score. (C) CD11b/CD11c staining of hepatic MNCs from each group 12 h after CCl₄ or olive oil treatment. Ratio and absolute number of CD11b⁺ macrophages in the hepatic MNCs. Data show the mean ± SEM. (D) TCRβ/NK1.1 staining of hepatic MNCs. Ratio and absolute number of NK cells in the hepatic MNCs. Data show the mean ± SEM. (E) SCA-1/Thy1 staining of Lin⁻ hepatic MNCs. Ratio of ILCs in the Lin⁻ fraction and absolute number of ILCs. Data show the mean ± SEM. Data are representative of two independent experiments.</p

Hepatic Mφ/cDCs cells under colitic conditions induce a Th1 inflammatory response.

<p>(<b>A</b>) FACS analysis of PDCA-1⁺CD11b⁻CD11c^int pDCs from the livers of WT (left column) mice. We also analyzed CD11b⁺CD11c⁻ Mφs from the livers of ConA-treated (middle) and IL-10^−/− (right) mice, respectively. Dead cells were excluded with 7AAD staining. (<b>B</b>) Proliferation of naïve CFSE-labeled splenic CD4⁺ T cells from OT-II mice, and co-cultured WT pDCs, ConA Mφs, or IL-10^−/− Mφs in the presence of OVA. Dead cells were excluded with 7AAD staining and CD4⁺ T cells gated on CD3⁺ CD4⁺ cells are shown (B and C). Data are representative of three independent experiments. (<b>C</b>) Intracellular IFN-γ and IL-17A expression in CD4⁺ T cells co-cultured with WT pDCs, ConA Mφs, or IL-10^−/− Mφs in the presence of OVA. Data are representative of three independent experiments. (<b>D</b>) Proportion of IFN-γ⁺IL-17A⁻, IFN-γ⁻IL-17A⁺, and IFN-γ⁺IL-17A⁺ cells among the Th cell population. (<b>E</b>) Cytokine concentrations in the culture supernatant of OT-II CD4⁺ T cells that were co-cultured with WT pDCs or ConA Mφs. Data are representative of three independent experiments. Each experiment was performed using duplicate samples. *<i>P</i><0.05.</p