Invariant NKT Cells Drive Hepatic Cytokinic Microenvironment Favoring Efficient Granuloma Formation and Early Control of Leishmania donovani Infection

The development of inflammatory granulomas around infected Kupffer cells is necessary for hepatic parasite clearance during visceral leishmaniasis. Invariant NKT (iNKT) cells are predominant T cells in the mouse liver and can synthesize large quantities of IL-4 and IFN-γ, two cytokines involved in granuloma formation. This study analyzed the role of iNKT cells in the hepatic immune response during Leishmania donovani infection, using a murine model of wild-type (WT) and iNKT cell-deficient (Jα18-/-) C57BL/6 mice sacrificed 15, 30 or 60 days post-infection. We recorded hepatic parasite loads, cytokine expression, and analyzed granulomatous response by immunohistochemistry and hepatic immune cell infiltration by flow cytometry. Whereas WT animals rapidly controlled the infection and developed an inflammatory response associated with a massive influx of iNKT cells observed by flow cytometry, Jα18-/- mice had significantly higher parasitic loads on all time points. This lack of control of parasite burden was associated with a delay in granuloma maturation (28.1% of large granulomas at day 60 versus 50.7% in WT). Cytokine transcriptome analysis showed that mRNA of 90/101 genes encoding chemokines, cytokines and their receptors, was underexpressed in Jα18-/- mice. Detection of IL-4 and TNF-α by ELISA in liver extracts was also significantly lower in Jα18-/- mice. Consistent with flow cytometry analysis, cytokinome profile in WT mice showed a bias of expression towards T cell-chemoattractant chemokines on D15, and displayed a switch towards expression of granulocytes and/or monocytes -chemoattractant chemokines on D60. In Jα18-/- mice, the significantly lower expression of CXCL5, MIP-2 and CCL2 mRNA was correlated with a defect in myeloperoxidase positive-cell attraction observed by immunohistochemistry and with a lower granulocyte and monocyte infiltration in the liver, as shown by flow cytometry. These data indicate that iNKT cells play a role in early and sustained pro-inflammatory cytokine response warranting efficient organization of hepatic granulomas and parasite clearance

Evaluation de l'expression des cytokératines 8 et 14 dans les tissus épithéliaux normaux de l'espèce canine

TOULOUSE3-BU Santé-Centrale (315552105) / SudocTOULOUSE-EN Vétérinaire (315552301) / SudocSudocFranceF

Phenotypic characterization of the iNKT cell infiltrate in the liver of WT mice on day 15 after infection with <i>L.donovani.</i>

<p>Hepatic cell infiltration of C57BL/6 WT was analyzed by flow cytometry using anti-CD3-V500, anti-NK1.1-PerCP-Cy-5.5, anti-TCRβ-V450, anti TCR γδ-FITC, and αGalCer/CD1d tetramer-APC, anti-CD4-PE-Cy7, anti-CD8-APC-Cy7. (A) Gating strategy allowing to estimate the low percentage of NK1.1- and NK1.1+ cells among TCRβ+/αGalCer/CD1d tetramer+ cells. (B) Gating strategy and evaluation of the percentage of γδ-T cells and non iNKT cells among CD3+/NK1.1+. (C) Percentage of CD4+ and CD4-/CD8- cells among iNKT cells (TCRβ+/αGalCer/CD1d tetramer+). This panel is representative of three independent experiments.</p

Jα18^-/- mice display a qualitatively and quantitatively impaired hepatic granulomatous response after infection with <i>L.donovani</i>.

<p>Quantification of granuloma formation in the liver of <i>L. donovani</i> infected WT and Jα18^-/-mice by microscopic examination of tissue sections stained with HES, at D15, D30 and D60 post-infection. Representative granuloma foci from WT mice at D15 (A) and D60 (B), and from Jα18^-/- mice at D15 (C) and D60 (D) (×100 magnification). (E) Total number of granulomas in 100 microscopic fields (×400) at various time points. (F) Relative percentage of large granulomas (>25 cells) detected in each group of mice. (G) Relative percentage of mature granulomas detected in each group of mice.</p

Quantification of myeloid cells in the liver of wild-type and Jα18^-/- mice.

<p>(A) Representative slides of small (Day 15) and large (Day 60) hepatic granulomas labeled by immunohistochemistry using an anti-MPO antibody, in WT and Jα18^-/- mice. (B) Proportion of MPO⁺ cells in granulomas, according to the size of the foci (i.e. small granulomas of <25 cells, and large granulomas >25 cells). (C) Quantification of the absolute number of MPO⁺ cells in granulomas in 100 consecutive microscopic fields (×400) at various time points. Data are the mean ± SEM for each group of mice (8–10 mice per group from two independent experiments). (D) Absolute quantification of myeloid cell infiltration in the liver of C57BL/6 WT and Jα18^-/- by flow cytometry on D60 after infection with <i>L. donovani</i>, using an anti-Gr1-FITC, anti-CD11b-PE-Cy7, and anti-F4/80-PE. Analysis was done on live cells after excluding lymphocyte cells. 10⁶ cells of liver homogenates were labeled and data were analyzed on 50.000 events. Data are the mean ± SEM of four mice per group. (E) Quantification of MPO mRNA expression in the liver of WT and Jα18^-/- mice. Data are the mean±SEM for each group of mice (8–10 mice per group from two independent experiments).</p

iNKT cells are recruited in the liver of WT mice on D15 after infection with <i>L.donovani</i>.

<p>Hepatic cell infiltration of C57BL/6 WT and Jα18^-/- mice was analyzed by flow cytometry 15 days after infection with <i>L. donovani</i>, using an anti-Gr1-FITC, anti-CD11b-PE-Cy7, anti-CD11c-APC, anti-CD4-PB, anti-CD3-FITC, anti-NK1.1-PerCP-Cy-5.5, and anti-CD8-APC-Cy7, and αGalCer-tetramer-PE. (A) Absolute quantification of hepatic NK (NK1.1+/CD3-), NKT (NK1.1+/CD3+), and TL (NK1.1-/CD3+) cell subsets in lymphocyte gated cells.(B) proportion of iNKT and non iNKT cell subsets in each group of mice in CD3+/NK1.1+ gated cells. (C) Absolute quantification of CD4⁺ and CD8⁺ cell subsets in gated NKT cells (CD3+/NK1.1+). (D) Absolute quantification of CD4⁺ and CD8⁺ cells in gated T lymphocytes (CD3+/NK1.1-). 10⁶ cells of liver homogenates were labeled and data were analyzed on 50.000 events. Data are the mean ± SEM of four mice per group.</p