Figure 1
From: MyD88-dependent dendritic and epithelial cell crosstalk orchestrates immune responses to allergens
Cd11c drives Cre-mediated recombination in lung DCs and AMs while Sftpc drives Cre-mediated recombination in ECs. (a) Fluorescent microscopic images of a PCLS prepared from progeny of Cd11c-cre x LoxP-Stop-LoxP-tdTomato cross. Top images show Tomato+ cells (red), CD103+ (green), Tomato+CD103+ DCs (yellow), and E-cadherin+ ECs (blue). Bottom images show Tomato+ cells (red), SIRP-1α+ (green), Tomato+SIRP-1α+ DCs (yellow), and E-cadherin+ ECs (blue). Low power images (left) include white squares marking an area also shown in higher power (right). (b) Real-time PCR analysis of Myd88 exon 3 in cDCs and AMs sorted from Myd88fx/fx (Cre–), Myd88fx/fx Cd11c-cre (Cd11c-cre+), Myd88fx/fx Sftpc-cre (Sftpc-cre+), and Myd88–/– mice at 6 h post-sensitization with OVA/FLA. Lung cDCs were gated on single cells that are CD11chi, nonautofluorescent, I-Ab+, Ly6C–, and CD88–. Data are representative of 3–6 separate cDC or AM sorts per genotype and are normalized to the housekeeping gene. (c) Fluorescent microscopic images of a frozen lung section from progeny of Sftpc-cre x LoxP-Stop-LoxP-tdTomato cross. Shown are a low power image of the whole lung, including the bifurcation of the trachea (top), and a high power image of a representative small airway, showing fluorescent ECs in the airway and alveoli (bottom). tdTomato cells (red), DAPI (blue), and Differential Interference Contrast (DIC)/ brightfield (grey). (d) Real-time PCR for the ‘floxed’ exon 3 region of the Myd88 gene in ECs sorted from Myd88fx/fx (Cre–), Myd88fx/fx Cd11c-cre (Cd11c-cre+), Myd88fx/fx Sftpc-cre (Sftpc-cre+), and Myd88–/– mice at 2 h post-sensitization with OVA/FLA. Data are representative of three different EC sorts per genotype. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AM, alveolar macrophage; cDC, conventional dendritic cells; EC, epithelial cells; FLA, flagellin; OVA, ovalbumin; PCLS, precision cut lung slices.
