Selective JAK inhibition decreases lymphocyte accumulation but differentially alters their parenchymal localization in the lung.

Aire−/− mice were treated for four weeks with selective JAK inhibitors; untreated Aire−/− and ruxolitinib-treated Aire−/− mice served as controls. Lung lymphocytes were analyzed by flow cytometry. (A) Absolute numbers of CD45+ leukocytes and (B) TCRαβ+ T cells. (C-D) Frequency (of total CD45+) and absolute numbers of CD4+ and CD8+ T cells. (E-F) Representative flow cytometry plots showing parenchymal (CD45 i.v.−) or intravascular (CD45 i.v.+) CD4+ and CD8+ T cells. (G-H) Frequency and absolute numbers of parenchymal and intravascular CD4+ and CD8+ T cells in Aire+/+and untreated Aire−/− mice. (I-J) Frequency and absolute numbers of parenchymal and intravascular CD4+ and CD8+ T cells in untreated and JAK inhibitor-treated Aire−/− mice. For total CD45+, TCRαβ+, CD4+ T, and CD8+ T cells: n = 15-18 mice per group from four independent experiments. For parenchymal/intravascular ratio analyses: n = 11-15 mice per group from three independent experiments. Statistical comparisons between Aire+/+ and untreated Aire−/− mice were performed using the Mann-Whitney test. Comparisons among groups were performed using one-way ANOVA or Kruskal-Wallis test with Dunn’s multiple comparisons to untreated Aire−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Selective JAK inhibition decreases TCRγδ+ T cell, innate lymphoid cell, and non-lymphoid cell accumulation in the lung.

Aire−/− mice were treated for four weeks with selective JAK inhibitors; untreated Aire−/− and ruxolitinib-treated Aire−/− mice served as controls. Lung lymphocytes were analyzed by flow cytometry. (A) Absolute numbers of TCRγδ+ T cells, (B) innate lymphoid cells (CD90.2+TCRβ−, TCRγ−), and (C) non-lymphoid immune cells (CD45+CD90.2−). n = 15-18 mice per group from four independent experiments. Statistical comparisons were performed using Kruskal-Wallis test with Dunn’s multiple comparisons to the untreated Aire−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Selective JAK3 inhibition uncouples lymphocyte burden from IFN-γ-dominant signaling.

Untreated Aire−/− mice and Aire−/− mice treated for four weeks with selective JAK1i, JAK2i, or JAK3i were analyzed. Lungs were processed for intracellular cytokine staining, qPCR, ELISA, and immunoblot analyses. (A-B) Representative flow cytometry plots showing IFN-γ production by CD4+ and CD8+ T cells. (C-D) Frequency (of total CD4+ and CD8+ T cells) and absolute numbers of IFN-γ+CD4+ and IFN-γ+CD8+ T cells in the lung. (E-F) Relative Ifng mRNA expression and IFN-γ protein concentrations in lung homogenates. (G) Relative Stat1 mRNA expression. (H) Representative immunoblots of phospho-STAT1 (pSTAT1), total STAT1, and GAPDH. (I-J) Quantification of total STAT1 and phospho-STAT1 normalized to GAPDH. For IFN- γ+CD4+ and IFN-γ+CD8+ T cells: n = 9-14 mice per group from four independent experiments. For Ifng and Cxcl9 mRNA: n = 15-22 mice per group from four independent experiments. For Stat1_mRNA: n = 10-17 mice per group from three independent experiments. For CXCL9 protein: n = 5-10 mice per group from two independent experiments. For STAT1 and pSTAT1 immunoblots: n = 15-22 mice per group from four independent experiments). Statistical analyses were performed using one-way ANOVA or Kruskal-Wallis test with Dunn’s multiple comparisons to the untreated Aire−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

JAK2 inhibition most effectively ameliorates autoimmune tissue injury in the lungs.

Untreated Aire−/− mice and Aire−/− mice treated for four weeks with selective JAK1i, JAK2i, or JAK3i were analyzed. Lungs were harvested and processed for histopathological assessment. (A) Representative hematoxylin and eosin (H&E)-stained sections of lung (0.5x; scale bar = 5 mm) from untreated Aire−/− mice and JAK inhibitor-treated Aire−/− mice. (B) Quantification of residual tissue inflammation in lungs expressed as percentage of inflammation relative to untreated Aire−/− mice. All samples were normalized to untreated Aire−/− controls within each independent experiment. n = 7-8 mice per group from 3 independent experiments. Statistical comparisons were performed using Kruskal-Wallis test with Dunn’s multiple comparisons to the untreated Aire−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Histopathological analysis of lungs with QuPath.

Hematoxylin and eosin (H&E)-stained sections of lungs from all groups were scanned, and a pixel threshold for the outline of lung tissue and areas of inflammation was created in QuPath. Panel A shows a representative lung section stained with H&E before pixel thresholds were applied. Panel B shows an inset of panel A before analysis. Panel C shows the inset with annotations created with the pixel thresholds for lung tissue (green outline) and areas of inflammation (black outline).

JAK2 inhibition most effectively ameliorates autoimmune tissue injury in the salivary glands.

Untreated Aire−/− mice and Aire−/− mice treated for four weeks with selective JAK1i, JAK2i, or JAK3i were analyzed. Salivary glands were harvested and processed for histopathological assessment. (A) Representative hematoxylin and eosin (H&E)-stained sections of salivary glands (0.75x; scale bar = 2.5 mm) from untreated Aire−/− mice and JAK inhibitor-treated Aire−/− mice. (B) Quantification of residual tissue inflammation in salivary glands expressed as percentage of inflammation relative to untreated Aire−/− mice. All samples were normalized to untreated Aire−/− controls within each independent experiment. n = 17-25 mice per group from six independent experiments. Statistical comparisons were performed using Kruskal-Wallis test with Dunn’s multiple comparisons to the untreated Aire−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

JAK2 inhibition most effectively ameliorates autoimmune tissue injury in the eyes.

Untreated Aire−/− mice and Aire−/− mice treated for four weeks with selective JAK1i, JAK2i, or JAK3i were analyzed. Eyes were harvested and processed for histopathological assessment. (A) Representative hematoxylin and eosin (H&E)-stained sections of eyes (2.5x; scale bar = 1 mm) from untreated Aire−/− mice and JAK inhibitor-treated Aire−/− mice. The insets provide a greater magnification of the ciliary body area of the inflamed eye (10x; scale bar = 250 µm). (B) Quantification of residual tissue inflammation in eyes expressed as percentage of inflammation relative to untreated Aire−/− mice. All samples were normalized to untreated Aire−/− controls within each independent experiment. n = 17-25 mice per group from six independent experiments. Statistical comparisons were performed using Kruskal-Wallis test with Dunn’s multiple comparisons to the untreated Aire−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.