Fra-2 Is a Dominant Negative Regulator of Natural Killer Cell Development

Abstract

Natural killer (NK) cells play an important role in recognizing and killing pathogen-infected or malignant cells. Changes in their numbers or activation can contribute to several diseases and pathologies including systemic sclerosis (SSc), an autoimmune disease characterized by inflammation and tissue remodeling. In these patients, increased expression of the AP-1 transcription factor, Fra-2 was reported. In mice ectopic overexpression of Fra-2 (TG) leads to SSc with strong pulmonary fibrosis, pulmonary hypertension, and inflammation. Analysis of the underlying immune cell profile in the lungs of young TG mice, which do not yet show any signs of lung disease, revealed increased numbers of eosinophils and T cells but strongly reduced NK numbers. Therefore, we aimed to identify the cause of the absence of NK cells in the lungs of these mice and to determine the potential role of Fra-2 in NK development. Examination of inflammatory cell distribution in TG mice revealed similar NK deficiencies in the spleen, blood, and bone marrow. Deeper analysis of the WT and TG bone marrow revealed a potential NK cell developmental defect beginning at the preNKP stage. To determine whether this defect was cell-intrinsic or extrinsic, mixed bone marrow chimera and in vitro differentiation experiments were performed. Both experiments showed that the defect caused by Fra-2 was primarily cell-intrinsic and minimally dependent on the environment. Closer examination of surface markers and transcription factors required for NK development, revealed the expected receptor distribution but changes in transcription factor expression. We found a significant reduction in Nfil3, which is essential for the transition of common lymphoid cells to NK committed precursor cells and an AP-1 binding site in the promotor of this gene. In Summary, our data demonstrates that regulation of Fra-2 is essential for NK development and maturation, and suggests that the early NK dysfunction plays an important role in the pathogenesis of systemic sclerosis.

Keywords: AP-1; Fra-2; Natural killer (NK) cell; activator protein 1; differentiation; innate immunity.

Figure 1

Fra-2 TG mice display an altered immune profile already before onset of disease. (A) Multicolour immunofluorescence of lung tissue of wild-type (WT) and Fra-2 TG mice, aged 8 and 16 weeks. White: CD45, Green: COL1, Red: aSMA, blue: DAPI. Scale bar 50µm. (B) Principal component analysis of the inflammatory cell profile in the lungs of 8 weeks old Fra-2 TG and WT mice as determined by flow cytometry. (C) Heatmap highlighting the changes in the abundance of immune cells. (D) tSNE plots of concatenated CD45⁺ immune cells; overlayed are populations of myeloid (left) and lymphoid (right) immune cells. (E) Significantly altered populations of immune cells in lung tissue. Statistical differences were determined with a Wilcoxon Rank Sum test, ^nsp>0.05, *p<0.05, **p<0.01, ***p<0.001, n=8.

Figure 2

Altered recruitment factor expression and NK cell turnover in the lungs of Fra-2 TG mice (A) Heatmap of mRNA levels of NK recruiting chemokines in the lungs of 8 week Fra2-TG and WT mice. (B) Flow cytometry analysis of NK cell chemokine receptors expression. (C) Representative plots of intracellular flow cytometry staining of Cleaved Caspase and the proliferation marker Ki67. (D) Respective quantification. Statistical differences were determined with a Wilcoxon Rank Sum test, *p<0.05, **p<0.01, n=8.

Figure 3

Fra-2 TG mice display reduced numbers of NK cells in all tissues. Flow cytometry analysis of lymphocytes in the blood (A), spleen (B) and bone marrow (C) of wild-type (WT) and Fra-2 TG mice. tSNE plots of concatenated CD45+ from the respective tissue with overlaid gated cell populations. Statistical differences were determined with a Wilcoxon Rank Sum test, ^nsp>0.05 *p<0.05, **p<0.01, ***p<0.001, n≥5.

Figure 4

Fra-2 overexpression leads to a cell-intrinsic defect in NK development. (A) Schematic representation of mixed bone marrow (BM) chimera experiment. BM was extracted from WT and Fra-2 TG mice, mixed 1:1 and injected into irradiated WT mice, and mice analysed six weeks post transfer. (B) Histograms of the GFP signal in total immune cells in different compartments of WT mice post BM reconstitution; y-axis represents percentage viable counts in the bone marrow (BM), and percentage CD45⁺ counts in the spleen and lung and (C) their quantification. (D) tSNE plots of concatenated CD45⁺ cells in the lung and overlaid lymphocyte populations split according to GFP positivity (negative = cells of WT origin and positive = TG origin). (E) Relative proportion of CD45⁺/GFP^- (blue) and CD45⁺/GFP⁺ (red) cells in the lungs of WT recipient mice, line shows median. Data was log₁₀₊₁ transformed to allow all cells to presented on the same axis. (F) Quantification of NK cell GFP positivity in the BM, spleen and lungs of WT mice. (G) schematic overview of the NK cell in vitro differentiation experiments from bone marrow precursors. NK precursors were enriched by magnetic depletion of lineage positive cells (CD3, B220, Gr1, Ter119, CD11c and CD49b positive cells) and stimulated with FLT3L, IL-7 and IL-15 to stimulate NK cell differentiation. Amount of in vitro generated NK cells over time, shown as percentage viable (G) or absolute cell count (H). Statistical differences in (C, F) were determined with a Wilcoxon Rank Sum test, and (H, I) using mixed effects models with Bonferroni’s multiple comparisons test. *p<0.05, ***p<0.001, ****p<0.0001, n≥5.

Figure 5

The defect caused by Fra-2 affects all NK committed precursors. (A) Schematic representation of surface marker expression and essential transcription factors during NK cell development in the bone marrow. (B–D) Flow cytometry analysis of NK cells in bone marrow of WT and Fra-2 TG mice. (B) Quantification of NK precursors in the bone marrow. (C) Surface Marker expression on NK precursors. (D) Quantification of NFIL3, Ets1 and Tcf1 on NK precursors via flow cytometry; Z-scored mean fluorescent intensity are shown, with a line at the median. (E) AP-1 consensus sequence. (F) In silico transcription factor binding site analysis for AP-1 in the promotor of Nfil3. Statistical differences were determined in (B) with a Wilcoxon Rank Sum test and in (D) with mixed models using mouse genotype and cell type as fixed factors and the individual mouse as a random factor, ^nsp>0.05 *p<0.05, **p<0.01, ****p<0.0001.