The transcription factor Gata6 links tissue macrophage phenotype and proliferative renewal

Abstract

Tissue-resident macrophages are heterogeneous as a consequence of anatomical niche-specific functions. Many populations self-renew independently of bone marrow in the adult, but the molecular mechanisms of this are poorly understood. We determined a transcriptional profile for the major self-renewing population of peritoneal macrophages in mice. These cells specifically expressed the transcription factor Gata6. Selective deficiency of Gata6 in myeloid cells caused substantial alterations in the transcriptome of peritoneal macrophages. Gata6 deficiency also resulted in dysregulated peritoneal macrophage proliferative renewal during homeostasis and in response to inflammation, which was associated with delays in the resolution of inflammation. Our investigations reveal that the tissue macrophage phenotype is under discrete tissue-selective transcriptional control and that this is fundamentally linked to the regulation of their proliferation renewal.

Fig.1. Selective myeloid cell alterations in the peritoneum of mice with myeloid Gata6-deficiency

A. Representative Flow-cytometric and immunofluorescent assessment of peritoneal-resident macrophages from WT and Gata6-KO^mye mice. F4/80^high (arrowhead) and F4/80^low (arrows) macrophages are indicated. Fluorescent images were captured with a 40x objective lens, the scale bar is indicated and the images are representative of 4 mice per group (Fig. S9). B. Representative flow-cytometric analysis of peritoneal myeloid cell (CD11b⁺CD19⁻) composition of the Gata6-WT and Gata6-KO^mye mice. Percentages indicate typical proportions of the cell types of all peritoneal cells. C. Quantification of peritoneal myeloid cells in the Gata6-WT (black bars, n=9♂/7♀) and Gata6-KO^mye mice (white bars, n=5♂/5♀) analyzed by flow-cytometry in (A) and (B) above. Data represents the mean±SEM of mice pooled from two independent experiments and was analyzed by two-way ANOVA (Int, Interaction statistic; Gata6, Gata6 effects; Sex, sex effects). Abbreviations used in this figure: MØ, macrophage; Res, tissue-resident; Eos, eosinophil; DC, dendritic cell.

Fig.2. Gata6 is fundamental to the peritoneal-resident macrophage phenotype

A. Volcano plot showing the differential gene expression between peritoneal macrophages from Gata6-KO^mye mice and WT. Significantly 2-fold down-regulated (green) and up-regulated (magenta) probesets are indicated. B. Same volcano plot as (A), overlaid (orange) with the 215 peritoneal macrophage-selective ‘cluster 15’ (Cl. 15) probesets see (Fig. S1-2 (14)), which were significantly (below) disproportionately down-regulated in the absence of Gata6. C-E. Representation (C, E) and quantification (D, E) of flow-cytometric validation of the array data from (A). Data (analyzed by t-test) represents the difference in median fluorescent intensity (ΔMFI) between receptor-specific and isotype-control antibodies (mean±SEM) of individual mice (n=4) from one of two experiments (solid bars denote WT, and hatched denote Gata6-KO^mye mice).

Fig.3. Dysregulated peritoneal macrophage proliferation in the absence of Gata6

A-C. Representative density plots (A) gated on resident-peritoneal macrophages (Fig. 1A) showing proliferation (SG₂M) and polyploidy, which were quantified (B) and visualized (arrowheads, C), respectively. Data in (A) and (B) is derived from one of two independent experiments (Gata6-KO^mye, n=5; ‘Het’, n=4; WT, n=3), represented as mean±SEM and analyzed by one-way ANOVA (P value as indicated) with Bonferroni post tests. Immune fluorescence is representative of 5 mice. D. Examination of proliferative differences between the majority F4/80^low (○) KO and the WT F4/80^high (●) macrophages (see Fig. S9) within the same Gata6-KO^mye mice. Lines denote paired samples from the same mice (n=9), which were pooled from two similar experiments and analyzed by paired t-test. E. The impact of Gata6 deletion on proliferation was examined 7 days after delivery of Cre-expressing lentiviruses to Gata6^tm2.1Sad/J mice intraperitoneally. The proportion of cells in the SG₂M phases of cell-cycle were compared between F4/80^lowCre⁺ (○) and F4/80^highCre⁻ (●) macrophages. Data is represented as 3 independent experiments (Exp) with lines denoting paired samples from the same mice and analyzed as indicated. F. Gata6 mRNA expression compared by qPCR between peritoneal and pleural leukocytes. Data shows mean±SEM from one of two independent experiments in (129S6 mice, n=≥3/group) is normalized to reflect the number of resident macrophages. G. Similar analysis to (D) except using pleural macrophages. Data from two similar experiments were pooled and analyzed by a paired t-test. Abbreviations used in this figure: Res, resident; MØ, macrophage; * = P<0.05, ** = P<0.01, *** = P<0.001.

Fig.4. Impaired proliferative recovery of peritoneal macrophages during inflammation in the absence of Gata6

A-B. Quantification of the numbers (A) and proliferation (B) of Tim4⁺ macrophages at the indicated times after intraperitoneal zymosan injection (2×10⁶ particles). Data were pooled from two independent experiments with Gata6-WT (black bars) and Gata6-KO^mye (white bars) mice (n=5-11/group). C. Comparison of the SG₂M and G₁ stages of cell cycle in the Gata6-KO (Tim4⁺F4/80^low, white bars) and WT (Tim4⁺F4/80^high, black bars) macrophages within the Gata6-KO^mye mice from (A-B). D. Verification of the proliferative alterations shown in (C) by in vivo incorporation of 5-ethynyl-2′-deoxyuridine (EdU) (n=8 mice, pooled from 2 independent experiments). E. Analysis of inflammatory parameters in the resolution phase of a higher dose zymosan (2×10⁷ zymosan particles) peritonitis model comparing cell counts from WT (black bars) and Gata6-KO^mye (white bars) mice (Data pooled from 3 independent experiments (n=11-13/group). All data in this figure represents mean±SEM of individual mice and were examined by ANOVA (as indicated) with pairing as appropriate and Bonferroni post-tests, except (D), which was analyzed by paired t-test. Abbreviations used in this figure: MØ, macrophage; * = P<0.05, ** = P<0.01, *** = P<0.001.