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. 2025 May 5;222(5):e20240379.
doi: 10.1084/jem.20240379. Epub 2025 Mar 12.

KLF family members control expression of genes required for tissue macrophage identities

Kathleen Pestal  1 , Leianna C Slayden  1   2 , Gregory M Barton  1   2
Affiliations

KLF family members control expression of genes required for tissue macrophage identities

Kathleen Pestal et al. J Exp Med. .

Abstract

Tissue-resident macrophages adopt distinct gene expression profiles and exhibit functional specialization based on their tissue of residence. Recent studies have begun to define the signals and transcription factors that induce these identities. Here we describe an unexpected and specific role for the broadly expressed transcription factor Krüppel-like factor 2 (KLF2) in the development of embryonically derived large cavity macrophages (LCMs) in the serous cavities. KLF2 not only directly regulates the transcription of genes previously shown to specify LCM identity, such as retinoic acid receptors and GATA6, but also is required for induction of many other transcripts that define the identity of these cells. Our results suggest that KLF4 may similarly regulate the identity of alveolar macrophages in the lung. These data demonstrate that broadly expressed transcription factors, such as group 2 KLFs, can play important roles in the specification of distinct identities of tissue-resident macrophages.

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Conflict of interest statement

Disclosures: The authors declare no competing interests exist.

Figures

Figure 1.
Figure 1.
Impaired development of LCMs in myeloid-specific KLF2-deficient mice. (A) Flow cytometry of cells in peritoneal cavity lavage from the indicated mice, gated on live, single, CD3−B220−CD19− cells. Numbers adjacent to gates indicate the percentage of total live events for each gate. (B) Bar graphs depicting the percentage of total live cells and total number of cells for the following populations (representative gates shown in A) as measured by flow cytometry: CD11b+F4/80+, SCM (CD11b+F4/80+MHCIIhiDNAM-1hi), LCMs (CD11b+F4/80+MHCII−DNAM-1−), ICAM2+GATA6+ LCM (CD11b+F4/80+MHCII−DNAM-1−ICAM2+GATA6+), and transitional (CD11b+F4/80+MHCIImidDNAM-1mid). (C) Dimensionality reduction via UMAP of combined CD11b+F4/80+ cells from LysM+/+Gata6fl/fl and LysM+/+Klf2fl/fl (gated together as LysM+/+ controls), LysMCre/+Gata6fl/fl, or LysMCre/+Klf2fl/fl mice, downsampled to normalize cells per genotype. The resulting projection was overlayed with supervised gates for LCMs, transitional, and SCMs as shown in A. The upper panel shows all cells, while the lower panels show only cells from the indicated genotypes. (D) Heatmaps of defining marker expression for SCM, LCM, and transitional cell clusters overlaid onto the UMAP projections from C. Data in A are from one experiment representative of six independent experiments; data in B are combined from six independent experiments, LysM+/+Gata6fl/fl (n = 9), LysMCre/+Gata6fl/fl (n = 6), LysM+/+Klf2fl/fl (n = 5), LysMCre/+Klf2fl/fl (n = 7), CX3CR1-Cre−Klf2fl/fl (n = 4), and CX3CR1-Cre+Klf2fl/fl (n = 5). Significance determined by ordinary two-way ANOVA with multiple comparisons and Šidák’s correction. Asterisks denote: ****P < 0.0001, ***P = 0.0006, **P = 0.0021, and *P = 0.033.
Figure S1.
Figure S1.
Effects of KLF2 deficiency on myeloid cells in the pleural and peritoneal cavities. (A) Flow cytometry gating strategy for cavities. (B) Flow cytometry of cells in pleural cavity lavage from the indicated mice, gated on live, single, CD3−B220−CD19− cells. Numbers adjacent to gates indicate the percentage of total live events for each gate. (C) Bar graphs depicting the percentage of total live cells and total number of cells for the following populations in the pleural cavities (representative gates shown in A) as measured by flow cytometry: CD11b+F4/80+, SCM (CD11b+F4/80+MHCIIhiDNAM-1hi), LCMs (CD11b+F4/80+MHCII−DNAM-1−), ICAM2+GATA6+ LCMs (CD11b+F4/80+MHCII−DNAM-1−ICAM2+GATA6+), and transitional (CD11b+F4/80+MHCIImidDNAM-1mid). (D) Bar graphs depicting the percent of total and total number of monocytes (CD11b+F4/80−Ly6C+MHCII+ or MHCII−) and neutrophils (CD11b+F4/80−, Ly6G+), as measured by flow cytometry. (E and F) FlowSOM and ClusterExplorer-derived unsupervised cluster identification (E) and heatmap of marker intensities defining identified clusters (F). Significance determined by ordinary two-way ANOVA with multiple comparisons and Šidák’s correction. Asterisks denote: ****P < 0.0001, ***P = 0.0006, **P = 0.0021, and *P = 0.033.
Figure 2.
Figure 2.
Inability of KLF2-deficient progenitors to develop into LCMs is cell intrinsic. (A) Frequency of cells of the indicated genotypes within SCM, LCM, and monocyte and resident macrophage subsets in the indicated tissues from radiation chimeras generated with LysM+/+Gata6fl/fl:B6.SJL or LysMCre/+Gata6fl/fl:B6.SJL mixtures of donor bone marrow into CD45.1 × CD45.2 hosts. (B) Frequency of cells of the indicated genotypes within SCM, LCM, and monocyte and resident macrophage subsets in the indicated tissues from radiation chimeras generated with LysM+/+Klf2fl/fl:B6.SJL or LysMCre/+Klf2fl/fl:B6.SJL mixtures of donor bone marrow into CD45.1 × CD45.2 hosts. Data in A and B are from one experiment representative of two independent experiments and presented as mean with SD of three to four chimeric animals per group. Significance determined by Fisher’s exact test of relative contributions of each genotype in B6.SJL:LysMCre/+ chimeras compared with B6.SJL:LysM+/+ chimeras. Asterisks denote: ****P < 0.0001, ***P = 0.0006, and *P = 0.033.
Figure S2.
Figure S2.
Identification of tissue myeloid populations and transferred cell phenotypes. (A–D) Flow cytometry gating for (A) liver, (B) lung, (C) small intestine lamina propria, and (D) spleen. (E) Flow cytometry gating for transferred cell experiments. (F) Representative flow cytometry analysis showing marker expression on purified bone marrow monocytes from LysM+/+Klf2fl/fl or LysMCre/+Klf2fl/fl mice 7 days after transfer into a precleared peritoneal cavity of congenitally marked (CD45.1 × CD45.2) mice. (G) Bar graph depicting the percentage of total transferred cells of gates from F. Data from three combined experiments of transferred bone marrow monocytes into peritoneal cavities. Significance determined by ordinary two-way ANOVA with multiple comparisons and Šidák’s correction.
Figure 3.
Figure 3.
KLF2 is required for macrophages to acquire expression of LCM identity genes. (A) Schematic of experimental design for transfer of BMMs into the peritoneal cavity of recipient mice for subsequent harvest and analysis 7 days later. (B and C) Representative flow cytometry analysis (B) and compiled results (C) of LysM+/+Klf2fl/fl or LysMCre/+Klf2fl/fl BMMs recovered on day 7 after transfer or prior to transfer (day 0). Data in B are gated on live, single, CD45.2+ cells. (D) Dimensionality reduction via UMAP of LysM+/+Klf2fl/fl- or LysMCre/+Klf2fl/fl-transferred BMMs gated on live, single, CD45.2+ cells from four mice, downsampled to equal cells per genotype, barcoded, and concatenated. The projection was then overlayed with supervised gates based on genotype. (E and F) Volcano plots of DEG comparing LysM+/+Klf2fl/fl to LysMCre/+Klf2fl/fl BMMs before (E) or after (F) transfer. Genes with a log2 fold change >|1| are colored grey. Genes with a P value of −log10 (P value) >4 are above the horizontal black line. Genes meeting both criteria are colored: teal, enriched in LysM+/+Klf2fl/fl; or pink, enriched in LysMCre/+Klf2fl/fl. Genes meeting neither criterion are colored black. (G) GSEA of unfiltered expressed genes between day 7 transferred LysM+/+Klf2fl/fl BMMs relative to LysMCre/+Klf2fl/fl BMMs. Gene sets for LCMs or SCMs were generated from DEG analysis of sorted WT LCMs or SCMs relative to each other. (H) Scatter plot of RNA-Seq expression data presenting the values of log2 fold change for shared DEGs in sorted small versus LCM (x axis) versus the DEGs between the day 7 sorted LysM+/+Klf2fl/fl versus LysMCre/+Klf2fl/fl transferred BMMs (y axis). Data in B are from one experiment representative of three independent experiments; data in C and D are combined from three independent experiments, LysM+/+Klf2fl/fl (n = 4), LysMCre/+Klf2fl/fl (n = 4) on day 0 and LysM+/+Klf2fl/fl (n = 4), LysMCre/+Klf2fl/fl (n = 4) on day 7. Significance determined by ordinary two-way ANOVA with multiple comparisons and Šidák’s correction. Asterisks denote: ***P = 0.0006 and *P = 0.033. Sequencing (E–H) was performed once with LysM+/+Klf2fl/fl (n = 3), LysMCre/+Klf2fl/fl (n = 3) on day 0, LysM+/+Klf2fl/fl (n = 3), and LysMCre/+Klf2fl/fl (n = 3) on day 7. FC; fold change.
Figure S3.
Figure S3.
KLF2 expression and gene regulation of surface markers in LCMs. (A) Representative flow cytometry of GFP expression in LCMs (gated on live, single cells, CD3−CD19−B220−F4/80+CD11b+DNAM1−MHCII−ICAM2+) from GFP-KLF2 mice. (B) Genome browser tracks of anti-GPFKLF2 (pink) or IgG control (grey) CUT&RUN peaks from selected LCM genes.
Figure 4.
Figure 4.
Lineage determining factors are directly regulated by KLF2 in LCMs. (A) Genome browser tracks of three independent samples showing anti-GFP KLF2 (pink) or IgG control (grey) CUT&RUN peaks from LCMs at the indicated genetic loci. Significant peaks (representing the top 1% of peak regions identified by SEACR) are indicated by blue bars. ENCODE cCREs (candidate cis-regulatory elements) are highlighted to indicate regulatory features that overlap with the peak region (red: promoter; orange: proximal enhancer; yellow: distal enhancer). All genes are shown 5′ → 3′, and peak signals of 0–10 are shown on the y axis. (B) Known motif enrichment and de novo motif analysis of LCM KLF2 CUT&RUN peaks compared with the IgG control. The top ranked KLF and ETS matches are shown along with the significance score (−logP) and enrichment of each motif, indicated as frequency of the motif within GFP peaks (target) relative to frequency of the motif within IgG peaks (Bkgrd). (C) Model of proposed function of KLF2 in LCMs. Data in A are from three samples of total LCMs (F4/80+CD11b+MHCII−DNAM-1−) split in half for anti-GFP or IgG control.
Figure 5.
Figure 5.
Ectopic expression of KLF2 confers LCM phenotype and transcriptional identity in vitro. (A) Graphical representation of experimental design for the RV OE of transcription factors in in vitro–derived BMMs. (B) GSEA of unfiltered expressed genes between KLF2 OE BMMs and EV BMMs. Gene sets for LCMs and SCMs are derived from DEG analysis of sorted WT LCMs or SCMs relative to each other. (C) Bar graphs depicting the log2 fold change of RNA-Seq data from transcription factor–transduced BMMs relative to EV of the indicated LCM relevant genes. (D) Dimensionality reduction via UMAP of live, single, virus+ cells from EV, KLF2 OE, or GATA6 OE cells and downsampled to normalize cells per genotype, which were barcoded and concatenated. The left panel depicts all concatenated events, the middle panel shows the overlay of untreated cells transduced as indicated, and the right panel shows the overlay of ATRA treated cells transduced as indicated. (E) UMAP plots generated as described in D showing expression intensities of the indicated markers. (F) GATA6 expression measured by intracellular staining and flow cytometry of the indicated BMM genotypes transduced with KLF2 OE RV with or without ATRA treatment. RNA-Seq was performed on samples generated in three independently performed transduction experiments (B and C). Data are from one experiment representative of three independent experiments (D–F). FC; fold change.
Figure S4.
Figure S4.
Similarity of shared DEGs in KLF2 OE BMMs and LCMs. Scatter plot of RNA-Seq expression data presenting the values of log2 fold change for shared DEGs in sorted LCMs versus SCMs (x axis) versus the DEGs between the EV versus KLF2-transduced BMMs (y axis).
Figure 6.
Figure 6.
KLF4 is required for AM development and identity. (A) Flow cytometry of cells in lungs from the indicated mice, gated on live, single, CD45+CD3−B220−CD19− cells. Numbers adjacent to gates indicate percentage of total live events for each gate. (B) Bar graphs depicting the percentage number of total live cells of the following lung populations (representative gates show in in Fig. S2 B) as measured by flow cytometry: CD11c+CD11b−Siglec-F+CD24− (AMs), CD11b+MHCII+CD24−CD64+ (interstitial macrophages, IM), MHCII−CD11b+CD24+Siglec-F+ (eosinophils, eos), MHCII−CD11b+CD24+Ly6G+ (neutrophils, neuts), MHCII−CD11b+CD64+Ly6C+ or Ly6Cmid/− (monocytes), CD11c+CD11b−Siglec-F−CD24+ (cDC1), and CD11b+MHCII+CD24+CD64− (cDC2). (C) Dimensionality reduction via UMAP of AMs was performed after downsampling to equivalent cells per genotype, barcoding, and concatenation. The resulting projection is color coded to distinguish WT from Klf4-deficient cells. (D) Heatmaps of defining marker expression for AMs from LysM-Cre (top) or CD11c-Cre (bottom) mice overlaid onto the UMAP projections from C. The WT or KO cell clusters based on C are indicated. Data in A are representative of four independent experiments. Data in B are combined from four independent experiments. Data in C and D are combined from three experiments. Significance determined by ordinary two-way ANOVA with multiple comparisons and Šidák’s correction; Asterisks denote: ****P = < 0.0001, ***P = 0.0006, and **P = 0.0021.
Figure S5.
Figure S5.
KLF4 dependency in BALF AM development and gene expression in AMs. (A) Representative flow cytometry of BALF AMs (live, single, CD19−CD11c+CD11b−SiglecF+CD24−) from the indicated mice. Numbers adjacent to gates indicate the percentage of total live events for each gate. (B) Bar graphs depict the percent of total and total number of the following BALF populations (representative gates shown in Fig. S2 B): CD11c+CD11b−SiglecF+CD24− (AMs); CD11b+MHCII+CD24−CD64+ (interstitial macrophages, IM); MHCII−CD11b+CD24+SiglecF+ (eosinophils, eos); MHCII−CD11b+CD24+Ly6G+ (neutrophils, neuts); CD11c+CD11b−SiglecF−CD24+ (cDC1); and CD11b+MHCII+CD24+CD64− (cDC2). (C) Volcano plot of expressed genes comparing sorted LysM+/+Klf4fl/fl to LysMCre/+Klf4fl/fl AM. Genes with a log2 fold change >|1| are colored grey. Genes with a P value of −log10 (P value) >6 are above the horizontal black line. Genes meeting both criteria are colored: teal, enriched in LysM+/+Klf4fl/fl; purple, enriched in LysMCre/+Klf4fl/fl. Genes meeting neither criterion are colored black. (D) Top 25 GO term analysis results of genes upregulated in LysM+/+Klf4fl/fl AMs. (E) Top 25 GO term analysis results of genes upregulated in LysMCre/+Klf4fl/fl AMs. Bulk RNA-Seq (E) was performed from one set of samples containing two biological replicates of each genotype. Asterisks denote: ****P < 0.0001.
Figure 7.
Figure 7.
The requirement for KLF4 in AM development is cell intrinsic. Frequency of cells of the indicated genotypes within lung AMs, interstitial macrophages, and monocyte and resident macrophage subsets in other tissues from radiation chimeras generated with LysM+/+Klf4fl/fl:B6.SJL or LysMCre/+Klf4fl/fl:B6.SJL mixtures of donor bone marrow into CD45.1 × CD45.2 hosts. Data in A and B are from one experiment representative of two independent experiments and presented as mean with SD of three to four chimeric animals per group. Significance determined by Fisher’s exact test of relative contributions of each genotype in B6.SJL:LysMCre/+Klf4fl/fl chimeras compared with B6.SJL:LysM+/+Klf4fl/fl chimeras. Asterisks denote: ****P < 0.0001 and *P = 0.033.
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