Lipin-1 Contributes to IL-4 Mediated Macrophage Polarization

Abstract

Macrophage responses contribute to a diverse array of pathologies ranging from infectious disease to sterile inflammation. Polarization of macrophages determines their cellular function within biological processes. Lipin-1 is a phosphatidic acid phosphatase in which its enzymatic activity contributes to macrophage pro-inflammatory responses. Lipin-1 also possesses transcriptional co-regulator activity and whether this activity is required for macrophage polarization is unknown. Using mice that lack only lipin-1 enzymatic activity or both enzymatic and transcriptional coregulator activities from myeloid cells, we investigated the contribution of lipin-1 transcriptional co-regulator function toward macrophage wound healing polarization. Macrophages lacking both lipin-1 activities did not elicit IL-4 mediated gene expression to levels seen in either wild-type or lipin-1 enzymatically deficient macrophages. Furthermore, mice lacking myeloid-associated lipin-1 have impaired full thickness excisional wound healing compared to wild-type mice or mice only lacking lipin-1 enzymatic activity from myeloid cell. Our study provides evidence that lipin-1 transcriptional co-regulatory activity contributes to macrophage polarization and influences wound healing in vivo.

Keywords: lipin-1; macrophage; polarization; transcriptional coregulator; wound healing.

FIGURE 1

Lipin-1 promotes IL-4 mediated gene expression. (A) Flow cytometry was used to quantify CD 11b surface expression of BMDMs from lipin-1^mKO and littermate controls. Each dot represents an independent experiment. (B) Lipin-1 was quantified by Western blot analysis, representative image of three independent experiments shown. (C) BMDMs generated from lipin-1^mKO, lipin-1^mEnzyKO and their respective littermate control mice. BMDMs were stimulated with 20 ng/ml IL-4 for 4 h. mRNA was isolated and wound healing associated genes were quantified by qRT-PCR. No difference was noted between littermate controls as such they were combined in WT. Each dot represented an individual experiment. Experiments were performed a minimum of three times. All data were normal except for WT IL-4 treated gene expression. Mann Whitney test was used for comparing WT and lipin-1^mKO; unpaired T test used for comparing lipin-1^mKO and lipin-1mEnzyKO Data presented is mean ± SEM, * p < 0.05.

FIGURE 2

Lipin-1 does not regulate IL-4 mediated STAT6 phosphorylation. (A) Flow cytometry was used to quantify surface expression of IL-4R in unstimulated BMDMs from lipin-1^mKO and litter mate controls (mean ± SEM n > 3 from 3 independent experiments). (B) BMDMs from lipin-1^mKO and litter mate controls were stimulated with 20 ng/ml IL-4 for 30 min. Protein was isolated and p-STAT6, STAT6 was quantified by Western blot analysis. A representative blot from three independent experiments is shown.

FIGURE 3

Lipin-1 contributes to IL-4 enhancement of phagocytosis. (A) Representative microscopic images of BMDMs from lipin-1^mKO and littermate control mice fed pHrodo-Green zymosan particles. (B) Quantification of number of beads (zymosan beads) divided by number of nuclei in a given image. Experiment was performed 3 times with 4 random image panels taken per group for a total of 12 images. Each dot represents analysis of a single image (mean ± SEM n = 12, *p < 0.05).

FIGURE 4

Loss of full lipin-1 delays wound closure. (A,C) Representative image of gross lesions. (B,D) Percent wound closure as [(area of original wound - area of current wound)/area of original wound] × 100. Wound measurements were made on days 0, 2, 5, 7, 9, 12, and 14 post-wounding. KO mice are shifted by a half day in graph in order to see differences (Experiment was performed a minimum of three times and each dot represents a single animal) *p < 0.05. Each symbol represents an individual mouse.

FIGURE 5

Loss of lipin-1 does not alter systemic immune responses. (A) Splenocytes and (B) blood cells were stained with a panel of antibodies to quantify monocyte/macrophage and PMN populations. Myeloid populations were defined as CD45⁺, CD3^–CD19^–CD11b⁺. PMNs were CD11b⁺ Ly6g⁺ and monocytes were CD11b⁺Ly6G^–. Each dot represents an individual animal. Experiment was performed twice (mean ± SEM). (C) Cytometric bead arrays to quantify cytokine concentrations in serum taken from mice 2 days after wounding Each dot represents an individual animal. Experiment was performed twice (mean ± SEM).

FIGURE 6

Loss of full lipin-1 delays wound closure. (A) H&E depicting epithelial closure of 5mm wounded skin in lipin-1^mKO and wild type mice post 2 and 5 days of wounding. Red arrow heads indicate host tissue-wound interphase; yellow arrow heads indicate tip of epithelial tongue. (B) representative micrographs depicting immune infiltration at the host tissue-wound interphase (highlighted in 5A in rectangular box) post 2 days of wounding. Red arrow points toward monocytes and black arrow points toward neutrophils. (C) Pathology score concerning immune cell infiltration and scab thickness. Tissue from 4 mice from each group (n = 4). Scale bar (A), 500 μm; (B), 50 μm.

FIGURE 7

Loss of full lipin-1 leads to reduce CD206 surface expression on macrophages within the wound. Cells were isolated from wounded tissue. Cells were stained with a panel of antibodies to identify (A) leukocytes, (B) neutrophils, and (C) macrophages. We also used an anti-CD206 antibody to characterize macrophage polarization within the wound. (D) A representative staining of macrophages from the wound and quantification of mice. Each dot represents an individual wound from 6 animals per group. The experiment was performed twice (n = 12) (mean ± SEM). Data was tested for normalcy and T test was used for analysis.