Small Heterodimer Partner Modulates Macrophage Differentiation during Innate Immune Response through the Regulation of Peroxisome Proliferator Activated Receptor Gamma, Mitogen-Activated Protein Kinase, and Nuclear Factor Kappa B Pathways

Abstract

Hepatic macrophages act as the liver's first line of defense against injury. Their differentiation into proinflammatory or anti-inflammatory subpopulations is a critical event that maintains a delicate balance between liver injury and repair. In our investigation, we explored the influence of the small heterodimer partner (SHP), a nuclear receptor primarily associated with metabolism, on macrophage differentiation during the innate immune response. During macrophage differentiation, we observed significant alterations in Shp mRNA expression. Deletion of Shp promoted M1 differentiation while interfering with M2 polarization. Conversely, overexpression of SHP resulted in increased expression of peroxisome proliferator activated receptor gamma (Pparg), a master regulator of anti-inflammatory macrophage differentiation, thereby inhibiting M1 differentiation. Upon lipopolysaccharide (LPS) injection, there was a notable increase in the proinflammatory M1-like macrophages, accompanied by exacerbated infiltration of monocyte-derived macrophages (MDMs) into the livers of Shp myeloid cell specific knockout (Shp-MKO). Concurrently, we observed significant induction of tumor necrosis factor alpha (Tnfa) and chemokine (C-C motif) ligand 2 (Ccl2) expression in LPS-treated Shp-MKO livers. Additionally, the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways were activated in LPS-treated Shp-MKO livers. Consistently, both pathways were hindered in SHP overexpression macrophages. Finally, we demonstrated that SHP interacts with p65, thereby influencing macrophage immune repones. In summary, our study uncovered a previously unrecognized role of SHP in promoting anti-inflammatory macrophage differentiation during the innate immune response. This was achieved by SHP acting as a regulator for the Pparg, MAPK, and NF-κB pathways.

Keywords: differentiation; knockout; macrophage; nuclear receptor; small heterodimer partner (SHP).

Figure 1

Macrophage differentiation alters Shp mRNA expression. The livers of C57BL/6 mice were perfused and digested to harvest nonparenchymal cells (NPCs). Hepatic macrophages were then isolated from NPCs by CD11b MicroBeads and differentiated into M1 or M2 macrophages using DMEM media supplemented with IFN-gamma (100 ng/mL) or IL4 (50 ng/mL) for 24 h, respectively. The relative mRNA levels of M1 markers (Tnfa and Nos2), M2 markers (Arg1 and Cd163), and Shp were determined using quantitative PCR (qPCR). Data are presented as mean ± SEM for 3 samples/group. * p < 0.05 and ** p < 0.01 between the indicated groups.

Figure 2

SHP regulates macrophage differentiation in vitro. (A) Peritoneal macrophages were isolated from WT and Shp-MKO mice, and the relative mRNA levels of Shp were determined by qPCR. (B) Bone marrow cells were isolated from WT and Shp-MKO mice and differentiated into macrophages using M-CSF (10 ng/mL) for 7 days. On the 7th day, the differentiated macrophages were cultured with IFN-gamma (100 ng/mL) or IL4 (50 ng/mL) for 24 h to induce M1 or M2 macrophage polarization, respectively. The mRNA expression of Tnfa, Nos2, and Cd206 was determined using qPCR. (C) Left, Western blot confirmed the overexpression of Flag-SHP in mouse macrophage RAW cells. Right, the expression of miR-34a and Pparg was determined by qPCR. (D) RAW cells with or without SHP overexpression were treated with IFN-gamma (100 ng/mL) for 24 h. qPCR was employed to measure mRNA levels of Nos2 and Pparg. The data are presented as mean ± SEM for 3 samples/group. * p < 0.05 and ** p < 0.01 between the indicated groups.

Figure 3

Shp knockout results in a persistent hepatic accumulation of macrophages following LPS challenge. (A) Shp myeloid cell specific knockout (Shp-MKO) was generated by breeding Shp^flox/flox with LysM-Cre mice. Both WT and Shp-MKO mice were subjected to intraperitoneal LPS (1 mg/kg body weight) injection, and samples were collected at 0-, 3-, and 7 h post-injection. (B) Mouse body weight, liver weight, and liver-to-body weight ratio. (C) Liver sections were stained with hematoxylin and eosin (H&E) to examine the histological changes in the liver. (D) Left, representative images of liver sections stained with macrophage marker F4/80. Original magnification, X40. Right, quantification of the DAB-positive staining area. n = 3/group. The data are presented as mean ± SEM for 3 samples/group. * p < 0.05 between the indicated groups.

Figure 4

Flow cytometry analysis of composition of hepatic macrophages and monocytes following LPS challenge. WT and Shp-MKO mice were intraperitoneally injected with or without LPS (1 mg/kg body weight). After 3 h, mouse livers were perfused and digested to isolate nonparenchymal cells (NPCs). Approximately 1 × 10⁶ NPCs were labeled with specific antibodies and prepared for flow cytometry analysis. Single cells were gated based on FSC–A and FSC–H to exclude doublets. Dead cells stained with Zombie Aqua were excluded from the analysis. Live cells positive for CD45 expression were gated, and the populations of interest were calculated, including F4/80⁺Ly6C^High proinflammatory M1 macrophages (A), CD11b⁺Ly6C^High proinflammatory monocytes (B), and CD11b^HighF4/80^Intermediate monocyte-derived macrophages (C). The data are presented as mean ± SEM for 3 samples/group. * p < 0.05 and ** p < 0.01 between the indicated groups.

Figure 5

Myeloid cell specific deletion of Shp leads to enhanced chemokine production in response to LPS challenge. WT and Shp-MKO mice were intraperitoneally injected with LPS (1 mg/kg body weight), and samples were collected at 0, 3, and 7 h post-injection. (A) The mRNA levels of Shp, Ccl2, Cd11b, and Ly6c in liver tissues were quantified using qPCR. (B) The serum levels of CCL2 and TNFα were measured using ELISA to evaluate the circulating levels of these chemokines. The data are presented as mean ± SEM for 3 samples/group. * p < 0.05 and ** p < 0.01 between the indicated groups.

Figure 6

Myeloid cell specific deletion of Shp results in hyperactivation of MAPK and NF-κB pathways in response to LPS challenge. WT and Shp-MKO mice were intraperitoneally injected with LPS (1 mg/kg body weight), and samples were collected at 0-, 3-, and 7 h post-injection. (A) Left, Western blot analysis of whole protein lysates from liver tissues revealed the expression and phosphorylation levels of proteins involved in MAPK and NF-κB signaling pathways. Right, the protein band density was quantified using Image Studio 5.2 software, and the relative expression levels were normalized to the loading control β-actin. (B) Left, Western blot analysis of cytoplasmic and nuclear fractions demonstrated the expression and phosphorylation levels of proteins involved in NF-κB signaling. Right, the protein band density was quantified using Image Studio software, and the relative levels of proteins were normalized to the nuclear loading control Histone H3 and the cytoplasmic loading control α-tubulin, respectively. The data are presented as mean ± SEM. Western blots were repeated 3 times and one represented image was included in the figure. * p < 0.05 and ** p < 0.01 between the indicated groups.

Figure 7

SHP overexpression hinders the activation of MAPK and NF-κB pathways in response to LPS challenge. (A) Left, mouse macrophage RAW cells with or without SHP overexpression were treated with 100 ng/mL LPS for different durations (0, 5, 10, 30, and 60 min). Whole cell lysates were collected and subjected to Western blot analysis to assess the expression and phosphorylation levels of proteins involved in MAPK and NF-κB signaling pathways. Right, the protein band density was quantified using Image Studio software, and the relative expression levels were normalized to the loading control β-actin. (B) Co-immunoprecipitation experiments were conducted using whole protein lysates from RAW cells with or without FLAG-SHP overexpression. The protein–protein interaction of SHP with p65 was detected by Western blot analysis. The data are presented as mean ± SEM. Western blots were repeated 3 times and one represented image was included in the figure. * p < 0.05 and ** p < 0.01 between the indicated groups.

Figure 8

Schematic diagram illustrating the crucial role of SHP in regulating macrophage polarization and its significant impact on the immune response during LPS-induced inflammation. The absence of Shp in myeloid cells results in the augmented infiltration of proinflammatory monocytes and their subsequent differentiation into proinflammatory M1 macrophages upon LPS challenge. These effects are attributed to the dysregulation of Pparg, MAPK, and NF-κB signaling pathways due to the loss of Shp in macrophages, further contributing to the persistent accumulation of proinflammatory M1 macrophages and the downregulation of hepatic Shp expression through the interactions between monocytes/macrophages and hepatocytes.