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. 2021 Jan;57(1):10-16.
doi: 10.1007/s11626-020-00535-5. Epub 2021 Jan 14.

Dexamethasone enhances CD163 expression in porcine IPKM immortalized macrophages

Takato Takenouchi  1 , Takeya Morozumi  2 , Emi Wada  2 , Shunichi Suzuki  3 , Yasutaka Nishiyama  2 , Shin Sukegawa  2 , Hirohide Uenishi  3
Affiliations

Dexamethasone enhances CD163 expression in porcine IPKM immortalized macrophages

Takato Takenouchi et al. In Vitro Cell Dev Biol Anim. 2021 Jan.

Abstract

In our previous study, we established a unique porcine macrophage cell line, immortalized porcine kidney-derived macrophages (IPKM). The purpose of the present study was to further elucidate the characteristics of IPKM. CD163 is a scavenger receptor for the hemoglobin-haptoglobin complex and is used as a phenotypic marker of anti-inflammatory M2 macrophages. The expression of CD163 is enhanced by dexamethasone (DEX), a potent steroidal anti-inflammatory drug, in human and rodent macrophages in vitro. Therefore, we investigated the effects of DEX on CD163 expression in porcine IPKM. Treatment with DEX markedly enhanced CD163 expression in the IPKM. In addition, we found that SB203580, a selective inhibitor of p38 mitogen-activated protein kinase (MAPK), blocked the effects of DEX, suggesting that the p38 MAPK signaling pathway is involved in the regulation of the DEX-induced enhancement of CD163 expression. Since CD163 is considered to be a putative receptor for the porcine reproductive and respiratory syndrome virus (PRRSV), the effects of DEX on the infection of IPKM by PRRSV were evaluated. Although the IPKM were susceptible to infection by the Fostera PRRSV vaccine strain, DEX treatment did not affect the propagation of the virus in the IPKM. This suggests that the DEX-induced enhancement of CD163 expression alone is not sufficient to facilitate the infection of IPKM by PRRSV.

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Figures

Figure 1.
Figure 1.
Effect of DEX on CD163 expression in IPKM. IPKM were treated with DEX at the indicated concentrations for 1 d (A–D) or 3 d (E–H). Then, the cells were fixed and immunostained with a specific antibody against CD163 (brown). All nuclei were counterstained with hematoxylin (blue). DEX increased the expression of CD163 in a dose-dependent manner.
Figure 2.
Figure 2.
Effect of DEX on CD169 expression in IPKM. IPKM were treated with DEX at the indicated concentrations for 1 d (A–D) or 3 d (E–H). Then, the cells were fixed and immunostained with a specific antibody against CD169 (brown). All nuclei were counterstained with hematoxylin (blue). DEX did not increase the expression of CD169.
Figure 3.
Figure 3.
Effects of SB203580 and U0126 on the DEX-induced upregulation of CD163 expression. IPKM were treated with (A–D) or without (E–H) 80 ng/ml DEX for 1 d in the presence or absence of 10 μM SB203580 (C, G) or 10 μM U0126 (D, H). DMSO (0.1%) was used as a vehicle control (B, F) because SB203580 and U0126 were dissolved in DMSO. Then, the cells were fixed and immunostained with a specific antibody against CD163 (brown). All nuclei were counterstained with hematoxylin (blue). DEX enhanced the expression of CD163 compared with that seen in the untreated cells (A, E), even in the presence of DMSO (B, F). SB203580 and U0126 had no effect on the basal CD163 expression level (C, D). However, SB203580 blocked the DEX-induced enhancement of CD163 expression (G), while U0126 did not exert any such inhibitory effect (H).
Figure 4.
Figure 4.
Effects of SB203580 and U0126 on the proliferation of DEX-treated IPKM and flow cytometric analyses of IPKM. IPKM (1 × 106) were pre-cultured in 90-mm dishes for 3 d, and treated with or without 80 ng/ml DEX for 1 d in the presence or absence of 10 μM SB203580 or 10 μM U0126. DMSO (0.1%) was used as a vehicle control because SB203580 and U0126 were dissolved in DMSO. Then, the cells were harvested and counted with a TC10 automated cell counter (A). Six independent experiments were performed, and the cell numbers are expressed as mean±SEM values (*p < 0.05 vs. DMSO-treated control) (A). Also, no treated (B) and DEX-treated (C) IPKM were reacted with mouse monoclonal anti-CD163 (red line), anti-CD169 (blue line), or anti-CD203a (green line) antibodies, before being labeled with Alexa Fluor 488-conjugated anti-mouse IgG antibodies (Alexa 488). The negative control cells were reacted with Alexa Fluor 488-conjugated anti-mouse IgG antibodies alone (B and C: black line). The frequency of CD163-positive cells was higher (B and C, red line), while the frequency of CD169-positive cells was slightly lower (B and C, blue line), among the DEX-treated IPKM. DEX had no effect on the frequency of CD203a-positive cells (B and C, green line).
Figure 5.
Figure 5.
Flow cytometric analyses of DEX-treated IPKM. IPKM were treated with or without 80 ng/ml DEX for 1 d in the presence or absence of 10 μM SB203580 or 10 μM U0126. DMSO (0.1%) was used as a vehicle control because SB203580 and U0126 were dissolved in DMSO. Then, the cells were reacted with mouse monoclonal anti-CD163 (A) or anti-CD169 (B) antibodies, before being labeled with Alexa Fluor 488-conjugated anti-mouse IgG antibodies (Alexa 488). The positive region was set up to contain about 0.5% of the negative control cells, which reacted with Alexa Fluor 488-conjugated anti-mouse IgG antibodies alone (A and B: black line). Three independent experiments were performed, and the data regarding the percentages of cells in the positive region are expressed as mean±SEM values (*p < 0.05, **p < 0.01 vs. DMSO-treated control) (C). The frequency of CD163-positive cells was significantly higher among the DEX-treated IPKM (A: purple line, and C). The administration of SB203580 completely blocked the effects of DEX (A: blue line, and C). Conversely, the administration of U0126 facilitated the DEX-induced increase in the frequency of CD163-positive cells (A: green line, and C). The frequency of CD169-positive cells was slightly lower among the DEX-treated IPKM (B: purple line, and C). Co-treatment with DEX and SB203580 (B: blue line) or U0126 (B: green line) significantly decreased the frequency of CD169-positive cells (C).
Figure 6.
Figure 6.
Detection of N protein in Fostera PRRSV–infected IPKM. IPKM were treated with or without 80 ng/ml DEX for 1 d in the presence or absence of 10 μM SB203580. DMSO (0.1%) was used as a vehicle control because SB203580 was dissolved in DMSO. Then, the cells were exposed to the Fostera PRRSV for 2 h, cultured for a further day, and lysed with extraction buffer. The N protein was detected in the Fostera PRRSV-exposed IPKM, even in the presence of DMSO. DEX treatment had no effect on the expression of the N protein, while SB203580 clearly reduced it. The experiments were performed in triplicate and produced similar results (upper three panels). An anti-actin antibody was used as a loading control for the immunoblotting (the lowest panel).
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