C-X3-C motif chemokine ligand 1/receptor 1 regulates the M1 polarization and chemotaxis of macrophages after hypoxia/reoxygenation injury

Abstract

Background: Macrophages play an important role in renal ischemia reperfusion injury, but the functional changes of macrophages under hypoxia/reoxygenation and the related mechanism are unclear and need to be further clarified.

Methods: The effects of hypoxia/reoxygenation on functional characteristics of RAW264.7 macrophages were analyzed through the protein expression detection of pro-inflammatory factors TNF-α and CD80, anti-inflammatory factors ARG-1 and CD206. The functional implications of C-X3-C motif chemokine receptor 1(CX3CR1) down-regulation in hypoxic macrophages were explored using small interfering RNA technology. Significance was assessed by the parametric t-test or nonparametric Mann-Whitney test for two group comparisons, and a one-way ANOVA or the Kruskal-Wallis test for multiple group comparisons.

Results: Hypoxia/reoxygenation significantly increased the protein expression of M1-related pro-inflammatory factors TNF-α, CD80 and chemokine C-X3-C motif chemokine ligand 1 (CX3CL1)/CX3CR1 and inhibited the protein expression of M2-related anti-inflammatory factors ARG-1 and CD206 in a time-dependent manner in RAW264.7 cells. However, the silencing of CX3CR1 in RAW264.7 cells using specific CX3CR1-siRNA, significantly attenuated the increase in protein expression of TNF-α (P < 0.05) and CD80 (P < 0.01) and the inhibition of ARG-1 (P < 0.01) and CD206 (P < 0.01) induced by hypoxia/reoxygenation. In addition, we also found that hypoxia/reoxygenation could significantly enhance the migration (2.2-fold, P < 0.01) and adhesion capacity (1.5-fold, P < 0.01) of RAW264.7 macrophages compared with the control group, and CX3CR1-siRNA had an inhibitory role (40% and 20% reduction, respectively). For elucidating the mechanism, we showed that the phosphorylation levels of ERK (P < 0.01) and the p65 subunit of NF-κB (P < 0.01) of the RAW264.7 cells in the hypoxic/reoxygenation group were significantly increased, which could be attenuated by down-regulation of CX3CR1 expression (P < 0.01, both). ERK inhibitors also significantly blocked the effects of hypoxic/reoxygenation on the protein expression of M1-related pro-inflammatory factors TNF-α, CD80 and M2-related anti-inflammatory factors ARG-1 and CD206. Moreover, we found that conditioned medium from polarized M1 macrophages induced by hypoxia/reoxygenation, notably increased the degree of apoptosis of hypoxia/reoxygenation-induced TCMK-1 cells, and promoted the protein expression of pro-apoptotic proteins bax (P < 0.01) and cleaved-caspase 3 (P < 0.01) and inhibited the expression of anti-apoptotic protein bcl-2 (P < 0.01), but silencing CX3CR1 in macrophages had a protective role. Finally, we also found that the secretion of soluble CX3CL1 in RAW264.7 macrophages under hypoxia/reoxygenation was significantly increased.

Conclusions: The findings suggest that hypoxia/reoxygenation could promote M1 polarization, cell migration, and adhesion of macrophages, and that polarized macrophages induce further apoptosis of hypoxic renal tubular epithelial cells by regulating of CX3CL1/CX3CR1 signaling pathway.

Keywords: C-X3-C motif chemokine ligand 1/receptor 1; Hypoxia/Reoxygenation; Macrophages; Phenotypic polarization.

Fig. 1

Hypoxia/reoxygenation increased the protein expressions of TNF-α, CX3CL1, and CX3CR1, and inhibited the protein expression of ARG-1 in a time-dependent manner in RAW264.7 cells. (A and B) The proteins of RAW264.7 macrophages were tested for TNF-α, ARG-1, CX3CL1, and CX3CR1 at different hypoxia times. (C) The protein expression of TNF-α and ARG-1 in RAW264.7 macrophages under hypoxia/reoxygenation combined with different concentrations of CX3CL1 recombinant protein. (D, E, and F) The quantitative analysis of A, B and C, respectively. ^aP < 0.05, ^bP < 0.01 vs control or H/R. H/R: hypoxia/reoxygenation, hypoxia at 2, 4, 6 or 8 h and then reoxygenates for 24 h.

Fig. 2

Down-regulation of CX3CR1 with siRNA attenuated the M1 polarization of macrophages induced by hypoxia/reoxygenation. (A and B) Representative Western blots and summarized data showing the protein levels of CX3CR1, TNF-α, and ARG-1 in different groups of RAW264.7 macrophages. (C) The expression analysis of CD80 and CD206 by flow cytometry in different groups of RAW264.7 macrophages. ^aP < 0.05, ^b P < 0.01 vs Control; ^cP < 0.05, ^dP < 0.01 vs H/R; ^eP < 0.05, ^fP < 0.01 vs CX3CL1 + H/R. H/R: hypoxia/reoxygenation. Cells were exposed to hypoxia condition for 6 h, then reoxygenation for 24 h. The concentration of CX3CL1 recombinant protein used was 50 ng/mL.

Fig. 3

CX3CR1-siRNA inhibited cell adhesion and migration capacity of macrophages in hypoxia/reoxygenation. (A and B) Representative photomicrographs and quantitative analysis of migratory RAW264.7 macrophages in different groups. The grouping situation included control (a), H/R (b), H/R + siRNA-NC (c), H/R + siRNA-CX3CR1 (d), CX3CL1 (e), CX3CL1 + H/R (f) and CX3CL1 + H/R + siRNA-CX3CR1 (g). (C) The adhesive capacity analysis of RAW264.7 macrophages of different groups is shown. ^aP < 0.05, ^bP < 0.01 vs Control; ^cP < 0.05, ^dP < 0.01 vs H/R; ^eP < 0.05, ^fP < 0.01 vs CX3CL1 + H/R. H/R: hypoxia/reoxygenation. NC: negative control. Cells were exposed to hypoxia condition for 6 h, then reoxygenation for 24 h. The concentration of CX3CL1 recombinant protein used was 50 ng/mL.

Fig. 4

Hypoxia/reoxygenation increased the phosphorylation levels of ERK and the p65 subunit of NF-κB of the RAW264.7 cells and the CX3CR1 siRNA had an inhibitory role. (A and B) Representative Western blots and summarized data showing phosphorylation levels of ERK and the p65 subunit of NF-κB in different groups of RAW264.7 macrophages. ^aP < 0.05, ^bP < 0.01 vs Control; ^cP < 0.05, ^dP < 0.01 vs H/R; ^eP < 0.01 vs CX3CL1 + H/R. H/R: hypoxia/reoxygenation. NC: negative control. Cells were exposed to hypoxia condition for 6 h, then reoxygenation for 24 h. The concentration of CX3CL1 recombinant protein used was 50 ng/mL.

Fig. 5

ERK protein inhibitor reversed the protein expression alteration of TNF-α and ARG-1 induced by hypoxia/reoxygenation. (A and B, C and D) Representative Western blots and summarized data showing the protein expressions of pro-inflammatory TNF-α, and anti-inflammatory marker ARG-1 in different groups of RAW264.7 macrophages. ^aP < 0.05, ^bP < 0.01 vs Control; ^cP < 0.05, ^dP < 0.01 vs H/R; ^eP < 0.01 vs CX3CL1 + H/R. H/R: hypoxia/reoxygenation. Cells were exposed to hypoxia condition for 6 h, then reoxygenation for 24 h. The concentration of CX3CL1 recombinant protein and ERK protein inhibitor PD98059 and U0126 used was 50 ng/mL, 20 μmol/L and 10 μmol/L, respectively.

Fig. 6

The apoptosis degree of renal tubular epithelial TCMK-1 cells was increased when exposed to conditioned medium from polarized M1 macrophages induced by hypoxia/reoxygenation. (A) The ratio analysis of apoptosis of TCMK-1 cells by flow cytometry in different groups is shown. (B and C) Representative Western blots and summarized data showing the protein expressions of cell apoptosis-related proteins in different groups of TCMK-1. ^aP < 0.01 vs Control; ^bP < 0.01 vs CM; ^cP < 0.01 vs H/R; ^dP < 0.01 vs H/R + CM. H/R: hypoxia/reoxygenation, CM: conditioned medium of hypoxic macrophages, CM-CX3CR1-siRNA: conditioned medium of hypoxic macrophages transfected with CX3CR1-siRNA.

Fig. 7

Hypoxia/reoxygenation promoted the secretion of soluble CX3CL1 of RAW264.7 macrophages, which could be inhibited by CX3CR1-siRNA or ERK inhibitors. (A) Summarized data showing cell supernatant CX3CL1 levels of RAW264.7 macrophages after different hypoxia time. (B and C) Summarized data show the effects of CX3CR1-siRNA and ERK inhibitors on the cell supernatant CX3CL1 levels of RAW264.7 macrophages under hypoxia/reoxygenation. ^aP < 0.01 vs Control; ^bP < 0.01 vs H/R. H/R: hypoxia/reoxygenation. Cells were exposed to hypoxia condition for the indicated time or 6 h, then reoxygenation for 24 h. The concentration of ERK protein inhibitor PD98059 and U0126 used was 20 μmol/L and 10 μmol/L, respectively.