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. 2022 May 9:13:865363.
doi: 10.3389/fphar.2022.865363. eCollection 2022.

Iguratimod Attenuates Macrophage Polarization and Antibody-Mediated Rejection After Renal Transplant by Regulating KLF4

Zhou Hang  1 , Jintao Wei  2 , Ming Zheng  3 , Zeping Gui  1 , Hao Chen  3 , Li Sun  3 , Shuang Fei  3 , Zhijian Han  3 , Jun Tao  3 , Zijie Wang  3 , Ruoyun Tan  3 , Min Gu  1   3
Affiliations

Iguratimod Attenuates Macrophage Polarization and Antibody-Mediated Rejection After Renal Transplant by Regulating KLF4

Zhou Hang et al. Front Pharmacol. .

Abstract

Background: This study aimed to explore the effect and mechanism of iguratimod (IGT) on M1 macrophage polarization and antibody-mediated rejection (ABMR) after renal transplant. Methods: Bioinformatics analysis was performed using three public databases derived from the GEO database. Sprague-Dawley (SD) rats were pre-sensitized with donors of Wistar rats in skin transplantation and a rat renal transplant ABMR model was established from the donors to skin pre-sensitized recipients. Subsequently, IGT was treated on the ABMR model. Routine staining and immunofluorescence (IF) staining were performed to observe the pathological changes in each group and flow cytometry was performed to detect the changes of DSA titers in peripheral blood. In addition, bone-marrow-derived macrophage (BMDM) was extracted and interfered with IGT to explore the effect of IGT in vivo. PCR, IF staining, and Western blot were used to detect the expression of related genes and proteins. Results: Bioinformatics analysis revealed that several immune cells were significantly infiltrated in the ABMR allograft, while M1 macrophage was noticed with the most significance. Results of IF staining and PCR proved the findings of the bioinformatics analysis. Based on this, IGT was observed to significantly attenuate the degree of peritubular capillary vasculitis and arteriolitis in the rat renal transplant ABMR model, whereas it decreases the expression of C4d and reduces the titer of DSA. Results in vitro suggested that M1 macrophage-related transcripts and proteins were significantly reduced by the treatment of IGT in a dose- and time-dependent manner. Furthermore, IGT intervention could remarkably decrease the expression of KLF4. Conclusion: Polarization of M1 macrophages may aggravate ABMR after renal transplant by promoting DSA-mediated endothelial cell injury, and IGT may attenuate the pathogenesis of ABMR by targeting KLF4.

Keywords: KLF4; antibody-mediated rejection; iguratimod; kidney transplant; macrophage polarization.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Bioinformatics analysis results of three public databases from the GEO database. (A) Heat map after the joint analysis of the three databases. (B) Cell distribution after the combined analysis of the three databases. (C) Analysis of immune cell infiltration in ABMR transplanted kidney tissue. (D) Correlation between immune cell infiltration and ABMR in renal transplantation.
FIGURE 2
FIGURE 2
Determination of macrophage infiltration in human renal allograft samples. (A–B) Renal pathology in STA and ABMR patients (400×). (C) Representative images of C4d immunostaining in kidney sections from STA and ABMR patients (400×). (D) Double indirect fluorescence staining of CD68 and iNOS in ABMR and STA groups (400×). (E) Double indirect fluorescence staining of CD68 and CD206 in ABMR and STA groups (400×). ***p < 0.001 compared with the STA group; ****p < 0.0001 compared with the STA group; NS: no significant difference compared with the STA group; a non-parametric test was used for statistical analysis in this figure.
FIGURE 3
FIGURE 3
Determination of macrophage infiltration in renal allograft samples derived from the rat renal transplant ABMR model. (A) Double indirect fluorescence staining of CD68 and iNOS in the sham operation group (sham), skin transplant group (STx), non-sensitive kidney transplant group (non-sensitive), and ABMR group (ABMR) (200×). (B) Double indirect fluorescence staining of CD68 and iNOS in the sham group, STx group, non-sensitive group, and ABMR group (200×). (C–F) Renal mRNA expression of (C) CD68 (D) CD86 (E) iNOS, and (F) CD206 in the sham group, STx group, non-sensitive group, and ABMR group. *p < 0.05 compared with the sham group; **p < 0.01 compared with the sham group; ***p < 0.001 compared with the sham group; ****p < 0.0001 compared with the sham group; NS: no significant difference compared with the sham group; a non-parametric test was used for the statistical analysis in this figure.
FIGURE 4
FIGURE 4
Effect of iguratimod intervention in the rat renal transplant ABMR model. (A–B) Representative images of renal pathology in sham, sham + IGT, STx, non-sensitive, ABMR, and ABMR + IGT groups (400×). (C) Indirect fluorescence staining of C4d in sham, sham + IGT, STx, non-sensitive, ABMR, and ABMR + IGT groups (400×). (D) Quantitative analysis of C4d immunostaining in each group. (E) DSA examination of sham, sham + IGT, STx, non-sensitive, ABMR, and ABMR + IGT groups. ****p < 0.0001 compared with the sham group; #p < 0.001 compared with the ABMR group; the non-parametric test was used for the statistical analysis in this figure.
FIGURE 5
FIGURE 5
Determination of macrophage infiltration after intervention of IGT in the rat model. (A) Double indirect fluorescence staining of CD68 and iNOS in sham, sham + IGT, STx, non-sensitive, ABMR, and ABMR + IGT groups (400×). (B–D) Renal mRNA expression of (B) CD68 (C) CD86, and (D) iNOS in the sham, STx, non-sensitive, and ABMR groups. The non-parametric test was used for statistical analysis in this figure.
FIGURE 6
FIGURE 6
IGT-inhibited M1 macrophage polarization in vitro. (A) CCK8 assay of IGT intervention with BMDM. (B) Representative images of different macrophages under a light microscope (400×). (C) mRNA expression of iNOS in BMDM after the time-dependent intervention of IGT. (D–G) mRNA expression of CD86, iNOS, IL1-β, and IL6 in BMDM after concentration-dependent intervention of IGT. (H) Flow cytometry for F4/80 and iNOS in BMDM after concentration-dependent intervention of IGT. (I) Double indirect fluorescence staining of F4/80 and iNOS in M0 (M0), M1 (M1), and M1 macrophages after IGT intervention (M1+IGT). The non-parametric test was used for statistical analysis in this figure.
FIGURE 7
FIGURE 7
KLF4 was involved in macrophage polarization regulation in ABMR. (A) Double indirect fluorescence staining of CD68 and KLF4 in ABMR rat models. (B) Double indirect fluorescence staining of CD68 and KLF4 in M0, M1, and M1+IGT. (C–D) Quantitative analysis of CD68 (C) and KLF4 (D) in ABMR rat models. (E–F) Quantitative analysis of CD68 (E) and KLF4 (F) in cell culture. (G) mRNA expression of KLF4 in macrophages. **p < 0.01 compared with the control group; ***p < 0.001 compared with the control group; ****p < 0.0001 compared with the control group; #p < 0.05 compared with the M1 group; ##p > 0.05 compared with the M1 group; non-parametric test was used for statistical analysis in this figure.
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