Journal: iMeta

Article Title: Decidual macrophage‐mediated ferroptosis in trophoblasts leads to recurrent spontaneous abortion

doi: 10.1002/imt2.70138

Figure Lengend Snippet: Recurrent spontaneous abortion (RSA) decidual macrophages train trophoblasts to modulate M1/M2 macrophages. (A) Co‐culture of trophoblast cells from patients with HC ( n = 3) and RSA ( n = 3) with decidual macrophages, followed by co‐culture with THP‐1‐derived macrophages, then detection of macrophages. (B) Representative fluorescence images of the M1 macrophage marker cluster of differentiation (CD)86 and quantified mean fluorescence intensity (MFI) of CD86 in macrophages ( n = 3). (C) Representative fluorescence images of CD206, an M2 macrophage marker, and quantified MFI of CD206 in macrophages ( n = 3). (D, E) The mRNA expression levels of M1 phenotype markers (tumor necrosis factor‐alpha ( TNF‐α ), CXC chemokine ligand 9 ( CXCL9 ), and CD86 ) and M2 phenotype markers ( CD206 , C‐C Motif Chemokine Ligand ( 8 CCL8 ), and CD163 ) in macrophages were detected by qRT‐PCR ( n = 3). (F) M1 (inducible nitric oxide synthase (iNOS)) and M2 (arginase‐1 (Arg‐1)) marker expression across experimental groups was analyzed by Western blot in macrophages ( n = 3). (G) Differential expression of C‐X‐C motif chemokine ligand 2 ( CXCL2 ), interleukin ( IL ) −1β , TNF‐α , IL‐10 , IL‐6 , IL‐4 , and IL‐13 in HTR‐8 cells was detected by quantitative real‐time polymerase chain reaction (qRT‐PCR) ( n = 3). (H) IL‐6 expression was quantified by enzyme‐linked immunosorbent assay (ELISA) in HTR‐8 cells ( n = 5). (I) Interleukin‐6 receptor (IL‐6R) protein expression in macrophages was quantified using Western blot ( n = 3). (J) M1 macrophage marker CD86 expression visualized by immunofluorescence with corresponding MFI values in macrophages ( n = 3). (K) M2 macrophage marker CD206 expression visualized by immunofluorescence with corresponding MFI values in macrophages ( n = 3). (L, M) The qRT‐PCR analysis quantified M1 ( TNF‐α , CXCL9 , CD86 ) and M2 ( CD206 , CCL8 , CD163 ) marker expression in macrophages ( n = 3). (N–P) Western blot analysis detected M1 (iNOS) and M2 (Arg‐1) marker expression in macrophages ( n = 3). (Q) Expression and phosphorylation levels of janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling were analyzed by Western blot with statistical quantification in macrophages ( n = 3). (R) Immunofluorescence staining showing M1 marker CD86 expression with corresponding MFI in macrophages ( n = 3). (S) Immunofluorescence staining showing M2 marker CD206 expression with corresponding MFI in macrophages ( n = 3). (T, U) The qRT‐PCR analysis quantified M1 ( TNF‐α , CXCL9 , CD86 ) and M2 ( CD206 , CCL8 , CD163 ) marker expression in macrophages ( n = 3). (V) M1 (iNOS) and M2 (Arg‐1) marker expression in macrophages was analyzed by Western blot ( n = 3). (W) Decidual macrophages from RSA patients educate trophoblastic cells to promote macrophage inflammatory activation by suppressing the JAK2/STAT3 axis via IL‐6. Student's t ‐test was employed for comparisons between two groups. * p < 0.05, ** p < 0.01, ns: not significant.

Article Snippet: The THP‐1 cell line was differentiated into M0 macrophages by treatment with 100 ng/ml phorbol 12‐myristate 13‐acetate (PMA, Sigma) for 24 h. For the inhibition of ferroptosis, Ferrostatin‐1(60 nM, HY‐100579, MCE, Shanghai, China, dissolved in dimethyl sulfoxide) was used to treat cells for 48 h. Inhibition of the NF‐κB pathway was achieved by treating cells with an NF‐κB inhibitor [pyrrolidinedithiocarbamate ammonium (PDTC ammonium) (10 μM, HY‐18738, MCE, dissolved in dimethyl sulfoxide)] for 48 h. For activation of JAK2/STAT3 signaling, a STAT3 agonist ML115 (10 μM, HY‐111152, MCE, dissolved in dimethyl sulfoxide) was used to treat cells for 48 h. For IL‐6 supplementation, the recombinant human IL‐6 (50 ng/mL, 200‐06, Peprotech, USA, dissolved in PBS) was added to treat cells for 48 h. For CXCL2 supplementation, the recombinant human CXCL2 (10 ng/mL, 300‐39, Peprotech, dissolved in PBS) was added to treat cells for 24 h,48 h, and 72 h. Eriodictyol (15 μM, HY‐N0637, MCE, dissolved in dimethyl sulfoxide) was used to treat cells for 48 h. For activation of the NF‐κB pathway, NF‐κΒ activator 1 (5 μM, HY‐134476, MCE, dissolved in dimethyl sulfoxide) was used to treat cells for 48 h.

Techniques: Co-Culture Assay, Derivative Assay, Fluorescence, Marker, Expressing, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Phospho-proteomics, Staining, Activation Assay