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. 2024 May 15;22(1):271.
doi: 10.1186/s12964-024-01571-4.

Ficolin-A induces macrophage polarization to a novel pro-inflammatory phenotype distinct from classical M1

Li-Wen Zhu  1   2 , Zihao Li  3 , Xiaohong Dong  4 , Huadong Wu  5 , Yifan Cheng  5 , Shengnan Xia  2 , Xinyu Bao  2 , Yun Xu  6   7 , Runjing Cao  8
Affiliations

Ficolin-A induces macrophage polarization to a novel pro-inflammatory phenotype distinct from classical M1

Li-Wen Zhu et al. Cell Commun Signal. .

Abstract

Background: Macrophages are key inflammatory immune cells that orchestrate the initiation and progression of autoimmune diseases. The characters of macrophage in diseases are determined by its phenotype in response to the local microenvironment. Ficolins have been confirmed as crucial contributors to autoimmune diseases, with Ficolin-2 being particularly elevated in patients with autoimmune diseases. However, whether Ficolin-A stimulates macrophage polarization is still poorly understood.

Methods: We investigated the transcriptomic expression profile of murine bone marrow-derived macrophages (BMDMs) stimulated with Ficolin-A using RNA-sequencing. To further confirm a distinct phenotype activated by Ficolin-A, quantitative RT-PCR and Luminex assay were performed in this study. Additionally, we assessed the activation of underlying cell signaling pathways triggered by Ficolin-A. Finally, the impact of Ficolin-A on macrophages were investigated in vivo through building Collagen-induced arthritis (CIA) and Dextran Sulfate Sodium Salt (DSS)-induced colitis mouse models with Fcna-/- mice.

Results: Ficolin-A activated macrophages into a pro-inflammatory phenotype distinct to LPS-, IFN-γ- and IFN-γ + LPS-induced phenotypes. The transcriptomic profile induced by Ficolin-A was primarily characterized by upregulation of interleukins, chemokines, iNOS, and Arginase 1, along with downregulation of CD86 and CD206, setting it apart from the M1 and M2 phenotypes. The activation effect of Ficolin-A on macrophages deteriorated the symptoms of CIA and DSS mouse models, and the deletion of Fcna significantly alleviated the severity of diseases in mice.

Conclusion: Our work used transcriptomic analysis by RNA-Seq to investigate the impact of Ficolin-A on macrophage polarization. Our findings demonstrate that Ficolin-A induces a novel pro-inflammatory phenotype distinct to the phenotypes activated by LPS, IFN-γ and IFN-γ + LPS on macrophages.

Keywords: Autoimmune disease; Ficolin-A; Macrophage; Phenotype.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
FCNA activates a distinct transcriptional program in macrophages. A Principal-component analysis of murine BMDMs stimulated with FCNA or LPS showing the divergence of transcriptomic programs. B Principal-component analysis of BMDMs stimulated with FCNA, IFN-γ or IFN-γ + LPS showing the differences in transcriptional program. C and D Clusted heatmap of normalized differentially expressed genes (DEGs) of BMDMs treated with FCNA, LPS, IFN-γ or IFN-γ + LPS. E. Venn diagram showing the overlap between genes upregulated in macrophages treated with FCNA, LPS, IFN-γ or IFN-γ + LPS
Fig. 2
Fig. 2
Inflammatory genes were activated in BMDMs by FCNA. A Volcano plot of gene expressions in FCNA-treated BMDMs. The grey dots and blue dots indicate upregulated and downregulated DEGs, respectively. B KEGG gene set analysis showing biological signaling pathways that are differentially activated by FCNA. C Heatmap shows DEGs enriched in Cytokine-cytokine receptor interaction pathway. D The KEGG enrichment chord diagram was constructed to visualize ranked DEGs corresponding to KEGG terms. E Selected gene expression levels examined by RNA-Seq analysis. Each dot represents an individual cell sample. Error bars show the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001
Fig. 3
Fig. 3
Validation of featured genes identified by RNA-Seq. A qPCR was performed to validate representative gene expressions regulated by FCNA (200ng/ml, 500ng/ml) in BMDMs. B Analysis of Luminex assay showing the secretion of cytokines in supernatants of FCNA-treated BMDMs. Error bars show the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001
Fig. 4
Fig. 4
FCNA activated inflammatory signaling pathways in macrophages. A Protein analysis by western blot in BMDMs treated with FCNA at various timepoints. B Representative genes expressions were assessed by qPCR in FCNA-treated BMDMs following signaling inhibitor treatments, including p38 inhibitor (SB202190), JAK-STAT inhibitor (Ruxolitinib), NF-κb inhibitor (Pyrrolidinedithiocarbamate ammonium, PDTC), ERK inhibitor (SCH772984), and JNK inhibitor (SP600125). Data are representative of three independent experiments. Error bars show the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001
Fig. 5
Fig. 5
FCNA treatment deteriorates CIA symptoms. A DBA/1 mice were immunized on Day0 and 21 to induce the CIA model, with the treatment of FCNA (5 µg/mouse) i.p. once every two days. B Arthritis scores are measured every two days (Control, n = 8; FCNA, n = 8). C Representative images of forelimbs and hindlimbs from different mice on Day42 post immunization. D The morphology of articular cartilage and synovial invasion were observed by H&E staining, safranine O-fast green staining and toluidine blue staining of mice ankle joints. Scale bars, 400 μm. E and F Quantification of H&E staining by HSS scores and OARSI scores (n = 8/group). The P value was determined by two-way ANOVA (B) and unpaired Student’s t test (E, F). Error bars show the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001
Fig. 6
Fig. 6
Deletion of FCNA attenuates CIA symptoms. A Arthritis scores are measured every two days (WT, n = 6; Fcna-/-, n = 8) after the induction of CIA model. B Representative images of forelimbs and hindlimbs from different mice on Day42 post immunization. C The morphology of articular cartilage and synovial invasion were observed by H&E staining, safranine O-fast green staining and toluidine blue staining of mice ankle joints. Scale bars, 400 μm. D and E Quantification of H&E staining by HSS scores and OARSI scores (n = 6/group). The P value was determined by two-way ANOVA and unpaired Student’s t test. Error bars show the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001
Fig. 7
Fig. 7
FCNA deletion ameliorates DSS-induced colitis. A Body weight changes of each experimental group during DSS treatment (WT control, n = 7; Fcna−/− Control, n = 5; WT DSS, n = 7; Fcna−/− DSS, n = 6). B Representative images of colon tissues from each group (n = 3/group). C Bar graph showing the analysis of colon lengths in each group. D Histological analysis of colon tissues showed by H&E staining. Scale bars, 200 μm. E Histological scores of the colon damages were evaluated. F, G MODE-K cells were cultured with conditioned medium (CM) from Control or FCNA-treated BMDMs, and stimulated with or without LPS. The cells were examined for apoptosis by flow cytometry (F), and cell viability by CCK-8 (G). The P value was determined by two-way ANOVA and unpaired Student’s t test. Error bars show the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001
Fig. 8
Fig. 8
Graphical summary of FCNA activates macrophages into a distinct phenotype. FCNA activates M0 type macrophage polarizing into a novel pro-inflammatory phenotype. This distinctive phenotype is characterized by upregulation of interleukins including IL-12b, IL-6, IL-1a, TNF-α etc., chemokines including CCL5, CXCL10 etc., and macrophage signature markers iNOS and Arginase 1. The novel phenotype is also recognized with downregulation of CD86 and CD206
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