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. 2022 Nov 11;5(1):1225.
doi: 10.1038/s42003-022-04211-0.

Niclosamide targets the dynamic progression of macrophages for the resolution of endometriosis in a mouse model

Liang Zhao  1 , Mingxin Shi  1 , Sarayut Winuthayanon  1 , James A MacLean 2nd  1 , Kanako Hayashi  2
Affiliations

Niclosamide targets the dynamic progression of macrophages for the resolution of endometriosis in a mouse model

Liang Zhao et al. Commun Biol. .

Abstract

Due to the vital roles of macrophages in the pathogenesis of endometriosis, targeting macrophages could be a promising therapeutic direction. Here, we investigated the efficacy of niclosamide for the resolution of a perturbed microenvironment caused by dysregulated macrophages in a mouse model of endometriosis. Single-cell transcriptomic analysis revealed the heterogeneity of macrophages including three intermediate subtypes with sharing characteristics of traditional "small" or "large" peritoneal macrophages (SPMs and LPMs) in the peritoneal cavity. Endometriosis-like lesions (ELL) enhanced the differentiation of recruited macrophages, promoted the replenishment of resident LPMs, and increased the ablation of embryo-derived LPMs, which were stepwise suppressed by niclosamide. In addition, niclosamide restored intercellular communications between macrophages and B cells. Therefore, niclosamide rescued the perturbed microenvironment in endometriosis through its fine regulations on the dynamic progression of macrophages. Validation of similar macrophage pathogenesis in patients will further promote the clinical usage of niclosamide for endometriosis treatment.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Single-cell transcriptomic profiling of peritoneal immune cells.
a Schematic of the experimental pipeline. Created with BioRender.com b UMAP visualization of peritoneal immune cells. c Ratio of each population in cell number. d VlnPlot of characteristic gene expressions for macrophage subpopulations. Sham sham control, ELL endometriosis-like lesions, ELL_N niclosamide administration to ELL-induced mouse, Pre-SPMs premature “small” peritoneal macrophages, SPMs “small” peritoneal macrophages, IM1-3 intermediate macrophages subtype 1-3, Timd4+ LPMs Timd4+ “large” peritoneal macrophages, PMs proliferating macrophages, B1a B1a cells, B1b B1b cells, B2 B2 cells, GCBs germinal-centre B cells, PBs plasma blast B cells, Cd4+ Ts Cd4+ T cells, Cd8+ Ts Cd8+ T cells, DN1 double negative T cells 1, DN2 double negative T cells 2, cDC1 conventional dendritic cells 1, MCs Mast cells, NTPs neutrophils.
Fig. 2
Fig. 2. Niclosamide finely reverses transcriptomic changes caused by endometriosis-like lesions (ELL) to peritoneal macrophages.
a Venn diagram shows the overlaps of enriched GSEA terms of biological processes between those enhanced in by ELL (ELL/sham) and those reduced by niclosamide (ELL_N/ELL). b Similar to (a) but shows the overlaps between enriched terms of those down-regulated in ELL (ELL/sham) and up-regulated by niclosamide (ELL_N/ELL). c Enriched GSEA terms of biological processes that were reduced within the ELL_N group compared to the sham group. d Representative GSEA terms that were enhanced by ELL (ELL/sham) and were decreased by niclosamide (ELL_N/ELL). e Representative GSEA terms that were inhibited by ELL (ELL/sham) and were reversed by niclosamide (ELL_N/ELL).
Fig. 3
Fig. 3. Genes regulated by ELL and niclosamide and their distributions within the perineal macrophage subpopulations (suppressed by niclosamide).
a Venn diagram shows that overlaps of genes that were enhanced by ELL (ELL/sham) but decreased by niclosamide (ELL_N/ELL). b Overlaps of genes that were inhibited by ELL (ELL/sham) but enhanced by niclosamide (ELL_N/ELL). c Vlnplot shows representative genes that were enhanced by ELL but were reduced by niclosamide treatment (ELL_N). All genes shown here were significantly (p < 0.05) differentially expressed as determined by the “wilcox” test within the Seurat package. d Verification of differential gene expression by RT-qPCR. *p < 0.05, **p < 0.01, mean ± SEM, n = 6 per group. e UMAP of computationally selected macrophage populations and the distribution of genes within them.
Fig. 4
Fig. 4. Reconstruction of a pseudo-temporal trajectory for the early maturation and differentiation of recruited macrophages.
a UMAP shows selected cells of recruited macrophages (left) and a trajectory path built by Monocle 3 (right). The selected root cell was labelled as ①. b Dynamic changes of genes along this trajectory path of differentiation. c GO terms of biological processes that were enriched by perturbed genes affected by virtual KO of Rentla. d Flow cytometer isolation and quantification of Ly6C+ recruited monocytes. e Flow cytometer results and quantification of CD206+ and FRβ+ macrophages. *p < 0.05, **p < 0.01, ***p < 0.001, mean ± SEM, n = 5 per group.
Fig. 5
Fig. 5. Genes regulated by ELL and niclosamide and their distributions within the perineal macrophage subpopulations (enhanced by niclosamide).
a Vlnplot shows representative genes that were reduced by ELL (ELL/sham) but were enhanced by niclosamide (ELL_N/ELL). All genes shown here were significantly (p < 0.05) differentially expressed as determined by the “wilcox” test within the Seurat package. b Verification of differential gene expression by RT-qPCR. *p < 0.05, **p < 0.01, ***p < 0.001, mean ± SEM, n = 6 per group. c UMAP of computationally selected macrophage populations and the distribution of genes within them.
Fig. 6
Fig. 6. Reconstruction of a pseudo-temporal trajectory for maturation of intermediate “large” macrophages.
a UMAP shows selected cells of “large” macrophages (left) and a trajectory path built by Monocle 3 (right). The selected root cell was labelled as ①. b Dynamic changes of genes along this trajectory path of maturation and replenishment. c GO terms of biological processes that were enriched by perturbed genes affected by virtual KO of Cfb.
Fig. 7
Fig. 7. Niclosamide preserves the population of embryo-derived resident “large” peritoneal macrophages.
a Vlnplot shows gene expression of Timd4 and Apoc1. All genes shown here were significantly (p < 0.05) differentially expressed as determined by the “wilcox” test within the Seurat package. b The featured plot shows the distribution of Timd4 and Apoc1. c Dynamic changes of genes along the trajectory path of maturation and replenishment. d GO terms of biological processes that were enriched by perturbed genes affected by virtual KO of Timd4. e Flow cytometer isolation and quantification of TIM4+ resident macrophages. **p < 0.01, mean ± SEM, n = 5 per group.
Fig. 8
Fig. 8. Niclosamide rescued the disrupted intercellular communications from macrophages to B cells.
a Heatmap showing the interactions between macrophages and B cells in terms of CCL signalling networks. b Heatmap showing the interactions between macrophages and B cells in terms of CXCL signalling networks. c Bubble plot showing all significant expressed ligand-receptor pairs identified between three subtypes of “large” macrophages (IM2, IM3, Timd4+ LPMs) and three subtypes of B cells (B1a, B1b, B2). d Vlnplot showing the differential expressed genes in macrophages. All genes shown here were significantly (p < 0.05) differentially expressed as determined by the “wilcox” test within the Seurat package. e Overlaps of GO biological processes in B cells that were suppressed by ELL (ELL/sham) but enhanced by niclosamide (ELL_N/ELL). f Overlaps of GO biological processes in B cells that were promoted by ELL (ELL/sham) but suppressed by niclosamide (ELL_N/ELL). g Overlaps of GO biological processes that were promoted by ELL by suppressed by niclosamide.
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