Macrophages inhibit and enhance endometriosis depending on their origin

Abstract

Macrophages are intimately involved in the pathophysiology of endometriosis, a chronic inflammatory disorder characterized by the growth of endometrial-like tissue (lesions) outside the uterus. By combining genetic and pharmacological monocyte and macrophage depletion strategies we determined the ontogeny and function of macrophages in a mouse model of induced endometriosis. We demonstrate that lesion-resident macrophages are derived from eutopic endometrial tissue, infiltrating large peritoneal macrophages (LpM) and monocytes. Furthermore, we found endometriosis to trigger continuous recruitment of monocytes and expansion of CCR2+ LpM. Depletion of eutopic endometrial macrophages results in smaller endometriosis lesions, whereas constitutive inhibition of monocyte recruitment significantly reduces peritoneal macrophage populations and increases the number of lesions. Reprogramming the ontogeny of peritoneal macrophages such that embryo-derived LpM are replaced by monocyte-derived LpM decreases the number of lesions that develop. We propose a putative model whereby endometrial macrophages are "proendometriosis" while newly recruited monocyte-derived macrophages, possibly in LpM form, are "antiendometriosis." These observations highlight the importance of monocyte-derived macrophages in limiting disease progression.

Keywords: lesion; ontogeny; phenotype.

Fig. 1.

Lesion-resident macrophages have different origins. Donor endometrial tissue from MacGreen mice was injected into the peritoneal cavity of wild-type recipient mice to assess incorporation of endometrial macrophages into lesions. Lesions were collected at 2 wk after tissue injection in each of the separate studies presented in this figure. (A) Expression of GFP by lesion-resident macrophages recovered from MacGreen (donor) to wild-type (recipient) endometrial transfers (n = 6). (B) Quantification of donor endometrial-derived (GFP+) macrophages vs. recipient-derived (GFP−) macrophages. Dual immunodetection for identification of LpM in lesions. (C) Dual immunodetection for F4/80 (red) and GATA6 (green; n = 7 mice [10 lesions]). Thick arrows indicate dual-positive cells and thin arrows indicate GATA6− macrophages. (D) Quantification of F4/80+, dual-positive and GATA6+ cells in lesions. Fewer than 1% of cells were dual-positive for F4/80 and GATA6. Adoptive transfers of MacGreen peritoneal macrophages into wild-type mice with GFP immunodetection to assess incorporation of LpM and SpM into lesions. (E and F) Immunofluorescence for GFP on lesions collected following adoptive transfer of approximately 1 × 10⁶ LpM (E) or SpM (F) isolated from MacGreen mice. Curved dotted line indicates the boundary between peritoneal and lesion tissue. In E, i and ii show magnified images; in F, i shows a negative control. (G) Quantification of GFP+ LpM and SpM in lesions. Dual immunodetection for Ly6C+ monocytes in lesions. (H) Dual immunodetection for F4/80 (red) and Ly6C (green) performed on mouse lesions. Data are presented as mean with 95% confidence intervals. Statistical significance was determined using a Student’s t test. ***P < 0.001.

Fig. 2.

Monocyte recruitment and replenishment of peritoneal macrophage pools in mice with induced endometriosis. (A) LpM (F4/80^hi, MHCII^lo) and SpM (F4/80^lo, MHCII^hi) populations in the peritoneal fluid of mice. (B) Quantification of LpM and SpM of sham (n = 6 to 8 at each time point) and endometriosis mice at 1 (n = 6), 2 (n = 8), and 3 wk (n = 16) after endometrial tissue injection compared to naïve mice (n = 12). (C) Flow plot indicating gating of monocytes (F4/80^lo, Ly6C^hi) in peritoneal lavage fluid. (D) Quantification of monocyte numbers of sham and endometriosis mice at 1, 2, and 3 wk after tissue injection. (E) Flow plot demonstrating expression of Ccr2 on F4/80^hi macrophages in peritoneal lavage fluid from sham vs. endometriosis mice (2 wk after tissue injection). (F) Quantification of CCR2+, F4/80^hi cells from endometriosis mice (n = 5) compared to sham (n = 4) and naïve (n = 4) mice. Data are presented as mean ± SEM. Statistics were determined using a one-way ANOVA and a Tukey post hoc test. *P < 0.05, **P < 0.01.

Fig. 3.

Endometrial macrophage depletion impacts lesion size. (A) Schematic demonstrating timing of doxycycline administration to iCsf1r-KO donor mice. Donor endometrium was generated in iCsf1r-KO mice, with doxycycline administered to donor mice from days 15 to 19 to deplete endometrial macrophages prior to recovery of endometrium and i.p. transfer to wild-type recipients. Lesions were recovered 2 wk after tissue injection. (B) Quantification of F4/80^hi, Ly6C^lo macrophages and Ly6C^hi, F4/80^lo monocytes in donor endometrium from wild-type and iCsf1r-KO mice. (C) Number of lesions recovered from wild-type recipient mice receiving either wild-type (n = 10) or iCsf1r-KO endometrium (n = 9) (from two independent experiments). (D) Area of lesions recovered from mice receiving either wild-type or iCsf1r-KO endometrium. Data are presented as mean ± SEM or 95% confidence intervals (C). Statistical significance was determined using a Student’s t test. *P < 0.05.

Fig. 4.

”Monocytopenic” mice with induced endometriosis establish more lesions. (A) Schematic demonstrating experimental design. Wild-type donor endometrium was generated as previously shown (Fig. 3A) and injected i.p. into ovariectomized Ccr2^−/− recipients. Wild-type recipients were also used as controls. Lesions were recovered 2 wk after tissue injection. (B) Flow plot indicating gating and number of LpM (F4/80^hi) and SpM (MHCII^hi) in peritoneal lavage fluid recovered from wild-type (n = 7) and Ccr2^−/− mice (n = 8) with induced endometriosis. (C–E) Quantification of (C) LpM, (D) SpM, and (E) monocytes (Ly6C^hi) in peritoneal lavage fluid. (F) Number of lesions recovered from wild-type (n = 11) and Ccr2^−/− (n = 13) mice with induced endometriosis (from three independent experiments). (G) Size of lesions recovered from wild-type and Ccr2^−/− mice with induced endometriosis. (H) Dual immunodetection for Ly6C (green) and F4/80 (red) on lesions recovered from wild-type and Ccr2^−/− mice. (I) Quantification of monocytes (Ly6C+; yellow bars) and monocyte-derived macrophages (Ly6C+, F4/80+; green bars) in lesions recovered from wild-type and Ccr2^−/− mice. Data are presented as mean ± SEM or 95% confidence intervals (F). Statistical significance was determined using a Student’s t test or Mann–Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

More lesions are evident in Ccl2^−/− mice. Wild-type donor endometrium was generated as previously shown (Fig. 3A) and injected i.p. into ovariectomized Ccl2^−/− recipients as in the previous figure. Wild-type recipients were also used as controls. Lesions were recovered 2 wk after tissue injection. (A) LpM and SpM populations in peritoneal lavage fluid from wild-type and Ccl2^−/− mice with induced endometriosis. (B–D) Quantification of (B) LpM, (C) SpM, and (D) monocytes in peritoneal lavage fluid from wild-type (n = 5) and Ccl2^−/− (n = 5) mice with induced endometriosis. (E) Number of lesions recovered from wild-type and Ccl2^−/− mice. (F) Size of lesions recovered from wild-type (n = 6) and Ccl2/- (n = 7) mice with induced endometriosis (from two independent experiments). (G) Dual immunodetection for Ly6C (green) and F4/80 (red) on lesions recovered from wild-type and Ccl2^−/− mice. (H) Quantification of monocytes (Ly6C+) and monocyte-derived macrophages (Ly6C+, F4/80+) in lesions recovered from wild-type and Ccl2^−/− mice. Data are presented as mean ± SEM or 95% confidence intervals (E). Statistical significance was determined using a Student’s t test or Mann–Whitney U test. *P < 0.05.

Fig. 6.

A function-blocking Ccr2 mAb reduces monocyte numbers without significant impact on lesion number. (A) Schematic showing the experimental design; mice with induced endometriosis were treated with a control IgG (MC67; n = 9) or a function-blocking CCR2 mAb (MC21; n = 10, from two independent experiments) 6 h prior to endometrial tissue injection and daily for an additional 4 d. Lesions were recovered 5 d after tissue injection. (B) Flow plot demonstrating the numbers of Ly6C^hi monocytes in mice with induced endometriosis treated with MC67 or MC21. (C–E) Quantification of (C) LpM, (D) SpM, and (E) monocytes in the peritoneal lavage fluid of mice with induced endometriosis. (F) Number of lesions recovered from mice treated with MC67 or MC21. (G) Size of lesions recovered from mice treated with MC67 or MC21. Data are presented as mean ± SEM or 95% confidence intervals (F). Statistical significance was determined using a Student’s t test or a Mann–Whitney U test. *P < 0.05.

Fig. 7.

Reprogramming the ontogeny of peritoneal macrophages leads to establishment of fewer lesions. (A) Schematic showing the experimental design; 7 d after ovariectomy experimental mice were administered i.p. with liposomal clodronate to deplete all peritoneal macrophages (n = 10). Nineteen days were allowed for replenishment of the niche prior to transfer of endometrial tissue on day 26. Lesions were recovered 5 d after tissue injection. Control mice (n = 10) did not receive liposomal clodronate (single independent experiment). (B) Flow plot demonstrating the numbers of TIM4^hi and TIM4^lo LpM in control mice with induced endometriosis or mice with reprogrammed cavities with induced endometriosis. (C–E) Quantification of (C) LpM, (D) SpM, and (E) TIM4^hi LpM in the peritoneal lavage fluid of mice with induced endometriosis. (F) Number of lesions recovered from control mice and those with reprogrammed cavities. (G) Size of lesions recovered from control mice and those with reprogrammed cavities. Data are presented as mean ± SEM or 95% confidence intervals (F). Statistical significance was determined using a Student’s t test or a Mann–Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 8.

Monocyte-derived macrophages are guardians of the peritoneal cavity in mice with induced endometriosis. Created with https://biorender.com/. Lesion-resident macrophages are a heterogenous population constituted by macrophages that have different origins; endometrial, peritoneal (LpM), and recruited monocytes that differentiate into macrophages in lesions. Wild-type mice with induced endometriosis exhibit increased monocyte recruitment and replenishment of LpM pools from monocytes. In mice where monocyte recruitment is constitutively limited (Ccr2^−/− or Ccl2^−/−), LpM and SpM pools are significantly reduced, consistent with the majority of LpM in the peritoneal cavity being embryo-derived. In these (monocytopenic) mice, more lesions develop. Mice with ontogenetically reprogrammed peritoneal cavities (embryo-derived LpM depleted using liposomal clodronate followed by a 19-d replenishment window) develop significantly fewer lesions. Collectively, these data suggest that monocyte-derived LpM protect the peritoneal cavity when challenged with ectopic endometrial tissue. We propose a putative model where endometrial macrophages promote lesion growth, while monocyte-derived macrophages (possibly monocyte-derived LpM) protect the peritoneal cavity against establishment of lesions.