Recruited macrophages that colonize the post-inflammatory peritoneal niche convert into functionally divergent resident cells

Abstract

Inflammation generally leads to recruitment of monocyte-derived macrophages. What regulates the fate of these cells and to what extent they can assume the identity and function of resident macrophages is unclear. Here, we show that macrophages elicited into the peritoneal cavity during mild inflammation persist long-term but are retained in an immature transitory state of differentiation due to the presence of enduring resident macrophages. By contrast, severe inflammation results in ablation of resident macrophages and a protracted phase wherein the cavity is incapable of sustaining a resident phenotype, yet ultimately elicited cells acquire a mature resident identity. These macrophages also have transcriptionally and functionally divergent features that result from inflammation-driven alterations to the peritoneal cavity micro-environment and, to a lesser extent, effects of origin and time-of-residency. Hence, rather than being predetermined, the fate of inflammation-elicited peritoneal macrophages seems to be regulated by the environment.

Fig. 1. Competition mediates inflammatory macrophage phenotype early post resolution.

a F4/80^HI PKH26-PCL^Hi resident macrophages, PKH26-PCL^Lo Ly6c⁺ monocytes and PKH26-PCL^Lo F4/80^Int inflammatory macrophages in the naïve peritoneal cavity, 4 h and 3 days post 10 μg zymosan. b Number of RMac, Monocytes, IMac, and Neutrophils the naïve peritoneal cavity (n = 8), 4 h post zymosan (n = 9) and 3 days post zymosan (n = 9). *p < 0.05, ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. c Experimental scheme for transfer of RMac^Z10 (blue) or IMac^Z10 (orange) into mirroring inflamed (native), naïve or depleted recipient mice. d Engraftment efficiency of RMac^Z10 (n = 6) and IMac^Z10 (n = 8) 8 days after transfer into inflamed recipients. p = 0.029(*), Mann–Whitney test. e F4/80 and MHCII expression by RMac^Z10 (n = 6), IMac^Z10 (n = 8) or host (n = 14) cells 8 days post transfer. Each comparison p < 0.0001(****), determined by two-way ANOVA and post hoc Tukey test. f Engraftment efficiency of RMac^Z10 (n = 7) and IMac^Z10 (n = 7) 8 days after transfer into naïve recipients. Significance determined using Mann–Whitney test. g F4/80 and MHCII expression by RMac^Z10 n = 7), IMac^Z10 (n = 7), or host (n = 14) cells 8 days after transfer. Each comparison p < 0.0001(****), two-way ANOVA and post hoc Tukey test. h Engraftment efficiency of RMac^Z10 (n = 8) and IMac^Z10 (n = 6) 8 days after transfer into clodronate-depleted recipients. p = 0.008(**), Mann–Whitney test. i F4/80 and MHCII expression by RMac^Z10 (n = 8), IMac^Z10 (n = 6), or host (n = 14) cells 8 days after transfer. Each comparison p < 0.0001(****), two-way ANOVA and post hoc Tukey test. j Proportion of RMac^Z10(n = 4), IMac^Z10 (n = 5) that are GATA6⁺ and MFI of GATA6 expression 8 days after transfer into inflamed recipients. p < 0.0001 (****), student’s t-test. k Proportion of RMac^Z10 (n = 5) and IMac^Z10 (n = 5) that are GATA6⁺ and MFI of GATA6 expression 8 days after transfer into naïve recipients. p < 0.0001 (****), student’s t-test. l Proportion of RMac^Z10 (n = 6) and IMac^Z10 (n = 5) that are GATA6⁺ and MFI of GATA6 expression 8 days after transfer into clodronate-depleted recipients. p = 0.023(*), student’s t-test. Experiments are presented as mean ± standard deviation. Each symbol represents an individual animal. Data were pooled from at least two independent experiments. Host cells represented by squares or circles are from recipients of RMac^Z10 or IMac^Z10, respectively.

Fig. 2. Inflammatory macrophages are responsive to niche factors in vitro.

a Expression of CD102 and Tim4 by cultured cells after 24 h culture with indicated treatment. b Proportion of macrophage subsets that express MHCII and F4/80 MFI after 24 h culture with control medium (n = 8), Omentum factor containing medium (n = 4) or ATRA containing medium (n = 8). MHCII⁺: p = 0.0046 (**), F4/80 MFI: p = 0.0028 (**), p = 0.032 (*), determined by one-way ANOVA and Dunnet’s multiple comparisons test for each subset individually, followed by Bonferroni adjustment. Data was pooled from 2 independent experiments and are presented as mean ± standard deviation. For each treatment group a symbol represents a culture well derived from an individual mouse.

Fig. 3. Long-lived colonizing inflammatory macrophages retain intrinsic and environment-dependent differences to RMac.

a Experimental scheme for transfer of RMac, RMac^Z10 or IMac^Z10 into naïve, inflamed or clodronate-depleted recipients. b Engraftment efficiency of RMac, RMac^Z10 and IMac^Z10 8 weeks after transfer into native (left; n = 13, n = 9, n = 9) or depleted recipients (right; n = 10, n = 10, n = 8) **p < 0.01, one-way ANOVA and Tukey’s multiple comparisons test. c Expression of F4/80 and MHCII 8 weeks after transfer into native or clodronate-depleted recipients. d Proportion of RMac, RMac^Z10, and IMac^Z10 that express MHCII 8 weeks after transfer into native (left; n = 13,11,11) or depleted recipients (right; 10, 10, 8). p = 0.00037 (***), one-way ANOVA and Tukey’s multiple comparisons test. e GATA6 MFI 8 weeks after transfer of RMac, RMac^Z10, or IMac^Z10 into native (left; n = 12,11,11) or depleted (right; n = 10,10,8) recipients. p < 0.0001(****), one-way ANOVA and Tukey’s multiple comparisons test. f Venn diagram indicating overlap between genes differentially expressed between RMac^Z10 and IMac^Z10 (adj p-value < 0.05), 8 weeks after transfer into native (blue) or depleted (red) recipients, and circus plot depicting differentially expressed genes that are cluster markers identified by Bain et al.. g Marker expression by CD102⁺ RMac (black), RMac^Z10 (blue) and IMac^Z10 (orange) 8 week after transfer into native (left; n = 7,6,6) or depleted recipients (right; n = 5, 5, 4). **p < 0.01, **p < 0.01 ***p < 0.001, one-way ANOVA and Dunnet’s multiple comparisons test for each marker, followed by Bonferroni adjustment. h Engraftment efficiency of RMac, RMac^Z10, and IMac^Z10 22 weeks after transfer into native recipients (n = 4, 5, 5). *p < 0.05, one-way ANOVA and Tukey’s multiple comparisons test. i Marker expression by CD102⁺ RMac (black), RMac^Z10 (blue) and IMac^Z10 (orange) 22 weeks after transfer into native (n = 4, 5, 5) recipients. **p < 0.01 ***p < 0.001, one-way ANOVA and Dunnet’s multiple comparisons test for each marker, followed by Bonferroni adjustment. j GATA6 MFI, 22 weeks after transfer of RMac, RMac^Z10, or IMac^Z10 into native (n = 2, 3, 3) recipients. p = 0.019 (*), one-way ANOVA and Tukey’s multiple comparisons test. Data are presented as mean ± standard deviation with each symbol representing an individual animal. Data were pooled from at least two independent experiments, except for j, which is a single experiment. Host cells represented by squares or circles are recipients of RMac^Z10 or IMac^Z10, respectively.

Fig. 4. Monocyte-derived LPM are functionally distinct from embryonically seeded LPM.

a Tim4 and Sema4a expression by RMac, RMac^Z10, and IMac^Z10 8 weeks post transfer and host cells. b Proportion of RMac^Z10 (blue, n = 6), IMac^Z10 (orange, n = 6), and host (gray) macrophages with Sema4a^LoTim4⁺(R1), Sema4a^HiTim4⁺(R2), Sema4a^HiTim4⁻ (R3), or Sema4a^LoTim4⁻ (R4) phenotype. ***p < 0.001, ****p < 0.0001, one-way ANOVA and Tukey’s multiple comparisons test. c Ki67 expression on naïve RM-LPM and Mo-LPM (n = 5) or 8-week post zymosan RM^Z10-LPM and Mo^Z10-LPM (n = 8). Naïve; p = 0.0001(***), Post zymosan; p = 0.008(**), paired student’s t-test. d Morphological appearance of RM^Z10-LPM and Mo^Z10-LPM 8 weeks post zymosan. Single experiment, scale bar 20 μm. e Mean side scatter of naïve RM-LPM, Mo-LPM, SPM (n = 5) or 8 weeks post zymosan RM^Z10-LPM, Mo^Z10-LPM, and SPM^Z10(n = 8). Naïve; both p < 0.0001(****), post zymosan: p = 0.0062(**), p = 0.0008(***), one-way ANOVA with Tukey’s multiple comparisons test. f Normalized Phrodo E.coli MFI (MFI 37 °C minus MFI 4 °C) on naïve (n = 6) or 8 weeks post zymosan (n = 9) Tim4⁺ and Tim4⁻ macrophages. Naïve; p = 0.0048(**), Post zymosan; p = 0.0002(***), paired student’s t-test. g Analytes secreted by RM-LPM (n = 6, teal) or Mo-LPM (n = 5, red) sourced from naïve animals following 14 h LPS (1 ng/ml) treatment. Results shown as log2 fold change in mean pg/ml over mean RM-LPM. Box extends from the 25th to the 75th percentile, middle line denotes median. Whiskers denote minima and maxima. **p < 0.001, ***p < 0.0001, repeated student’s t-test with Holm-Sidak correction. h Analytes secreted by RM^Z10-LPM (n = 8, teal) or Mo^Z10-LPM (n = 8, red) sourced 8 weeks post zymosan following 14 h LPS (1 ng/ml) treatment. Results shown as log2 fold change in mean pg/ml over mean RM^Z10-LPM. Box extends from the 25th to the 75th percentile, middle line denotes median. Whiskers denote minima and maxima. *p < 0.05 **p < 0.001, ***p < 0.0001, repeated student’s t-test with Holm-Sidak correction. i Experimental scheme for purification of RM^Z10-LPM and Mo^Z10-LPM from donor mice treated 8 weeks prior with 10 μg zymosan and transfer into naïve-recipient mice followed by injection of 5 μg LPS IP. j Proportion of donor CD45.2⁺ F4/80^Hi RM^Z10-LPM (n = 7) and Mo^Z10-LPM (n = 6) 8 h post injection of LPS that express TNF. p = 0.0206(*), student’s t-test. Data are presented as mean ± standard deviation with each symbol representing an individual animal. Naïve animals were age matched to zymosan-treated (15–18-week) animals. For (c, g), animals were 10–12-week at time of analysis. Data pooled from two independent experiments.

Fig. 5. Ontogeny does not control monocyte phenotype after severe peritonitis and leads to impaired B1 cell expansion.

a Normalized chimerism of Tim4⁺ macrophages in tissue-protected BM chimeric mice left naïve or treated with 10 μg or 1000 μg zymosan 17 days prior (n = 3/group). **p < 0.01, one-way ANOVA and Tukey’s multiple comparisons test. b Engraftment efficiency of RMac^Z10 (n = 6), IMac^Z10 (n = 5), and IMac^Z1000 (n = 6), after transfer into the mirroring recipients. p = 0.005(**), p = 0.00038(***), one-way ANOVA and Tukey’s multiple comparisons test. c Expression of F4/80 and MHCII on indicated populations 8 weeks post after transfer. d GATA6 MFI of donor RMac^Z10, IMac^Z10 and IMac^Z10 after transfer into native recipients (n = 3, 2, 3). e Marker expression by CD102⁺ or F4/80⁺ donor RMac^Z10 (blue), IMac^Z10 (orange), and IMac^Z1000 (purple) 8 weeks after transfer into mirroring recipients (n = 6, 5, 6). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA and Dunnet’s multiple comparisons test for each marker, followed by Bonferroni adjustment. f Principal component analysis based on all markers assessed in e. g Scheme for transfer of F4/80^Hi MHCII^Lo naïve resident macrophages (RMac) into mice injected 3 days prior with 1000 μg zymosan. h F4/80, Tim4, and MHCII expression on RMac and host myeloid cells 8 days post transfer. i Proportion of RMac that express MHCII and high levels of F4/80 before transfer and 8 days post transfer (n = 5; left). Number of host Tim4⁺ or Tim4^- macrophages 8 days post transfer (n = 5). In order: p < 0.0001 (****), p < 0.0001 (****), p = 0.021 (*), student’s t-test. j Number of CXCL13⁺ host macrophages 8 weeks after 10 μg (n = 11) or 1000 μg zymosan (n = 6) treatment. p = 0.0020 (**), student’s t-test. k Number of peritoneal B1 cells in naïve female mice at indicated age in weeks (n = 5/timepoint). p = 0.00047(***), one-way ANOVA and Tukey’s multiple comparisons test. l Number of peritoneal CD11b⁺ B1 cells at indicated timepoints in naïve mice (black; n = 5, 6, 10) or mice treated with 10 μg (gray; n = 6, 11, 20) or 1000 μg zymosan (white; n = 3, 3, 4). *p < 0.05, ***p < 0.001, ****p < 0.0001, two-way ANOVA and post hoc Tukey test. m Serum anti-phosphorylcholine IgM and IgG in naïve mice (n = 6), or mice treated with 10 μg (n = 16) or 1000 μg (n = 2) zymosan 8 weeks prior. Data are presented as mean ± standard deviation with each symbol representing an individual animal. Data were pooled from at least two independent experiments except high-dose treatment (l, m), which is a single experiment. Host cells represented by squares or circles are recipients of RMac^Z10 or IMac^Z10, respectively.