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

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.

(c) Representative PKH26-PCL labelling and quantification of donor F4/80 hi macrophages prior to transfer (top) and 2hrs post transfer (red; n=3) compared to recipient F4/80 Hi macrophages (black). p<0.0001 (****) determined by one-way ANOVA with Tukey's multiple comparisons test.       Includes data presented in Figure 2b and Figure 4b. Each symbol refers to an experimental run. p=0.043 (*) determined by student's t test. For all experiments, data are presented as mean ± standard deviation with each symbol representing an individual animal. All data were pooled from at least 2 independent experiments, except for (k) which is from a single experiments.   (e) Expression of Tim4 and PKH26-PCL labelling on donor RMac following purification and prior to transfer.
(g) Expression of Tim4 and PKH26-PCL labelling on donor RMac following 8 days following transfer into clodronate-depleted recipients.
Data are presented as mean ± standard deviation with each symbol representing an individual animal.
Data were pooled from at least 2 independent experiments.  For all experiments, data are presented as mean ± standard deviation with each symbol representing an individual animal. All data were pooled from at least 2 independent experiments except (a) where high dose datapoints where from a single experiment.

Supplementary
Supplementary Figure 6. Severe peritonitis leads to long-term perturbations of peritoneal immune cells.
(a) Gating strategy to identify peritoneal CD11b + B1 cells in naïve female mice of indicated age.
(b) Gating strategy to identify peritoneal CD11b + B1 in mice 8 weeks after indicated treatment.
(d) Representative gating strategy to identify T-cell subsets and Neutrophils. respectively. Statistical significance determined using repeated t test with Holm-Sidak correction.
(g) Identification of zymosan particles on the basis of SSC-A/FCS-A relative to peritoneal cells.
For all experiments, data are presented as mean ± standard deviation with each symbol representing an individual animal. All data were pooled from at least 2 independent experiments except (g,h) originate from a single experiment.