Characterization of human peritoneal monocyte/macrophage subsets in homeostasis: Phenotype, GATA6, phagocytic/oxidative activities and cytokines expression

Peritoneal macrophages play a critical role in the control of infectious and inflammatory diseases. Although recent progress on murine peritoneal macrophages has revealed multiple aspects on their origin and mechanisms involved in their maintenance in this compartment, little is known on the characteristics of human peritoneal macrophages in homeostasis. Here, we have studied by flow cytometry several features of human peritoneal macrophages obtained from the peritoneal cavity of healthy women. Three peritoneal monocyte/macrophage subsets were established on the basis of CD14/CD16 expression (CD14++CD16−, CD14++CD16+ and CD14highCD16high), and analysis of CD11b, CD11c, CD40, CD62L, CD64, CD80, CD86, CD116, CD119, CD206, HLA-DR and Slan was carried out in each subpopulation. Intracellular expression of GATA6 and cytokines (pro-inflammatory IL-6 and TNF-α, anti-inflammatory IL-10) as well as their phagocytic/oxidative activities were also analyzed, in an attempt to identify genuine resident peritoneal macrophages. Results showed that human peritoneal macrophages are heterogeneous regarding their phenotype, cell complexity and functional abilities. A direct relationship of CD14/CD16 expression, intracellular content of GATA6, and activation/maturation markers like CD206 and HLA-DR, support that the CD14highCD16high subset represents the mature phenotype of steady-state human resident peritoneal macrophages. Furthermore, increased expression of CD14/CD16 is also related to the phagocytic activity.

Macrophages are a versatile and heterogeneous cell population that interconnects innate and adaptive immunity and plays a crucial role in many inflammatory diseases, tissue remodelling and wound healing, among others. These cells reside in all tissues in homeostasis, and are also rapidly recruited and differentiated from circulating blood monocytes in response to local danger signals provided by inflammation or microbial invasion 1,2 . Peritoneal macrophages are key players in the control of infectious and inflammatory diseases 3 through a variety of effector and regulatory functions 4 . Currently, the majority of studies on phenotypic and functional characteristics of tissue resident macrophages have been performed in murine peritoneal macrophages (pMϕ) 5 or in vitro differentiated human blood monocyte-derived macrophages 6 . However, the population of blood monocytes from healthy people contains three subpopulations displaying different phenotypes and functions 7 named as "classical" CD14 ++ CD16 − , "intermediate" CD14 ++ CD16 + and "non-classical" CD14 +/low CD16 + monocytes [8][9][10] . The CD16 + subpopulations are considered as pro-inflammatory monocytes since they are increased in acute 11 and chronic [12][13][14][15] inflammatory pathologies. These cells produce in vitro TNF-α, IL-6, MIP1α and MIP1β pro-inflammatory cytokines in response to LPS 7,14,16 and they are probably expanded from the classical to the intermediate subset and from this to the non-classical subpopulation 10 . Nowadays, a great interest has been focused to uncover the specific monocyte subpopulation that gives rise to tissue resident macrophages in steady-state or in different clinical scenarios. We have recently identified a new subset of monocyte/macrophages from ascites of cirrhotic patients displaying high expression of CD14 and CD16 referred to as CD14 high CD16 high , bigger size and more complexity than CD16 −/low cells, which is not detected in peripheral blood monocytes 17 .
On the other hand, all knowledge on the origin and development of tissue resident macrophages has been obtained from experimental mouse models. In this regard, it has been recently demonstrated the existence of several types of tissue-resident macrophages independent of hematopoietic stem cell origin, which are directly derived from embryonic progenitors (yolk sac and foetal liver) and have the intrinsic capacity to proliferate and self-renew 18,19 . Furthermore, it has also been described that the expression of transcription factor GATA-binding protein 6 (GATA6) is mainly restricted to the long-lived murine F4/80 hi CD11b hi large peritoneal-resident macrophages (LPM), which retain cells of embryonic origin for at least four months, while the other subset of F4/80 low MHC-II hi referred to as small peritoneal macrophages (SPM) is derived from inflammatory monocytes 20 . Given that the new human CD14 high CD16 high subpopulation of ascitic cells is not detected in peripheral blood monocytes 17 , we hypothesize that it could represent the phenotypic signature of genuine human resident-peritoneal macrophages. To assess this, we have analyzed the phenotype and the intracellular expression of GATA6 together with the phagocytic and oxidative activity, the intracellular expression of pro-inflammatory cytokines IL-6 and TNF-α, and the anti-inflammatory IL-10 in the three CD14/CD16 subsets of peritoneal monocytes/macrophages (pMo/Mϕ) from healthy women. Our results confirm the presence of three CD14/ CD16 subpopulations of monocytes/macrophages in steady-state. Although, the percentage of GATA6 expressing cells is similar among the three described subpopulations of pMo/Mϕ, the more complex CD14 high CD16 high subset contains the highest number of GATA6-expressing cells, as well as higher phagocytic/oxidative activities and expression of membrane receptors involved in antigen presentation, activation, co-stimulation and phagocytosis. The intermediate subset expresses a medium level of those markers, while the classical-like subset is more similar to the corresponding population of blood monocytes. Finally, among them a similar set of pro-inflammatory M1 and anti-inflammatory M2 polarization markers was observed, which is compatible with a basal pre-activated state to rapidly respond against any challenge to maintain peritoneal homeostasis.

Subjects baseline characteristics.
A consecutive series of 79 women was initially recruited for the study.
Seven patients were excluded after diagnosis of endometriosis (n = 5) and carcinoma (n = 2). Additionally, 21 patients were excluded due to technical difficulties in the collection of peritoneal samples, such as blood contamination (n = 8) or very low amount of cell content (n = 13). Finally, 51 women fulfilling inclusion and exclusion criteria were included in the study. Clinical and analytical characteristics of blood leukocytes from people analysed are detailed in Table 1. As it can be observed, the values of analysed data correspond to the normal range for the sex and age of the group studied.

Human peritoneal macrophages subpopulations based on the expression of CD14 and CD16.
First, we studied the distribution of pMo/Mϕ according to the expression pattern of membrane markers CD14 (co-receptor of LPS) and CD16 (FcγRIII) in comparison to the subsets of monocytes found in blood ( Fig. 1a-h). The gating strategy of blood monocytes and pMo/Mϕ was carried out as follows: Initially, cells were selected according to morphology (forward vs. side scatter). Afterwards, cellular aggregates were excluded based on FSC-W vs. FSC-A. Finally, cells were gated based on CD14/CD16 expression ( Supplementary Fig. S1). Results showed that, similarly to blood monocytes, pMo/Mϕ are distributed into three different subpopulations in terms of CD14/CD16 expression. Results showed that differently to blood monocytes (Fig. 1a), which display the known classical CD14 ++ CD16 − , intermediate CD14 ++ CD16 + , and non-classical CD14 +/low CD16 + subsets with uniform morphology and low complexity (Fig. 1c-e), pMo/Mϕ, as it is displayed in Fig. 1b, are distributed into: (a) a classical-like CD14 ++ CD16 − subset (Fig. 1f) similar to their counterparts found in blood (Fig. 1c); (b) an intermediate CD14 ++ CD16 + subset displaying higher level of CD14 expression, and higher heterogeneity related to both granularity/complexity and size (Fig. 1g) than intermediate blood monocytes (Fig. 1d); and (c) a totally different CD14 high CD16 high subset (Fig. 1h), which presents not only higher expression of both CD14 and CD16, but also includes cells with the biggest size and highest granularity. Quantitative analysis of the MFI for CD14 (Fig. 1i) and CD16 (Fig. 1j) within each pMo/pMϕ subpopulation was performed. Frequencies of the three subsets in blood vs. peritoneum (Mean ± SEM) are shown in Fig. 1k. As expected, the predominant subpopulation in blood was the classical subset (86.5 ± 1.0%), followed by the non-classical subset (8.5 ± 0.8%), and finally the intermediate subset with the lowest proportion (5.0 ± 0.4%); differences were significant in all cases (#). Besides, differences between each CD14 ++ CD16 − and CD14 ++ CD16 + subset of blood monocytes with those of pMo/Mϕ were significant in both cases (*). Percentages of peritoneal classical-like and intermediate subpopulations were similar (43.3 ± 4.1% and 42.0 ± 3.0%, respectively), while the most complex subset represented a 14.7 ± 1.8% of the whole monocyte/macrophage population. Differences with percentages of the former two subpopulations were significant (#).
Phenotypic characteristics of human peritoneal subpopulations compared to blood monocytes. To explore the functionality of pMo/Mϕ, we analyzed by flow cytometry the expression of several surface markers on the whole population of pMo/Mϕ comparatively to blood monocytes. Concretely, we analyzed receptors involved in activation: CD64 (FcγRI), CD40, CD80, CD86, CD116 (GM-CSFR) and CD119 (IFNγR1 or IFNγ chain α receptor); phagocytosis of complement-opsonised particles and adherence: CD11b and CD11c (the α subunit of CR3 and CR4 respectively); adhesion and migration: CD62L (Selectin-L) and P-selectin glycoprotein ligand-1(PSGL-1) carbohydrate modified Slan (6-sulfo LacNAc); antigen presentation: HLA-DR, and the M2 macrophage-polarized marker involved in phagocytosis: CD206 (mannose receptor). Results showed that some of them, such as CD11b, CD64, CD86, CD116, CD119 and HLA-DR were widely expressed in both peritoneal cells and blood monocytes with percentages ranging from 76.0 ± 4.7% for CD86 to 94.7 ± 1.3% for CD116 ( Fig. 2A). Among them, CD116, CD119 and HLA-DR presented slightly higher, but significant, percentages in peritoneal cells. Others like CD11b and CD86, followed the same trend, although did not reach significant differences or were close to reach them, as it was the case of CD64 (p = 0.055) ( Fig. 2A). Other markers presented remarkable differences between cells isolated from each location (Fig. 2B). The majority of those membrane molecules were significantly higher expressed in peritoneal cells than in blood monocytes, in which most of those markers could be actually considered absent. That was the case of CD11c, the co-stimulatory molecules CD40 and CD80, the mannose receptor CD206 and Slan (Fig. 2B). On the contrary, the percentage of cells expressing the adhesion molecule CD62L was significantly higher in blood monocytes (75.9 ± 3.7%) than in pMo/Mϕ (27.4 ± 5.1%) (Fig. 2B).
A deeper analysis of the phenotypic profile of each CD14/CD16 subpopulation of pMo/Mϕ and blood monocytes is summarized in Table 2. These data showed that the higher cell complexity and expression of CD16 in peritoneal cells goes parallel to the higher expression of all markers assayed, not only for those that already were higher in peritoneal cells than in blood monocytes (CD116, CD119 and HLA-DR), but also for those that did not show significant differences (CD11b, CD64 and CD86) (Fig. 2C). Even in the case of CD62L, that displayed percentages of CD62L+ cells higher in blood than in peritoneal cells (Fig. 2B), the percentage of CD62L+ peritoneal cells increased along with the cell complexity and expression of CD16 (Table 2). Nevertheless, in blood monocytes subsets the trend was variable depending on the marker analyzed. The majority of them showed a similar trend to peritoneal cells, increasing their frequencies as the expression of CD16 raised, except for CD11b, CD62L, CD64 and CD116, which showed the opposite trend, i.e., the higher percentage of positive cells for those markers appeared into the classic CD14 ++ CD16 − subset. As displayed in Table 2 the percentages of positive cells for the analyzed markers were significantly different when comparing the corresponding peritoneum and blood cell subsets (*p < 0.05, **p < 0.01, ***p < 0.001) with some exceptions. Differences between subsets located in each compartment were significant for those molecules that were practically absent in blood monocytes, such as CD11c, CD40, CD80, CD206 and the exclusive and partial marker of the non-classical subset Slan, but also for CD62L, which was poorly expressed in peritoneal cells and enriched in blood monocytes. Nevertheless, differences between subsets in each location were also observed for widely expressed molecules, especially for those with an opposite trend of expression in blood and peritoneum, such as CD11b, CD64, CD116 and HLA-DR.
We also analyzed the density of each receptor expressed per cell (MFI) in every cell subset in both blood and peritoneal cells (Table 3). A representative side by side experiment of markers expressed in cell subsets from both compartments is shown in Fig. 2C. Results confirm the gradual increased amount of molecules expressed on the membrane of pMo/Mϕ as the complexity and expression of CD16 also increase. Hence, histograms corresponding to the more complex CD14 high CD16 high peritoneal subpopulation (dark blue colour in Fig. 2C) are the most displaced to the right in all cases. The figure also remarks that percentages of cells expressing each marker analyzed are related to the density of receptors expressed per cell. Hence, those markers that showed a similar (CD11c, CD86, CD119 and HLA-DR) or opposite (CD11b, CD62L, CD64 and CD116) trend in the frequencies of expressing cells on each subset from both locations, followed a similar/opposite tendency for the intensity expressed per cell (MFI), respectively. ***p < 0.001, between the classic-like CD16 negative subset and the two other CD16 positive subsets present in peritoneum; ### p < 0.001, between the two CD16 positive subsets (CD14 ++ CD16 + vs. CD14 high CD16 high ). Percentages of cell subsets were comparatively analyzed in blood (N = 29) and peritoneum (N = 36) from healthy individuals (k). Histograms represent mean ± SEM of every cell subset. White bars are used for blood monocyte subsets and grey bars for peritoneal monocyte/macrophage subsets. Mann-Whitney U test: ***p < 0.001, between classic and intermediate subsets present in blood vs. peritoneum; ## p < 0.01, ### p < 0.001, between phenotypic subsets inside each location. Expression of GATA6 in human peritoneal macrophages. Trying to discriminate between mature resident peritoneal macrophages and newly migrated blood monocytes, we analyzed the intracellular expression of transcription factor GATA6, which has been described as a central regulator of the murine peritoneal macrophage phenotype. The results showed that while GATA6 was absent in peripheral blood monocytes (Fig. 3a,b), the percentage of GATA6+ cells in the peritoneal cavity was rather high (80.1 ± 11.4%) (Fig. 3c). Next, we analyzed the percentages of GATA6+ cells within each CD14/CD16 peritoneal subset. As displayed in Fig. 3d, the results did not show significant differences in the ratio of positive cells among the three peritoneal subpopulations; although there was a trend toward a higher number of GATA6+ cells within the more complex CD14 high CD16 high subpopulation (86.1 ± 8.6%), followed by the intermediate subset (83.3 ± 10.0%), and the classic-like CD14 ++ CD16 − subpopulation (79.2 ± 12.1%). Representative FACS histograms and quantitative analysis of GATA6 MFI within each pMo/pMϕ subpopulation are displayed in Fig. 3d. Similarly to the percentages of GATA6 positive cells, the intensity level of cell expression within each peritoneal subset was directly related to the cell complexity, and therefore to the expression of CD14 and CD16. The analysis of GATA6 intensity expression (MFI) related to the corresponding MFI of CD14 and CD16 showed a clear correlation (R 2 = 0.9957 and R 2 = 0.9992, respectively) (Fig. 3e). Thus, taking into account the results reported from the mouse model, these data reveal that, in spite of their phenotypic differences, the majority of resident pMo/Mϕ in homeostasis highly express the transcription factor GATA6. The mature and more active cells would mostly correspond to the complex CD14 high CD16 high and,   Table 3. Median fluorescence intensity of total pMo/Mϕ and CD14/CD16 subsets compared to blood of healthy humans. Data represent the Mean ± SEM of median fluorescence intensity (MFI) for each membrane marker analyzed. *p < 0.05, **p < 0.01, and ***p < 0.001, ratios of positive monocyte/macrophages from peritoneum compared to monocytes from blood of healthy subjects.  Phagocytic activity of human peritoneal CD14/CD16 subpopulations. Flow cytometry analysis of phagocytic activity of pMo/Mϕ subpopulations showed that the percentage of phagocytic cells in the classical-like subset was 26.0 ± 3.7% after 10 min incubation with E. coli at 37 °C, and 27.9 ± 8.7% after 1 hour at 37 °C. Subsets of CD16 + cells engulfed significantly more bacteria than CD16 − cells did. Thus, the intermediate subset showed percentages of phagocytic cells of 73.5 ± 11.5% and 77.4 ± 11.2% for 10 min and 1 hour assays, respectively; while the more complex subset displayed the highest frequency of phagocytic cells (96.6 ± 3.0%) even after only 10 min assays, which was maintained (96.4 ± 1.8%) after 1 hour incubations (Fig. 4a). Concomitantly to the percentages of phagocytic cells, intracellular bacterial load, measured as MFI, was higher as it was the complexity and the size of the cell subsets. Thus, as it can be observed in the representative histograms displayed in Fig. 4b,e the classic-like subset showed the lowest MFI, both after 10 min (bright aquamarine histogram in Fig. 4b) and 1 hour assays (dark aquamarine histogram in Fig. 4e), the intermediate subset showed an intermediate MFI (Fig. 4c,f), and the complex subset presented the highest MFI levels (Fig. 4d,g).
Oxidative potential of pMo/Mϕ. Oxidative capacity of pMo/Mϕ was also measured by flow cytometry.
To this purpose, dihydrorhodamine 123 (DHR) was added to cell samples, which displays green fluorescence when is oxidized to rhodamine 123 (Rho) by the ROS generated by cells, indicating their oxidative potential. Two parallel assays were performed with each sample, one without stimulation, to test the basal state of pMo/Mϕ regarding its oxidative activity, and another one with cells stimulated with PMA. In the first case a 75.5 ± 2.8% of cells showed oxidative potential (Fig. 5a), and no significant differences were found with those stimulated with PMA (81.8 ± 5.3%) (Fig. 5b).  Surprisingly, when pMo/Mϕ were classified in terms of rhodamine oxidative capacity three different subpopulations were observed, i.e., a population with no oxidative capacity: Rho − , and two subpopulations with different level of oxidation: Rho low , and Rho high . These three subpopulations were detected both in basal conditions (Fig. 5a) and after PMA stimulation (Fig. 5b). Percentages of Rho high cells were significantly higher in both basal and PMA-activated conditions, while the frequency of Rho − cells was low and similar in both cases. However, while percentages of Rho low showed a trend to be slightly lower than the corresponding to Rho − cells in basal conditions, this tendency was opposite in PMA-treated cells (Fig. 5c).
When cellular baseline oxidative potential was separately analyzed within each established CD14/CD16 subset, we found that the complex CD14 high CD16 high cells showed a lower percentage of oxidative cells (66.9 ± 4.4%) than both the classic-like CD14 ++ CD16 − (82.7 ± 1.1%, p = 0.0251) and the intermediate CD14 ++ CD16 + (78.3 ± 3.4%, p = 0.0274) subsets. Contrarily, when peritoneal cells were stimulated with PMA differences between the percentages of Rho positive cells within each CD14/CD16 subset were not significant, revealing a similar distribution of oxidative cells under this condition (Fig. 5d). Nonetheless, in terms of MFI values, differences among the three subpopulations of pMo/Mϕ were not significant (Fig. 5e).
When percentages of positive cells for these cytokines were analysed and compared among each CD14/CD16 subpopulation (Fig. 6h), we found that the highest ratios of cytokine-positive cells corresponded to the intermediate CD14 ++ CD16 + subset for the three cytokines. Furthermore, differences between the CD14 ++ CD16 + subset compared with their counterparts were significant for the pro-inflammatory cytokines IL-6 and TNF-α; although for the anti-inflammatory IL-10 this difference was only significant compared with percentages of IL-10+ CD14 ++ CD16 − cells. In turn, differences between percentages of cytokine-positive cells within the classic-like CD14 ++ CD16 − compared with those obtained within the complex CD14 high CD16 high subsets were only significant for TNF-α.

Discussion
Although tissue-resident macrophages are known for more than 100 years, the studies on the physiology of this cell type in humans have been hindered by the difficulty of isolating macrophages in conditions of health and disease from peripheral tissues, which endures aggressive surgical interventions in which besides that few cells are obtained they are difficult to grow in vitro. For this reason, the majority of research on resident macrophages in both steady-state and disease has been carried out in experimental animals, especially in the mouse model. However, extrapolation from mice to man is not always feasible, which is especially true in this particular subject 21,22 . Furthermore, studies on the role of the immune system in inflammatory diseases in humans largely rely on findings obtained from the blood compartment. However, studying macrophages from an inflammatory scenario can make valuable contributions to a better understanding of the physiopathology of those human diseases 2,19 . Hence, it seems necessary to assess whether the local tissue mediators and macrophage transcription factors that have been identified in mice play equal roles in the biology and development of humans tissue-resident macrophages. Human pMo/Mϕ are a good source of this cell type to study their biological properties under steady-state conditions. These samples must be obtained from healthy people or patients whose disease does not directly involve the peritoneal cavity. The most frequent examples are women subjected to gynaecological laparoscopy/laparotomy or continuous ambulatory peritoneal dialysis (CAPD) patients not affected by spontaneous bacterial peritonitis (SBP), a complication that occurs frequently in patients with cirrhosis and ascites and less frequently in CAPD [23][24][25][26] . Nevertheless, the required surgical procedures to obtain peritoneal samples from healthy people usually yield very low cell numbers, so that performing functional studies based on cell cultures remains as a challenging task. For this reason, there is little information about the phenotype and function of pMo/Mϕ from normal individuals. In our aim to characterize normal human pMϕ according to level 1 (developmental characteristics) and level 2 (surface antigen expression, phagocytosis and oxidative activity and intracellular cytokines content) proposed by Guilliams et al. 27 to define a new myeloid cell type, we have compared several features of pMo/Mϕ with the well defined CD14/CD16 blood monocytes subsets to describe common features or tissue-specific characteristics and differences. We found pMo/Mϕ to represent a heterogeneous cell type based on both morphology and expression of CD14/CD16. It is integrated by similar proportions (approx. 42%) of small classical-like (CD14 ++ CD16 − ) and intermediate-like (CD14 ++ CD16 + ) cells, and a new subset of highly complex CD14 high CD16 high cells (approx. 16%), which is absent in peripheral blood. In turn, cells similar to non-classical blood monocytes are not found into the peritoneal cavity. In regards to the staining pattern for both CD14 and CD16, pMo/Mϕ exhibit a higher expression level than blood monocytes, which would make them more efficient, or better equipped, to detect LPS and phagocyte microorganisms. Of note, the proportions of these subsets result modulated under inflammatory conditions, thus we have previously found in ascites of decompensated cirrhotic patients that percentages of CD14 high CD16 high cells were increased up to 33 ± 2.4%, the CD14 ++ CD16 + intermediate subset reached up to 49 ± 2.0%, while the CD14 ++ CD16 − classical subset, went down to 18 ± 1.3% 17 , these modifications in pathology vs. steady-state conditions strength the relevance of the results presented here.
Furthermore, we tested the expression of various monocyte/macrophage-associated surface antigens involved in phagocytosis of IgG-opsonised (CD64, high affinity FcγRI) and complement-opsonised particles (CD11b and CD11c, the α chains of Complement receptors, CR3 and CR4); adherence to stimulated endothelium and migration (CR3, CR4, CD62L and Slan), antigen presentation (MHC class II molecule HLA-DR), co-stimulatory molecules (CD80, CD86, CD40), cytokines receptors (CD116, GM-CSFR and CD119 IFNγR1 or IFNγ chain α receptor) and the mannose receptor (CD206) described as a M2 macrophage polarized marker and indicative of activation/maturation 28 . Compared with the whole population of blood monocytes, the expression of CD11b, CD64, and CD86 on pMo/Mϕ was not statistically different, while we found small but significant differences for a higher expression of CD116, CD119 and HLA-DR on pMo/Mϕ. The most striking differences were observed for CD11c, CD40, CD80, CD206, Slan and CD62L, with the exception that the last one was the only marker showing higher expression on blood monocytes. In view of these phenotypic results, one might assume that human pMo/ Mϕ would be able to exhibit a high antimicrobial (also supported by the phagocytic and oxidative capacity displayed by these cells), antigen-presenting and T-cell co-stimulatory capacity, although this remains to be further studied. On the other hand, the gradual increment of percentages of expression and density per cell of CD206 observed from 28.2% in CD14 ++ CD16 − to 60.3% in CD14 ++ CD16 + and 92.8% in CD14 high CD16 high suggests that human resident pMo/Mϕ may also display phenotypic and functional properties of M2 polarized macrophages previously reported from CAPD patients 29,30 and endometriosis 31 . In this regard, a recent work carried out on experimental endometriosis in a mouse model has demonstrated the dynamic changes in the proportions and polarization profile (M1 and M2) of F4/80 hi CD11b hi large and F4/80 low CD11 blow low pMϕ subsets along with the development of the endometriosis lesions 32 . Nevertheless, the most striking differences between blood and peritoneal monocytes/macrophages subsets were found on percentages and MFI of cells expressing the selectin CD62L, i.e., blood monocytes decrease CD62L expression as CD16 increases, while percentages of pMϕ expressing CD62L in each subset increase along with the expression of CD16. Furthermore, results obtained for the SCIENtIFIC REPoRTS | (2018) 8:12794 | DOI:10.1038/s41598-018-30787-x expression of Slan (6-sulfo LacNAc), reported as a marker differentiating new subsets of CD16 + monocytes that are expanded in patients with sarcoidosis (Slan-negative, CD16 + ) or depleted in hereditary diffuse leukodystrophy (Slan-positive, CD16 + ) 33 , showed that although the tendency is similar in both blood and peritoneal subsets, the overall percentages of Slan expressing cells were statistically higher in the latter compartment. Differences on expression of these adhesion molecules could be related with a very different pattern of cell-migration on each compartment (endothelium/mesothelium). In this regard, it has been recently described that GATA6+, F4/80+ mature pMϕ from mice, rapidly infiltrate the injured liver through a non-vascular route, adopting an alternatively M2 activated phenotype and protecting against acute liver damage 34 .
Expression of GATA6 is absent in blood monocytes, while the percentage of GATA6 expressing cells among the three described subpopulations of pMo/Mϕ is similar. Nevertheless there is a strong correlation between the gradual increase of GATA6 intracellular expression and the cellular membrane expression of both CD14 (R 2 = 0.9957) and CD16 (R 2 = 0.9992). These findings could either suggest that the migration of monocytes to the peritoneal cavity under steady-state conditions is very low, or that the expression of GATA6 in newly arrived peritoneal monocytes is very rapid. Altogether, these data and those reported from mice, point out to the more complex population of CD14 high CD16 high pMo/Mϕ as the phenotypic signature of mature differentiated human resident pMϕ, while the intermediate subset CD14 ++ CD16 + could represent a mixed transitional cell type also integrated by recruited blood monocytes developing phenotypic and functional characteristics of resident peritoneal macrophages. However, taken into account the results of Bain et al. 20 , on the origin, self-renewal and age-dependent replacement of F4/80 hi CD11b hi large pMϕ by the F4/80 low MHC-II hi subset of small pMϕ derived from inflammatory monocytes 20 , and without the possibility to perform fate-mapping experiments in humans, we can neither assume nor exclude the embryonic-derived origin of the CD14 high CD16 high subset.
The low frequency of cells expressing intracellular IL-6, TNF-α and IL-10 cytokines confirms the homeostatic state of this cell population. Interestingly, the intermediate subset displays the highest proportions of intracellular cytokines, while the CD14 high CD16 high subset presents a higher frequency of IL-10 positive cells compared to those of pro-inflammatory cytokines, which favours the hypothesis of its M2 polarization trend.
Finally, the linear relationship between the expression of CD14/CD16 and activation/maturation markers, such as CD206 and HLA-DR, the intracellular content of GATA6, the phagocytic/oxidative activity, and the intracellular content of IL-6, TNF-α and IL-10, support that the CD14 high CD16 high subset is the mature phenotype of human resident pMϕ in steady-state conditions, which would play a main role in the maintenance and recovery of homeostasis after injuries and pathogen challenges. The present results on healthy human pMo/Mϕ provide a useful information for those researchers studying peritoneal macrophages under inflammatory conditions or in the presence of tumors.

Material and Methods
Study subjects and cell collection. Steady-state peritoneal cells of 79 healthy women were obtained from the Gynecological Unit of the Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain. Cell samples (from blood and peritoneal cavity) were obtained during exploratory or therapeutic laparoscopies for benign gynaecological pathology (simple ovarian cysts or uterine fibroids) or tubal ligation. Both during surgery and in the analysis of peritoneal lavage cells there was no evidence of involvement of the peritoneal space in any one of the patients. After opening the abdomen by incision, the peritoneal cavity was instilled with 50 mL phosphate-buffered saline (PBS) solution that was collected from the rectouterine pouch, or pouch of Douglas, strictly avoiding contamination by blood. Nevertheless red-coloured samples indicative of peripheral blood contamination were excluded. Abdominal surgery continued after this brief lavage procedure. Samples were then maintained at 4 °C to avoid cell attachment to plastic. Cells were finally washed with RPMI-1640 (GIBCO Invitrogen, Paisley, UK) and processed for flow cytometry analysis.
The ethics committees (Hospital Clínico Universitario Virgen de la Arrixaca, and Comité de Bioética de la Universidad de Murcia) approved the study protocol according to the 1975 Declaration of Helsinki and all peritoneal cells donors gave informed written consent to be included in this study.
Alternatively, after following the same washing and preparation procedure, 0.4·10 6 white cells were stained intracellularly with the IntraStain Kit (Dako, Glostrup, Denmark) following manufacturer instructions.