The cohort for our inflammatory cell assessment included 21 control and 57 prolapsed individuals that were separated by the POP-HQ system into 19 POP-adipose (POP-A), 22 POP-inflammation (POP-I), and 16 POP-vascular (POP-V). Subjects were chosen for the current study, based upon the integrity of the USL tissue, the completeness of the subject’s demographic data, and their POP-HQ phenotype.

Table 1 shows the patient demographics means and intergroup statistics, according to POP-HQ phenotypes, of the subjects used for investigation of inflammatory cell types. Subjects in the POP-A and POP-V phenotypes were older, had more vaginal deliveries, and were more likely to be post-menopausal than control subjects. Subjects in the POP-I phenotype were older and more likely to be post-menopausal than control subjects. Almost all the Control, POP-A and POP-I group subjects identified racially as white, whereas only 44% of the POP-V group did so, the remaining POP-V subjects identified as Hispanic or Latino, Asian, or African American. Importantly, no statistically significant differences were noted in anti-inflammatory drug use between the 4 groups. The demographic data for each subject has been uploaded to Figshare: https://figshare.com/s/365b699d5d8078c90ff0.

Table 2 shows the means, standard deviations, and intergroup statistics for the POP-HQ histopathological scoring features of the subjects used for investigation of inflammatory cell types. The POP-A phenotype had less smooth muscle and more muscle fiber dropout than the control group, the POP-I phenotype had more muscle fiber dropout and inflammation and less NIH than the control group, and the POP-V phenotype had more NIH and less inflammation than the control group. These differences are similar to those we have previously reported for these POP-phenotypes [16]. The POP-HQ data for each subject has been uploaded to Figshare: https://figshare.com/s/365b699d5d8078c90ff0.

IHC staining for myeloperoxidase identified neutrophils (Fig. 1, and SupFig 1). Neutrophils were observed as individual cells and clumps and were observed throughout the various USL connective tissues although less commonly inside smooth muscle fascicles and never inside neural bundles. Only neutrophils (and all inflammatory cells) found outside of vessels (Fig. 1, bottom row, left hand side) were considered during quantification scoring (Table 5). The POP-I phenotype had an approximately ten-fold increase in neutrophils compared to the other phenotypes (p ≤ 0.05, Table 5). Data for neutrophil quantification for all subjects, as well as the quantification of all other inflammatory cells has been uploaded to Figshare: https://figshare.com/s/365b699d5d8078c90ff0.

IHC staining for CD3 identified T-lymphocytes (Fig. 1, and SupFig 1). Besides their presence in loose connective tissue, T-lymphocytes were observed in all three layers of arterial walls of subjects with NIH ( discussed below). The POP-I phenotype had an approximate doubling of the number of T-lymphocytes than the other phenotypes (p ≤ 0.05, Table 5).

IHC staining for CD68 identified all macrophages (Fig. 1, and SupFig 1). Besides their presence in loose connective tissue, macrophages were also observed in the arterial walls of subjects with NIH (Fig. 4, discussed below). The POP-I phenotype had approximately 2–threefold more macrophages than the other phenotypes (p ≤ 0.05, Table 5).

Immunohistochemical staining for inflammatory cells. IHC staining for neutrophils, T-lymphocytes, macrophages, and resolving macrophages are demonstrated, as well as toluidine blue staining of mast cells. B-lymphocytes are not demonstrated due to their very low abundance. All images shown were shot at the same magnification (400x); yellow arrows point to positive cells; size bars are 50 microns.  Data for quantification of all inflammatory cell types are found in Table 5

Toluidine blue staining was used to identify mast cells (Fig. 1, and SupFig 1). We hypothesized that since mast cells are increased in remodeling tissues, they might be increased in some of the POP-phenotypes. Mast cells were often observed at the interface of different connective tissue types, for instance between adipose tissue and vessels. Subjects in the POP-I phenotype had approximately 50% more mast cells than control subjects but did not have greater numbers of mast cells than POP-A and POP-V subjects (p ≤ 0.05, Table 5).

IHC staining for CD20 identified B-lymphocytes (SupFig 1). Very few B-lymphocytes were observed in the USL and therefore images of B-lymphocytes are not shown (Fig. 1). No statistical differences were observed in NKT cell numbers between the control and POP-phenotypes (p ≤ 0.05, Table 5).

The hypothesis that inflammageing played a role in the increased levels of inflammatory cells in the POP-I phenotype was examined. In the POP-A and POP-I phenotypes, the relative incidence of neutrophils increased with age although only very slightly between 30 and 70 years of age (Fig. 2a) whereas it did not increase in the control and POP-V phenotypes. The relative incidence of the POP-I phenotype T-lymphocytes, CD68-positive macrophages, and mast cells did not increase with age (Fig. 2b). The relative incidence of CD206-resolving macrophages increased with age but with considerable variation in cell quantity (Fig. 2b).

a and b. Inflammatory Cell Scoring data plotted as a function of Age. a Neutrophil scoring data plotted versus age in the various subgroups including control subjects, POP-I subjects, POP-A subjects, and POP-V subjects. Only a very slight increase with age is observed for the POP-I and POP-A subjects. b Inflammatory cell scoring data for CD3, CD68, Toluidine blue, and CD206 is plotted vs age for the subgroup POP-I. CD3, CD68, and toluidine blue data do not increase with increasing age whereas the data for CD206 does increase

Results from all patients’ myeloperoxidase, CD3, CD68, CD20, and toluidine blue quantification data were used in principal component analysis (PCA) to determine relatedness or not of USL inflammation. PCA separated all subjects of the POP-I subgroup away from all subjects of the control, POP-A and POP-V phenotypes (Fig. 3) although heterogeneity was observed within the POP-I subgroup.

PCA diagram of inflammatory cell abundance data. Shown here is the dimension 1 versus dimension 2 distribution from the PCA using myeloperoxidase, CD3, CD68, toluidine blue, and CD20 quantification data for all the subjects. The vector diagram at the top indicates the relative strength and direction vectors for the data. The dashed outline surrounds the POP-I phenotype subjects (magenta squares) and is included to show how they relatively separate from subjects of the other phenotypes. Control subjects (green circles) completely overlap the POP-A (grey triangles) and POP-V (tan squares) subjects

In addition to the quantification of the inflammatory cells, other characteristics of them were of interest. T-lymphocytes and macrophages were observed loosely clustered together in the arterial walls in 5 of the 16 POP-V subjects (31%) showing NIH (Fig. 4a). These co-occurring clusters were also present in 1 of the 21 control subjects. (5%), 0 of the 19 POP-A  subjects (0%), and 0 of the 22 POP-I subjects (0%) (Table 6). CD68 positive cells without the co-occurrence of CD-3 cells were also found loosely associated with the arterial walls in 0 of the 21 Control subjects (5%), 0 of the 19 POP-A subjects (0%), 1 of the 22 POP-I subjects (0%), and 3 of the 16 POP-V subjects (19%). Two of the POP-V subjects (13%) and 1 of the control subjects (5%) possessed foamy macrophages in the tunica intima of a large USL artery (Fig. 4b).

Immunohistochemical staining CD3, CD68 and myeloperoxidase positive cells in the walls of POP-V arteries. a IHC staining for CD3 (T-lymphocytes), CD68 (macrophages), and CD206 (resolving macrophages) show their presence in the walls of POP-V arteries and the lack of neutrophils. Abundant tunica intima hyperplasia is also present in these images. The purple bracket denotes the tunica adventitia, green bracket denotes the tunica media, and black bracket denotes the tunica intima. All images shown were shot at the same magnification (400x); Size bars are 200 microns. b IHC staining for CD68 (macrophages) shows the presence of “foamy macrophages” in the tunica intima of a few arteries. Right side image is an enlargement of the red boxed area seen in the left-hand image. Size bars: left side, 100 microns, right side, 25 microns

Stains for a few of the specialized inflammatory cell types were then undertaken to further define the inflammatory cells which were observed. IHC staining for PD-1 (SupFig 1) was used as a marker for activated lymphocytes [21, 22]. We hypothesized that the T-cells in the arterial walls of arteries with NIH might be activated, however only very low numbers of PD-1 positive cells were observed, and none were detected in the walls of arteries (data not shown).

Macrophages can be broadly divided into two major types with opposing functions; M1 macrophages inhibit cell proliferation and cause tissue damage whereas M2 macrophages promote cell proliferation and tissue repair [23]. IHC staining for CD206 (SupFig 1) was used as a marker for M2 macrophages that were actively promoting wound healing [24]. We hypothesized that the clusters of CD68-positive macrophages and CD3-positive T-cells in the walls of arteries with tunica intima hyperplasia were indicative of previous injuries that were now resolving and that the presence of resolving macrophages (M2) would be evidence in favor of this idea. Indeed, we did observe a few resolving macrophages in the arterial walls of 3 of the 9 POP-V USLs (33%) which contained clusters of CD68 cells, but zero in the arterial walls of control, POP-A, and POP-I USLs (0%) (Table 6).

IHC staining for CD11c (SupFig 1) was used as a marker for dendritic cells, a cell type that activates T-lymphocytes [25]. We hypothesized that there might be dendritic cells in the POP-I tissue due to the broader inflammatory cell presence observed in POP-I USLs, but that there would be very few dendritic cells in the arterial walls of arteries with NIH (POP-V) due to ongoing remodeling. Very few dendritic cells were observed in any of the USLs in this study (Table 5).

IHC staining for CD56, together with a lymphocyte morphology, revealed Natural killer cells (NKT) cells (SupFig1). NKT cells are a subset of restricted T cells now thought to be between the innate and adaptive immune systems [26]. Since NKT cells can have a major regulatory effect upon other immune cell types through their secretion of cytokines, we hypothesized they might amplify or dampen any immune response(s) in the USL. However, very few NKT cells were observed in the USL and therefore images of NKT cells are not shown (Fig. 1). No statistical differences were observed in NKT cell numbers between the control and POP-phenotypes (p ≤ 0.05, Table 5).