Correlation of the expression of hyaluronan and CD44 with the presence of gingival inflammatory infiltrate in advanced generalized periodontitis

Aim: To investigate the association between the expression of hyaluronan (HA) and its main receptor CD44 with the presence of inflammatory infiltrate in gingiva samples of patients suffering from advanced generalized periodontitis. 
 Material and Methods: Samples of gingival tissue from healthy donors (controls, n = 20) and patients suffering from advanced generalized periodontitis stage 3 and 4 (test group, n = 20) were obtained. For immunofluorescence (IF) staining, primary antibodies against HA, CD44 and general inflammatory cell marker CD45 were used. The microscopic slides were photographed and panoramic images were edited in Adobe Photoshop®. The quantification of IF signal expression domains and spatial gradients was performed in ImageJ. The Microsoft Office Excel 2016 and GraphPad v8 software ware used for statistical analysis. 
 Results: HA and CD44 were found to be widely expressed in the epithelium and subepithelial stroma of both healthy and diseased gingiva, including the areas of the gingiva containing the inflammatory infiltrate. No difference in the total expression of HA and CD44 in healthy and diseased gingiva was found. However, the pattern of distribution of HA and CD44 signals was somewhat different between the two groups of samples with regard to a statistically significant increase in stromal expression of HA and CD44 in diseased gingiva compared to healthy gingiva. No spatial correlation between HA and CD44 expression with the presence of inflammatory infiltrate was found in diseased gingiva. Moderate spatial correlation between HA and CD44 was found in diseased gingiva. 
 Conclusion: HA and CD44 might play a role in the regulation of inflammatory response in advanced generalized periodontitis. However, further studies are needed in order to properly characterize such role of both investigated factors.

Material and Methods: Samples of gingival tissue from healthy donors (controls, n = 20) and patients suffering from advanced generalized periodontitis stage 3 and 4 (test group, n = 20) were obtained. For immunofluorescence (IF) staining, primary antibodies against HA, CD44 and general inflammatory cell marker CD45 were used. The microscopic slides were photographed and panoramic images were edited in Adobe Photoshop®. The quantification of IF signal expression domains and spatial gradients was performed in ImageJ. The Microsoft Office Excel 2016 and GraphPad v8 software ware used for statistical analysis.
Results: HA and CD44 were found to be widely expressed in the epithelium and subepithelial stroma of both healthy and diseased gingiva, including the areas of the gingiva containing the inflammatory infiltrate. No difference in the total expression of HA and CD44 in healthy and diseased gingiva was found. However, the pattern of distribution of HA and CD44 signals was somewhat different between the two groups of samples with regard to a statistically significant increase in stromal expression of HA and CD44 in diseased gingiva compared to healthy gingiva. No spatial correlation between HA and CD44 expression with the presence of inflammatory infiltrate was found in diseased gingiva. Moderate spatial correlation between HA and CD44 was found in diseased gingiva.
Conclusion: HA and CD44 might play a role in the regulation of inflammatory response in advanced generalized periodontitis. However, further studies are needed in order to properly characterize such role of both investigated factors.

Hyaluronan (HA) is a glycosaminoglycan (GAG) component of the extracellular matrix that
is present in tissues in two main forms -as native high molecular weight HA (HMWHA) and various low molecular weight HA (LMWHA) fragments. The degree of fragmentation is an important determinant of the physiological function of HA, where HMWHA and LMWHA may play opposite roles in the maintenance of tissue homeostasis as well as in processes such as wound healing and the regulation of inflammatory response in tissues.
Research has shown that HA is directly involved in innate and adaptive immunity mechanisms, from the activation of various subpopulations of leukocytes, their migration and the formation of gradients of inflammatory mediators and inflammatory cell infiltrate in the tissue, to the resolution of the inflammatory response [3]. The regulatory role of HA in the inflammatory response is highly dependent on the HMWHA to LMWHA ratio (in general, inflammatory conditions promote HMWHA fragmentation), but the availability of various cellular HA receptors is of equal importance, as HMWHA and LMWHA affect cell behavior in response to events in their immediate microenvironment through these receptors. Several cellular HA receptors, such as HARE, LYVE-1 and RHAMM have been described so far, but the ubiquitously expressed CD44 receptor is regarded as the major HA receptor. Numerous studies have confirmed the key importance of the interaction between CD44 and HA in all aspects of HA metabolism as well as in cellular processes involved in the inflammatory response in the tissue (such as leukocyte adhesion and migration, macrophage and T-lymphocyte activation, and the resolution of inflammation) [4][5][6]. Generally speaking, the accumulation of HA and increased CD44 expression during the acute inflammatory response have been described in various tissues [1,[7][8][9][10].
The regulatory role of HA and of the factors functionally related to it in the pathogenesis of periodontitis as a chronic inflammatory disease has been poorly investigated. Most research has been concerned with studying the effectiveness of HA preparations as auxiliary substances in various forms of periodontal treatment (initial and surgical treatment) and has not differed conceptually from researching the role of HA in the acute inflammatory response during wound healing. This has resulted in a limited understanding of HA as an inhibitor of inflammatory cell activation and general contributor to the resolution of inflammation and the regeneration of the periodontal tissue [11]. A literature review identified only two 1990s studies that analyzed the expression of HA and its major receptor, CD44, by immune-histochemical (IHC) staining of gingival samples [12,13]. Both factors were expressed in all gingival tissue sections, including the regions of subepithelial stroma with inflammatory cell infiltrate. However, HA and CD44 expressions between groups of samples were not compared statistically as methods of precise quantification of IHC staining were not used in these studies.
In this study, the HA and CD44 expressions in human gingival samples ware examined by immuno-fluorescence (IF) and analyzed using computerized methods of IF signal quanti-  [14]. All subjects were adults in good general health (free from concomitant chronic or systemic diseases). Gingival samples were collected from patients that had been referred for clinical crown extension (controls) and tooth extraction due to extensive periodontal tissue degradation (test group).

Samples
The sampling and initial processing of gingiva samples have been described in detail [15].
The gingiva specimens used in this study included the free gingiva region extending from the gingival margin (marginally) to the interface with the alveolar bone edge (apically). 40 samples of the free gingiva (20 per group) were processed for the purposes of this study.
The gingiva samples were fixed in 4% paraformaldehyde solution for 24-48 h and then stained with tissue dye on the side of the oral epithelium (vestibular side) for correct orientation when embedded into paraffin blocks. The samples were then serially cut by microtome into 5 μm thick sections and mounted on slides. The sections were examined under a light microscope to confirm their correct orientation -the oral and sulcus epithelium of the free gingiva and the subepithelial stroma located between the two epithelia had to be visible in every section. In addition to the existing orientation slides from the archives that were stained with hematoxylin/eosin, new slides were additionally selected for Alcian further processing under the criteria listed above [15].

Immunofluorescence staining
Immuno-fluorescence (IF) staining was performed according to the standardized laboratory protocol at the Department of Anatomy, Histology and Embryology [16,17]. The fol-lowing primary antibodies were used: sheep polyclonal HA antibody (Anti-Hyaluronan) to correlate the intensity of double IF staining based on the previously described procedure [18,19]. In each sample, three sections were stained for the test factor and the best section (free from tissue damage, staining artifacts, with clearly visible IF signals) was selected for panoramic imaging.

Acquisition and processing of panoramic images
The slides were examined and photographed using the panoramic imaging technique with ×10 (IF) and ×20 (Alcian Blue) magnification under an invert epifluorescence microscope (Carl Zeiss Microscopy GmbH; Jena, Germany) with full-frame digital camera Zeiss Axiocam 506 (resolution: 2752×2208 pixels (px)) and fixed settings for sensor sensitivity (ISO: 100).
For IF panoramic images, the exposure was set at 500 ms (anti-HA and anti-CD44) and 35 ms (DAPI) and 8 ms for Alcian Blue staining (light microscopy). The resulting micrographic tiles were then merged into panoramic images with ZEN 2.5 (Carl Zeiss Microscopy GmbH; Jena, Germany). The original black-and-white panoramic TIFF images (24-bit depth; sRGB color mode) were used for the IF staining analysis. The DAPI channel of panoramic images that were selected to demonstrate IF staining here was pseudo-colorized (ImageJ).
Panoramic images were edited, rotated, and resized in Adobe Photoshop® CC (2019) on a high-resolution (600 dpi) background, as previously described, to preserve data resolution when reducing the image size [15].
The Wizard Tool was used to measure histometric parameters such as the surface area of the whole-section and the area of the epithelial and stromal compartments in panoramic images (Adobe Photoshop® CC (2019)). A digital graphic pen tablet (Wacom Intuos PRO; Wacom Co, Saitama, Japan) was used to mask the sections. The measures of the above histometric parameters were originally expressed in pixels (px) and then converted into

Statistical analysis
Analysis of variance (ANOVA) was used to analyze the expression domains of the examined factors. Group averages were used to compare the HA and CD44 expression domains, shown as fractions in histogram tables (one histogram for controls and one for the test group, respectively). As the total, epithelial, and stromal expression domains of the examined factors were compared separately, no post hoc testing for the sources of potential statistically significant differences was necessary. ANOVA was also used to analyze the Jolla, CA, USA). The statistical significance (α) for the analysis of expression domains was set at 0.01 (P < 0.01). The statistical significance (α) for the IF signal spatial gradient distribution analysis and regression models was set at 10 -8 (P < 10 -8 ).

Results
In the healthy gingiva, the HA and CD44 expression is primarily noticeable in the spinous layer of the gingival epithelium. The HA and CD44 expression is also visible in the subepithelial stroma, although not as intensely as in the gingival epithelium. The pattern of the HA and CD44 expression in the gingiva of subjects with advanced generalized periodontitis is similar to that of the healthy gingiva; however, the expression of both factors is more intense in the subepithelial stroma of diseased gingiva compared to healthy gingiva (Figure 1; Figure 2) and this difference is statistically significant ( Table 1; Table 2; Figure   3). The comparison of the ratio of epithelial and stromal IF signals in the total expression within each group of gingiva samples revealed that the HA to CD44 ratio in tissue compartments (epithelium, stroma) was similar in the control and test group and was approxi-     Based on an analysis of the general inflammatory cell marker CD45 expression from a previous study, the presence of stromal inflammatory infiltrate was significantly higher in gingival samples collected from subjects with advanced generalized periodontitis when compared to that of the healthy gingiva, where inflammatory infiltrate was scarce and narrowly limited to the perivascular tissue [15]. Since HA and CD44 play a regulatory role in the formation and maintenance of inflammatory infiltrate, the correlation between the spatial gradients of total HA and CD44 expression and the spatial gradient of total CD45 expression was analyzed for gingival samples collected from subjects with advanced generalized periodontitis (Figure 6; Figure 7; Table 3  *Comparison of the expression domains of HA and CD44 receptor calculated as relative values from the whole-section area (total) and tissue compartments (epithelium, stroma) between individual sections (n = 20) of gingival samples from the control and test groups. The statistically significant difference between the total expression domains is mainly a result of differences in the structure of individual sections (relative ratio of the epithelium and stroma in the whole-section area) and has less to do with the actual variation in expression of investigated factors between samples. No statistically significant difference within group variation was found for expression of HA and CD44 receptor in either epithelial or stromal tissue compartments. †One-Way ANOVA test for within-group consistency. The significance level was set to α = 0.01 (*P < 0.01) at Fcrit = 1.908 (F > Fcrit); degrees of freedom (df = 19).    †The level of statistical significance for the regression models was set to α = 10 -8 (P < 10 -8 ). ‡The confidence interval for the coefficients is set to 99%. Figure 6. Correlation of the spatial gradients of hyaluronan (HA), CD44, and CD45 expression in gingiva samples collected from subjects with advanced generalized periodontitis. The x-axis shows the independent variables and the y-axis shows the dependent variables based on complete pooling of spatially paired entries from 2D plots (100,000 entries). The significance level (α) is set to α = 10 -8 . No correlation was found between the spatial gradients of HA and CD45 (R = 0.0573; R 2 = 0.0033; P = 7.908×10 -57 ), and CD44 and CD45 (R = 0.0195; R 2 = 0.0004; P = 6.571×10 -8 ). A moderate correlation was found between the spatial gradients of HA and CD44 expression (R = 0.5508; R 2 = 0.3034; P = 0).

Figure 7.
Coefficients of determination in regression models for the correlation between the spatial gradient of the expression of examined factors and the general inflammatory cell marker CD45 in gingiva samples (n = 20) from the test group. All models are statistically significant (P < 10 -8 ), with P values ranging from 7.281×10 -278 to 9.669×10 -12 (for HA-CD45 correlation) and from 1.336×10 -12 to 3.146×10 -9 (for CD44-CD45 correlation). As shown above, the values of coefficients of determination (R 2 ) are in most cases closer to zero, which means that the expression of the examined factors weakly or very weakly correlates with the presence of inflammatory infiltrate in gingiva samples of subjects with advanced generalized periodontitis.

Discussion
Based on the reviewed literature, the total expression of HA and CD44 was expected to be increased in the gingiva of subjects with advanced generalized periodontitis when compared to the gingiva of healthy subjects, but the results of expression domain comparison showed comparable total expression of HA and CD44 in the healthy and diseased gingiva [1,[7][8][9][10]. Accordingly, the overall availability of the ligand (HA) compared to that of its major receptor (CD44) is comparable in the healthy and diseased gingiva. However, the analysis of signal distribution in tissue compartments (epithelial and stromal expression domains) found a slightly reduced HA and CD44 expression in the epithelium of the diseased gingiva (this reduction was not statistically significant) and, simultaneously, a statistically significant increase of HA and CD44 expression in the subepithelial stroma in diseased gingiva when compared to the healthy gingiva. Accordingly, based on results from our previous study, cumulative changes in the stromal expression of HA and CD44 in the subepithelial stroma of the gingiva of subjects with advanced generalized periodontitis occur simultaneously with the increased presence of inflammatory infiltrate [15]. On the other hand, no correlation was found between the overall spatial distribution of either HA or CD44 and the presence of inflammatory infiltrate in the diseased gingiva, which can be Immunohistochemical staining for HA cannot determine the exact relationship between HMWHA and LMWHA since the primary anti-HA antibody used in this study does not bind specifically to either HMWHA or LMWHA. Therefore, it was impossible to determine a difference in the HMWHA to LMWHA ratio (and thus a difference in HA metabolism) between the healthy gingiva and the gingiva of subjects with advanced generalized periodontitis. However, this aspect could be examined indirectly by staining gingival tissue with hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2) antibodies, which act as major enzymes in HA catabolism. An increased expression of Hyal-1 and Hyal-2 could indicate an increase in HA degradation and the presence of various LMWHA fragments that are generally known to have pro-inflammatory properties. Additionally, to elucidate the composition of HA, it is necessary to stain for some hyaladherins, such as TSG-6 (tumor necrosis factor-stimulated gene-6). More specifically, TSG-6 binding to HA can modulate the affinity of HA for its cell surface receptors, as well as the adhesiveness of HA itself, which impacts the activation, migration, and retention of inflammatory cells in the tissue [3,20].
Since there is an increase in inflammatory cells in the gingiva of subjects with advanced generalized periodontitis, the presence and/or spatial distribution of hyaladherin-containing HA complexes can be expected to differ from that of the healthy gingiva.
Similar to HA, the structure of its major cell surface receptor, CD44, is heterogeneous and depends on the type of CD44-expressing cells and the dynamics of physiological processes in the tissue. CD44 is extremely susceptible to post-translational modifications, which implies differing degrees of glycosylation of this receptor -in addition to HA, other types of GAGs can bind to the extracellular domain of CD44 and thus modulate the affinity of the HA-CD44 bond [4]. Additionally, various CD44 isoforms have been described. Apart from the most common, standard isoform of CD44 (CD44s), there are other, variable isoforms (CD44v1-10) resulting from alternative splicing during the CD44 gene transcription [4].
Epithelial cells and activated inflammatory cells can (in addition to CD44s) simultaneously express combinations of several different variable isoforms, some of which (such as CD44v7) are associated with the pathogenesis of chronic inflammatory diseases and malignant tissue alteration [21]. In this study, a primary antibody that binds to an epitope on the cytoplasmic domain of this receptor and therefore non-specifically stains all CD44 isoforms was used to stain gingival samples for CD44. To provide a more accurate description of the spatial relationships between the expression of this receptor and the presence of inflammatory infiltrate in the gingiva during advanced generalized periodontitis, additional staining for variable CD44 isoforms with commercially available antibodies should be carried out. Consequently, the prediction of anti-inflammatory role of CD44 in advanced generalized periodontitis based on virtual knockout as presented in this study should be carefully re-examined in the future by more comprehensive regression models. Additional staining with antibodies against HA biosynthesis and metabolism enzymes, as well as with antibodies against CD44 isoforms and other HA receptors could improve the existing model and make predictions more accurate. It should also be noted that previous research on models of various inflammatory diseases in experimental animals has shown that CD44 (similar to HA) may play a dual role in the regulation of inflammation. More specifically, both anti-inflammatory and pro-inflammatory effects may be achieved, depending on the mechanisms of the introduction of noxious stimuli and methods of blocking the activity of CD44 (either by antibodies or by classical gene knockout) (9). Interestingly, the described effects of CD44 blocking on the course of inflammation are not always accompanied by changes in HA metabolism/catabolism in terms of total HA in tissues, although CD44 does have a significant role in catabolism/elimination of HA [22].
In conclusion, the absence of differences in HA and CD44 expression between healthy and