primary keratinocyte passages  (PromoCell)

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Creation of the first draft of an integrated periodontitis meta-atlas. (a-e) (a) Human teeth are supported by a diverse collection of specialized tissues, including the periodontium consisting of the gingiva (blue: epithelial and stromal tissues), periodontal ligament, and mineralized tissues (cementum and alveolar bone). (b) Four studies to date have profiled the soft tissue components of human periodontium in health and disease states – . Datasets were first reprocessed and using the Biomage-hosted community instance of Cellenics®, broad cell classes were compared between the four studies, underscoring the complementary nature of cell capture based on unique dissociation strategies among the studies. (c) It is known that the epithelial attachment of the gingiva is also specialized but remains poorly understood. This niche is a stark example of transitional epithelia, and depending on the site, changes from non-keratinized alveolar mucosa (AM), to keratinized attached gingiva (AG), begins to alter expression profiles at the gingival margin (GM) before specializing in the gingival sulcus epithelial keratinocytes (SK) and junctional epithelial keratinocytes (JK). (d) Each study was first integrated using Harmony and assigned Tier 1 cell type annotation. (e) Each dataset was further integrated and harmonized tier annotation was performed between epithelial, stromal, endothelial, neural, and immune cell populations. No single study fully represented each cell type. (f-h) Integrated UMAP, cell assignments (f), and cell signatures (g; Supplementary Table 1) were generated for the meta-atlas; epithelial cells (blue) are highlighted in the shaded box. For public use, the entire dataset was uploaded to cellxgene. (h-j) SK and JKs were grouped in the Tier 3 analysis as co-expressing Keratin 14 ( KRT14 ) and Keratin 19 ( KRT19 ); Supplementary Table 1. (h) To validate this, healthy human gingival tissues were preserved on the tooth surface after extraction and fixed. (i) IHC reveals the gradual transition from tooth-facing JK and SKs to GM, AG, and AM keratinocytes using KRT19; however, (j) KRT19-high and -low epithelial stem cells proliferate in the basal layer in health. Abbreviations: Innate Lymphoid Cells (ILCs); Keratinocytes (KCs); Vascular Endothelial Cells (VECs); Vascular Smooth Muscle (VSM); Lymphatic Endothelial Cells (LECs); Neutrophils (Neut); Masticatory (Keratinized) Mucosa (Mast Muc; MM); Lining (Non-Keratinized Mucosa (Lining Muc; LM); Suprabasal (Differentiated) Keratinocytes (SB); Fibrolast (Fib); Arterial Endothelial Cells (AECs); Postcapillary Venule (PCV); Venule Endothelial Cells (VECs); Macrophage/Monocytes (Mac/Mono); Conventional Dendritic Cells (cDCs); Plasmacytoid Dendritic Cells (pDC); Cytotoxic T Cells (Tc); Gamma Delta T Cells (gdT); Regulatory T Cells (Treg); Mucosal Associated Invariant T Cells (MAIT); Helper T cells (Th). Illustration from (a) created with https://bioicons.com ; illustration from (b) created with BioRender.com. Scale bars: 250 and 50 μm.

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques: Expressing, Generated, Extraction

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Highly multiplexed, spatial immunofluorescence assays of periodontitis reveal innate and adaptive immune cell foci and phenotypes in peri-epithelial niches. (a-c) (a) The orientation of periodontal tissues is critical to show a tooth-facing (junctional, sulcular epithelial keratinocytes; SK/JKs) and oral-facing (gingival margin, GM; attached gingiva, AG; and alveolar mucosal, AM keratinocytes) attachment for highly multiplexed immunofluorescence (mIF) assays of periodontitis. (b) By doing this in sequential sections, we first confirmed orientation and noted highly localized inflammatory profiles near tooth-facing epithelial keratinocytes. As discovered in the initial analysis , tooth-facing JK and SKs uniquely express Keratin 19 (KRT19) in every cell type, highlighting the transition zone. An initial analysis of Tier 1 cell assignments using mIF (PhenoCycler-Fusion (Akoya Biosciences) revealed adaptive immune foci concentrated near SKs and more diverse, innate immune populated foci near JKs. Cell segmentation was performed using StarDist. (c) Expanding the assay to include 32 antibodies revealed more heterogeneity at the cell type and cell state level. Antibodies are grouped and zoomed-in regions from a periodontitis sample is featured; more regions from (b) are also shown in . (d-g) (d) Manual thresholding was performed to show individual marker heterogeneity along the peri-epithelial niches, Cell assignment algorithm for this study is shown in Extended Data 2. (e) Single marker analysis of CD45+ cells revealed general increases in both GM and AG stroma. (f) Comparison of cell identities and cell states shows minimal difference between peri-junctional and peri-sulcular niches in health. (g) While both peri-junctional and peri-sulcular immune infiltrate increase in disease, peri-junctional foci are biased toward more innate and relatively more CD4+ T cells compared to peri-sulcular niches, which are biased toward adaptive immune cells (T and B Cells). Both junctional stromal and JKs also express more PD-L1 compared to sulcular cell types. (h-k) (h) Spatial analysis of peri-epithelial regions was broken into four specific and one broad classification. (i) Segmented immune cells were assigned identities in health and disease. (j) Periodontitis displays more diverse heterogeneity considering the whole tissue. (k) However, cell-cell interactions among immune cells revealed diverse enrichment of immune cell types in peri-junctional and peri-sulcular immune foci in periodontitis. (l-n) Cell states are also spatially distinct in periodontitis, with the peri-junctional immune cells expressing more immune exhaustion. Abbreviations: Antibodies (see Methods); also see legend. Scale bars: 250 and 50 μm. Statistical test (e): p<0.05, paired Student’s T-test (e); chi-square test (j). Illustration from (a) created with BioRender.com.

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques: Immunofluorescence, Marker, Comparison, Expressing

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Investigation of the “Structural Immune” Contribution to Periodontitis using Pseudobulk and Cell-specific Analyses. (a-d) Pseudobulk analysis of differentially expressed genes (DEGs) in periodontitis using all (a) and Tier 1 cell annotations of (b) Fibroblasts (c) Endothelial Cells/Vasculature, and Keratinocytes (d) are shown via volcano plots. Only some DEGs are highlighted; the full list is in Supplementary Table 1. (e) Using a Venn diagram revealed that many genes in these three cell types appear in the pseudobulk DEGs from (a). Abbreviations: see legend. Illustration from (b) created using Interactivenn.net.

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques:

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Keratinocytes play a prominent role in the host response to periodontitis. (a-b) (a) Assessing biological pathways using g:Prolifer, key processes that are upregulated in disease include cell migration, cell signaling, cell death, and cell responses to bacteria. (b). Key processes that are downregulated include tissue differentiation and development, protein translation, and stress responses. This is just an example of the g:Profiler data and key genes attributed to these pathways; however, these data suggest an active immune signaling role for keratinocytes in periodontitis besides just wound healing. (c) Using CellPhoneDB and the integrated periodontitis meta-atlas, all Tier 3 cell types were assessed for inferred receptor-ligand interactions. As expected, the most active cell types in periodontitis predicted include fibroblasts, vascular endothelial cells, and keratinocytes which are also predicted to interact with innate immune cells over adaptive immune cell populations. This full list is included in Supplementary Data 2. (d) Using dot plots to assess some key signaling genes in periodontitis as shown by the DEGs from (Extended Data 2), SK and JKs demonstrate higher expression of these genes, even in health. Furthermore, the patterns appear complementary to vascular and fibroblast populations, suggesting these cells may be responding to something in the clinical “healthy” state. (e) CellPhoneDB on the same scale as (c) shows less receptor-ligand activity in health but SK and JKs are relatively higher and focused on interactions between vascular endothelial cells (VECs), lymphatic endothelial cells, and Schwann cells— and less on fibroblasts and other cell types. This full list is included in Supplementary Data 2. (f) Using g:Profiler, the reactome of keratinocytes further emphasizes their active role via cytokine signaling, immunoregulation, and immune cell recruitment which was observed as presented in . Abbreviations: see legend.

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques: Migration, Bacteria, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Discovery and validation of novel human keratinocyte populations in vivo and in vitro. (a-b) (a) Using Cellenics®, all keratinocytes ( KRT14+ ) were subclustered from the integrated periodontitis meta-atlas (30 samples, 8584 cells) and assigned annotations (b) based on Louvain clustering. (b) Cell signatures for these populations are plotted and included in Supplementary Table 1. (c) Using these signatures, a HiPlex panel of 12 markers was used to profile all keratinocytes from this niche (AG, GM, and JK here; SK and AM as in ). Markers like CXCL14 mark the AG basal epithelium in the opposite pattern of KRT19, ODAM, RHCG, IL18, and SAA1 . Furthermore, keratinized epithelial populations (AG) share expression of CXCL14, KRT15, and LGR6, SFRP1. (d) Primary human gingival keratinocytes were cultured over multiple passages and (e) KRT19 -high basal and larger suprabasal keratinocytes are found mixed population at (e) first passage and over (f) multiple passages. (g) Using RNA ISH and additional markers, cell subpopulations can be identified, suggesting a heterogeneous model of tooth-facing and oral-facing keratinocytes can be utilized for assays and that these markers are more likely cell identities than cell states (see ). Abbreviations: Passage (P2); see legend. Scale bars: (c, e) 50 μm; (f) 25 μm; (g) 10 μm. Illustration from (d) created with BioRender.com.

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques: Biomarker Discovery, In Vivo, In Vitro, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Human gingival keratinocytes contain distinct subpopulations. (a) Using UMAPs to demonstrate cell marker enrichment, keratinocytes are defined by KRT14 expression. JK and SKs are defined by KRT19, FDCSP, RHCG, SAA1, IL1A, and ODAM. (b) Validation of markers using a 12-plex ISG in non-keratinized oral mucosa (alveolar mucosal keratinocytes, AM) and non-keratinized oral mucosa near the tooth surface (sulcular keratinocytes, SK). (c) Single-cell RNA sequencing (scRNAseq) was performed on adult healthy gingival tissues from mice; the UMAP represents the subclustering of murine keratinocytes. (d) Using Louvain clustering, Krt14+; Krt19+ cells were found in small proportion to differentiated keratinized mucosal cells. (e) The majority of cells are basal ( Krt14 with and without Ki67 ) and suprabasal keratinocytes ( Krt4 and Krt1 ). (d) Though Krt19 was expressed as mRNA, Krt19 was not detected at the protein level. (f) Looking at similar cell signatures discovered in humans, many markers are not specific, though there appears to be some expression of immune markers in Krt19 (Cluster 7), including Cxcl1, Cxcl16, Cxcl17, and Cxcl3 . Another cluster (6, pink box) expressed some interleukin inflammatory markers unique to cluster 7 (yellow box) that are shared in humans. Abbreviations: Molar (M); see legend. Scale bars: (b) 50 μm, (c) 100 μm. Illustration from (a) created with BioRender. com.

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques: Marker, Expressing, Biomarker Discovery, RNA Sequencing

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Discovery of polybacterial coinfection of human keratinocytes in vivo and in vitro. (a) Using a 16S in situ probe, we segmented cells using StarDist in health and disease tissues. We found bacterial signals primarily focused on the most terminally differentiated suprabasal keratinocytes across each region of the oral and tooth-associated keratinocytes. The most bacterial burden was found to be in JKs. In disease states, epithelial barrier integrity appears compromised as we see more stromal and epithelial stem cells associated with bacteria, especially in JK and SKs. (b-f) (b) Using a modified pipeline for single-cell analysis of host-microbiome interactions (SAHMI) and a custom Kraken2 database, we used the unmapped reads from our integrated single-cell periodontitis meta-atlas. (c) Using a broad Tier 1 annotation of cell types, this analysis revealed 37 distinct species across Keratinocytes (KC), Fibroblasts (Fib), Vascular Endothelial Cells (VEC), Lymphatic Endothelia Cells (LEC), Pericyte/Vascular Smooth Muscle (PC/VSM), Glial Cells (Glia), Monocyte/ Dendritic Cell Lineages (Mono/DC), T and Natural Killer Cells (T/NK), B Cells (B), and Mast Cells (Mast). In health, using a fraction of microbial reads to the averaged reads per human cell class, we find low read counts across most of the 37 bacterial species. (d) In disease cells, we find large shifts in associated reads. (e) Performing an analysis of c,d, we find dramatic increases in many bacteria comparing disease to health, with large increases in known periopathogens (i.e., P. gingivalis, T. vincentii, P. aeruginosa [P. sp. CIP-10] ). (f) Focusing on all keratinocytes, we find variable numbers of bacteria associated with these cell types, ranging from 0.1% to 15% of all KCs. (g-h) (g) Utilizing broad cell classification of our multiplex immunofluorescence data (mIF; ), we show the innate versus adaptive immune foci in disease. (h) By using the same mIF slides and targets predicted from our SAHMI pipeline, we apply in situ hybridization against 16S and four periopathogens ( F. nucleatum, P. aeruginosa, T. denticola, and P. gingivalis ) and find polybacterial coinfection of all four species in some epithelial stem cells of the JKs using Nyquist optimized, three-dimensional imaging. (i-j) (i) Using primary human gingival keratinocytes, we find polybacterial coinfection in basal (i) and suprabasal KCs (j) for F. nucleatum and P. gingivalis . We also find this phenomenon for P. aeruginosa and T. denticola (k). Abbreviations: see legend. Scale bars: 100 μm; 50 μm; 10 μm; 5 μm. Illustration from (b) created with BioRender.com.

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques: In Vivo, In Vitro, In Situ, Bacteria, Modification, Single-cell Analysis, Multiplex Assay, Immunofluorescence, In Situ Hybridization, Imaging

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Proinflammatory profiles coincide with altered differentiation trajectories of tooth-associated epithelial stem/progenitor cells. (a-c) (a) Due to the single-cell annotation and in situ validation, a draft model of the oral-to-tooth transition zone in humans is presented with basal and suprabasal keratinocyte markers. (b) These markers allowed for KRT19 -high keratinocyte (KCs) cell subclustering for the first time (2504 cells in total), including gingival margin keratinocytes (GM), sulcular keratinocytes (SK), and junctional keratinocytes (JK). (c). This also permitted a more granular draft annotation of cells of the gingival attachment. (d-e) (d) Assaying differentially expressed genes in periodontitis, SK and JKs only share about a quarter of upregulated genes, further underscoring spatial differences in their response in vivo. JKs display nearly 125 unique upregulated genes in diseased cells. (e) Further analysis considering basal versus suprabasal (differentiating) keratinocytes reveals unique cell signatures between basal and suprabasal cell types. This full list is included in Supplementary Data 1. (f-i) To understand SK and JK trajectories, we used partitioned-based graph abstraction (PAGA) on these cell types, comparing these cells between basal and suprabasal cells and in health and disease states using pseudotime (f) Looking at the basal to suprabasal transition, aligned cells from JKs display altered gene expressed comparing health to disease cell types including broader expression of KRT17 , more expression of JUND , COL17A1 , and CDH3 . Key differentiation genes such as SPRR family members are downregulated. (g) JKs display robust cell signaling and inflammatory phenotypes, which are exacerbated in disease states. (h). In SKs, differentiation genes are uniquely expressed compared to JKs in health but also appear altered in disease along basal to suprabasal trajectory. (i) SKs appear generally less reactive compared to JKs in disease. (j) CellChat was used to understand cell signaling pathways in health and disease considering tooth-associated keratinocytes. Circle plots highlight the significant receptor-ligand interactions between any cell populations, including same-cell type signaling interactions (i.e., JK-JK, SK-SK, etc.). The proportion of interactions increased across more detailed immune cell type annotations (Tier 4 annotations; see Extended Data 7). (k) Relative information flow in health and disease shows a preference for cell adhesion (NECTIN, COLLAGEN, JAM, LAMININ) and other pathways such as APP, CXCL, and MIF pathways. In disease, more preference for cell signaling pathways is preferred, such as TGFB, TIGIT, CCL, CD45, and EGF. Innate (l) and adaptive (m) immune cell communication was measured gene by gene using a chord diagram for visualizing cell-cell communication. Abbreviations: Cycling Keratinocytes (Cycle; KCs); Spinous Layer (Spin); Granular Layer (Granular); Intermediate Layer (Inter); Superficial (Super); Merkel Cells (Merk); Melanocytes (Mela); Langerhans Cells (LC); Migratory Dendritic Cells (MigDC); Mast Cells (Mast); also see legend. Illustration from (a) created with BioRender.com.

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques: In Situ, Biomarker Discovery, In Vivo, Expressing, Protein-Protein interactions

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Tier 4 annotation of epithelial and immune cell types of the periodontium. (a-c) KRT19 -high JK, SK, and GM keratinocytes (KCs) were subclustered for further annotation using PAGA. (d-e) PAGA broken out for health (d) and periodontitis (e). (f-g) Using CellTypist, we created another Tier 4 UMAP (f) and refined the first draft annotation of innate and adaptive immune cell subpopulations (g) that were included in the CellChat analysis74 from . (h) Markers are included for each cell subpopulation. Abbreviations: Tissue Resident Memory T Cell (TRM T); also see legend.

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques:

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Polybacterial coinfection is not specific to gingiva but can be enriched in JKs. (a) Some periopathogens are enriched in JKs where the obligate anaerobes such as T. denticola survive in an oxygen-poor environment. Facultative anaerobes such as P. aeruginosa can tolerate JK, SK, ad GM environments, making coinfection dependent on niche environmental conditions. (b) Evidence for polybacterial infection in tonsil epithelial stem cells from classically defined “periopathogens” P. gingivalis and F. nucleatum . (c-f) Further evidence at a single-cell level that CCL28, CXCL8, and CXCL17 are positively correlated with 16S levels (c), independent of keratinocyte spatial localization. Quantification phenotypes in Figure 8 reveal statistically significant associations between bacterial burden and CXCL8 (d), CCL28 (e), and CXCL17 (f). Abbreviations: see legend. Scale bars: (a,c) 10 μm; (b) 100 μm (insets: 25 μm); p<0.05, paired Student’s T-test (d-f).

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques: Infection

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Polybacterial coinfection uniquely affects human gingival keratinocytes and synergizes with LPS. (a) Human gingival keratinocytes (HGK) harboring polybacterial coinfection were cultured with and without LPS (P. gingivalis). These cells were assessed for 16S , and cytokines were predicted to increase in disease states using Hi-Plex in situ hybridization in three rounds. (b-h) (b) HGKs were imaged in xyz using Nyquist-optimized parameters and showed a correlation of 16S burden and cytokine mRNA production. This happens without LPS (b-c) and increases in HGKs with LPS. The first round of images highlights this phenomenon for CCL28, CXCL17, and CXCL8 . (g-h) (g) Examples of 16S and the other cytokine panels ( IL36G, IL1B, CCL20 ; (g)) and ( TNFSF15, IL6, CXCL3 ; (h)) with and without LPS. (i-j) (i) Quantification of all cytokines considering 16S signals per cell. For CXCL17, CXCL8, IL36G, IL1B, CCL20, TNFSF15, IL6, and CXCL8 , polybacterial coinfection elicits mRNA expression without LPS. Only CCL28 , which is known to be chemoattractant to T and B cells, is downregulated with increasing bacterial burden. Binning cells in 16S groups with and without LPS challenge, CXCL17, TNFSF15, and CXCL8 expression demonstrates a synergy between LPS and polybacterial co-infection. Abbreviations: In situ hybridization (ISH); Human Gingival Keratinocytes (HGK); Lipopolysaccharide (LPS). Scale bars: (f) 100 μm; 10 μm. p<0.05, paired Student’s T-test (j).

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques: Cell Culture, In Situ Hybridization, Expressing, Infection

Journal: bioRxiv

Article Title: Polybacterial intracellular coinfection of epithelial stem cells in periodontitis

doi: 10.1101/2023.08.23.554343

Figure Lengend Snippet: Polybacterial coinfection of human keratinocytes in vivo similarly affects cells in health and disease. (a) 12-plex in situ hybridization panel of mRNA targets and 16S were overlaid using Warpy. All 11 cytokines are shown simultaneously in GM, SK, and JKs. (b) Without 16S , there is a distinct patterning of each cytokine. Some, such as IL1B, are broadly expressed in epithelial and stroma. Others such as CXCL8 and CXCL3 appeared to be cell-specific and enriched in JK over SK. (c) Considering infected keratinocytes at a cell-specific level, we find that 16S alone is positively associated with most cytokines in health and disease states. (d) There appears to be polybacterial infection patterns in disease that spread to all epithelial regions, including in terminally differentiated keratinocytes of the attached gingiva (i.e., keratinized mucosa). Epithelial stem cell infection was found in each region. (e-f) (e) Ridge plots (n=3) from JKs in health and disease were quantified without consideration of 16S, showing general increases in IL1A, IL1B, CXCL8, CXCL17, and CCL20 . Assessing both JKs and GM, all keratinocytes were plotted on a normalized heatmap relative to 16S expression, and we quantify that CXCL8, CXCL17, CCL28, IL1A, IL1B , and CCL20 are associated with microbial burden in healthy GM keratinocytes. In JKs, nearly all cytokines are positively associated with microbial burden in heath, suggesting that some bacteria (i.e., gram-positive in GM, gram-negative in JK) may have cell-specific effects in vivo. (f) Patterns in disease between GM and JK align in disease, suggesting an effect by periopathogens as shown in D. Scale bars: (a,d) 100 μm (insets; 50 μm); (b-c) 25 μm. Abbreviations: In situ hybridization (ISH); Human Gingival Keratinocytes (HGK); Lipopolysaccharide (LPS); also see legend. Illustration from (a) created with BioRender.com.

Article Snippet: Human gingival keratinocyte (HGK) culture passaging, cryopreservation, and fixation: All reagents in this section were purchased and used as received from ATCC (Extended Data 4) or Lifeline Cell Technologies ( ).

Techniques: In Vivo, In Situ Hybridization, Infection, Expressing, Bacteria

Journal: Development (Cambridge, England)

Article Title: Age-related changes in expression and function of Toll-like receptors in human skin

doi: 10.1242/dev.083477

Figure Lengend Snippet: Bacterial stimuli and TLR ligands induce distinct immune responses in fetal, neonatal and adult keratinocytes. ( A ) Primary keratinocytes from fetal, neonatal and adult skin were cultured in 12-well plates to ~75% confluence and stimulated in triplicates for 24 hours with the indicated bacterial stimuli and ligands at concentrations described in the Materials and methods. IL1α was used as positive control, cell culture medium (untreated) and TSB as negative controls. ( B ) CXCL8 ( C ) CXCL10 and ( D ) TNFα levels in supernatants were quantified by ELISA. Experiments were performed as three biological replicates/age group (different donors) and the data represent the average (mean±s.e.m.) of these donors. * P <0.05, ** P <0.01, *** P <0.001. ( E ) Western blot analysis of untreated primary keratinocytes showed an almost equal expression of TLR3 and TLR6 proteins in all indicated age groups. Stimulation of keratinocytes with poly (I:C) did not affect TLR3 expression, whereas TLR6 expression was upregulated in fetal and neonatal, but not in adult, keratinocytes.

Article Snippet: Second passage primary keratinocytes from fetal (20-23 weeks EGA; n =3; tebu-bio, Offenbach, Germany), neonatal (3-4 days after birth; n =3; Lonza, Basel, Switzerland) and adult skin (19-37 years; n =3; Cell Systems, Troisdorf, Germany) were cultured in 12-well-plates (Costar, Cambridge, MA, USA) and grown to approximately 75% confluence in a serum-free, keratinocyte growth medium (KGM, Lonza) at 37°C and 5% CO 2 .

Techniques: Cell Culture, Positive Control, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Development (Cambridge, England)

Article Title: Age-related changes in expression and function of Toll-like receptors in human skin

doi: 10.1242/dev.083477

Figure Lengend Snippet: Poly (I:C) activates differential expression of chemokines, cytokines and angiogenic factors in fetal keratinocytes in comparison with neonates and adults. Keratinocytes from fetal, neonatal and adult donors ( n =3 donors/group) were cultured in KGM and stimulated with poly (I:C) or left untreated (negative control) for 24 hours. Supernatants were collected, pooled and analysed for the indicated chemokines, cytokines and angiogenic factors using commercially available protein arrays.

Article Snippet: Second passage primary keratinocytes from fetal (20-23 weeks EGA; n =3; tebu-bio, Offenbach, Germany), neonatal (3-4 days after birth; n =3; Lonza, Basel, Switzerland) and adult skin (19-37 years; n =3; Cell Systems, Troisdorf, Germany) were cultured in 12-well-plates (Costar, Cambridge, MA, USA) and grown to approximately 75% confluence in a serum-free, keratinocyte growth medium (KGM, Lonza) at 37°C and 5% CO 2 .

Techniques: Expressing, Cell Culture, Negative Control

Journal: Development (Cambridge, England)

Article Title: Age-related changes in expression and function of Toll-like receptors in human skin

doi: 10.1242/dev.083477

Figure Lengend Snippet: Expression pattern of TLRs 3 and 6 in skin equivalents generated from fetal, neonatal and adult keratinocytes. ( A ) Cryostat sections of skin equivalents generated with keratinocytes from the indicated age groups ( n =3 donors/group), and adult breast skin (positive control), were stained for TLR 3 and 6 protein expression using immunohistochemistry. ( B ) Immunofluorescence double labeling revealed K10 − TLR3 + TLR6 + basal keratinocytes (red) and K10 + TLR3 − TLR6 − suprabasal keratinocytes (green). Nuclei were stained with Hoechst dye (blue). Scale bars: 50 μm in A,B (immunohistochemistry); 40 μm in B (immunofluorescence).

Article Snippet: Second passage primary keratinocytes from fetal (20-23 weeks EGA; n =3; tebu-bio, Offenbach, Germany), neonatal (3-4 days after birth; n =3; Lonza, Basel, Switzerland) and adult skin (19-37 years; n =3; Cell Systems, Troisdorf, Germany) were cultured in 12-well-plates (Costar, Cambridge, MA, USA) and grown to approximately 75% confluence in a serum-free, keratinocyte growth medium (KGM, Lonza) at 37°C and 5% CO 2 .

Techniques: Expressing, Generated, Positive Control, Staining, Immunohistochemistry, Immunofluorescence, Labeling