Intracellular calprotectin (S100A8/A9) facilitates DNA damage responses and promotes apoptosis in head and neck squamous cell carcinoma.

OBJECTIVES: In head and neck squamous cell carcinoma (HNSCC), poor prognosis and low survival rates are associated with downregulated calprotectin. Calprotectin (S100A8/A9) inhibits cancer cell migration and invasion and facilitates G2/M cell cycle arrest. We investigated whether S100A8/A9 regulates DNA damage responses (DDR) and apoptosis in HNSCC after chemoradiation. MATERIALS AND METHODS: Human HNSCC cases in TCGA were analyzed for relationships between S100A8/A9 and expression of apoptosis-related genes. Next, S100A8/A9-expressing and non-expressing carcinoma lines (two different lineages) were exposed to genotoxic agents and assessed for 53BP1 and γH2AX expression and percent of viable/dead cells. Finally, S100A8/A9-wild-type and S100A8/A9null C57BL/6j mice were treated with 4-NQO to induce oral dysplastic and carcinomatous lesions, which were compared for levels of 53BP1. RESULTS: In S100A8/A9-high HNSCC tumors, apoptosis-related caspase family member genes were upregulated, whereas genes limiting apoptosis were significantly downregulated based on TCGA analyses. After X-irradiation or camptothecin treatment, S100A8/A9-expressing carcinoma cells (i.e., TR146 and KB-S100A8/A9) showed significantly higher 53BP1 and γH2AX expression, DNA fragmentation, proportions of dead cells, and greater sensitivity to cisplatin than wild-type KB or TR146-S100A8/A9-KD cells. Interestingly, KB-S100A8/A9Δ113-114 cells showed similar 53BP1 and γH2AX levels to S100A8/A9-negative KB and KB-EGFP cells. After 4-NQO treatment, 53BP1 expression in oral lesions was significantly greater in calprotectin+/+ than S100A8/A9null mice. CONCLUSIONS: In HNSCC cells, intracellular calprotectin is strongly suggested to potentiate DDR and promote apoptosis in response to genotoxic agents. Hence, patients with S100A8/A9-high HNSCC may encounter more favorable outcomes because more tumor cells enter apoptosis with increased sensitivity to chemoradiation therapy.

Antimicrobial peptides: Defending the mucosal epithelial barrier.

The recent epidemic caused by aerosolized SARS-CoV-2 virus illustrates the importance and vulnerability of the mucosal epithelial barrier against infection. Antimicrobial proteins and peptides (AMPs) are key to the epithelial barrier, providing immunity against microbes. In primitive life forms, AMPs protect the integument and the gut against pathogenic microbes. AMPs have also evolved in humans and other mammals to enhance newer, complex innate and adaptive immunity to favor the persistence of commensals over pathogenic microbes. The canonical AMPs are helictical peptides that form lethal pores in microbial membranes. In higher life forms, this type of AMP is exemplified by the defensin family of AMPs. In epithelial tissues, defensins, and calprotectin (complex of S100A8 and S100A9) have evolved to work cooperatively. The mechanisms of action differ. Unlike defensins, calprotectin sequesters essential trace metals from microbes, which inhibits growth. This review focuses on defensins and calprotectin as AMPs that appear to work cooperatively to fortify the epithelial barrier against infection. The antimicrobial spectrum is broad with overlap between the two AMPs. In mice, experimental models highlight the contribution of both AMPs to candidiasis as a fungal infection and periodontitis resulting from bacterial dysbiosis. These AMPs appear to contribute to innate immunity in humans, protecting the commensal microflora and restricting the emergence of pathobionts and pathogens. A striking example in human innate immunity is that elevated serum calprotectin protects against neonatal sepsis. Calprotectin is also remarkable because of functional differences when localized in epithelial and neutrophil cytoplasm or released into the extracellular environment. In the cytoplasm, calprotectin appears to protect against invasive pathogens. Extracellularly, calprotectin can engage pathogen-recognition receptors to activate innate immune and proinflammatory mechanisms. In inflamed epithelial and other tissue spaces, calprotectin, DNA, and histones are released from degranulated neutrophils to form insoluble antimicrobial barriers termed neutrophil extracellular traps. Hence, calprotectin and other AMPs use several strategies to provide microbial control and stimulate innate immunity.

Streptococcus gordonii Poised for Glycan Feeding through a MUC5B-Discriminating, Lipoteichoic Acid-Mediated Outside-In Signaling Circuit.

Many oral bacteria employ cell wall-anchored adhesins to bind to the salivary films coating the teeth and mucosal surfaces. Surface binding prevents clearance and facilitates catabolism of salivary film glycoproteins. We asked whether Streptococcus gordonii adhesin expression changes in response to surface salivary cues using a eukaryote-like, outside-in recognition and signaling circuit. To determine whether the cues were discriminated, S. gordonii was tested during cell adhesion and biofilm formation on a MUC5B-rich or lower-molecular-mass salivary fraction or an uncoated abiotic surface. Cells were recovered and analyzed for differences in gene expression and proteins in cell wall fractions. In salivary-free conditions, planktonic S. gordonii presented three prominent cell wall LPXTG-motif proteins, SGO_1487, SGO_0890, and MbpA (mucin-binding protein A; SGO_0707). During biofilm formation on MUC5B-coated surfaces, MbpA, a MUC5B-binding protein, and key genes in the tagatose and quorum-sensing pathways were strongly promoted. The response to MUC5B required the two-component system (TCS), streptococcal regulator of adhesins sensor and regulator (SraSR, SGO_1180/81), lipoteichoic acid (LTA), and the homologous paired adhesins, SspA and SspB (SspAB). LTA appears to link the outside signal (MUC5B) to intramembrane SraSR. Tagatose pathway gene expression may poise cells to metabolize MUC5B glycans and, with a quorum-sensing gene (luxS), may direct formation of a consortium to facilitate glycan cross-feeding by S. gordonii. We now show that a Gram-positive bacterium discriminates specific surface environmental cues using an outside-in signaling mechanism to apparently optimize colonization of saliva-coated surfaces. IMPORTANCE All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism.

Calprotectin (S100A8/A9) Is an Innate Immune Effector in Experimental Periodontitis.

Upregulated in inflammation, calprotectin (complexed S100A8 and S100A9; S100A8/A9) functions as an innate immune effector molecule, promoting inflammation, and also as an antimicrobial protein. We hypothesized that antimicrobial S100A8/A9 would mitigate change to the local microbial community and promote resistance to experimental periodontitis in vivo. To test this hypothesis, S100A9-/- and wild-type (WT; S100A9+/+) C57BL/6 mice were compared using a model of ligature-induced periodontitis. On day 2, WT mice showed fewer infiltrating innate immune cells than S100A9-/- mice; by day 5, the immune cell numbers were similar. At 5 days post ligature placement, oral microbial communities sampled with swabs differed significantly in beta diversity between the mouse genotypes. Ligatures recovered from molar teeth of S100A9-/- and WT mice contained significantly dissimilar microbial genera from each other and the overall oral communities from swabs. Concomitantly, the S100A9-/- mice had significantly greater alveolar bone loss than WT mice around molar teeth in ligated sites. When the oral microflora was ablated by antibiotic pretreatment, differences disappeared between WT and S100A9-/- mice in their immune cell infiltrates and alveolar bone loss. Calprotectin, therefore, suppresses emergence of a dysbiotic, proinflammatory oral microbial community, which reduces innate immune effector activity, including early recruitment of innate immune cells, mitigating subsequent alveolar bone loss and protecting against experimental periodontitis.