S100A8 and S100A9 Are Important for Postnatal Development of Gut Microbiota and Immune System in Mice and Infants.

BACKGROUND & AIMS: After birth, the immune system matures via interactions with microbes in the gut. The S100 calcium binding proteins S100A8 and S100A9, and their extracellular complex form, S100A8-A9, are found in high amounts in human breast milk. We studied levels of S100A8-A9 in fecal samples (also called fecal calprotectin) from newborns and during infancy, and their effects on development of the intestinal microbiota and mucosal immune system. METHODS: We collected stool samples (n = 517) from full-term (n = 72) and preterm infants (n = 49) at different timepoints over the first year of life (days 1, 3, 10, 30, 90, 180, and 360). We measured levels of S100A8-A9 by enzyme-linked immunosorbent assay and analyzed fecal microbiomes by 16S sRNA gene sequencing. We also obtained small and large intestine biopsies from 8 adults and 10 newborn infants without inflammatory bowel diseases (controls) and 8 infants with necrotizing enterocolitis and measured levels of S100A8 by immunofluorescence microscopy. Children were followed for 2.5 years and anthropometric data and medical information on infections were collected. We performed studies with newborn C57BL/6J wild-type and S100a9-/- mice (which also lack S100A8). Some mice were fed or given intraperitoneal injections of S100A8 or subcutaneous injections of Staphylococcus aureus. Blood and intestine, mesenterial and celiac lymph nodes were collected; cells and cytokines were measured by flow cytometry and studied in cell culture assays. Colon contents from mice were analyzed by culture-based microbiology assays. RESULTS: Loss of S100A8 and S100A9 in mice altered the phenotypes of colonic lamina propria macrophages, compared with wild-type mice. Intestinal tissues from neonatal S100-knockout mice had reduced levels of CX3CR1 protein, and Il10 and Tgfb1 mRNAs, compared with wild-type mice, and fewer T-regulatory cells. S100-knockout mice weighed 21% more than wild-type mice at age 8 weeks and a higher proportion developed fatal sepsis during the neonatal period. S100-knockout mice had alterations in their fecal microbiomes, with higher abundance of Enterobacteriaceae. Feeding mice S100 at birth prevented the expansion of Enterobacteriaceae, increased numbers of T-regulatory cells and levels of CX3CR1 protein and Il10 mRNA in intestine tissues, and reduced body weight and death from neonatal sepsis. Fecal samples from term infants, but not preterm infants, had significantly higher levels of S100A8-A9 during the first 3 months of life than fecal samples from adults; levels decreased to adult levels after weaning. Fecal samples from infants born by cesarean delivery had lower levels of S100A8-A9 than from infants born by vaginal delivery. S100 proteins were expressed by lamina propria macrophages in intestinal tissues from infants, at higher levels than in intestinal tissues from adults. High fecal levels of S100 proteins, from 30 days to 1 year of age, were associated with higher abundance of Actinobacteria and Bifidobacteriaceae, and lower abundance of Gammaproteobacteria-particularly opportunistic Enterobacteriaceae. A low level of S100 proteins in infants' fecal samples associated with development of sepsis and obesity by age 2 years. CONCLUSION: S100A8 and S100A9 regulate development of the intestinal microbiota and immune system in neonates. Nutritional supplementation with these proteins might aide in development of preterm infants and prevent microbiota-associated disorders in later years.

S100A8/A9, a potent serum and molecular imaging biomarker for synovial inflammation and joint destruction in seronegative experimental arthritis.

BACKGROUND: Seronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis. METHODS: Serum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)-/- mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs. RESULTS: Serum levels of S100A8/A9 were significantly increased in IL-1Ra-/- mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra-/- mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs. CONCLUSIONS: Expression of S100A8 and S100A9 in IL-1Ra-/- mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction.

Proteins S100A8 and S100A9 are potential biomarkers for renal cell carcinoma in the early stages: results from a proteomic study integrated with bioinformatics analysis.

In order to investigate the two members of the EF­hand Ca2+ binding protein S100 family, S100A8 and S100A9, in renal cell carcinoma (RCC), serum samples were collected from patients with RCC, transitional cell carcinoma in the kidney, benign renal masses and normal controls. The samples were analyzed by isobaric tags for relative and absolute quantification technology to identify the differential expression of S100A8 and S100A9 in the respective groups. Hierarchical clustering analysis was then conducted for the samples and the relevant selected gene. The cross­platform analysis for the external validation was performed by means of The Cancer Genome Atlas database, containing the gene/microRNA expression pattern and clinical information of patients with RCC. Immunohistochemical staining was used to verify the expression of S100A8 and S100A9 in the four groups. As a result, serum and mRNA expression levels of S100A8 and S100A9 were found to be upregulated in patients with RCC compared with the other three groups, which was consistent with the result of the upregulated expression of mRNA levels in RCC tissue. The overexpression of S100A8 and S100A9 in cancer cells was also confirmed by immunohistochemistry. In addition, bioinformatics revealed that let­7, a microRNA formerly identified as an inhibiting factor of RCC was downregulated in RCC, which contrasted with S100A8. It was also complementary to the sequence at the 3' untranslated region terminal of S100A8. Therefore, indicating that S100A8 and S100A9 may serve as biomarkers for the detection of RCC.

High density of S100A9 positive inflammatory cells in prostate cancer stroma is associated with poor outcome.

PURPOSE: To elucidate if the density of inflammatory cells expressing S100A9 in malignant and surrounding non-malignant prostate tissues is a prognostic marker for outcome in prostate cancer patients. EXPERIMENTAL DESIGN: Tissue was obtained from 358 men diagnosed with prostate cancer at transurethral resection of the prostate due to obstructive voiding problems, of which 260 were then followed with watchful waiting. Tissue microarrays of both malignant and non-malignant tissues were stained with an antibody against S100A9. The number of stained inflammatory cells was scored and related to clinical outcome and histopathological parameters of known prognostic value. RESULTS: A high number of inflammatory cells expressing S100A9 in both malignant and surrounding non-malignant tissues were associated with significantly shorter cancer-specific survival. This association remained significant when Gleason score and local tumour stage were analysed together with S100A9 in a Cox regression model. Low number of S100A9 positive cells in non-malignant stroma was correlated to significantly longer cancer-specific survival also in patients with Gleason score 8-10 tumours. S100A9 positive cells in tumour stroma were correlated with Gleason score, hyaluronan, platelet-derived growth factor receptor beta (PDGFR-ß), and androgen receptor (inverse correlation) in tumour stroma. S100A9 positive cells in non-malignant stroma correlated with androgen receptor in this tissue compartment (inverse correlation). CONCLUSIONS: A high number of S100A9 positive inflammatory cells in tumour stroma and in non-malignant stroma were associated with shorter cancer-specific survival in prostate cancer patients.

Proteomic approach reveals FKBP4 and S100A9 as potential prediction markers of therapeutic response to neoadjuvant chemotherapy in patients with breast cancer.

Although doxorubicin (Doxo) and docetaxel (Docet) in combination are widely used in treatment regimens for a broad spectrum of breast cancer patients, a major obstacle has emerged in that some patients are intrinsically resistant to these chemotherapeutics. Our study aimed to discover potential prediction markers of drug resistance in needle-biopsied tissues of breast cancer patients prior to neoadjuvant chemotherapy. Tissues collected before chemotherapy were analyzed by mass spectrometry. A total of 2,331 proteins were identified and comparatively quantified between drug sensitive (DS) and drug resistant (DR) patient groups by spectral count. Of them, 298 proteins were differentially expressed by more than 1.5-fold. Some of the differentially expressed proteins (DEPs) were further confirmed by Western blotting. Bioinformatic analysis revealed that the DEPs were largely associated with drug metabolism, acute phase response signaling, and fatty acid elongation in mitochondria. Clinical validation of two selected proteins by immunohistochemistry found that FKBP4 and S100A9 might be putative prediction markers in discriminating the DR group from the DS group of breast cancer patients. The results demonstrate that a quantitative proteomics/bioinformatics approach is useful for discovering prediction markers of drug resistance, and possibly for the development of a new therapeutic strategy.

Shosaikoto increases calprotectin expression in human oral epithelial cells.

BACKGROUND AND OBJECTIVE: Oral epithelial cells help to prevent against bacterial infection in the oral cavity by producing antimicrobial peptides (AMPs). A broad-spectrum AMP, calprotectin (a complex of S100A8 and S100A9 proteins), is expressed by oral epithelial cells and is up-regulated by interleukin-1alpha (IL-1alpha). Shosaikoto (SST) is a traditional Japanese herbal medicine that has immunomodulatory effects and is reported to enhance the levels of IL-1alpha in epithelial cells. The purpose of this study was to investigate the effect of SST on the expression of calprotectin and other AMPs through the regulation of IL-1alpha in oral epithelial cells. MATERIAL AND METHODS: Human oral epithelial cells (TR146) were cultured with SST (at concentrations ranging from 10 to 250 microg/mL) in the presence or absence of anti-IL-1alpha or IL-1 receptor antagonist. The expression of S100A8- and S100A9-specific mRNAs was examined by northern blotting. Calprotectin expression and IL-1alpha secretion were investigated by immunofluorescent staining or ELISA. The expression of other AMPs and IL-1alpha was analyzed by RT-PCR and by quantitative real-time PCR. RESULTS: Shosaikoto (25 microg/mL) significantly increased the expression of S100A8- and S100A9-specific mRNAs and calprotectin protein. Shosaikoto increased S100A7 expression, but had no effect on the expression of other AMPs. The expression of IL-1alpha-specific mRNA and its protein were slightly increased by SST. A neutralizing antibody against IL-1alpha or IL-1 receptor antagonist inhibited SST up-regulated S100A8/S100A9 mRNA expression. CONCLUSION: These results suggest that SST increases the expression of calprotectin and S100A7 in oral epithelial cells. In response to SST, up-regulation of calprotectin may be partially induced via IL-1alpha.

Inflammatory peritoneal reaction after perforated appendicitis: continuous peritoneal lavage versus non lavage.

INTRODUCTION: Bacterial peritonitis is a severe medical condition associated with a natural mortality rate of 80-100%. Progress in surgical techniques, new developments in intensive care medicine and antibiotic therapy reduced this rate significantly. Aim of this study was to evaluate sepsis parameter in perforated appendicitis and different postoperative management. METHODS: In 50 consecutive patients with diffuse bacterial peritonitis and perforated appendicitis, laparotomy was performed. Subsequently, 25 patients were treated with adjuvant, continuous peritoneal lavage (CPL) using standard peritoneal dialysis (CAPD)-solution. The remaining 25 patients were peritoneally drained without postoperative irrigation (Non-CPL). In all patients endotoxin, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL-6), C-reactive protein (CRP) and myeloid-related protein (MRP-8, MRP-14 and Heterocomplex) were determined. RESULTS: No difference in clinical outcome between CPL and Non-CPL could be established. An uncomplicated clinical outcome was associated with lower levels of inflammation markers. Furthermore, clinical data revealed that mortality depended on co-morbidity, and patient's age. SUMMARY: In perforated appendicitis a faster decrease of mediator release could not be achieved with either method. In addition, no difference could be established for the clinical parameters like hospitalization, duration of intensive care and morbidity.

Regulation of calprotectin expression by interleukin-1alpha and transforming growth factor-beta in human gingival keratinocytes.

BACKGROUND AND OBJECTIVE: Calprotectin, a heterodimer of S100A8 and S100A9 with antimicrobial properties, is expressed in gingival keratinocytes and plays an important role in innate immunity. Because calprotectin expression is localized in the spinous cell layer of the gingival epithelium, we hypothesized that the expression of calprotectin in keratinocytes is related to the differentiation stage. The aim of the present study was to investigate the relationship between calprotectin expression and keratinocyte differentiation using some factors that regulated its differentiation. MATERIAL AND METHODS: Normal human gingival keratinocytes were isolated from gingival tissues obtained at the extraction of wisdom teeth, and were cultured in serum-free keratinocyte medium supplemented with interleukin-1alpha or calcium, which promote keratinocyte differentiation, and transforming frowth factor-beta (TGF-beta) or retinoic acid, which suppress its differentiation. The expression of S100A8/A9 mRNA and the production of calprotectin in normal human gingival keratinocytes were examined by northern blotting and enzyme-linked immunosorbent assay, respectively. The expression of cytokeratin 14, involucrin and filaggrin (marker proteins of keratinocyte differentiation) was investigated by immunohistochemical staining, and the DNA-binding activity of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor, was examined by electrophoretic mobility shift assay. RESULTS: The expression of S100A8/A9 mRNA and the production of calprotectin were increased by interleukin-1alpha and calcium, but decreased by TGF-beta. RA inhibited the expression of S100A8/A9 and keratinocyte differentiation, which were induced by interleukin-1alpha. C/EBPalpha DNA-binding activity in normal human gingival keratinocytes was enhanced by interleukin-1alpha and calcium, but suppressed by TGF-beta. CONCLUSION: The present study suggests that calprotectin expression is related to keratinocyte differentiation and that C/EBPalpha is a regulator of calprotectin expression in keratinocytes.