S100A9 induces tissue remodeling of human nasal epithelium in chronic rhinosinusitis with nasal polyp.

BACKGROUND: Chronic inflammation triggers tissue remodeling in human nasal epithelial (HNE) cells. S100A9, a protein secreted by inflammatory cells, exhibits potent proinflammatory activity. However, its effect on HNE cell remodeling, such as squamous metaplasia, remains unclear. Therefore, this study aimed to determine the effects and underlying pathways of S100A9 on HNE cell remodeling and investigate its clinical implications in chronic rhinosinusitis (CRS). METHODS: Cultured HNE cells were treated with S100A9. Bulk RNA sequencing was performed to analyze gene ontology (GO). Ingenuity pathway analysis (IPA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were also analyzed. Additionally, immunohistochemistry and multiplex immunofluorescence were performed on tissue samples obtained from 60 patients, whose clinical informations were also reviewed. RESULTS: GO enrichment analysis indicated that S100A9 induced tissue remodeling in HNE cells toward squamous metaplasia. IPA and KEGG commonly showed that S100A9 affected HNE cells associated with the IL-17 signaling pathway, including target molecules such as matrix metalloproteinase 1 (MMP1) and small proline-rich protein 2A (SPRR2A). Squamous metaplasia with a marked expression of S100A9 was observed in 50% of CRS with nasal polyps (CRSwNPs). In addition, in multiplex immunofluorescence, the S100A9 in sub-epithelium was co-expressed with myeloperoxidase, a neutrophil marker, and MMP1 and SPRR2A were strongly expressed in epithelial remodeling. Clinically, the expression of S100A9 correlated with sino-nasal outcome test-22 (r = 0.294, p = 0.022) and Lund-Mackay scores (r = 0.348, p = 0.006). CONCLUSION: S100A9 induces tissue remodeling in HNE cells. Its increased expression in CRSwNP, particularly squamous epithelium, correlates with disease severity. This suggests the clinical potential of S100A9 as a biomarker for CRS severity.

BAL Proteomic Signature of Lung Adenocarcinoma in IPF Patients and Its Transposition in Serum Samples for Less Invasive Diagnostic Procedures.

Idiopathic pulmonary fibrosis (IPF) is a form of chronic and irreversible fibrosing interstitial pneumonia of unknown etiology. Although antifibrotic treatments have shown a reduction of lung function decline and a slow disease progression, IPF is characterize by a very high mortality. Emerging evidence suggests that IPF increases the risk of lung carcinogenesis. Both diseases show similarities in terms of risk factors, such as history of smoking, concomitant emphysema, and viral infections, besides sharing similar pathogenic pathways. Lung cancer (LC) diagnosis is often difficult in IPF patients because of the diffuse lung injuries and abnormalities due to the underlying fibrosis. This is reflected in the lack of optimal therapeutic strategies for patients with both diseases. For this purpose, we performed a proteomic study on bronchoalveolar lavage fluid (BALF) samples from IPF, LC associated with IPF (LC-IPF) patients, and healthy controls (CTRL). Molecular pathways involved in inflammation, immune response, lipid metabolism, and cell adhesion were found for the dysregulated proteins in LC-IPF, such as TTHY, APOA1, S10A9, RET4, GDIR1, and PROF1. The correlation test revealed a relationship between inflammation- and lipid metabolism-related proteins. PROF1 and S10A9, related to inflammation, were up-regulated in LC-IPF BAL and serum, while APOA1 and APOE linked to lipid metabolism, were highly abundant in IPF BAL and low abundant in IPF serum. Given the properties of cytokine/adipokine of the nicotinamide phosphoribosyltransferase, we also evaluated its serum abundance, highlighting its down-regulation in LC-IPF. Our retrospective analyses of BAL samples extrapolated some potential biomarkers of LC-IPF useful to improve the management of these contemporary pathologies. Their differential abundance in serum samples permits the measurement of these potential biomarkers with a less invasive procedure.

Deletion of the inflammatory S100-A9/MRP14 protein does not influence survival in hSOD1G93A ALS mice.

Neuroinflammation is a hallmark of Amyotrophic Lateral Sclerosis (ALS) in hSOD1G93A mouse models where microglial cells contribute to the progressive motor neuron degenerative process. S100-A8 and S100-A9 (also known as MRP8 and MRP14, respectively) are cytoplasmic proteins expressed by inflammatory myeloid cells, including microglia and macrophages. Mainly acting as a heterodimer, S100-A8/A9, when secreted, can activate Toll-like Receptor 4 on immune cells, leading to deleterious proinflammatory cytokine production. Deletion of S100a9 in Alzheimer's disease mouse models showed a positive outcome, reducing pathology. We now assessed its role in ALS. Unexpectedly, our results show that deleting S100a9 in hSOD1G93A ALS mice had no impact on mouse survival, but rather accelerated symptoms with no impact on microglial activation and motor neuron survival, suggesting that blocking S100-A9 would not be a valuable strategy for ALS.

Deviant proteome profile of equine granulocytes associates to latent activation status in organ specific autoimmune disease.

Equine recurrent uveitis (ERU) is a spontaneous, remitting-relapsing autoimmune disease driven by the adaptive immune system. Although T cells are described as the main effector cells in pathogenesis, granulocytes have also emerged as possible disease mediators. To explore the role of these innate immune cells, we investigated the whole cell proteome of granulocytes from equine recurrent uveitis cases and healthy controls. Among the 2362 proteins identified by mass spectrometry, we found 96 proteins with significantly changed abundance between groups (p < 0.05, fold change >1.2), representing 4.1% of total granulocyte proteome. Within these differential identifications, calgranulin B, a protein associated with pathogenesis in other autoimmune diseases, showed highest abundance in equine recurrent uveitis (18 fold). For a better interpretation of the results from our hypothesis-generating approach, we added a threshold for biological significance (ratio ERU/controls >2: 36 proteins) to the proteins with increased abundance in equine recurrent uveitis and analyzed their allocation to the subsets within the Immune System superpathway. The 36 differentially abundant proteins predominantly associated to RAF/MAP kinase cascade, MHC-I-mediated antigen presentation and neutrophil degranulation, suggesting a latently activated phenotype of these innate immune cells in disease. Raw data are available via ProteomeXchange with identifier PXD013648. SIGNIFICANCE: Our study provides new insights into the protein repertoire of primary equine granulocytes and identifies protein abundance changes associated to equine recurrent uveitis (ERU), an organ specific, spontaneously occurring autoimmune disease. We show that granulocyte proteins with increased abundance in ERU strongly associate to RAF/MAP kinase signaling, MHC-I antigen presentation and neutrophil degranulation, pointing to a more activated state of these cells in ERU cases. Since cells were obtained in quiescent stage of disease, latent activation of granulocytes underlines the role of these innate immune cells in ERU. These findings are highly relevant for veterinary medicine, further establishing the importance of granulocytes in this T cell-driven autoimmune disease. Moreover, they have translational quality for autoimmune uveitis in man, due to strong similarity in disease occurrence, progression and pathogenesis.

[S100A8/A9 and periodontal inflammatory diseases].

Calprotectin S100A8/A9, a heterodimer composed of S100A8 and S100A9, is the main component of cytoplasmic proteins in neutrophils. It plays multiple roles in the immuno-inflammatory reactions intracellularly and extracellularly. Recent studies find that S100A8/A9 is closely related with the initiation and progression of periodontal inflammatory diseases. S100A8/A9 is expected to be a new biomarker for diagnosing periodontal inflammatory diseases, monitoring inflammatory activities in patients with periodontitis, evaluating the outcome of periodontal treatments and predicting the susceptibility of individual patient to periodontitis. In this literature review, we summarize the clinical research progress on the relation between S100A8/A9 and periodontal inflammatory diseases.

Biological variation of serum canine calprotectin concentrations as measured by ELISA in healthy dogs.

Calprotectin is a useful biomarker of inflammation in dogs. However, the biological variation of serum canine calprotectin is unknown. Indices of biological variation were determined in serial serum samples (n=147) from 11 healthy dogs (males/females: 4/7, median age: 5 years): analytical (3.0%), intra-individual (29.9%), and inter-individual variation (33.2%), reciprocal index of individuality (1.1), and index of heterogeneity (4.9). Serum calprotectin concentrations measured by ELISA and by the previous radioimmunoassay were highly correlated, but a constant and proportional bias exists between both assays. A de novo ELISA-reference interval (RI) for serum calprotectin concentration was established (0.6-11.8mg/L). Moderate changes in serum calprotectin (minimum critical difference: 6.4mg/L) between sequential measurements are needed to be considered relevant, and a population-based RI may or may not be appropriate for serum calprotectin.

The guardians of the periodontium-sequential and differential expression of antimicrobial peptides during gingival inflammation. Results from in vivo and in vitro studies.

AIM: To evaluate the sequential and differential expression of a variety of antimicrobial peptides (AMPs) during the development of an experimentally induced gingivitis in humans. MATERIAL AND METHODS: In twenty healthy volunteers, gingival inflammation was induced by abstention from oral hygiene at 6 teeth. Bleeding on probing (BOP) and plaque index (PI) were assessed, and gingival biopsies and gingival crevicular fluid (GCF) were collected at 8 different time points (t0-t35). Gingival epithelial cells (GECs) were stimulated with various receptor agonists. In biopsies and GECs, mRNA expression of human beta-defensins (hBD-2, hBD-3), CC-chemokine ligand 20 (CCL20), S100A7/psoriasin (S100A7), and calgranulin A/B (S100A8, S100A9) was evaluated using real-time PCR, and protein profiles were measured by ELISA. Statistical analysis was performed using non-parametric tests. RESULTS: The clinical parameters BOP, PI and GCF increased over time (p < 0.0001). Tissue AMP mRNA expression was elevated, but at different and AMP-specific time points (p < 0.05). Protein analysis revealed a similar expression pattern for hBD-2 and CCL20 in GCF (p < 0.05). In GECs, multiple receptor stimulation was required to induce AMP gene expression (p < 0.0001). CONCLUSIONS: For the first time, this study showed the sequential and differential expression of AMPs during a developing inflammation in vivo providing further evidence for their role as guardians of a healthy periodontium.

Measuring calprotectin in plasma and blood with a fully automated turbidimetric assay.

Calprotectin in plasma and blood might prove to be a useful biomarker of inflammation and infection; however, automated methods for analysing the concentration of calprotectin in those materials are lacking. We have validated a fully automated turbidimetric method and present health-related reference limits. Calprotectin was measured by Siemens Advia XPT with the Bühlmann fCAL® turbo test (Bühlmann Laboratories AG, Schönenbuch, Switzerland), a particle enhanced turbidimetric immunoassay for quantification of calprotectin in fecal extracts. Plasma and serum samples were analysed directly, while whole blood was first extracted with M-PER® Mammalian Protein Extraction Reagent (ThermoFisher) and diluted with B-CAL-EX (Bühlmann). We studied analytical imprecision, estimated health-related reference limits and examined the correlation between neutrophil-calprotectin (blood-calprotectin adjusted for plasma-calprotectin) and the neutrophil count. The intermediate ('day-to-day') coefficient of variation was 3.5 and 1.0% for heparin-plasma-calprotectin at 0.52 mg/L and 3.53 mg/L, respectively, and 4.9% for heparin-blood-calprotectin at 50.2 mg/L. Health-related reference limits were 0.470-3.02 mg/L for calprotectin in heparin-plasma, 50.8-182 mg/L for calprotectin in heparin-blood, 0.534-2.41% for the ratio between them and 24.7-33.3 pg for the mean amount of calprotectin per neutrophil. Compared to heparin-plasma, calprotectin concentrations were significantly lower in EDTA-plasma and higher in serum (p < .05). Correlation between neutrophil-calprotectin and the neutrophil count was excellent. We have shown that the Bühlmann fCAL® turbo test can be used to measure calprotectin in plasma and blood.

Calgranulin B and KL-6 in Bronchoalveolar Lavage of Patients with IPF and NSIP.

Idiopathic pulmonary fibrosis (IPF) and non-specific interstitial pneumonia (NSIP) are the most frequent idiopathic interstitial pneumonias. The aim of this study was to evaluate concentrations of calgranulin B and Krebs von den Lungen 6 (KL-6) in bronchoalveolar lavage (BAL) fluid of patients with IPF and idiopathic NSIP (i-NSIP) with fibrotic pattern. Thirty patients with IPF (68.73 ± 8.63 years), 30 with i-NSIP (68.33 ± 7.45 years), and healthy controls were included in the study. Calgranulin B and KL-6 both proved to be significantly higher in BAL of IPF and i-NSIP patients than in healthy controls (p < 0.05). Calgranulin B showed several significant correlations with functional parameters (oxygen demand at rest, 6-min walking test (6MWT), and PFTs); KL-6 was correlated with oxygen demand at rest and during 6MWT. Patients with higher concentrations of both biomarkers (> 75th percentile) had more advanced disease with lower values of FEV1%, FVC%, RV%, TLC%, DLCO% of predicted, distance walked in 6MWT, and BAL neutrophil percentage. Calgranulin B and KL-6 in BAL proved to be reliable biomarkers of IPF and i-NSIP and to have prognostic meaning, discriminating severe and advanced patients. The combination of the two biomarkers can facilitate the stratification of severity.

The role of calgranulin B gene on the biological behavior of squamous cervical cancer in vitro and in vivo.

OBJECTIVE: The objective of the study was to explore the role of calgranulin B gene on the biological behavior of squamous cervical cancer. METHODS: Differential transcription in calgranulin B gene between human papillomavirus (HPV)-positive and negative cervical cancer groups was identified, and the relationship between calgranulin B gene and matrix metalloproteinase (MMP) genes were explored using The Cancer Genome Atlas database. Subsequently, the role of calgranulin B on the cell proliferation, apoptosis, invasion and migration was investigated, through overexpression and/or underexpression of calgranulin B in cervical cancer cells. In addition, the effect of calgranulin B on the growth of the cervical cancer was studied via constructing xenograft model in BALB/c nude mice that either overexpressed or underexpressed calgranulin B. RESULTS: Calgranulin B gene transcription in cervical cancer was highly correlated with the high-risk HPV-16 and HPV-45. In addition, overexpression of calgranulin B increased cell proliferation, invasion and migration, whereas it did not significantly affect cell apoptosis. This effect was also confirmed by calgranulin B knockdown assay. Additionally, we found that the transcription of calgranulin B gene was negatively correlated with MMP15 and MMP24 genes, but positively associated with MMP25 genes in cervical cancer. Furthermore, calgranulin B significantly promoted the growth of cervical cancer in vivo. CONCLUSION: Calgranulin B promotes cell proliferation, migration and invasion of squamous cervical cancer, possibly via regulation of MMPs. Whether there are synergistic actions between calgranulin B and HPV-16/HPV-45 infection on the squamous cervical carcinogenesis or progression need further study.

Role of S100A9 in the development of neutrophilic inflammation in asthmatics and in a murine model.

S100A9 is an endogenous danger signal that promotes and exacerbates the neutrophilic inflammatory response. To investigate the role of S100A9 in neutrophilic asthma, S100A9 levels were measured in sputum from 101 steroid-naïve asthmatics using an ELISA kit and the levels were significantly correlated with percentages of neutrophils in sputum. Intranasal administration of recombinant S100A9 markedly increased neutrophil numbers at 8h and 24h later with concomitant elevation of IL-1ß, IL-17, and IFN-γ levels. Treatment with an anti-S100A9 antibody restored the increased numbers of neutrophils and the increased airway resistance in OVA/CFA mice toward the levels of sham-treated mice. Concomitantly, the S100A9 and neutrophil elastase double positive cells were markedly reduced with attenuation of IL-1ß, IL-17, and IFN-γ levels by the treatment with the anti-S100A9 antibody. Our data support a role of S100A9 to initiate and amplify the neutrophilic inflammation in asthma, possibly via inducing IL-1ß, IL-17 and IFN-γ.

Significance of S100A8, S100A9 and calprotectin levels in bladder cancer.

OBJECTIVES: Members of the S100 protein family, S100A8, S100A9 and their heterodimer complex known as calprotectin are thought to be involved not only in inflammatory pathways but also in tumorigenesis and cancer progression. Therefore, they have been widely studied in various types of cancer; however, there is limited knowledge about their role in bladder cancer. In this study, our aim was to determine the levels of S100A8 and S100A9 in the sera, and calprotectin levels in the sera and urines of bladder cancer patients and compare it to urinary BTA, a tumor marker that can be used in the diagnosis of bladder cancer. MATERIALS AND METHODS: The study was comprised of two major groups: 52 healthy controls and 82 patients with bladder cancer. The patient group was also divided into subgroups according to tumor stage and grade. Urine BTA levels, serum S100A8 and S100A9 levels, and serum and urine calprotectin levels in healthy controls and patients were determined using commercially available ELISA kits. RESULTS: While serum S100A8 and S100A9 levels did not differ between the controls and patients significantly, serum and urine calprotectin levels and urine BTA levels were significantly elevated in patients compared to controls. Serum calprotectin or urine BTA levels did not differ significantly among the patient subgroups. However, urine calprotectin levels were significantly elevated in muscle-invasive tumors (T2-4) compared to lower stages (Ta and T1). CONCLUSIONS: Urine calprotectin levels can be used in the diagnosis and staging of bladder cancer as a marker for muscle invasion.

Proteins that interact with calgranulin B in the human colon cancer cell line HCT-116.

Calgranulin B is released from immune cells and can be internalized into colon cancer cells to prevent proliferation. The present study aimed to identify proteins that interact with calgranulin B to suppress the proliferation of colon cancer cells, and to obtain information on the underlying anti-tumor mechanism(s) of calgranulin B. Calgranulin B expression was induced in colon cancer cell line HCT-116 by infection with calgranulin B-FLAG expressing lentivirus, and it led to a significant suppression of cell proliferation. Proteins that interacted with calgranulin B were obtained by immunoprecipitation using whole homogenate of lentivirus-infected HCT-116 cells which expressing calgranulin B-FLAG, and identified using liquid chromatography-mass spectrometry/mass spectrometry analysis. A total of 454 proteins were identified that potentially interact with calgranulin B, and most identified proteins were associated with RNA processing, post-transcriptional modifications and the EIF2 signaling pathway. Direct interaction of calgranulin B with flotillin-1, dynein intermediate chain 1, and CD59 glycoprotein has been confirmed, and the molecules N-myc proto-oncogene protein, rapamycin-insensitive companion of mTOR, and myc proto-oncogene protein were shown to regulate calgranulin B-interacting proteins. Our results provide new insight and useful information to explain the possible mechanism(s) underlying the role of calgranulin B as an anti-tumor effector in colon cancer cells.

Antitumor effects of calgranulin B internalized in human colon cancer cells.

Calgranulin B is a small, calcium-binding protein expressed in neutrophils that is secreted into the tumor microenvironment in cancer cases. We previously showed that calgranulin B levels are increased in the stools of colorectal cancer patients. In patient tumor tissues, calgranulin B protein levels correlated with the presence of stromal inflammatory cells surrounding tumor cells, and calgranulin B promoter methylation was observed in both paired human tissues and colon cancer cell lines. Cell lines did not express calgranulin B, but in vitro studies showed that colon cancer cells internalized extracellular calgranulin B, while other types of cancer cells did not. Calgranulin B internalization led to reduced cell proliferation and increased apoptotic cell death. AKT and ERK signals were also increased after calgranulin B treatment, as were p53, ß-catenin, E-cadherin and cleaved caspase-3 levels. Additionally, a human protein microarray identified aurora A kinase as a calgranulin B binding partner, and binding inhibited aurora A kinase activity in a dose-dependent manner. Our findings demonstrate the antitumor effects of calgranulin B in the inflammatory microenvironment and suggest that calgranulin B could be potentially efficacious in the treatment of colon cancer.

Calprotectin as a biomarker for rheumatoid arthritis: a systematic review.

OBJECTIVE: Calprotectin (myeloid-related protein 8/14), a heterodimeric complex of calcium-binding proteins, is expressed in granulocytes and monocytes. Calprotectin levels are high in synovial tissue, particularly in activated cells adjacent to the cartilage-pannus junction. This systematic review evaluates the use of calprotectin as an indicator of disease activity, therapeutic response, and prognosis in rheumatoid arthritis (RA). METHODS: Medline, Scopus, and the Cochrane Library (1970-2013) were searched for studies containing original data from patients with RA in whom calprotectin levels were measured in plasma/serum and/or synovial fluid (SF). We included studies examining associations between calprotectin levels and clinical and laboratory assessments, disease progression, and therapeutic response. There were no restrictions for sample size, disease duration, or length of followup. RESULTS: We evaluated 17 studies (1988-2013) with 1065 patients enrolled; 11 were cross-sectional and 8 had longitudinal designs with 2 studies reporting cross-sectional and longitudinal data. Systemic and SF levels of calprotectin were raised in RA. There was a wide range of levels and marked interstudy and intrastudy variability. Calprotectin levels were high in active disease and were particularly high in rheumatoid factor (RF)-positive patients. Levels fell with effective treatment. Longitudinal data showed that calprotectin was a significant and independent predictor of erosive progression and therapeutic responses, particularly in patients who received effective biological treatments. CONCLUSION: SF calprotectin levels are high, suggesting there is substantial local production by inflamed synovium. Blood calprotectin levels, though highly variable, are elevated in active RA and fall with effective therapy. High baseline calprotectin levels predict future erosive damage.

Imaging mass spectrometry for assessing cutaneous wound healing: analysis of pressure ulcers.

Imaging mass spectrometry (IMS) was employed for the analysis of frozen skin biopsies to investigate the differences between stage IV pressure ulcers that remain stalled, stagnant, and unhealed versus those exhibiting clinical and histological signs of improvement. Our data reveal a rich diversity of proteins that are dynamically modulated, and we selectively highlight a family of calcium binding proteins (S-100 molecules) including calcyclin (S100-A6), calgranulins A (S100-A8) and B (S100-A9), and calgizzarin (S100-A11). IMS allowed us to target three discrete regions of interest: the wound bed, adjacent dermis, and hypertrophic epidermis. Plots derived using unsupervised principal component analysis of the global protein signatures within these three spatial niches indicate that these data from wound signatures have potential as a prognostic tool since they appear to delineate wounds that are favorably responding to therapeutic interventions versus those that remain stagnant or intractable in their healing status. Our discovery-based approach with IMS augments current knowledge of the molecular signatures within pressure ulcers while providing a rationale for a focused examination of the role of calcium modulators within the context of impaired wound healing.

Role of inflammasome activation in development and exacerbation of asthma.

Human airways contact with pathogen-associated molecular patterns and danger-associated molecular patterns present in many environments. Asthmatic's airways may be more susceptible to these patterns and lead to inflammasome activation; however, the participation of inflammasome in the development and exacerbation of asthma is not fully understood and remains controversial. Asthma is a heterogeneous group composed of different airway inflammation patterns with different underlying immune mechanisms. One mechanism is neutrophilic airway inflammation based on the axis of inflammasome activation, interleukin (IL) 1ß/IL-18 production, T helper 17 activation, IL-8/IL-6 overproduction, and neutrophilic inflammation. The role of inflammasome activation has been highlighted in experimental asthma models and some evidence of inflammasome activation has been recently demonstrated in human neutrophilic asthmatic airways. In addition to caspase-1 activation, proteinase 3 and other protease from activated neutrophils directly cleave pro-IL-1ß and pro-IL-18 to IL-1ß and IL-18, which contribute to the phenotype of subsequent adaptive immune responses without inflammasome activation. Data suggests that neutrophilics in asthmatic airways may have an additional effect in initiating inflammasome activation and amplifying immune responses. Among the mediators from neutrophils, S100A9 seems to be one candidate mediator to explain the action of neutrophils in amplifying the airway inflammation in concert with inflammasome.