Histidine-Rich Glycoprotein Suppresses the S100A8/A9-Mediated Organotropic Metastasis of Melanoma Cells.

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis.

Study of calprotectin gene polymorphism and serum level in acne vulgaris patients.

BACKGROUND: Acne vulgaris (AV) is an inflammatory skin disease of the pilosebaceous unit. S100A8 and S100A9 (the light subunits of calprotectin) gene polymorphisms have been known to be associated with inflammatory disorder. Until now, no study investigated calprotectin gene polymorphism in acne patients. OBJECTIVE: This study was designed to investigate calprotectin serum levels and gene polymorphism (rs3806232) in acne vulgaris patients and to correlate them with different clinical aspects of them. METHODS: This case-control study included 50 patients having variable degrees of acne vulgaris (AV) severity, in addition to a control group of 26 age- and gender-matched seemingly healthy volunteers. RESULTS: Acne vulgaris patients had considerably greater (P < 0.001) mean serum calprotectin levels than the control group (3.86 ± 2.58 pg/ml vs. 0.29 ± 0.14). AA genotype of calprotectin S100 A8 (rs3806232) was significantly predominant over AG or GG genotypes in patients compared to the controls, and the A allele was significantly (P < 0.001) predominant in patients (80%), while A and G alleles were equally distributed in controls; also, there was a significantly higher serum calprotectin level in calprotectin AA genotype than in AG or GG (P < 0.001) in acne vulgaris patients. CONCLUSION: The serum levels of calprotectin were considerably greater in AV patients than in controls. AA genotype and A allele of the S100 A8 gene were significantly higher in patients, which was associated with significantly higher calprotectin serum levels. Thus, calprotectin, both gene and serum level, might participate in disease pathogenesis, which needs further studies.

Neutrophil-Derived Protein S100A8/A9 Alters the Platelet Proteome in Acute Myocardial Infarction and Is Associated With Changes in Platelet Reactivity.

OBJECTIVE: Platelets are central to acute myocardial infarction (MI). How the platelet proteome is altered during MI is unknown. We sought to describe changes in the platelet proteome during MI and identify corresponding functional consequences. Approach and Results: Platelets from patients experiencing ST-segment-elevation MI (STEMI) before and 3 days after treatment (n=30) and matched patients with severe stable coronary artery disease before and 3 days after coronary artery bypass grafting (n=25) underwent quantitative proteomic analysis. Elevations in the proteins S100A8 and S100A9 were detected at the time of STEMI compared with stable coronary artery disease (S100A8: FC, 2.00; false discovery rate, 0.05; S100A9: FC, 2.28; false discovery rate, 0.005). During STEMI, only S100A8 mRNA and protein levels were correlated in platelets (R=0.46, P=0.012). To determine whether de novo protein synthesis occurs, activated platelets were incubated with 13C-labeled amino acids for 24 hours and analyzed by mass spectrometry. No incorporation was confidently detected. Platelet S100A8 and S100A9 was strongly correlated with neutrophil abundance at the time of STEMI. When isolated platelets and neutrophils were coincubated under quiescent and activated conditions, release of S100A8 from neutrophils resulted in uptake of S100A8 by platelets. Neutrophils released S100A8/A9 as free heterodimer, rather than in vesicles or extracellular traps. In the community-based Bruneck study (n=338), plasma S100A8/A9 was inversely associated with platelet reactivity-an effect abrogated by aspirin. CONCLUSIONS: Leukocyte-to-platelet protein transfer may occur in a thromboinflammatory environment such as STEMI. Plasma S100A8/A9 was negatively associated with platelet reactivity. These findings highlight neutrophils as potential modifiers for thrombotic therapies in coronary artery disease.

Danger-associated molecular pattern molecules and the receptor for advanced glycation end products enhance ANCA-induced responses.

OBJECTIVES: The pro-inflammatory activities of the calgranulins and HMGB1 can be counteracted by sRAGE, the soluble form of their shared receptor. To understand the role of these molecules in AAV and their potential as therapeutic targets we have studied (i) the relationship between these DAMPS and disease activity; (ii) the expression of RAGE and sRAGE in biopsy tissue and peripheral blood; and (iii) the effect of these molecules on ANCA-mediated cytokine production. METHODS: We examined circulating levels of calgranulins (S100A8/A9 and S100A12), HMGB1 and sRAGE by ELISA. RAGE was examined in AAV kidney and lung biopsies by immunohistochemistry and RAGE expression was monitored in peripheral blood by qPCR. In vitro, the effect of co-stimulating PBMC with ANCA and S100A8/A9 on cytokine production was studied by ELISA. RESULTS: We found significantly raised levels of calgranulins and HMGB1 in active AAV regardless of clinical phenotype (PR3+/MPO+ AAV). Levels of calgranulins showed significant correlations with each other. RAGE protein and message was raised in peripheral blood and in cells infiltrating kidney and lung biopsy tissue, while sRAGE was lowered. Furthermore, ANCA-mediated production of IL-8 from PBMC was significantly enhanced by the presence of S100A8/A9 in a RAGE/TLR4-dependent manner. CONCLUSIONS: Raised circulating calgranulins provide a good marker of disease activity in AAV and are unlikely to be counteracted by sRAGE. Increased RAGE expression in AAV indicates receptor stimulation in active disease that may exacerbate ANCA-induced cytokine production. Targeting the RAGE pathway may provide a useful therapeutic approach in AAV.

Clinical parameters and biological markers associated with acute severe ocular complications in Stevens-Johnson syndrome and toxic epidermal necrolysis.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are severe cutaneous adverse drug reactions with high mortality rates. Its sequelae, such as blindness, persist even after recovery. Patients with SJS/TEN should be accurately diagnosed and receive appropriate treatment as soon as possible. Therefore, identifying the factors for severity prediction is necessary. We aimed to clarify the clinical parameters and biological markers that can predict acute severe ocular complications (SOCs) in SJS/TEN. This retrospective cross-sectional study enrolled 47 patients with SJS/TEN who were divided into two groups according to ocular severity at acute onset: non-severe ocular complications group (n = 27) and severe ocular complications group (n = 20). Multivariate logistic regression analysis revealed that disease severity (body surface area detachment ≥ 10%) was a predictive factor for acute SOCs, and older age (≥ 60 years) was marginally significantly predictive of SOCs. Serum biomarker levels of S100A8/A9 and granulysin were marginally significant and tended to increase in the SOC group. Therefore, during the early acute stage, focusing on disease severity, patient age, and serum inflammatory biomarkers (S100A8/A9 and granulysin) might help predict SOC progression in patients with SJS/TEN who need prompt and aggressive ocular management to prevent severe ocular sequelae.

Differential Regulation of Circulating Soluble Receptor for Advanced Glycation End Products (sRAGEs) and Its Ligands S100A8/A9 Four Weeks Post an Exercise Intervention in a Cohort of Young Army Recruits.

Apart from its beneficial effects on cardiovascular risk factors, an anti-inflammatory effect of exercise is strongly implicated. Yet, data regarding the effect of an exercise intervention on healthy individuals are limited and contradictory. The present study aimed to investigate the effects of a physical activity intervention on the soluble form of the receptor for advanced glycation end products (sRAGEs) and its ligands S100A8/A9. A total of 332 young army recruits volunteered and 169 completed the study. The participants underwent the standard basic training of Greek army recruits. IL-6, IL-1ß, S100A8/A9, and sRAGEs were measured at the beginning and at the end of the training period. Primary rodent adult aortic smooth muscle cells (ASMCs) were analyzed for responsiveness to direct stimulation with S100A8/A9 alone or in combination with sRAGEs. At the end of the training period, we observed a statistically significant reduction in S100A8/A9 (630.98 vs. 472.12 ng/mL, p = 0.001), IL-1ß (9.39 [3.8, 44.14] vs. 5.03 [2.44, 27.3] vs. pg/mL, p = 0.001), and sRAGEs (398.38 vs. 220.1 pg/mL, p = 0.001). IL-6 values did not change significantly after exercise. S100A8/A9 reduction was positively correlated with body weight (r = 0.236 [0.095, 0.370], p = 0.002) and BMI (r = 0.221 [0.092, 0.346], p = 0.004). Direct stimulation of ASMCs with S100A8/A9 increased the expression of IL-6, IL-1ß, and TNF-α and, in the presence of sRAGEs, demonstrated a dose-dependent inhibition. A 4-week military training resulted in significant reduction in the pro-inflammatory cytokines IL-1ß and S100A8/A9 complex. The observed reduction in sRAGEs may possibly reflect diminished RAGE axis activation. Altogether, our findings support the anti-inflammatory properties of physical activity.

S100A8 may govern hyper-inflammation in severe COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic threatens human species with mortality rate of roughly 2%. We can hardly predict the time of herd immunity against and end of COVID-19 with or without success of vaccine. One way to overcome the situation is to define what delineates disease severity and serves as a molecular target. The most successful analogy is found in BCR-ABL in chronic myeloid leukemia, which is the golden biomarker, and simultaneously, the most effective molecular target. We hypothesize that S100 calcium-binding protein A8 (S100A8) is one such molecule. The underlying evidence includes accumulating clinical information that S100A8 is upregulated in severe forms of COVID-19, pathological similarities of the affected lungs between COVID-19 and S100A8-induced acute respiratory distress syndrome (ARDS) model, homeostatic inflammation theory in which S100A8 is an endogenous ligand for endotoxin sensor Toll-like receptor 4/Myeloid differentiation protein-2 (TLR4/MD-2) and mediates hyper-inflammation even after elimination of endotoxin-producing extrinsic pathogens, analogous findings between COVID-19-associated ARDS and pre-metastatic lungs such as S100A8 upregulation, pulmonary recruitment of myeloid cells, increased vascular permeability, and activation coagulation cascade. A successful treatment in an animal COVID-19 model is given with a reagent capable of abrogating interaction between S100A8/S100A9 and TLR4. In this paper, we try to verify our hypothesis that S100A8 governs COVID-19-associated ARDS.

A clinical study on plasma biomarkers for deciding the use of adjuvant corticosteroid therapy in bronchopulmonary dysplasia of premature infants.

Objective: The study was designed to investigate some plasma markers which help us to decide the use of adjuvant corticosteroid therapy in bronchopulmonary dysplasia (BPD) of premature infants. Methods: Thirty BPD infants were treated by dexamethasone. Among these cases, dexamethasone was significant effective in 10 cases, and no significant effective in 20 cases. These patients were divided into two groups as the significant effect (SE) group (n=10) and the non-significant effect (NE) group (n=20) according to the curative effect of dexamethasone. Fifteen non-BPD infants with gestational age and gender matching were selected as the control group. Plasma samples before and after dexamethasone treatment were collected from three infants chosen randomly from SEG for the data-independent acquisition (DIA) analysis. ELISA was further used to detect the levels of differential proteins LRP1 and S100A8 in all individuals, including SE, NE and control groups. Results: DIA analysis results showed that after dexamethasone treatment, there were a total of 52 plasma proteins that showed significant differences, of which 43 proteins were down-regulated and 9 proteins were up-regulated. LRP1 and S100A8 were two plasma proteins that were significantly changed after dexamethasone treatment. Compared with the control group, plasma LRP1 was significantly increased in BPD. Interestingly, the plasma concentration of LRP1 in the NE group was significantly higher than that in the SE group. S100A8, as an indicator of plasma inflammation, was significantly higher in BPD than the control group. Unlike LRP1, there was no significantly difference between the SE and NE group (P=0.279) before dexamethasone treatment. Conclusion: Elevated plasma LRP1 and S100A8 in BPD infants are two indicators that correlated with the efficacy of dexamethasone, and might be used as biomarkers for deciding the use of adjuvant corticosteroids therapy in the BPD.

Serum calprotectin (S100A8/A9): a promising biomarker in diagnosis and follow-up in different subgroups of juvenile idiopathic arthritis.

INTRODUCTION: In the management of juvenile idiopathic arthritis (JIA), there is a lack of diagnostic and prognostic biomarkers. This study assesses the use of serum calprotectin (sCal) as a marker to monitor disease activity, and as a classification and prognosis tool of response to treatment or risk of flares in patients with JIA. METHODS: Eighty-one patients with JIA from the CAP48 multicentric cohort were included in this study, as well as 11 non-paediatric healthy controls. An ELISA method was used to quantify sCal with a commercial kit. RESULTS: Patients with an active disease compared with healthy controls and with patients with inactive disease showed an eightfold and a twofold increased level of sCal, respectively. sCal was found to be correlated with the C-reactive protein (CRP) and even more strongly with the erythrocyte sedimentation rate. Evolution of DAS28 scores correlated well with evolution of sCal, as opposed to evolution of CRP. With regard to CRP, sCal could differentiate forms with active oligoarthritis from polyarthritis and systemic forms. However, sCal brought an added value compared with the CRP as a prognosis marker. Indeed, patients with active disease and reaching minimal disease activity (according to Juvenile Arthritis Disease Activity Score) at 6 months following the test had higher sCal levels, while patients with inactive disease had higher sCal levels if a flare was observed up to 3-9 months following the test. CONCLUSIONS: This study confirms the potential uses of sCal as a biomarker in the diagnosis and follow-up of JIA.

Serum calprotectin (S100A8/A9) levels as a new potential biomarker of treatment response in Hodgkin lymphoma.

INTRODUCTION: Hodgkin lymphoma (HL) is unusual among malignancies, with inflammation playing such a prominent role in its pathogenesis. S100A8/A9 (calprotectin) is a heterodimeric protein, which has a role in the inflammatory response and oncogenesis. In this study in HL patients, the correlation between serum S100A8/A9 levels and treatment responses was investigated along with whether this marker is correlated with other inflammatory markers. MATERIALS AND METHODS: Thirty-three HL patients and 20 healthy volunteers were included. Demographic and clinical characteristics were recorded. Calprotectin levels were measured with Human S100A8/A9 Heterodimer Quantikine ELISA kit. Calprotectin levels were measured twice in patients, before and after treatment, and once in the control group. Treatment responses were evaluated with positron emission tomography-computed tomography (PET-CT). RESULTS: The mean age of patients was 44.3 ± 18.1 (66.3% male). The median (IQR) values of S100A8/A9 before and after treatment in the patient group were 4.98 (2.6-7.8) and 1.87 (1.1-4.8)µg/mL. Median (IQR) S100A8/A9 concentration in the control group was 1.41 (0.98-2.73)µg/mL. In patients, pretreatment values were significantly higher than in controls (P < .001). However, median values of patients after treatment and controls were similar. Patient median S100A8/A9 levels were significantly lower post-treatment compared with pretreatment values (P = .001). When inflammatory markers were examined within groups, no relationship was found between markers. In ROC analysis, a S100A8/A9 cutoff value of ≥3.31µg/mL accurately discriminated end-of-treatment PET positivity (AUC = 0.78; 95% CI 0.58-0.98; accuracy = 76.2%). CONCLUSION: S100A8/A9 may be a potential biomarker for treatment response in HL independent of inflammation. This is the first study to investigate and show this finding. However, further large-scale studies are still required.

Circulating Calprotectin as a Biomarker of COVID-19 Severity.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although demographic and clinical parameters such as sex, age, comorbidities, genetic background and various biomarkers have been identified as risk factors, there is an unmet need to predict the risk and onset of severe inflammatory disease leading to poor clinical outcomes. In addition, very few mechanistic biomarkers are available to inform targeted treatment of severe (auto)-inflammatory conditions associated with COVID-19. Calprotectin, also known as S100A8/S100A9, MRP8/14 (Myeloid-Related Protein) or L1, is a heterodimer involved in neutrophil-related inflammatory processes. In COVID-19 patients, calprotectin levels were reported to be associated with poor clinical outcomes such as significantly reduced survival time, especially in patients with severe pulmonary disease. AREAS COVERED: Pubmed was searched using the following keywords: Calprotectin + COVID19, S100A8/A9 + COVID19, S100A8 + COVID-19, S100A9 + COVID-19, MRP8/14 + COVID19; L1 + COVID-19 between May 2020 and 8 March 2021. The results summarized in this review provide supporting evidence and propose future directions that define calprotectin as an important biomarker in COVID-19. EXPERT OPINION: Calprotectin represents a promising serological biomarker for the risk assessment of COVID-19 patients.

Phenotypical and Functional Characterization of Neutrophils in Two Pyrin-Associated Auto-inflammatory Diseases.

PURPOSE: Familial Mediterranean Fever (FMF) and Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND) are clinically distinct autoinflammatory disorders caused by mutations in the pyrin-encoding gene MEFV. We investigated the transcriptional, phenotypical, and functional characteristics of patient neutrophils to explore their potential role in FMF and PAAND pathophysiology. METHODS: RNA sequencing was performed to discover transcriptional aberrancies. The phenotypical features, degranulation properties, and phagocytic capacity of neutrophils were assessed by flow cytometry. Production of reactive oxygen species (ROS), myeloperoxidase (MPO) release, and chemotactic responses were investigated via chemiluminescence, ELISA, and Boyden chamber assays, respectively. RESULTS: Neutrophils from PAAND and FMF patients showed a partially overlapping, activated gene expression profile with increased expression of S100A8, S100A9, S100A12, IL-4R, CD48, F5, MMP9, and NFKB. Increased MMP9 and S100A8/A9 expression levels were accompanied by high plasma concentrations of the encoded proteins. Phenotypical analysis revealed that neutrophils from FMF patients exhibited an immature character with downregulation of chemoattractant receptors CXCR2, C5aR, and BLTR1 and increased expression of Toll-like receptor 4 (TLR4) and TLR9. PAAND neutrophils displayed an increased random, but reduced CXCL8-induced migration. A tendency for enhanced random migration was observed for FMF neutrophils. PAAND neutrophils showed a moderately but significantly enhanced phagocytic activity as opposed to neutrophils from FMF patients. Neutrophils from both patient groups showed increased MPO release and ROS production. CONCLUSIONS: Neutrophils from patients with FMF and PAAND, carrying different mutations in the MEFV gene, share a pro-inflammatory phenotype yet demonstrate diverse features, underscoring the distinction between both diseases.

The Inflammatory Factors Associated with Disease Severity to Predict COVID-19 Progression.

Coronavirus disease 2019 (COVID-19) is associated with immune dysregulation and cytokine storm. Exploring the immune-inflammatory characteristics of COVID-19 patients is essential to reveal pathogenesis and predict progression. In this study, COVID-19 patients showed decreased CD3+, CD4+, and CD8+ T cells but increased neutrophils in circulation, exhibiting upregulated neutrophil-to-lymphocyte and neutrophil-to-CD8+ T cell ratio. IL-6, TNF-α, IL-1ß, IL-18, IL-12/IL-23p40, IL-10, Tim-3, IL-8, neutrophil extracellular trap-related proteinase 3, and S100A8/A9 were elevated, whereas IFN-γ and C-type lectin domain family 9 member A (clec9A) were decreased in COVID-19 patients compared with healthy controls. When compared with influenza patients, the expressions of TNF-α, IL-18, IL-12/IL-23p40, IL-8, S100A8/A9 and Tim-3 were significantly increased in critical COVID-19 patients, and carcinoembryonic Ag, IL-8, and S100A8/A9 could serve as clinically available hematologic indexes for identifying COVID-19 from influenza. Moreover, IL-6, IL-8, IL-1ß, TNF-α, proteinase 3, and S100A8/A9 were increased in bronchoalveolar lavage fluid of severe/critical patients compared with moderate patients, despite decreased CD4+ T cells, CD8+ T cells, B cells, and NK cells. Interestingly, bronchoalveolar IL-6, carcinoembryonic Ag, IL-8, S100A8/A9, and proteinase 3 were found to be predictive of COVID-19 severity and may serve as potential biomarkers for predicting COVID-19 progression and potential targets in therapeutic intervention of COVID-19.

Changes in inflammatory factors in the Brown Norway rat model of food allergy.

BACKGROUND: The role of serum S100A8/A9 in intestinal inflammation has been confirmed, and its role in food allergy is currently being investigated. OBJECTIVE: To explore the levels of S100A8/A9 and inflammatory factors, including Toll-like receptors 4 (TLR4), Nuclear transcription factors (NF-κB) and Tumor necrosis factor α (TNF-α), in mild food allergies. METHODS: Eighty 3-week-old male Brown Norway rats were used. Forty rats were randomly assigned to the ovalbumin-sensitized experimental group, while 40 rats were assigned to the normal saline sham-sensitized control group. Body weight and length and the levels of serum ovalbumin-specific IgE (OVA-IgE), histamine, Th1-associated and Th2-associated factors, S100A8/A9 and inflammation-associated cytokines were compared. RESULTS: Through the evaluation of OVA-IgE level and Th1/Th2 balance in the experimental group, a successful IgE-mediated food allergy model was constructed. Compared with the control group, the experimental group had higher serum S100A8/A9 levels on days 21, 28, 35 and 42 (all P < 0.05); higher TLR4 levels on days 28, 35 and 42 (all P < 0.05); higher TNF-α levels on days 28, 35 and 42 (all P < 0.05); higher NF-κB levels on days 35 and 42 (all P < 0.05); and higher IL-1ß and IL-6 levels on days 7 to 42 (all P < 0.05). Moreover, positive correlations were found between the serum levels of S100A8/A9 and inflammation-associated cytokines [TNF-α: r = 0.378, P = 0.039; IL-1ß: r = 0.679, P = 0.000; IL-6: r = 0.590, P = 0.001]. CONCLUSION: S100A8/A9 and inflammatory-related factors, including TLR4, NF-κB, TNF-α, IL-6 and IL-1ß, is closely related to food allergies. Moreover, immune and inflammatory factors interact with each other in food allergies, which may provide insight into food allergy causes and treatments.

The heterodimer S100A8/A9 is a potent therapeutic target for idiopathic pulmonary fibrosis.

In patients with interstitial pneumonia, pulmonary fibrosis is an irreversible condition that can cause respiratory failure. Novel treatments for pulmonary fibrosis are necessary. Inflammation is thought to activate lung fibroblasts, resulting in pulmonary fibrosis. Of the known inflammatory molecules, we have focused on S100A8/A9 from the onset of inflammation to the subsequent progression of inflammation. Our findings confirmed the high expression of S100A8/A9 in specimens from patients with pulmonary fibrosis. An active role of S100A8/A9 was demonstrated not only in the proliferation of fibroblasts but also in the fibroblasts' differentiation to myofibroblasts (the active form of fibroblasts). S100A8/A9 also forced fibroblasts to upregulate the production of collagen. These effects were induced via the receptor of S100A8/A9, i.e., the receptor for advanced glycation end products (RAGE), on fibroblasts. The anti-S100A8/A9 neutralizing antibody inhibited the effects of S100A8/A9 on fibroblasts and suppressed the progression of fibrosis in bleomycin (BLM)-induced pulmonary fibrosis mouse model. Our findings strongly suggest a crucial role of S100A8/A9 in pulmonary fibrosis and the usefulness of S100A8/A9-targeting therapy for fibrosis interstitial pneumonia. HIGHLIGHTS: S100A8/A9 level is highly upregulated in the IPF patients' lungs as well as the blood. S100A8/A9 promotes not only the growth of fibroblasts but also differentiation to myofibroblasts. The cell surface RAGE acts as a crucial receptor to the extracellular S100A8/A9 in fibroblasts. The anti-S100A8/A9 antibody effectively suppresses the progression of IPF in a mouse model. In idiopathic pulmonary fibrosis (IPF), S100A8/A9, a heterodimer composed of S100A8 and S100A9 proteins, plays a crucial role in the onset of inflammation and the subsequent formation of a feed-forward inflammatory loop that promotes fibrosis. (1) The local, pronounced increase in S100A8/A9 in the injured inflammatory lung region-which is provided mainly by the activated neutrophils and macrophages-exerts strong inflammatory signals accompanied by dozens of inflammatory soluble factors including cytokines, chemokines, and growth factors that further act to produce and secrete S100A8/A9, eventually making a sustainable inflammatory circuit that supplies an indefinite presence of S100A8/A9 in the extracellular space with a mal-increased level. (2) The elevated S100A8/A9 compels fibroblasts to activate through receptor for advanced glycation end products (RAGE), one of the major S100A8/A9 receptors, resulting in the activation of NFκB, leading to fibroblast mal-events (e.g., elevated cell proliferation and transdifferentiation to myofibroblasts) that actively produce not only inflammatory cytokines but also collagen matrices. (3) Finally, the S100A8/A9-derived activation of lung fibroblasts under a chronic inflammation state leads to fibrosis events and constantly worsens fibrosis in the lung. Taken together, these findings suggest that the extracellular S100A8/A9 heterodimer protein is a novel mainstay soluble factor for IPF that exerts many functions as described above (1-3). Against this background, we herein applied the developed S100A8/A9 neutralizing antibody to prevent IPF. The IPF imitating lung fibrosis in an IPF mouse model was effectively blocked by treatment with the antibody, leading to enhanced survival. The developed S100A8/A9 antibody, as an innovative novel biologic, may help shed light on the difficulties encountered with IPF therapy in clinical settings.

Phenotype of Innate Immune Cells in Uveitis Associated with Axial Spondyloarthritis- and Juvenile Idiopathic Arthritis-associated Uveitis.

Purpose: To analyze circulating immune cells in patients with anterior uveitis (AU) associated to axial spondyloarthritis (SpA), or juvenile idiopathic arthritis (JIA).Methods: Venous blood samples were collected from healthy controls (n = 16), and either SpA (n = 19) or JIA (n = 23) patients with associated anterior uveitis (AU) during active flare, or after ≥3 months of inactivity. Frequencies of CD56+, MHC-I+, and S100A9+ monocytes, CCR7+ dendritic cells, CD56+dim natural killer (NK) cells and CD3+CD56bright T-cells were analyzed via flow cytometry. Serum S100A8/A9 levels were determined via ELISA.Results: SpA patients showed a reduced frequency of CD56+dim NK cells during uveitis activity, a constitutively activated monocyte phenotype, and elevated S100A8/A9 serum levels. In contrast, JIAU patients showed elevated frequencies of CD56+ monocytes and CCR7+ DC.Conclusion: Phenotype of peripheral immune cells differ between patients, probably contributing to different courses of acute onset AU in SpA and insidious onset AU in JIAU patients.Abbreviations: AU: anterior uveitis, AR: arthritis, JIA: juvenile idiopathic arthritis, SpA: axial spondyloarthritis.

Biomarkers of platelet activation and cardiovascular risk in the DAPT trial.

Prolonged use of dual antiplatelet therapy (DAPT) post-percutaneous coronary intervention (PCI) has been shown to reduce the risk of major adverse cardiovascular events (MACE), but with increased bleeding. It remains unknown whether biomarkers of platelet activation may be useful for identifying patients at increased risk of MACE. The DAPT study was a randomized trial of 12 versus 30 months of DAPT in patients who underwent PCI. Serum biomarkers [myeloid-related protein (MRP)-8/14, P-selectin, soluble CD-40 ligand (sCD40L)] were assessed in 1399 patients early post-PCI. On-treatment platelet reactivity index (PRI) using VASP phosphorylation was assessed in 443 patients randomized to continued DAPT at 1 year. MACE was defined as CV death, MI, or ischemic stroke. Multivariable models were adjusted for baseline characteristics, index event, and stent type. A stepwise increase in the risk of MACE was observed with increasing tertiles of both MRP-8/14 and P-selectin (p-trend = 0.04 for both). After multivariable adjustment, the adjusted HR (95% CI) for MACE in patients in the top tertile was 1.94 (1.14-3.30) for MRP-8/14 and 1.62 (0.99-2.64) for P-selectin. In contrast, baseline sCD40L was not associated with CV risk. Among patients randomized to continued DAPT, higher on-treatment platelet reactivity was not significantly associated with risk of MACE (p-trend = 0.32; adj-HR T3 vs. T1 1.54, 95% CI 0.20-12.18) or bleeding (P-trend = 0.17; adj-HR 0.25, 95% CI 0.05-1.21). MRP-8/14 and soluble P-selectin may be useful for identifying patients at increased risk of MACE after PCI. The utility of on-treatment platelet function testing requires further study.Clinical Trial Registration https://www.clinicaltrials.gov . Unique identifier NCT00977938.

Neutrophil calprotectin identifies severe pulmonary disease in COVID-19.

Severe cases of coronavirus disease 2019 (COVID-19) are regularly complicated by respiratory failure. Although it has been suggested that elevated levels of blood neutrophils associate with worsening oxygenation in COVID-19, it is unknown whether neutrophils are drivers of the thrombo-inflammatory storm or simple bystanders. To better understand the potential role of neutrophils in COVID-19, we measured levels of the neutrophil activation marker S100A8/A9 (calprotectin) in hospitalized patients and determined its relationship to severity of illness and respiratory status. Patients with COVID-19 (n = 172) had markedly elevated levels of calprotectin in their blood. Calprotectin tracked with other acute phase reactants including C-reactive protein, ferritin, lactate dehydrogenase, and absolute neutrophil count, but was superior in identifying patients requiring mechanical ventilation. In longitudinal samples, calprotectin rose as oxygenation worsened. When tested on day 1 or 2 of hospitalization (n = 94 patients), calprotectin levels were significantly higher in patients who progressed to severe COVID-19 requiring mechanical ventilation (8039 ± 7031 ng/ml, n = 32) as compared to those who remained free of intubation (3365 ± 3146, P < 0.0001). In summary, serum calprotectin levels track closely with current and future COVID-19 severity, implicating neutrophils as potential perpetuators of inflammation and respiratory compromise in COVID-19.

Plasma levels of S100A8/A9, histone/DNA complexes, and cell-free DNA predict adverse outcomes of immune thrombotic thrombocytopenic purpura.

BACKGROUND: Immune thrombotic thrombocytopenic purpura (iTTP) is a life-threatening blood disorder, primarily resulting from autoantibodies against ADAMTS13. Infection or inflammation often precedes acute iTTP. However, the association of inflammation and inflammatory mediators with disease severity and outcome of acute iTTP is not fully assessed. OBJECTIVES: Here, we determined plasma levels of S100A8/A9, histone/DNA complexes, citrullinated histone H3 (CitH3), and cell-free DNA (cfDNA) in a cohort of 108 acute episodes from 94 unique iTTP patients and healthy controls, and assessed the association of each of these biomarkers with the disease severity and mortality. RESULTS: All acute iTTP patients had significantly increased plasma levels of S100A8/A9 (median 84.8, interquartile range [IQR] 31.2-157.4 µg/mL), histone/DNA complexes (median 55.7, IQR 35.8-130.8 U/mL), CitH3 (median 3.8, IQR 2.2-6.4 ng/mL), and cfDNA (median 937.7, IQR 781.3-1420.0 ng/mL) on the admission blood samples when compared with healthy controls. An increased plasma level of S100A8/A9, histone/DNA complex and cfDNA was associated with organ damage, coagulopathy, and mortality in iTTP. After being adjusted for age and history of hypertension, Cox proportional hazard regression analysis demonstrated that a hazard ratio (95% confidence interval) for an elevated plasma level of S100A8/A9, histone/DNA complexes, and cfDNA was 11.5 (1.4-90.9) (P = .021), 10.3 (2.7-38.5) (P = .001), and 12.8 (3.9-42.0) (P = .014), respectively. CONCLUSION: These results indicate that inflammation or plasma inflammatory mediators such as S100A8/A9 or NETosis markers such as histone/DNA complexes and cfDNA may play a role in pathogenesis of iTTP, which may help stratify patients with a high risk of death during acute iTTP episodes.