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Neutrophil Extracellular Traps (NETs) are implicated in the development of auto-immunity in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) through the externalization of intracellular neoepitopes e.g., dsDNA and nuclear proteins in SLE and citrullinated peptides in RA. The aim of this work was to use quantitative proteomics to identify and measure NET proteins produced by neutrophils from healthy controls, and from patients with RA and SLE to determine if NETs can be differentially-generated to expose different sets of neoepitopes. Ultra-pure neutrophils (>99%) from healthy individuals ( = 3) and patients with RA or SLE ( = 6 each) were incubated ± PMA (50 nM, PKC super-activator) or A23187 (3.8 μM, calcium ionophore) for 4 h. NETs were liberated by nuclease digestion and concentrated onto Strataclean beads prior to on-bead digestion with trypsin. Data-dependent LC-MS/MS analyses were conducted on a QExactive HF quadrupole-Orbitrap mass spectrometer, and label-free protein quantification was carried out using Progenesis QI. PMA-induced NETs were decorated with annexins, azurocidin and histone H3, whereas A23187-induced NETs were decorated with granule proteins including CAMP/LL37, CRISP3, lipocalin and MMP8, histones H1.0, H1.4, and H1.5, interleukin-8, protein-arginine deiminase-4 (PADI4), and α-enolase. Four proteins were significantly different between PMA-NETs from RA and SLE neutrophils ( < 0.05): RNASE2 was higher in RA, whereas MPO, leukocyte elastase inhibitor and thymidine phosphorylase were higher in SLE. For A23187-NETs, six NET proteins were higher in RA ( < 0.05), including CAMP/LL37, CRISP3, interleukin-8, MMP8; Thirteen proteins were higher in SLE, including histones H1.0, H2B, and H4. This work provides the first, direct comparison of NOX2-dependent (PMA) and NOX2-independent (A23187) NETs using quantitative proteomics, and the first direct comparison of RA and SLE NETs using quantitative proteomics. We show that it is the nature of the stimulant rather than neutrophil physiology that determines NET protein profiles in disease, since stimulation of NETosis in either a NOX2-dependent or a NOX2-independent manner generates broadly similar NET proteins irrespective of the disease background. We also use our proteomics pipeline to identify an extensive range of post-translationally modified proteins in RA and SLE, including histones and granule proteins, many of which are known targets of auto-antibodies in each disease.

Psychosocial stress has profound effects on the body, including the immune system and the brain 1 , 2 . Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD) 3 , the underlying mechanisms are not well understood. Here we show that expression of a circulating myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is increased in the serum of humans with MDD as well as in stress-susceptible mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), as well as altered social behaviour. Using a combination of mass cytometry and single-cell RNA sequencing, we performed high-dimensional phenotyping of immune cells in circulation and in the brain and demonstrate that peripheral monocytes are strongly affected by stress. In stress-susceptible mice, both circulating monocytes and monocytes that traffic to the brain showed increased Mmp8 expression following chronic social defeat stress. We further demonstrate that circulating MMP8 directly infiltrates the NAc parenchyma and controls the ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behaviour and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a mechanism by which peripheral immune factors can affect central nervous system function and behaviour in the context of stress. Targeting specific peripheral immune cell-derived matrix metalloproteinases could constitute novel therapeutic targets for stress-related neuropsychiatric disorders. Serum MMP8 is increased in stress-susceptible mice following chronic stress and leads to brain structure and behavioural changes in mice.

Idiopathic pulmonary fibrosis (IPF) is a restrictive lung disease that is associated with high morbidity and mortality. Current medical therapies are not fully effective at limiting mortality in patients with IPF, and new therapies are urgently needed. Matrix metalloproteinases (MMPs) are proteinases that, together, can degrade all components of the extracellular matrix and numerous nonmatrix proteins. MMPs and their inhibitors, tissue inhibitors of MMPs (TIMPs), have been implicated in the pathogenesis of IPF based upon the results of clinical studies reporting elevated levels of MMPs (including MMP-1, MMP-7, MMP-8, and MMP-9) in IPF blood and/or lung samples. Surprisingly, studies of gene-targeted mice in murine models of pulmonary fibrosis (PF) have demonstrated that most MMPs promote (rather than inhibit) the development of PF and have identified diverse mechanisms involved. These mechanisms include MMPs: (1) promoting epithelial-to-mesenchymal transition (MMP-3 and MMP-7); (2) increasing lung levels or activity of profibrotic mediators or reducing lung levels of antifibrotic mediators (MMP-3, MMP-7, and MMP-8); (3) promoting abnormal epithelial cell migration and other aberrant repair processes (MMP-3 and MMP-9); (4) inducing the switching of lung macrophage phenotypes from M1 to M2 types (MMP-10 and MMP-28); and (5) promoting fibrocyte migration (MMP-8). Two MMPs, MMP-13 and MMP-19, have antifibrotic activities in murine models of PF, and two MMPs, MMP-1 and MMP-10, have the potential to limit fibrotic responses to injury. Herein, we review what is known about the contributions of MMPs and TIMPs to the pathogenesis of IPF and discuss their potential as therapeutic targets for IPF.

Colorectal cancer (CRC) is the third and second cancer for incidence and mortality worldwide, respectively, and is becoming prevalent in developing countries. Most CRCs derive from polyps, especially adenomatous polyps, which can gradually transform into CRC. The family of Matrix Metalloproteinases (MMPs) plays a critical role in the initiation and progression of CRC. Prominent MMPs, including MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, MMP-14, and MMP-21, have been detected in CRC patients, and the expression of most of them correlates with a poor prognosis. Moreover, many studies have explored the inhibition of MMPs and targeted therapy for CRC, but there is not enough information about the role of MMPs in polyp malignancy. In this review, we discuss the role of MMPs in colorectal cancer and its pathogenesis

Skin aging is a complex process, and alterations in human skin due to aging have distinct characteristic as compared to other organs. The aging of dermal cells and the biological mechanisms involved in this process are key areas to understand skin aging. A large number of biological mechanisms, such as decreasing of protein synthesis of extracellular matrix or increasing of degradation, are known to be altered through skin aging. However, environmental influence can accelerate this characteristic phenotype. In this study, we analyzed primary human dermal fibroblasts in three different in-vitro aging models-UVB irradiation and accelerated proliferation of human dermal fibroblasts from young donors as well as from elderly donors-for the gene expression of COL1A1, COL1A2, COL3A1, COL4A1, COL7A1, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12, MMP13, MMP14, TIMP1, TIMP2, TIMP3, TIMP4, IL1B, IL1A, IL6, IL8, IL10, PTGS2, TP53, CASP3, LMNA, SIRT1. We compared the gene expression levels with young control. Furthermore, the behavior of skin fibroblasts was also evaluated using cell growth rate. The findings reveal that the gene expression levels in skin fibroblasts was altered in the process of aging in all three in-vitro aging models, and the cell growth rate was reduced, suggesting that these methods can be employed to understand skin aging mechanisms as well as drug discovery screening method.

Periodontitis is a complex condition. Left untreated, it leads to tooth loss and the need for prosthetic treatment. The incidence of periodontitis is steadily increasing, so new methods are being sought to aid in the diagnosis of the disease. Among the methods postulated is the determination of concentrations of bioactive compounds which include extracellular matrix metalloproteinases (MMPs). These enzymes are present in various structural elements of the stomatognathic system. The most promising enzyme of this group appears to be metalloproteinase 8 (MMP-8). MMP-8 assays are performed in gingival fluid or saliva, and MMP-8 levels have been shown to be higher in patients with periodontitis compared to healthy subjects and correlated with some clinical parameters of the condition and the severity of the disease. In addition, the preliminary usefulness of this enzyme in evaluating the effectiveness of periodontal treatment and doxycycline therapy has been demonstrated. Determination of the active form of MMP-8 (aMMP-8) in oral rinse fluid using off-the-shelf assays shows the highest potential. Despite reports about aMMP-8 and promising data on the role of MMP-8 in periodontal diagnosis, a clear determination of the usefulness of this enzyme requires further research.

In rheumatoid arthritis(RA) pathogenesis, activated RA fibroblast-like synoviocytes (RA-FLSs) exhibit similar proliferative features as tumor cells and subsequent erosion to cartilage will eventually lead to joint destruction. Therefore, it is imperative to search for compounds, which can effectively inhibit the abnormal activation of RA-FLSs, and retard RA progression.3'3-Diindolylmethane (DIM), the major product of the acid-catalyzed oligomerization of indole-3-carbinol from cruciferous vegetables, has been reported to be functionally relevant to inhibition of migration, invasion and carcinogenesis in some solid tumors. In this study, we explored the anti-proliferation, anti-metastasis and anti-inflammation effects of DIM on RA-FLSs as well as the underlying molecular mechanisms. To do this, primary RA-FLSs were isolated from RA patients and an animal model. Cell proliferation, migration and invasion were measured using CCK-8, scratch, and Transwell assays, respectively. The effects of DIM on Matrix metalloproteinases (MMPs) and some inflammatory factors mRNA and key molecules such as some inflammatory factors and those involved in aberrantly-activated signaling pathway in response to tumor necrosis factor α(TNF-α), a typical characteristic mediator in RA-FLS, were quantitatively measured by real-time PCR and western blotting. Moreover, the effect of DIM on adjuvant induced arthritis(AIA) models was evaluated with C57BL/6 mice . The results showed that DIM inhibited proliferation, migration and invasion of RA-FLS . Meanwhile, DIM dramatically suppressed TNF-α-induced increases in the mRNA levels of , and ; as well as the proinflammatory factors , and β. Mechanistic studies revealed that DIM is able to suppress phosphorylated activation not only of p38, JNK in MAPK pathway but of AKT, mTOR and downstream molecules in the AKT/mTOR pathway. Moreover, DIM treatment decreased expression levels of proinflammatory cytokines in the serum and alleviated arthritis severity in the knee joints of AIA mice. Taken together, our findings demonstrate that DIM could inhibit proliferation, migration and invasion of RA-FLSs and reduce proinflammatory factors induced by TNF-α by blocking MAPK and AKT/mTOR pathway and prevent inflammation and knee joint destruction , which suggests that DIM might have therapeutic potential for RA.

This study analyzed the association of functionally significant SNPs of matrix metalloproteinase ( MMP ) genes in the development of peptic ulcer disease (PUD) in Caucasians from Central Russia. Ten SNPs of the MMP-1, MMP-2, MMP-3, MMP-8 , and MMP-9 genes were analyzed for association with PUD in a cohort of 798 patients with PUD (including 404 H. pylori -positive and 394 H. pylori -negative) and 347 H. pylori -negative controls using logistic regression and assuming the additive, recessive, and dominant genetic models. The variants of MMP-1 , MMP-2 , MMP-3 , and MMP-8 did not manifest any significant associations with the diseases. Five SNPs of the MMP-9 gene demonstrated such association. Allele G of the rs17576 MMP-9 locus conferred a higher risk for PUD (OR adj  = 1.31, p perm  = 0.016), haplotype AACG of loci rs17576-rs3787268-rs2250889-rs17577 of the MMP-9 gene decreased risk for PUD (OR adj  = 0.17, p perm  = 0.003). Also, allele C of rs3918249, allele G of rs17576 and haplotype CG of rs3918249-rs17576 of the MMP-9 gene increased risk for H. pylori -positive PUD (OR adj  = 1.82, p perm  = 0.002; OR adj  = 1.53–1.95 p perm  = 0.001–0.013 and OR adj  = 1.49 p perm  = 0.009 respectively). The above loci and 50 linked to them possess significant regulatory effects and may affect the alternative splicing of four genes and the expression of 17 genes in various organs and tissues related to the PUD pathogenesis.

Cetuximab therapy is the major treatment for colorectal cancer (CRC), but drug resistance limits its effectiveness. Here, we perform longitudinal and deep proteomic profiling of 641 plasma samples originated from 147 CRC patients (CRCs) undergoing cetuximab therapy with multi-course treatment, and 90 healthy controls (HCs). COL12A1, THBS2, S100A8, and S100A9 are screened as potential proteins to distinguish CRCs from HCs both in plasma and tissue validation cohorts. We identify the potential biomarkers (RRAS2, MMP8, FBLN1, RPTOR, and IMPDH2) for the initial response prediction. In a longitudinal setting, we identify two clusters with distinct fluctuations and construct the model with high accuracy to predict the longitudinal response, further validated in the independent cohort. This study reveals the heterogeneity of different biomarkers for tumor diagnosis, the initial and longitudinal response prediction respectively in the first course and multi-course cetuximab treatment, may ultimately be useful in monitoring and intervention strategies for CRC. Resistance to cetuximab is a common feature of colorectal cancer progression. Here, the authors utilize longitudinal proteomic profiling of 147 colorectal cancer patients and find potential biomarkers which can predict treatment response.

Oral health is important not only due to the diseases emerging in the oral cavity but also due to the direct relation to systemic health. Thus, early and accurate characterization of the oral health status is of utmost importance. There are several salivary biomarkers as candidates for gingivitis and periodontitis, which are major oral health threats, affecting the gums. These need to be verified and validated for their potential use as differentiators of health, gingivitis and periodontitis status, before they are translated to chair-side for diagnostics and personalized monitoring. We aimed to measure 10 candidates using high sensitivity ELISAs in a well-controlled cohort of 127 individuals from three groups: periodontitis (60), gingivitis (31) and healthy (36). The statistical approaches included univariate statistical tests, receiver operating characteristic curves (ROC) with the corresponding Area Under the Curve (AUC) and Classification and Regression Tree (CART) analysis. The main outcomes were that the combination of multiple biomarker assays, rather than the use of single ones, can offer a predictive accuracy of > 90% for gingivitis versus health groups; and 100% for periodontitis versus health and periodontitis versus gingivitis groups. Furthermore, ratios of biomarkers MMP-8, MMP-9 and TIMP-1 were also proven to be powerful differentiating values compared to the single biomarkers.