Myeloid-related Protein Research Articles

Myeloid-Related Protein S100A9 Induces Cellular Differentiation in Acute Myeloid Leukemia through TLR2 and TLR4 Receptors

The myeloid-related proteins S100A8 (MRP8) and S100A9 (MRP14) are endogenous alarmins abundantly and constitutively expressed by myeloid cells (neutrophils, monocytes and immature myeloid cells). S100A8 and S100A9 proteins exist as homodimers but also associate to form the heterodimer calprotectin (S100A8/A9) and are up-regulated in several inflammatory diseases and human cancers. In patients with acute myeloid leukemia (AML), the concentration of S100A8/A9 in serum is elevated and the expression of S100A8 correlates with poor prognosis. However, the role of these proteins in hematologic malignancies is largely unknown.Here, we studied the roles of S100A8 and S100A9 in a mouse model ofAML induced by overexpression of Hoxa9 and Meis1 (H9M1). As observed in human, mice developing AML have a substantial increase in S100A8/A9 their serum (5.0µg/ml ± 1.0µg/mL vs 0.5µg/mL ± 0.1µg/mL for the control, p<0.0001). Using S100A8KO and S100A9KO mice, we demonstrated that S100A8/A9 proteins found in the sera are secreted by leukemic cells and not by the micro-environment.To investigate if secreted S100A8 and S100A9 proteins play a role in leukemogenesis, H9M1 AML were transplanted to secondary recipients were treated intraperitoneally (i.p) with anti-S100A8 or anti-S100A9 antibodies. Blocking S100A8 led to a marked delay in leukemia progression and significantly extended survival compared to control immunoglobulins (IgG) (31 days vs 39.5 days, p=0.010) with an increase of the CD11b+ Gr-1+ double positive population (78.8%±1.4 vs 90.1%±3, p=0.038). In contrast, no differences in overall survival were observed between control IgG and anti-S100A9 treated mice. In addition, we demonstrate that anti-S100A8 treatments reduced AML cell proliferation through the G0/G1 cell cycle arrest. Thus, blocking S100A8 reduces leukemogenesis and induced leukemic blast maturation in AML.To further investigate the roles of S100A8 and S100A9 in AML, we treated secondary H9M1 mice with S100A8 or S100A9 proteins i.p three times per week. Interestingly, injection of S100A8 had no effect on AML latency, but S100A9 treatment resulted in significant delays of leukemia symptoms suggesting an anti-leukemic activity (32 days vs 41 days, p=0.0045). The extent of increased survival induced by S100A9 treatment was similar to standard induction chemotherapy using combination of doxorubicin and cytarabine. Furthermore, S100A9 treatment led to significant cell cycle arrest and an increase of mature cells marker CD11band Gr-1 in bone marrow (76.6%± 1.0% vs 94.8 ± 1.2%, p<0.0001). Analysis of leukemic cells morphology confirmed that S100A9 modulates AML cells maturation.Since injection of the S100A9 protein and anti-S100A8 antibody had similar effect on AML progression and cellular differentiation, we postulated that cell differentiation is regulated by the balance between S100A9 and S100A8. To test the hypothesis, cells were cultured in vitro in presence of different ratio of S100A9 on S100A8. At high ratio (S100A9>S100A8), the percentage of CD11b+ Gr-1+ was increased compared to the control suggesting that leukemic cells underwent differentiation. Nevertheless, the augmentation of S100A8 level prevented the increases of CD11b+ Gr-1+ mediated by S100A9.To test the ability of S100A9 protein to promote terminal cell differentiation of human leukemia, human cord blood (CB) CD34+ cells were transduced with retrovirus expressing the oncogene MLL-AF9. In vitro, S100A9 induced a 10-fold up-regulation of CD14 expression in MLL-AF9 cells. More importantly, the increase of CD14 was associated with morphological changes typical of terminal differentiation into monocytes and then macrophages. To determine the receptor(s) involved in regulation of cellular differentiation induced by S100A9 in human AML, we followed CD14 expression in presence of anti-TLR neutralizing antibodies. Blockage of TLR4 and TLR2 prevented the differentiation of human leukemic cells mediated by S100A9.Taken together, we show that increasing the S100A9/S100A8 ratio in murine AML, either by anti-S100A8 antibody or recombinant S100A9 protein, prolong the survival of secondary mice in vivo by inducing differentiation on AML cells. We corroborated these data in human MLL-AF9 cells in vitro and show that S100A9 protein induces terminal differentiation through TLR receptors which could represent a new therapeutic target to explore. DisclosuresNo relevant conflicts of interest to declare.

Gout Is a Chronic Inflammatory Disease in Which High Levels of Interleukin-8 (CXCL8), Myeloid-Related Protein 8/Myeloid-Related Protein 14 Complex, and an Altered Proteome Are Associated With Diabetes Mellitus and Cardiovascular Disease.

The frequent association of gout with metabolic syndrome and cardiovascular disease (CVD) suggests that it has a systemic component. Our objective was to study whether circulating proinflammatory cytokines are associated with comorbidities in gout patients. We studied 330 gout patients from 3 independent cohorts and compared them with 144 healthy individuals and 276 disease controls. We measured circulating levels of interleukin-8 (IL-8)/CXCL8, IL-1β, IL-6, IL-10, IL-12, and tumor necrosis factor, after which we performed proteome-wide analysis in a selection of samples to identify proteins that were possibly prognostic for the development of comorbidities. Replication analysis was performed specifically for myeloid-related protein 8 (MRP-8)/MRP-14 complex. Compared to healthy controls and disease control patients, patients with gouty arthritis (n = 48) had significantly higher mean levels of CXCL8 (P < 0.001), while other cytokines were almost undetectable. Similarly, patients with intercritical gout showed high levels of CXCL8. CXCL8 was independently associated with diabetes mellitus in patients with intercritical gout (P < 0.0001). Proteome-wide analysis in gouty arthritis (n = 18) and intercritical gout (n = 39) revealed MRP-8 and MRP-14 as the proteins with the greatest differential expression between low and high levels of CXCL8 and also showed a positive correlation of MRP8/MRP14 complex with CXCL8 levels (R(2) = 0.49, P < 0.001). These findings were replicated in an independent cohort. The proteome of gout patients with high levels of CXCL8 was associated with diabetes mellitus (odds ratio 16.5 [95% confidence interval 2.8-96.6]) and CVD (odds ratio 3.9 [95% confidence interval 1.0-15.3]). Circulating levels of CXCL8 are increased during both the acute and intercritical phases of gout, and they coincide with a specific circulating proteome that is associated with risk of diabetes mellitus and CVD. Further research focused on the roles of CXCL8 and MRP8/MRP14 complex in patients with gout is warranted.

Sleep Fragmentation and Biomarkers in Juvenile Idiopathic Arthritis.

(1) To compare sleep (nighttime sleep duration and sleep efficiency) and sleep fragmentation (movement and fragmentation index), as measured by actigraphy, and symptoms (pain and fatigue) in 8- to 14-year-old children with polyarticular and extended oligoarticular juvenile idiopathic arthritis (JIA) and (2) to examine the associations between sleep fragmentation (movement and fragmentation index) and the calcium-binding protein biomarkers S100A12 and myeloid-related protein (MRP8/14). Participants included 40 children with extended oligoarticular (n = 15) or polyarticular (n = 25) JIA and their parents. Serum protein samples were obtained during routine rheumatology clinic visits. Children completed the PedsQL Multidimensional Fatigue Scale and daily pain and sleep diaries and wore actigraphy monitors for 9 consecutive days. Parents completed the Children's Sleep Habits Questionnaire (CSHQ). Of the 40 children, 68% scored above the CSHQ clinical cutoff score for sleep disturbances. Mean nighttime sleep duration was 7.5 hr, and mean sleep efficiency was 85.3%. Group differences were not found for nighttime sleep duration, sleep efficiency, movement and fragmentation index, or S100A12 and MRP8/14 protein concentrations. In a stepwise regression, medications, joint count, and movement and fragmentation index explained 21% of the variance in MRP8/14 concentration. Decreased nighttime sleep duration, poor sleep efficiency, and fragmented sleep were observed in our sample, regardless of JIA category. Sleep fragmentation was a significant predictor of MRP8/14 protein concentration. Additional research is needed to understand the interrelations among sleep fragmentation, effects of medication, and S100A12 and MRP8/14 protein biomarkers in JIA.

Interaction of titanium, zirconia and lithium disilicate with peri-implant soft tissue: study protocol for a randomized controlled trial.

BackgroundAgainst the background of increasing use of dental implants, and thus an increasing prevalence of implant-associated complications, a deeper understanding of the biomolecular mechanisms in the peri-implant tissue is needed. Peri-implant soft tissue is in direct contact with transmucosal dental implant abutments. The aim of this trial is to distinguish the biomolecular and histological interactions of various dental abutment materials with peri-implant soft tissue.Methods/DesignThe study is designed as a prospective, randomized, investigator-initiated clinical pilot trial with blinded assessment. We will ultimately include 24 eligible patients who opt for implant treatment to replace a single missing posterior tooth. Three months after implantation (submerged procedure), the study begins with the second-stage surgery. Each of the 24 patients will be given three different transmucosal abutments (zirconia, lithium disilicate, titanium) consecutively. The sequence in which the three materials are used is randomized. Peri-implant crevicular fluid is sampled weekly around the respective abutment for biomolecular analyses. After one month of wearing time, the stamping press from the second-stage surgery is used to gain a narrow gingival ring biopsy around the abutment for immunohistochemical analyses. The next abutment is then inserted. The same procedure is used for all three abutments. After sampling is completed, the patients will receive a definitive crown. The primary outcome measure of the trial is biomolecular detection of specific markers in the peri-implant crevicular fluid: matrix metalloproteinase 8, interleukin- 1β, polymorphonuclear elastase, and myeloid-related protein MRP8/14 (calprotectin). Secondary outcome measures include immunohistochemical analyses and clinical parameters.DiscussionThe study design will allow us to perform correlation analyses between the clinical indices with biomarkers’ expression in the interface of the transmucosal abutments and the peri-implant soft tissue. A deeper understanding of the three abutment materials’ interactions with peri-implant soft tissue will help us understand the formation mechanisms of implant-associated complications and then develop prevention strategies.Trial registrationThe trial is registered at the German Clinical Trial Register and the International Clinical Trials Registry Platform by the WHO under DRKS00006555 (Registered on 27 October 2014).

The diagnostic and prognostic value of MRP8/MRP14 in intrahepatic cholangiocarcinoma.

Myeloid-related protein 8 (MRP8) and 14 (MRP14) are abundantly expressed in several kinds of benign and malignant tumors. However, little is known about their clinicopathological significance in intrahepatic cholangiocarcinoma (ICC), biliary intraepithelial neoplasia (BilIN), intraductal papillary neoplasm of bile duct (IPNB), or inflammatory hepatic biliary ducts epithelium (IHBD). This study aimed to investigate the diagnostic and prognostic values of MRP8 and MRP14 as new biomarkers for ICC. We examined MRP8 and MRP14 expression levels by immunohistochemistry in IHBD (n = 15), BilIN (BilIN1 = 24, BilIN2 = 9, BilIN3 = 5), IPNB (n = 18) and ICC (n = 416). The differential diagnostic and prognosis values were also evaluated. The results showed that the ratio of tumor-infiltrating MRP8 and MRP14 positive immune cells, relative to biliary epithelial cells, was significantly increased in ICC tissues compared with nonmalignant tissues, including IHBD, BilIN1, BilIN2, BilIN3, and IPNB (P value < 0.05). In addition, over-expression levels of MRP8 and MRP14 were correlated with overall survival (OS) and time to recurrence (TTR) by univariate analysis; MRP8/MRP14 combination was an independent prognostic factor for OS and TTR. MRP8 and MRP14 expression might help to identify the benign bile duct diseases from ICC, as high expression of MRP8 and MRP14 suggests a poor prognosis after surgical resection.

Role of polymorphonucleates in the pathogenesis of systemic juvenile idiopathic arthritis and Still's disease: a proof of concept study

Background Systemic juvenile idiopathic arthritis (sJIA) and adult-onset Still9s disease (AOSD) are autoinflammatory disorders characterized by neutrophilia and abnormal innate immunity response [1]. It has been hypothesized a pathogenic role of neutrophils, because of patients neutrophilia, maybe related to the typical higher production of pro-inflammatory cytokines, like IL-1β, whose role in these disorders should explain the efficacy of IL-1 blockers [2, 3]. IL-1β is synthesized as inactive form and its activation is mediated by the NLRP3 inflammasome [4]. Increased release of this cytokine in the extracellular environment lead to a positive feedback loop that perpetuates and amplifies itself stimulation [5]. This mechanism as well as neutrophils9 activation state could be modified in sJIA and AOSD. Objectives Our aim was to verify possible differences between sJIA and Still9s patients compared to healthy donors, in term of PMNs responsiveness to the extracellular environment and activation state. Methods PMNs were obtained from heparinised venous blood of sJIA patients (n=6), AOSD patients (n=4) and healthy controls (HC, n=8). All patients9 samples were collected during stages of active or non-active disease, according to international disease activity criteria used for the assessment of each disease. After lipopolysaccharide (LPS) treatment, IL-1β content in PMNs supernatants was measured by ELISA assay. CD11b expression levels were measured by flow cytometry, together with intracellular ROS levels through the H 2 DCFDA ROS-indicator. Results In comparison with HC, sJIA PMNs showed an increased IL-1β secretion after LPS stimulation (p Conclusions Data from our proof of concepts study suggest a possible involvement of PMNs in the pathogenesis of sJIA and AOSD, since they seem more active respect healthy ones and more sensitive to pro-inflammatory stimuli. Although further studies are necessary to confirm and validate this hypothesis, the trend observed may have a potential role in the direction of future therapeutic studies. References Pay, S., et al., A multicenter study of patients with adult-onset Still9s disease compared with systemic juvenile idiopathic arthritis. Clin Rheumatol, 2006. 25(5): p. 639-44. Mellins, E.D., et al., Pathogenesis of systemic juvenile idiopathic arthritis: some answers, more questions. Nat Rev Rheumatol, 2011. 7(7): p. 416-26. Mavragani, C.P., et al., Adult-Onset Still9s Disease: From Pathophysiology to Targeted Therapies. Int J Inflam, 2012. 2012: p. 879020. Martinon, F., et al., The inflammasomes: guardians of the body. Annu Rev Immunol, 2009. 27: p. 229-65. Frosch, M., et al., The myeloid-related proteins 8 and 14 complex, a novel ligand of toll-like receptor 4, and interleukin-1beta form a positive feedback mechanism in systemic-onset juvenile idiopathic arthritis. Arthritis Rheum, 2009. 60(3): p. 883-91. Disclosure of Interest None declared

Myeloid-related protein 8 induces self-tolerance and cross-tolerance to bacterial infection via TLR4- and TLR2-mediated signal pathways.

Myeloid-related protein 8 (Mrp8) is the active component of Mrp8/14 protein complex released by phagocytes at the site of infection and stimulates inflammatory responses. However, it is unclear whether Mrp8 could induce self-tolerance and cross-tolerance to bacterial infection. Here we report that Mrp8 triggered TNF-α and IL-6 release via a Toll-like receptor 4 (TLR4)-dependent manner. Pre-stimulation of murine macrophages and human monocytes with Mrp8 induced self-tolerance to Mrp8 re-stimulation and cross-tolerance to lipopolysaccharide (LPS), bacterial lipoprotein (BLP), gram-negative and gram-positive bacterial challenges, with substantially attenuated TNF-α and IL-6 release. Moreover, Mrp8 tolerisation significantly reduced serum TNF-α and IL-6, increased polymorphonuclear neutrophil (PMN) recruitment and accelerated bacterial clearance, thus protecting mice against LPS-induced lethality and cecal ligation and puncture (CLP)-induced polymicrobial sepsis. In addition to TLR4, TLR2 also contributed to Mrp8-induced inflammatory response and tolerance. Down-regulation of phosphorylated p38 by Mrp8 pre-stimulation was predominantly responsible for the intracellular mechanism of Mrp8-induced tolerance. Thus, our findings of Mrp8-induced self-tolerance and cross-tolerance may provide a potential strategy for attenuating an overwhelming proinflammatory cascade and enhancing antimicrobial responses during microbial sepsis.

Gender-Related Differences in Patients Presenting with Suspected Acute Coronary Syndromes: Clinical Presentation, Biomarkers and Diagnosis

Objectives: Gender differences in patients presenting with suspected acute coronary syndromes (ACS) have not yet been fully characterized. The aim of this study was to assess gender-related disparities in clinical profiles, biomarkers and diagnoses of patients with suspected ACS. Methods: This single-centre, prospective cohort study included 377 consecutive patients presenting with suspected ACS to the emergency department. Suspected ACS was defined as a request for conventional troponin T (c-cTnT) measurements on clinical grounds. Results: Women were older than men (p = 0.004), and had a lower prevalence of known coronary artery and peripheral vascular disease (p < 0.05). c-cTnT was positive in 8% of female and in 14% of male patients (p = 0.16), TIMI risk score and cardiac biomarkers including c-cTnT, hs-cTnT, myoglobin, creatine kinase, N-terminal pro-brain natriuretic peptide, myeloid-related protein 8/14 and pregnancy-associated plasma protein A were lower in women (p < 0.05). Women were less frequently diagnosed with ACS (30 vs. 51%), and were not referred for urgent coronary angiography as often as men (p < 0.001). In multivariate analysis, female gender was associated with a lower referral for coronary angiography (HR 0.41, 95% CI 0.23-0.78, p = 0.006). Conclusions: In patients with suspected ACS, women presented with different biomarker profiles, and were less often diagnosed with ACS and referred to coronary angiography.

Elevated expressions of myeloid-related proteins-8 and -14 are danger biomarkers for lupus nephritis.

Myeloid-related proteins, MRP-8 and -14, which have been identified as molecules that mediate the danger signaling in innate immune response, are also known as the DAMPs (damage associated molecular pattern molecules). The proteins were found in infiltrating macrophages and neutrophils at inflammatory sites. Their expression was correlated with severe forms of glomerulonephritis. Therefore, this study examined whether or not MRP-8 and -14 can be used as biomarkers for identifying severely active lupus nephritis (LN). Total blood leukocyte samples and renal biopsy tissues from a prospective cohort of LN patients were used to determine mRNA and protein expression levels of MRP-8 and -14. The mRNA levels of MRP-8 and -14 in total blood leukocytes were significantly higher in active LN patients than quiescent LN patients and healthy controls. Moreover, the mRNA levels of MRP-8 and -14 in the total blood leukocytes and kidney tissues were significantly correlated with therapeutic response and the mRNA expression levels in the kidney were associated with an early loss of the kidney function. MRP-8 and -14 can be used as non-invasive prognostic biomarkers in patients with LN.

The role of interleukin-1 beta in the pathophysiology of Schnitzler's syndrome.

IntroductionSchnitzler’s syndrome (SchS) is a disabling autoinflammatory disorder, characterized by a chronic urticarial rash, an M-protein, arthralgia, and other signs of systemic inflammation. Anti-interleukin-1 (IL-1) beta antibodies are highly effective, but the pathophysiology is still largely unknown. Here we studied the effect of in-vivo IL-1 inhibition on serum markers of inflammation and cellular immune responses.MethodsEight patients with SchS received monthly subcutaneous (s.c.) injections with 150 mg canakinumab for six months. Blood was drawn for measurement of serum markers of inflammation (12 times per patient) and for functional and phenotypic analysis of both freshly isolated and toll-like receptor (TLR)-ligand-stimulated peripheral blood mononuclear cells (PBMCs) (five times per patient). All data were compared to results of healthy controls.ResultsIL-6 levels in serum and in lysates of freshly isolated PBMCs and serum myeloid-related protein (MRP8)/14 and S100A12 levels correlated with disease activity. In vitro, LPS stimulation resulted in higher IL-6 and IL-1 beta production in PBMCs from symptomatic SchS patients compared to healthy controls, whereas patient cells were relatively hyporesponsive to poly:IC and Pam3Cys. The mRNA microarray of PBMCs showed distinct transcriptomes for controls, symptomatic patients and anti-IL-1-treated patients. Numbers of T- and B-cell subsets as well as M-protein concentrations were not affected by IL-1 inhibition. Free light chain levels were elevated in 4 out of 8 patients.ConclusionsIn conclusion, patient PBMCs are hyperresponsive to LPS, and clinical efficacy of IL-1 beta inhibition in patients with SchS is associated with in-vivo and ex-vivo suppression of inflammation. Interestingly, patient PBMCs showed divergent responses to TLR2/6, TLR3 and TLR4 ligands. Our data underscore that IL-1 beta plays a pivotal role in SchS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0696-0) contains supplementary material, which is available to authorized users.

Diagnostic and prognostic value of myeloid-related protein complex 8/14 for sepsis

ObjectiveThe objective of this study is to evaluate the diagnostic and prognostic value of myeloid-related protein complex 8/14 (MRP8/14) in septic patients in intensive care units. MethodsFifty-three cases of healthy people, 72 cases of systemic inflammatory response syndrome patients, 74 cases of sepsis patients, 78 cases of severe sepsis patients, and 76 cases of septic shock patients were enrolled in our hospital. Serum MRP8/14 was detected by enzyme-linked immunosorbent assay, and Mortality in Emergency Department Sepsis (MEDS) score was calculated at enrollment. The receiver operating characteristic curve was used to analyze the diagnostic value of MRP8/14 for sepsis. The prognostic values of MRP8/14 were compared with MEDS score. A 28-day follow-up was performed for all patients. ResultsSerum MRP8/14 level of control group, systemic inflammatory response syndrome group, sepsis group, severe sepsis group, and septic shock group was (834.135 ± 251.662), (2343.213 ± 901.850), (3163.617 ± 912.821), (3941.326 ± 850.490), and (4131.326 ± 950.439) ng/mL, respectively. Myeloid-related protein complex 8/14 level increased with sepsis severity. The area under the receiver operating characteristic curves of MRP8/14 for sepsis was 0.901 (95% confidence interval, 0.852-0.942).The area under the receiver operating characteristic curves of MRP8/14 was greater than that of MEDS score in predicting 28-day mortality. Serum MRP8/14 level was significantly higher in nonsurvivors than survivors at 28 days' follow-up. In addition, the level of MRP8/14 was much higher in septic patients with acute kidney injury (AKI) than those without AKI. ConclusionsMyeloid-related protein complex 8/14 is valuable for diagnosis and prognostication of sepsis and septic patients with AKI in the intensive care unit.

Single amino acid charge switch defines clinically distinct proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1)–associated inflammatory diseases

Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 μg/mL) compared with those with PAPA syndrome (116 ± 74 μg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. Mutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.

The Effect of miR-132, miR-146a, and miR-155 on MRP8/TLR4-Induced Astrocyte-Related Inflammation.

Astrocyte activation, associated with the release of pro-inflammatory cytokines interleukin 1-β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α), is a hallmark of multiple brain diseases, including mesial temporal lobe epilepsy. In recent years, several microRNAs have emerged as important controllers of Toll-like receptor (TLR) signaling. In this study, we investigated the effect of miR-132, miR-146a, and miR-155 on myeloid-related protein-8 (MRP8) induced astrocyte-related inflammation. Using quantitative polymerase chain reaction (qPCR) and western blot, we found clear upregulation of TLR4 and downstream inflammatory cytokines, along with dysregulation of miR-132, miR-146a, and miR-155 in in vitro astrocytes after exposing them to different concentrations of MRP8. In addition, we focused on the effect of miR-132 on astrocyte-related inflammation induced by MRP8 via lentiviral infection then evaluated the expression of its possible target genes: acetylcholinesterase (AChE) and interleukin-1 receptor-associated kinase (IRAK4). Our results show that miR-132 is a negative feedback regulator of IL-1β and IL-6, but not TNF-α, by targeting IRAK4. Together, our findings demonstrate the novel role of TLR4-related microRNAs, especially miR-132, in the regulation of MRP8-induced astrocyte activation and highlight the importance of miR-132 in the modulation of innate immune response induced by endogenous ligands in neurological diseases.

PP28. Myeloid-related proteins 8 and 14: potential biomarkers of disease activity of arthritis in children with trisomy 21

PP28. Myeloid-related proteins 8 and 14: potential biomarkers of disease activity of arthritis in children with trisomy 21

Myeloid-related protein-14 deficiency promotes inflammation in staphylococcal pneumonia.

Staphylococcus aureus has evolved as an important cause of pneumonia in both hospital and community settings. Staphylococcal lung infection can lead to overwhelming pulmonary inflammation. During infection, neutrophils release complexes of myeloid-related protein (MRP)8 and MRP14 (MRP8/14). MRP8/14 has been shown to exert pro-inflammatory and chemotactic activity, and to assist in the killing of S. aureus. In the current study we sought to determine the role of MRP8/14 in the host response during S. aureus pneumonia.Pneumonia was induced in wildtype and MRP14-deficient mice (mice unable to form MRP8/14) by intranasal inoculation of 1×10(7) CFU of S. aureus USA300. Mice were sacrificed at 6, 24, 48 or 72 h after infection for analyses.S. aureus pneumonia was associated with a strong rise in MRP8/14 in bronchoalveolar lavage fluid and lung tissue. Surprisingly, MRP14 deficiency had a limited effect on bacterial clearance and was associated with increased cytokine levels in bronchoalveolar lavage fluid and aggravated lung histopathology. MRP14 deficiency in addition was associated with a diminished transmigration of neutrophils into bronchoalveolar lavage fluid at late time-points after infection together with reduced release of nucleosomes.MRP8/14 serves in an unexpected protective role for the lung in staphylococcal pneumonia.

S100A8/A9 (Calprotectin) Is Critical for Development of Glomerulonephritis and Promotes Inflammatory Leukocyte–Renal Cell Interactions

Glomerulonephritis is a common cause of end-stage renal disease. Infiltrating leukocytes interacting with renal cells play a critical role during the initiation and progression of glomerulonephritis, but the exact mechanisms are not clearly defined. By using the murine model of nephrotoxic nephritis, we investigated the role of S100A8/A9 [myeloid-related protein (MRP) 8/14, calprotectin] in promoting glomerulonephritis. In nephrotoxic nephritis, wild-type (WT) mice with glomerulonephritis have elevated serum levels of S100A8/A9, whereas mice deficient in MRP14 (S100a9(-/-)), and hence S100A8/A9, are significantly protected from disease. By using bone marrow transplants, we showed that MRP14 deficiency is required in both the hemopoietic and intrinsic cells for the protective effect. In vitro, both the WT bone marrow-derived macrophages and renal mesangial cells stimulated with S100A8/A9 secrete IL-6, CXCL1, and tumor necrosis factor α; however, Mrp14(-/-) cells exhibit significantly blunted proinflammatory responses. The interaction of WT bone marrow-derived macrophages with renal microvascular endothelial cells results in increased levels of monocyte chemotactic protein 1, IL-8, and IL-6 cytokines, which is attenuated in Mrp14(-/-) bone marrow-derived macrophages. Data shows that S100A8/A9 plays a critical role during glomerulonephritis, exerting and amplifying autocrine and paracrine proinflammatory effects on bone marrow-derived macrophages, renal endothelial cells, and mesangial cells. Therefore, complete S100A8/A9 blockade may be a new therapeutic target in glomerulonephritis.

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

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). 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. 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. 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.

Myeloid-related protein 14 promotes inflammation and injury in meningitis.

Neutrophilic inflammation often persists for days despite effective antibiotic treatment and contributes to brain damage in bacterial meningitis. We propose here that myeloid-related protein 14 (MRP14), an abundant cytosolic protein in myeloid cells, acts as an endogenous danger signal, driving inflammation and aggravating tissue injury. The release pattern of MRP14 was analyzed in human and murine cerebrospinal fluid (CSF), as well as in isolated neutrophils. Its functional role was assessed in a mouse meningitis model, using MRP14-deficient mice. We detected large quantities of MRP14 in CSF specimens from patients and mice with pneumococcal meningitis. Immunohistochemical analyses and a cell-depletion approach indicated neutrophils as the major source of MRP14. In a meningitis model, MRP14-deficient mice showed a better resolution of inflammation during antibiotic therapy, which was accompanied by reduced disease severity. Intrathecal administration of MRP14 before infection reverted the phenotype of MRP14-deficient mice back to wild type. Moreover, intrathecal injection of MRP14 alone was sufficient to induce meningitis in a Toll-like receptor 4 (TLR4)-CXCL2-dependent manner. Finally, treatment with the MRP14 antagonist paquinimod reduced inflammation and disease severity significantly, reaching levels comparable to those achieved after genetic depletion of MRP14. The present study implicates MRP14 as an essential propagator of inflammation and potential therapeutic target in pneumococcal meningitis.

Consolidated and emerging inflammatory markers in coronary artery disease.

Coronary artery disease is an event of atherosclerosis characterized by a chronic vascular inflammation. Risk factors like obesity, diabetes mellitus, hypertension, smoking, hypercholesterolemia and positive family history sometimes are not sufficiently adequate to the enhancement of cardiovascular risk assessment. In the past years numerous biomarkers, like C reactive protein, cytokines and adhesion molecules, have been observed to be related to adverse cardiovascular prognosis. Recently, several studies found an association among inflammatory biomarkers and cardiovascular diseases suggesting their utility to identify the risk of an acute ischemic event and the detection of vulnerable plaques. The emerging inflammatory markers are well divided for diagnosis and prognosis and plaque instability of coronary artery disease. Some of them, the lectin-like oxidized low density lipoprotein receptor-1 can be important both in diagnosis and in the evaluation of plaque instability, other are inserted in the above reported classification. The emerging inflammatory markers in acute-phase include amyloid A, fibrinogen and pentraxin 3 while myeloperoxidase, myeloid-related protein 8/14 and pregnancy-associated plasma protein-A are recognize markers of plaque instability. Lastly, some studies demonstrated that circulating miRNAs are involved in coronary artery disease, acute myocardial infarction and heart failure.

Coronary neutrophil extracellular trap burden and deoxyribonuclease activity in ST-elevation acute coronary syndrome are predictors of ST-segment resolution and infarct size.

Mechanisms of coronary occlusion in ST-elevation acute coronary syndrome are poorly understood. We have previously reported that neutrophil (polymorphonuclear cells [PMNs]) accumulation in culprit lesion site (CLS) thrombus is a predictor of cardiovascular outcomes. The goal of this study was to characterize PMN activation at the CLS. We examined the relationships between CLS neutrophil extracellular traps (NETs), bacterial components as triggers of NETosis, activity of endogenous deoxyribonuclease, ST-segment resolution, and infarct size. We analyzed coronary thrombectomies from 111 patients with ST-elevation acute coronary syndrome undergoing primary percutaneous coronary intervention. Thrombi were characterized by immunostaining, flow cytometry, bacterial profiling, and immunometric and enzymatic assays. Compared with femoral PMNs, CLS PMNs were highly activated and formed aggregates with platelets. Nucleosomes, double-stranded DNA, neutrophil elastase, myeloperoxidase, and myeloid-related protein 8/14 were increased in CLS plasma, and NETs contributed to the scaffolds of particulate coronary thrombi. Copy numbers of Streptococcus species correlated positively with dsDNA. Thrombus NET burden correlated positively with infarct size and negatively with ST-segment resolution, whereas CLS deoxyribonuclease activity correlated negatively with infarct size and positively with ST-segment resolution. Recombinant deoxyribonuclease accelerated the lysis of coronary thrombi ex vivo. PMNs are highly activated in ST-elevation acute coronary syndrome and undergo NETosis at the CLS. Coronary NET burden and deoxyribonuclease activity are predictors of ST-segment resolution and myocardial infarct size.