Main Mediators Research Articles

Intranasal administration of exosomes derived from mesenchymal stem cells ameliorates autistic-like behaviors of BTBR mice

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. They have been steadily increasing in children over the past several years, with no effective treatment. BTBR T+tf/J (BTBR) mice are an accepted model of evaluating autistic-like behaviors as they present all core symptoms of ASD. We have previously shown that transplantation of human bone marrow mesenchymal stem cells (MSC) to the lateral ventricles of BTBR mice results in long lasting improvement in their autistic behavioral phenotypes. Recent studies point exosomes as the main mediators of the therapeutic effect of MSC. Here, we tested whether treatment with the exosomes secreted from MSC (MSC-exo) will show similar beneficial effects. We found that intranasal administration of MSC-exo increased male to male social interaction and reduced repetitive behaviors. Moreover, the treatment led to increases of male to female ultrasonic vocalizations and significant improvement in maternal behaviors of pup retrieval. No negative symptoms were detected following MSC-exo intranasal treatments in BTBR or healthy C57BL mice. The marked beneficial effects of the exosomes in BTBR mice may translate to a novel, non-invasive, and therapeutic strategy to reduce the symptoms of ASD.

GLI2 promotes cell proliferation and migration through transcriptional activation of ARHGEF16 in human glioma cells

BackgroundThe Hedgehog (Hh) signaling pathway plays critical roles in modulating embryogenesis and maintaining tissue homeostasis, with glioma-associated oncogene (GLI) transcription factors being the main mediators. Aberrant activation of this pathway is associated with various human malignancies including glioblastoma, although the mechanistic details are not well understood.MethodsWe performed a microarray analysis of genes that are differentially expressed in glioblastoma U87 cells overexpressing GLI2A, the active form of GLI2, relative to the control cells. Chromatin immunoprecipitation and dual-luciferase assays were used to determine whether Rho guanine nucleotide exchange factor 16 (ARHGEF16) is a downstream target of GLI2. Then, transwell migration, EdU and soft-agar colony formation assays were employed to test effects of ARHGEF16 on glioma cancer cell migration and proliferation, and the effects of GLI2/ARHGEF16 signaling on tumor growth were examined in vivo. Finally, we performed yeast two-hybrid assay, Co-IP and GST-pull down to identify factors that mediate effects of ARHGEF16.ResultsWe found that ARHGEF16 mRNA level was upregulated in U87 cells overexpressing GLI2A relative to control cells. GLI2 binds to the ARHGEF16 promoter and activates gene transcription. Glioma cells U87 and U118 overexpressing ARHGEF16 showed enhanced migration and proliferation relative to the control cells, while knockdown of ARHGEF16 in H4 cells led to decreased cell proliferation compared to the control H4 cells. In contrast to the promoting effect of GLI2A overexpression on glioma xenograft growth, both GLI2 inhibition and ARHGEF16 knockdown retarded tumor growth. Cytoskeleton-associated protein 5 (CKAP5) was identified as an interaction protein of ARHGEF16, which is important for the stimulatory effects of ARHGEF16 on glioma cell migration and proliferation.ConclusionsThese results suggest that therapeutic strategies targeting the GLI2/ARHGEF16/CKAP5 signaling axis could inhibit glioma progression and recurrence.

Role of GLI Transcription Factors in Pathogenesis and Their Potential as New Therapeutic Targets.

GLI transcription factors have important roles in intracellular signaling cascade, acting as the main mediators of the HH-GLI signaling pathway. This is one of the major developmental pathways, regulated both canonically and non-canonically. Deregulation of the pathway during development leads to a number of developmental malformations, depending on the deregulated pathway component. The HH-GLI pathway is mostly inactive in the adult organism but retains its function in stem cells. Aberrant activation in adult cells leads to carcinogenesis through overactivation of several tightly regulated cellular processes such as proliferation, angiogenesis, EMT. Targeting GLI transcription factors has recently become a major focus of potential therapeutic protocols.

IsoDGR-Peptides for Integrin Targeting: Is the Time Up for RGD?

Integrins are the main cell adhesion mediators on the cell surface. Especially integrin αvβ3 has gained a lot of attention as a target in cancer therapy because it mediates diverse angiogenic processes during tumor development. The peptide sequence Arg-Gly-Asp (RGD), which is present in a number of endogenous integrin ligands like fibronectin, vitronectin, and related proteins of the extracellular matrix (ECM), has been extensively used as a targeting vector for therapeutic as well as diagnostic purposes, and cilengitide, a cyclic RGD peptide, has entered clinical trials for the treatment of various cancers. However, recent advancements utilizing isoDGR, a sequence that was found in aged fibronectin, already show that RGD-based targeting is not the end of the line. Novel developments and a closer investigation of the binding mode of these peptides now show promising results for the future use of such compounds.

Targeting FXR and FGF19 to Treat Metabolic Diseases-Lessons Learned From Bariatric Surgery.

Bariatric surgery procedures, such as Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG), are the most effective interventions available for sustained weight loss and improved glucose metabolism. Bariatric surgery alters the enterohepatic bile acid circulation, resulting in increased plasma bile levels as well as altered bile acid composition. While it remains unclear why both VSG and RYGB can alter bile acids, it is possible that these changes are important mediators of the effects of surgery. Moreover, a molecular target of bile acid synthesis, the bile acid–activated transcription factor FXR, is essential for the positive effects of VSG on weight loss and glycemic control. This Perspective examines the relationship and sequence of events between altered bile acid levels and composition, FXR signaling, and gut microbiota after bariatric surgery. We hypothesize that although bile acids and FXR signaling are potent mediators of metabolic function, unidentified downstream targets are the main mediators behind the benefits of weight-loss surgery. One of these targets, the gut-derived peptide FGF15/19, is a potential molecular and therapeutic marker to explain the positive metabolic effects of bariatric surgery. Focusing research efforts on identifying these complex molecular mechanisms will provide new opportunities for therapeutic strategies to treat obesity and metabolic dysfunction.

Preferential use of Siglec-1 or Siglec-10 by type 1 and type 2 PRRSV strains to infect PK15S1\u2013CD163 and PK15S10\u2013CD163 cells

Cellular entry mediators define whether the cell is permissive to PRRSV infection. Porcine sialoadhesin (pSn, Siglec-1) and CD163 are main entry mediators facilitating infection of porcine macrophages by PRRSV. Recently, Siglec-10 was demonstrated to be an alternative receptor for PRRSV. To examine if virulence and pathogenicity of PRRSV strains could be correlated with the use of different Siglecs, a PK15 cell line recombinantly expressing Siglec-1 and CD163 (PK15S1–CD163) and a PK15 cell line recombinantly expressing Siglec-10 and CD163 (PK15S10–CD163) were used to compare the virus replication of 7 genotype 1 subtype 1 strains (G1s1), 2 genotype 1 subtype 3 (G1s3) strains and 5 genotype 2 (G2) strains. Some strains (08VA (G1s1), 13V117 (G1s1), 17V035 (G1s1), VR2332 (G2)) were poor virus producers (<104 TCID50/mL), while other strains (07V063 (G1s1), 13V091 (G1s1), Su1-Bel (G1s3), MN-184 (G2), Korea17 (G2) and SDSU-73 (G2)) easily grew up to ≥106 TCID50/mL. PK15S10–CD163 cells exhibited a higher efficiency in virus production per infected cell than the PK15S1–CD163 cells. The G1s1 strains LV and 07V063 infected more cells in the PK15S1–CD163, whereas the 94V360 and 08VA strains preferred PK15S10–CD163. The highly virulent G1s3 strains Lena and Su1-Bel showed a strong preference for PK15S1–CD163. The G2 strains MN-184, SDSU-73, Korea17 had a much higher infection rate in PK15S10–CD163, while the reference strain VR2332 and the NADC30 strain had a slight preference for PK15S1–CD163. Differences in receptor use may influence the outcome of a PRRSV infection in pigs and explain in part the virulence/pathogenicity of PRRSV strains.

Targeting the NLRP3 Inflammasome-Related Pathways via Tianeptine Treatment-Suppressed Microglia Polarization to the M1 Phenotype in Lipopolysaccharide-Stimulated Cultures.

An increasing body of evidence postulates that microglia are the main mediators of inflammation-related disorders, including depression. Since activated microglia produce a wide range of pro- and anti-inflammatory factors, the modulation of M1/M2 microglial polarization by antidepressants may be crucial in the treatment of depression. The current paper aimed to investigate the impact of tianeptine on the microglia’s viability/death parameters, and on M1/M2 microglial activation in response to lipopolysaccharide (LPS) stimulation. Furthermore, the molecular mechanisms via which tianeptine affected the LPS-evoked changes were investigated. The results revealed that tianeptine had partially protective effects on the changes in microglia viability/death evoked by LPS. Tianeptine attenuated microglia activation by decreasing the expression of cluster of differentiation 40 (CD40), and major histocompatibility complex class II (MHC II) markers, as well as the release of pro-inflammatory factors: interleukin (IL)-1β, IL-18, IL-6, tumor necrosis factor alpha (TNF-α), and chemokine CC motif ligand 2 (CCL2), and the production of nitric oxide and reactive oxygen species. In contrast, we did not observe an impact of tianeptine on M2 microglia measured by IL-4, IL-10, TGF-β, and insulin-like growth factor 1 (IGF-1) expression. Moreover, we demonstrated an inhibitory effect of tianeptine on the LPS-induced activation of the nucleotide-binding oligomerization domain-like (NOD-like) receptor pyrin-containing 3 inflammasome (NLRP3) inflammasome subunits, NLRP3 and caspase-1, as well as the ability of tianeptine to reduce Toll-like receptor 4 (TLR4) levels, as well as the phosphorylation of extracellular signal-related kinases 1 and 2 (ERK1/2) and of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Collectively, we demonstrated that tianeptine has protective properties and inhibits M1 polarization, thus attenuating the production of inflammatory mediators. Moreover, we found that M1 microglia suppression may be related to the NLRP3 inflammasome and TLR4 signaling. These findings suggest that a better understanding of the multifaceted mechanisms of tianeptine action on microglia may increase the effectiveness of therapy, where inflammation is a central hallmark.

Adherence to the Mediterranean diet and inflammatory markers in children with asthma

IntroductionThere is accumulated evidence supporting a beneficial role of Mediterranean diet (MD) in the control of asthma symptoms. The aim of this study was to investigate the relationships between adherence to MD and serum levels of certain cytokines namely, interleukin (IL)-4, and IL-17 known to have a pathogenetic role in the airway changes associated with asthma. MethodsWe measured serum IL-4, IL-33, and IL-17, in 44 asthmatic and 26 healthy children, 5–15 years old. Their adherence to MD was estimated with the Mediterranean Diet Quality Index for children and adolescents (KIDMED) score. ResultsKIDMED score did not differ between the two groups (P=0.59) and was not correlated with any of the three measured cytokines. However, when the analysis was restricted only to asthmatic children, the KIDMED score was correlated with IL-4, IL-33, and IL-17 (Beta: −0.56, P=0.007; Beta: 0.57, P=0.010; Beta: −0.62, P=0.017, respectively). ConclusionOur results indicate that MD can modulate the production of some of the main inflammatory mediators of asthma, in asthmatic children.

PO-370 A co-culture assay system using engineered anti-CD3 tumour cells to assess tumour cell sensitivity to CD8 +T cell killing

IntroductionImmunotherapy has had a significant impact on the landscape of cancer treatment. However, with only a 20%–30% response rate, identifying novel tumour intrinsic mechanisms of resistance is key to advancing treatment success. Activation of CD8+ T cells, the main cellular mediators of adaptive tumour immunity, requires both T cell receptor (TCR) binding of MHC bound peptides and co-stimulation by antigen presenting cells (APCs). On tumour recognition, antigen-specific T cells become reactivated, leading to tumour cell lysis. Therefore, cytotoxic assays that measure CD8+ T cell responses against tumours, are crucial for understanding therapeutic outcomes.Material and methodsWe have developed an in vitro system in which we have engineered tumour cells to express single-chain (scFv) anti-CD3 (clone OKT3) which provides signal 1 to CD8+ T cells, in a co-culture assay. This system allows for understanding the relative contributions of MHC-independent mechanisms to T-cell killing of a panel of tumour cell lines, in parallel. As a model, we have transduced the EGFR mutant non-small cell lung cancer (NSCLC) cell lines PC-9, NCH-H1975 and NCI-H3255 to stably express anti-CD3.Results and discussionsImmune checkpoint blockade yields a clinical response in a subset of NSCLC patients. However, EGFR mutant NSCLC patients show less favourable responses. This assay allows us to investigate how EGFR genotype might influence tumour cell susceptibility to T cell killing. The pre-activated CD8+ T cells used in the co-culture assay underwent two rounds of anti-CD3/CD28 stimulation to optimise effector function. This resulted in increased CD45RO but decreased CD45RA expression and a heightened ability to lyse P815 target cells. Co-cultures of these T cells with tumour cells showed that these cell lines differ in their sensitivities to T cell killing as NCI-H1975 cells appeared to be the most sensitive whilst PC-9 and NCI-H3255 showed similar levels of killing. The flow cytometry based readout also allows for exploring tumour effects on T cell phenotype. Our data showed that all three cell lines significantly induced Granzyme B production in T cells in co-culture, which decreased with increasing E:T ratios and was the lowest with NCI-H1975 cells.ConclusionTaken together, our results demonstrate that our co-culture system is a robust assay for evaluating T cell-mediated cytotoxicity across cell lines and can potentially be used to investigate the effects of immunomodulatory drugs on T cell killing of different tumour cell lines.

Association of Vitamin D Receptor Gene Variation With Osteoporosis Risk in Belarusian and Lithuanian Postmenopausal Women.

Vitamin D receptor (VDR) is one of the main mediators of vitamin D biological activity. VDR dysfunction might substantially contribute to development of postmenopausal osteoporosis (PMO). Numerous studies have revealed the effects of several VDR gene variants on osteoporosis risk, although significant variation in different ethnicities have been suggested. The main purpose of this work was to assess the frequency of distribution of VDR genetic variants with established effect and evaluate their haplotype association with the risk of PMO in a cohort of Belarusian and Lithuanian women. Case group included women with PMO (n = 149), the control group comprised women with normal bone mineral density (BMD) and without previous fragility fractures (n = 172). Both groups were matched for age, height, sex, and BMI—no statistically significant differences observed. VDR gene polymorphic variants (ApaI rs7975232, BsmI rs1544410, TaqI rs731236, and Cdx2 rs11568820) were determined using polymerase chain reaction and restriction fragment length polymorphism. The lumbar spine (L1-L4) and femoral neck BMD was measured using dual-energy X-ray absorptiometry. Association between each VDR variant and PMO risk was assessed using multiple logistic regression. The genotyping revealed statistically significant difference in the rs7975232 genotype frequencies between the patients and the controls (homozygous C/C genotype was overrepresented in patients, p = 0.008). Patients with osteoporosis were also three times more likely to carry the rs1544410 G/G genotype, when compared to controls. We found that rs7975232, rs1544410, and rs731236 variants were in a strong direct linkage disequilibrium (p < 0.0001), suggesting that risk alleles of these markers are preferably inherited jointly. For the bearers of C-G-C haplotype (consisting of rs7975232, rs1544410, and rs731236 unfavorable alleles), the risk of PMO was significantly higher (OR = 4.7, 95% CI 2.8–8.1, p < 0.0001) compared to controls. This haplotype was significantly over-represented in PMO group compared to all other haplotypes. Our findings highlight the importance of identified haplotypes of VDR gene variants. Complex screening of these genetic markers can be used to implement personalized clinical approach for prevention, treatment, and rehabilitation programs.

Hypoxia/Reoxygenation of Rat Renal Arteries Impairs Vasorelaxation via Modulation of Endothelium-Independent sGC/cGMP/PKG Signaling.

Ischemia/reperfusion injury holds a key position in many pathological conditions such as acute kidney injury and in the transition to chronic stages of renal damage. We hypothesized that besides a reported disproportional activation of vasoconstrictor response, hypoxia/reoxygenation (H/R) adversely affects endothelial dilatory systems and impairs relaxation in renal arteries. Rat renal interlobar arteries were studied under isometric conditions. Hypoxia was induced by application of 95% N2, 5% CO2 for 60 min to the bath solution, followed by a 10 min period of reoxygenation (95% O2, 5% CO2). The effect of H/R on relaxation was assessed using various inhibitors of endothelial dilatory systems. mRNA expression of phosphodiesterase 5 (PDE5), NADPH oxidases (NOX), and nitric oxide synthase (NOS) isoforms were determined using qRT-PCR; cGMP was assayed with direct cGMP ELISA. Acetylcholine induced relaxation was impaired after H/R. Inhibition of the NOS isoforms with L-NAME, and cyclooxygenases (COXs) by indomethacin did not abolish the H/R effect. Moreover, blocking the calcium activated potassium channels KCa3.1 and KCa2.1, the main mediators of the endothelium-derived hyperpolarizing factor, with TRAM34 and UCL1684, respectively, showed similar effects in H/R and control. Arterial stiffness did not differ comparing H/R with controls, indicating no impact of H/R on passive vessel properties. Moreover, superoxide was not responsible for the observed H/R effect. Remarkably, H/R attenuated the endothelium-independent relaxation by sodium nitroprusside, suggesting endothelium-independent mechanisms of H/R action. Investigating the signaling downstream of NO revealed significantly decreased cGMP and impaired relaxation during PDE5 inhibition with sildenafil after H/R. Inhibition of PKG, the target of cGMP, did not normalize SNP-induced relaxation following H/R. However, the soluble guanylyl cyclase (sGC) inhibitor ODQ abolished the H/R effect on relaxation. The mRNA expressions of the endothelial and the inducible NOS were reduced. NOX and PDE5 mRNA were similarly expressed in H/R and control. Our results provide new evidence that impaired renal artery relaxation after H/R is due to a dysregulation of sGC leading to decreased cGMP levels. The presented mechanism might contribute to an insufficient renal reperfusion after ischemia and should be considered in its pathophysiology.

Tumor necrosis factor α-308 G/A and interleukin 1 β-511 C/T gene polymorphisms in patients with scarring acne.

Acne is a chronic inflammatory skin disorder which may heal with scarring. Tumor necrosis factor alpha (TNF α) and interleukin 1 β (IL-1β) are considered as the main responsible proinflammatory mediators of acne pathogenesis. Oversecretion of these cytokines was found to be associated with TNF α-308G>A and IL-1β-511 C<T polymorphisms. To evaluate the association of TNF α-308 and IL-1β-511 gene polymorphisms with acne and postacne scarring susceptibility and acne severity. Study subjects included 90 patients with acne vulgaris (31 males, 59 females; mean age: 19.6±3.7years) and 30 healthy controls (11 males, 19 females; mean age: 19.2±5.1years). Patients were sub-grouped on the basis of acne severity into mild, moderate, and severe acne groups and on the presence postacne scarring into scarring acne and nonscarring acne groups. Peripheral venous blood samples were obtained for performing real-time PCR analysis for detecting TNF α-308 and IL-1β-511 genotypic variants. Among patients, 21.7% (n=26) had mild, 22.5% (n=27) had moderate, 30.8% (n=37) had severe, and 30% (n=36) had scarring acne. Genotypic variants of TNF α-308 and IL-1β-511 did not statistically differ between acne patients and controls (P values: .245 and .466). When compared in terms of acne severity and the presence of postacne scarring, no statistical significance was observed regarding frequencies of genotypic variants related to the both TNF α-308 and IL-1β polymorphisms (P>.05). TNF α-308 and IL-1β polymorphic variants are not associated with acne and postacne scarring susceptibility and acne severity.

PGE2 and NO Mediate the Inhibitory Effect Exerted by FTY720P at 15 Minutes on Colonic Na+/K+ ATPase

Inflammatory Bowel Disease was shown to be accompaniedwith higher levels of sphingosine‐1‐ phosphate (S1P) and lower Na+/K+ ATPase activity. This work was undertaken to see if a cause‐effect relationship exists between S1P and the ATPase, and in case it does to determine the main mediators involved, using Caco‐2 cells as a model and fingolimod phosphate (FTY720P), a S1P analogue. The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the Na+/K+ATPase. Western blot analysis showed that sphingosine‐1‐phosphate receptor 2 (S1PR2) is the mostly expressed receptor in Caco‐2 cells. FTY720P, applied for 15 min, reduced significantly the activity of the Na+/K+ ATPase, with a maximal inhibition observed at a concentration of 7.5nM. The effect of FTY720P disappeared completely in presence of JTE‐013 (a specific blocker of S1PR2) and indomethacin (an inhibitor of cyclooxygenase enzymes), and was mimicked by CYM5520 (a S1PR2 agonist), prostaglandin E2 (PGE2), and sulprostone (an agonist of E prostanoid receptor 3 (EP3)). Carboxy‐PTIO a nitric oxide scavenger, abrogated the effect of FTY720P as well as that of PGE2. Treating the cells with Glyco‐SNAP (nitric oxide donor) reduced significantly the ATPase activity. It was concluded that FTY720‐P inhibits the ATPase by binding to S1PR2 leading to PGE2 production. PGE2 binds to EP3 receptors and increases nitric oxide levels resulting in an inhibitory effect on the Na+/K+ ATPase.Support or Funding InformationUniversity Research BoardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tumor-derived extracellular vesicles activate primary monocytes.

Tumor cells educate immune effector cells in their vicinity by releasing factors that manipulate their phenotype and function. In fact, the thus generated immunosuppressive tumor microenvironment constitutes an integral part and a hallmark of solid tumors and contributes significantly to tumor development and immune escape. It has long been thought that soluble factors like prostaglandin E2 and TGF‐β are the main mediators of these effects. But tumor cells also constantly release large number of extracellular vesicles (EVs), which are important conveyors of immune responses. We show here that tumor‐derived EVs interact with primary monocytes and induce an activated phenotype, which is also observed in tumor‐associated macrophages. Thus, both tumor‐derived EVs and soluble factors together collaborate to form the immunosuppressive milieu of the tumor environment.

Micro-Economics of Apoptosis in Cancer: ncRNAs Modulation of BCL-2 Family Members.

In the last few years, non-coding RNAs (ncRNAs) have been a hot topic in cancer research. Many ncRNAs were found to regulate the apoptotic process and to play a role in tumor cell resistance to treatment. The apoptotic program is on the frontline as self-defense from cancer onset, and evasion of apoptosis has been classified as one of the hallmarks of cancer responsible for therapy failure. The B-cell lymphoma 2 (BCL-2) family members are key players in the regulation of apoptosis and mediate the activation of the mitochondrial death machinery in response to radiation, chemotherapeutic agents and many targeted therapeutics. The balance between the pro-survival and the pro-apoptotic BCL-2 proteins is strictly controlled by ncRNAs. Here, we highlight the most common mechanisms exerted by microRNAs, long non-coding RNAs and circular RNAs on the main mediators of the intrinsic apoptotic cascade with particular focus on their significance in cancer biology.

THE ROLE OF CYTOKINES OF INTERLEUKIN 36 FAMILY IN IMMUNOPATHOGENESIS OF PSORIASIS

Over last decade, the importance of immune mechanisms for development of psoriasis has expanded considerably. This review describes biological properties of the cytokines from the interleukin-36 (IL-36) family, and their role in pathogenesis of plaque psoriasis, generalized pustular psoriasis and psoriatic arthritis. Keratinocytes and dendritic cells (DC) are the main sources of IL-36 in the skin. Production of this cytokine is greatly enhanced by inflammation. The studies have shown that, in affected skin of psoriatic patients, an increased expression of IL-36α and IL-36γ is observed. IL-36γ can be considered a specific marker of psoriasis, which is not found in other inflammatory skin diseases (neurodermatitis, lichen planus, eczema). The levels of this cytokine correlate with severity of psoriasis and decreases after anti-TNFα therapy. High level of IL-36 cytokines in the areas of psoriatic eruptions correlates with increased concentrations of pro-inflammatory TNFα, IL-17, IL-22 and IFNγ in the skin. We present literature data on the role of IL-36RN mutation which encodes the IL-36ra receptor antagonist, playing a role in the development of generalized pustular psoriasis (GPP). Excessive activity of IL-36 agonists leading to accumulation of neutrophilic granulocytes in epidermis may be a key event in the GPP pathogenesis. The article deals with modern ideas about the role of neutrophilic granulocytes in development of chronic inflammatory processes underlying pathogenesis of psoriasis. Proteases of neutrophils (cathepsin G, elastase and proteinase-3) are potent IL-36 activating enzymes that increase activity of IL-36 cytokines up to 500 times. In this review, a new mechanism (netosis) is suggested for the role of neutrophils in immune response. During the netosis, a large number of cytokines, antimicrobial peptides, intracellular components, serine proteases are released into the extracellular space. Thus, they may be involved into initiation and maintenance of a chronic inflammatory process in psoriasis. The present information concerning a role of new IL-36 family cytokines as one of the main psoriasis mediators has opened new therapeutic goals for the treatment of this disease and development of new methods of targeted therapy. Specific blocking of IL-36R signaling, or inhibition of neutrophil proteases of cathepsin G, neutrophil elastase that activate members of the IL-36 family, is likely to be a successful strategy in the therapy of psoriasis.

Sustained release of endothelial progenitor cell-derived extracellular vesicles from shear-thinning hydrogels improves angiogenesis and promotes function after myocardial infarction.

Previous studies have demonstrated improved cardiac function following myocardial infarction (MI) after administration of endothelial progenitor cells (EPCs) into ischaemic myocardium. A growing body of literature supports paracrine effectors, including extracellular vesicles (EVs), as the main mediators of the therapeutic benefits of EPCs. The direct use of paracrine factors is an attractive strategy that harnesses the effects of cell therapy without concerns of cell engraftment or viability. We aim to reproduce the beneficial effects of EPC treatment through delivery of EPC-derived EVs within a shear-thinning gel (STG) for precise localization and sustained delivery. EVs were harvested from EPCs isolated from adult male Rattus norvegicus (Wistar) rats and characterized by electron microscopy, nanoparticle tracking analysis (NTA), and mass spectrometry. EVs were incorporated into the STG and injected at the border zone in rat models of MI. Haemodynamic function, angiogenesis, and myocardial remodelling were analyzed in five groups: phosphate buffered saline (PBS) control, STG control, EVs in PBS, EVs in STG, and EPCs in STG. Electron microscopy and NTA of EVs showed uniform particles of 50-200 nm. EV content analysis revealed several key angiogenic mediators. EV uptake by endothelial cells was confirmed and followed by robust therapeutic angiogenesis. In vivo animal experiments demonstrated that delivery of EVs within the STG resulted in increased peri-infarct vascular proliferation, preservation of ventricular geometry, and improved haemodynamic function post-MI. EPC-derived EVs delivered into ischaemic myocardium via an injectable hydrogel enhanced peri-infarct angiogenesis and myocardial haemodynamics in a rat model of MI. The STG greatly increased therapeutic efficiency and efficacy of EV-mediated myocardial preservation.

Th1, Th17, and Th1Th17 Lymphocytes during Tuberculosis: Th1 Lymphocytes Predominate and Appear as Low-Differentiated CXCR3+CCR6+ Cells in the Blood and Highly Differentiated CXCR3+/-CCR6- Cells in the Lungs.

Th1 lymphocytes are considered the main mediators of protection against tuberculosis (TB); however, their phenotypic characteristics and relationship with Th17 and Th1Th17 populations during TB are poorly understood. We have analyzed Th1, Th17, and Th1Th17 lymphocytes in the blood and pulmonary lesions of TB patients. The populations were identified based on the production of IFN-γ and/or IL-17 and the coexpression of CXCR3 (X3) and CCR6 (R6). In the blood, IL-17+ and IFN-γ+IL-17+ lymphocytes were barely detectable (median, <0.01% of CD4+ lymphocytes), whereas IFN-γ+ lymphocytes predominated (median, 0.45%). Most IFN-γ+ lymphocytes (52%) were X3+R6+, suggesting their "nonclassical" (ex-Th17) nature. In the lungs, IL-17+ and IFN-γ+IL-17+ lymphocytes were more frequent (0.3%, p < 0.005), yet IFN-γ+ cells predominated (11%). Phenotypically, lung CD4+ cells were X3+/loR6- The degree of differentiation of blood effector CD4+ lymphocytes (evaluated based on CD62L/CD27/CD28 coexpression) increased as follows: X3+R6+ < X3+R6- < X3-R6-, with X3-R6- cells being largely terminally differentiated CD62L-CD27-CD28- cells. Lung CD4+ lymphocytes were highly differentiated, recalling blood X3+/-R6- populations. Following in vitro stimulation with anti-CD3/anti-CD28 Abs, X3+R6+CD4+ lymphocytes converted into X3+R6- and X3-R6- cells. The results demonstrate that, during active TB, Th1 lymphocytes predominate in blood and lungs, document differences in X3/R6 expression by blood and lung CD4+ cells, and link the pattern of X3/R6 expression with the degree of cell differentiation. These findings add to the understanding of immune mechanisms operating during TB and are relevant for the development of better strategies to control it.

Distinct signalling properties of insulin receptor substrate (IRS)-1 and IRS-2 in mediating insulin/IGF-1 action

Insulin/IGF-1 action is driven by a complex and highly integrated signalling network. Loss-of-function studies indicate that the major insulin/IGF-1 receptor substrate (IRS) proteins, IRS-1 and IRS-2, mediate different biological functions in vitro and in vivo, suggesting specific signalling properties despite their high degree of homology. To identify mechanisms contributing to the differential signalling properties of IRS-1 and IRS-2 in the mediation of insulin/IGF-1 action, we performed comprehensive mass spectrometry (MS)-based phosphoproteomic profiling of brown preadipocytes from wild type, IRS-1−/− and IRS-2−/− mice in the basal and IGF-1-stimulated states. We applied stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of changes in protein phosphorylation. We found ~10% of the 6262 unique phosphorylation sites detected to be regulated by IGF-1. These regulated sites included previously reported substrates of the insulin/IGF-1 signalling pathway, as well as novel substrates including Nuclear Factor I X and Semaphorin-4B. In silico prediction suggests the protein kinase B (PKB), protein kinase C (PKC), and cyclin-dependent kinase (CDK) as the main mediators of these phosphorylation events. Importantly, we found preferential phosphorylation patterns depending on the presence of either IRS-1 or IRS-2, which was associated with specific sets of kinases involved in signal transduction downstream of these substrates such as PDHK1, MAPK3, and PKD1 for IRS-1, and PIN1 and PKC beta for IRS-2. Overall, by generating a comprehensive phosphoproteomic profile from brown preadipocyte cells in response to IGF-1 stimulation, we reveal both common and distinct insulin/IGF-1 signalling events mediated by specific IRS proteins.

BCG vaccination drives accumulation and effector function of innate lymphoid cells in murine lungs

The tuberculosis (TB) vaccine bacille Calmette-Guérin (BCG) prevents disseminated childhood TB; however, it fails to protect against the more prevalent pulmonary TB. Limited understanding of the immune response to Mycobacterium tuberculosis, the causative agent of TB, has hindered development of improved vaccines. Although memory CD4 T cells are considered the main mediators of protection against TB, recent studies suggest there are other key subsets that contribute to antimycobacterial immunity. To that end, innate cells may be involved in the protective response. In this study, we investigated the primary response of innate lymphoid cells (ILCs) to BCG exposure. Using a murine model, we showed that ILCs increased in number in the lungs and lymph nodes in response to BCG vaccination. Additionally, there was significant production of the antimycobacterial cytokine IFN-γ by ILCs. As ILCs are located at mucosal sites, it was investigated whether mucosal vaccination (intranasal) stimulated an enhanced response compared to the traditional vaccination approach (intradermal or subcutaneous). Indeed, in response to intranasal vaccination, the number of ILCs, and IFN-γ production in NK cells and ILC1s in the lungs and lymph nodes, were higher than that provoked through intradermal or subcutaneous vaccination. This work provides the first evidence that BCG vaccination activates ILCs, paving the way for future research to elucidate the protective potential of ILCs against mycobacterial infection. Additionally, the finding that lung ILCs respond rigorously to mucosal vaccination may have implications for the delivery of novel TB vaccines.