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This study aimed to evaluate the effect of direct pulp capping on the expression of erythropoietin (Epo) and Epo-receptor (Epor) genes in relation to the expression of inflammatory and osteogenic genes in rat pulp. Dental pulps of the first maxillary molars of Wistar Albino rats were exposed and capped with either calcium hydroxide or mineral trioxide aggregate, or were left untreated. After 4 wk, animals were euthanized, and maxillae were prepared for histological and real-time polymerase chain reaction analysis. Histological scores of pulp inflammation and mineralization, and relative expressions of Epo, Epor, inflammatory cytokines, and pulp osteogenic genes were evaluated. The capped pulps showed higher expressions of Epo, while the untreated pulps had the highest expression of Epor. Both calcium hydroxide and mineral trioxide aggregate downregulated the expression of tumor necrosis factor alpha compared to untreated controls, and upregulated transforming growth factor beta compared to healthy controls. Alkaline phosphatase expression was significantly higher in experimental groups. Relative expression of Epo negatively correlated with pulp inflammation, and positively correlated with pulp mineralization. Pulp exposure promoted expression of Epor and pro-inflammatory cytokines, while pulp capping promoted expression of Epo, alkaline phosphatase, and downregulated Epor and pro-inflammatory cytokines.

Interleukin-33 (IL-33) regulates innate and acquired immune response to pathogens, self-antigens and tumors. IL-33 effects on tumors depend on the dose and mode of administration along with the type of malignancy. We studied the effects of IL-33 on the development of primary and metastatic melanoma induced by B16-F1 cell line in C57BL/6 mice. Intraperitoneally applied IL-33 restricts primary tumor growth. When administered intranasally 3 days prior to the intravenous injection of the tumor cells, IL-33 promoted growth of B16-F1 melanoma metastases, while B16-F10 gave massive metastases independently of IL-33. To mimic natural dissemination, we next used a limited number (5 × 104) of B16-F1 cells intravenously followed by application of IL-33 intraperitoneally. IL-33 increased the size of metastases (10.96 ± 3.96 mm2) when compared to the control group (0.86 ± 0.39 mm2), without changing incidence and number of metastases. IL-33 increased expression of ST2 on both tumor and immune cells in metastases. Also, IL-33 enhanced eosinophils and anti-tumor NK cells in the lung. The striking finding was reduced cytotoxicity of CD8+ T cells derived from metastatic lung of IL-33 injected mice. IL-33 reduced the percentage of TNF-α+ and IFN-γ+ CD8+ T cells while increasing the frequency of CD8+ T cells that express inhibitory molecules (PD-1, KLRG-1 and CTLA-4). There was a significant accumulation of CD11b+Gr-1+ myeloid suppressor cells and FoxP3+, IL-10+ and CTLA-4+ regulatory T cells in the metastatic lung of IL-33 injected mice. The relevance of IL-33 for melanoma metastases was also documented in a significantly increased level of serum IL-33 in stage III melanoma patients.

The study investigates conformational analysis and the in vitro cytokine-mediated immunomodulatory and insulin-releasing activities of rhinophrynin-27 (ELRLPEIARPVPEVLPARLPLPALPRN; RP-27), a proline-arginine-rich peptide first isolated from skin secretions of the Mexican burrowing toad Rhinophrynus dorsalis (Rhinophrynidae). In both water and 50% trifluoroethanol-water, the peptide adopts a polyproline type II helical conformation with a high degree of deviation from the canonical collagen-like folding and a pronounced bend in the molecule at the Glu13 residue. Incubation of mouse peritoneal cells with RP-27 significantly (P

Galectin-3 (Gal-3) has diverse roles in inflammatory and autoimmune diseases. There is evidence that Gal-3 plays a role in both type 1 and type 2 diabetes. While the role of Gal-3 expression in immune cells invading the pancreatic islets in the experimental model of type 1 diabetes mellitus has been already studied, the importance of the overexpression of Gal-3 in the target β cells is not defined. Therefore, we used multiple low doses of streptozotocin (MLD–STZ)–induced diabetes in C57Bl/6 mice to analyze the effect of transgenic (TG) overexpression of Gal-3 in β cells. Our results demonstrated that the overexpression of Gal-3 protected β cells from apoptosis and attenuated MLD–STZ–induced hyperglycemia, glycosuria, and ketonuria. The cellular analysis of pancreata and draining lymph nodes showed that Gal-3 overexpression significantly decreased the number of pro-inflammatory cells without affecting the presence of T-regulatory cells. As the application of exogenous interleukin 33 (IL-33) given from the beginning of MLD–STZ diabetes induction attenuates the development of disease, by increasing the presence of regulatory FoxP3+ST2+cells, we evaluated the potential synergistic effect of the exogenous IL-33 and TG overexpression of Gal-3 in β cells at the later stage of diabetogenesis. The addition of IL-33 potentiated the survival of β cells and attenuated diabetes even when administered later, after the onset of hyperglycemia (12–18 days), suggesting that protection from apoptosis and immunoregulation by IL-33 may attenuate type 1 diabetes.

AIM To investigate the influence of strain differences in immune responses on the pathogenesis of experimental periapical lesions in Dark Agouti (DA) and Albino Oxford (AO) inbred strains of rats. METHODOLOGY Periapical lesions were induced in male DA and AO rats by pulp exposure of the first mandibular right molars to the oral environment. Animals were killed 21 days after pulp exposure. The mandibular jaws were retrieved and prepared for radiographic, pathohistological, immunohistochemical analysis, real-time PCR and flow cytometry. Blood samples and the supernatant of periapical lesions were collected for measurement of cytokines and oxidative stress marker levels. Statistical analysis was performed using the Kruskal-Wallis H and Mann-Whitney U non-parametric tests or parametric One-Way anova and Independent Samples T-test to determine the differences between groups depending on the normality of the data. A significant difference was considered when p values were

When it comes to tumor immunology, understanding of molecular pathways is rather important. During oncogenesis, many molecules should be taken in consideration altogether in context of a single malignancy. It is of a great significance to determine whether these molecules act synergistically or contrary, whether to understand a malignant disease more thoroughly, or even more important, to reveal new approaches of therapy. In this review, we discuss whether and how IL-33/ST2 and PD-1/PDL axis involve in antitumor immunity.

Galectin-3 (Gal-3), a member of lectin family that binds to oligosaccharides, is involved in several biological processes, including maturation and function of nervous system. It had been reported that Gal-3 regulates oligodendrocytes differentiation and that Gal-3/Toll-like receptor-4 (TLR4) axis is involved in neuroinflammation. As both, central nervous system (CNS) maturation and neuroinflammation may affect behavior, the principle aim of this study was to examine the effects of Gal-3 gene deletion on behavior. Here we provide the evidence that Gal-3 deficiency shows clear anxiogenic effect in mature untreated animals (basal conditions). This was accompanied with lower interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) relative gene expression and hippocampal content, with no effect on TLR4 expression. Gal-3 deficiency was also accompanied with lower brain-derived neurotrophic factor (BDNF) relative gene expression and immunoreactivity in hippocampus (predominantly in CA1 region). Besides, the Gal-3 gene deletion resulted in attenuation of the hippocampal relative gene expression of GABA-A receptor subunits 2 and 5 (GABA-AR2S and GABA-AR5S), On the other hand, Gal-3 deficiency attenuates LPS-induced neuroinflammation. The anxiogenic effect of acute neuroinflammation was accompanied with increased hippocampal IL-6, TNF-α and TLR4 gene expression, as well as decreased gene and immunohistochemical BDNF expression in hippocampus, with significant decline in GABA-AR2S in wild type (WT) mice in comparison to basal conditions. Gal-3 gene deletion prevented the increase in IL-6, the decline in BDNF gene expression and immunoreactivity, and reduction in hippocampal GABA-AR2S, and therefore attenuated the anxiogenic effect of neuroinflammation. In summary, our data demonstrate that apparently opposite effects of Gal-3 deficiency on anxiety levels (anxiogenic effect under basal conditions and anxiolytic action during neuroinflammation) seem to be related to the shift in IL-6, TNF-α and hippocampal BDNF.

Strategies targeting cross-talk between immunosuppressive renal dendritic cells (DCs) and T regulatory cells (Tregs) may be effective in treating cisplatin (CDDP)-induced acute kidney injury (AKI). Galectin 3 (Gal-3), expressed on renal DCs, is known as a crucial regulator of immune response in the kidneys. In this study, we investigated the role of Gal-3 for DCs-mediated expansion of Tregs in the attenuation of CDDP-induced AKI. Methods: AKI was induced in CDDP-treated wild type (WT) C57BL/6 and Gal-3 deficient (Gal-3-/-) mice. Biochemical, histological analysis, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, real-time PCR, magnetic cell sorting, flow cytometry and intracellular staining of renal-infiltrated immune cells were used to determine the differences between CDDP-treated WT and Gal-3-/- mice. Newly synthesized selective inhibitor of Gal-3 (Davanat) was used for pharmacological inhibition of Gal-3. Recombinant Gal-3 was used to demonstrate the effects of exogenously administered soluble Gal-3 on AKI progression. Pam3CSK4 was used for activation of Toll-like receptor (TLR)-2 in DCs. Cyclophosphamide or anti-CD25 antibody were used for the depletion of Tregs. 1-Methyl Tryptophan (1-MT) was used for pharmacological inhibition of Indoleamine 2,3-dioxygenase-1 (IDO1) in TLR-2-primed DCs which were afterwards used in passive transfer experiments. Results: CDDP-induced nephrotoxicity was significantly more aggravated in Gal-3-/- mice. Significantly reduced number of immunosuppressive TLR-2 and IDO1-expressing renal DCs, lower serum levels of KYN, decreased presence of IL-10-producing Tregs and significantly higher number of inflammatory IFN-γ and IL-17-producing neutrophils, Th1 and Th17 cells were observed in the CDDP-injured kidneys of Gal-3-/- mice. Pharmacological inhibitor of Gal-3 aggravated CDDP-induced AKI in WT animals while recombinant Gal-3 attenuated renal injury and inflammation in CDDP-treated Gal-3-/- mice. CDDP-induced apoptosis, driven by Bax and caspase-3, was aggravated in Gal-3-/- animals and in WT mice that received Gal-3 inhibitor (CDDP+Davanat-treated mice). Recombinant Gal-3 managed to completely attenuate CDDP-induced apoptosis in CDDP-injured kidneys of Gal-3-/- mice. Genetic deletion as well as pharmacological inhibition of Gal-3 in renal DCs remarkably reduced TLR-2-dependent activation of IDO1/KYN pathway in these cells diminishing their capacity to prevent transdifferentiation of Tregs in inflammatory Th1 and Th17 cells. Additionally, Tregs generated by Gal-3 deficient DCs were not able to suppress production of IFN-γ and IL-17 in activated neutrophils. TLR-2-primed DCs significantly enhanced capacity of Tregs for attenuation of CDDP-induced AKI and inflammation and expression of Gal-3 on TLR-2-primed DCs was crucially important for their capacity to enhance nephroprotective and immunosuppressive properties of Tregs. Adoptive transfer of TLR-2-primed WTDCs significantly expanded Tregs in the kidneys of CDDP-treated WT and Gal-3-/- recipients resulting in the suppression of IFN-γ and IL-17-driven inflammation and alleviation of AKI. Importantly, this phenomenon was not observed in CDDP-treated WT and Gal-3-/- recipients of TLR-2-primed Gal-3-/-DCs. Gal-3-dependent nephroprotective and immunosuppressive effects of renal DCs was due to the IDO1-induced expansion of renal Tregs since either inhibition of IDO1 activity in TLR-2-primed DCs or depletion of Tregs completely diminished DCs-mediated attenuation of CDDP-induced AKI. Conclusions: Gal-3 protects from CDDP-induced AKI by promoting TLR-2-dependent activation of IDO1/KYN pathway in renal DCs resulting in increased expansion of immunosuppressive Tregs in injured kidneys. Activation of Gal-3:TLR-2:IDO1 pathway in renal DCs should be further explored as new therapeutic approach for DC-based immunosuppression of inflammatory renal diseases.

Galectin-3 (Gal-3) is the only member of galectin family able to form pentamers and heterodimers with chemokines. Its presence in various cells and tissues suggests variety of regulatory functions in physiological conditions, but increasing body of evidence indicates involvement of Gal-3 in pathological cascades of many diseases. Gal-3 exerts different, sometimes opposite, effects in various disorders or in different phases of the same disease. These differences in action of Gal-3 are related to the localization of Gal-3 in the cell, types of receptors through which it acts, or the types of cells that secrete it. As a regulator of immune response and T-cell activity, Gal-3 appears to have important role in development of autoimmunity mediated by T cells. Absence of Gal-3 in C57Bl6 mice favors Th2 mediated inflammatory myocarditis but attenuate fibrosis. Recent data also indicate Gal-3 involvement in development atherosclerosis. In pathogenesis of diabetes type 1 and autoimmune components of diabetes type 2 Gal-3 may have detrimental or protective role depending on its intracellular or extracellular localization. Gal-3 mediates autoimmune hepatic damage through activation of T-cells or natural killer T cells. Gal-3 is an important mediator in neurodevelopment, neuropathology and behavior due to its expression both in neurons and glial cells. All together, assessing the role of Gal-3 in immunopathology and autoimmunity it could be concluded that it is an important participant in pathogenesis, as well as promising monitoring marker and therapeutic target.

Experimental autoimmune myocarditis (EAM) is a mouse model of immune-mediated myocarditis and cardiomyopathy. The role of Galectin-3 (Gal-3), a β-galactoside-binding lectin, in autoimmune myocarditis has not been studied. Therefore, the aim of this study was to delineate the role of Gal-3 in myosin peptide-induced autoimmune myocarditis in mice. EAM was induced in relatively resistant C57BL/6J mice (wild type, WT) and in mice with a targeted deletion of Gal-3 gene (Gal-3KO) by immunization with myosin peptide MyHCα334-352. Gal-3KO mice developed more severe myocarditis and more pronounced heart hypertrophy than WT mice. Increased infiltration of CD45+ leucocytes, CD3+ T cells, F4/80+ macrophages, and eosinophils was observed in hearts of Gal-3KO mice compared to WT mice on day 21 after EAM induction. Moreover, hearts of Gal-3KO mice had more T helper type 2 (Th2) cells, alternatively activated M2 macrophages, higher amounts of IgG deposits, and higher serum levels of IL-4 and IL-33 than WT mice. Ablation of Gal-3 in Th1-dominant C57BL/6J mice that are relatively resistant to EAM resulted in more severe disease characterized by type 2 cardiac inflammation. The complex effects of Gal-3 on EAM progression might be important in the consideration of therapeutic options for the treatment of EAM.

One of the therapeutic options for the treatment of fulminant hepatitis is repopulation of intrahepatic regulatory cells because their pool is significantly reduced during acute liver failure. Although it is known that mesenchymal stem cells (MSCs), which have beneficent effects in the therapy of fulminant hepatitis, may promote expansion of regulatory T cells (Tregs) and regulatory B cells (Bregs), the role of these regulatory cells in MSC-mediated attenuation of acute liver injury is unknown. Herewith, we described the molecular mechanisms involved in the crosstalk between MSCs and liver regulatory cells and analyzed the potential of MSC-based therapy for the expansion of intrahepatic regulatory cells in mouse model of acute liver failure. MSC-dependent attenuation of α-galactosylceramide (α-GalCer)-induced acute liver injury in mice was accompanied with an increased presence of interleukin (IL) 10-producing CD4+ CD25+ forkhead box P3+ Tregs and IL10- and transforming growth factor β-producing marginal zone-like Bregs in the liver. Depletion of Bregs did not alter MSC-based alleviation of acute liver failure, whereas depletion of Tregs completely abrogated hepatoprotective effects of MSCs and inhibited their capacity to attenuate hepatotoxicity of liver natural killer T cells (NKTs), indicating that Tregs, and not Bregs, were critically involved in MSC-based modulation of acute liver inflammation. MSCs, in a paracrine, indoleamine 2,3-dioxygenase-dependent manner, significantly increased the capacity of Tregs to produce immunosuppressive IL10 and to suppress hepatotoxicity of liver NKTs. Accordingly, adoptive transfer of MSC-primed Tregs resulted in the complete attenuation of α-GalCer-induced acute liver failure. In conclusion, our findings highlighted the crucial importance of Tregs for MSC-based attenuation of acute liver failure and indicated the significance of MSC-mediated priming of Tregs as a new therapeutic approach in Treg-based therapy of acute liver injury. Liver Transplantation 24 687-702 2018 AASLD.

An altered immune response to normal gut microflora is important for the pathogenesis of ulcerative colitis (UC). Galectin- 3 (Gal-3) is an endogenous lectin that plays an important pro-inflammatory role in the induction phase of acute colitis by promoting activation of the NLRP3 infl ammasome and production of IL-1β in macrophages. By using dextran sulphate sodium (DSS) induced colitis, a well-established animal model of UC, we determined whether Gal-3 affects the function of colon infiltrating macrophages by interfering with intestinal microfl ora. Our results showed that genetic deletion of Gal-3 significantly attenuates DSS-induced colitis by down-regulating infiltration of phagocytic cells (neutrophils, macrophages and dendritic cells) in colon tissue of DSS-treated mice, and this correlated with differences in bacterial flora of the gut. Antibiotic treatment attenuates DSS-induced colitis in WT and Gal-3-/- mice without affecting differences between the groups. In conclusion, Gram negative bacterial flora play an important role in DSS-induced acute colitis of mice but are not involved in Gal-3 dependent modulation of colon inflammation.

Frog skin is a source of peptides with various biological properties. Frenatin 2.1S, derived from norepinephrine-stimulated skin secretions of the Orinoco lime tree frog Sphaenorhynchus lacteus, exhibits immunostimulatory effects as demonstrated by the promotion of proinflammatory phenotypes of mononuclear cells in mouse peritoneal cavity and spleen. The aim of this study was to identify the populations of host cells sensitive to the action of frenatin 2.1S in vivo and to study its effects on their functional antitumor capacity. A single injection of frenatin 2.1S (100 μg) in BALB/c mice increased the presence of peritoneal CD11c+ dendritic cells and CD3+ T cells 24 h after administration and there was a significant increase in the number of IL-17 and CXCR3 expressing inflammatory T cells. Frenatin 2.1S treatment also increased the number of TNF-α expressing F4/80+ proinflammatory M1 macrophages. The most striking finding of the study is the marked increase of the number of peritoneal natural killer (NK) cells following frenatin 2.1S injection. Further, frenatin 2.1S administration led to activation of NK cells as evaluated by increased expression of NKG2D, FasL, CD69 and CD107a. The increased ratio of interferon-γ vs. IL-10 producing NK cells is further indication of the proinflammatory action of frenatin 2.1S. Peptide treatment enhanced the tumoricidal action of peritoneal NK cells on 4T1 mouse mammary carcinoma cells as revealed by the real-time automated monitoring of cell status. Our data demonstrate that frenatin 2.1S promotes activation and cytotoxic capacity of NK cells and should be regarded as a candidate for antitumor immunotherapy.

Mesenchymal stem cells (MSCs) are, due to their immunomodulatory characteristics, utilized in therapy of immune-mediated diseases. We used murine model of cisplatin nephrotoxicity to explore the effects of MSCs on immune cells involved in the pathogenesis of this disease. Intraperitoneal application of MSCs significantly attenuated cisplatin nephrotoxicity, decreased inflammatory cytokines TNF-α and IL-17, and increased anti-inflammatory IL-10, IL-6, nitric oxide (NO), and kynurenine in sera of cisplatin-treated mice. MSC treatment significantly attenuated influx of leukocytes, macrophages, dendritic cells (DCs), neutrophils, CD4+ T helper (Th), and CD8+ cytotoxic T lymphocytes (CTLs) in damaged kidneys and attenuated the capacity of renal-infiltrated DCs, CD4+ Th, and CD8+ CTLs to produce TNF-α and IL-17. Similar effects were observed after intraperitoneal injection of MSC-conditioned medium (MSC-CM) indicating that MSCs exert their beneficial effects in paracrine manner. Inhibition of inducible nitric oxide synthase (iNOS) in MSC-CM resulted with increased number of TNF-α-producing DCs and IL-17-producing CTLs, decreased number of IL-10-producing tolerogenic DCs and regulatory CD4+FoxP3+ T cells, and completely diminished renoprotective effects of MSC-CM. In conclusion, MSCs, in iNOS-dependent manner, attenuated inflammation in cisplatin nephrotoxicity by reducing the influx and capacity of immune cells, particularly DCs and T lymphocytes, to produce inflammatory cytokines.

Galectin-3 regulates numerous biological processes in the gut. We investigated molecular mechanisms responsible for the Galectin-3-dependent regulation of colon inflammation and evaluated whether Galectin-3 may be used as biomarker for monitoring the progression of ulcerative colitis (UC). The differences in disease progression between dextran sodium sulphate-treated wild type and Galectin-3-deficient mice were investigated and confirmed in clinical settings, in 65 patients suffering from mild, moderate, and severe colitis. During the induction phase of colitis, Galectin-3 promoted interleukin-1&beta, induced polarization of colonic macrophages towards inflammatory phenotype. In the recovery phase of colitis, Galectin-3 was required for the immunosuppressive function of regulatory dendritic cells (DCs). Regulatory DCs in Galectin-3:Toll-like receptor-4:Kynurenine-dependent manner promoted the expansion of colon-infiltrated T regulatory cells (Tregs) and suppressed Th1 and Th17 cell-driven colon inflammation. Concentration of Galectin-3 in serum and stool samples of UC patients negatively correlated with clinical, endoscopic, and histological parameters of colitis. The cutoff serum values of Galectin-3 that allowed the discrimination of mild from moderate and moderate from severe colitis were 954 pg/mL and 580 pg/mL, respectively. Fecal levels of Galectin-3 higher than 553.44 pg/mL indicated attenuation of UC. In summing up, Galectin-3 regulates the cross-talk between colon-infiltrating DCs and Tregs and represents a new biomarker for monitoring the progression of UC.

International audience; The aim of the study was to determine the in vitro immunomodulatory, cytotoxic, and insulin‐releasing activities of seven phylloseptin‐TR peptides and plasticin‐TR, first isolated from the frog Phyllomedusa trinitatis. The most cationic peptides, phylloseptin‐1.1TR and phylloseptin‐3.1TR, showed greatest cytotoxic potency against A549, MDA‐MB231, and HT‐29 human tumor‐derived cells and against mouse erythrocytes. Phylloseptin‐4TR was the most hydrophobic and the most effective peptide at inhibiting production of the proinflammatory cytokines TNF‐α and IL‐1β by mouse peritoneal cells but was without effect on production of the antiinflammatory cytokine IL‐10. Phylloseptin‐2.1TR and phylloseptin‐3.3TR were the most effective at stimulating the production of IL‐10. The noncytotoxic peptide, plasticin‐TR, inhibited production of TNF‐α and IL‐1β but was without effect on IL‐10 production. The results of CD spectroscopy suggest that the different properties of plasticin‐TR compared with the immunostimulatory activities of the previously characterized plasticin‐L1 from Leptodactylus laticeps may arise from greater ability of plasticin‐TR to oligomerize and adopt a stable helical conformation in a membrane‐mimetic environment. All peptides stimulated release of insulin from BRIN‐BD11 rat clonal β cells with phylloseptin‐3.2TR being the most potent and effective and phylloseptin‐2.1TR the least effective suggesting that insulinotropic potency correlates inversely with helicity. The study has provided insight into structure‐activity relationships among the phylloseptins. The combination of immunomodulatory and insulinotropic activities together with low cytotoxicity suggests that phylloseptin‐3.3TR and plasticin‐TR may represent templates for the development of agents for use in antiinflammatory and type 2 diabetes therapies.

Inflammation plays a crucial role in the progression of atherosclerotic plaques. The aim of the study was to investigate serum levels and expression of Interleukin-33 (IL-33) and ST2 receptor in atherosclerotic plaques and to analyze correlation with the type of the carotid plaques in patients with carotid disease.This study included 191 consecutive patients submitted for carotid endarterectomy (CEA). Preoperative serum levels of IL-33 and soluble ST2 (sST2) were measured. Atherosclerotic plaques obtained during surgery were initially histologically classified and immunohistochemical analyzes of IL-33, IL-33R, CD68 and alpha-SMA expression was performed. Ultrasound assessment of the level of carotid stenosis in each patient was performed prior to carotid surgery. Demographic and clinical data such as gender, age, smoking status, blood pressure, glycaemia, hemoglobin and creatinine levels, and comorbidities were collected and the comparisons between variables were statistically evaluated.Serum levels of IL-33 (35.86 ± 7.93 pg/ml vs.12.29 ± 1.8 pg/ml, p 0.05) and sST2 (183 ± 8.03 pg/ml vs. 122.31 ± 15.89 pg/ml, p 0.05) were significantly higher in the group of CEA patients vs. healthy subjects. We demonstrated abundant tissue expression of IL-33 and ST2 in atherosclerotic carotid artery lesions. The levels of IL-33 and IL-33R expression were significantly higher in vulnerable plaques and significantly correlated with the degree of inflammatory cells infiltration in these plaques (R = 0.579, p = 0.049). Immunohistochemical analysis also revealed that cells responsible for IL-33 expression are not only mononuclear cells confined to inflammatory atherosclerotic lesions, but also smooth muscle cells which gained phenotypic characteristics of foam cells and were loaded with lipid droplets.The obtained results confirm the importance of IL-33/ST2 axis in the process of atherosclerosis, and indicate its ambiguous function in immune response, whether as proinflammatory cytokine in advanced atherosclerotic lesions, or as profibrotic, in early lesions.

Carotid atherosclerosis may be associated with neurosymptoms including cerebral infarction. IL-10 exerts atheroprotective effects, but its role in carotid disease is not fully defined. We aimed to investigate serum IL-10 levels in patients undergoing endarterectomy and their relation to the degree of carotid stenosis, plaque types and neurosymptoms. Two hundred consecutive patients with atherosclerotic carotid stenosis and 29 healthy controls were enrolled in this study. Plaque types were classified according to AHA criteria. Serum IL-10 levels were determined by ELISA. Patients undergoing endarterectomy had significantly higher circulating IL-10 levels (18.7 ± 3.2 pg/ml) in comparison with healthy controls (7.2 ± 1.8pg/ml; P =0.0001) and IL- 10 has good discriminatory efficacy between these two groups (ROC curve, AUC = 0.723, P=0.0001). Patients with < 70% and those with > 70% of carotid stenosis did not differ in terms of age, sex, cardiovascular risk factors except hypertension, neurosymptoms and AHA plaque types. Circulating IL-10 levels differed significantly among patients with different carotid plaque types (P = 0.002). Patients with uncomplicated plaques had significantly higher serum levels of IL-10 (23.0 ± 6.1 pg/ml) compared to those with complicated plaques (13.0 ±1.4 pg/ml, P=0.035) and IL-10 can differentiate patients between these two groups (ROC curve, AUC = 0.413, P= 0.035). Our findings reveal an important role for IL-10 in carotid atherosclerosis. IL-10 might be a potential biomarker in discriminating patients with carotid disease from healthy controls. Decreased serum levels of IL-10 are related to complicated carotid plaques.

Interleukin-33 (IL-33) regulates innate and acquired immune response to pathogens, self-antigens and tumors. IL-33 effects on tumors depend on the dose and mode of administration along with the type of malignancy. We studied the effects of IL-33 on the development of primary and metastatic melanoma induced by B16-F1 cell line in C57BL/6 mice. Intraperitoneally applied IL-33 restricts primary tumor growth. When administered intranasally 3 days prior to the intravenous injection of the tumor cells, IL-33 promoted growth of B16-F1 melanoma metastases, while B16-F10 gave massive metastases independently of IL-33. To mimic natural dissemination, we next used a limited number (5 × 10

Four host-defense peptides belonging to the tigerinin family (tigerinin-1O: RICTPIPFPMCY; tigerinin-2O: RTCIPIPLVMC; tigerinin-3O: RICTAIPLPMCL; and tigerinin-4O: RTCIPIPPVCF) were isolated from skin secretions of the African crowned bullfrog Hoplobatrachus occipitalis. In aqueous solution at pH 4.8, the cyclic domain of tigerinin-2O adopts a rigid amphipathic conformation that incorporates a flexible N-terminal tail. The tigerinins lacked antimicrobial (MIC100 μM) and hemolytic (LC50500 μM) activities but, at a concentration of 20 μg/mL, significantly (P0.05) inhibited production of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice without affecting production of IL-10 and IL-17. Tigerinin-2O and -4O inhibited IFN-γ production at concentrations as low as 1 μg/mL. The tigerinins significantly (P ≤ 0.05) stimulated the rate of insulin release from BRIN-BD11 clonal β-cells without compromising the integrity of the plasma membrane. Tigerinin-1O was the most potent (threshold concentration 1 nM) and the most effective (395% increase over basal rate at a concentration of 1 μM). Tigerinin-4O was the most potent and effective peptide in stimulating the rate of glucagon-like peptide-1 release from GLUTag enteroendocrine cells (threshold concentration 10 nM; 289% increase over basal rate at 1 μM). Tigerinin peptides have potential for development into agents for the treatment of patients with type 2 diabetes.

The phenomenological uniqueness of each patient with schizophrenia is determined by complex symptomatology, particularly the overlapping of symptoms and their prominence in certain phases of this mental disorder. Establishing biological markers is an important step in the further objectivisation and quantification of schizophrenia. Identifying the cytokine profiles that precede a psychotic episode could direct the strategies for relapse prevention and be useful in predicting disease progression and treatment response. In the context of infl ammation, TGF-β exerts potent anti-inflammatory and immunosuppressive functions by inhibiting pro-inflammatory cytokine synthesis, but it can also have pro-inflammatory functions through its stimulatory effects on inflammatory Th17 cells. It has been shown that the T helper cell type-1 and type-17 responses are reduced and type-2 response is increased in patients with schizophrenia. Both data from the literature and our results also indicate the presence of an anti-inflammatory response through production of the TGF-β regulatory cytokine. A meta-analysis of plasma cytokine alterations suggested that TGF-β is the state marker for acute exacerbation of schizophrenia, and we showed that TGF-β can also be a valuable marker for psychosis. Hyperactivity of TGF-β signalling pathways in schizophrenia may be both a neuroprotective mechanism and a possible therapeutic target.

Diabetes mellitus is chronic disorder characterized by hyperglycaemia. Hyperglycaemia induces mitochondrial dysfunction, enhances oxidative stress and thus promotes reactive oxygen species (ROS) production. Earlier studies suggested that reactive oxygen species (ROS) are involved in the pathogenesis of many diseases. Previous studies have revealed that hyperglycaemia changes the functional phenotype of monocytes, macrophages, neutrophils, NK cells and CD8+ T cells. The aim of this study was to investigate whether diabetes affects the functional phenotype of NKT cells. Diabetes mellitus was induced in BALB/c mice by intraperitoneal injection of streptozotocin at a single dose of 170 mg/kg body weight. The number and functional phenotype of splenic NKT cells was assessed by fl ow cytometry, 28 days after diabetes induction. The diabetic condition facilitated the production of antioxidant enzymes, including catalase (p+CD49+ NKT cells that express the activatory receptor NKP46 or produce IFN-γ. However, hyperglycaemia increased the frequency of splenic NKT cells that express KLRG-1 and produce TGF-β, IL-4, and IL-5, and it decreased the frequency of IL-17+ NKT cells. Our study indicates that diabetes mellitus induces oxidative stress and switches the functional phenotype of NKT cells towards type 2 (IL-4 and IL-5 producing NKTs) and regulatory (TGF-β Thproducing NKTs) phenotypes. These findings are correlated with the clinical observation in humans that diabetic patients are more prone to infections and tumours.

// Milos Z. Milosavljevic 1,* , Ivan P. Jovanovic 2,* , Nada N. Pejnovic 2 , Slobodanka L. J. Mitrovic 1 , Nebojsa N. Arsenijevic 2 , Bojana J. Simovic Markovic 2 and Miodrag L. Lukic 2 1 Department of Pathology, University Medical Center Kragujevac, Kragujevac, Serbia 2 Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia * These authors have contributed equally to this work Correspondence to: Miodrag L. Lukic, email: // Keywords : breast neoplasm, IL-33, IL-33R, neoangiogenesis, necrosis Received : October 26, 2015 Accepted : February 11, 2016 Published : February 23, 2016 Abstract Interleukin-33 (IL-33)/IL-33 receptor (IL-33R, ST2) signaling pathway promotes mammary cancer growth and metastasis by inhibiting anti-tumor immunity. However, the role of IL-33/IL-33R axis in neoangiogenesis and tumor necrosis is not elucidated. Therefore, the aim of this study was to investigate the role of IL-33/IL-33R axis in mammary tumor necrosis. Deletion of IL-33R (ST2) gene in BALB/c mice enhanced tumor necrosis and attenuated tumor growth in 4T1 breast cancer model, which was associated with markedly decreased expression of vascular endothelial growth factor (VEGF) and IL-33 in mammary tumor cells. We next analyzed IL-33, IL-33R and VEGF expression and microvascular density (MVD) in breast tumors from 40 female patients with absent or present tumor necrosis. We found significantly higher expression of IL-33, IL-33R and VEGF in breast cancer tissues with absent tumor necrosis. Both, IL-33 and IL-33R expression correlated with VEGF expression in tumor cells. Further, VEGF expression positively correlated with MVD in perinecrotic zone. Taking together, our data indicate that IL-33/IL-33R pathway is critically involved in mammary tumor growth by facilitating expression of pro-angiogenic VEGF in tumor cells and attenuating tumor necrosis. These data add an unidentified mechanism by which IL-33/IL-33R axis facilitates tumor growth.

There is a plethora of evidence to suggest that Galectin-3 plays an important role in normal functions of mammalian cells, as well as in different pathogenic conditions. This review highlights recent data published by researchers, including our own team, on roles of Galectin-3 in the nervous system. Here, we discuss the roles of Galectin-3 in brain development, its roles in glial cells, as well as the interactions of glial cells with other neural and invading cells in pathological conditions. Galectin-3 plays an important role in the pathogenesis of neuroinflammatory and neurodegenerative disorders, such as multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. On the other hand, there is also evidence of the protective role of Galectin-3 due to its anti-apoptotic effect in target cells. Interestingly, genetic deletion of Galectin-3 affects behavioral patterns in maturing and adult mice. The results reviewed in this paper and recent development of highly specific inhibitors suggests that Galectin-3 may be an important therapeutic target in pathological conditions including the disorders of the central nervous system.

Acute pancreatitis is characterized by autodigestion of pancreatic cells followed by acute inflammation leading to pathology and death. In experimental acute pancreatitis, pancreatic acinar cells and infiltrating macrophages express Galectin-3 but its role in pathology of this disease is unknown. Therefore, we studied its role using Galectin-3 deficient mice. Deletion of Galectin-3 prolonged the survival of mice, led to attenuation of histopathology, and decreased infiltration of mononuclear cells and neutrophils that express TLR-4, in particular, pro-inflammatory N1 neutrophils. Galectin-3 and TLR-4 are also colocalized on infiltrating cells. Lack of Galectin-3 reduced expression of pro-inflammatory TNF-α and IL-1β in F4/80+ CD11c- and CD11c+ F4/80- cells. Thus, deletion of Galectin-3 ameliorates acute pancreatitis by attenuating early influx of neutrophils and inflammatory mononuclear cells of innate immunity. These findings provide the basis to consider Galectin-3 as a therapeutic target in acute pancreatitis.

Background: Dendritic cell (DC)-derived indolamine 2,3-dioxygenase (IDO) degrades tryptophan to kynurenine, which promotes conversion of inflammatory T cells in immunosuppressive regulatory T cells (Tregs). We analyzed the significance of the IDO:Treg axis for inducing and maintaining mucosal healing in ulcerative colitis (UC). Methods: Dextran sodium sulphate (DSS)-induced colitis in BALB/c mice (model for mucosal healing) and C57BL/6 mice (model for persistent disease) was used. Serum, fecal samples and colon-infiltrating immune cells of 65 patients with UC with mucosal healing or persistent colitis were analyzed. Results: Significantly higher serum levels of kynurenine and downregulated inflammatory cytokines were noticed in DSS-treated BALB/c mice compared with C57BL/6 mice. Increased IDO activity and attenuated capacity for antigen presentation and production of inflammatory cytokines, observed in BALB/c DCs, was followed by a significantly lower number of inflammatory T helper 1 (Th1) and Th17 cells and a notably increased number of Tregs in the colons of DSS-treated BALB/c mice. DCs and Tregs were crucially important for the maintenance of mucosal healing since their depletion aggravated colitis. Mucosal healing, followed by an increase in kynurenine and intestinal Tregs, was re-established when BALB/c DCs were transferred into DC-depleted or Treg-depleted DSS-treated BALB/c mice. This phenomenon was completely abrogated by the IDO inhibitor. Significantly higher serum and fecal levels of kynurenine, accompanied by an increased presence of intestinal Tregs, were noticed in patients with UC with mucosal healing and negatively correlated with disease severity, fecal calprotectin, colon-infiltrating interferon γ and interleukin-17-producing cells, serum and fecal levels of inflammatory cytokines. Conclusion: IDO-dependent expansion of endogenous Tregs should be further explored as a new approach for the induction and maintenance of mucosal healing in patients with UC.

Background Sepsis is a life-threatening disease mediated by profound disturbances in systemic inflammatory response to infection. IL-33 is multifunctional regulator of numerous aspects of innate and adaptive immune response. The aim of this article was to further evaluate the role of IL-33 receptor (ST2) in different pathways of innate immunity during early polymicrobial sepsis. Methods Polymicrobial sepsis was induced using cecal ligation and puncture (CLP) model in ST2 deficient (ST2−/−) and wild type BALB/c mice. Peritoneal and spleen cells were isolated for further phenotyping. Apoptosis was determined by immunohistochemistry and flow cytometry. Results Deletion of ST2 leads to increased susceptibility to early manifestations of sepsis as evaluated by clinical signs and survival. These are accompanied by decrease in the total number of neutrophils, eosinophils and mast cells in peritoneal cavity 12 h after CLP. In early sepsis there was also low number of precursors of myeloid cells in particular CD11b+Ly6G+Ly6Clow cells in spleen of ST2−/− mice. Although the number of NK cells in the spleen was similar, there were significant differences in the presence of inflammatory IFN-γ and IL-17 producing NK cells. Further, ST2 deletion affects the phenotype and maturation of dendritic cell in sepsis. The total number of dendritic cells in the spleen was lower as well as IL-12 expressing dendritic cells. Finally, there was higher frequency of active caspase-3 positive and early apoptotic cells, in particular CD11c positive cells, in spleen of septic ST2−/− mice. Conclusion Taken together, our data provide the evidence that ST2 deficiency in early phase of sepsis downregulates myeloid precursors, inflammatory NK and dendritic cells.

Schizophrenia and treatment of this disorder are often accompanied with metabolic syndrome and cardiovascular issues. Alterations in the serum level of innate immune mediators, such as interleukin-33 (IL-33) and its receptor IL-33R (ST2) and Galectin-3 (Gal-3) were observed in these conditions. Moreover, these parameters are potential prognostic and therapeutic markers. There is also accumulating evidence that these molecules play a role in neuroinflammation. Therefore, in this study we have investigated the serum level of Gal-3, IL-33 and soluble ST2 (sST2) in different stages of schizophrenia. Gal-3 levels were elevated in remission and lower in schizophrenia exacerbation in comparison with controls. Levels of IL-33 and sST2 are higher in schizophrenia exacerbation in comparison with controls and patients in remission. This initial analysis of new markers of neuroinflammation suggested their involvement in schizophrenia pathophysiology and/or cardiometabolic comorbidity.

Diabetic patients have higher incidence and mortality of cancer. Recent study revealed that hyperglycemia-induced oxidative stress is involved in the acceleration of tumor metastasis. We used model of high-dose streptozotocin-induced diabetes to investigate its effect on tumor growth and modulation of antitumor immune response of 4T1 murine breast cancer in BALB/c mice. Diabetes accelerated tumor appearance, growth and weight, which was associated with decreased NK cells cytotoxicity against 4T1 tumor cells in vitro. Diabetes reduced frequencies of systemic NKG2D+, perforin+, granzyme+, IFN-γ+ and IL-17+ NK cells, while increased level of PD-1 expression and production of IL-10 in NK cells. Diabetes decreased percentage of NKG2D+NK cells and increased percentage of PD-1+ NK cells also in primary tumor. Diabetes increased accumulation of IL-10+ Tregs and TGF-β+ myeloid-derived suppressor cells (MDSCs) in spleen and tumor. Diabetic sera in vitro significantly increased the percentage of KLRG-1+ and PD-1+ NK cells, decreased the percentage of IFN-γ+NK cells, expression of NKp46 and production of perforin, granzyme, CD107a and IL-17 per NK cell in comparison to glucose-added mouse sera and control sera. Significantly increased percentages of inducible nitric oxide synthase (iNOS) and indoleamine 2,3-dioxygenase (IDO) producing MDSCs and dendritic cells (DC) were found in the spleens of diabetic mice prior to tumor induction. 1-methyl-DL-tryptophan, specific IDO inhibitor, almost completely restored phenotype of NK cells cultivated in diabetic sera. These findings indicate that diabetes promotes breast cancer growth at least in part through increased accumulation of immunosuppressive cells and IDO-mediated attenuation of NK cells.

Dendritic cells (DCs) have important pathogenic role in the induction and progression of ulcerative colitis (UC), but their role in mesenchymal stem cells (MSCs)-mediated suppression of colon injury and inflammation is not revealed. By using dextran sodium sulfate (DSS)-induced colitis, a well-established murine model of UC, we examined effects of MSCs on phenotype and function of colon infiltrating DCs. Clinical, histological, immunophenotypic analysis and passive transfer of MSCs-primed DCs were used to evaluate capacity of MSC to suppress inflammatory phenotype of DCs in vivo. Additionally, DCs:MSCs interplay was also investigated in vitro, to confirmed in vivo obtained findings. Intraperitoneally administered MSCs (2 × 106) significantly reduced progression of DSS-induced colitis and reduced serum levels of inflammatory cytokines (IL-1β, IL-12, and IL-6). Passive transfer of in vivo MSCs-primed DCs reduced severity of colitis while passive transfer of MSCs-non-primed DCs aggravated DSS-induced colitis. Through the secretion of immunomodulatory Galectin 3, MSCs, in paracrine manner, down-regulated production of inflammatory cytokines in DCs and attenuated expression of co-stimulatory and major histocompatibility complex class II molecules on their membranes. Taken together, these results indicate that MSCs achieved their beneficial effects in DSS-induced colitis by suppressing inflammatory phenotype of DCs in Gal-3 dependent manner. Therapeutic targeting of DCs by MSCs should be explored in future studies as a useful approach for the treatment of UC.

Innate lymphoid cells (ILCs) represent the most recently identified members of the innate immune system. These cells play important roles in inflammation, tissue remodelling and metabolic disease. ILCs can be subdivided into three major groups according to their cytokine production. The role of ILCs in tumourigenesis and tumour progression is not completely clarified. In this review, we discuss whether and how ILCs are involved in tumour genesis, growth and metastasis.

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with obesity, but the molecular mechanisms of liver steatosis and its progression to non-alcoholic steatohepatitis and fibrosis are incompletely understood. Immune reactivity plays an important role in the pathogenesis of NAFLD. The IL-33/ST2 axis has a protective role in adiposity and atherosclerosis, but its role in obesity-associated metabolic disorders requires further clarification. To investigate the unresolved role of IL-33/ST2 signalling in NAFLD, we used ST2-deficient (ST2-/-) and wild type (WT) BALB/c mice maintained on a high-fat diet (HFD) for 24 weeks. HFD-fed ST2-/- mice exhibited increased weight gain, visceral adipose tissue weight and triglyceridaemia and decreased liver weight compared with diet-matched WT mice. Compared with WT mice on an HFD, ST2 deletion significantly reduced hepatic steatosis, liver inflammation and fibrosis and downregulated the expression of genes related to lipid metabolism in the liver. The frequency of innate immune cells in the liver, including CD68+ macrophages and CD11c+ dendritic cells, was lower in HFD-fed ST2-/- mice, accompanied by lower TNFα serum levels compared with diet-matched WT mice. Less collagen deposition in the livers of ST2-/- mice on an HFD was associated with lower numbers of profibrotic CD11b+Ly6clow monocytes and CD4+IL-17+ T cells in the liver, lower hepatic gene expression of procollagen, IL-33 and IL-13, and lower serum levels of IL-33 and IL-13 compared with diet-matched WT mice. Our findings suggest that the IL-33/ST2 axis may have a complex role in obesity-associated metabolic disorders. Although it is protective in HFD-induced adiposity, the IL-33/ST2 pathway promotes hepatic steatosis, inflammation and fibrosis.

The importance of Galectin-3 (Gal-3) in obesity-associated liver pathology is incompletely defined. To dissect the role of Gal-3 in fibrotic nonalcoholic steatohepatitis (NASH), Gal-3-deficient (LGALS3(-/-)) and wild-type (LGALS3(+/+)) C57Bl/6 mice were placed on an obesogenic high fat diet (HFD, 60% kcal fat) or standard chow diet for 12 and 24 wks. Compared to WT mice, HFD-fed LGALS3(-/-) mice developed, in addition to increased visceral adiposity and diabetes, marked liver steatosis, which was accompanied with higher expression of hepatic PPAR-γ, Cd36, Abca-1 and FAS. However, as opposed to LGALS3(-/-) mice, hepatocellular damage, inflammation and fibrosis were more extensive in WT mice which had an elevated number of mature myeloid dendritic cells, proinflammatory CD11b(+)Ly6C(hi) monocytes/macrophages in liver, peripheral blood and bone marrow, and increased hepatic CCL2, F4/80, CD11c, TLR4, CD14, NLRP3 inflammasome, IL-1β and NADPH-oxidase enzymes mRNA expression. Thus, obesity-driven greater steatosis was uncoupled with attenuated fibrotic NASH in Gal-3-deficient mice. HFD-fed WT mice had a higher number of hepatocytes that strongly expressed IL-33 and hepatic CD11b(+)IL-13(+) cells, increased levels of IL-33 and IL-13 and up-regulated IL-33, ST2 and IL-13 mRNA in liver compared with LGALS3(-/-) mice. IL-33 failed to induce ST2 upregulation and IL-13 production by LGALS3(-/-) peritoneal macrophages in vitro. Administration of IL-33 in vivo enhanced liver fibrosis in HFD-fed mice in both genotypes, albeit to a significantly lower extent in LGALS3(-/-) mice, which was associated with less numerous hepatic IL-13-expressing CD11b(+) cells. The present study provides evidence of a novel role for Gal-3 in regulating IL-33-dependent liver fibrosis.