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We used transgenic mice to investigate the effect selective Notch inhibition or forced activation in activated hepatic stellate cells (aHSCs) has on liver fibrosis. We used two models, carbon tetrachloride (CCl4) treatment and DDC- supplemented diet. The effect of Notch inhibition was investigated in SMACreERT2+/-/RBP- Jfl/fl mice, while Notch activation was studied using SMACreERT2+/- /RosaNICD1 mice. Mice were treated with 75μg/g of tamoxifen i.p. twice weekly. Identically treated Cre- littermates served as controls. CCl4 and DDC caused a similar degree of fibrosis (Sirius red stained area, hydroxyproline content, aminotransferase activities, gene expression of Col1a1 and Acta2) in experimental and control groups in Notch-inhibition as well as Notch-activation experiments. We further tested whether forced Notch activation in aHSCs delays resolution of fibrosis. SMACreERT2+/-/RosaNICD1 mice were treated with either CCl4 or DDC, and tamoxifen for 10 days, and then recovered for one month. Recovery was similar in both SMACreERT2+/-/RosaNICD1 and SMACreERT2-/-/RosaNICD1 mice, suggesting that forced Notch activation doesn’t impair recovery. We also found that tamoxifen per se protects against DDC- induced fibrosis. This was evidenced by a lower Sirius red-stained area, lower hydroxyproline content, and further confirmed by qPCR showing a lower expression of genes for Col1a1, Acta2, Sox9, Pdgf, and Krt19, indicating the inhibitory effect on HSCs, collagen production, and biliary duct proliferation. This effect highlights the importance of a proper Cre- littermate control in this investigation. We conclude that modulation of Notch activity in aHSCs neither changes the degree of liver fibrosis nor does it impair recovery once the noxious stimulus is withdrawn.

CroatiaOsteoclasts are multinuclear bone resorbing cells differentiated from monocyte/macrophage lineage of hematopoietic cells. They form by fusion of mononuclear osteoclast progenitors (OCPs), and mediate increased bone loss in arthritis. Our research group identified murine periarticular bone marrow OCPs as CD45+Ly6G-CD3- B220-NK1.1-CD11b-/loCD115+ cells using flow cytometry. They were further sepa-rated by the level of chemokine receptor CCR2 expression, into CCR2hi and CCR2lo subsets. We previ-ously showed that these subsets are significantly expanded in collagen-induced arthrtis (CIA). Our aim was to characterize these OCP subsets by RNA-sequencing. Therefore, we sorted CCR2hi and CCR2lo OCPs from control and CIA mice (n=4 for every group), and performed RNA extraction, library prepara-tion and RNA sequencing. For transcriptome profiling, we first aligned raw sequencing reads to the mouse reference genome, and then performed gene set enrichment analysis to evaluate the differential gene expression profile across samples. Principal component analysis showed that transcriptomes differed mostly based on CCR2 expression, and less depending on intervention (CIA/control). Our stringent analysis criteria (log2 fold change≥2) revealed total of 862 differentially expressed genes between CCR2hi and CCR2lo OCPs, included within several biological pathways. Top enriched path-ways in CCR2hi OCPs in both CIA and control were osteoclast differentiation, chemokine and NOD-like receptor signaling pathways, while ribosomal biosynthetic pathways were downregulated compared to CCR2lo OCPs. In addition, several inflammatory pathways were enriched in CCR2hi subset only in CIA, including Staphylococcus aureus infection, Toll-like receptor and TNF signaling pathways. Genes selected from the enriched pathways were further validated by qPCR. To conclude, CCR2hi was more altered than CCR2lo subset in CIA compared to control. CCR2hi OCPs posses greater osteoclastogenic and migratory potential than CCR2lo OCPs. Furthermore, CCR2hi subset becomes specifically respon-sive to inflammatory conditions in CIA. These results indicate that CCR2hi OCPs could be mainly responsible for enhanced osteoresorption in arthritis.

Activated hepatic stellate cells (aHSCs) are a major source of myofibroblasts and modulating their signaling pathways may affect fibrogenesis. Recent findings suggested involvement of Notch signaling pathway in pathogenesis of liver fibrosis. We aimed to characterize the role of Notch overactivation in aHSCs in the development and resolution of liver fibrosis. SMACreERT/NICD (with Cre- littermates as control) mice were given CCl4 (0.5 mL/kg i.p., 2x weekly) for 6 weeks or were fed 0.025%-DDC- supplemented diet for 4 weeks. Tamoxifen was injected twice weekly (75 μg/kg i.p.) in order to activate the Cre recombinase in aHSCs. In the second set of experiments, mice were allowed a recovery period of 4 weeks following the 2 weeks of fibrosis induction. Liver tissue and sera were taken for analysis. ALT and AST activities were not significantly different between groups, and neither was gene expression of Col1a1 and Acta2. Percentage of sirius red stained area, calculated with ImageJ, did not differ between groups. This finding was confirmed by liver tissue hydroxyproline content, which was similar in both groups. Similar results were found in the recovery set of experiments, with no significant difference between groups biochemically (ALT, AST, hydroxyproline), histologically (red area percentage) nor on gene expression (Col1a1, Acta2). Overexpression of Notch1 in activated HSCs does not lead to greater fibrosis, nor does it inhibit its resolution upon removal of CCl4 or DDC.

As the existence of different osteoclast progenitor (OCP) subsets has been confirmed, their detailed characterization is required to understand the pathophysiology of increased osteoclast activity causing periarticular and systemic bone resorption in arthritis. We previously defined OCP subsets based on the level of CCR2 expression, as circulatory-like committed CCR2hi OCPs, substantially expanded in arthritis, and marrow-resident CCR2lo OCPs of immature phenotype and behaviour. We sorted CCR2hi and CCR2lo OCPs of mice with collagen-induced arthritis (CIA) and control mice (n=4 for each group) and performed next-generation RNA sequencing. We confirmed a disparity between the transcriptomes of CCR2hi and CCR2lo OCP subsets, and identified pathway enrichment for osteoclast differentiation, chemokine and NOD-like receptor signaling in the CCR2hi OCP subset and ribosome biogenesis in eukaryotes and pyrimidine metabolism in the CCR2lo OCP subset. Genes associated with the osteoclastogenic pathway (Fcgr1, Socs3), and genes involved in cell adhesion and migration (F11r, CD38, Lrg1) were used to both identify the CCR2hi subset and distinguish CIA from control group, as validated by qPCR (n=6 for control, n=9 for CIA). The latter set positively correlated with arthritis clinical score and percentage of CCR2hi OCPs, indicating potential disease markers. Moreover, osteoclast pathway-identifying genes remained upregulated in committed preosteoclasts cultured for two days, suggesting their importance for enhanced osteoclastogenesis of CCR2hi OCPs in arthritis. Our approach identified differentially expressed genes that allow detection of distinct OCP subsets associated with arthritis and well as indicate possible therapeutic targets aimed to modulate progenitor migration, proliferation, differentiation, or activity.

Osteoclasts (OC), dendritic cells (DC) and macrophages (MA) may be derived from common trilineage myeloid progenitor of hematopoietic origin. Progenitor commitment is susceptible to regulation through Notch signaling. Moreover, Notch dysregulation has been implicated in a number of inflammatory diseases. We aimed to determine the effects of Notch signaling modulation on common progenitor commitment and functional properties of differentiated cells. We used CX3CR1CreERT2NICD1 (Cre+NICD1, overexpressed Notch 1 signal) and CX3CR1CreERT2RBPJκ (Cre+RBPJκ, silenced Notch signal) mice as a source of common bone marrow progenitor (CD45+CD3-B220-NK1.1-CD11bloCD115+). Cre recombinase, under the control of CX3CR1 promoter expressed in monocytes/macrophages, was induced in vitro by 4-hydroxytamoxifen. OCs differentiation was induced by M-CSF/RANKL ; MAs by M-CSF ; DCs by IL-4/GM-CSF ; inflammation by E. coli lipopolysaccharide. Functionally, DCs were tested for the ability to process and present antigen ; MAs to phagocytose E. coli particles, and OCs to resorb bone and express TRAP. Among tested lineages, Notch1 overexpression suppressed OC formation, whereas Notch deletion enhanced osteoclastogenesis, resulting in a greater number and larger osteoclasts. In accordance, RANK protein expression was upregulated in osteoclastogenic cultures from Cre+RBPJκ mice, as well as the gene expression of cFos and CatK. Notch modulation did not seem to affect the number of MAs or DCs. Functional assays under inflammatory conditions confirmed that Notch silencing amplifies antigen presentation by DCs and TRAP expression by OCs, whereas the effect on phagocytosis by MAs was less prominent. Although Notch signaling modulation affected functional properties of all three lineages, the major effect was observed in OCs, with enhanced differentiation and function by Notch signal silencing. Our results indicate that Notch signal participates in the regulation of OC activity in inflammatory diseases.

Background: Osteoclasts mediate periarticular and systemic bone loss in rheumatoid arthritis (RA). Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45 ⁺ Ly6G ⁻ CD3 ⁻ B220 ⁻ NK1.1 ⁻ CD11b lo/+ CD115 ⁺ CCR2 ⁺ cells, are specifically increased in arthritis. We previously identified an increased frequency of OCPs expressing Notch receptors in arthritic mice. Objectives Several studies suggested that Notch signaling modulation affects the course of experimental arthritis. We aimed to determine the effects of Notch receptor signaling inhibition on OCP activity and arthritis severity in murine collagen-induced arthritis (CIA). Methods: Male C57/Bl6 and DBA mice were immunized with chicken type II collagen and treated with i.p. injections of anti-Notch 1 neutralizing antibodies (1mg/kg). Notch receptor 1 through 4 expression on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). Gene expression of Notch receptors, ligands and transcription targets as well as osteoclast differentiation genes RANK, cFos and cFms was determined by qPCR from tissues and sorted OCPs. FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL), in control, IgG, Jagged (Jag)1 or Delta-like (DLL)1 coated wells, with or without anti-Notch 1 antibodies. Research was approved by the Ethics Committee. Results: We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 25% and 40% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Seeding OCPs on DLL1 coated wells significantly increased while seeding on Jag1 coated wells significantly decreased osteoclastogenesis as reflected on the number of TRAP+ osteoclasts and expression of osteoclast differentiation genes. The addition of anti-Notch 1 antibodies to ligand- stimulated OCPs resulted in an increased number of TRAP+ osteoclasts, partially reversing Jag1 inhibition. In vivo treatment with anti-Notch 1 antibodies did not affect total OCP frequency, but increased expression of Notch 4 both in PBM and SPL as seen by flow cytometry and qPCR. Additionally, anti-Notch 1 treatment stimulated Notch transcription factors HES and HEY. Both PBM and SPL cultured OCPs from anti-Notch 1 treated mice produced a higher number of large TRAP+ osteoclasts, doubling the area covered with osteoclasts in the latter compared to untreated mice. Increased osteoclastogenesis in vitro was further confirmed by an increased expression of osteoclast differentiation genes in the treated group. Conclusion: Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo anti-Notch 1 neutralizing antibodies enhanced osteoclastogenesis in CIA model, implying an inhibitory role of Notch 1 signaling in osteoclast differentiation. As Notch 2 expression is increased on OCPs of arthritic mice, we next plan to determine the effects of Notch 2 neutralization on osteoclast activity and arthritis severity. Acknowledgements: The work has been supported by Croatian Science Foundation projects IP-2018-01- 2414, UIP-2017-05-1965 and DOK-2018-09-4276. Disclosure of Interests None declared.

Obesity represents a state of low‐grade chronic systemic inflammation, potentially leading to insulin resistance and endothelial damage, causing metabolic and vascular complications. We aimed to investigate metabolic and inflammatory parameters in obese children, by analyzing laboratory data and peripheral blood chemokine/chemokine receptor profile. Upon Ethical approval, mononuclear cells were isolated from peripheral blood of healthy controls (n=29, 14 M, age 15.46+/‐1.51 years) and obese children (n=34, 19 M, age 14.69+/‐ 1.60 years). B‐cell (CD19+), T‐cell (CD3+) and monocyte (CD14+) frequencies were determined in regard to chemokine receptor expression (CCR2, CCR4, CXCR3 and CXCR4) by flow cytometry. Chemokines (CCL2, CCL5, CXCL10, CXCL11) were determined by LEGENDplex™ and CXCL12 by ELISA. Data were correlated with clinical and laboratory parameters (HOMA‐ IR, ALT, lipid profile, diastolic blood pressure (DBP), CRP, fibrinogen). Obese children have higher inflammatory markers (CRP, fibrinogen) and parameters of metabolic complications (ALT, LDL‐cholesterol, triglycerides, DBP, HOMA IR). Their peripheral blood samples have decrease monocyte expression of CCR4 and CXCR3, and lower concentration of CXCL12. CXCR3+ monocytes showed negative correlation with CRP, fibrinogen, ALT, LDL‐cholesterol and HOMA‐IR, while CCR4+ monocytes positively correlated with HDL‐cholesterol, and negatively with HOMA‐IR and triglycerides. We also found correlation between CCR2+ B‐cells and HDL‐ cholesterol, and CCR4+ B‐calls and DBP. CCL2, CRP and fibrinogen positively correlated with metabolic parameters. Obesity is associated with systemic inflammation and metabolic complications even in young age. Precise identification of inflammatory molecules associated with these complications may indicate therapeutic targets for preventing obesity related morbidity and mortality.

Osteomyelitis (OM) is a debilitating infection of the bone primarily caused by the opportunistic pathogen Staphylococcus aureus (SA). SA exploits several strategies to evade the immune response and subvert bone homeostasis, yet the underlying mechanisms are largely unclear. Here we studied the SA/bone interface with a focus on the soluble pattern recognition molecule long pentraxin 3 (PTX3), which is emerging as a new player in bone physiology and pathology. A murine model of SA intrabone infection was developed that recapitulates surgery/trauma‐OM in humans. Local and systemic infection and inflammation were assessed by means of flow cytometry, RT‐PCR, ELISA, histochemistry, and microCT. At 14 days from SA challenge, >95% of mice developed infection in the injected limb only, which underwent extensive remodeling with loss of trabecular and apposition of periosteal bone. Larger bacterial burdens were found in the bone of WT than in that of PTX3 KO mice at 6 and 14 days from infection. Accordingly, WT animals had more severe inflammation, in terms of expansion of innate immune cells in the spleen, and increase of inflammatory cytokines in the serum. Also, PTX3 levels were augmented in SA‐ infected mice in the serum and bone. Genetic deficiency of PTX3 protects from infection in a murine model of locally‐induced SA‐ OM. Our findings point to an involvement of this long pentraxin in the pathogenesis of OM, and open unforeseen windows on the molecular mechanisms of this disease, with therapeutic and diagnostic potentials

Hepatic fibrosis is a common feature of various liver diseases characterized by activation of hepatic stellate cells (HSC), a principal source of alpha smooth muscle actin (αSMA) liver myofibroblasts. The pathophysiological role of Notch activation has been well established, but the role of Notch1 in activated HSCs is still not sufficiently investigated. In the present research we first used two common murine models of liver fibrosis, carbon tetrachloride (CCL4) treatment for 6 weeks and 0.1% DDC‐supplemented diet for 4 weeks to analyse the expression of Notch‐related genes. In CCL4 model, qPCR analysis showed an upregulation of Notch2, Hey1, HeyL, and Jag2, while DDC‐induced fibrosis was associated with increased expression of Notch2, Notch3, Hey1, Hes1, HeyL, Jag1 and Jag2. In the next set of experiments, we used double transgenic SMACre∆Rbpjκ∆ mice in which Notch1 signalling pathway was specifically inhibited in myofibroblasts by tamoxifen injections during the fibrosis development. Notch1 inhibition, however, did not change the degree of liver fibrosis, as evidenced by no significant difference in histological score on Sirius red liver staining and no difference in tissue expression of COL1A1 and ACTA2 between the control (SMACre‐∆Rbpjκ∆) and Notch1 inhibited (SmaCre+∆Rbpjκ∆) mice. So far, our data show that canonical Notch1 signalling in myofibroblasts is not a crucial contributor to liver fibrosis development in CCL4 and DDC model.

Hepatic fibrosis is a common feature of various liver diseases characterized by activation of hepatic stellate cells (HSC), a principal source of alpha smooth muscle actin (αSMA) liver myofibroblasts. The pathophisiological role of Notch activation has been well established, but the role of Notch pathway in activated HSCs is still not sufficiently investigated. In the present research we first used two common murine models of liver fibrosis, carbon tetrachloride (CCL4) treatment for 6 weeks and 0.1% DDC- supplemented diet for 4 weeks to analyse expression of Notch-related genes. In CCL4 model, PCR analysis showed an upregulation of Notch2, Hey1, HeyL, and Jag2, while DDC-induced fibrosis was associated with increased expression of Notch2, Notch3, Hey1, Hes1, HeyL, Jag1 and Jag2. In the next set of experiments we used double transgenic SmaCre∆Rbpjκ∆ and SmaCreNICD1 mice in which Notch signaling pathway was specifically inhibited or activated in myofibroblasts by tamoxifen injections during the fibrosis development. However, Notch inhibition did not change significantly the degree of fibrosis, as evidenced by similar histological Sirius red liver staining and similar tissue expression of COL1A1 and ACTA2 between the control (SmaCre-∆Rbpjκ∆) and Notch inhibited (SmaCre+∆Rbpjκ∆) mice. Furthermore forcefull activation of Notch in myofibrroblasts did not change the degree of liver foibrosis. So far, our data do not support conclusion that Notch signaling in myofibroblasts contribute to liver fibrosis development in CCL4 and DDC model.

Background: Systemic and periarticular bone loss in rheumatoid arthritis (RA) is mediated by osteoclasts, multinucleated cells originating from the myeloid lineage. Recently, Notch signaling pathway has emerged as a potential regulator of osteoclast progenitor (OCP) differentiation and activation. Objectives: The exact role of Notch signaling in the context of arthritis is still unknown ; however, its inhibition has beneficial effects in animal arthritis models. We aimed to determine the expression of Notch receptors and ligands on specific OCP subpopulations and define changes that occur in murine collagen-induced arthritis (CIA) and RA patients. Methods: Peripheral blood, synovial tissue and subchondral bone marrow were collected from RA patients, and periarticular bone marrow (PBM) and spleen (SPL) were harvested from male C57/Bl6 mice immunized with chicken type II collagen. Notch 1 to 4 receptor expression on OCPs was analyzed by flow cytometry. Gene expression of Notch receptors/ligands was determined by qPCR from tissues and sorted OCPs. Sorted OCPs were cultured, with addition of MCSF and RANKL, in control, IgG, Jagged (Jag) 1 or Delta (DLL) 1 coated wells. Immunohistochemistry (IHC) for Notch 1 and 2 was performed on sections of murine hind paws. Research was approved by Ethics committee. Results: We previously identified peripheral and periarticular subpopulations of murine and human OCPs, as CD45+CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ and CD45+ CD3-CD19-CD56-CD11b+CD14+CCR2+ respectively, specifically associated with arthritis. Flow cytometry revealed that majority of murine splenic and periarticular OCPs express Notch 2, whereas Notch 1 and 4 were expressed on approximately 10% of cells. In CIA, this highly osteoclastogenic population is expanded as is the expression of Notch 4 in PBM and Notch 3 in SPL. Majority of human peripheral-blood OCPs express Notch 2 and 4, with a specific increase in the expression of Notch 1 and 3 in RA. In contrast, RA synovial-derived OCPs mostly express Notch 1 to 3, whereas subchondral OCPs mostly express Notch 1 and 4. Notch ligands were analyzed at mRNA level and revealed expression of Jag1, Jag2 and DLL4 in murine sorted OCPs and Jag1 and DLL1 in human sorted OCPs. Expression of Notch 1 and 2 was confirmed by IHC on arthritic murine hind paws, with Notch 2 expressed by bone marrow, synovial tissue and chondrocytes and Notch 1 expressed by chondrocytes and synovial tissue. Increased expression of Notch 1, Notch 2 and Jag1 was also confirmed in murine arthritic periarticular tissue by qPCR. During osteoclastogenic culture, murine and human OCPs exhibit a similar gene expression pattern with higher initial expression of Notch 1 and 2, and increase in the expression of Notch 3 and 4 with differentiation. Osteoclasts were also differentiated under Notch-ligand stimulation. Coating with DLL1 results in a greater number of cells expressing osteoclast- specific TRAP, whereas Jag1 seemed to inhibit osteoclastogenesis. Conclusion: Our results indicate that murine and human OCPs express a distinct tissue-specific pattern of Notch receptors. Notch signaling in OCPs is increased in arthritis and may contribute to the osteoclastogenic potential and increased bone resorption. Our next aim would be to determine the effect of Notch inhibition on OCP activity and arthritis severity.

Hepatic fibrosis is a common feature of various liver diseases characterized by activation of hepatic stellate cells (HSC), a principal source of alpha smooth muscle actin (αSMA) liver myofibroblasts. Recent studies suggested a possible role of Notch signaling pathway in pathogenesis of fibrosis. In our research, we have studied the expression of Notch-related molecules during fibrogenesis and analyzed contribution of various αSMA+ cell populations to the pool of liver myofibroblasts. Common murine model of liver fibrosis, 6- week carbon tetrachloride (CCl4) treatment, was used. PCR analysis of whole liver tissue showed upregulation of genes for Hey1, HeyL, Notch2 and Jag2, while downregulation of Notch1 and Notch4, and upregulation of Notch2 was shown on isolated HSCs. Notch1 downregulation in HSCs was further confirmed by flow cytometry where it was shown that 42.3% of freshly isolated HSCs from normal mice express Notch1, whereas only 13.9% of those from fibrotic liver express it. We used tamoxifen inducible Cre mice (αSMA- CreERT2/Ai9) to assess contribution of various αSMA+ cell populations to myofibroblast pool. In normal, nonfibrotic liver, only vascular smooth muscle cells (VSMCs) were labeled after tamoxifen application. In further experiments, tamoxifen was given either before (to label VSMCs) or after (to label activated HSCs and VSMCs) the initiation of CCl4 treatment. Immunohistochemical analysis excluded VSMCs as a major source of myofibroblasts in fibrosis and confirmed that majority of myofibroblasts stem from activated HSCs. In the upcoming experiments we aim to modulate Notch signaling pathway specifically in αSMA+ cells to clarify its importance in fibrotic processes.

INTRODUCTION: Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by insulin deficiency, hyperglycemia and metabolic disturbances. Chemokines contribute to low-grade inflammation, which predisposes to the development of vascular complications. Our aim was to determine the chemokine receptor phenotype on peripheral blood mononuclear cells (PBMCs) in children with T1D. METHODS: Mononuclear cells were isolated from peripheral blood of healthy controls (n=13) and children with T1D (n=22). B-cell (CD19+), T-cell (CD3+) and monocyte (CD14+) phenotype was determined using flow cytometry for the following chemokine receptors: CCR2, CCR4, CXCR3 and CXCR4. Frequencies of PBMC subpopulations expressing chemokine receptors were correlated with clinical parameters indicating disease activity (HbA1c and insulin dose), vascular complications (blood pressure and lipid profile) and inflammation (CRP and fibrinogen). RESULTS: Major immune population frequencies did not differ between groups, however there was a decrease of CCR2+ monocytes in T1D group (p=0.019) as well as expansion, though nonsignificant, of CCR4+ T-cells. Expression of CXCR3 and CXCR4 on PBMCs was similar among T1D patients and controls. Monocytes expressing CCR2 were negatively associated with HbA1c levels (ρ=-0.459) and T-cells expressing CCR4 were negatively associated with systolic blood pressure (ρ=-0.389). CONCLUSION: Because of the migratory role of CCR2, we propose the decrease in CCR2+ monocyte subpopulation is due to the increased peripheral sequestration that contributes to the disease pathogenesis and its vascular complications. In contrast, T-cells expressing CCR4 may have a protective role and are therefore, decreased, in children with vascular complications. SUPPORT: Croatian Science Foundation projects IP- 2018-01-2414, IP-2014-09-7406 and DOK2018-09-4276.

Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune joint disease characterized by subchondral bone destruction not reversible by currently available therapeutics. We have shown that mice deficient for Fas gene (Fas-/-) are protected from local bone resorption in antigen-induced arthritis (AIA), a murine model of RA, and have a lower frequency of synovial myeloid cells, which downregulate Mid1 gene. The objective of the study was to evaluate the role of Mid1 in bone resorption in AIA. Materials and methods: After receiving ethical approval, arthritis was induced by immunization of mice with methylated bovine serum albumin (mBSA) with subsequent intra articular injection of mBSA. Synovial myeloid (CD11b + Gr- 1 +) cell transcriptome was analyzed by Affymetrix ST 2.0 arrays. Bioinformatics analysis was performed using Bioconductor. Differences in gene expression were confirmed by qRT-PCR. WT-AIA mice were treated in vivo with metformin, which inhibits proinflammatory effect of Mid1, at daily dose 1 g/kg, to assess effects on arthritis development. Results: Mid1 gene was up-regulated in myeloid cells (logFC = 2.01, p(BH-adjusted) = 0.0003, limma BH-adjustment) and bulk joint tissue (logFC = 8.74, p = 0.02, Welch-test) of WT mice in comparison to Fas-/- mice with non-resorptive arthritis. Despite its position on X chromosome Mid1 expression in joints was not sexually dimorphic and was up-regulated in WT- AIA in both male (logFC = 1.92, p = 0.006, T-test) and female mice (logFC = 8.74, p = 0.02, Welch-test). Furthermore, expression positively correlated with knee diameter (r = 0.68, p = 0.03, Spearman's rank correlation) and levels of pro-inflammatory cytokines in arthritis joints (IL-1: r = 0.78, p = 0.008 ; IL-6: r = 0.70, p = 0.025 ; TNF:r = 78, p = 0.008, Spearman's rank correlation). Metformin treatment of WT-AIA mice ameliorated the severity of arthritis assessed by knee diameter (3.69 ± 0.21 mm WT-AIA vs. 3.36 ± 0.12 mm WT-AIA ? metformin, p = 0.008, T-test). Conclusions: Mid1 is a novel mediator of subchondral bone destruction in arthritis and its inhibition might present a new therapeutic target for inflammation-mediated joint destruction.

Introduction: The exact effects of acute inflammation on the liver apoptotic processes are still not well elucidated. We explored the nature of inflammation- apoptosis crosstalk in liver and to evaluate underlying mechanisms. Methods: Male C57BL/6 mice were treated with ruxolitinib, selective JAK1/2 inhibitor, or vehicle followed by intraperitoneal injection of LPS (0.025 µg/g) 2 hours after. Liver samples were collected after 90 minutes for determination of phosphorylated Stat3 (pStat3) and Stat3 expressions by western blot. The gene expression of proinflammatory cytokines and apoptosis-related factors were analyzed (qPCR). To explore the possible involvement of JAK/Stat3 pathway in the anti-apoptotic effect of the inflammation, mice were treated with ruxolitinib or vehicle, followed by LPS and anti-Fas (JO2) activating antibody (0.25 μg/g, intravenously) 2 hours after LPS. Samples were collected 6 hours after anti-Fas application. Results: LPS treatment induced the phosphorylation of Stat3 in hepatocytes. Immunoblots revealed an increase in pStat3 expression in liver samples after LPS treatment, while immunohistochemical analysis showed an increase in pStat3 positive hepatocytes when compared to saline treated mice. Ruxolitinib pretreatment successfully inhibited the LPS-induced Stat3 phosphorilation. Densitometry showed lower pStat3/Stat3 ratio in ruxolitinib/LPS treated mice in comparison with vehicle/LPS group (p=0.008). Finally, in-vivo experiments showed that ruxolitinib pretreatment abrogates the anti-apoptotic effect of LPS. Ruxolitinib/LPS/anti-Fas treated mice had significantly higher levels of ALT when compared with vehicle/LPS/anti-Fas treated group. Finally, ruxolitinib pretreatment augmented the LPS-induced gene expression of proinflammatory cytokines (TNF- alpha, IL-1 and IL-6), and significantly reduced the expression of Bcl-xL in comparison with LPS treated mice (p=0.02). Conclusion: Acute inflammation induced by LPS protects mice from Fas-mediated liver injury. Stat3 could be one of the crucial mediators involved in this protective effect, as ruxolitinib-induced inhibition of JAK signaling significantly reversed the anti-apoptotic feature of LPS.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic autoimmune joint disease which often causes structural joint damage not reversible by currently available therapeutics, urging discovery of new molecular mediators for therapeutic targeting. In a murine model of RA, antigen- induced arthritis(AIA) we have previously shown that destructive AIA is characterized by accumulation of synovial myeloid cells which up- regulate Mid1 gene. Due to the previously described involvement of Mid1 in pathogeneses of immune-mediated diseases, the objective of this study was to evaluate the role of Mid1 in AIA. MATERIALS AND METHODS: AIA was induced in C57BL6 mice by immunization with methylated bovine serum albumin (mBSA) and subsequent intra-articular injection of mBSA. Expression of Mid1 and pro- inflammatory cytokines was assessed by qRT-PCR. Mice were treated with metformin, which prevents Mid1-mediated ubiquitinilation of PP2A, and thus has an anti-inflamatory effect, at a daily dose of 1g/kg, to evaluate the effects of blocking the Mid1-mediated inflammation on arthritis development and subchondral bone resorption (assesed by μ-CT). RESULTS: Mid1 was up-regulated in knee joints early after arthritis induction. Its expression positively correlated with severity of arthritis assesed by knee diameter measurement and levels of pro-inflammatory cytokines, IL-1, IL-6 and TNF. Although Mid1 is positioned on the X-chromosome, its expression was up regulated in arthritic joints of both male and female mice. Metformin treatment ameliorated the severity of arthritis and arthritis-induced subchondral bone resorption. CONCLUSION: Inhibition of Mid1- mediated PP2A degradation by metformin might aid to therapeutic management of inflammatory arthritides.