Abstract
The transmembrane aminopeptidase CD13 is highly expressed in cells of the myeloid lineage, regulates dynamin-dependent receptor endocytosis and recycling and is a necessary component of ...actin cytoskeletal organization. Here, we show that CD13-deficient mice present a low bone density phenotype with increased numbers of osteoclasts per bone surface, but display a normal distribution of osteoclast progenitor populations in the bone marrow and periphery. In addition, the bone formation and mineral apposition rates are similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Lack of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells containing remarkably high numbers of nuclei. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP1 must be downregulated for fusion to proceed, these are aberrantly sustained at high levels even in CD13-deficient mature multi-nucleated osteoclasts. Further, the stability of fusion-promoting proteins is maintained in the absence of CD13, implicating CD13 in protein turnover mechanisms. Together, we conclude that CD13 may regulate cell–cell fusion by controlling the expression and localization of key fusion regulatory proteins that are critical for osteoclast fusion.
The peripheral blood (PB) monocyte pool contains osteoclast progenitors (OCPs), which contribute to osteoresorption in inflammatory arthritides and are influenced by the cytokine and chemokine ...milieu. We aimed to define the importance of chemokine signals for migration and activation of OCPs in rheumatoid arthritis (RA) and psoriatic arthritis (PsA).
PB and, when applicable, synovial fluid (SF) samples were collected from 129 patients with RA, 53 patients with PsA, and 110 control patients in parallel to clinical parameters of disease activity, autoantibody levels, and applied therapy. Receptors for osteoclastogenic factors (CD115 and receptor activator of nuclear factor-κB RANK) and selected chemokines (CC chemokine receptor 1 CCR1, CCR2, CCR4, CXC chemokine receptor 3 CXCR3, CXCR4) were determined in an OCP-rich subpopulation (CD3
CD19
CD56
CD11b
CD14
) by flow cytometry. In parallel, levels of CC chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, CXC chemokine ligand 9 (CXCL9), CXCL10, and CXCL12 were measured using cytometric bead array or enzyme-linked immunosorbent assay. Sorted OCPs were stimulated in culture by macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, and they were differentiated into mature osteoclasts that resorb bone. Selected chemokines (CCL2, CCL5, CXCL10, and CXCL12) were tested for their osteoclastogenic and chemotactic effects on circulatory OCPs in vitro.
The OCP population was moderately enlarged among PB cells in RA and correlated with levels of tumor necrosis factor-α (TNF-α), rheumatoid factor, CCL2, and CCL5. Compared with PB, the RANK
subpopulation was expanded in SF and correlated with the number of tender joints. Patients with PsA could be distinguished by increased RANK expression rather than total OCP population. OCPs from patients with arthritis had higher expression of CCR1, CCR2, CCR4, CXCR3, and CXCR4. In parallel, patients with RA had increased levels of CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10, with significant elevation in SF vs PB for CXCL10. The subset expressing CXCR4 positively correlated with TNF-α, bone resorption marker, and rheumatoid factor, and it was reduced in patients treated with disease-modifying antirheumatic drugs. The CCR4
subset showed a significant negative trend during anti-TNF treatment. CCL2, CCL5, and CXCL10 had similar osteoclastogenic effects, with CCL5 showing the greatest chemotactic action on OCPs.
In our study, we identified distinct effects of selected chemokines on stimulation of OCP mobilization, tissue homing, and maturation. Novel insights into migratory behaviors and functional properties of circulatory OCPs in response to chemotactic signals could open ways to new therapeutic targets in RA.
Pentraxin 3 (PTX3) is an inflammatory mediator acting as a fluid-phase pattern recognition molecule and playing an essential role in innate immunity and matrix remodeling. Inflammatory mediators also ...contribute to skeletal homeostasis, operating at multiple levels in physiological and pathological conditions. This study was designed to investigate the role of PTX3 in physiological skeletal remodeling and bone healing. Micro-computed tomography (μCT) and bone histomorphometry of distal femur showed that PTX3 gene-targeted female and male mice (
) had lower trabecular bone volume than their wild-type (
) littermates (BV/TV by μCT: 3.50 ± 1.31 vs 6.09 ± 1.17 for females,
< 0.0001; BV/TV 9.06 ± 1.89 vs 10.47 ± 1.97 for males,
= 0.0435). In addition, μCT revealed lower trabecular bone volume in second lumbar vertebra of
mice. PTX3 was increasingly expressed during osteoblast maturation
and was able to reverse the negative effect of fibroblast growth factor 2 (FGF2) on osteoblast differentiation. This effect was specific for the
-terminal domain of PTX3 that contains the FGF2-binding site. By using the closed transversal tibial fracture model, we found that
female mice formed significantly less mineralized callus during the anabolic phase following fracture injury compared to
mice (BV/TV 17.05 ± 4.59 vs 20.47 ± 3.32,
= 0.0195). Non-hematopoietic periosteal cells highly upregulated PTX3 expression during the initial phase of fracture healing, particularly CD51
and αSma
osteoprogenitor subsets, and callus tissue exhibited concomitant expression of PTX3 and FGF2 around the fracture site. Thus, PTX3 supports maintenance of the bone mass possibly by inhibiting FGF2 and its negative impact on bone formation. Moreover, PTX3 enables timely occurring sequence of callus mineralization after bone fracture injury. These results indicate that PTX3 plays an important role in bone homeostasis and in proper matrix mineralization during fracture repair, a reflection of the function of this molecule in tissue homeostasis and repair.
Osteoclasts, macrophages and dendritic cells (DCs) can be derived from a common trilineage myeloid progenitor of hematopoietic origin. Progenitor commitment is susceptible to regulation through Notch ...signaling. Our aim was to determine the effects of Notch modulation on trilineage progenitor commitment and functional properties of differentiated cells under inflammatory conditions. We used the conditional inducible CX3CR1CreERT2 mouse strain to achieve overexpression of the Notch 1 intracellular domain (NICD1) or to inhibit Notch signaling
via
deletion of the transcription factor RBP-J in a bone marrow population, used as a source of the trilineage progenitor (CD45
+
Ly6G
−
CD3
−
B220
−
NK1.1
−
CD11b
–/lo
CD115
+
). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced
in vitro
by 4-hydroxytamoxifen. Differentiation of osteoclasts was induced by M-CSF/RANKL; macrophages by M-CSF; DCs by IL-4/GM-CSF, and inflammation by LPS. Functionally, DCs were tested for the ability to process and present antigen, macrophages to phagocytose
E. coli
particles, and osteoclasts to resorb bone and express tartrate-resistant acid phosphatase (TRAP). We found that Notch 1 signal activation suppressed osteoclast formation, whereas disruption of the Notch canonical pathway enhanced osteoclastogenesis, resulting in a higher number and size of osteoclasts. RANK protein and
Ctsk
gene expression were upregulated in osteoclastogenic cultures from RBP-J
+
mice, with the opposing results in NICD1
+
mice. Notch modulation did not affect the number of
in vitro
differentiated macrophages and DCs. However, RBP-J deletion stimulated
Il12b
and
Cd86
expression in macrophages and DCs, respectively. Functional assays under inflammatory conditions confirmed that Notch silencing amplifies TRAP expression by osteoclasts, whereas the enhanced phagocytosis by macrophages was observed in both NICD1
+
and RBP-J
+
strains. Finally, antigen presentation by LPS-stimulated DCs was significantly downregulated with NICD1 overexpression. This experimental setting allowed us to define a cell-autonomous response to Notch signaling at the trilineage progenitor stage. Although Notch signaling modulation affected the activity of all three lineages, the major effect was observed in osteoclasts, resulting in enhanced differentiation and function with inhibition of canonical Notch signaling. Our results indicate that Notch signaling participates as the negative regulator of osteoclast activity during inflammation, which may be relevant in immune and bone diseases.
Introduction
The existence of different osteoclast progenitor (OCP) subsets has been confirmed by numerous studies. However, pathological inflammation-induced osteoclastogenesis remains incompletely ...understood. Detailed characterization of OCP subsets may elucidate the pathophysiology of increased osteoclast activity causing periarticular and systemic bone resorption in arthritis. In our study, we rely on previously defined OCP subsets categorized by the level of CCR2 expression as circulatory-like committed CCR2
hi
OCPs, which are substantially expanded in arthritis, and marrow-resident CCR2
lo
OCPs of immature phenotype and behavior.
Methods
In order to perform transcriptome characterization of those subsets in the context of collagen-induced arthritis (CIA), we sorted CCR2
hi
and CCR2
lo
periarticular bone marrow OCPs of control and arthritic mice, and performed next-generation RNA sequencing (n=4 for each group) to evaluate the differential gene expression profile using gene set enrichment analysis with further validation.
Results
A disparity between CCR2
hi
and CCR2
lo
subset transcriptomes (863 genes) was detected, with the enrichment of pathways for osteoclast differentiation, chemokine and NOD-like receptor signaling in the CCR2
hi
OCP subset, and ribosome biogenesis in eukaryotes and ribosome pathways in the CCR2
lo
OCP subset. The effect of intervention (CIA) within each subset was greater in CCR2
hi
(92 genes) than in CCR2
lo
(43 genes) OCPs. Genes associated with the osteoclastogenic pathway (
Fcgr1
,
Socs3
), and several genes involved in cell adhesion and migration (
F11r
,
Cd38
,
Lrg1
) identified the CCR2
hi
subset and distinguish CIA from control group, as validated by qPCR (n=6 for control mice, n=9 for CIA mice). The latter gene set showed a significant positive correlation with arthritis clinical score and frequency of CCR2
hi
OCPs. Protein-level validation by flow cytometry showed increased proportion of OCPs expressing F11r/CD321, CD38 and Lrg1 in CIA, indicating that they could be used as disease markers. Moreover, osteoclast pathway-identifying genes remained similarly expressed (
Fcgr1
) or even induced by several fold (
Socs3
) in preosteoclasts differentiated
in vitro
from CIA mice compared to pre-cultured levels, suggesting their importance for enhanced osteoclastogenesis of the CCR2
hi
OCPs in arthritis.
Conclusion
Our approach detected differentially expressed genes that could identify distinct subset of OCPs associated with arthritis as well as indicate possible therapeutic targets aimed to modulate osteoclast activity.
Purpose: To investigate the aqueous humor and serum levels of selected cytokines and vascular endothelial growth factor (VEGF) in diabetic patients, implicating their role in the pathogenesis of ...diabetic eye complications.
Materials and methods: Atotal of 65 patients (27 males and 38 females) who underwent cataract surgery were recruited into the study. The study group consisted of 30 cataract patients with type 2 diabetes mellitus, and this group was divided into two subgroups: 14 patients with diabetic retinopathy (DR group) and 16 patients without DR (NDR group). The control group consisted of 35 non-diabetic cataract subjects.
Results: Patients in the DR group had significantly higher aqueous humor concentrations of interleukin (IL)-1β, IL-6, IL-8, IL-10, monocyte chemotactic protein (MCP-1) and VEGF. Likewise, serum concentrations of IL-1β, IL-6, IL-8, IL-12, TNF-α and IFN-γ were significantly higher in the DR group as compared to the controls. Aqueous humor concentrations of IL-1β, IL-8, MCP-1 and VEGF were significantly higher in the DR group as compared with the NDR group.
Conclusion: Our findings support the hypothesis that chronic inflammation and a disturbance of the immune system play important roles in the pathogenesis of diabetic cataract and DR.
The inflammatory milieu favors recruitment and activation of osteoclasts, and leads to bone destruction as a serious complication associated with arthritis and with other inflammatory processes. The ...frequency and activity of osteoclast progenitors (OCPs) correspond to arthritis severity, and may be used to monitor disease progression and bone resorption, indicating the need for detailed characterization of the discrete OCP subpopulations. Collectively, current studies suggest that the most potent murine bone marrow OCP population can be identified among lymphoid negative population within the immature myeloid lineage cells, as B220
−
CD3
−
CD11b
–/lo
CD115
+
CD117
+
CX3CR1
+
and possibly also Ter119
−
CD11c
−
CD135
lo
Ly6C
+
RANK
−
. In peripheral blood the OCP population bears the monocytoid phenotype B220
−
CD3
−
NK1.1
−
CD11b
+
Ly6C
hi
CD115
+
CX3CR1
+
, presumably expressing RANK in committed OCPs. Much less is known about human OCPs and their regulation in arthritis, but the circulating OCP subset is, most probably, comprised among the lymphoid negative population (CD3
−
CD19
−
CD56
−
), within immature monocyte subset (CD11b
+
CD14
+
CD16
−
), expressing receptors for M-CSF and RANKL (CD115
+
RANK
+
). Our preliminary data confirmed positive association between the proportion of peripheral blood OCPs, defined as CD3
−
CD19
−
CD56
−
CD11b
+
CD14
+
, and the disease activity score (DAS28) in the follow-up samples from patients with psoriatic arthritis receiving anti-TNF therapy. In addition, we reviewed cytokines and chemokines which, directly or indirectly, activate OCPs and enhance their differentiation potential, thus mediating osteoresorption. Control of the activity and migratory behaviour of OCPs as well as the identification of crucial bone/joint chemotactic mediators represent promising therapeutic targets in arthritis.
Detailed characterization of medullary and extramedullary reservoirs of osteoclast progenitors (OCPs) is required to understand the pathophysiology of increased periarticular and systemic bone ...resorption in arthritis. In this study, we focused on identifying the OCP population specifically induced by arthritis and the role of circulatory OCPs in inflammatory bone loss. In addition, we determined the relevant chemokine axis responsible for their migration, and targeted the attraction signal to reduce bone resorption in murine collagen-induced arthritis (CIA). OCPs were expanded in periarticular as well as circulatory compartment of arthritic mice, particularly the CCR2
hi
subset. This subset demonstrated enhanced osteoclastogenic activity in arthritis, whereas its migratory potential was susceptible to CCR2 blockade
in vitro
. Intravascular compartment of the periarticular area contained increased frequency of OCPs with the ability to home to the arthritic bone, as demonstrated
in vivo
by intravascular staining and adoptive transfer of splenic LysMcre/Ai9 tdTomato-expressing cells. Simultaneously, CCL2 levels were increased locally and systemically in arthritic mice. Mouse cohorts were treated with the small-molecule inhibitor (SMI) of CCR2 alone or in combination with methotrexate (MTX). Preventive CCR2/CCL2 axis blockade
in vivo
reduced bone resorption and OCP frequency, whereas combining with MTX treatment also decreased disease clinical score, number of active osteoclasts, and OCP differentiation potential. In conclusion, our study characterized the functional properties of two distinct OCP subsets in CIA, based on their CCR2 expression levels, implying that the CCR2
hi
circulatory-like subset is specifically induced by arthritis. Signaling through the CCL2/CCR2 axis contributes to OCP homing in the inflamed joints and to their increased osteoclastogenic potential. Therefore, addition of CCL2/CCR2 blockade early in the course of arthritis is a promising approach to reduce bone pathology.
Gastric cancer remains one of the deadliest types of cancer despite the improvements in therapy regimens. The knowledge about specific molecular and histopathologic features of gastric tumors leads ...toward targeted therapy protocols. Patients with microsatellite instability-high cancer exhibit special characteristics regarding the response to therapy. These patients benefit from specific immunotherapy regimens and respond poorly to conventional therapy. Our study aimed to evaluate the effect of chemotherapy and chemoradiotherapy on the survival of patients with microsatellite instability gastric cancer in our institution. The results of our study show an increase in survival in the microsatellite instability-high group of patients who received radiotherapy (p=0.04). Since all the recent studies recommend radiotherapy only in patients with incompletely resected disease or those with less than D2 lymphadenectomy, our study offers a novelty suggestion in consideration of therapeutic options. If the benefit of radiotherapy can be confirmed in a larger sample of patients, radiotherapy could be a part of tailored therapy for microsatellite instability-high (MSI-H) gastric cancer.
The biological effects of drug vehicles are often overlooked, often leading to artifacts in acetaminophen-induced liver injury assessment. Therefore, we decided to investigate the effect of ...dimethylsulfoxide, dimethylformamide, propylene glycol, ethanol, and Tween 20 on acetaminophen-induced liver injury. C57BL/6 male mice received a particular drug vehicle (0.6 or 0.2 mL/kg, i.p.) 30 min before acetaminophen administration (300 mg/kg, i.p.). Control mice received vehicle alone. Liver injury was assessed by measuring the concentration of alanine aminotransferase in plasma and observing histopathological changes. The level of reduced glutathione (GSH) was assessed by measuring total nonprotein hepatic sulfhydrils. Dimethylsulfoxide and dimethylformamide (at both doses) almost completely abolished acetaminophen toxicity. The higher dose of propylene glycol (0.6 mL/kg) was markedly protective, but the lower dose (0.2 mL/kg) was only slightly protective. These solvents also reduced acetaminophen-induced GSH depletion. Dimethylformamide was protective when given 2 h before or 1 h after acetaminophen administration, but was ineffective if given 2.5 h after acetaminophen. Ethanol at the higher dose (0.6 mL/kg) was partially protective, whereas ethanol at the lower dose (0.2 mL/kg) as well as Tween 20 at any dose had no influence. None of the vehicles (0.6 mL/kg) was hepatotoxic per se, and none of them was protective in a model of liver injury caused by
d
-galactosamine and lipopolysaccharide.
Celotno besedilo
Dostopno za:
DOBA, FSPLJ, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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