Macrophage activation and inflammatory priming by anti-MAA antibodies in rheumatoid arthritis.

We studied the effects of rheumatoid arthritis (RA) autoantibodies that target malondialdehyde-acetaldehyde protein adducts (anti-MAA) on inflammation and macrophage functions. We detected a profound reprogramming of gene expressions and the production of chemokines, such as CCL22 and CCL24, in anti-MAA exposed macrophages. Moreover, anti-MAA pretreatment promoted a more inflammatory cytokine profile upon TLR activation. Although anti-MAA are typically multi-reactive, we observed a prominent clonal diversity in inducing macrophage activation. Anti-MAA antibodies were not arthritogenic in mice, but altered a set of cytokine and growth factor encoding genes in the joints. In individuals at risk of RA anti-MAA IgG levels correlated with circulating inflammatory mediators prior to and at arthritis onset. Certain IgG anti-MAA clones may thus contribute to an inflammatory priming of the joint prior to the onset of systemic inflammation via inducing FcγR-mediated macrophage pre-activation and setting the stage for augmented responses to subsequent inflammatory stimuli.

Inhibition of Notch Signaling Stimulates Osteoclastogenesis From the Common Trilineage Progenitor Under Inflammatory Conditions.

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-/loCD115+). 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.

Tamoxifen Ameliorates Cholestatic Liver Fibrosis in Mice: Upregulation of TGFß and IL6 Is a Potential Protective Mechanism.

The available treatments for cholestatic liver fibrosis are limited, and the disease often progresses to liver cirrhosis. Tamoxifen is a selective modulator of estrogen receptors, commonly used in breast cancer therapy. A recent in vitro study showed that tamoxifen deactivates hepatic stellate cells, suggesting its potential as an antifibrotic therapeutic, but its effects in vivo remain poorly investigated. In the present study, we show that tamoxifen protects against the cholestatic fibrosis induced by a diet supplemented with 0.025% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Mice fed with a DDC-supplemented diet for four weeks and treated with tamoxifen developed a significantly milder degree of liver fibrosis than vehicle-treated mice, as evidenced by a lower percentage of Sirius red-stained area (60.4% decrease in stained area in male and 42% decrease in female mice, p < 0.001 and p < 0.01, respectively) and by lower hydroxyproline content. The finding was further confirmed by qPCR analysis, which showed a lower expression of genes for Col1a1, Acta2, Sox9, Pdgf, and Krt19, indicating the inhibitory effect on hepatic stellate cells, collagen production, and biliary duct proliferation. The degree of protection was similar in male and female mice. Tamoxifen per se, injected into standard-diet-fed mice, increased the expression of genes for Il6 (p < 0.01 and p < 0.001 in male and female mice, respectively) and Tgfß (p < 0.01 for both sexes), and had no adverse effects. We showed that tamoxifen sex-independently protects against cholestatic DDC-induced liver fibrosis. The increased expression of Il6 and Tgfß seems to be a plausible protective mechanism that should be the primary focus of further research.

Transcriptome profiling of osteoclast subsets associated with arthritis: A pathogenic role of CCR2hi osteoclast progenitors.

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 CCR2hi OCPs, which are substantially expanded in arthritis, and marrow-resident CCR2lo 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 CCR2hi and CCR2lo 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 CCR2hi and CCR2lo subset transcriptomes (863 genes) was detected, with the enrichment of pathways for osteoclast differentiation, chemokine and NOD-like receptor signaling in the CCR2hi OCP subset, and ribosome biogenesis in eukaryotes and ribosome pathways in the CCR2lo OCP subset. The effect of intervention (CIA) within each subset was greater in CCR2hi (92 genes) than in CCR2lo (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 CCR2hi 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 CCR2hi 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 CCR2hi 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.

Preventive CCL2/CCR2 Axis Blockade Suppresses Osteoclast Activity in a Mouse Model of Rheumatoid Arthritis by Reducing Homing of CCR2hi Osteoclast Progenitors to the Affected Bone.

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 CCR2hi 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 CCR2hi 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.

RNA sequencing data from osteochondroprogenitor populations in synovial joints of mice during murine model of rheumatoid arthritis.

The aim of this study was to analyze the transcriptome of TER119-CD31-CD45-CD51+CD200+CD105- population (further, CD200+), potential early osteocondroprogenitors, whose frequency is reduced in the joints of mice with antigen-induced arthritis (AIA) [1]. A population defined by similar surface markers has been previously identified as murine skeletal stem cells in bone [2]. In order to confirm their identity this population was compared to TER119-CD31-CD45-CD51+CD200-CD105+ (further, CD105+) cells, which possibly represent committed progenitors, or other non-progenitor population such as synovial fibroblasts. In order to asses changes in CD200+ population in inflammatory setting, it was also compared to the same population from healthy mice. AIA was induced by immunization of mice with methylated bovine serum albumin (mBSA) and subsequent intra-articular injection of mBSA, while non-immunized mice were injected with phosphate-buffered saline at all timepoints. Ten days after intra-articular injection, knee joints were harvested and synovial cells were released by collagenase digestion. Using fluorescence-activated cell sorting, 200-500 cells from selected populations were sorted directly into cell lysis buffer, RNA was reversely transcribed, and first strand cDNA product was amplified. cDNA amplicons were used for library preparation. Bioinformatics analysis was performed using cutadapt [3], HISAT2 [4], Samtools [5] and StringTie [6] tools, and egdeR [7], limma [8], and ClusterProfiler [9] Bioconductor packages. In addition to access to raw data at the NCBI Gene Expression Omnibus repository, this article also provides sample similarity analysis, tables of differentially expressed genes, graphic visualisations of differential expression and gene set enrichment analysis performed on publicly available GO terms. Interpretation of osteochondroprogenitor phenotype of CD200+ population based on analysis of presented data is provided in the article "What do we know about bone morphogenetic proteins and osteochondroprogenitors in inflammatory conditions?" [10]. Reuse of this data may help researchers elucidate alterations of synovial stromal and osteochondroprogenitor populations in inflammatory settings and define their role in structural damage in rheumatoid arthritis.

What do we know about bone morphogenetic proteins and osteochondroprogenitors in inflammatory conditions?

Osteochondroprogenitors are crucial for embryonic bone development and postnatal processes such as bone repair in response to fracture injury, and their dysfunction may contribute to insufficient repair of structural damage in inflammatory arthritides. In the fracture healing, the early inflammatory phase is crucial for normal callus development and new bone formation. This process involves a complex interplay of many molecules and cell types, responsible for recruitment, expansion and differentiation of osteochondroprogenitor populations. In inflammatory arthritides, inflammation induces bone resorption and causes insufficient bone formation, which leads to local and systemic bone loss. While bone loss is a predominant feature in rheumatoid arthritis, inflammation also induces pathologic bone formation at enthesial sites in seronegative spondyloarthropathies. Bone morphogenetic proteins (BMP) are involved in cell proliferation, differentiation and apoptosis, and have fundamental roles in maintenance of postnatal bone homeostasis. They are crucial regulators of the osteochondroprogenitor pool and drive their proliferation, differentiation, and lifespan during bone regeneration. In this review, we summarize the effects of inflammation on osteochondroprogenitor populations during fracture repair and in inflammatory arthritides, with special focus on inflammation-mediated modulation of BMP signaling. We also present data in which we describe a population of murine synovial osteochondroprogenitor cells, which are reduced in arthritis, and characterize their expression of genes involved in regulation of bone homeostasis, emphasizing the up-regulation of BMP pathways in early progenitor subset. Based on the presented data, it may be concluded that during an inflammatory response, innate immune cells induce osteochondroprogenitors by providing signals for their recruitment, by producing BMPs and other osteogenic factors for paracrine effects, and by secreting inflammatory cytokines that may positively regulate osteogenic pathways. On the other hand, inflammatory cells may secrete cytokines that interfere with osteogenic pathways, proapoptotic factors that reduce the pool of osteochondroprogenitor cells, as well as BMP and Wnt antagonists. The net effect is strongly context-dependent and influenced by the local milieu of cells, cytokines, and growth factors. Further elucidation of the interplay between inflammatory signals and BMP-mediated bone formation may provide valuable tools for therapeutic targeting.

Notch receptors and ligands in inflammatory arthritis - a systematic review.

BACKGROUND: Notch pathway is highly conserved across species and is involved in the regulation of cell differentiation and activity both in embryonic development and adult life. Notch signaling has an important role in the development of hematopoietic stem cells and their differentiation to committed lineages, as well as in the regulation of several non-hematopoietic cell lines. OBJECTIVE: As Notch signaling has been implicated in various inflammatory and autoimmune diseases, it is of interest to elucidate what role do Notch receptors and ligands have in inflammatory arthritides. METHODS: We performed a search on the role of Notch receptors (1-4) and Notch ligands Delta-like (DLL) 1, 3, 4 and Jagged (Jag) 1 and 2 in animal models of inflammatory arthritis and most common types of human inflammatory arthritis (rheumatoid arthritis, psoriatic arthritis or ankylosing spondylitis). The initial search identified 135 unique articles, of which 24 were ultimately deemed relevant and included in this systematic review. RESULTS: Overall, identified articles describe roles for Notch ligands and receptors in inflammatory arthritis, with Notch activation resulting in enhanced Th1/17 polarization, osteoclast differentiation, macrophage activation and fibroblast-like synoviocyte proliferation. However, the inhibitory role of Notch signaling, especially by Jag1 is also described. CONCLUSION: There is evidence that Notch pathway activation affects multiple cell lineages present within the arthritic environment, therefore potentially acting as one of the drivers of disease pathogenesis. Since cell lineage-selective transgenic mouse models and specific Notch receptor inhibitors are becoming increasingly available, it can be expected that future research will evaluate whether Notch signaling components initiate crucial pathogenic impulses and, therefore, present viable therapeutic targets in inflammatory arthritis.

Exploring causality in the association between circulating 25-hydroxyvitamin D and colorectal cancer risk: a large Mendelian randomisation study.

BACKGROUND: Whilst observational studies establish that lower plasma 25-hydroxyvitamin D (25-OHD) levels are associated with higher risk of colorectal cancer (CRC), establishing causality has proven challenging. Since vitamin D is modifiable, these observations have substantial clinical and public health implications. Indeed, many health agencies already recommend supplemental vitamin D. Here, we explore causality in a large Mendelian randomisation (MR) study using an improved genetic instrument for circulating 25-OHD. METHODS: We developed a weighted genetic score for circulating 25-OHD using six genetic variants that we recently reported to be associated with circulating 25-OHD in a large genome-wide association study (GWAS) meta-analysis. Using this score as instrumental variable in MR analyses, we sought to determine whether circulating 25-OHD is causally linked with CRC risk. We conducted MR analysis using individual-level data from 10,725 CRC cases and 30,794 controls (Scotland, UK Biobank and Croatia). We then applied estimates from meta-analysis of 11 GWAS of CRC risk (18,967 cases; 48,168 controls) in a summary statistics MR approach. RESULTS: The new genetic score for 25-OHD was strongly associated with measured plasma 25-OHD levels in 2821 healthy Scottish controls (P = 1.47 × 10- 11), improving upon previous genetic instruments (F-statistic 46.0 vs. 13.0). However, individual-level MR revealed no association between 25-OHD score and CRC risk (OR 1.03/unit log-transformed circulating 25-OHD, 95% CI 0.51-2.07, P = 0.93). Similarly, we found no evidence for a causal relationship between 25-OHD and CRC risk using summary statistics MR analysis (OR 0.91, 95% CI 0.69-1.19, P = 0.48). CONCLUSIONS: Despite the scale of this study and employing an improved score capturing more of the genetic contribution to circulating 25-OHD, we found no evidence for a causal relationship between circulating 25-OHD and CRC risk. Although the magnitude of effect for vitamin D suggested by observational studies can confidently be excluded, smaller effects sizes and non-linear relationships remain plausible. Circulating vitamin D may be a CRC biomarker, but a causal effect on CRC risk remains unproven.