Presence Of M-CSF Research Articles

P026 Differential effects of tofacitinib on macrophage activation may contribute to lack of response in ulcerative colitis patients

Abstract Background We previously showed that ulcerative colitis (UC) patients refractory to tofacitinib present an increase in the activation and the pro-inflammatory profile of intestinal macrophages1. However, the mechanism by which tofacitinib promotes further activation of macrophages in these group of patients remains unknown. Methods Blood monocytes from healthy individuals were cultured in the presence of M-CSF as previously described2. The differentiated macrophages were treated with LPS (10ng/ml), TNF (20ng/ml) or IFNγ (5ng/ml) in the presence or absence of tofacitinib (300nM). The effect of tofacitinib on gene expression and protein production in response to each stimulus was measured by qPCR and ELISA, respectively. Single-cell RNAseq (scRNAseq) data generated in a cohort of UC patients treated with tofacitinib1 was compared to the bulk transcriptome of monocyte-derived macrophages stimulated with LPS +/- anti-IL-10 mAb3. Results Monocyte-derived macrophages treated with tofacitinib significantly reduced the expression of activation markers downstream from IFNγ or TNFα. In contrast, the response to LPS was either maintained or intensified by tofacitinib as seen by the increase in expression of inflammatory marker genes including chemokines and cytokines CXCL1, CXCL5, IL1B, TNFA and IL6, and M1-like markers INHBA and CLEC5A (Figure 1A). Protein analysis of cell supernatants confirmed these observations. Compared to IFNγ and TNF, LPS drives the production of high concentrations of the anti-inflammatory IL-10 (Figure 1B) which can act on macrophages through a JAK-dependent signaling pathway. We hypothesized that by inhibiting IL-10 autocrine signaling on LPS-activated macrophages tofacitinib would promote their hyperactivation. Indeed, the transcriptional profile of LPS-stimulated macrophages treated with a blocking anti-IL-10 antibody3 highly overlapped with those genes that were upregulated in macrophages from patients refractory to tofacitinib, including IL1B, IL6, INHBA and IDO1 (Figure 1C). This overlap was not observed in responder patients. Conclusion Based on the combination of biopsy scRNAseq from tofacitinib-treated patients and in vitro macrophage cultures, we suggest that lack of response to tofacitinib is driven by its effect on IL-10 signaling. While IL-10 is essential to control macrophage activation when stimulated with the pathogen molecular pattern LPS, it plays no significant role when macrophages are activated with IFNγ or TNF. Hence, we hypothesize that LPS-dependent inflammation may predispose to lack of response to tofacitinib.

DCIR suppresses osteoclastic proliferation and resorption by downregulating M-CSF and RANKL signaling.

Dendritic cell immunoreceptor (DCIR) is an inhibitory C-type lectin receptor that acts as a negative regulator in the immune system and bone metabolism. We previously revealed that DCIR deficiency enhanced osteoclastogenesis and antigen presentation of dendritic cells, and that asialo-biantennary N-glycan (NA2) functions as a ligand for DCIR. NA2 binding to DCIR suppressed murine and human osteoclastogenesis that occurs in the presence of M-CSF and RANKL. The DCIR-NA2 axis, therefore, plays an important role in regulating osteoclastogenesis in both mice and humans, although the underlying mechanisms remain unclear. Here we found that Dcir -/- bone marrow-derived macrophages (BMMs) exhibited greater proliferative and differentiation responses to M-CSF and RANKL, respectively, than wild-type (WT) BMMs. Moreover, Dcir -/- osteoclasts (OCs) increased resorptive activity and cell fusion more significantly than WT OCs. DCIR deficiency affects gene expression patterns in OCs, and we found that the expression of neuraminidase 4 was increased in Dcir -/- OCs. Furthermore, DCIR-NA2 interaction in WT BMMs, but not Dcir -/- BMMs, decreased Akt phosphorylation in response to M-CSF and RANKL. These data suggest that DCIR regulates osteoclastogenesis by downregulating M-CSF and RANKL signaling, and that DCIR-mediated signaling may contribute to the terminal modification of oligosaccharides by controlling the expression of glycosylation enzymes.

Brain-Type Creatine Kinase Release from Cultured Osteoclasts Exposed to Neridronate in Children Affected by Osteogenesis Imperfecta Type 1

Brain-type creatine kinase (CK-BB) increases during osteoclastogenesis, with high circulating amounts in type I osteogenesis imperfecta (OI) following treatment with neridronate, a bisphosphonate able to inhibit osteoclast activity and survival. The aim of this study was to demonstrate the correlation between osteoclastogenesis and CK-BB release from OI patients' osteoclasts treated with different concentrations of neridronate. Our patients showed reduced bone quality, increased levels of CTX I, a marker of bone resorption, and decreased levels of OPG, an inhibitor of osteoclastogenesis. In OI patients, the presence of MCSF and RANKL determined an increased secretion of CK-BB from osteoclasts (p = 0.04) compared with control conditions without these cytokines; interestingly, in the absence of these factors, the secretion of CK-BB is significantly elevated at 3 µmol/L compared with 0.03 and 1 µmol/L (p = 0.007). In healthy donors' cultures, the higher concentration of CK-BB can be detected following stimulation with 3 µmol/L neridronate compared with the untreated condition both with and without MCSF and RANKL (p = 0.03 and p = 0.006, respectively). Consistently, in osteoclast cultures, neridronate treatment is associated with a decrease in multinucleated TRAP+ cells, together with morphology changes typical of apoptosis. Consistently, in the media of the same osteoclast cultures, we demonstrated a significant increase in caspase-3 levels. In conclusion, our findings support the idea that CK-BB levels increase in the serum of OI-treated patients.

Genesis of osteoclasts on calcium phosphate ceramics and their role in material-induced bone formation.

Innate immune responses play important roles in material-induced bone formation and such roles were further explored in the current study with an emphasis on M2 macrophages and osteoclastogenesis. With the presence of M-CSF and RANKL, M0 macrophages from FVB mouse bone marrow-derived monocytes (BMMs) fused to osteoclasts with both M2 marker and osteoclast marker at day 5, and such osteoclast formation at day 5 was enhanced when the cells were treated with IL-4 at day 3. With IL-4 treatment alone for 24 h, M0 polarized into M2 macrophages. Conditioned medium of M2 macrophages enhanced osteogenic differentiation of MC3T3-E1 (pre-osteoblasts) while osteoclast conditioned medium enhanced osteogenic differentiation of CRL-12424 (osteogenic precursors). TCPs (a typical osteoinductive material) supported M2 macrophage polarization at day 4 and osteoclast formation at day 5, while TCPb (a typical non-osteoinductive material) was less effective. Moreover, osteoclasts formed on TCPs produced osteogenic factors including S1P, Wnt10B and BMP-6, resulting osteogenic differentiation of CRL-12424 cells. Similar to in vitro testing, TCPs favored M2 macrophage polarization followed by the formation of osteoclasts in vivo, as compared to TCPb. The overall data provided evidence of a coupling between M2 macrophages, osteoclasts and material-induced bone formation: osteoclasts formed from M2 macrophages secrete osteogenic cytokines to induce osteogenic differentiation of osteogenic precursor cells to finally form bone. The current findings outlined a biological mechanism of material-induced bone formation and further rationalized the use of osteoinductive materials for bone regeneration. STATEMENT OF SIGNIFICANCE: This paper provides evidence for finding out the relationship between M2 macrophages, osteoclasts and osteogenesis in material-induced bone formation. It suggested that osteoinductive materials enhanced macrophage polarization to M2 macrophages which fuses to osteoclasts, osteoclasts subsequently secret osteogenic cytokines to differentiate finally osteogenic precursors to form bone in osteoinductive materials. The data supports scientifically the superiority of osteoinductive materials for bone regeneration in clinics.

TLR7 Activation in M-CSF-Dependent Monocyte-Derived Human Macrophages Potentiates Inflammatory Responses and Prompts Neutrophil Recruitment

Toll-like receptor 7 (TLR7) is an endosomal pathogen-associated molecular pattern (PAMP) receptor that senses single-stranded RNA (ssRNA) and whose engagement results in the production of type I IFN and pro-inflammatory cytokines upon viral exposure. Recent genetic studies have established that a dysfunctional TLR7-initiated signaling is directly linked to the development of inflammatory responses. We present evidence that TLR7 is preferentially expressed by monocyte-derived macrophages generated in the presence of M-CSF (M-MØ). We now show that TLR7 activation in M-MØ triggers a weak MAPK, NFκB, and STAT1 activation and results in low production of type I IFN. Of note, TLR7 engagement reprograms MAFB+ M-MØ towards a pro-inflammatory transcriptional profile characterized by the expression of neutrophil-attracting chemokines (CXCL1-3, CXCL5, CXCL8), whose expression is dependent on the transcription factors MAFB and AhR. Moreover, TLR7-activated M-MØ display enhanced pro-inflammatory responses and a stronger production of neutrophil-attracting chemokines upon secondary stimulation. As aberrant TLR7 signaling and enhanced pulmonary neutrophil/lymphocyte ratio associate with impaired resolution of virus-induced inflammatory responses, these results suggest that targeting macrophage TLR7 might be a therapeutic strategy for viral infections where monocyte-derived macrophages exhibit a pathogenic role.

P231 Targeting the Rho/MRTF-A pathway inhibits growth factor and cytokine release but enhances efferocytosis in scleroderma macrophages

Abstract Background/Aims In the systemic sclerosis (SSc) microenvironment, accumulation of extracellular matrix (ECM) components induces changes in the matrix's mechanical properties, which triggers Rho/MRTFF-A activation and the downstream signalling cascades, inducing the expression of fibrotic and inflammatory genes in fibroblasts. However, macrophages also play a key role in tissue homeostasis and wound repair. It is possible that these cells are also influenced by ECM stiffness and therefore we investigated the effects of altering substrate stiffness and tested the effects of an inhibitor of the mechano-sensing Rho/MRTF-A pathway on the activation state of SSc macrophages. Methods SSc and healthy control macrophages (both n = 4 donors) were derived by culture of blood monocytes in the presence of M-CSF (4ng/ml) for 7 days. Soft substrates (4kPa) or stiff ECM substrates (50kPa) were used to model the effect of soft healthy skin tissue and stiff fibrotic SSc skin (Softwell, Matrigen, polymer gels, collagen I coated). Morphology, gene expression (qPCR for CD86 for M1-like, CD206 for M2-like, MerTK for efferocytosis), Multiplex assay, labelled E. Coli uptake assay and Rho/MRTF/SRF pathway inhibition (small molecule CCG-257081) were used to assess the polarisation and activation state of these cells. Results Morphology and CD86 did not vary with matrix stiffness, but CD206 expression was significantly lower in stiff compared to soft gels (Mean+/- SEM; soft 38.2+/- 4.2 vs stiff 16.2+/-3.7 relative expression, p value 0.0167). CCG-257081 strongly inhibited secretion of cytokines, growth factors and chemokines. Factors present at > 50 pg/ml suppressed by CCG-257081 include TNFα, GM-CSF, IL-1RA, IP-10, RANTES, MIP-1α, MIP-1β, MCP-1, MCP-3, PDGF-BB (all p < 0.05). SSc macrophages secreted at high levels on soft, and were attenuated by culture on stiff, whereas healthy control macrophages showed the opposite trend inducing more secretion on stiff, inhibited by CCG-257081. Although polarisation markers CD206 and CD86 did not alter with the CCG-257081 treatment, there was a significant increase in MerTK with treatment (p < 0.0001). Endocytosis was moderately yet significantly suppressed by the CCG treatment (basal endocytosis 80.2+/- 4.0 vs CCG-257081 treated 62+/-2.5; % endocytosis; p < 0.01), whereas cell viability was not significantly altered. Conclusion Although differences are observed amongst patients, this data indicates that stiffness of the matrices, as used in this study, did not greatly alter the morphology or polarisation of the macrophages. However, transfer to stiff matrices enhanced the secretion of certain cytokines by control macrophages. Strong effects were seen with the Rho/MRTF-A inhibitor, which appeared to block the secretion of multiple factors, including inflammatory cytokines, pro-fibrotic factors, as well as regulatory factors and to strongly induce MerTK, a marker of efferocytosis. One possibility is that the inhibitor converts these cells from secretory to resolving phenotype. Based on these findings, CCG-257081 has promising effects against activated macrophages, which could be utilised in inflammatory fibrotic diseases. Disclosure S. Lopez Garces: None. B. Ahmed Abdi: None. C. MacFadyen: None. R. Smillie: None. L. Nagib: None. S. Ghani: None. S. Shah: None. D. Abraham: None. C. Denton: None. R.J. Stratton: None.

GM-CSF Protects Macrophages from DNA Damage by Inducing Differentiation.

At inflammatory loci, pro-inflammatory activation of macrophages produces large amounts of reactive oxygen species (ROS) that induce DNA breaks and apoptosis. Given that M-CSF and GM-CSF induce two different pathways in macrophages, one for proliferation and the other for survival, in this study we wanted to determine if these growth factors are able to protect against the DNA damage produced during macrophage activation. In macrophages treated with DNA-damaging agents we found that GM-CSF protects better against DNA damage than M-CSF. Treatment with GM-CSF resulted in faster recovery of DNA damage than treatment with M-CSF. The number of apoptotic cells induced after DNA damage was higher in the presence of M-CSF. Protection against DNA damage by GM-CSF is not related to its higher capacity to induce proliferation. GM-CSF induces differentiation markers such as CD11c and MHCII, as well as the pro-survival Bcl-2A1 protein, which make macrophages more resistant to DNA damage.

Inhibitor of protein kinase N3 suppresses excessive bone resorption in ovariectomized mice

The long-term inhibition of bone resorption suppresses new bone formation because these processes are coupled during physiological bone remodeling. The development of anti-bone-resorbing agents that do not suppress bone formation is urgently needed. We previously demonstrated that Wnt5a-Ror2 signaling in mature osteoclasts promoted bone-resorbing activity through protein kinase N3 (Pkn3). The p38 MAPK inhibitor SB202190 reportedly inhibited Pkn3 with a low Ki value (0.004μM). We herein examined the effects of SB202190 on osteoclast differentiation and function in vitro and in vivo. Bone marrow cells were cultured in the presence of M-csf and GST-Rankl to differentiate into multinucleated osteoclasts. Osteoclasts were treated with increasing concentrations of SB202190. For in vivo study, 10-week-old female mice were subjected to ovariectomy (OVX). OVX mice were intraperitoneally administered with a Pkn3 inhibitor at 2mg/kg or vehicle for 4weeks, and bone mass was analyzed by micro-CT. SB202190 suppressed the auto-phosphorylation of Pkn3 in osteoclast cultures. SB202190 significantly inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation. SB202190 reduced c-Src activity in osteoclast cultures without affecting the interaction between Pkn3 and c-Src. A treatment with SB202190 attenuated OVX-induced bone loss without affecting the number of osteoclasts or bone formation by osteoblasts. Our results showed that Pkn3 has potential as a therapeutic target for bone loss due to increased bone resorption. SB202190 is promising as a lead compound for the development of novel anti-bone-resorbing agents.

Histamine Increases Th2 Cytokine-Induced CCL18 Expression in Human M2 Macrophages.

The chemokine CCL18 is produced in cells of the myelomonocytic lineage and represents one of the most highly expressed chemokines in lesional skin and serum of atopic dermatitis patients. We investigated the role of histamine in CCL18 production in human monocyte-derived M2 macrophages differentiated in the presence of M-CSF and activated with IL-4, IL-13 or with IL-10. Since expression and regulation of histamine H1 receptor (H1R), H2R and H4R by IL-4 and IL-13 on human M2 macrophages were described, we analyzed expression of the histamine receptors in response to IL-10 stimulation by quantitative RT-PCR. IL-10 upregulated H2R and downregulated H4R mRNA expression by trend in M2 macrophages. IL-10, but in a more pronounced manner, IL-4 and IL-13, also upregulated CCL18. Histamine increased the cytokine-induced upregulation of CCL18 mRNA expression by stimulating the H2R. This effect was stronger in IL-10-stimulated M2 macrophages where the upregulation of CCL18 was confirmed at the protein level by ELISA using selective histamine receptor agonist and antagonists. The histamine-induced CCL18 upregulation in IL-10-activated M2 macrophages was almost similar in cells obtained from atopic dermatitis patients compared to cells from healthy control persons. In summary, our data stress a new function of histamine showing upregulation of the Th2 cells attracting chemokine CCL18 in human, activated M2 macrophages. This may have an impact on the course of atopic dermatitis and for the development of new therapeutic interventions.

Neoplastic Monocyte-Derived Osteoclasts Are Highly Proliferative, but Sub-Functional in MPN-Associated Myelofibrosis

Background: Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the proliferation of myeloid progenitors. A defining hallmark of MPN-associated myelofibrosis (MF) is the deposition of fibrous tissue inside the bone marrow (BM) stroma, arising either as a primary process (PMF), or secondary to the essential thromocythemia (post-ET MF) or polycythemia vera. In at least 1/3 of cases, BM fibrosis is accompanied by the osteosclerotic thickening of the bone trabeculae. It was generally held that these changes are a reaction of mesenchymal cells to the stimuli from the malignant clone. However, the recent discovery of neoplastic monocyte-derived fibrocytes capable of directly inducing BM fibrosis in situ and in vivo has led us to hypothesize that other tissue remodeling cells of the myeloid lineage may play similar roles in the pathogenesis of MPN-associated MF. Osteoclasts (OC) are giant multinuclear cells formed by the fusion of multiple monocytic progenitors. Apart from being exclusively bone-resorbing cells, OCs have a critical role in regulating bone formation by the mesenchymal cell-derived osteoblasts (OB) and maintaining the hematopoietic BM microenvironment. The aim of this study was to examine the status of these cells in MF patients and explore the effects that the neoplastic OC progenitors exert on the development of MPN-associated osteosclerosis.Methods: In order to quantitate bone remodeling cells, trephine biopsies were obtained from patients with PMF (n = 32) and post-ET MF (n = 27), as well as healthy controls (n = 3). A tyramide signal amplification-based fluorescence IHC approach was employed using TRAP and cathepsin K as OC-specific and CD56and osterix as OB-specific markers. In addition, CD14+ low-density peripheral blood cells from the representative MF patients and healthy controls were cultured in the presence of M-CSF and RANKL. After 3 weeks mature OCs were visualized using fluorescently labeled F-actin and TRAP with DAPI as the nuclear counterstain, and the JAK2V617F allele frequency was assessed via RT-PCR. For the OC functional assay, cells were plated on a mineralized surface, which was subsequently stained using a modified von Kossa method. To assess the OC fusion capacity, two populations of progenitors labeled with different lipophilic probes were mixed in a 1:1 ratio and the colocalized signal was quantified for each mature cell. All imaging was conducted using an automated multispectral or a dual spinning disk confocal system.Results: Based on the multiplex BM imaging analysis, we observed a significant increase in the number of OCs in MF patients compared to healthy controls (median: 21.8 vs. 7.0 per 100 mm of bone perimeter; p < 0.0001) that was followed by a substantial increase in the total number of OBs and the density of trabecular bone. After inducing differentiation of JAK2+ monocytes from MF patients into mature TRAP+ OCs in vitro, these cells consistently showed a high mutant allele frequency. Compared to OCs cultured from healthy monocytes, MF patients' OCs exhibited a 40.5% decrease in the median number of nuclei per cell (p= 0.024) and a tendency to incompletely develop F-actin rings. Simultaneously, there was no change in the mean TRAP positivity between the two groups. Finally, MF OCs were significantly less apt at resorbing mineral-coated areas in contrast to healthy cells (median: 21.5% vs. 7.3%; p= 0.016), and there was a 3-fold decrease in the proportion of fused mature OCs from MF monocytes compared to healthy ones (p= 0.008).Conclusions: The development of OCs is highly skewed towards proliferation in both primary and secondary MPN-associated MF. We were able to demonstrate that neoplastic monocytic progenitors retain their aberrant genetic constitution even after differentiating into mature OCs. However, fusion of such progenitors seems to be profoundly impaired, and MF OCs are unable to fully acquire the phenotypical features associated with efficient bone resorption, particularly multinucleation and the development of actin-rich structures. At least to a certain extent, this process happens independently of the non-malignant stroma, although it seems to be impacting the OBs as well. Taken together, we show evidence that even though MF OCs are hyperproliferative, their function is intrinsically supressed due to the inherited neoplastic burden, which in turn contributes to the osteosclerotic dysplasia of the MPN-affected BM. DisclosuresVerstovsek:Blueprint Medicines Corp: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Galena BioPharma: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Roche: Research Funding; Pfizer: Research Funding; Lilly Oncology: Research Funding; Astrazeneca: Research Funding; Incyte: Research Funding; Promedior: Research Funding; Genentech: Research Funding; Galena BioPharma: Research Funding; Pfizer: Research Funding; Gilead: Research Funding; NS Pharma: Research Funding; Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Lilly Oncology: Research Funding; Roche: Research Funding; CTI BioPharma Corp: Research Funding; NS Pharma: Research Funding; Blueprint Medicines Corp: Research Funding; Gilead: Research Funding; CTI BioPharma Corp: Research Funding; Celgene: Research Funding; Promedior: Research Funding.

CD13 is a critical regulator of cell\u2013cell fusion in osteoclastogenesis

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.

Human dendritic cell-derived osteoclasts with high bone resorption capacity and T cell stimulation ability.

Osteoclasts are typically differentiated from monocytes (Mo-OC). A subset of osteoclasts (DC-OC) that are differentiated from dendritic cells (DC) has been reported in the arthritic mice model. However, little information is available on DC-OC in humans. The present study applied both in vitro and in vivo experiments to determine the function and pathological significance of DC-OC. DC-OC were differentiated from human monocyte-derived DC and their bone resorption and antigen-presenting functions were investigated. Synovial tissue samples from patients with rheumatoid arthritis were examined for the presence and characteristics of DC-OC. DC-OC differentiated from DC in the presence of M-CSF and RANKL in vitro were demonstrated to be cathepsin K-positive and TRAP-positive multinucleated giant cells. The DC-OC showed stronger bone resorption ability than monocyte-derived osteoclast (Mo-OC) as observed with the pit formation assay. The DC-OC retained CD11c positivity and expressed costimulatory molecules, unlike Mo-OC. T-cells proliferated when co-cultured with DC-OC, but not with Mo-OC. The addition of abatacept to the cocultures reduced T-cell stimulating activity of DC-OC. Abatacept inhibited the differentiation of monocytes into Mo-OC but did not suppress the differentiation of DC into DC-OC. TRAP-positive and CD86-positive DC-OC were detected in the synovial membranes of rheumatoid arthritis patients but not in patients with osteoarthritis. Human DC-OC demonstrated T-cell stimulating activity in addition to osteolytic activity. We further observed this subset of osteoclasts in the inflammatory synovial membrane of patients with rheumatoid arthritis. Such deviations from normal bone metabolism contribute to the inflammation and bone destruction in chronic inflammatory diseases such as rheumatoid arthritis.

Abstract 4532A: Development of selective small molecule STING agonists suitable for systemic administration

Abstract Background: Stimulator of Interferon Genes (STING) is a major player in the activation of robust innate immune response leading to initiation and enhancement of tumor-specific adaptive immunity. Several clinical and pre-clinical programs are developing cyclic dinucleotides - analogues of endogenous STING ligands. However their chemical nature and stability limit their use as systemic immuno-therapeutics. Herein, we present potent and selective non-nucleotide, non-macrocyclic, small molecule direct STING agonists, structurally unrelated to known chemotypes and suitable for systemic administration. Methods: Binding to recombinant STING protein was examined using FTS, MST, FP and crystallography studies. Phenotypic screen was performed in THP-1 Dual reporter cells. Human macrophages (HMDM) and dendritic cells (HMDC) were differentiated from monocytes (obtained from PBMC) in the presence of M-CSF and GM-CSF/IL-4 for HMDM and HMDC, respectively. Mouse bone marrow-derived dendritic cells (BMDC) were obtained from C57BL/6 or STING KO mice and differentiated with mIL-4 and mGM-CSF. STING agonists were administered into BALB/c mice and cytokine release was measured in plasma. Additionally, mice were inoculated with CT26 murine colon carcinoma cells and the compound was administered, followed by the regular tumor growth monitoring. Finally, the compound was administered to C57BL/6 WT and STING KO mice in several escalating doses. Results: Ryvu's agonists demonstrate a strong binding affinity to recombinant STING proteins across tested species. They trigger pro-inflammatory cytokine release from human PBMC and HMDC and induce dendritic cell maturation regardless of the STING haplotype. Systemic in vivo administration leads to dose-dependent upregulation of STING-dependent pro-inflammatory cytokines, suggesting immune activation which translates into efficacy in vivo in CT26 mouse colorectal cancer model and complete tumor remissions. Furthermore, cured animals develop lasting immunological response demonstrated by diminished tumor growth or lack of palpable tumors in re-challenged mice. Conclusion: Ryvu's STING agonists selectively activate STING-dependent signaling in both mouse and human immune cells promoting anti-tumor immunity. Treatment with Ryvu's STING agonists leads to engagement of the immune system which results in complete tumor remission and development of immunological memory against cancer cells. The compounds show good selectivity and ADME properties enabling development for systemic administration as a single agent or in combinations with immunotherapies or targeted agents. Citation Format: Stefan Chmielewski, Magdalena Zawadzka, Jolanta Mazurek, Maciej K. Rogacki, Karolina Gluza, Katarzyna Wójcik-Jaszczyńska, Aleksandra Poczkaj, Grzegorz Ćwiertnia, Grzegorz Topolnicki, Maciej Kujawa, Eliza Zimoląg, Urszula Głowniak-Kwitek, Magdalena Mroczkowska, Agnieszka Gibas, Marcin Leś, Sylwia Sudoł, Marek Wronowski, Kinga Michalik, Katarzyna Banaszak, Katarzyna Wiklik, Federico Malusa, Michał Combik, Karolina Wiatrowska, Łukasz Dudek, Jose Alvarez, Anna Rajda, Faustyna Gajdosz, Aniela Gołas, Katarzyna Wnuk-Lipińska, Kamil Kuś, Ewelina Gabor-Worwa, Charles Fabritius, Luigi Stasi, Peter Littlewood, Krzysztof Brzózka, Monika Dobrzańska. Development of selective small molecule STING agonists suitable for systemic administration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4532A.

3106 – DIRECT INDUCTION OF HEMOGENIC ENDOTHELIUM AND MYELOID CELLS FROM HUMAN IPSCS USING ETV2 MODIFIED RNA

Previously, we demonstrated that overexpressing transcription factors ETV2 and GATA2 is sufficient to induce a pan-myeloid program in human induced pluripotent stem cells (hiPSCs), which proceeds through a hemogenic endothelium (HE) stage (Elcheva et al., 2014). Although we have found that constitutive overexpression of ETV2 using lentiviral vectors induces predominantly non-hemogenic endothelium. We also noted that ETV2 induces GATA2 expression in human pluripotent stem cells (hPSCs) and very few HE with macrophage potential. In addition, our recent studies suggest that molecular mechanisms upstream of GATA2 are sufficient to specify hematoendothelial program in hPSCs, while GATA2 is required for endothelial-to-hematopoietic transition (EHT) (Kang et al., 2018). Given these findings and studies demonstrating the critical role of ETV2 threshold for hematoendothelial commitment (Zhao and Choi, 2017) and obligatory downregulation of ETV2 during subsequent stages of hematopoietic development in the embryo (Hayashi et al., 2012), we explored whether transitional expression of ETV2 with modified mRNA (mmRNA) alone is sufficient for hematoendothelial programming in hiPSCs. We found that overexpression of modified ETV2 mmRNA (mmETV2) following culture of transfected hiPSCs in StemLine II serum-free medium with FGF2, rapidly induces CD144+ expressing endothelial cells that, upon addition of GM-CSF, form floating CD43+ blood cells most of which co-express CD45. Culture of ETV2-induced hematoendothelial progenitors in the presence of GM-CSF, FGF2 and UM171 led to production of CD34+CD33+ myeloid progenitors which could be harvested every 8-10 days for up to 30 days of culture. Subsequently, myeloid progenitors were differentiated into functional neutrophils in the presence of G-CSF and the retinoic acid agonist Am580 or macrophages in presence of M-CSF and IL3 or IL-1β and M-CSF. Overall, this technology is suitable for generating functional neutrophils and macrophages from iPSCs to interrogate the role of genes in a myeloid cell development and function when coupled with genetic engineering technologies.

MO058INHIBITION OF OSTEOCLAST DIFFERENTIATION BY 1.25-D AND THE CALCIMIMETIC KP2326 REVEALS 1.25-D RESISTANCE IN ADVANCED CKD

Abstract Background and Aims Active vitamin D analogs and calcimimetics are cornerstones for managing secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD). Their direct effects on bone cells remain to be determined. Method Peripheral blood mononuclear cells (PBMCs) of 19 pediatric CKD patients and 6 healthy donors (HD) were differentiated into osteoclasts in presence of M-CSF and RANKL. Effect of combined or single treatment with active vitamin D (1.25-D) and/or the calcimimetic KP2326 were evaluated onto osteoclast differentiation and osteoclast mediated bone resorption. Results 1.25-D inhibited osteoclastic differentiation, a significant resistance to 1.25-D was observed when CKD worsens. A significant albeit less important inhibitory effect of KP2326 on osteoclastic differentiation was also found both in cells derived from HD and CKD patients, through an activation of the Erk pathway. This inhibitory effect was not modified by CKD stage. Combinatorial treatment with 1.25-D and KP2326 did not result in synergistic effects. Last, KP2326 significantly inhibited human osteoclast-mediated bone resorption. Conclusion Both 1.25-D and KP2326 inhibit osteoclastic differentiation, however to a different extent. Whilst 1.25-D has no significant effect on bone resorption, KP2326 inhibits bone resorption. Recent data showed that calcimimetics also have a direct anabolic effect on bone, through the stimulation of osteoblastic differentiation and mineralization in human mesenchymal stem cells in vitro. All these results provide a strong rationale for a global positive effect of calcimimetics on bone remodeling. Calcimimetics also significantly decrease FGF23 levels. In the setting of global systematic deleterious effects of high FGF23 levels in CKD, and keeping in mind that active vitamin D analogs stimulate FGF 23 production, all these data could favor the use of decreased doses of 1.25-D with low-doses of calcimimetics in SHPT in dialysis, the combination of these two therapies already being proposed in the 2017 K-DIGO guidelines.

Regulation of human macrophage phenotype and GPBAR1 expression by cell seeding density and glucocorticosteroids

Abstract Macrophages are key mediators of tissue homeostasis and inflammation and do so by virtue of differential expression of secreted and cell surface proteins. Cytokines such as IFNγ or IL-4 and IL-13 are known to generate classical proinflammatory M1 and alternatively activated M2 macrophages, respectively, but it is less clear how cell density, cell-cell contact and corticosteroids work together to affect macrophage function. Corticosteroids such as cortisol are known to downregulate the inflammatory signaling but they can also alter the expression of cell surface proteins, such as CD163, on steady-state macrophages. Here we examined the effect of monocyte seeding density and cortisol on the expression of cell surface proteins on human macrophages. The expression of the macrophage markers CD16, CD163, CD204 and VSIG4 were found to correlate with monocyte seeding density such that at low seeding densities, monocyte-derived macrophages were found to express minimal amounts of CD16, CD163, CD204 and VSIG4 while at high seeding densities, expression of these markers was restored. When cultured in the presence of cortisol, macrophages seeded at low densities were found to upregulate expression of these markers, but not CD204, to levels comparable to macrophages seeded at high densities grown in the presence of MCSF. Additionally, in the presence of cortisol, macrophages expressed higher levels of GPBAR1 which has also been shown to reduce classical proinflammatory macrophage activation in colitis models. This data suggests that corticosteroids may regulate macrophage function by modulating the expression of cell surface proteins such as GPBAR1 and not just interfering with morer conventional inflammatory cell signaling pathways..

Altered distribution and enhanced osteoclastogenesis of mucosal-associated invariant T cells in gouty arthritis.

This study was designed to investigate the role of mucosal-associated invariant T (MAIT) cells in gouty arthritis (GA) and their effects on osteoclastogenesis. Patients with GA (n = 61), subjects with hyperuricaemia (n = 11) and healthy controls (n = 30) were enrolled in this study. MAIT cells, cytokines, CD69, programmed death-1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) levels were measured by flow cytometry. In vitro osteoclastogenesis experiments were performed using peripheral blood mononuclear cells in the presence of M-CSF and RANK ligand. Circulating MAIT cell levels were significantly reduced in GA patients. However, their capacities for IFN-γ, IL-17 and TNF-α production were preserved. Expression levels of CD69, PD-1 and LAG-3 in MAIT cells were found to be elevated in GA patients. In particular, CD69 expression in circulating MAIT cells was increased by stimulation with MSU crystals, suggesting that deposition of MSU crystals might contribute to MAIT cell activation. Interestingly, MAIT cells were found to be accumulated in synovial fluid and infiltrated into gouty tophus tissues within joints. Furthermore, activated MAIT cells secreted pro-resorptive cytokines (i.e. IL-6, IL-17 and TNF-α) and facilitated osteoclastogenesis. This study demonstrates that circulating MAIT cells are activated and numerically deficient in GA patients. In addition, MAIT cells have the potential to migrate to inflamed tissues and induce osteoclastogenesis. These findings provide an important role of MAIT cells in the pathogenesis of inflammation and bone destruction in GA patients.

IL-23 induces the expression of pro-osteogenic factors in osteoclasts

AbstractBackground The mechanism for the new bone formation in ankylosing spondylitis (AS) is still unclear. Although it has been demonstrated that IL-23 plays a pivotal role in the pathophysiology of AS, IL-23 has no direct effects on osteoblasts but modulates the function of osteoclasts.Aims To explore whether IL-23 indirectly facilitates new bone formation through osteoclasts in AS, here we analyzed whether IL-23 enhances the expression levels of pro-osteogenic factors by osteoclasts.Methods Mononuclear cells were harvested from mouse bone marrow and cultured in the presence of M-CSF (50 ng/ml) and RANKL (30 ng/ml) to trigger the production of osteoclasts. Protein and mRNA expression levels of Semaphorin 4D, Ephrin B2, BMP2, BMP6, SPHK1, HtrA1 and Wnt10b were measured using Western blot and qRT-PCR.Results Primary mononuclear cells were transformed into osteoclasts with RANKL and M-CSF. The increased expression of NFATc1 and TRAP together with TRAP staining of&gt;3 nuclei were used to identify mature osteoclasts. The mRNA expression levels of BMP2, Ephrin B2 and SPHK1 were enhanced by 1.46, 2.1 and 2.46 folds after exposure to IL-23. Confirmation of increased levels of Ephrin B2 and SPHK1 in IL-23-stimulated osteoclasts was provided by Western blot analysis. IL-23 had no effects on the expression of BMP6 or Wnt10b, or on the anti-osteogenic factors Semaphorin 4D or HtrA1.Conclusions IL-23 induces osteoclasts to express pro-osteogenic factors rather than anti-osteogenic factors, suggesting IL-23 might indirectly promote the differentiation of osteoblasts through activated osteoclasts in ankylosing spondylitis.

Water Extracts of Hull-less Waxy Barley (Hordeum vulgare L.) Cultivar 'Boseokchal' Inhibit RANKL-induced Osteoclastogenesis.

Osteoporosis is a disease that leads to reduced bone mineral density. The increase in patient and medical costs because of global aging is recognized as a problem. Decreased bone mass is a common symptom of bone diseases such as Paget’s disease, rheumatoid arthritis, and multiple myeloma. Osteoclasts, which directly affect bone mass, show a marked increase in differentiation and activation in the aforementioned diseases. Moreover, these multinucleated cells made from monocytes/macrophages under the influence of RANKL and M-CSF, are the only cells capable of resorbing bones. In this study, we found that the water extracts of Boseokchal (BSC-W) inhibited osteoclast differentiation in vitro and investigated its inhibitory mechanism. BSC-W was obtained by extracting flour of Boseokchal using hexane and water. To osteoclast differentiation, bone marrow-derived macrophage cells (BMMs) were cultured with the vehicle (0.1% DMSO) or BSC-W in the presence of M-CSF and RANKL for 4 days. Cytotoxicity was measured by CCK-8. Gene expression of cells was confirmed by real-time PCR. Protein expression of cells was observed by western blot assay. Bone resorption activity of osteoclast evaluated by bone pit formation assay using an Osteo Assay Plate. BSC-W inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner without exerting a cytotoxic effect on BMMs. BSC-W decreased the transcriptional and translational expression of c-Fos and NFATc1, which are regulators of osteoclastogenesis and reduced the mRNA expression level of TRAP, DC-STAMP, and cathepsin K, which are osteoclast differentiation marker. Furthermore, BSC-W reduced the resorption activity of osteoclasts. Taken together, our results indicate that BSC-W is a useful candidate for health functional foods or therapeutic agents that can help treat bone diseases such as osteoporosis.

Finding a Toll on the Route: The Fate of Osteoclast Progenitors After Toll-Like Receptor Activation.

M-CSF and RANKL are two crucial cytokines stimulating differentiation of mature, bone resorbing, multinucleated osteoclasts from mononucleated progenitor cells in the monocyte/macrophage lineage. In addition to the receptors for M-CSF and RANKL, osteoclast progenitor cells express receptors for several other pro- and anti-osteoclastogenic cytokines, which also regulate osteoclast formation by affecting signaling downstream M-CSF and RANKL receptors. Similar to many other cells originating from myeloid hematopoetic stem cells, also osteoclast progenitors express toll-like receptors (TLRs). Nine murine TLRs are expressed in the progenitors and all, with the exception of TLR2 and TLR4, are downregulated during osteoclastogenesis. Activation of TLR2, TLR4, and TLR9, but not TLR5, in osteoclast progenitors stimulated with M-CSF and RANKL arrests differentiation along the osteoclastic lineage and keeps the cells at a macrophage stage. When the progenitors are primed with M-CSF/RANKL and then stimulated with agonists for TLR2, TLR4, or TLR9 in the presence of M-CSF, but in the absence of RANKL, the cells differentiate to mature, bone resorbing osteoclasts. TLR 2, 4, 5, and 9 are also expressed on osteoblasts and their activation increases osteoclast differentiation by an indirect mechanism through stimulation of RANKL. In mice, treatment with agonists for TLR2, 4, and 5 results in osteoclast formation and extensive bone loss. It remains to be shown the relative importance of inhibitory and stimulatory effects by TLRs on osteoclast progenitors and the role of RANKL produced by TLR stimulated osteoblasts, for the bone resorbing effects in vivo.