Karakterizacija reakcije osteoklastnih progenitora i pojačane osteoresorpcije u mišjem modelu reumatoidnoga artritisa [Characterization of osteoclast progenitor responses and increased osteoresorption in mouse model of rheumatoid arthritis]

Osteoclasts, specialized bone resorbing cells of hematopoietic origin, become overactive in rheumatoid arthritis, leading to bone loss and joint destruction. Osteoclast progenitor cells (OCP) arise from myeloid precursors of monocyte/macrophage lineage and are normally present in the bone marrow and among circulatory monocytes. Under inflammatory conditions their subpopulations get attracted to inflamed sites by yet unknown mechanisms and chemotactic signals. Here, we investigated frequencies and chemokine receptor expression of OCP subpopulations in circulation and periarticular bone marrow (PBM) of C57BL/6 mice with collagen induced arthritis (CIA). We found both lymphoid-negative CD45+CD11b+CD115+ and lymphoid-negative CD45+CD11b-/loCD115+ subsets of OCPs to be increased in blood and PBM of affected joints in CIA. Chemokine receptors were increasingly expressed by both mentioned OCP subsets in CIA, with substantial expression of CCR2 and CX3CR1, and lower expression of CCR1, CCR3, CCR5, CCR9 and CXCR4. CCL2 serum levels, and gene expression for TNF-α, IL-1β, IL-6 and CCL2 in affected areas and spleen were significantly increased in CIA. PBM population of OCP increasingly migrated toward CCL2 chemotactic gradient, whereas down-regulation of CCR2 expression by siRNA significantly reduced the migration potential. Intravascular in vivo staining demonstrated increased recirculation of CD45+CD11b+ cells through PBM and hind paws in CIA, with the ability of tissue homing after adoptive transfer. These results suggest that highly induced OCP subpopulations in CIA migrate to inflamed joints due to chemotaxis and therefore blocking of chemokine signaling may reduce osteoclast activity in arthritis

Osteoporosis in the view of osteoimmunology: common feature underlined by different pathogenic mechanisms

Osteoporosis is a skeletal disorder characterized by low bone mineral density and microarchitectural changes with increased susceptibility to fractures, resulting in significant morbidity and mortality. Although it predominantly affects postmenopausal women, it is now well known that systemic bone loss is a common underlying feature of different metabolic, endocrine and inflammatory diseases. Investigations of osteoporosis as a complication of chronic inflammatory conditions revealed immune mechanisms behind the increased osteoclast bone resorption and impaired osteoblast bone formation. This concept was particularly emphasized after the research field of osteoimmunology emerged, focusing on the interaction between the immune system and bone. It is increasingly becoming evident that immune cells and mediators critically regulate osteoclast and osteoblast development, function and coupling activity. Among other mediators, receptor activator of nuclear factor-kB ligand (RANKL), receptor activator of nuclear factor-kB (RANK) and soluble decoy receptor osteoprotegerin (OPG) form a key functional link between the immune system and bone, regulating both osteoclast formation and activity as well as immune cell functions. Excessive production of inflammatory mediators exerts autocrine, paracrine and endocrine signaling effects on bone remodeling with the net increase in bone resorption locally, in diseases primarily affecting joints, bones or surrounding tissues, and systemically, causing osteoporosis in various chronic inflammatory diseases. This brief review particularly focuses on bone pathology in rheumatoid arthritis, as one of the most extensively studied conditions accompanied by local and systemic inflammation-induced bone loss

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

Characterization of osteoclast progenitor responses and increased osteoresorption in mouse model of rheumatoid arthritis

Osteoklasti, stanice hematopoetskog podrijetla specijalizirane za resorpciju kosti, pojačano su aktivni u reumatoidnom artritisu, dovodeći do gubitka kosti i razaranja zglobova. Osteoklastni progenitori (OCP) potječu iz mijeloidnih progenitora monocitno/makrofagne loze te su normalno prisutni u koštanoj srži i među cirkulirajućim monocitima. U upalnim uvjetima, zasad još nedovoljno razjašnjeni mehanizmi i kemotaktični signali privlače subpopulacije OCP na mjesta upale. U ovom radu istražili smo brojnost i izražaj kemokinskih receptora na subpopulacijama OCP u cirkulaciji i periartikularnoj koštanoj srži (PBM) C57Bl/6 miševa s artritisom potaknutim kolagenom (CIA). Uočili smo da su subpopulacije CD3-B220-NK1.1-CD45+CD11b+CD115+ i CD3-B220-NK1.1-CD45+CD11b-/loCD115+ povećane brojnosti u krvi i PBM zahvaćenih zglobova u CIA, sa značajnim izražajem CCR2 i CX3CR1, te slabijim izražajem CCR1, CCR3, CCR5, CCR9 i CXCR4. Serumska koncentracija CCL2 i genski izražaj TNF-α, IL-1β, IL-6 te CCL2 značajno su povećani u zahvaćenim područjima i slezeni u CIA. Subpopulacija OCP iz PBM pojačano migrira prema CCL2-kemotaktičnom gradijentu, dok utišavanje CCR2, uporabom siRNA, smanjuje njezin migracijski potencijal. Intravaskularno in vivo bojenje pokazuje povećanu recirkulaciju CD45+CD11b+ stanica kroz PBM i stražnje šape u CIA, sa sposobnošću navođenja u tkiva nakon adoptivnog transfera. Ovi rezultati sugeriraju mogućnost privlačenja umnožene OCP-subpopulacije u upaljene zglobove kemokinima, stoga blokada kemokinske signalizacije predstavlja obećavajući terapijski pristup za smanjenje osteoklastne aktivnosti u artitisu.Osteoclasts, specialized bone resorbing cells of hematopoietic origin, become overactive in rheumatoid arthritis, leading to bone loss and joint destruction. Osteoclast progenitor cells (OCP) arise from myeloid precursors of monocyte/macrophage lineage and are normally present in the bone marrow and among circulatory monocytes. Under inflammatory conditions their subpopulations get attracted to inflamed sites by yet unknown mechanisms and chemotactic signals. Here, we investigated frequencies and chemokine receptor expression of OCP subpopulations in circulation and periarticular bone marrow (PBM) of C57BL/6 mice with collagen induced arthritis (CIA). We found both lymphoid-negative CD45+CD11b+CD115+ and lymphoid-negative CD45+CD11b-/loCD115+ subsets of OCPs to be increased in blood and PBM of affected joints in CIA. Chemokine receptors were increasingly expressed by both mentioned OCP subsets in CIA, with substantial expression of CCR2 and CX3CR1, and lower expression of CCR1, CCR3, CCR5, CCR9 and CXCR4. CCL2 serum levels, and gene expression for TNF-α, IL-1β, IL-6 and CCL2 in affected areas and spleen were significantly increased in CIA. PBM population of OCP increasingly migrated toward CCL2 chemotactic gradient, whereas down-regulation of CCR2 expression by siRNA significantly reduced the migration potential. Intravascular in vivo staining demonstrated increased recirculation of CD45+CD11b+ cells through PBM and hind paws in CIA, with the ability of tissue homing after adoptive transfer. These results suggest that highly induced OCP subpopulations in CIA migrate to inflamed joints due to chemotaxis and therefore blocking of chemokine signaling may reduce osteoclast activity in arthritis

Characterization of osteoclast progenitor responses and increased osteoresorption in mouse model of rheumatoid arthritis

Osteoklasti, stanice hematopoetskog podrijetla specijalizirane za resorpciju kosti, pojačano su aktivni u reumatoidnom artritisu, dovodeći do gubitka kosti i razaranja zglobova. Osteoklastni progenitori (OCP) potječu iz mijeloidnih progenitora monocitno/makrofagne loze te su normalno prisutni u koštanoj srži i među cirkulirajućim monocitima. U upalnim uvjetima, zasad još nedovoljno razjašnjeni mehanizmi i kemotaktični signali privlače subpopulacije OCP na mjesta upale. U ovom radu istražili smo brojnost i izražaj kemokinskih receptora na subpopulacijama OCP u cirkulaciji i periartikularnoj koštanoj srži (PBM) C57Bl/6 miševa s artritisom potaknutim kolagenom (CIA). Uočili smo da su subpopulacije CD3-B220-NK1.1-CD45+CD11b+CD115+ i CD3-B220-NK1.1-CD45+CD11b-/loCD115+ povećane brojnosti u krvi i PBM zahvaćenih zglobova u CIA, sa značajnim izražajem CCR2 i CX3CR1, te slabijim izražajem CCR1, CCR3, CCR5, CCR9 i CXCR4. Serumska koncentracija CCL2 i genski izražaj TNF-α, IL-1β, IL-6 te CCL2 značajno su povećani u zahvaćenim područjima i slezeni u CIA. Subpopulacija OCP iz PBM pojačano migrira prema CCL2-kemotaktičnom gradijentu, dok utišavanje CCR2, uporabom siRNA, smanjuje njezin migracijski potencijal. Intravaskularno in vivo bojenje pokazuje povećanu recirkulaciju CD45+CD11b+ stanica kroz PBM i stražnje šape u CIA, sa sposobnošću navođenja u tkiva nakon adoptivnog transfera. Ovi rezultati sugeriraju mogućnost privlačenja umnožene OCP-subpopulacije u upaljene zglobove kemokinima, stoga blokada kemokinske signalizacije predstavlja obećavajući terapijski pristup za smanjenje osteoklastne aktivnosti u artitisu.Osteoclasts, specialized bone resorbing cells of hematopoietic origin, become overactive in rheumatoid arthritis, leading to bone loss and joint destruction. Osteoclast progenitor cells (OCP) arise from myeloid precursors of monocyte/macrophage lineage and are normally present in the bone marrow and among circulatory monocytes. Under inflammatory conditions their subpopulations get attracted to inflamed sites by yet unknown mechanisms and chemotactic signals. Here, we investigated frequencies and chemokine receptor expression of OCP subpopulations in circulation and periarticular bone marrow (PBM) of C57BL/6 mice with collagen induced arthritis (CIA). We found both lymphoid-negative CD45+CD11b+CD115+ and lymphoid-negative CD45+CD11b-/loCD115+ subsets of OCPs to be increased in blood and PBM of affected joints in CIA. Chemokine receptors were increasingly expressed by both mentioned OCP subsets in CIA, with substantial expression of CCR2 and CX3CR1, and lower expression of CCR1, CCR3, CCR5, CCR9 and CXCR4. CCL2 serum levels, and gene expression for TNF-α, IL-1β, IL-6 and CCL2 in affected areas and spleen were significantly increased in CIA. PBM population of OCP increasingly migrated toward CCL2 chemotactic gradient, whereas down-regulation of CCR2 expression by siRNA significantly reduced the migration potential. Intravascular in vivo staining demonstrated increased recirculation of CD45+CD11b+ cells through PBM and hind paws in CIA, with the ability of tissue homing after adoptive transfer. These results suggest that highly induced OCP subpopulations in CIA migrate to inflamed joints due to chemotaxis and therefore blocking of chemokine signaling may reduce osteoclast activity in arthritis

Characterization of osteoclast progenitor responses and increased osteoresorption in mouse model of rheumatoid arthritis

Osteoklasti, stanice hematopoetskog podrijetla specijalizirane za resorpciju kosti, pojačano su aktivni u reumatoidnom artritisu, dovodeći do gubitka kosti i razaranja zglobova. Osteoklastni progenitori (OCP) potječu iz mijeloidnih progenitora monocitno/makrofagne loze te su normalno prisutni u koštanoj srži i među cirkulirajućim monocitima. U upalnim uvjetima, zasad još nedovoljno razjašnjeni mehanizmi i kemotaktični signali privlače subpopulacije OCP na mjesta upale. U ovom radu istražili smo brojnost i izražaj kemokinskih receptora na subpopulacijama OCP u cirkulaciji i periartikularnoj koštanoj srži (PBM) C57Bl/6 miševa s artritisom potaknutim kolagenom (CIA). Uočili smo da su subpopulacije CD3-B220-NK1.1-CD45+CD11b+CD115+ i CD3-B220-NK1.1-CD45+CD11b-/loCD115+ povećane brojnosti u krvi i PBM zahvaćenih zglobova u CIA, sa značajnim izražajem CCR2 i CX3CR1, te slabijim izražajem CCR1, CCR3, CCR5, CCR9 i CXCR4. Serumska koncentracija CCL2 i genski izražaj TNF-α, IL-1β, IL-6 te CCL2 značajno su povećani u zahvaćenim područjima i slezeni u CIA. Subpopulacija OCP iz PBM pojačano migrira prema CCL2-kemotaktičnom gradijentu, dok utišavanje CCR2, uporabom siRNA, smanjuje njezin migracijski potencijal. Intravaskularno in vivo bojenje pokazuje povećanu recirkulaciju CD45+CD11b+ stanica kroz PBM i stražnje šape u CIA, sa sposobnošću navođenja u tkiva nakon adoptivnog transfera. Ovi rezultati sugeriraju mogućnost privlačenja umnožene OCP-subpopulacije u upaljene zglobove kemokinima, stoga blokada kemokinske signalizacije predstavlja obećavajući terapijski pristup za smanjenje osteoklastne aktivnosti u artitisu.Osteoclasts, specialized bone resorbing cells of hematopoietic origin, become overactive in rheumatoid arthritis, leading to bone loss and joint destruction. Osteoclast progenitor cells (OCP) arise from myeloid precursors of monocyte/macrophage lineage and are normally present in the bone marrow and among circulatory monocytes. Under inflammatory conditions their subpopulations get attracted to inflamed sites by yet unknown mechanisms and chemotactic signals. Here, we investigated frequencies and chemokine receptor expression of OCP subpopulations in circulation and periarticular bone marrow (PBM) of C57BL/6 mice with collagen induced arthritis (CIA). We found both lymphoid-negative CD45+CD11b+CD115+ and lymphoid-negative CD45+CD11b-/loCD115+ subsets of OCPs to be increased in blood and PBM of affected joints in CIA. Chemokine receptors were increasingly expressed by both mentioned OCP subsets in CIA, with substantial expression of CCR2 and CX3CR1, and lower expression of CCR1, CCR3, CCR5, CCR9 and CXCR4. CCL2 serum levels, and gene expression for TNF-α, IL-1β, IL-6 and CCL2 in affected areas and spleen were significantly increased in CIA. PBM population of OCP increasingly migrated toward CCL2 chemotactic gradient, whereas down-regulation of CCR2 expression by siRNA significantly reduced the migration potential. Intravascular in vivo staining demonstrated increased recirculation of CD45+CD11b+ cells through PBM and hind paws in CIA, with the ability of tissue homing after adoptive transfer. These results suggest that highly induced OCP subpopulations in CIA migrate to inflamed joints due to chemotaxis and therefore blocking of chemokine signaling may reduce osteoclast activity in arthritis

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

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 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 CD115 ). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced 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 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 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 differentiated macrophages and DCs. However, RBP-J deletion stimulated and 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 TGFβ 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 p 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 p Tgfβ (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 Tgfβ seems to be a plausible protective mechanism that should be the primary focus of further research

Combined manual and automated immunophenotypisation identified disease-specific peripheral blood immune subpopulations in rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis

Objectives: Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are associated with abnormal immune cell functions. We combined manual and automated profiling in subpopulations of T-cells, B-cells and monocytes, in parallel to functional testing and clinical correlation. ----- Methods: Using flow cytometry, we analysed the expression of CCR4, CCR6 and CXCR5 on helper and cyotoxic T-cells, CD32B and CD86 on naïve and memory B-cells, and CCR1, CCR2, CCR4 and CXCR4 on monocytes in chronic high-disease activity patients to identify peripheral blood subpopulations. Cell activation, proliferative capability and osteoclastogenic effects were tested in vitro. Comparison with synovial compartment, clinical data and anti-TNF treatment were added to peripheral blood analysis. ----- Results: PsA had lower double-negative T-cell frequency, while RA had lower double-positive T-cell frequency and expanded Th1-like and cytotoxic T-cell subsets. CD32B expression was increased on naïve and memory B-cells in AS and associated with disease activity. CCR6+ and CXCR5+ cytotoxic T-cells and CD32B+ naïve and memory B-cells were highly enriched within the synovial compartment. T-cells and B-cells from AS exhibited enhanced activation and proliferation in vitro, whereas T-cell conditioned medium from RA produced an increased osteoclastogenic effect. CCR1 and CXCR4 were upregulated on osteoclastogenic monocyte subsets of RA, AS and PsA patients. Bioinformatic Citrus analysis identified additional T-cell, B-cell and monocyte clusters specifically associated with each disease. ----- Conclusions: By combining manual and automated data analysis, our study revealed several disease-specific immune cell subpopulations, particularly cytotoxic T-cell subsets in RA and memory B-cell subsets in AS, which may serve as an indicator of active disease or possible therapeutic target