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Background: Ulcerative colitis (UC) is characterized by altered chromogranin-A (CHGA), alternatively activated macrophages (M2) and intestinal epithelial cells (IECs). We previously demonstrated that CHGA is implicated in colitis progression by regulating the macrophages. Here, we investigated the interplay between CHGA, M2, tight junctions (TJ) and IECs in an inflammatory environment. Methods: Correlations between CHGA mRNA expression of and TJ proteins mRNA expressions of (Occludin [OCLN], zonula occludens-1 [ZO1], Claudin-1 [CLDN1]), epithelial associated cytokines (interleukin [IL]-8, IL-18), and collagen (COL1A2) were determined in human colonic mucosal biopsies isolated from active UC and healthy patients. Acute UC-like colitis (5% dextran sulphate sodium [DSS], five days) was induced in Chga-C57BL/6-deficient (Chga-/-) and wild type (Chga+/+) mice. Col1a2 TJ proteins, Il-18 mRNA expression and collagen deposition were determined in whole colonic sections. Naïve Chga-/- and Chga+/+ peritoneal macrophages were isolated and exposed six hours to IL-4/IL-13 (20 ng/mL) to promote M2 and generate M2-conditioned supernatant. Caco-2 epithelial cells were cultured in the presence of Chga-/- and Chga+/+ non- or M2-conditioned supernatant for 24 h then exposed to 5% DSS for 24 h, and their functional properties were assessed. Results: In humans, CHGA mRNA correlated positively with COL1A2, IL-8 and IL-18, and negatively with TJ proteins mRNA markers. In the experimental model, the deletion of Chga reduced IL-18 mRNA and its release, COL1A2 mRNA and colonic collagen deposition, and maintained colonic TJ proteins. Chga-/- M2-conditioned supernatant protected caco-2 cells from DSS and oxidative stress injuries by improving caco-2 cells functions (proliferation, viability, wound healing) and by decreasing the release of IL-8 and IL-18 and by maintaining the levels of TJ proteins, and when compared with Chga+/+ M2-conditioned supernatant. Conclusions: CHGA contributes to the development of intestinal inflammation through the regulation of M2 and epithelial cells. Targeting CHGA may lead to novel biomarkers and therapeutic strategies in UC.
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Cromogranina A/genética , Colite Ulcerativa/imunologia , Citocinas/genética , Macrófagos/imunologia , Proteínas de Junções Íntimas/genética , Animais , Células CACO-2 , Estudos de Casos e Controles , Células Cultivadas , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/genética , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Humanos , Interleucina-18/genética , Interleucina-8/genética , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Macrophages are professional innate immune cells that are broadly disseminated throughout the body, shape various innate and adaptive immune responses, and play crucial roles in inflammation, homeostasis, wound healing, and tissue remodelling. According to their surrounding microenvironments, macrophages can differentiate themselves in different phenotypes. Over the last two decades, gene expression profiling has been used to decipher new transcripts associated with macrophage phenotypes. This chapter outlines protocols used to isolate and culture murine macrophages and how they can be "polarized" to obtain a specific phenotype. Furthermore, we describe a protocol for gene expression profiling using a quantitative real-time polymerase chain reaction (qPCR), a high-standard technology in the field of gene expression.
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Polaridade Celular/genética , Expressão Gênica/genética , Macrófagos/fisiologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Microambiente Celular/genética , Perfilação da Expressão Gênica/métodos , Inflamação/genética , Ativação de Macrófagos/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fenótipo , Células RAW 264.7RESUMO
BACKGROUND: Manual palpation of rabbit spine levels has been used to assess fusion status. This method of testing is subject to inter-observer differences in assessment. We attempted to quantify fusion based on the amount of movement between rabbit vertebrae at the level of fusion. METHODS: Rabbits were divided into three groups. The first underwent a sham surgery; the second underwent a unilateral spinal fusion; and the third underwent a bilateral spinal fusion. All groups were sacrificed at either 5- or 10-week post-procedure. Each spine was tested for fusion using standard manual palpation techniques. The spines were also placed on a specially designed apparatus and moved through 10°, 20°, and 30° of extension/flexion. RESULTS: Out of 10 rabbits, 2 underwent sham surgery, 2 underwent a fusion procedure at L4-L5 and 6 underwent a fusion at L5-L6. We only included rabbits that underwent a L5-L6 fusion surgery. Our apparatus did not always rotate the spine the intended amount with up to 30% error. When rabbits graded as fused were compared to sham rabbits, there was a trend towards reduction in percent of overall measured angle within the fused group as compared to the sham group (8.77% vs. 13.84%, P=0.14). CONCLUSIONS: Our model attempted to quantify the amount of displacement between vertebrae during the manual palpation exam. There is a trend towards reduced measured angle between vertebrae between fused and non-fused spines and no statistically significant difference in overall measured angle between unilaterally and bilaterally fused spines.
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Osteoarthritis (OA) is a progressive disease associated with cartilage injury and its inherently limited repair capability. Synovium-based cellular constructs (sConstructs) are proposed as possible treatments. Equine sConstructs were produced from decellularized synovium-based extracellular matrix scaffolds (sECM) seeded with synovium-derived mesenchymal stem cells (sMSC), and engineered to express green fluorescent protein (GFP), or bone morphogenetic protein-2 (BMP-2). Survival, distribution, and chondrogenic potential of the sConstructs in vitro and in vivo were assessed. sConstructs in co-culture with chondrocytes increased chondrocyte proliferation, viability, and Col II production, greatest in BMP-2-sConstructs. Chondrocyte presence increased the production of hyaluronic acid (HA), proteoglycan (PG), and BMP-2 by the sConstructs in a positive feedback loop. sECM alone, or GFP- or BMP-2-sConstructs were implanted in synovium adjacent to clinically created full-thickness rat-knee cartilage lesions. At 5 weeks, the lesion area and implants were resected. Gross anatomy, adjacent articulate cartilage growth and subchondral bone repair were scored; and peripheral, central and cartilage lesion measurements taken. For all scores and measurements, sConstruct implants were significantly greater than controls, greatest with the BMP-2-sConstructs. Immunohistochemistry demonstrated migration of endogenous cells into the sECM, with greater cellularity in the constructs with intense positive GFP staining confirming engraftment of implanted sMSC and continued gene expression. In summary, exposing cartilage to sConstructs was chondrogenic in vitro and in vivo, and resulted in substantially increased growth in vivo. This effect was mediated, in part, by soluble ECM and cell factors and upregulation of anabolic growth proteins, such as BMP-2. This work is "proof of concept" that sConstructs surgically implanted adjacent to cartilage damage can significantly improve cartilage and subchondral bone repair, and potentially prevent the progression of OA.
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Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Condrogênese , Matriz Extracelular/metabolismo , Articulação do Joelho/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Osteoartrite do Joelho/terapia , Animais , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Cartilagem Articular/patologia , Condrócitos/patologia , Colágeno Tipo II/biossíntese , Colágeno Tipo II/genética , Modelos Animais de Doenças , Matriz Extracelular/patologia , Cavalos , Articulação do Joelho/patologia , Células-Tronco Mesenquimais/patologia , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Ratos , Transdução GenéticaRESUMO
OBJECTIVE To create a bioactive synovium scaffold by infusing decellularized synovial-derived extracellular matrix (synECM) with synovial-derived mesenchymal stem cells (synMSCs). SAMPLE Synovium from the femoropatellar and medial femorotibial joints of equine cadavers. PROCEDURES The synMSCs were cultured in monolayer and not treated or cotransduced to enhance expression of green fluorescent protein (GFP) and human bone morphogenetic protein (BMP)-2. The synECM was decellularized with 0.1% peracetic acid and then seeded with synMSCs (0.5 × 106 cells/0.5 mL) by use of a 30% serum gradient. Samples were evaluated on days 0, 3, 7, and 14. Cell migration, differentiation, and distribution into the synECMs were determined by cell surface marker CD90, viability, histologic morphology, and fluorescence microscopy results and expression of GFP, BMP-2, hyaluronan (HA), and proteoglycan (PG). RESULTS At day 14, synMSCs were viable and had multiplied 2.5-fold in the synECMs. The synECMs seeded with synMSCs had a significant decrease in CD90 expression and significant increases in HA and PG expression. The synECMs seeded with synMSCs cotransduced with GFP, or BMP-2 had a significant increase in BMP-2 expression. CONCLUSIONS AND CLINICAL RELEVANCE The synECM seeded with synMSCs or synMSCs cotransduced with GFP, or BMP-2 yielded a bioactive synovial scaffold. Expression of BMP-2 by synMSCs cotransduced to enhance expression of BMP-2 or GFP and an accompanying increase in both HA and PG expression indicated production of anabolic agents and synoviocyte differentiation in the scaffold. Because BMP-2 can promote repair of damaged cartilage, such a bioactive scaffold could be useful for treatment of injured cartilage.
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Matriz Extracelular , Células-Tronco Mesenquimais , Membrana Sinovial/citologia , Animais , Cartilagem , Diferenciação Celular , Células Cultivadas , Cavalos , Humanos , Ácido Hialurônico , Células-Tronco Mesenquimais/citologia , Proteoglicanas/metabolismoRESUMO
Chromogranin-A (CHGA) is elevated in inflammatory bowel disease (IBD), but little is known about its role in colonic inflammation. IBD is associated with impaired functions of macrophages and increased apoptosis of intestinal epithelial cells. We investigated CHGA expression in human subjects with active ulcerative colitis (UC) and the underlying mechanisms in Chga -/- mice. In UC, CHGA, classically activated macrophage (M1) markers, caspase-3, p53, and its associated genes were increased, while alternatively activated macrophage (M2) markers were decreased without changes in the extrinsic apoptotic pathway. CHGA correlated positively with M1 and the apoptotic pathway and negatively with M2. In the murine dextran sulfate sodium (DSS)-induced colitis, Chga deletion reduced the disease severity and onset, pro-inflammatory mediators, M1, and p53/caspase-3 activation, while it upregulated anti-inflammatory cytokines and M2 markers with no changes in the extrinsic apoptotic markers. Compared to Chga +/+ , M1 and p53/caspase-3 activation in Chga -/- macrophages were decreased in vitro, while M2 markers were increased. CHGA plays a critical role during colitis through the modulation of macrophage functions via the caspase-3/p53 pathway. Strategies targeting CHGA to regulate macrophage activation and apoptosis might be developed to treat UC patients. KEY MESSAGES: ⢠Chromogranin-A (CHGA) is pro-hormone and is secreted in the gut. CHGA is elevated in colitis and is associated with the disease severity. The lack of GHGA has beneficial immunomodulatory properties during the development of intestinal inflammation. The lack of CHGA regulates the plasticity of macrophages and p53/caspase activation in colitis. Functional analysis of CHGA may lead to a novel therapy for IBD.
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Apoptose , Cromogranina A/metabolismo , Colite/metabolismo , Macrófagos/metabolismo , Animais , Células Cultivadas , Cromogranina A/genética , Colite/induzido quimicamente , Colite/patologia , Colo/metabolismo , Colo/patologia , Sulfato de Dextrana , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND CONTEXT: Adipokines are secreted by white adipose tissue and have been associated with fracture healing. Our goal was to report the temporal expression of adipokines during spinal fusion in an established rabbit model. PURPOSE: Our goal was to report the temporal expression of adipokines during spinal fusion in an established rabbit model. STUDY DESIGN: The study design included a laboratory animal model. METHODS: New Zealand white rabbits were assigned to either sham surgery (n=2), unilateral posterior spinal fusion (n=14), or bilateral posterior spinal fusion (n=14). Rabbits were euthanized 1-6 and 10 weeks out from surgery. Fusion was evaluated by radiographs, manual palpation, and histology. Reverse transcription-polymerase chain reaction on the bone fusion mass catalogued the gene expression of leptin, adiponectin, resistin, and vascular endothelial growth factor (VEGF) at each time point. Results were normalized to the internal control gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2^ΔCt), and control bone sites (2^ΔΔCt). Quantitative data were analyzed by two-factor analysis of variance (p<.05). RESULTS: Manual palpation scores, radiograph scores, and histologic findings showed progression of boney fusion over time (p<.0003). The frequency of fusion by palpation after 4 weeks was 68.75%. Leptin expression in decortication and bone graft sites peaked at 5 weeks after the fusion procedure (p=.0143), adiponectin expression was greatest 1 week after surgery (p<.001), VEGF expression peaked at 4 weeks just after initial increases in leptin expression (p<.001), and resistin decreased precipitously 1 week after the fusion procedure (p<.001). CONCLUSIONS: Leptin expression is likely associated with the maturation phase of bone fusion. Adiponectin and resistin may play a role early on during the fusion process. Our results suggest that leptin expression may be upstream of VEGF expression during spinal fusion, and both appear to play an important role in bone spinal fusion.
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Adipocinas/metabolismo , Transplante Ósseo/efeitos adversos , Vértebras Lombares/metabolismo , Complicações Pós-Operatórias/metabolismo , Fusão Vertebral/efeitos adversos , Adipocinas/genética , Animais , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Leptina/genética , Leptina/metabolismo , Vértebras Lombares/cirurgia , Coelhos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Retinoic acid (RA), an active metabolite of vitamin A, has shown potential therapeutic immunomodulatory properties. Allogeneic mesenchymal stem cells (MSCs)-based therapy is an effective approach to induce tissue healing and regeneration in many equine orthopedic conditions. However, MSCs-based therapies induced inflammatory responses in vivo. This study aimed to: 1. Determine the effect of RA cell culture treatment on inflammatory responses of lipopolysaccharides (LPS)- and allogeneic MSCs-stimulated peripheral blood mononuclear cells (PBMCs). 2. Determine the effect of RA on stimulated MSCs viability and morphology. Allogeneic MSCs-stimulated PBMCs had significant decreases in the anti-inflammatory cytokines (IL-10, IL-1ra, TGF-ß1), increases in the pro-inflammatory mediators (IL-1ß, IL-6, TNF-α, SAA), and increases of CD14 and MHC II percent positive cells compared to LPS- and non-stimulated PBMCs. Retinoic acid treatment of LPS- and allogeneic MSCs-stimulated PBMCs counterbalanced the induced inflammatory responses. Moreover, RA significantly improved the viability and morphology of stimulated MSCs. These findings highlighted the potential complications of equine allogeneic MSCs-based therapies and the immuno-modulatory effect of RA on equine stimulated cells. In conclusion, the use of RA to ameliorate allogeneic MSCs therapy associated inflammation may offer advantages that would require further investigations.
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Citocinas/genética , Cavalos/imunologia , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/farmacocinética , Células-Tronco Mesenquimais/fisiologia , Tretinoína/metabolismo , Animais , Citocinas/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologiaRESUMO
OBJECTIVE To investigate effects of hyaluronic acid (HA) or HA combined with chondroitin sulfate (CS) and N-acetyl-d-glucosamine (NAG) by use of a lipopolysaccharide (LPS) in vitro method. SAMPLE Monolayer cultures of synovial cells from 4 adult horses. PROCEDURES Synovial cell cultures were untreated or treated with HA alone or HA-CS-NAG for 24 hours, subsequently unchallenged or challenge-exposed with 2 LPS concentrations (20 and 50 ng/mL) for 2 hours, and retreated with HA or HA-CS-NAG for another 24 hours. Cellular morphology of cultures was evaluated at 0, 24 (before LPS), 26 (after LPS), and 50 (24 hours after end of LPS) hours. At 50 hours, cell number and viability and prostaglandin (PG) E2, interleukin (IL)-6, matrix metalloproteinase (MMP)-3, and cyclooxygenase (COX)-2 production were measured. RESULTS LPS challenge exposure induced a significant loss of characteristic synovial cell morphology, decrease in cell viability, and increases in concentrations of PGE2, IL-6, MMP-3, and COX-2. Cells treated with HA or HA-CS-NAG had significantly better viability and morphology scores and lower concentrations of PGE2, MMP-3, IL-6, and COX-2 than untreated LPS challenge-exposed cells. Cells treated with HA had significantly better morphology scores at 50 hours than cells treated with HA-CS-NAG. Cells treated with HA-CS-NAG had significantly superior suppression of LPS-induced production of PGE2, IL-6, and MMP-3 than cells treated with HA alone. CONCLUSIONS AND CLINICAL RELEVANCE HA and HA-CS-NAG protected synovial cells from the effects of LPS. Treatment with HA-CS-NAG had the greatest anti-inflammatory effect. These results supported the protective potential of HA and HA-CS-NAG treatments.
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Acetilglucosamina/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Sulfatos de Condroitina/farmacologia , Ácido Hialurônico/farmacologia , Sinoviócitos/efeitos dos fármacos , Animais , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Interações Medicamentosas , Cavalos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologiaRESUMO
There are several animal models routinely used for study of the spinal fusion process and animal selection largely depends on the scientific question to be answered. This review outlines the advantages and disadvantages of various animal models used to study spinal fusion and describes the New Zealand White (NSW) rabbit which is the most popular preclinical model to study spinal fusion. We outline critical steps required in planning and performing spinal fusion surgery in this model. This includes determination of the required animal number to obtain statistical significance, an outline of appropriate technique for posterolateral fusion and other components of completing a study. As advances in drug delivery move forward and our understanding of the cascade of gene expression occurring during the fusion process grows, performing and interpreting preclinical animal models will be vital to validating new therapies to enhance spinal fusion.
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Modelos Animais , Coelhos , Fusão Vertebral/métodos , Animais , Cuidados Pós-Operatórios , Complicações Pós-OperatóriasRESUMO
Selective inhibition of Cathepsin K (CatK) has a promising therapeutic potential for diseases associated with bone loss and osseous inflammation, such as osteoarthritis, periodontitis, and osteoporosis. In horses, stress-related bone injuries are common and accompanied by bone pain and inflammation resulting in excessive bone resorption and periostitis. VEL-0230 is a highly selective inhibitor of CatK that significantly decreased bone resorption and increased bone formation biomarkers. The goal of this study was to demonstrate the presence of CatK in equine bone and a simultaneous influence on the bone marrow cellular components including function and differentiation. Our objectives were: 1) to investigate the tissue localization of CatK protein in equine bone using immunohistochemistry, and 2) to determine the effect of CatK inhibition on osteoclastogenic, chondrogenic and osteogenic differentiation potential of equine stem and progenitor cells in vitro using histochemical staining and differentiation-related gene expression analyses. Bone biopsies, harvested from the tuber coxae and proximal phalanx of six healthy horses, were processed for immunostaining against CatK. Sternal bone marrow aspirates were cultured in 0, 1, 10, or 100 µM of VEL-0230 and subsequent staining scoring and gene expression analyses performed. All cells morphologically characterized as osteoclasts and moderate number of active bone lining osteoblasts stained positive for CatK. Histochemical staining and gene expression analyses revealed a significant increase in the osteoclastogenic, chondrogenic and osteogenic differentiation potential of equine bone marrow cells, which was VEL-0230-concentration dependent for the latter two. These results suggested that CatK inhibition may have anabolic effects on bone and cartilage regeneration that may be explained as a feedback response to CatK depletion. In conclusion, the use of CatK inhibition to reduce inflammation and associated bone resorption in equine osseous disorders may offer advantages to other therapeutics that would require further study.
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BACKGROUND: Many animal models have been developed to characterize the complexity of colonic inflammation. In dextran sodium sulfate (DSS) experimental colitis in mice the choice of reference genes is critical for accurate quantification of target genes using quantitative real time PCR (RT-qPCR). No studies have addressed the performance of reference genes in mice DSS-experimental colitis. This study aimed to determine the stability of reference genes expression (RGE) in DSS-experimental murine colitis. METHODS: Colitis was induced in male C57BL/6 mice using DSS5% for 5 days, control group received water. RNA was extracted from inflamed and non-inflamed colon. Using RT-qPCR, comparative analysis of 13 RGE was performed according to predefined criteria and relative colonic TNF-α and IL-1ß gene expression was determined by calculating the difference in the threshold cycle. RESULTS: Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), ß-actin (Actb), or ß2-microglobulin (ß2m) showed the highest variability within the inflamed and control groups. Conversely, TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. Normalization of colonic TNF-α and IL-1ß mRNA levels was dependent on the reference gene used. Depending on the genes used to normalize the data, statistical significance varied from significant when TBP / Eef2 were used to non-significant when Gapdh, Actb or ß2m were used. CONCLUSIONS: This study highlights the appropriate choice of RGE to ensure adequate normalization of RT-qPCR data when using this model. Suboptimal RGE may explain controversial results from published studies. We recommend using Tbp and Eef2 instead of Gapdh, Actb or ß2m as reference genes.
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Colite/genética , RNA Mensageiro/genética , Animais , Colite/metabolismo , Colite/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Interleucina-1beta/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 de Elongação de Peptídeos/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteína de Ligação a TATA-Box/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Cathepsin K (CatK) is an important enzyme regulating bone degradation and has been shown to contribute to the immune response. We have studied two inflammatory models in equine bone marrow nucleated cells (BMNCs); the LPS and the unmethylated CpG stimulation with the following objectives to: 1.determine whether CatK inhibition will alter the cytokine secretion by stimulated BMNCs; specifically IL-1ß, IL-6, and TNF-α, and 2.determine the changes in BMNCs surface markers' expression and MHC II molecule under CatK inhibition. Cathepsin K inhibition promoted BMNCs viability and reduced cell apoptosis. Moreover, CatK inhibition significantly decreased cytokine secretion of either naïve or stimulated BMNCs, and altered their MHC II molecule expression. In conclusion, CatK inhibition in horses did affect BMNCs other than mature osteoclasts rendering them hypo-responsive to both TLR4- and TLR9-induced inflammation, predicting a proteolytic activity for CatK within the MyD88 pathway and/or the following proteolytic events required for the cytokines secretion.