RESUMEN
Kingella kingae is an emerging pathogen that causes septic arthritis, osteomyelitis, and bacteremia in children from 6 to 48 months of age. The presence of bacteria within or near the bone is associated with an inflammatory process that results in osteolysis, but the underlying pathogenic mechanisms involved are largely unknown. To determine the link between K. kingae and bone loss, we have assessed whether infection per se or through the genesis of a pro-inflammatory microenvironment can promote osteoclastogenesis. For that purpose, we examined both the direct effect of K. kingae and the immune-mediated mechanism involved in K. kingae-infected macrophage-induced osteoclastogenesis. Our results indicate that osteoclastogenesis is stimulated by K. kingae infection directly and indirectly by fueling a potent pro-inflammatory response that drives macrophages to undergo functional osteoclasts via TNF-α and IL-1ß induction. Such osteoclastogenic capability of K. kingae is counteracted by their outer membrane vesicles (OMV) in a concentration-dependent manner. In conclusion, this model allowed elucidating the interplay between the K. kingae and their OMV to modulate osteoclastogenesis from exposed macrophages, thus contributing to the modulation in joint and bone damage.
Asunto(s)
Microambiente Celular/fisiología , Infecciones por Neisseriaceae/inmunología , Infecciones por Neisseriaceae/patología , Osteoclastos/inmunología , Osteogénesis/fisiología , Animales , Línea Celular , Humanos , Kingella kingae , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Osteoclastos/metabolismo , Células RAW 264.7RESUMEN
Brucellosis is a prevalent global zoonotic infection but has far more impact in developing countries. The adipocytes are the most abundant cell type of adipose tissue and their secreted factors play an important role in several aspects of the innate and adaptive immune response. Here, we demonstrated the ability of Brucella abortus to infect and replicate in both adipocytes and its precursor cells (pre-adipocytes) derived from 3T3-L1 cell line. Additionally, infection of pre-adipocytes also inhibited adipogenesis in a mechanism independent of bacterial viability and dependent on lipidated outer membrane protein (L-Omp19). B. abortus infection was able to modulate the secretion of IL-6 and the matrix metalloproteases (MMPs) -2 and-9 in pre-adipocytes and adipocytes, and also modulated de transcription of adiponectin, leptin, and resistin in differentiated adipocytes. B. abortus-infected macrophages also modulate adipocyte differentiation involving a TNF-α dependent mechanism, thus suggesting a plausible interplay between B. abortus, adipocytes, and macrophages. In conclusion, B. abortus is able to alter adipogenesis process in adipocytes and its precursors directly after their infection, or merely their exposure to the B. abortus lipoproteins, and indirectly through soluble factors released by B. abortus-infected macrophages.
Asunto(s)
Adipocitos/citología , Adipogénesis , Brucelosis/complicaciones , Diferenciación Celular , Inflamación/inmunología , Macrófagos/inmunología , Células 3T3-L1 , Adipocitos/inmunología , Adipocitos/metabolismo , Adipocitos/microbiología , Animales , Brucella abortus/fisiología , Citocinas/metabolismo , Inflamación/metabolismo , Inflamación/microbiología , Mediadores de Inflamación/metabolismo , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , RatonesRESUMEN
In Brucellosis, the role of hepatic stellate cells (HSCs) in the induction of liver fibrosis has been elucidated recently. Here, we study how the infection modulates the antigen-presenting capacity of LX-2 cells. Brucella abortus infection induces the upregulation of class II transactivator protein (CIITA) with concomitant MHC-I and -II expression in LX-2 cells in a manner that is independent from the expression of the type 4 secretion system (T4SS). In concordance, B. abortus infection increases the phagocytic ability of LX-2 cells and induces MHC-II-restricted antigen processing and presentation. In view of the ability of B. abortus-infected LX-2 cells to produce monocyte-attracting factors, we tested the capacity of culture supernatants from B. abortus-infected monocytes on MHC-I and -II expression in LX-2 cells. Culture supernatants from B. abortus-infected monocytes do not induce MHC-I and -II expression. However, these supernatants inhibit MHC-II expression induced by IFN-γ in an IL-10 dependent mechanism. Since hepatocytes constitute the most abundant epithelial cell in the liver, experiments were conducted to determine the contribution of these cells in antigen presentation in the context of B. abortus infection. Our results indicated that B. abortus-infected hepatocytes have an increased MHC-I expression, but MHC-II levels remain at basal levels. Overall, B. abortus infection induces MHC-I and -II expression in LX-2 cells, increasing the antigen presentation. Nevertheless, this response could be modulated by resident or infiltrating monocytes/macrophages.
RESUMEN
Osteoarticular brucellosis is the most frequent complication of active disease. A large amount of cells in bone are osteocytes. Since bone remodeling process is regulated by hormones we sought to study the effect of cortisol and DHEA in Brucella abortus-infected osteocytes. Cortisol treatment inhibited the expression of IL-6, TNF-α, MMP-2 and RANKL in B. abortus-infected osteocytes. DHEA could reverse the inhibitory effect of cortisol on MMP-2 production. B. abortus infection inhibited connexin 43 (Cx43) expression in osteocytes. This expression was increased when cortisol was incorporated during the infection and DHEA treatment partially reversed the effect of cortisol. Osteocytes-infected with B. abortus induced osteoclast's differentiation. Yet, the presence of cortisol, but not DHEA, during osteocyte infection inhibited osteoclastogenesis. Glucocorticoid receptor (GR) is implicated in the signaling of cortisol. Infection with B. abortus was able to increase GRα/ß ratio. Levels of intracellular cortisol are not only dependent on GR expression but also a result of the activity of the isoenzymes 11ß-hydroxysteroid dehydrogenase (11ß-HSD)-1 (cortisone to cortisol conversion), 11ß-HSD2 (cortisol to cortisone conversion). B. abortus infection increased 11ß-HSD 1/2 ratio and cortisone mimicked the effect of cortisol. Our results indicated that cortisol and DHEA could modulate osteocyte responses during B. abortus infection.
Asunto(s)
Brucella abortus/fisiología , Brucelosis/patología , Osteocitos/microbiología , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasas/genética , Animales , Brucella abortus/crecimiento & desarrollo , Brucella abortus/metabolismo , Brucelosis/metabolismo , Células Cultivadas , Conexina 43/metabolismo , Cortisona/farmacología , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Deshidroepiandrosterona/farmacología , Hidrocortisona/metabolismo , Hidrocortisona/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Viabilidad Microbiana , Osteocitos/citología , Osteocitos/efectos de los fármacos , Osteocitos/metabolismo , Osteogénesis/efectos de los fármacos , Osteoprotegerina/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Transducción de SeñalRESUMEN
In human brucellosis, the liver is frequently affected. Brucella abortus triggers a profibrotic response on hepatic stellate cells (HSCs) characterized by inhibition of MMP-9 with concomitant collagen deposition and TGF-ß1 secretion through type 4 secretion system (T4SS). Taking into account that it has been reported that the inflammasome is necessary to induce a fibrotic phenotype in HSC, we hypothesized that Brucella infection might create a microenvironment that would promote inflammasome activation with concomitant profibrogenic phenotype in HSCs. B. abortus infection induces IL-1ß secretion in HSCs in a T4SS-dependent manner. The expression of caspase-1 (Casp-1), absent in melanoma 2 (AIM2), Nod-like receptor (NLR) containing a pyrin domain 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC) was increased in B. abortus-infected HSC. When infection experiments were performed in the presence of glyburide, a compound that inhibits NLRP3 inflammasome, or A151, a specific AIM2 inhibitor, the secretion of IL-1ß was significantly inhibited with respect to uninfected controls. The role of inflammasome activation in the induction of a fibrogenic phenotype in HSCs was determined by performing B. abortus infection experiments in the presence of the inhibitors Ac-YVAD-cmk and glyburide. Both inhibitors were able to reverse the effect of B. abortus infection on the fibrotic phenotype in HSCs. Finally, the role of inflammasome in fibrosis was corroborated in vivo by the reduction of fibrotic patches in liver from B. abortus-infected ASC, NLRP, AIM2, and cCasp-1/11 knock-out (KO) mice with respect to infected wild-type mice.
Asunto(s)
Brucella abortus/fisiología , Brucelosis/inmunología , Células Estrelladas Hepáticas/inmunología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Animales , Brucella abortus/genética , Brucelosis/genética , Brucelosis/microbiología , Caspasa 1/genética , Caspasa 1/inmunología , Fibrosis/genética , Fibrosis/inmunología , Fibrosis/microbiología , Células Estrelladas Hepáticas/microbiología , Humanos , Inflamasomas/genética , Interleucina-1beta/genética , Hígado/inmunología , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunologíaRESUMEN
Osteoarticular brucellosis is the most common localization of human active disease. Osteoblasts are specialized mesenchymal-derived cells involved in bone formation and are considered as professional mineralizing cells. Autophagy has been involved in osteoblast metabolism. The present study demonstrates that Brucella abortus infection induces the activation of the autophagic pathway in osteoblast cells. Autophagy was revealed by upregulation of LC3II/LC3I ratio and Beclin-1 expression as well as inhibition of p62 expression in infected cells. Induction of autophagy was also corroborated by using the pharmacological inhibitors wortmannin, a PI 3-kinase inhibitor, and leupeptin plus E64 (inhibitors of lysosomal proteases). Autophagy induction create a microenvironment that modifies osteoblast metabolism by the inhibition of the deposition of organic and mineral matrix, the induction of matrix metalloproteinase (MMP)-2, osteopontin, and RANKL secretion leading to bone loss. Accordingly, autophagy is also involved in the down-modulation of the master transcription factor in bone formation osterix during B. abortus infection. Taking together our results indicate that B. abortus induces the activation of autophagy pathway in osteoblast cells and this activation is involved in the modulation of osteoblast function and bone formation.
Asunto(s)
Autofagia , Brucella abortus/patogenicidad , Brucelosis , Osteoblastos/metabolismo , Osteoblastos/microbiología , Beclina-1/metabolismo , Matriz Ósea/metabolismo , Matriz Ósea/microbiología , Brucelosis/patología , Diferenciación Celular , Línea Celular , Colágeno/metabolismo , Citocinas/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Osteogénesis , Osteopontina , Fosfatidilinositol 3-Quinasas , Ligando RANK/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Brucella abortus induces an inflammatory response that stimulates the endocrine system resulting in the secretion of cortisol and dehydroepiandrosterone (DHEA). Osteoarticular brucellosis is the most common presentation of the active disease in humans, and we have previously demonstrated that B. abortus infection inhibits osteoblast function. We aimed to evaluate the role of cortisol and DHEA on osteoblast during B. abortus infection. B. abortus infection induces apoptosis and inhibits osteoblast function. DHEA treatment reversed the effect of B. abortus infection on osteoblast by increasing their proliferation, inhibiting osteoblast apoptosis, and reversing the inhibitory effect of B. abortus on osteoblast differentiation and function. By contrast, cortisol increased the effect of B. abortus infection. Cortisol regulates target genes by binding to the glucocorticoid receptor (GR). B. abortus infection inhibited GRα expression. Cell responses to cortisol not only depend on GR expression but also on its intracellular bioavailability, that is, dependent on the activity of the isoenzymes 11ß-hydroxysteroid dehydrogenase (HSD) type-1, 11ß-HSD2 (which convert cortisone to cortisol and vice versa, respectively). Alterations in the expression of these isoenzymes in bone cells are associated with bone loss. B. abortus infection increased 11ß-HSD1 expression but had no effect on 11ß-HSD2. DHEA reversed the inhibitory effect induced by B. abortus infection on osteoblast matrix deposition in an estrogen receptor- and ERK1/2-dependent manner. We conclude that DHEA intervention improves osteoblast function during B. abortus infection making it a potential candidate to ameliorate the osteoarticular symptoms of brucellosis.
Asunto(s)
Brucella abortus/fisiología , Brucelosis Bovina/metabolismo , Brucelosis Bovina/microbiología , Deshidroepiandrosterona/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteoblastos/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Apoptosis , Biomarcadores , Brucelosis Bovina/genética , Brucelosis Bovina/patología , Bovinos , Diferenciación Celular , Línea Celular , Proliferación Celular , Expresión Génica , Ratones , Viabilidad Microbiana , Osteoblastos/citología , Osteogénesis/efectos de los fármacos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismoRESUMEN
The liver is frequently affected in patients with active brucellosis. The present study demonstrates that Brucella abortus infection induces the activation of the autophagic pathway in hepatic stellate cells to create a microenvironment that promotes a profibrogenic phenotype through the induction of transforming growth factor-ß1 (TGF-ß1), collagen deposition, and inhibition of matrix metalloproteinase-9 (MMP-9) secretion. Autophagy was revealed by upregulation of the LC3II/LC3I ratio and Beclin-1 expression as well as inhibition of p62 expression in infected cells. The above-described findings were dependent on the type IV secretion system (VirB) and the secreted BPE005 protein, which were partially corroborated using the pharmacological inhibitors wortmannin, a phosphatidyl inositol 3-kinase inhibitor, and leupeptin plus E64 (inhibitors of lysosomal proteases). Activation of the autophagic pathway in hepatic stellate cells during Brucella infection could have an important contribution to attenuating inflammatory hepatic injury by inducing fibrosis. However, with time, B. abortus infection induced Beclin-1 cleavage with concomitant cleavage of caspase-3, indicating the onset of apoptosis of LX-2 cells, as was confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and Hoechst staining. These results demonstrate that the cross talk of LX-2 cells and B. abortus induces autophagy and fibrosis with concomitant apoptosis of LX-2 cells, which may explain some potential mechanisms of liver damage observed in human brucellosis.
Asunto(s)
Autofagia/fisiología , Brucella abortus/patogenicidad , Fibrosis/microbiología , Fibrosis/patología , Células Estrelladas Hepáticas/microbiología , Células Estrelladas Hepáticas/patología , Apoptosis/fisiología , Beclina-1/metabolismo , Brucelosis/metabolismo , Brucelosis/microbiología , Brucelosis/patología , Caspasa 3/metabolismo , Línea Celular , Colágeno/metabolismo , Fibrosis/metabolismo , Células Estrelladas Hepáticas/metabolismo , Humanos , Hígado/metabolismo , Hígado/microbiología , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/microbiología , Cirrosis Hepática/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Fenotipo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Sistemas de Secreción Tipo IV/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Brucella abortus is an intracellular bacterium that establishes lifelong infections in livestock and humans although the mechanisms of its chronicity are poorly understood. Activated B cells have long lifespan and B. abortus infection activates B cells. Our results indicate that the direct infection of B cells with B. abortus induced matrix metalloproteinase-9 (MMP-9), receptor activator for NF κB ligand (RANKL), tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion. In addition, supernatants from B. abortus-infected B cells induced bone marrow-derived monocytes to undergo osteoclastogenesis. Using osteoprotegerin, RANKL's decoy receptor, we determined that RANKL is involved in osteoclastogenesis induced by supernatants from B. abortus-infected B cells. The results presented here shed light on how the interactions of B. abortus with B cells may have a role in the pathogenesis of brucellar osteoarticular disease.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/microbiología , Brucella abortus/inmunología , Citocinas/metabolismo , Activación de Linfocitos , Monocitos/metabolismo , Osteogénesis , Animales , Células Cultivadas , Ratones Endogámicos BALB C , Monocitos/efectos de los fármacos , Ligando RANK/metabolismoRESUMEN
Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-ß, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage.
Asunto(s)
Brucella abortus/patogenicidad , Conexina 43/biosíntesis , Integrinas/biosíntesis , Osteocitos/metabolismo , Ligando RANK/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis , Brucelosis/microbiología , Brucelosis/patología , Diferenciación Celular , Línea Celular , Quimiocina CXCL1/metabolismo , Macrófagos/microbiología , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Osteoclastos/citología , Osteocitos/microbiología , Osteoprotegerina/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway.