RESUMEN

Background: Chronic rhinosinusitis (CRS) is an inflammatory disease affecting more than 10% of the global adult population. It is classified into Th1, Th2, and Th17 endotypes and eosinophilic and non-eosinophilic types. Th2-based inflammation and eosinophilic CRS (ECRS) are associated with tissue remodeling and fibrinolytic system impairment. Objective: To elucidate the role of eosinophils in inducing fibrin deposition in CRS nasal polyp tissues and explore potential regulatory mechanisms. Methods: We analyzed the expression of genes related to the serpin family and fibrinolytic system using Gene Expression Omnibus and Next-generation sequencing data. Differentially expression genes (DEGs) analysis was used to compare control and nasal polyp tissues, followed by KEGG and Gene ontology (GO) analysis. We measured the expression and correlation of plasminogen activator-1 (PAI-1), tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and urokinase plasminogen activator surface receptor (u-PAR) in CRS tissues, and evaluated the effect of eosinophils on the fibrinolytic system using a cytokine array and co-culture. Results: Nasal polyp tissues showed upregulated PAI-1, u-PA, and u-PAR expression and downregulated t-PA expression. Fibrinolytic system-related genes positively correlated with Th2 cytokines, except for t-PA. Eosinophil-derived Chitinase-3-like protein 1 (CHI3L1) increased PAI-1 expression and decreased t-PA levels in fibroblasts and epithelial cells. The inhibition of CHI3L1 suppresses these alterations. Conclusion: CHI3L1 contributes to fibrin deposition by impairing the fibrinolytic system during nasal polyp formation. The regulation of CHI3L1 expression may inhibit fibrin deposition and edema in ECRS, presenting a potential treatment for this condition.

Asunto(s)

Proteína 1 Similar a Quitinasa-3 , Eosinófilos , Fibrinólisis , Pólipos Nasales , Inhibidor 1 de Activador Plasminogénico , Rinitis , Sinusitis , Humanos , Pólipos Nasales/metabolismo , Pólipos Nasales/inmunología , Sinusitis/metabolismo , Sinusitis/inmunología , Rinitis/metabolismo , Rinitis/inmunología , Enfermedad Crónica , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Proteína 1 Similar a Quitinasa-3/metabolismo , Proteína 1 Similar a Quitinasa-3/genética , Adulto , Femenino , Masculino , Persona de Mediana Edad , Eosinófilos/inmunología , Eosinófilos/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Activador de Tejido Plasminógeno/genética , Citocinas/metabolismo , Rinosinusitis

RESUMEN

Degenerative cascades of the intervertebral disc (IVD) are characterized by the presence of immune cells like monocytes, macrophages, and leukocytes, which contribute to inflammation. Previous in vitro studies on monocyte chemotaxis in the presence of chemical or mechanical stimulation were unable to establish the effects of endogenous stimulating factors from resident IVD cells, or fully understand macrophage and monocyte differentiation pathways in IVD degeneration. Our study simulates monocyte extravasation using a fabricated microfluidic chemotaxis IVD organ-on-a-chip (IVD organ chip), which models the geometry of IVD, chemoattractant diffusion, and infiltration of immune cells. Additionally, the fabricated IVD organ chip mimics stepwise monocyte infiltration and differentiation into macrophages in the degenerative nucleus pulposus (NP) induced by IL-1ß. We find that naïve NP cells do not recruit THP-1 monocyte-like cells, but degenerative NP cells recruit and accumulate macrophages through chemo-gradient channels. Furthermore, the differentiated and migrated THP-1 cells show phagocytic activity around inflammatory NP cells. Our in vitro model of monocyte chemotaxis with degenerative NP on an IVD organ chip depicts the sequential processes of monocyte migration/infiltration, monocyte-to-macrophage differentiation, and accumulation. Using this platform to gain a deeper understanding of monocyte infiltration and differentiation processes can provide insights into the pathophysiology of the immune response in degenerative IVD.

Asunto(s)

Degeneración del Disco Intervertebral , Disco Intervertebral , Núcleo Pulposo , Humanos , Núcleo Pulposo/metabolismo , Monocitos , Degeneración del Disco Intervertebral/metabolismo , Sistemas Microfisiológicos , Disco Intervertebral/metabolismo

RESUMEN

Degeneration of the intervertebral disc (IVD) is a major contributor to low back pain (LBP). IVD degeneration is characterized by abnormal production of inflammatory cytokines secreted by IVD cells. Although the underlying molecular mechanisms of LBP have not been elucidated, increasing evidence suggests that LBP is associated particularly with microglia in IVD tissues and the peridiscal space, aggravating the cascade of degenerative events. In this study, we implemented our microfluidic chemotaxis platform to investigate microglial inflammation in response to our reconstituted degenerative IVD models. The IVD models were constructed by stimulating human nucleus pulposus (NP) cells with interleukin-1ß and producing interleukin-6 (129.93 folds), interleukin-8 (18.31 folds), C-C motif chemokine ligand-2 (CCL-2) (6.12 folds), and CCL-5 (5.68 folds). We measured microglial chemotaxis (p < 0.05) toward the conditioned media of the IVD models. In addition, we observed considerable activation of neurodegenerative and deactivation of protective microglia via upregulated expression of CD11b (p < 0.001) and down-regulation of CD206 protein (p < 0.001) by soluble factors from IVD models. This, in turn, enhances the inflammatory milieu in IVD tissues, causing matrix degradation and cellular damage. Our findings indicate that degenerative IVD may induce degenerative microglial proinflammation, leading to LBP development.

Asunto(s)

Degeneración del Disco Intervertebral , Disco Intervertebral , Dolor de la Región Lumbar , Humanos , Microglía/metabolismo , Interleucina-1beta/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Interleucina-8/metabolismo , Medios de Cultivo Condicionados/metabolismo , Interleucina-6/metabolismo , Ligandos , Disco Intervertebral/metabolismo , Citocinas/metabolismo

RESUMEN

To evaluate dominant cell-to-cell paracrine interactions, including those of human annulus fibrosus (AF), nucleus pulposus (NP), and endothelial cells (ECs), in the production of inflammatory mediators and catabolic enzymes, ECs was cultured in soluble factors derived from AF or NP cells (AFCM or NPCM, respectively) and vice versa. We analysed IL-6 and -8, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and -3, nerve growth factor (NGF)-ß, and brain-derived neurotrophic factors (BDNFs) with qRT-PCR and ELISA. We implement a microfluidic platform to analyse migration properties of AF and NP cells and ECs in 3D cultures. Our results show that IL-1ß-stimulated AF cells produced significantly higher levels of IL-6 and -8, VEGF, and MMP-1 than IL-1ß-stimulated NP cells. However, production of IL-6 and -8, VEGF, and MMP-3 was significantly higher in NP cells than in AF cells, under the presence of ECs conditioned medium. We observed considerable migration of NP cells co-cultured with ECs through the microfluidic platform. These results suggest that AF cells may play a major role in the initial degeneration of intervertebral disc. Furthermore, it was found that interactions between NP cells and ECs may play a significant role in the development or progression of diseases.

Asunto(s)

Anillo Fibroso/patología , Células Endoteliales/patología , Inflamación/patología , Degeneración del Disco Intervertebral/patología , Núcleo Pulposo/patología , Anillo Fibroso/citología , Anillo Fibroso/metabolismo , Línea Celular , Células Cultivadas , Técnicas de Cocultivo/métodos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Humanos , Inflamación/complicaciones , Inflamación/metabolismo , Degeneración del Disco Intervertebral/complicaciones , Degeneración del Disco Intervertebral/metabolismo , Masculino , Persona de Mediana Edad , Núcleo Pulposo/citología , Núcleo Pulposo/metabolismo

RESUMEN

Intervertebral disc (IVD) degeneration with chronic low back pain is associated with neo-vascularisation into the deeper IVD regions. During this process, endothelial cells (ECs), which are primarily responsible for angiogenesis, interact with the adjacent annulus fibrosus (AF) cells, which are the first line of defence against the invasion of vascular structures into deeper IVD regions. However, the accumulation of inflammatory and catabolic enzymes that results from this interaction promotes matrix degradation and an inflammatory response. Thus, regulating the production of these mediators and catabolic enzymes could ameliorate IVD degeneration. Photobiomodulation (PBM) therapy is a non-invasive stimulation known to have biologically beneficial effects on wound healing, tissue repair, and inflammation. Here, we examined the effects of PBM, administered at various wavelengths (645, 525, and 465 nm) and doses (16, 32, and 64 J/cm2), on EC-stimulated human AF cells. Our results show that PBM selectively inhibited the EC-mediated production of inflammatory mediators, catabolic enzymes, and neurotrophins by human AF cells in a dose- and wavelength-dependent manner. These results suggest that PBM could be a superior and advanced treatment strategy for IVD degeneration.

Asunto(s)

Anillo Fibroso/citología , Medios de Cultivo Condicionados/química , Células Endoteliales/citología , Matriz Extracelular/metabolismo , Degeneración del Disco Intervertebral/metabolismo , Terapia por Luz de Baja Intensidad/métodos , Neovascularización Patológica/metabolismo , Adulto , Anillo Fibroso/metabolismo , Anillo Fibroso/efectos de la radiación , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Células Endoteliales/química , Matriz Extracelular/genética , Femenino , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/radioterapia , Masculino , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Persona de Mediana Edad , Modelos Biológicos , Neovascularización Patológica/radioterapia

RESUMEN

Intervertebral disc (IVD) degeneration is associated with imbalances between catabolic and anabolic responses, regulated by extracellular matrix (ECM)-modifying enzymes such as matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors of metalloproteinases (TIMPs). Potential contributing factors, such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, derived from infiltrated, activated macrophages within IVD tissues, can trigger abnormal production of ECM-modifying enzymes and progression of IVD degeneration. Novel therapies for regulating ECM-modifying enzymes can prevent or ameliorate IVD degeneration. Photobiomodulation (PBM), known to regulate wound repair, exhibits regenerative potential by modulating biological molecules. This study examined the effects of PBM, administered at various wavelengths (630, 525, and 465 nm) and energy densities (16, 32, and 64 J/cm2), on the production of ECM-modifying enzymes in replicated degenerative IVD. Our results showed that PBM selectively inhibited the production of ECM-modifying enzymes in a dose- and wavelength-dependent manner, suggesting that it could be a novel tool for treating symptomatic IVD degeneration.

Asunto(s)

Matriz Extracelular/enzimología , Degeneración del Disco Intervertebral/terapia , Terapia por Luz de Baja Intensidad , Núcleo Pulposo/enzimología , Progresión de la Enfermedad , Matriz Extracelular/efectos de la radiación , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Interleucina-1beta/genética , Disco Intervertebral/enzimología , Disco Intervertebral/patología , Disco Intervertebral/efectos de la radiación , Degeneración del Disco Intervertebral/genética , Degeneración del Disco Intervertebral/patología , Macrófagos/patología , Macrófagos/efectos de la radiación , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/efectos de la radiación , Núcleo Pulposo/patología , Núcleo Pulposo/efectos de la radiación , Cultivo Primario de Células , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/efectos de la radiación , Factor de Necrosis Tumoral alfa/genética

RESUMEN

The etiology of intervertebral disc (IVD) degeneration accompanied by low back pain (LBP) is largely unknown, and there are no curative therapies. Painful IVD degeneration is associated with infiltrated macrophage-mediated inflammatory response of human nucleus pulposus (NP) cells. The present study aimed to address the hypothesis that pro-inflammatory cytokines derived from macrophages lead to the altered molecular phenotype of human NP cells and to investigate the effects of phototherapy (630, 525, 465 nm with 16, 32, 64 J/cm2) on pain-related cytokine interleukin (IL)-6 and chemokine IL-8 under inflammatory conditions in human NP cells. Human NP cells were treated with soluble factors derived from macrophages in an inflammatory microenvironment, similar to that found in degenerative IVD. Human NP cells were also treated with phototherapy (630, 525, 465 nm with 16, 32, 64 J/cm2), and their cytokine and chemokine levels were detected. The soluble factors caused modulated expression of IL-6, IL-8, and matrix metalloproteinases (MMPs) at the gene and protein levels, causing a shift toward matrix catabolism through the expression of MMPs and increased pain-related factors via preferential activation of the nuclear factor-kappa B (NF-κB) p50 protein. Importantly, phototherapy attenuated the protein and gene expression of pain-related factor IL-6 at all doses and wavelengths. Interestingly, phototherapy also modulated the protein and gene expression of IL-8, which is responsible for the anabolic response, at a wavelength of 465 nm at all doses, in human NP cells. These findings suggested that phototherapy, at an optimal dose and wavelength, might be a useful therapeutic tool to treat IVD degeneration.

Asunto(s)

Degeneración del Disco Intervertebral/terapia , Núcleo Pulposo/patología , Fototerapia , Línea Celular , Citocinas/metabolismo , Femenino , Expresión Génica/efectos de la radiación , Humanos , Inflamación/metabolismo , Dolor de la Región Lumbar/metabolismo , Dolor de la Región Lumbar/terapia , Macrófagos/metabolismo , Masculino , FN-kappa B/metabolismo , Núcleo Pulposo/inmunología , Núcleo Pulposo/metabolismo

RESUMEN

The aetiology of intervertebral disc (IVD) degeneration accompanied by low back pain (LBP) is largely unknown, and there are no effective fundamental therapies. Symptomatic IVD is known to be associated with nerve root compression. However, even in the absence of nerve compression, LBP occurs in patients with IVD degeneration. We hypothesize that this phenomenon is associated with a concentration of pro-inflammatory cytokines such as interleukin (IL)-1ß and tumour necrosis factor-alpha (TNF-α), which can lead to altered histologic features and cellular phenotypes observed during IVD degeneration. This study investigated the effects of the concentration of IL-1ß and macrophage derived soluble factor including IL-1ß and TNF-α on the painful response of human annulus fibrosus (AF) cells using a newly developed spine-on-a-chip. Human AF cells were treated with a range of concentrations of IL-1ß and macrophage soluble factors. Our results show that increasing the concentration of inflammatory initiator caused modulated expression of pain-related factors, angiogenesis molecules, and catabolic enzymes. Furthermore, accumulated macrophage derived soluble factors resulted in morphological changes in human AF cells and kinetic alterations such as velocity, dendritic length, cell area, and growth rate, similar to that reported within degenerative IVD. Thus, a better understanding of the relationships between molecular and kinetic alterations can provide fundamental information regarding the pathology of IVD degenerative progression.