Unraveling the neuroimmune interface in chronic pain-the association between cytokines in the cerebrospinal fluid and pain in patients with lumbar disk herniation or degenerative disk disease.

ABSTRACT: Recent evidence highlights the importance of the neuroimmune interface, including periphery-to-central nervous system (CNS) neuroimmune crosstalk, in chronic pain. Although neuroinflammatory processes have been implicated in central sensitization for a long time, their potential neuroprotective and analgesic effects remain relatively elusive. We have explored the relationships between cytokine expression and symptom severity, and candidates for periphery-to-CNS crosstalk. Patients with degenerative disk disease (DDD) (nociceptive pain) or patients with lumbar disk herniation (LDH) with radiculopathy (predominantly neuropathic pain) completed questionnaires regarding pain and functional disability, underwent quantitative sensory testing, and provided blood and cerebrospinal fluid (CSF) samples. Proximity extension assay (PEA) was used to measure the levels of 92 inflammatory proteins in the CSF and serum from a total of 160 patients and controls, and CSF/serum albumin quotients was calculated for patients with DDD and patients with LDH. We found signs of neuroimmune activation, in the absence of systemic inflammation. Regarding periphery-to-CNS neuroimmune crosstalk, there were significant associations between several cytokines and albumin quotient, despite the latter being primarily at subclinical levels. The cytokines CCL11, CD5, IL8, and MMP-10 were elevated in the CSF, had positive correlations between CSF and serum levels, and associated in a nonlinear manner with back, but not leg, pain intensity in the LDH, but not the DDD, group. In conclusion, we found evidence for neuroimmune activation in the CNS of both patient groups in the absence of systemic inflammation and signs of a communication between CSF and serum. Complex and disease-specific associations were found between cytokines in CSF and back pain intensity.

Adipokines as potential pharmacological targets for immune inflammatory rheumatic diseases: Focus on rheumatoid arthritis, osteoarthritis, and intervertebral disc degeneration.

Adipokines are a heterogeneous group of signalling molecules secreted prevalently by adipose tissue. Initially considered as regulators of energy metabolism and appetite, adipokines have been recognized for their substantial involvement in musculoskeletal disorders, including osteoarthritis, rheumatoid arthritis, and many others. Understanding the role of adipokines in rheumatic inflammatory and autoimmune diseases, as well as in other musculoskeletal diseases such as intervertebral disc degeneration, is crucial for the development of novel therapeutic strategies. Targeting adipokines, or their signalling pathways, may offer new opportunities for the treatment and management of these conditions. By modulating adipokines levels or activity, it may be possible to regulate inflammation, to maintain bone health, and preserve muscle mass, thereby improving the outcomes and quality of life for individuals affected by musculoskeletal diseases. The aim of this review article is to update the reader on the multifaceted role of adipokines in the main rheumatic diseases such as osteoarthritis and rheumatoid arthritis and to unravel the complex interplay among adipokines, cartilage metabolism, bone remodelling and muscles, which will pave the way for innovative therapeutic intervention in the future. For completeness, the role of adipokines in intervertebral disc degeneration will be also addressed.

Mrgprb2-mediated mast cell activation exacerbates Modic changes by regulating immune niches.

Modic changes are radiographic features associated with microfracture, low-virulence organism infection and chronic inflammation with inflammatory cell infiltration in the vertebral endplate region. Mast cells, as innate immune cells similar to macrophages, are present in painful degenerated intervertebral discs. However, the involvement and mechanisms of mast cells in the development of Modic changes remain unclear. Herein, we found increased mast cell infiltration in samples from patients with Modic changes and in mouse models of Modic changes. To clarify the role of mast cells in the progression of Modic changes, we used mast cell-deficient (KITW-SH/W-SH) mice to construct a model of Modic changes and found that the severity of Modic changes in KITW-SH/W-SH mice was significantly lower than that in WT mice. These findings were further supported by the use of a mast cell-specific activator (compound 48/80) and a stabilizer (cromolyn). Furthermore, we found that mast cells were not activated via the classic IgE pathway in the Modic change models and that Mrgprb2 is the specific receptor for mast cell activation reported in recent studies. Then, we utilized Mrgprb2 knockout mice to demonstrate that Mrgprb2 knockout inhibited mast cell activation and thus reduced the degree of Modic changes. Transcriptomic sequencing revealed aberrant PI3K-AKT and MAPK pathway activation in the Mrgprb2-deficient mast cells. Additionally, Mrgpbrb2-activated mast cells regulate immune niches by recruiting macrophages, promoting M1 polarization and reducing M2 polarization, thereby promoting the progression of Modic changes. These findings suggest that mast cells may serve as a novel therapeutic target for addressing Modic changes.

Research on the role and mechanism of IL-17 in intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is one of the primary causes of low back pain (LBP), which seriously affects patients' quality of life. In recent years, interleukin (IL)-17 has been shown to be highly expressed in the intervertebral disc (IVD) tissues and serum of patients with IDD, and IL-17A has been shown to promote IDD through multiple pathways. We first searched databases such as PubMed, Cochrane, Embase, and Web of Science using the search terms "IL-17 or interleukin 17″ and "intervertebral discs". The search period ranged from the inception of the databases to December 2023. A total of 24 articles were selected after full-text screening. The main conclusion of the clinical studies was that IL-17A levels are significantly increased in the IVD tissues and serum of IDD patients. The results from the in vitro studies indicated that IL-17A can activate signaling pathways such as the NF-κB and MAPK pathways; promote inflammatory responses, extracellular matrix degradation, and angiogenesis; and inhibit autophagy in nucleus pulposus cells. The main finding of the in vivo experiments was that puncture of animal IVDs resulted in elevated levels of IL-17A within the IVD, thereby inducing IDD. Clinical studies, in vitro experiments, and in vivo experiments confirmed that IL-17A is closely related to IDD. Therefore, drugs that target IL-17A may be novel treatments for IDD, providing a new theoretical basis for IDD therapy.

Identification of Characteristic Genes in Whole Blood of Intervertebral Disc Degeneration Patients by Weighted Gene Coexpression Network Analysis (WGCNA).

Intervertebral disc degeneration (IDD) is a major cause of lower back pain. However, to date, the molecular mechanism of the IDD remains unclear. Gene expression profiles and clinical traits were downloaded from the Gene Expression Omnibus (GEO) database. Firstly, weighted gene coexpression network analysis (WGCNA) was used to screen IDD-related genes. Moreover, least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine (SVM) algorithms were used to identify characteristic genes. Furthermore, we further investigated the immune landscape by the Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) algorithm and the correlations between key characteristic genes and infiltrating immune cells. Finally, a competing endogenous RNA (ceRNA) network was established to show the regulatory mechanisms of characteristic genes. A total of 2458 genes were identified by WGCNA, and 48 of them were disordered. After overlapping the genes obtained by LASSO and SVM-RFE algorithms, genes including LINC01347, ASAP1-IT1, lnc-SEPT7L-1, B3GNT8, CHRNB3, CLEC4F, LOC102724000, SERINC2, and LOC102723649 were identified as characteristic genes of IDD. Moreover, differential analysis further identified ASAP1-IT1 and SERINC2 as key characteristic genes. Furthermore, we found that the expression of both ASAP1-IT1 and SERINC2 was related to the proportions of T cells gamma delta and Neutrophils. Finally, a ceRNA network was established to show the regulatory mechanisms of ASAP1-IT1 and SERINC2. In conclusion, the present study identified ASAP1-IT1 and SERINC2 as the key characteristic genes of IDD through integrative bioinformatic analyses, which may contribute to the diagnosis and treatment of IDD.

A new immunometabolic perspective of intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration is a common finding on spine imaging that increases in prevalence with age. IVD degeneration is a frequent cause of low back pain, which is a leading cause of disability. The process of IVD degeneration consists of gradual structural change accompanied by severe alterations in metabolic homeostasis. IVD degeneration, like osteoarthritis, is a common comorbidity in patients with obesity and type 2 diabetes mellitus, two metabolic syndrome pathological conditions in which adipokines are important promoters of low-grade inflammation, extracellular matrix degradation and fibrosis. Impairment in white adipose tissue function, due to the abnormal fat accumulation in obesity, is characterized by increased production of specific pro-inflammatory proteins such as adipokines by white adipose tissue and of cytokines such as TNF by immune cells of the stromal compartment. Investigations into the immunometabolic alterations in obesity and type 2 diabetes mellitus and their interconnections with IVD degeneration provide insights into how adipokines might affect the pathogenesis of IVD degeneration and impair IVD function and repair. Toll-like receptor-mediated signalling has also been implicated as a promoter of the inflammatory response in the metabolic alterations associated with IVD and is thus thought to have a role in IVD degeneration. Pathological starvation, obesity and adipokine dysregulation can result in immunometabolic alterations, which could be targeted for the development of new therapeutics.

An Oxidative Stress-Related Gene Pair (CCNB1/PKD1), Competitive Endogenous RNAs, and Immune-Infiltration Patterns Potentially Regulate Intervertebral Disc Degeneration Development.

Oxidative stress (OS) irreversibly affects the pathogenesis of intervertebral disc degeneration (IDD). Certain non-coding RNAs act as competitive endogenous RNAs (ceRNAs) that regulate IDD progression. Analyzing the signatures of oxidative stress-related gene (OSRG) pairs and regulatory ceRNA mechanisms and immune-infiltration patterns associated with IDD may enable researchers to distinguish IDD and reveal the underlying mechanisms. In this study, OSRGs were downloaded and identified using the Gene Expression Omnibus database. Functional-enrichment analysis revealed the involvement of oxidative stress-related pathways and processes, and a ceRNA network was generated. Differentially expressed oxidative stress-related genes (De-OSRGs) were used to construct De-OSRG pairs, which were screened, and candidate De-OSRG pairs were identified. Immune cell-related gene pairs were selected via immune-infiltration analysis. A potential long non-coding RNA-microRNA-mRNA axis was determined, and clinical values were assessed. Eighteen De-OSRGs were identified that were primarily related to intricate signal-transduction pathways, apoptosis-related biological processes, and multiple kinase-related molecular functions. A ceRNA network consisting of 653 long non-coding RNA-microRNA links and 42 mRNA-miRNA links was constructed. Three candidate De-OSRG pairs were screened out from 13 De-OSRG pairs. The abundances of resting memory CD4+ T cells, resting dendritic cells, and CD8+ T cells differed between the control and IDD groups. CD8+ T cell infiltration correlated negatively with cyclin B1 (CCNB1) expression and positively with protein kinase D1 (PKD1) expression. CCNB1-PKD1 was the only pair that was differentially expressed in IDD, was correlated with CD8+ T cells, and displayed better predictive accuracy compared to individual genes. The PKD1-miR-20b-5p-AP000797 and CCNB1-miR-212-3p-AC079834 axes may regulate IDD. Our findings indicate that the OSRG pair CCNB1-PKD1, which regulates oxidative stress during IDD development, is a robust signature for identifying IDD. This OSRG pair and increased infiltration of CD8+ T cells, which play important roles in IDD, were functionally associated. Thus, the OSRG pair CCNB1-PKD1 is promising target for treating IDD.

M2a Macrophage-Secreted CHI3L1 Promotes Extracellular Matrix Metabolic Imbalances via Activation of IL-13Rα2/MAPK Pathway in Rat Intervertebral Disc Degeneration.

The accumulation of macrophages in degenerated discs is a common phenomenon. However, the roles and mechanisms of M2a macrophages in intervertebral disc degeneration (IDD) have not been illuminated. This study investigated the expression of the M2a macrophage marker (CD206) in human and rat intervertebral disc tissues by immunohistochemistry. To explore the roles of M2a macrophages in IDD, nucleus pulposus (NP) cells were co-cultured with M2a macrophages in vitro. To clarify whether the CHI3L1 protein mediates the effect of M2a macrophages on NP cells, siRNA was used to knock down CHI3L1 transcription. To elucidate the underlying mechanisms, NP cells were incubated with recombinant CHI3L1 proteins, then subjected to western blotting analysis of the IL-13Rα2 receptor and MAPK pathway. CD206-positive cells were detected in degenerated human and rat intervertebral disc tissues. Notably, M2a macrophages promoted the expression of catabolism genes (MMP-3 and MMP-9) and suppressed the expression of anabolism genes (aggrecan and collagen II) in NP cells. These effects were abrogated by CHI3L1 knockdown in M2a macrophages. Exposure to recombinant CHI3L1 promoted an extracellular matrix metabolic imbalance in NP cells via the IL-13Rα2 receptor, along with activation of the ERK and JNK MAPK signaling pathways. This study elucidated the roles of M2a macrophages in IDD and identified potential mechanisms for these effects.

Revealing the Immune Infiltration Landscape and Identifying Diagnostic Biomarkers for Lumbar Disc Herniation.

Intervertebral disc (IVD) degeneration and its inflammatory microenvironment ultimately led to discogenic pain, which is thought to originate in the nucleus pulposus (NP). In this study, key genes involved in NP tissue immune infiltration in lumbar disc herniation (LDH) were identified by bioinformatic analysis. Gene expression profiles were downloaded from the Gene Expression Omnibus (GEO) database. The CIBERSORT algorithm was used to analyze the immune infiltration into NP tissue between the LDH and control groups. Hub genes were identified by the WGCNA R package in Bioconductor and single-cell sequencing data was analyzed using R packages. Gene expression levels were evaluated by quantitative real-time polymerase chain reaction. The immune infiltration profiles varied significantly between the LDH and control groups. Compared with control tissue, LDH tissue contained a higher proportion of regulatory T cells and macrophages, which are associated with the macrophage polarization process. The most significant module contained three hub genes and four subclusters of NP cells. Functional analysis of these genes was performed, the hub gene expression pattern was confirmed by PCR, and clinical features of the patients were investigated. Finally, we identified TGF-ß and MAPK signaling pathways as crucial in this process and these pathways may provide diagnostic markers for LDH. We hypothesize that the hub genes expressed in the specific NP subclusters, along with the infiltrating macrophages play important roles in the pathogenesis of IVD degeneration and ultimately, disc herniation.

Effects of vertebral fusion on levels of pro-inflammatory and catabolic mediators in a rabbit model of intervertebral disc degeneration.

OBJECTIVE: The aim of this study was to explore the alterations in levels of pro-inflammatory and catabolic mediators following vertebral fusion in a rabbit model of intervertebral disc degeneration. METHODS: In this study, 24 female New Zealand albino rabbits (aged 4 to 5 months and weighing 3 to 3.5 kg) were used. All the animals were randomly categorized into four groups, and dorsal spinal exposure of all lumbar vertebrae was routinely performed in each group. While disc degeneration was created in groups B, C, and D, spinal fusion was added to disc degeneration in groups C and D. Disc degeneration was typically created by puncturing the discs with an 18-gauge needle under the guidance of C-arm imaging. Fusion was achieved with posterior/posterolateral decortication and iliac bone grafts. The rabbits in groups A, B, and C were euthanized, and the discs were removed in the first week after the surgery. The rabbits in Group D were sacrificed, and the discs were harvested at 5 weeks after the surgery. The levels of Interleukin (IL)-1ß, IL-6, Nitric Oxide (NO), Matrix Metalloproteinase (MMP)-3, MMP-13, and Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) in the discs were analyzed using enzyme-linked immunosorbent assay kits. RESULTS: Significant increase was observed in the protein levels of both pro-inflammatory and catabolic mediators in disc degeneration groups (Group B, C, and D) compared to Group A. In the fusion groups (Group C and D), these increased mediators decreased, compared to non-fusion group (Group B), (IL1-ß P = 0.017, TIMP-1 P = 0.03, NO P = 0.03). However, there was no statistically significant difference in mediator levels between the short- and long-term fusion (Group C versus D). CONCLUSION: The results of this study have shown that a significant decrease in pro-inflammatory and catabolic mediators may be expected after vertebral fusion whereas there may be no significant difference between the first and fourth week of fusion surgery. These findings may contribute to clarifying the mechanism of action of vertebral fusion in the treatment of low back pain.

Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway.

Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1ß stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin ß1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin ß1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration.

Role of microRNA-15a-5p/Sox9/NF-κB axis in inflammatory factors and apoptosis of murine nucleus pulposus cells in intervertebral disc degeneration.

OBJECTIVE: MicroRNAs are well-established players in post-transcriptional gene modulation. We aim to explore the role of microRNA-15a-5p (miR-15a-5p)/sex determining region Y-box 9 (Sox9)/nuclear factor-κB (NF-κB) axis in inflammation and apoptosis of murine nucleus pulposus cells (NPCs) in intervertebral disc degeneration (IVDD). METHODS: Expression levels of miR-15a-5p and Sox9 in disc tissues from IVDD patients were determined. The IVDD mouse models were established by disc puncture, and the modeled mice were accordingly injected with miR-15a-5p antagomir and/or overexpressed Sox9 plasmid, or their negative controls. Then, the expression of miR-15a-5p, Sox9 and p-p65, pathological changes and the apoptosis of NPCs in IVDD mouse intervertebral disc tissues were measured. The NPCs were isolated and cultured, which were then transfected with miR-15a-5p inhibitor, overexpressed or silenced Sox9 plasmids, or the NCs. Next, the expression of miR-15a-5p and Sox9, apoptosis, proliferation and cell cycle distribution of NPCs, and the contents of inflammatory factors in the NPCs were evaluated. RESULTS: MiR-15a-5p expression was increased while Sox9 expression was reduced in intervertebral disc tissues from IVDD patients and mice. Mouse NPCs were successfully isolated. The down-regulated miR-15a-5p could elevate Sox9 to activate p-p65 expression, suppress NPC apoptosis and inflammatory factor contents, promote proliferation of NPCs, and arrest the NPCs at S and G2/M phases. However, these effects could be reversed by silencing Sox9. CONCLUSION: Reduction of miR-15a-5p elevated Sox9 to inhibit the inflammatory response and apoptosis of NPCs in IVDD mice through the NF-κB pathway. This study may be helpful for IVDD treatment.

Substance P Mediates Estrogen Modulation Proinflammatory Cytokines Release in Intervertebral Disc.

Intervertebral disc degeneration (IDD) is a main contributor to low back pain. A close relationship exists between inflammation and pain. Estrogen can affect inflammation and may play a crucial role in IDD and pain. Substance P (SP) can also regulate the expression of pro-inflammatory cytokines in intervertebral disc (IVD). This study aimed to investigate the potential role of SP in estrogen regulation of IDD. Nine-week-old C57BL/6 female mice were divided into four groups as follows: sham surgery (sham), ovariectomy (OVX), ovariectomy plus estrogen replacement therapy (ERT) group (OVX+E2), and ovariectomy, ERT plus neurokinin 1 receptor (NK1R) agonist (OVX+E2+G). Serum E2, body, and uterus weight were recorded. Immunohistochemistry study and quantitative real-time PCR were used for SP, NK1R, IL-1ß, IL-6, and TNF-α examination and comparison in IVD at protein and gene levels. After OVX, the gene and protein expression of TNF-α, IL-1ß, IL-6, SP, and NK1R in NP cells significantly increased compared with the sham group. ERT can reverse these impacts. ERT plays anti-inflammatory and anti-hyperalgesic roles in IDD of OVX mice. The estrogen-induced changes of the pro-inflammatory cytokines, TNF-α, IL-1ß, and IL-6, are significantly inhibited by NK1R agonists. SP may be a mediator of estrogen regulating pro-inflammatory factors in IDD. Estrogen may affect IVD inflammation through two ways: one is to directly affect the level of pro-inflammatory cytokines and the other is by means of modulation of SP.

Interleukin-17A Promotes Human Disc Degeneration by Inhibiting Autophagy Through the Activation of the Phosphatidylinositol 3-Kinase/Akt/Bcl2 Signaling Pathway.

BACKGROUND: Previous studies have suggested that interleukin (IL)-17A is a key factor that contributes to intervertebral disc degeneration (IDD), whereas autophagy has been shown to be a protective mechanism in IDD. However, the relationship between IL-17A and autophagy in IDD remains to be fully elucidated. This study sought to evaluate the association between IL-17 and autophagy and the potential mechanism through which IL-17A affects autophagy in IDD. METHODS: Intervertebral disc specimens were collected from 10 patients with lumbar disc herniation. Human degenerated nucleus pulposus (NP) cells were cultured in the presence or absence of IL-17A treatment. Western blot and monodansylcadaverine staining were used to measure autophagy levels in human degenerated NP cells. Subsequently, phosphatidylinositol 3-kinase (PI3K)/Akt/Bcl-2 pathway inhibitors were used to reveal the potential mechanism. RESULTS: IL-17A treatment inhibited the autophagic activity in human NP cells in a time- and dose-dependent manner. Moreover, monodansylcadaverine staining showed that cells treated with IL-17A had significantly fewer changes in their autophagic vacuoles compared with control-treated cells. After IL-17A treatment, expression levels of PI3K, p-Akt, and Bcl-2 in NP cells were significantly increased. Further assays with PI3K/Akt/Bcl-2 inhibitors revealed that IL-17A suppressed autophagy in NP cells by activating the PI3K/Akt/Bcl-2 signaling pathway. CONCLUSIONS: These data suggest that IL-17A promotes IDD by inhibiting autophagy through activation of the PI3K/Akt/Bcl-2 signaling pathway and may offer new insights for targeted therapy of this disease.

Chemerin facilitates intervertebral disc degeneration via TLR4 and CMKLR1 and activation of NF-kB signaling pathway.

Now days, obesity is a major risk factor for intervertebral disc degeneration (IDD). However, adipokine, such as chemerin is a novel cytokine, which is secreted by adipose tissue, and are thought to be played major roles in various degenerative diseases. Obese individuals are known to have high concentration of serum chemerin. Our purpose was to study whether chemerin acts as a biochemical relationship between obesity, and IDD. In this study, we found that the expression level of chemerin was significantly increased in the human degenerated nucleus pulposus (NP) tissues, and had higher level in the obese people than the normal people. Chemerin significantly increased the inflammatory mediator level, contributing to ECM degradation in nucleus pulposus cells (NPCs). Furthermore, chemerin overexpression aggravates the puncture-induced IVDD progression in rats, while knockdown CMKLR1 reverses IVDD progression. Chemerin activates the NF-kB signaling pathway via its receptors CMKLR1, and TLR4 to release inflammatory mediators, which cause matrix degradation, and cell aging. These findings generally provide novel evidence supporting the causative role of obesity in IDD, which is essentially important to literally develop novel preventative or generally therapeutic treatment in the disc degenerative disorders.

IL-38 alleviates the inflammatory response and the degeneration of nucleus pulposus cells via inhibition of the NF-κB signaling pathway in vitro.

Previous studies have suggested that the inflammatory response contributes to the onset of intervertebral disc degeneration (IVDD). Interleukin (IL)-38, a newly discovered cytokine of the IL-1 family, has been demonstrated to play an anti-inflammatory role in autoimmune diseases, such as Crohn's disease, rheumatoid arthritis and psoriasis. However, whether IL-38 participates in the pathogenesis of IVDD remains unknown. In this study, human disc tissues from IVDD patients and rat disc tissues from an IVDD model were collected to measure the expression of IL-38 in the IVDD groups and the control groups by western blot and immunohistochemical staining. To further determine the role of IL-38 in IVDD, human nucleus pulposus cells (HNPCs) were stimulated with TNF-α to generate an in vitro model of inflammation to mimic the local inflammatory environment of the lumbar disc. The inflammatory response and HNPC degeneration markers were measured after stimulation with TNF-α and IL-38. IL-38 was upregulated in both the human and rat degenerated disc tissues compared with the control tissues. In vitro, IL-38 significantly decreased the TNF-α-induced expression of IL-1ß, IL-6, COX-2, MMP-13 and ADAMTS-5 in the HNPCs, and IL-38 also alleviated the TNF-α-induced reductions in type II collagen and aggrecan. Moreover, IL-38 inhibited the activation of the NF-κB signaling pathway in the HNPC-based model of inflammation by reducing the expression level of the NF-κB P-P65 protein. In conclusion, IL-38 could alleviate the inflammatory response and HNPC degeneration in vitro via the inhibition of the NF-κB signaling pathway. These results suggest that IL-38 may be a new strategy for the treatment of IVDD.

Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration.

Inflammation plays an essential role in the development of lumbar disc degeneration (LDD), although the exact effects of macrophage subtypes on LDD remain unclear. Based on previous studies, we hypothesized that M2-polarization of local macrophages and simultaneous suppression of their production of fibrotic transforming growth factor beta 1 (TGFß1) could inhibit progression of LDD. Thus, we applied an orthotopic injection of adeno-associated virus (AAV) carrying shRNA for DNA Methyltransferase 1 (DNMT1) and/or shRNA for TGFß1 under a macrophage-specific CD68 promoter to specifically target local macrophages in a mouse model for LDD. We found that shDNMT1 significantly reduced levels of the pro-inflammatory cytokines TNFα, IL-1ß and IL-6, significantly increased levels of the anti-inflammatory cytokines IL-4 and IL-10, significantly increased M2 macrophage polarization, significantly reduced cell apoptosis in the disc degeneration zone and significantly reduced LDD-associated pain. The anti-apoptotic and anti-pain effects were further strengthened by co-application of shTGFß1. Together, these data suggest that M2 polarization of macrophages induced by both epigenetic modulation and suppressed production and release of TGFß1 from polarized M2 macrophages, may have a demonstrable therapeutic effect on LDD.

Modulation of the In Vivo Inflammatory Response by Pro- Versus Anti-Inflammatory Intervertebral Disc Treatments.

Inflammation is central in intervertebral disc (IVD) degeneration/regeneration mechanisms, and its balance is crucial to maintain tissue homeostasis. This work investigates the modulation of local and systemic inflammatory response associated with IVD degeneration/herniation by administration of PRO- versus ANTI-inflammatory treatments. Chitosan/poly-γ-glutamic acid nanocomplexes, known as pro-inflammatory (PRO), and soluble diclofenac, a non-steroidal anti-inflammatory drug (ANTI), were intradiscally administered in a rat IVD injury model, 24 h after lesion. Two weeks after administration, a reduction of disc height accompanied by hernia formation was observed. In the PRO-inflammatory treated group, IL-1ß, IL-6 and COX-2 IVD gene expression were upregulated, and loss of nucleus pulposus (NP) structure and composition was observed. Systemically, lower T-cell frequency was observed in the lymph nodes (LN) and spleen (SP) of the PRO group, together with an increase in CD4+ T cells subset in the blood (BL) and LN. In contrast, the ANTI-group had higher proteoglycans/collagen ratio and collagen type 2 content in the NP, while an increase in the frequency of myeloid cells, M1 macrophages and activated macrophages (MHCII+) was observed at the systemic level. Overall, this study illustrates the dynamics of local and systemic inflammatory and immune cell responses associated with intradiscal therapies, which will contribute to designing more successful immunomodulatory treatments for IVD degeneration.

Investigation of resident and recruited macrophages following disc injury in mice.

Macrophages, particularly M1 macrophages, produce proinflammatory cytokines and contribute to the degenerative process in injured intervertebral discs (IVDs). We previously showed that macrophages in both intact and injured IVDs increased following IVD injury. Resident macrophages and macrophages recruited from the peripheral blood have distinct roles in tissue. However, it remains to be determined whether increased macrophages derive from resident or recruited macrophages. We investigated the origin of M1 macrophages in injured IVDs using green fluorescent protein (GFP) transgenic bone marrow chimeric mice. The M1 macrophage marker, CD86, increased in both disc-derived resident macrophages and bone marrow-derived macrophages (BMMs) after lipopolysaccharide/interferon γ stimulation in vitro. Following IVD injury, the proportion of cells positive for the CD86 ligand, the F4/80 antigen, and the surface glycoprotein CD11b (CD86+ CD11b+ F4/80+) significantly increased in GFP+ populations at days 3, 7, and 14. In contrast, CD86+ CD11b+ F4/80+ cells in GFP- populations significantly increased on day 3, and thereafter decreased on days 7 and 14. The proportion of CD86+ CD11b+ F4/80+ cells in the GFP+ populations was significantly higher than that in the GFP- populations at days 1, 3, 7, and 14. Monocyte chemoattractant protein-1 expression in disc-derived macrophages, but not in BMMs, increased following interleukin-1ß stimulation. Our results suggest M1 macrophages following IVD injury originate from recruited macrophages. Resident macrophages may behave differently in IVD injury. The role of resident macrophages needs to be clarified. Further investigation is needed.

Sequential CCL2 Expression Profile After Disc Injury in Mice.

Macrophages produce proinflammatory cytokines in injured intervertebral discs (IVDs). We recently showed that macrophage-derived inflammatory cytokines contribute to the production of pain-related factors. However, the mechanism by which macrophages are recruited to injured IVDs has not been fully clarified. Here, we examined the expression dynamics of the chemokine CCL2 in a mouse IVD injury model and the mechanisms of its regulation. The percentage of macrophages increased from day 1 after injury and persisted up until day 28. At 1 and 3 days after injury, the expression of both Ccl2 messenger RNA (mRNA) and CCL2 protein was elevated in the IVD injury group, after which expression decreased to basal levels. Consistent with the increase in CCL2 expression, Ccr2 and Tnfa expression and various types of macrophages were also immediately elevated following disc injury. Further, tumor necrosis factor-α (TNF-α) stimulated Ccl2 mRNA and CCL2 protein expression in IVD cells in vitro. The expressions of M1 (Cd86 and Nos2) and M2a (Ym1) macrophage markers were all significantly elevated from day 1 following injury in injured compared with control mice. Meanwhile, the expression of Cd206 (M2a and M2c marker) was significantly elevated on days 3, 7, 14, and 28 following injury. These results suggest that in IVD injury, TNF-α stimulates CCL2, which, in turn, mediates the recruitment of macrophages with the recruited macrophages subsequently differentiating into M1 and M2 subtypes. CCL2 signaling may, therefore, play an important role in IVD pathology via macrophage recruitment. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:895-901, 2020.