A novel aquaporin-4-associated optic neuritis rat model with severe pathological and functional manifestations.

BACKGROUND: Optic neuritis (ON) is a common manifestation of aquaporin-4 (AQP4) antibody seropositive neuromyelitis optica (NMO). The extent of tissue damage is frequently severe, often leading to loss of visual function, and there is no curative treatment for this condition. To develop a novel therapeutic strategy, elucidating the underlying pathological mechanism using a clinically relevant experimental ON model is necessary. However, previous ON animal models have only resulted in mild lesions with limited functional impairment. In the present study, we attempted to establish a feasible ON model with severe pathological and functional manifestations using a high-affinity anti-AQP4 antibody. Subsequently, we aimed to address whether our model is suitable for potential drug evaluation by testing the effect of minocycline, a well-known microglia/macrophage inhibitor. METHODS: AQP4-immunoglobulin G (IgG)-related ON in rats was induced by direct injection of a high-affinity anti-AQP4 monoclonal antibody, E5415A. Thereafter, the pathological and functional characterizations were performed, and the therapeutic potential of minocycline was investigated. RESULTS: We established an experimental ON model that reproduces the histological characteristics of ON in seropositive NMO, such as loss of AQP4/glial fibrillary acidic protein immunoreactivity, immune cell infiltration, and extensive axonal damage. We also observed that our rat model exhibited severe visual dysfunction. The histological analysis showed prominent accumulation of macrophages/activated microglia in the lesion site in the acute phase. Thus, we investigated the possible effect of the pharmacological inhibition of macrophages/microglia activation by minocycline and revealed that it effectively ameliorated axonal damage and functional outcome. CONCLUSIONS: We established an AQP4-IgG-induced ON rat model with severe functional impairments that reproduce the histological characteristics of patients with NMO. Using this model, we revealed that minocycline treatment ameliorates functional and pathological outcomes, highlighting the usefulness of our model for evaluating potential therapeutic drugs for ON in NMO.

Tumor-Associated Macrophages/Microglia in Glioblastoma Oncolytic Virotherapy: A Double-Edged Sword.

Oncolytic virotherapy is a rapidly progressing field that uses oncolytic viruses (OVs) to selectively infect malignant cells and cause an antitumor response through direct oncolysis and stimulation of the immune system. Despite demonstrated pre-clinical efficacy of OVs in many cancer types and some favorable clinical results in glioblastoma (GBM) trials, durable increases in overall survival have remained elusive. Recent evidence has emerged that tumor-associated macrophage/microglia (TAM) involvement is likely an important factor contributing to OV treatment failure. It is prudent to note that the relationship between TAMs and OV therapy failures is complex. Canonically activated TAMs (i.e., M1) drive an antitumor response while also inhibiting OV replication and spread. Meanwhile, M2 activated TAMs facilitate an immunosuppressive microenvironment thereby indirectly promoting tumor growth. In this focused review, we discuss the complicated interplay between TAMs and OV therapies in GBM. We review past studies that aimed to maximize effectiveness through immune system modulation-both immunostimulatory and immunosuppressant-and suggest future directions to maximize OV efficacy.

Ketone Metabolite ß-Hydroxybutyrate Ameliorates Inflammation After Spinal Cord Injury by Inhibiting the NLRP3 Inflammasome.

Ketogenic diet (KD) has been shown to be beneficial in a range of neurological disorders, with ketone metabolite ß-hydroxybutyrate (ßOHB) reported to block the nucleotide oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in bone marrow-derived macrophages. In this study, we show that pretreatment with KD or in situ ßOHB suppressed macrophages/microglia activation and the overproduction of inflammatory cytokines, while KD downregulated the expression of NLRP3 inflammasome. Moreover, KD promoted macrophages/microglia transformation from the M1 phenotype to the M2a phenotype following spinal cord injury (SCI) in the in vivo study. Rats in the KD group demonstrated improved behavioral and electrophysiological recovery after SCI when compared to those rats in the standard diet group. The in vitro study performed on BV2 cells indicated that ßOHB inhibited an LPS+ATP-induced inflammatory response and decreased NLRP3 protein levels. Our data demonstrated that pretreatment with KD attenuated neuroinflammation following SCI, probably by inhibiting NLRP3 inflammasome and shifting the activation state of macrophages/microglia from the M1 to the M2a phenotype. Therefore, the ketone metabolite ßOHB might provide a potential future therapeutic strategy for SCI.

Molecular signature of slowly expanding lesions in progressive multiple sclerosis.

Multiple sclerosis is an immune-mediated chronic inflammatory disease of the CNS that leads to demyelinated lesions in the grey and white matter. Inflammatory, active demyelinating white matter lesions predominate in the relapsing-remitting disease stages, whereas in the progressive stage the so-called slowly expanding lesion is characteristic. These lesions show an accumulation of macrophages/microglia at their borders, mediating the ongoing myelin breakdown and axonal degeneration. The exact pathogenetic mechanisms of lesion progression in chronic multiple sclerosis are still not clear. In the present study, we performed a detailed immunological and molecular profiling of slowly expanding lesions (n = 21) from 13 patients aged between 30 to 74 years (five females and eight males), focusing on macrophage/microglia differentiation. By applying the microglia-specific marker TMEM119, we demonstrate that cells accumulating at the lesion edge almost exclusively belonged to the microglia lineage. Macrophages/microglia can be subdivided into the M1 type, which are associated with inflammatory and degenerative processes, and M2 type, with protective properties, whereby also intermediate polarization phenotypes can be observed. By using a panel of markers characterizing M1- or M2-type macrophages/microglia, we observed a preferential accumulation of M1-type differentiated cells at the lesion edge, indicating a crucial role of these cells in lesion progression. Additionally, unbiased RNA microarray analyses of macrodissected lesion edges from slowly expanding and chronic inactive lesions as well as normal-appearing white matter were performed. In slowly expanding lesions, we identified a total of 165 genes that were upregulated and 35 genes that were downregulated. The upregulated genes included macrophage/microglia-associated genes involved in immune defence and inflammatory processes. Among the upregulated genes were ALOX15B, MME and TNFRSF25. We confirmed increased expression of ALOX15B by quantitative PCR, and of all three genes on the protein level by immunohistochemistry. In conclusion, the present study characterized in detail slowly expanding lesions in progressive multiple sclerosis and demonstrated a preferential accumulation of resident microglia with M1 differentiation at the lesion edge. Microarray analysis showed an increased expression of genes related to immune function, metabolic processes as well as transcription/translation. Thus, these genes may serve as future therapeutic targets to impede lesion progression.

Macrophages/Microglia in the Glioblastoma Tumor Microenvironment.

The complex interaction between glioblastoma and its microenvironment has been recognized for decades. Among various immune profiles, the major population is tumor-associated macrophage, with microglia as its localized homolog. The present definition of such myeloid cells is based on a series of cell markers. These good sentinel cells experience significant changes, facilitating glioblastoma development and protecting it from therapeutic treatments. Huge, complicated mechanisms are involved during the overall processes. A lot of effort has been dedicated to crack the mysterious codes in macrophage/microglia recruiting, activating, reprogramming, and functioning. We have made our path. With more and more key factors identified, a lot of new therapeutic methods could be explored to break the ominous loop, to enhance tumor sensitivity to treatments, and to improve the prognosis of glioblastoma patients. However, it might be a synergistic system rather than a series of clear, stepwise events. There are still significant challenges before the light of truth can shine onto the field. Here, we summarize recent advances in this field, reviewing the path we have been on and where we are now.

Inhibiting HMGB1-RAGE axis prevents pro-inflammatory macrophages/microglia polarization and affords neuroprotection after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) favors a persistent pro-inflammatory macrophages/microglia-mediated response with only a transient appearance of anti-inflammatory phenotype of immune cells. However, the mechanisms controlling this special sterile inflammation after SCI are still not fully elucidated. It is known that damage-associated molecular patterns (DAMPs) released from necrotic cells after injury can trigger severe inflammation. High mobility group box 1(HMGB1), a ubiquitously expressed DNA binding protein, is an identified DAMP, and our previous study demonstrated that reactive astrocytes could undergo necroptosis and release HMGB1 after SCI in mice. The present study aimed to explore the effects and the possible mechanism of HMGB1on macrophages/microglia polarization, as well as the neuroprotective effects by HMGB1 inhibition after SCI. METHODS: In this study, the expression and the concentration of HMGB1 was determined by qRT-PCR, ELISA, and immunohistochemistry. Glycyrrhizin was applied to inhibit HMGB1, while FPS-ZM1 to suppress receptor for advanced glycation end products (RAGE). The polarization of macrophages/microglia in vitro and in vivo was detected by qRT-PCR, immunostaining, and western blot. The lesion area was detected by GFAP staining, while neuronal survival was examined by Nissl staining. Luxol fast blue (LFB) staining, DAB staining, and western blot were adopted to evaluate the myelin loss. Basso-Beattie-Bresnahan (BBB) scoring and rump-height Index (RHI) assay was applied to evaluate locomotor functional recovery. RESULTS: Our data showed that HMGB1 can be elevated and released from necroptotic astrocytes and HMGB1 could induce pro-inflammatory microglia through the RAGE-nuclear factor-kappa B (NF-κB) pathway. We further demonstrated that inhibiting HMGB1 or RAGE effectively decreased the numbers of detrimental pro-inflammatory macrophages/microglia while increased anti-inflammatory cells after SCI. Furthermore, our data showed that inhibiting HMGB1 or RAGE significantly decreased neuronal loss and demyelination, and improved functional recovery after SCI. CONCLUSIONS: The data implicated that HMGB1-RAGE axis contributed to the dominant pro-inflammatory macrophages/microglia-mediated pro-inflammatory response, and inhibiting this pathway afforded neuroprotection for SCI. Thus, therapies designed to modulate immune microenvironment based on this cascade might be a prospective treatment for SCI.

The Chinese herb Fructus Broussonetiae aids learning and memory in chronic cerebral hypoperfusion by reducing proinflammatory microglia activation in rats.

The neuroprotective role of Fructus Broussonetiae in a model of chronic cerebral hypoperfusion with cognitive decline was focused on neural plasticity and microglia/macrophage polarization. Chronic cerebral hypoperfusion was induced by bilateral common carotid artery ligation. Fructus Broussonetiae shortened escape latency and added the number of platform crossings of rats, up-regulated the expression of synaptophysin in the gray matter and increased myelin basic protein expression in the white matter. Further mechanistic experiments were conducted to examine microglia activation and M1/M2 polarization. It was shown that Fructus Broussonetiae reduced the activation of microglia revealed by decreased expression of ionized calcium-binding adapter molecule-1, inhibited M1 polarization of microglia and improved microglial M2 polarization shown by down-regulated the expression of inducible nitric oxide synthase and Fc fragment of IgG receptor IIIa and up-regulated the expression of arginase-1. In conclusion, the Chinese herb Fructus Broussonetiae can improve cognitive function following chronic cerebral hypoperfusion by down-regulating the activation of microglia, inhibiting microglial M1 polarization, and improving neural plasticity.

Quercetin prevents necroptosis of oligodendrocytes by inhibiting macrophages/microglia polarization to M1 phenotype after spinal cord injury in rats.

BACKGROUND: Oligodendrocytes (OLs) death after spinal cord injury (SCI) contributes to demyelination, even leading to a permanent neurological deficit. Besides apoptosis, our previous study demonstrated that OLs underwent receptor-interacting serine-threonine kinase 3(RIP3)/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis. Considering that necroptosis is always accompanied with pro-inflammatory response and quercetin has long been used as anti-inflammatory agent, in the present study we investigated whether quercetin could inhibit necroptosis of OLs and suppress the M1 macrophages/microglia-mediated immune response after SCI as well as the possible mechanism. METHODS: In this study, we applied quercetin, an important flavonoid component of various herbs, to treat rats with SCI and rats injected with saline were employed as the control group. Locomotor functional recovery was evaluated using Basso-Beattie-Bresnahan (BBB) scoring and rump-height Index (RHI) assay. In vivo, the necroptosis, apoptosis, and regeneration of OLs were detected by immunohistochemistry, 5'-bromo-2'-deoxyuridine (BrdU) incorporation. The loss of myelin and axons after SCI were evaluated by Luxol fast blue (LFB) staining, immunohistochemistry, and electron microscopic study. The polarization of macrophages/microglia after SCI and the underlying mechanisms were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry. In vitro, the ATP and reactive oxygen species (ROS) level examination, propidium iodide (PI) labeling, and Western blotting were used to analyze the necroptosis of cultured OLs, while the signaling pathways-mediated polarization of cultured macrophages/microglia was detected by qRT-PCR and Western blotting. RESULTS: We demonstrated that quercetin treatment improved functional recovery in rats after SCI. We then found that quercetin significantly reduced necroptosis of OLs after SCI without influencing apoptosis and regeneration of OLs. Meanwhile, myelin loss and axon loss were also significantly reduced in quercetin-treated rats, as compared to SCI + saline control. Further, we revealed that quercetin could suppress macrophages/microglia polarized to M1 phenotype through inhibition of STAT1 and NF-κB pathway in vivo and in vitro, which contributes to the decreased necroptosis of OLs. CONCLUSIONS: Quercetin treatment alleviated necroptosis of OLs partially by inhibiting M1 macrophages/microglia polarization after SCI. Our findings suggest that necroptosis of OLs may be a potential therapeutic target for clinical SCI.

PI3K: A master regulator of brain metastasis-promoting macrophages/microglia.

Mutations and activation of the PI3K signaling pathway in breast cancer cells have been linked to brain metastases. However, here we describe that in some breast cancer brain metastases samples the protein expression of PI3K signaling components is restricted to the metastatic microenvironment. In contrast to the therapeutic effects of PI3K inhibition on the breast cancer cells, the reaction of the brain microenvironment is less understood. Therefore we aimed to quantify the PI3K pathway activity in breast cancer brain metastasis and investigate the effects of PI3K inhibition on the central nervous system (CNS) microenvironment. First, to systematically quantify the PI3K pathway activity in breast cancer brain metastases, we performed a prospective biomarker study using a reverse phase protein array (RPPA). The majority, namely 30 out of 48 (62.5%) brain metastatic tissues examined, revealed high PI3K signaling activity that was associated with a median overall survival (OS) of 9.41 months, while that of patients, whose brain metastases showed only moderate or low PI3K activity, amounted to only 1.93 and 6.71 months, respectively. Second, we identified PI3K as a master regulator of metastasis-promoting macrophages/microglia during CNS colonization; and treatment with buparlisib (BKM120), a pan-PI3K Class I inhibitor with a good blood-brain-barrier penetrance, reduced their metastasis-promoting features. In conclusion, PI3K signaling is active in the majority of breast cancer brain metastases. Since PI3K inhibition does not only affect the metastatic cells but also re-educates the metastasis-promoting macrophages/microglia, PI3K inhibition may hold considerable promise in the treatment of brain metastasis and the respective microenvironment.

DRα1-MOG-35-55 treatment reduces lesion volumes and improves neurological deficits after traumatic brain injury.

Traumatic brain injury (TBI) results in severe neurological impairments without effective treatments. Inflammation appears to be an important contributor to key pathogenic events such as secondary brain injury following TBI and therefore serves as a promising target for novel therapies. We have recently demonstrated the ability of a molecular construct comprised of the human leukocyte antigen (HLA)-DRα1 domain linked covalently to mouse (m)MOG-35-55 peptide (DRα1-MOG-35-55 construct) to reduce CNS inflammation and tissue injury in animal models of multiple sclerosis and ischemic stroke. The aim of the current study was to determine if DRα1-MOG-35-55 treatment of a fluid percussion injury (FPI) mouse model of TBI could reduce the lesion size and improve disease outcome measures. Neurodeficits, lesion size, and immune responses were determined to evaluate the therapeutic potential and mechanisms of neuroprotection induced by DRα1-MOG-35-55 treatment. The results demonstrated that daily injections of DRα1-MOG-35-55 given after FPI significantly reduced numbers of infiltrating CD74+ and CD86+ macrophages and increased numbers of CD206+ microglia in the brain concomitant with smaller lesion sizes and improvement in neurodeficits. Conversely, DRα1-MOG-35-55 treatment of TBI increased numbers of circulating CD11b+ monocytes and their expression of CD74 but had no detectable effect on cell numbers or marker expression in the spleen. These results demonstrate that DRα1-MOG-35-55 therapy can reduce CNS inflammation and significantly improve histological and clinical outcomes after TBI. Future studies will further examine the potential of DRα1-MOG-35-55 for treatment of TBI.

Tumor necrosis factor-α inhibition attenuates middle cerebral artery remodeling but increases cerebral ischemic damage in hypertensive rats.

Hypertension causes vascular inflammation evidenced by an increase in perivascular macrophages and proinflammatory cytokines in the arterial wall. Perivascular macrophage depletion reduced tumor necrosis factor (TNF)-α expression in cerebral arteries of hypertensive rats and attenuated inward remodeling, suggesting that TNF-α might play a role in the remodeling process. We hypothesized that TNF-α inhibition would improve middle cerebral artery (MCA) structure and reduce damage after cerebral ischemia in hypertensive rats. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with the TNF-α inhibitor etanercept (ETN; 1.25 mg·kg(-1)·day(-1) ip daily) or PBS (equivolume) for 6 wk. The myogenic tone generation, postischemic dilation, and passive structure of MCAs were assessed by pressure myography. Cerebral ischemia was induced by MCA occlusion (MCAO). Myogenic tone was unchanged, but MCAs from SHRSP + ETN had larger passive lumen diameter and reduced wall thickness and wall-to-lumen ratio. Cerebral infarct size was increased in SHRSP + ETN after transient MCAO, despite an improvement in dilation of nonischemic MCA. The increase in infarct size was linked to a reduction in the number of microglia in the infarct core and upregulation of markers of classical macrophage/microglia polarization. There was no difference in infarct size after permanent MCAO or when untreated SHRSP subjected to transient MCAO were given ETN at reperfusion. Our data suggests that TNF-α inhibition attenuates hypertensive MCA remodeling but exacerbates cerebral damage following ischemia/reperfusion injury likely due to inhibition of the innate immune response of the brain.

Quercetin Inhibits Neuronal Pyroptosis and Ferroptosis by Modulating Microglial M1/M2 Polarization in Atherosclerosis.

Atherosclerosis (AS) with iron and lipid overload and systemic inflammation is a risk factor for Alzheimer's disease. M1 macrophage/microglia participate in neuronal pyroptosis and recently have been reported to be the ferroptosis-resistant phenotype. Quercetin plays a prominent role in preventing and treating neuroinflammation, but the protective mechanism against neurodegeneration caused by iron deposition is poorly understood. ApoE-/- mice were fed a high-fat diet with or without quercetin treatment. The Morris water maze and novel object recognition tests were conducted to assess spatial learning and memory, and nonspatial recognition memory, respectively. Prussian blue and immunofluorescence staining were performed to assess the iron levels in the whole brain and in microglia, microglia polarization, and the degree of microglia/neuron ferroptosis. In vitro, we further explored the molecular biological alterations associated with microglial polarization, neuronal pyroptosis, and ferroptosis via Western blot, flow cytometry, CCK8, LDH, propidium iodide, and coculture system. We found that quercetin improved brain lesions and spatial learning and memory in AS mice. Iron deposition in the whole brain or microglia was reversed by the quercetin treatment. In the AS group, the colocalization of iNOS with Iba1 was increased, which was reversed by quercetin. However, the colocalization of iNOS with PTGS2/TfR was not increased in the AS group, suggesting a character resisting ferroptosis. Quercetin induced the expression of Arg-1 and decreased the colocalizations of Arg-1 with PTGS2/TfR. In vitro, ox-LDL combined with ferric ammonium citrate treatment (OF) significantly shifted the microglial M1/M2 phenotype balance and increased the levels of free iron, ROS, and lipid peroxides, which was reversed by quercetin. M1 phenotype induced by OF caused neuronal pyroptosis and was promoted to ferroptosis by L-NIL treatment, which contributed to neuronal ferroptosis as well. However, quercetin induced the M1 to M2 phenotype and inhibited M2 macrophages/microglia and neuron pyroptosis or ferroptosis. In summary, quercetin alleviated neuroinflammation by inducing the M1 to M2 phenotype to inhibit neuronal pyroptosis and protected neurons from ferroptosis, which may provide a new idea for neuroinflammation prevention and treatment.

Sphingosine-1-Phosphate Recruits Macrophages and Microglia and Induces a Pro-Tumorigenic Phenotype That Favors Glioma Progression.

Glioblastoma is the most aggressive brain tumor in adults. Treatment failure is predominantly caused by its high invasiveness and its ability to induce a supportive microenvironment. As part of this, a major role for tumor-associated macrophages/microglia (TAMs) in glioblastoma development was recognized. Phospholipids are important players in various fundamental biological processes, including tumor-stroma crosstalk, and the bioactive lipid sphingosine-1-phosphate (S1P) has been linked to glioblastoma cell proliferation, invasion, and survival. Despite the urgent need for better therapeutic approaches, novel strategies targeting sphingolipids in glioblastoma are still poorly explored. Here, we showed that higher amounts of S1P secreted by glioma cells are responsible for an active recruitment of TAMs, mediated by S1P receptor (S1PR) signaling through the modulation of Rac1/RhoA. This resulted in increased infiltration of TAMs in the tumor, which, in turn, triggered their pro-tumorigenic phenotype through the inhibition of NFkB-mediated inflammation. Gene set enrichment analyses showed that such an anti-inflammatory microenvironment correlated with shorter survival of glioblastoma patients. Inhibition of S1P restored a pro-inflammatory phenotype in TAMs and resulted in increased survival of tumor-bearing mice. Taken together, our results establish a crucial role for S1P in fine-tuning the crosstalk between glioma and infiltrating TAMs, thus pointing to the S1P-S1PR axis as an attractive target for glioma treatment.

Combination of single-cell and bulk RNA seq reveals the immune infiltration landscape and targeted therapeutic drugs in spinal cord injury.

Background: In secondary spinal cord injury (SCI), the immune microenvironment of the injured spinal cord plays an important role in spinal regeneration. Among the immune microenvironment components, macrophages/microglia play a dual role of pro-inflammation and anti-inflammation in the subacute stage of SCI. Therefore, discovering the immune hub genes and targeted therapeutic drugs of macrophages/microglia after SCI has crucial implications in neuroregeneration. This study aimed to identify immune hub genes and targeted therapeutic drugs for the subacute phase of SCI. Methods: Bulk RNA sequencing (bulk-RNA seq) datasets (GSE5296 and GSE47681) and single-cell RNA sequencing (scRNA-seq) dataset (GSE189070) were obtained from the Gene Expression Omnibus database. In the bulk RNA-seq, the R package 'limma,' 'WGCNA,' and 'CIBERSORT' were used to jointly screen key immune genes. Subsequently, the R package 'Seurat' and the R package 'celldex' were used to divide and annotate the cell clusters, respectively. After using the Autodock software to dock immune hub genes and drugs that may be combined, the effectiveness of the drug was verified using an in vivo experiment with the T9 SCI mouse model. Results: In the bulk-RNA seq, B2m, Itgb5, and Vav1 were identified as immune hub genes. Ten cell clusters were identified in scRNA-seq, and B2m and Itgb5 were mainly located in the microglia, while Vav1 was mainly located in macrophages. Molecular docking results showed that the proteins corresponding to these immune genes could accurately bind to decitabine. In decitabine-treated mice, the pro-inflammatory factor (TNF-α, IL-1ß) levels were decreased while anti-inflammatory factor (IL-4, IL-10) levels were increased at 2 weeks post-SCI, and macrophages/microglia transformed from M1 to M2. At 6 weeks post-SCI, the neurological function score and electromyography of the decitabine treatment group were also improved. Conclusion: In the subacute phase of SCI, B2m, Itgb5, and Vav1 in macrophages/microglia may be key therapeutic targets to promote nerve regeneration. In addition, low-dose decitabine may promote spinal cord regeneration by regulating the polarization state of macrophages/microglia.

Chronic spinal cord compression associated with intervertebral disc degeneration in SPARC-null mice.

Chronic spinal cord compression (CSCC) is induced by disc herniation and other reasons, leading to movement and sensation dysfunction, with a serious impact on quality of life. Spontaneous disc herniation rarely occurs in rodents, and therefore establishing a chronic spinal cord compression (CSCC) animal model is of crucial importance to explore the pathogenesis and treatment of CSCC. The absence of secreted protein, acidic, and rich in cysteine (SPARC) leads to spontaneous intervertebral disc degeneration in mice, which resembles human disc degeneration. In this study, we evaluated whether SPARC-null mice may serve as an animal model for CSCC. We performed rod rotation test, pain threshold test, gait analysis, and Basso Mouse Scale score. Our results showed that the motor function of SPARC-null mice was weakened, and magnetic resonance images revealed compression at different spinal cord levels, particularly in the lumbar segments. Immunofluorescence staining and western blot assay showed that the absence of SPARC induced apoptosis of neurons and oligodendrocytes, activation of microglia/macrophages with M1/M2 phenotype and astrocytes with A1/A2 phenotype; it also activated the expression of the NOD-like receptor protein 3 inflammasome and inhibited brain-derived neurotrophic factor/tyrosine kinase B signaling pathway. Notably, these findings are characteristics of CSCC. Therefore, we propose that SPARC-null mice may be an animal model for studying CSCC caused by disc herniation.

Gold nanoclusters conjugated berberine reduce inflammation and alleviate neuronal apoptosis by mediating M2 polarization for spinal cord injury repair.

Spinal cord injury (SCI) leads to nerve cell apoptosis and loss of motor function. Herein, excessive activation of the M1 phenotype macrophages/microglia is found to be the main reason for the poor prognosis of SCI, but the selective activation phenotype (M2) macrophages/microglia facilitates the recovery of SCI. Thereafter, we used gold nanoclusters loaded berberine (BRB-AuNCs) to reduce inflammation by inhibiting the activation of M1 phenotype macrophages/microglia, which simultaneously inhibited neuronal apoptosis after SCI. In vitro and in vivo experiments showed that BRB-AuNCs reduced M1 protein marker CD86, increased M2 protein marker CD206, reduced inflammation and apoptotic cytokines (IL-1ß, IL-6, TNF-α, Cleaved Caspase-3 and Bax). These results indicate that BRB-AuNCs have excellent anti-inflammatory and anti-apoptotic effects by inducing the polarization of macrophages/microglia from M1 phenotype to M2 phenotype. Thereafter, the motor functions of SCI rats were significantly improved after treatment with BRB-AuNCs. This work not only provides a new way for the treatment of SCI but also broadens BRB utilization strategies.

The GBM Tumor Microenvironment as a Modulator of Therapy Response: ADAM8 Causes Tumor Infiltration of Tams through HB-EGF/EGFR-Mediated CCL2 Expression and Overcomes TMZ Chemosensitization in Glioblastoma.

Standard chemotherapy of Glioblastoma multiforme (GBM) using temozolomide (TMZ) frequently fails due to acquired chemoresistance. Tumor-associated macrophages and microglia (TAMs) as major immune cell population in the tumor microenvironment are potential modulators of TMZ response. However; little is known about how TAMs participate in TMZ induced chemoresistance. Members of the metzincin superfamily such as Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloprotease (ADAM) proteases are important mediators of cellular communication in the tumor microenvironment. A qPCR screening was performed to identify potential targets within the ADAM and MMP family members in GBM cells. In co-culture with macrophages ADAM8 was the only signature gene up-regulated in GBM cells induced by macrophages under TMZ treatment. The relationship between ADAM8 expression and TAM infiltration in GBM was determined in a patient cohort by qPCR; IF; and IHC staining and TCGA data analysis. Moreover; RNA-seq was carried out to identify the potential targets regulated by ADAM8. CCL2 expression levels were determined by qPCR; Western blot; IF; and ELISA. Utilizing qPCR; IF; and IHC staining; we observed a positive relationship between ADAM8 expression and TAMs infiltration level in GBM patient tissues. Furthermore; ADAM8 induced TAMs recruitment in vitro and in vivo. Mechanistically; we revealed that ADAM8 activated HB-EGF/EGFR signaling and subsequently up-regulated production of CCL2 in GBM cells in the presence of TMZ treatment; promoting TAMs recruitment; which further induced ADAM8 expression in GBM cells to mediate TMZ chemoresistance. Thus; we revealed an ADAM8 dependent positive feedback loop between TAMs and GBM cells under TMZ treatment which involves CCL2 and EGFR signaling to cause TMZ resistance in GBM.

Berberine-loaded M2 macrophage-derived exosomes for spinal cord injury therapy.

Spinal cord injury (SCI) causes immune activation of resident macrophages/microglia. Activated macrophages/microglia have two different phenotypes, the pro-inflammatory classically activated (M1) phenotype and the anti-inflammatory alternatively activated (M2) phenotype. M1 phenotype macrophages/microglia are the key factor in inflammation. The treatment of SCI remains a huge challenge due to the nontargeting and inefficiency of anti-inflammatory drugs through the blood-brain barrier (BBB). The purpose of this experiment was to design M2-type primary peritoneal macrophages exosomes (Exos) as a drug carrier for berberine (Ber), which can be efficiently targeted to deliver drugs to the injured spinal cord due to the natural advantage of Exos across the BBB. The Exos with particle size of 125±12 nm were loaded with by an ultrasonic method and the drug loading reached 17.13 ±1.64%. The Ber release experiment showed that the loaded sample (Exos-Ber) exhibited sustained release effect, and the cumulative release amount reached 71.44±2.86% within 48 h. In vitro and in vivo experiments confirmed that the Exos-Ber could decrease the M1 protein marker iNOS, elevate the M2 protein marker CD206 and reduce inflammatory and apoptotic cytokines (TNF-α, IL-1ß, IL-6, Caspase 9, Caspase 8), which showed that Exos-Ber had a good anti-inflammatory and anti-apoptotic effect by inducing macrophages/microglia from the M1 phenotype to M2 phenotype polarization. Moreover, the motor function of SCI mice was significantly improved after Exos-Ber treatment, indicating that Exos-Ber is a potential agent for SCI therapy. STATEMENT OF SIGNIFICANCE: Efficient targeting strategy for drug delivery. In addition to good biocompatibility and stealth ability, M2 macrophage-derived Exosomes present natural inflammatory targeting ability. The inflammatory microenvironment after spinal cord injury provides motivation for the targeting of exosomes. Natural drug carrier with higher safety. With the rapid development of nanomaterials, drug carriers have become more selective. However, due to the special microenvironment after central nervous system damage, some non-degradable inorganic materials will increase the pressure of self-healing and even secondary damage to neurons, which has been solved by the emergence of exosomes. Some previous studies used tumor cell line exosomes as drug carriers, but the carcinogenic factors carried by themselves have extremely high hidden dangers, and endogenous macrophage exosomes have absolute advantages over their safety.

Endogenous Regulatory T Cells Promote M2 Macrophage Phenotype in Diabetic Stroke as Visualized by Optical Imaging.

Regulatory T cells (Tregs) play an immunosuppressive role in various diseases, yet their function remains controversial in stroke and obscure in diabetic stroke. In the present study, Tregs were found downregulated in the peripheral blood of type 2 diabetes mellitus (T2DM) stroke models and patients compared with controls. In ischemic stroke mice (both T2DM and wild type), endogenous Tregs boosted by CD28SA increased CD206+ M2 macrophage/microglia cells, decreased infarct volumes, and improved neurological recovery. Our results demonstrated the potential of boosting Tregs for treating T2DM stroke. Furthermore, we utilized an optical imaging probe (IRD-αCD206) to target M2 macrophage/microglia cells and demonstrated its effect in visualizing M2 macrophage/microglia cells in vivo in ischemic brain tissue.

Up-regulated inflammatory signatures of the spinal cord in canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is an adult-onset fatal disease characterized by progressive degeneration of the spinal cord. Affected dogs have homozygous mutations in superoxide dismutase 1, and thus DM is a potential spontaneous animal model of human familial amyotrophic lateral sclerosis (ALS). Neuroinflammation is the pathological hallmark of ALS, whereby proinflammatory cytokines and chemokines are overproduced by activated glial cells such as astrocytes and microglia. However, the detailed pathogenesis of spinal cord degeneration in DM remains unknown. To further characterize the pathological mechanism of DM, we analyzed the caudal cervical cords of ten Pembroke Welsh Corgis pathologically diagnosed with DM by quantitative real-time reverse transcription polymerase chain reaction, immunohistochemistry (IHC), and double immunofluorescence. Compared to control spinal cord tissues, we found significantly enhanced transcriptions of interleukin-1ß, tumor necrosis factor-α, CC motif chemokine ligand (CCL) 2 and vascular cell adhesion molecule -1 mRNA in the spinal cords of DM dogs. Moreover, IHC for the class II major histocompatibility complex molecules HLA-DR and CCL2 indicated that the immunopositive areas of activated macrophages/microglia and CCL2 protein were significantly increased in DM, and CCL2 protein was mainly overproduced by astrocytes. Our results suggest a proinflammatory state of the microenvironment in the DM spinal cord in which activated microglia and astrocytes play important roles by secreting a set of cytokines, chemokines, and expressing adhesion molecules.