The present study aimed to evaluate the effect of the cX3cR1 inhibitor AZd8797 in early recovery after acute ScI and elucidate its potential mechanism in blocking inflammation and apoptosis. Adult rats were sacrificed after 3, 7, 10, or 14 days of ScI. The injured spinal tissues were collected for assessing c-X3-c motif chemokine ligand 1(cX3cL1)/c-X3-c motif chemokine receptor 1 (cX3cR1) expression at each time point via western blotting (WB) and quantitative PcR. The cellular localization of the proteins was detected by immunofluorescence. Another batch of rats (subdivided into sham, injury model, AZd8797 and methylprednisolone groups) were used to evaluate locomotive recovery with a Basso Beattie Bresnahan score. Based on the expression level of cX3cR1, these rats were sacrificed at the most prominent stage of cX3cR1 expression (10 days after ScI), for assessing the serum levels of tumor necrosis factor-α/interleukin (IL)-6/IL-1β and the expression of cX3cL1/cX3cR1/caspase 3/Bcl-2/Bax in the spinal cord tissues through WB and ELISA. Additionally, apoptosis and necrosis in the injured spinal cord were evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining/fluoro-jade B staining. Expression levels of both cX3cR1 and cX3cL1 reached their peak 10 days after the injury, followed by a dramatic downward trend at 14 days. The enhanced expression of cX3cR1 was detected in astrocytes and microglia of the injured spinal cord. AZd8797 improved locomotive recovery after 10 days of ScI and was as effective as methylprednisolone. The effect of AZd8797 was mediated by suppressing apoptosis, necrosis and inflammatory responses, as assessed by WB/ELISA and morphological examinations. The current study has demonstrated that AZd8797 can effectively block overwhelming inflammation, apoptosis and necrosis after SCI and facilitate early recovery of locomotive function.

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Surgical brain injury (SBI) triggers microglia to release numerous inflammatory factors, leading to brain edema and neurological dysfunction. Reducing neuroinflammation and protecting the blood-brain barrier (BBB) are key factors to improve the neurological function and prognosis after SBI. Na+-K+-Cl– cotransporter 1 (NKCC1) and nuclear factor κB (NF-κB) have been implicated in the secretion of inflammatory cytokines by microglia in brain injury. This study aimed to establish the role of NKCC1 in inducing inflammation in SBI, as well as to determine whether NKCC1 controls the release of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) via phosphorylation of NF-κB in microglia, thus affecting BBB permeability and neuronal cell apoptosis. Male Sprague-Dawley (SD) rats were used to establish an SBI model. This study revealed that compared with the sham group, the expression levels of p-NKCC1, p-p65-NF-κB, and related inflammatory factor proteins in SBI model group significantly increased. After p-NKCC1 was inhibited, p-p65-NF-κB, IL-6, IL-1β, and TNF-α were downregulated, and nerve cell apoptosis and BBB permeability were significantly reduced. These findings suggest that the SBI-induced increase in p-NKCC1 exacerbates neuroinflammation, brain edema, and nerve function injury, which may be mediated by regulating the activity of p65-NF-κB that in turn influences the release of inflammatory factors.

BackgroundIn triple-negative breast cancer (TNBC), PDL1/PD1-directed immunotherapy is effective in less than 20% of patients. In our preliminary study, we have found CSPG4 to be highly expressed together with PDL1 in TNBCs, particularly those harboring TP53 aberrations. However, the clinical implications of co-expressed CSPG4 and PDL1 in TNBCs remain elusive.MethodsA total of 85 advanced TNBC patients treated in the Hunan Cancer Hospital between January 2017 and August 2019 were recruited. The expressions of CSPG4 and PDL1 in TNBC tissues were investigated using immunohistochemistry (IHC). The RNA-seq dataset from the TCGA-BRCA project was further used to analyze the mRNA expression of CSPG4 and PDL1 in TP53-aberrant TNBCs. Cox proportional hazards model and Kaplan–Meier curves with Logrank test was used to analyze the effects of CSPG4 and PDL1 on survival. TNBC cell lines were further used to investigate the molecular mechanism that were involved.ResultsTP53 aberrations occurred in more than 50% of metastatic TNBCs and were related to higher tumor mutation burden (TMB). In TCGA-BRCA RNA-seq dataset analysis, both CSPG4 and PDL1 levels were high in TNBCs, especially in TP53-aberrant TNBCs. IHC assay showed nearly 60% of advanced TNBCs to be CSPG4-positive and about 25% to be both CSPG4-positive and PDL1-positive. The levels of CSPG4 and PDL1 were high in TNBC cell lines as revealed by flow cytometry and immunoblotting compared with non-TNBC cells. Univariate Cox regression analysis indicated that CSPG4 positivity was a significant risk factor for progression-free survival in metastatic TNBCs, with a hazard ratio (HR) of 2.26 (P = 0.05). KM curves with Logrank test also identified high level of CSPG4 as a significant risk factor for overall survival in advanced breast cancers in TCGA-BRCA samples (P = 0.02). The immunoblotting assays showed that EMT-related pathways were involved in CSPG4-mediated invasion.ConclusionsCSPG4 expression level is associated with PDL1 positivity in TP53-aberrant TNBC cells. Patients with CSPG4 expression have poor treatment response and poor overall survival. Co-expressed CSPG4 and PDL1 may have an important prognostic value and provide new therapeutic targets in TNBC patients. CSPG4 might mediate tumor invasion and PDL1 overexpression through EMT-related pathway.

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer, with limited therapeutic options readily available. Immunotherapy such as immune checkpoint inhibition has been investigated in TNBC but still encounters low overall response. Neutrophils, the most abundant leukocytes in the body, are increasingly recognized as an active cancer-modulating entity. In the bloodstream, neutrophils escort circulating tumor cells to promote their survival and stimulate their proliferation and metastasis. In the tumor microenvironment, neutrophils modulate the immune milieu through polarization between the anti-tumor and the pro-tumor phenotypes. Through a comprehensive review of recently published literature, it is evident that neutrophils are an important player in TNBC immunobiology and can be used as an important prognostic marker of TNBC. Particularly, in their pro-tumor form, neutrophils facilitate TNBC metastasis through formation of neutrophil extracellular traps and the pre-metastatic niche. These findings will help advance the potential utilization of neutrophils as a therapeutic target in TNBC.