Osteocalcin-expressing neutrophils from skull bone marrow exert immunosuppressive and neuroprotective effects after TBI.

Neutrophils from skull bone marrow (Nskull) are activated under some brain stresses, but their effects on traumatic brain injury (TBI) are lacking. Here, we find Nskull infiltrates brain tissue quickly and persistently after TBI, which is distinguished by highly and specifically expressed osteocalcin (OCN) from blood-derived neutrophils (Nblood). Reprogramming of glucose metabolism by reducing glycolysis-related enzyme glyceraldehyde 3-phosphate dehydrogenase expression is involved in the antiapoptotic and proliferative abilities of OCN-expressing Nskull. The transcription factor Fos-like 1 governs the specific gene profile of Nskull including C-C motif chemokine receptor-like 2 (CCRL2), arginase 1 (Arg1), and brain-derived neurotrophic factor (BDNF) in addition to OCN. Selective knockout of CCRL2 in Nskull demonstrates that CCRL2 mediates its recruitment, whereas high Arg1 expression is consistent with its immunosuppressive effects on Nblood, and the secretion of BDNF facilitating dendritic growth contributes to its neuroprotection. Thus, our findings provide insight into the roles of Nskull in TBI.

Metabotropic glutamate receptor 5 promotes blood-brain barrier recovery after traumatic brain injury.

Blood-brain barrier (BBB) impairment and glutamate release are two pathophysiological features of traumatic brain injury (TBI), contributing to secondary brain damage and neuroinflammation. However, our knowledge of BBB integrity damage and dysfunction are still limited due to the diverse and fluctuating expression of glutamate receptors after trauma. Here, we confirmed the downregulation of metabotropic glutamate receptor 5 (mGluR5) on microvascular endothelial cell within the acute phase of TBI, and the recovered mGluR5 levels on BBB was positively associated with blood perfusion and neurological recovery. In whole body mGluR5-knockout mice, BBB dysfunction and neurological deficiency were exacerbated after TBI compared with wild type mice. In terms of mechanism, the amino acid sequence 201-259 of cytoskeletal protein Alpha-actinin-1 (ACTN1) interacted with mGluR5, facilitating mGluR5 translocation from cytoplasmic compartment to plasma membrane in endothelial cells. Activation of plasma membrane mGluR5 triggers the PLC/PKCµ/c-Jun signaling pathway, leading to increased expression of the tight junction-actin cytoskeleton connecting protein zonula occludens-1 (ZO-1). Our findings uncover a novel mechanism mediated by membrane and cytoplasmic mGluR5 in endothelial cell integrity maintenance and repair, providing the potential therapeutic target for TBI treatment targeting at mGluR5 and mGluR5/ACTN1 complex in BBB.

Change and challenge: An online course in Medical Biochemistry and Molecular Biology.

The development of information technology and portable devices has sparked a revolution in the field of education, facilitating access to diverse educational resources and lifelong learning. In particular, the COVID-19 pandemic has accelerated the transition from face-to-face to distance teaching, which requires online education to be provided worldwide. Biochemistry and Molecular Biology are key basic medical courses in laboratory-based science that cover complicated theories and applications. The balance between traditional and online courses, and the effectiveness of online courses, are fundamental to the teaching quality of Biochemistry and Molecular Biology. In this study, we explored the concepts, designs, and practices of a new blended online course and identified potential challenges. We hope that our experiences will provide new ideas for online teaching and promote teaching reform and the development of Medical Biochemistry and Molecular Biology education.

Sinomenine alleviates lipopolysaccharide-induced acute lung injury via a PPARß/δ-dependent mechanism.

Evidence is mounting that sinomenine and peroxisome proliferator-activated receptor ß/δ (PPARß/δ) are effective against lipopolysaccharide (LPS)-induced acute lung injury (ALI) via anti-inflammatory properties. However, it is unknown whether PPARß/δ plays a role in the protective effect of sinomenine on ALI. Here, we initially observed that preemptive administration of sinomenine markedly alleviated lung pathological changes, pulmonary edema and neutrophil infiltration, accompanied by inhibition of the expression of the pro-inflammatory cytokines Tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6), which were largely reversed following the addition of a PPARß/δ antagonist. Subsequently, we also noticed that sinomenine upregulated adenosine A2A receptor expression in a PPARß/δ-dependent manner in LPS-stimulated bone marrow-derived macrophages (BMDMs). Further investigation indicated that PPARß/δ directly bound to the functional peroxisome proliferator responsive element (PPRE) in the adenosine A2A receptor gene promoter region to enhance the expression of the adenosine A2A receptor. Sinomenine was identified as a PPARß/δ agonist. It could bind with PPARß/δ, and promote the nuclear translocation and transcriptional activity of PPARß/δ. In addition, combined treatment with sinomenine and an adenosine A2A receptor agonist exhibited synergistic effects and better protective roles than their single use against ALI. Taken together, our results reveal that sinomenine exerts advantageous effects on ALI by activating of PPARß/δ, with the subsequent upregulation of adenosine A2A receptor expression, and provide a novel and potential therapeutic application for ALI.

Histones of Neutrophil Extracellular Traps Induce CD11b Expression in Brain Pericytes Via Dectin-1 after Traumatic Brain Injury.

The brain pericyte is a unique and indispensable part of the blood-brain barrier (BBB), and contributes to several pathological processes in traumatic brain injury (TBI). However, the cellular and molecular mechanisms by which pericytes are regulated in the damaged brain are largely unknown. Here, we show that the formation of neutrophil extracellular traps (NETs) induces the appearance of CD11b+ pericytes after TBI. These CD11b+ pericyte subsets are characterized by increased permeability and pro-inflammatory profiles compared to CD11b- pericytes. Moreover, histones from NETs by Dectin-1 facilitate CD11b induction in brain pericytes in PKC-c-Jun dependent manner, resulting in neuroinflammation and BBB dysfunction after TBI. These data indicate that neutrophil-NET-pericyte and histone-Dectin-1-CD11b are possible mechanisms for the activation and dysfunction of pericytes. Targeting NETs formation and Dectin-1 are promising means of treating TBI.

Glutamate blunts cell-killing effects of neutrophils in tumor microenvironment.

Neutrophils are the first defenders of the innate system for injury and infection. They have gradually been recognized as important participants in tumor initiation and development due to their heterogeneity and plasticity. In the tumor microenvironment (TME), neutrophils can exert antitumor and protumor functions, depending on the surroundings. Tumor cells systemically alter intracellular amino acid (AA) metabolism and extracellular AA distribution to meet their proliferation need, leading to metabolic reprogramming and TME reshaping. However, the underlying mechanisms that determine how altered AAs affect neutrophils in TME are less-explored. Here, we identified that abundant glutamate releasing from tumor cells blunted neutrophils' cell-killing effects toward tumor cells in vitro and in vivo. Mass spectrometric detection, flow cytometry, and western blot experiments proved that increased levels of pSTAT3/RAB10/ARF4, mediated by glutamate, were accompanied with immunosuppressive phenotypes of neutrophils in TME. We also discovered that riluzole, an FDA-approved glutamate release inhibitor, significantly inhibited tumor growth by restoring neutrophils' cell-killing effects and decreasing glutamate secretion from tumor cells. These findings highlight the importance of tumor-released glutamate on neutrophil transformation in TME, providing new possible cancer treatments targeting altered glutamate metabolism.

Targeting metabotropic glutamate receptor 4 for cancer immunotherapy.

In this study, we report a novel role of metabotropic glutamate receptor 4 (GRM4) in suppressing antitumor immunity. We revealed in three murine syngeneic tumor models (B16, MC38, and 3LL) that either genetic knockout (Grm4−/−) or pharmacological inhibition led to significant delay in tumor growth. Mechanistically, perturbation of GRM4 resulted in a strong antitumor immunity by promoting natural killer (NK), CD4+, and CD8+ T cells toward an activated, proliferative, and functional phenotype. Single-cell RNA sequencing and T cell receptor profiling further defined the clonal expansion and immune landscape changes in CD8+ T cells. We further showed that Grm4−/− intrinsically activated interferon-γ production in CD8+ T cells through cyclic adenosine 3',5'-monophosphate (cAMP)/cAMP response element binding protein­mediated pathway. Our study appears to be of clinical significance as a signature of NKhigh-GRM4low and CD8high-GRM4low correlated with improved survival in patients with melanoma. Targeting GRM4 represents a new approach for cancer immunotherapy.

Transplantation with mGluR5 deficiency bone marrow displays antidepressant-like effect in C57BL/6J mice.

Antidepressant-like effects of metabotropic glutamate receptor 5 (mGluR5) have been verified by specific antagonists or whole body knock-out (KO) mice. Previous experiments indicate that blocking mGluR5 exerts antidepressant-like effects through neuronal mechanisms, like modulating NMDA receptor activity or 5-HT system. Here we found that transplanting bone marrow from mGluR5 KO mice to WT mice could also show antidepressant-like effects, which were confirmed by sucrose preference test and tail suspension test. Furthermore, mGluR5 deficiency dramatically inhibits cytokines release from bone marrow cells, such as IL-1ß, TNF-α and IL-6, alleviating proinflammatory responses in LPS-induced depression model. In addition, inhibited cytokines could decrease the activation of brain endothelial cells in ERK-dependent manner. These data provide the evidence that blocking mGluR5 could improve depression through inhibiting peripheral immune responses, confirming the causal relationship between peripheral immune phenotype and brain behavior.

Neutrophils in traumatic brain injury (TBI): friend or foe?

Our knowledge of the pathophysiology about traumatic brain injury (TBI) is still limited. Neutrophils, as the most abundant leukocytes in circulation and the first-line transmigrated immune cells at the sites of injury, are highly involved in the initiation, development, and recovery of TBI. Nonetheless, our understanding about neutrophils in TBI is obsolete, and mounting evidences from recent studies have challenged the conventional views. This review summarizes what is known about the relationships between neutrophils and pathophysiology of TBI. In addition, discussions are made on the complex roles as well as the controversial views of neutrophils in TBI.

Metabotropic glutamate receptor 5 deficiency inhibits neutrophil infiltration after traumatic brain injury in mice.

Both brain native inflammatory cells and infiltrated peripheral white blood cells (WBCs) are primary participants in the brain inflammatory damage post-TBI. Metabotropic glutamate receptor 5 (mGluR5) has been reported to regulate microglias and astrocytes to affect inflammation after TBI, but its effect on modulating infiltrated peripheral WBCs remains unclear. In a mouse moderate TBI model, we found that mGluR5 knockout (KO) significantly reduced neutrophil infiltration and inflammatory cytokine expression in the brain at 24 hours post TBI, which was accompanied by improved neurological dysfunction. Further investigation indicated that mGluR5 KO reduced the permeability of blood-brain barrier (BBB), the entrance for neutrophils to enter brain, and markedly decreased the mRNA levels of neutrophil-associated chemokines in brain tissue, including CXCL1, CXCL2, CCL2, CCL4 and CCL5. Using brain microvascular endothelial cells (BMECs), neutrophils and a BBB model in vitro, we confirmed the inhibitory effect of mGluR5 deficiency on neutrophil infiltration and demonstrated that blockade of protein kinase C (PKC) signaling was involved in it. These results provide insight into the role of mGluR5 in the regulation of inflammation in the acute phase of TBI, which may provide novel clues for TBI therapy.

Activation of Adenosine 2A receptor inhibits neutrophil apoptosis in an autophagy-dependent manner in mice with systemic inflammatory response syndrome.

Systemic inflammatory response syndrome (SIRS) is an overwhelming whole body inflammation caused by infectious diseases or sterile insults. Neutrophils are the dominant participants during inflammation, and their survival and death determine the initiation as well as resolution of SIRS. Apoptosis and autophagy are two fundamental cellular processes that modulating cell fate, but their correlation and regulators in neutrophils under SIRS condition have not been elucidated. In this study, we demonstrated that high dose of LPS induced both apoptosis and autophagy of neutrophils in a mouse SIRS model and LPS-stimulated neutrophils in vitro. Moreover, we found that the adenosine 2A receptor (A2AR), a known anti-inflammatory G protein-coupled receptor (GPCR), could inhibit LPS-induced neutrophil apoptosis by suppressing the LPS-induced autophagy. Activation of A2AR suppressed LPS-induced autophagy by inhibiting the ROS-JNK pathway as well as promoting GPCR ßϒ subunit-AKT signaling. The A2AR-inhibited autophagy suppressed apoptosis of neutrophils by blocking caspase8, caspase3 and PARP signaling. These findings not only increase our understandings of neutrophils' fate and function in response to systemic inflammation, but also identify a novel anti-inflammatory role of A2AR in modulating neutrophils' survival during inflammation.

The mutual regulation between miR-214 and A2AR signaling plays an important role in inflammatory response.

Inflammation is a pathological course involved in several diseases. Both adenosine A2A receptor (A2AR) and miR-214 play important roles in regulation of inflammation. However, the internal link between them and their synergic modulation in inflammatory response has not been elucidated. In this study, we found that miR-214 and A2AR activation could downregulate the expressions of each other in murine macrophages. Comparing with the well known anti-inflammatory role of A2AR, miR-214 promoted the release of inflammatory cytokines TNF-α and IL-6. Further investigation demonstrated that miR-214 downregulated A2AR expression by directly targeting the 3'-untranslated region of A2AR mRNA. Instead of directly interacting with miR-214, A2AR activation repressed miR-214 expression by stimulating PKA signaling to suppress the nuclear translocation of NF-κB which could enhance the transcript activity of miR-214 gene promoter. Then using an LPS-induced ALI mouse model, in which inflammation is a hallmark, we confirmed their negative relationship and demonstrated that combination of miR-214 antagomir and A2AR agonist CGS21680 exerts more anti-inflammatory effect including alleviating the pathological changes, suppressing the neutrophil infiltration and the expression of inflammatory cytokines than using one of them alone. These findings for the first time uncovered a mutual suppression feedback loop between A2AR signaling and miR-214 in inflammation, which may provide new insight of inflammatory regulation and potential therapeutic significance for some inflammation-associated diseases.