Terminally differentiated cytotoxic CD4+ T cells were clonally expanded in the brain lesion of radiation-induced brain injury.

BACKGROUND: Accumulating evidence supports the involvement of adaptive immunity in the development of radiation-induced brain injury (RIBI). Our previous work has emphasized the cytotoxic function of CD8+ T cells in RIBI. In this study, we aimed to investigate the presence and potential roles of cytotoxic CD4+ T cells (CD4+ CTLs) in RIBI to gain a more comprehensive understanding of adaptive immunity in this context. MAIN TEXT: Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed 3934 CD4+ T cells from the brain lesions of four RIBI patients and identified six subclusters within this population. A notable subset, the cytotoxic CD4+ T cells (CD4+ CTLs), was marked with high expression of cytotoxicity-related genes (NKG7, GZMH, GNLY, FGFBP2, and GZMB) and several chemokine and chemokine receptors (CCL5, CX3CR1, and CCL4L2). Through in-depth pseudotime analysis, which simulates the development of CD4+ T cells, we observed that the CD4+ CTLs exhibited signatures of terminal differentiation. Their functions were enriched in protein serine/threonine kinase activity, GTPase regulator activity, phosphoprotein phosphatase activity, and cysteine-type endopeptidase activity involved in the apoptotic signaling pathway. Correspondingly, mice subjected to gamma knife irradiation on the brain showed a time-dependent infiltration of CD4+ T cells, an increase of MHCII+ cells, and the existence of CD4+ CTLs in lesions, along with an elevation of apoptotic-related proteins. Finally, and most crucially, single-cell T-cell receptor sequencing (scTCR-seq) analysis at the patient level determined a large clonal expansion of CD4+ CTLs in lesion tissues of RIBI. Transcriptional factor-encoding genes TBX21, RORB, and EOMES showed positive correlations with the cytotoxic functions of CD4+ T cells, suggesting their potential to distinguish RIBI-related CD4+ CTLs from other subsets. CONCLUSION: The present study enriches the understanding of the transcriptional landscape of adaptive immune cells in RIBI patients. It provides the first description of a clonally expanded CD4+ CTL subset in RIBI lesions, which may illuminate new mechanisms in the development of RIBI and offer potential biomarkers or therapeutic targets for the disease.

A Synthetic Steroid 5α-Androst-3ß, 5, 6ß-triol Alleviates Radiation-Induced Brain Injury in Mice via Inhibiting GBP5/NF-κB/NLRP3 Signal Axis.

Radiotherapy for head and neck tumors can lead to a severe complication known as radiation-induced brain injury (RIBI). However, the underlying mechanism of RIBI development remains unclear, and limited prevention and treatment options are available. Neuroactive steroids have shown potential in treating neurological disorders. 5α-Androst-3ß, 5, 6ß-triol (TRIOL), a synthetic neuroprotective steroid, holds promise as a treatment candidate for RIBI patients. However, the neuroprotective effects and underlying mechanism of TRIOL on RIBI treatment are yet to be elucidated. In the present study, our findings demonstrate TRIOL's potential as a neuroprotective agent against RIBI. In gamma knife irradiation mouse model, TRIOL treatment significantly reduced brain necrosis volume, microglial activation, and neuronal loss. RNA-sequencing, immunofluorescence, real-time quantitative polymerase chain reaction, siRNA transfection, and western blotting techniques revealed that TRIOL effectively decreased microglial activation, proinflammatory cytokine release, neuron loss, and guanylate-binding protein 5 (GBP5) expression, along with its downstream signaling pathways NF-κB and NLRP3 activation in vitro. In summary, TRIOL effectively alleviate RIBI by inhibiting the GBP5/NF-κB/NLRP3 signal axis, reducing microglia activation and pro-inflammation cytokines release, rescuing neuron loss. This study highlights the potential of TRIOL as a novel and promising therapy drug for RIBI treatment.

Comparative proteomic analysis of edible bird's nest from different origins.

Edible bird's nest (EBN) mainly made of saliva that secreted by a variety of swiftlets is a kind of precious traditional Chinese medicine. EBNs from different biological and geographical origins exhibit varieties in morphology, material composition, nutritive value and commercial value. Here, we collected four different EBN samples from Huaiji, China (Grass EBN), Nha Trang, Vietnam (Imperial EBN) and East Kalimantan, Indonesia (White EBN and Feather EBN) respectively, and applied label-free quantitative MS-based proteomics technique to identify its protein composition. First, phylogenetic analysis was performed based on cytb gene to identify its biological origin. Second, a total of 37 proteins of EBNs were identified, among which there were six common proteins that detected in all samples and exhibited relatively higher content. Gene ontology analysis revealed the possible function of EBN proteins, and principal component analysis and hierarchical clustering analysis based on 37 proteins were performed to compare the difference of various EBNs. In summary, our study deciphered the common and characteristic protein components of EBNs of different origins and described their possible functions by GO enrichment analysis, which helps to establish an objective and reliable quality evaluation system.

Microglia drive transient insult-induced brain injury by chemotactic recruitment of CD8+ T lymphocytes.

The crosstalk between the nervous and immune systems has gained increasing attention for its emerging role in neurological diseases. Radiation-induced brain injury (RIBI) remains the most common medical complication of cranial radiotherapy, and its pathological mechanisms have yet to be elucidated. Here, using single-cell RNA and T cell receptor sequencing, we found infiltration and clonal expansion of CD8+ T lymphocytes in the lesioned brain tissues of RIBI patients. Furthermore, by strategies of genetic or pharmacologic interruption, we identified a chemotactic action of microglia-derived CCL2/CCL8 chemokines in mediating the infiltration of CCR2+/CCR5+ CD8+ T cells and tissue damage in RIBI mice. Such a chemotactic axis also participated in the progression of cerebral infarction in the mouse model of ischemic injury. Our findings therefore highlight the critical role of microglia in mediating the dysregulation of adaptive immune responses and reveal a potential therapeutic strategy for non-infectious brain diseases.

Notch Signaling Mediates Radiation-Induced Smooth Muscle Cell Hypermuscularization and Cerebral Vasculopathy.

BACKGROUND: Emerging evidence highlighted vascular injury in aggravating radiation-induced brain injury (RIBI), a common complication of radiotherapy. This study aimed to delineate the pathological feature of cerebral small vessel and investigate the functional roles of Notch signaling in RIBI. METHODS: Brain tissue and functional MRI from RIBI patients were collected and analyzed for radiation-induced vasculopathy. A RIBI mouse model was induced by a single dose of 30-Gy cranial irradiation. Vascular morphology, pulsatility, and reactivity to pharmacological interventions, such as nimodipine and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, were monitored by 2-photon imaging in mice at 6 weeks postirradiation. Western blot, real-time quantitative PCR, immunofluorescence staining, and behavioral tests were performed. The effect of N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester, a Notch inhibitor, was used to investigate the vascular pathogenesis of RIBI mouse model. RESULTS: Morphologically, radiation resulted in vascular malformation featured by focal contractile rings together with general stenosis. Functionally, radiation also led to hypoperfusion, attenuated vascular pulsatility, and decreased dilation to nimodipine and 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid. Mechanically, Notch activation and increased expression of α-SMA protein were found in both surgical specimens of RIBI patients and the irradiated mice. Importantly, Notch inhibition by N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester significantly alleviated cerebral hypoperfusion, vasculopathy, and cognitive deficits in the RIBI mouse model. CONCLUSIONS: Radiation-induced cerebral vasculopathy showed bead-like shape and increased contractile state. Inhibition of Notch signaling by N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-s-phenylglycinet-butyl ester effectively attenuated vasculopathy and relieved cognitive impairment, suggesting Notch signaling as a therapeutic target for the treatment of RIBI.

Molecular analysis of edible bird's nest and rapid authentication of Aerodramus fuciphagus from its subspecies by PCR-RFLP based on the cytb gene.

Edible bird's nest (EBN), for its great nutritional value, is widely used around the world, especially in China and Singapore. EBNs of different origins and types may vary in price and quality. Nowadays, birds' nests are difficult to identify morphologically, except for some whole bird's nests of which origins can be roughly identified. In this study, forty-two samples were collected from different regions for sequencing analysis and phylogenetic classification to initially determine their origins. Two stable enzyme digestion sites were found in the analysis of restriction maps of the species. Then, a quick and specific PCR-RFLP method was established to identify the EBN samples' origins. The genetic identification results indicated that the forty-two samples were from five origins. With the Af/g-486bp-F/R primer and restriction enzyme Taq I, Aerodramus fuciphagus (A. fuciphagus) was efficiently differentiated from the other species. Furthermore, the cytb-592bp-F/R primer and the BamH I enzyme were found to be useful in distinguishing Aerodramus fuciphagus (A. fuciphagus) from its subspecies (Aerodramus germani, A. germani). The PCR-RFLP method provides a potential tool for the rapid discrimination of A. fuciphagus at the species and even the subspecies levels to ensure the quality of the EBN products.

Asprellcosides B of Ilex asprella Inhibits Influenza A Virus Infection by Blocking the Hemagglutinin- Mediated Membrane Fusion.

Ilex asprella is routinely used in China as a traditional medicinal herb to treat influenza (Flu). However, its specific antiviral activity and underlying molecular mechanism have not yet been determined. In this study, we sought to determine the antiviral activity and mechanism of Asprellcosides B, an active component extracted from Ilex asprella, and used against the influenza A virus cell culture. We also performed a computer-assisted structural modeling analysis and carried out surface plasmon resonance (SPR) experiments in the hope of determining the viral target of Asprellcosides B. Results from our studies show that Asprellcosides B reduced virus replication by up to 63% with an IC50 of about 9 µM. It also decreased the low pH-induced and virus-mediated hemolysis by 71% in vitro. Molecular docking simulation analysis suggested a possible binding of Asprellcosides B to the hemagglutinin (HA), which was confirmed by a surface plasmon resonance (SPR) assay. Altogether, our findings demonstrate that Asprellcosides B inhibits the influenza A virus, through a specific binding to the HA, resulting in the blockade of the HA-mediated membrane fusion.