RNA-Seq Reveals Th17 Cell Differentiation Pathway as a Mechanism of Radiation-Induced Brain Injury

Summary

This study aims to investigate the activation of the Th17 cell differentiation pathway in mice with radiation-induced brain injury.

Abstract

Radiotherapy is a prevalent therapeutic modality for head and neck malignancies; however, it invariably results in radiation-induced damage to normal cerebral tissue, culminating in radiation-induced brain injury (RBI). Despite extensive research on radiation-induced neuroinflammation, the association between RBI and the Th17 cell differentiation pathway remains inadequately understood. C57BL/6 mice underwent a single administration of 30 Gy cranial irradiation to develop the RBI model. Cognitive function was evaluated through the Morris water maze (MWM), open field test, novel object recognition test, and rotarod test. Histopathological alterations in brain tissue were analyzed using hematoxylin and eosin (H&E) staining. Immunofluorescence staining was employed to assess the activation of microglia (IBA-1) and astrocytes (GFAP). RNA sequencing was conducted to identify differentially expressed genes, while Simple Western and qPCR were utilized to examine key signaling molecules involved in Th17 cell differentiation. RBI mice demonstrated marked cognitive deficits, particularly in spatial learning and memory retention. Histological examination indicated activation of microglia and astrocytes within the cortex and hippocampus of irradiated mice. RNA sequencing analysis identified a significant enrichment of the Th17 cell differentiation pathway in the cortex of the RBI group. Further validation through Simple Western and qPCR analyses confirmed the upregulation of TGF-β, IL-6, RORγt, IL-17, and P-STAT3 in the cortex of RBI mice. These findings suggested that the Th17 cell differentiation pathway played a pivotal role in the pathogenesis of radiation-induced brain injury. Neuroinflammation mediated by Th17 cells may be a critical mechanism underlying radiation-induced cognitive dysfunction.

Introduction

Radiotherapy is a crucial treatment for both primary and metastatic head and neck tumors, but radiation-induced brain injury (RBI) can occur as a severe complication, characterized by central nervous system damage. RBI incidence varies by tumor type and treatment modality, with reported rates ranging from 28%-50% following stereotactic radiotherapy for meningiomas¹, 1.9%-5% for nasopharyngeal carcinoma^2,3, 1%-24% for low-grade gliomas^4,5, and 8%-20% for brain metastases^6,7

Protocol

The experimental procedures conducted here are in strict accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). Approval was obtained from the Institutional Animal Care and Use Committee of the Beijing Institute of Radiation Medicine (Approval No. AF/SC-08/02; Date: Feb 25, 2024).

1. Mice cranial irradiation

1. Use a total of 10 male C57BL/6 mice aged 6-8 weeks in the study, allocating five animals to the RBI group and five to the control group, where the control group consists of blank control mice that did not receive RBI. Maintain the mice under controlled environmental condit....

Results

Cognitive impairment in RBI mice
Following exposure to a 30 Gy whole-brain irradiation, the body weight of the mice was monitored on a weekly basis. Cognitive function was evaluated during the 4^th week (Figure 1A). Initial measurements indicated that the body weights of the control and RBI groups were similar. However, between 1- and 3-weeks post-irradiation, the RBI group demonstrated a significant decrease in body weight (

Discussion

Critical steps within the protocol
This protocol involves the induction of RBI in mice, followed by comprehensive assessments of neuroinflammation and the Th17 cell differentiation pathway (Figure 6). A critical step is cranial irradiation of C57BL/6 mice with a 30 Gy dose, a well-established model for RBI¹. Accurate dose calibration using ionization chambers ensures reproducibility²⁸. Behavioral assays, including the MWM.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12-230 kDa Separation Module, 8 x 25 capillary cartridges	ProteinSimple	SM-W004	for SimpleWestern
anti-GAPDH antibody	Proteintech	HRP-60004	1:500 dilution/antibody for SimpleWestern
anti-GFAP antibody	Abcam	ab7260	1:2000 dilution/antibody for Immunofluorescence
anti-IBA-1 antibody	Abcam	ab178846	1:2000 dilution/antibody for Immunofluorescence
anti-IL-17 A/F antibody	Thermo	MA5-23748	1:10 dilution/antibody for SimpleWestern
anti-IL-1β antibody	CST	12242	1:50 dilution/antibody for SimpleWestern
anti-IL-6 antibody	Thermo	MA5-23800	1:5 dilution/antibody for SimpleWestern
Anti-Mouse Detection Module	ProteinSimple	DM-002	secondary antibody for SimpleWestern
anti-P-STAT3(Try705) antibody	Thermo	MA5-15193	1:2 dilution/antibody for SimpleWestern
Anti-Rabbit Detection Module	ProteinSimple	DM-001	secondary antibody for SimpleWestern
anti-RORγt antibody	Thermo	14-6988-82	1:5 dilution/antibody for SimpleWestern
anti-STAT3 antibody	Thermo	710077	1:20 dilution/antibody for SimpleWestern
anti-TGF β-1,2,3 antibody	Thermo	MA5-23795	1:10 dilution/antibody for SimpleWestern
BAC assay	Thermo	A55860	protein assays
EZ Standard Pack 1 12-230 kDa	ProteinSimple	PS-ST01EZ-8	for SimpleWestern
Goat Anti-Rabbit IgG H&L	Abcam	ab150077	1:1000 dilution/antibody for Immunofluorescence
RNeasy Kit	Qiagen	74134	RNA Extraction
SuperScript IV First-Strand Synthesis System	Thermo Fisher Scientific	18091050	Reverse Transcription
SYBR Green Master Mix	Takara Bio	RR820A	Q-PCR
TRIzol Reagent	Invitrogen	15596026	RNA Extraction