Elevated IL-6 Levels and Upregulation of Iron-Regulatory Biomarkers Promote Japanese Encephalitis Virus Infection

In this research paper, the authors explore changes in inflammatory response and iron-regulatory biomarkers during Japanese Encephalitis Virus (JEV) infection. A comprehensive analysis of iron-regulatory biomarkers and inflammatory response strategies for JEV infection may be an important approach to disease management.

Research Background

Iron pathway regulation in the host is an essential component of the innate immune response against invading pathogens. During infection, any disruption of iron homeostasis (such as iron deficiency or iron overload) can lead to cellular and organ dysfunction and significantly affect the outcome of infectious diseases. JEV infection causes hypoferremia and iron accumulation in splenic macrophages, but this iron redistribution does not correspond to changes in total iron-binding capacity in serum or total iron content in the body. This suggests abnormalities in iron metabolism and immune cell accumulation after JEV infection.

The authors believe that many pathways involve fine mechanisms of iron metabolism regulation that have not yet been validated through clinical studies. In particular, the role of iron-regulatory biomarkers and their related genes in the pathophysiology of Japanese encephalitis virus infection deserves in-depth study.

Research Source

This article was co-authored by several scholars including Alok Kumar, Anjali Singh, Sneha Ghildiyal, Prabhaker Mishra, and Gajendra Singh from Sanjay Gandhi Post Graduate Institute of Medical Sciences and King George’s Medical University in India, along with Himanshu Dandu. The paper was published in the journal “Neuromolecular Medicine” on October 31, 2023.

Research Methods

Study Subjects and Samples

The study included a total of 148 participants, with 74 JEV-positive acute encephalitis syndrome patients and 74 healthy controls matched for gender and age. Symptoms such as fever, headache, vomiting, abnormal behavior, mental state, neck stiffness, and Glasgow Coma Scale (GCS) scores were recorded. Patients were followed up 6 months after discharge to record their prognosis.

Experimental Methods

1. Cytokine Detection (ELISA):

   • Blood samples were collected from healthy controls and JEV patients, serum was separated and stored at -80°C until use.
   • Commercial enzyme-linked immunosorbent assay (ELISA) kits were used to detect IL-6 levels in serum.

2. HAMP, TF, and TFR1 mRNA Level Determination:

   • Total RNA was isolated from blood samples of JEV patients and healthy controls using the QIAamp RNA Blood Mini Kit (Qiagen).
   • Real-time PCR was performed using the ABI 7500 Sequence Detection System, and relative gene expression was calculated using the 2−ΔΔCt comparative CT method.

3. Genotyping Analysis (PCR-RFLP):

   • PCR-RFLP method was used to analyze tf (rs4481157) gene polymorphisms.
   • PCR amplification products were analyzed on 2% agarose gel electrophoresis and genotyped using restriction endonucleases.

4. Classification and Regression Tree (CART) Analysis:

   • Decision tree analysis was used for prediction, exploring the predictive role of major genotype indicators in JEV susceptibility and severity.

Data Analysis

• SPSS-23 and GraphPad Prism 8 were used for statistical analysis.
• Chi-square test was used to compare relationships between genotypes and symptoms.
• Binary logistic regression analysis was used for genotype association risk assessment.
• p-value <0.05 was considered statistically significant.

Research Results

Demographic and Clinical Characteristics of JEV Patients

The study included 74 JEV-positive patients and 74 healthy controls. Patient symptoms were recorded and compared in detail with controls. Among these patients, 73 (98.6%) presented with fever, 56 (75.6%) with headache, 54 (72.97%) with altered sensations, and other symptoms.

IL-6, HAMP, TF, TFR1 mRNA Levels in JEV Patients

ELISA detection showed that serum IL-6 levels in JEV patients were significantly higher than in the healthy control group. RT-PCR showed that HAMP, TF, and TFR1 mRNA levels in JEV patients were significantly higher than in the control group, suggesting the role of iron-regulatory biomarkers in JEV infection.

Association of TF Genotype with JEV Clinical Manifestations

TF (rs4481157) gene polymorphism was significantly associated with JEV clinical symptoms. CART analysis showed that patients with wild-type genotype were more susceptible to JEV infection, while homozygous type patients had a higher risk of developing severe JEV conditions.

Discussion

The authors point out that in previous studies, pathogen invasion of host cells led to the release of various molecules, triggering high inflammatory responses. The current study shows that HAMP mRNA expression and IL-6 protein levels are significantly elevated in JEV patients’ serum, and the IL-6-HAMP axis plays a key role. Additionally, increased iron load is associated with inflammation, such as the IL-6 upregulation induced by JEV infection in this study leading to increased HAMP expression.

Furthermore, many iron-sensing genes (such as TF) were found to be important regulators of HAMP expression. JEV infection affects HAMP production by inducing an inflammatory state, thereby affecting TF and TFR1 expression to maintain serum iron balance.

Conclusion

The study reveals changes in IL-6 levels and iron-regulatory processes during JEV infection, which play a crucial role in JEV pathophysiological development. The TF genotype (rs4481157) can serve as an important tool for assessing JEV infection susceptibility and severity.

Research Value

This study provides new insights into understanding the inflammatory and iron-regulatory mechanisms in JEV infection and proposes potential applications of corresponding biomarkers (such as IL-6 and HAMP). Future research is needed to uncover the fine interactions between these regulatory proteins and genes, as well as the impact of gene sequence changes on overall transcription levels.