Antibiotic-Induced Intestinal Immunomodulation Alleviates Experimental Autoimmune Neuritis (EAN)

Academic Background

Guillain-Barré syndrome (GBS) is an acute autoimmune disease that causes inflammatory demyelination of peripheral nerves. As the most common cause of acute flaccid paralysis, its global annual incidence is 1-4 cases per 100,000 people. The pathological features of GBS include the accumulation of muscle myelin antigen-reactive T cells and macrophages in the endoneurium. Although certain pathological features of GBS vary among different subtypes, its core mechanism is believed to be driven by molecular mimicry, leading to antibody-mediated immune responses that attack peripheral nerve gangliosides and other unknown neural epitopes. This mechanism is typically associated with respiratory and especially gastrointestinal infections (such as Campylobacter enteritis).

Recent studies have shown a correlation between the composition of gut microbiota and GBS and its animal model, experimental autoimmune neuritis (EAN). Gut microbiota plays an important role in regulating intestinal homeostasis and immune tolerance, and adverse changes in gut flora can exacerbate symptoms of various autoimmune diseases, such as multiple sclerosis (MS) and Crohn’s disease. Based on these foundations, the authors hypothesized that eliminating gut bacteria through antibiotic treatment might modulate intestinal and systemic cellular immune effects, thereby beneficially affecting EAN. This new research direction has not yet been explored in peripheral neuropathies. Given that existing treatments (such as intravenous immunoglobulin or plasma exchange) cannot fully control GBS, and the disability rate remains high, new therapeutic approaches are needed.

Research Source

This study, titled “Antibiotic-Induced Intestinal Immunomodulation Alleviates Experimental Autoimmune Neuritis (EAN),” was authored by Alina Sprenger-Svačina, Ines Klein, Martin K. R. Svačina, et al. The researchers are affiliated with the University of Cologne Medical School and related institutions. The paper was published in the “Journal of Neuroimmune Pharmacology” in 2024, with the received date of November 30, 2023, and the accepted date of April 21, 2024.

Research Methods

Animal Experiments

The experiments were conducted in accordance with the guidelines of the North Rhine-Westphalia State Office for Environment and Consumer Protection, Germany. A total of 17 adult Lewis rats (7 males, 10 females, 12 weeks old) were used, with 10 for the EAN model and 7 as healthy controls. To induce EAN, rats were subcutaneously injected with 100μg myelin P0 peptide 180-199 and complete Freund’s adjuvant in the hind limbs. Immediately after immunization, one group of rats received antibiotic treatment (colistin, metronidazole, vancomycin added to drinking water) for 14 days. Daily control of water intake and replacement of antibiotic-containing drinking water ensured antibiotic intake.

Fecal Sample Collection and Genetic Analysis

Fecal samples were collected from each animal before EAN induction, on day 9 and day 14 of the experiment, and stored at -80°C for further analysis. Bacterial genomic DNA was extracted using the FastDNA Spin Kit for Soil, and 16S rRNA gene amplification and sequencing were performed using the Illumina MiSeq platform. Data were processed using DADA2 software and QIIME 2 for taxonomic sample inference and diversity analysis.

Tissue Collection and Immunohistochemistry

Rats underwent cardiac perfusion under deep anesthesia, and tissues were fixed with 4% paraformaldehyde. Sciatic nerve and duodenum tissues were obtained and processed for frozen sections and paraffin embedding, respectively. Immunofluorescence staining was used to assess intestinal mucosal permeability and various immune cells, such as Zonulin, CD3/CD8, and CD3/FoxP3 positive cells.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 9.2.0, employing Mann-Whitney U test, Kruskal-Wallis or One-Way ANOVA test to detect inter-group differences. R software was used to generate relevant data charts. Adjusted p-values were calculated for the relative abundance of each bacterial taxon to correct for statistical errors from multiple comparisons.

Research Results

Antibiotic Treatment Improves EAN Severity

EAN rats receiving antibiotic treatment showed significantly milder neuroinflammatory symptoms starting from day 3 post-immunization, as evidenced by significantly lower average neuritis scores.

Antibiotic Treatment Reduces T Cell Infiltration in Endoneurium

EAN rats without antibiotic treatment showed significantly higher CD3+ T cell counts in the sciatic nerve, while the antibiotic-treated group showed T cell counts not significantly different from healthy controls. Simultaneously, the antibiotic treatment group showed a decrease in CD8+ T cells in the intestinal mucosa and an increase in gut bacteria with anti-inflammatory properties, such as Lactobacillus and Parasutterella.

Antibiotic Treatment Increases Anti-inflammatory Bacteria

Antibiotic treatment significantly reduced the α-diversity of gut microbiota and caused significant changes in β-diversity. The gut microbiota of the antibiotic group showed significant increases in Sutterellaceae family, Lactobacillus genus, and Parasutterella genus, while Bacillaceae family, Anaerotruncus genus, and UBA1819 genus were significantly reduced.

Intestinal Mucosal Barrier Dysfunction in EAN

In the non-antibiotic EAN group, intestinal mucosal zonulin levels were significantly higher than in healthy controls, indicating increased intestinal barrier permeability. However, the antibiotic group showed intermediate levels of mucosal zonulin, not significantly different from healthy controls.

Antibiotic Treatment Reduces CD8+ Cytotoxic T Cells in the Intestine

Non-antibiotic treated mice showed significantly higher CD3+CD8+ cytotoxic T cells in the intestine compared to the antibiotic group and healthy controls, while antibiotic treatment reduced cytotoxic T cells in diseased mice to healthy control levels.

Discussion

The results indicate that EAN exhibits unique immune characteristics in the intestinal mucosa, including increased CD8+ cytotoxic T cells in the lamina propria and increased mucosal permeability. Antibiotic treatment led to a reduction in CD8+ cytotoxic T cells in the intestinal mucosa of diseased rats, along with significantly reduced T cell infiltration in the sciatic nerve, suggesting the role of the gut immune-peripheral nerve axis in EAN pathology.

Notably, antibiotics promoted an increase in the relative abundance of gut bacteria with known anti-inflammatory effects (such as Sutterellaceae and Lactobacillus), suggesting that antibiotic-induced intestinal immune regulation may play a key role in modulating neuritis. This study demonstrates that antibiotic treatment may improve peripheral neuritis through intestinal immune regulation, showing potential clinical translational value.

This paper provides the first preliminary evidence that antibiotic-induced intestinal immune regulation may improve immune neuropathy simulation. Future translational studies should further confirm its efficacy in patients.