Alcaligenes faecalis Induces Intestinal T Helper 17 Cells by Promoting E3 Ubiquitin Ligase TRIM21-Mediated FBXW7 Degradation

Rheumatology and Immunology Life Sciences Medicine Immunology Microbiology Cell Biology Biochemistry
Alcaligenes faecalis Th17 cells TRIM21 FBXW7 ubiquitination intestinal immunity cell differentiation

1. Research Background

Intestinal Th17 cells play a central role in maintaining mucosal immune homeostasis and defending against pathogen infections. Previous studies identified segmented filamentous bacteria (SFB) as key microbes inducing intestinal Th17 cells, but controversies exist regarding SFB colonization in adult intestines: 1. Clinical Paradox: SFB detection rates in human intestines decline sharply with age (24% in children under 3 years vs. 0% in adults), making it difficult to explain the widespread enrichment of Th17 cells in human intestines. 2. Mechanistic Limitations: Known SFB pathways involve dendritic cell-dependent mechanisms or CDC42-mediated endocytosis by epithelial cells to indirectly induce Th17 cells, but whether microbes can directly regulate intrinsic T cell mechanisms remains unclear.

This study, led by Zhijian Cai’s team at Zhejiang University School of Medicine in collaboration with Indiana University and other institutions, was published in Immunity (June 2025, DOI:10.1016/j.immuni.2025.03.008). It reveals a novel mechanism by which the ubiquitous Alcaligenes faecalis directly induces Th17 cells through the TRIM21-FBXW7-JUNB-AHR-RORγt axis.

2. Research Process and Findings

1. FBXW7 Deficiency Promotes Th17 Cell Differentiation (Basic Mechanism Exploration)

Experimental Design: - Genetically engineered mouse model: Constructed T cell-specific FBXW7 knockout mice (FBXW7fl/fl*dLck-Cre). - Sample processing: Isolated splenocytes, mesenteric lymph nodes (MLNs), and lamina propria (LP) lymphocytes. - Key experiments: - Flow cytometry analysis of Th17 cell proportion (IL-17A+CD4+). - qPCR detection of Th17 signature genes (Il17a, Rorc, Il23r). - In vitro Th17 polarization culture (TGF-β1 + IL-6 + IL-23).

Key Findings: - FBXW7 deletion increased intestinal LP Th17 cells by 2.3-fold (p < 0.001). - Non-pathogenic Th17 marker Il10 was upregulated, while pathogenic marker Tbx21 was downregulated. - Human validation: Negative correlation between FBXW7 and IL-17A expression in colonic CD4+ T cells (r = -0.82).

2. Gut Microbiota Induces FBXW7 Ubiquitination Degradation via TRIM21

Innovative Methods: - Mass spectrometry: Identified 8 E3 ligases interacting with FBXW7. - Conditional gene silencing: shRNA screening for key regulators.

Key Data: - Microbiota lysate treatment shortened FBXW7 half-life from 8.5 hours to 3.2 hours. - TRIM21 specifically mediated K48-linked polyubiquitination of FBXW7 (7 ubiquitination sites identified by mass spectrometry). - After antibiotic clearance of microbiota, Th17 cells decreased by 67% in wild-type mice but only 19% in FBXW7-/- mice.

3. Unique Mechanisms of Alcaligenes faecalis

Microbiota Screening: - Metagenomic sequencing: Identified 4,494 bacterial species from LB agar-cultured gut microbiota. - Functional screening: Only A. faecalis and A. haemolyticus among 10 commercial strains induced Th17 cells.

Dual Regulatory Pathways: 1. Protein Endocytosis Pathway: - CDC42-dependent endocytosis: Confocal microscopy showed colocalization of biotin-labeled A. faecalis proteins with EEA1+ endosomes. - Inhibition of TRIM21 self-ubiquitination: UBE2W-TRIM21 interaction reduced by 50% (co-IP verified).

  1. Outer Membrane Vesicles (OMVs) Pathway:
    • Nanoparticle tracking analysis (NTA): Isolated 80–200 nm diameter vesicles.
    • Macropinocytosis inhibitor Baf-A1 blocked OMV internalization (>85% efficiency).

Clinical Relevance: - Human fecal detection: A. faecalis detection rates reached 86% in adults and 63% in children. - Intravenous OMV injection increased intestinal Th17 cells by 3.1-fold in germ-free mice.

4. Molecular Axis Mechanism (JUNB-AHR-RORγt)

Innovative Experiments: - Chromatin immunoprecipitation (ChIP): Confirmed AHR directly binds to the RORC promoter region (-8567 to -8549 bp). - Dual-luciferase reporter system: JUNB overexpression enhanced AHR promoter activity by 4.8-fold.

Key Findings: - FBXW7 suppresses AHR transcription by degrading JUNB. - AHR directly activates RORγt expression (72% reduction in luciferase activity upon binding site mutation). - Lineage tracing: FBXW7 deficiency did not affect survival of differentiated Th17 cells (no difference in cell numbers after adoptive transfer).

3. Research Significance and Highlights

Scientific Value

  1. Theoretical Breakthrough:

    • Identified a new paradigm of non-SFB microbes directly regulating T cell differentiation.
    • Elucidated the novel TRIM21-FBXW7-JUNB-AHR-RORγt signaling axis.

  2. Technical Innovation:

    • Established a “microbial protein-host E3 ligase” interaction screening platform.
    • Developed microbiota intervention strategies based on CDC42 inhibitors.

Application Prospects

  1. Disease Treatment:

    • A. faecalis OMVs could serve as prophylactic agents against Citrobacter rodentium infection.
    • TRIM21 inhibitors may be used for targeted therapy of inflammatory bowel disease.

  2. Technology Translation:

    • Patent data: Filed for invention patent on “A. faecalis-based immunomodulators” (WO2025/086532).

4. Research Limitations

  1. Mechanistic Depth: The specific protein component(s) of A. faecalis responsible for the effect remain unidentified.
  2. Clinical Translation: Further validation is needed for the consistency of A. faecalis immunomodulation across different human populations.

This study provides a new perspective on gut microbiota-immune system interactions. The Graphical Abstract was featured on the cover of Immunity.