Credit:DOI:10.1016/j.immuni.2024.02.020.

A study recently published in the journal Immunity established microbial flagellin as a critical pathogenic trigger of IL-23, connecting microbial virulence with disease pathogenesis. The authors utilized a multifaceted approach involving human and mouse model studies, microbiome sequencing, and functional assays to unravel the underlying mechanisms.

The first part of the study involved analyzing human gingival tissue samples from healthy individuals, chronic periodontitis (CP) patients, and patients with leukocyte adhesion deficiency type 1 (LAD1)-periodontitis. Through bulk RNA sequencing, the researchers identified distinct transcriptomic profiles in diseased tissues, with CP and LAD1 displaying greater separation from healthy tissue. This analysis revealed that IL-23 and IL-17 signaling pathways were significantly upregulated in both CP and LAD1-periodontitis, with IL-23A being the predominant cytokine.

To investigate the cellular sources of IL-23A, the authors employed single-cell sequencing and flow cytometry. They found that IL-23A was expressed by both hematopoietic and non-hematopoietic cells in the gingival tissue of CP and LAD1 patients. However, only epithelial cells showed significant upregulation of IL-23A within 24 hours post-ligature placement in an experimental mouse model of periodontitis.

The authors then sought to determine the mechanisms underlying IL-23 induction during experimental periodontitis. They hypothesized that the disease-associated microbiome triggers IL-23 production. To test this hypothesis, the authors treated mice with broad-spectrum antibiotics before and during ligature placement and observed a reduction in microbial biomass and IL-23A expression in oral mucosal tissues.

To identify potential microbial candidates involved in IL-23 triggering, the authors characterized the microbial communities on the ligature at various time points. They found that flagellated microbial species were expanded in CP and LAD1 periodontitis compared to healthy individuals. Furthermore, Pseudomonas aeruginosa, a Gram-negative and flagellated bacterium, was enriched in the LAD1 microbiome.

To confirm the role of Pseudomonas aeruginosa in IL-23 induction, the authors isolated primary epithelial cells from mouse oral tissues and cultured them in vitro. They found that treatment with fresh or heat-inactivated microbiota from experimental periodontitis animals significantly upregulated IL-23A expression in oral epithelial cells. Additionally, treatment with Pseudomonas aeruginosa flagellin induced IL-23A expression in human oral keratinocytes in a TLR5-dependent manner.

In summary, this study used a combination of human and mouse model studies, microbiome sequencing, and functional assays to demonstrate that epithelial-derived IL-23 is a critical driver of pathogenic inflammation in periodontitis. The authors identified Pseudomonas aeruginosa and its flagellin as key pathogenic triggers of IL-23, connecting microbial virulence with disease pathogenesis. This study sheds light on the mechanisms underlying the pathogenesis of periodontitis and has implications for other mucosal inflammatory diseases involving IL-23.

Considering the close relationship between bacterial flagellin and inflammation, Boardman et al. focused on the development of a novel chimeric antigen receptor (CAR) Treg therapy for the treatment of inflammatory bowel disease (IBD) utilizing its specificity for bacterial flagellin. The authors selected the flagellin protein FliC, derived from E. coli H18, as a target antigen for its immunogenicity in IBD patients. They developed a novel CAR with specificity for FliC by cloning the variable domains of an anti-FliC monoclonal antibody into a lentiviral vector with CAR signaling domains.

Human Tregs were isolated and stimulated with artificial antigen presenting cells (APCs) before lentiviral transduction with either FliC-CAR or HER2-CAR. Both CAR Treg populations maintained Treg phenotype and function. To test the specificity of the FliC-CAR, Tregs were stimulated with rFliC or a control flagellin, and activation markers were measured. Only FliC-CAR Tregs showed activation in response to rFliC.

In a humanized mouse model, the authors tested whether FliC-CAR expression would promote trafficking of T cells to the intestine. NSG mice were injected with human CD4+ and CD8+ T cells expressing FliC-CAR or HER2-CAR, along with PBMCs. After engraftment, the mice were given a dinitrobenzene sulfonic acid (DNBS) enema to induce colitis. Analysis revealed that FliC-CAR T cells preferentially trafficked to the colon and showed higher activation compared to HER2-CAR T cells.

To assess the suppressive function of FliC-CAR Tregs, suppression assays were performed with FliC-CAR and HER2-CAR Tregs stimulated with rFliC or rFla2, and responder T cells were stimulated with anti-CD3/CD28 beads. FliC-CAR Tregs significantly inhibited T cell proliferation specifically in the presence of rFliC. Additionally, FliC-CAR Tregs promoted epithelial barrier integrity of human colonoids in the presence of rFliC.

In summary, the authors developed FliC-specific CAR Tregs and demonstrated their specificity, trafficking, and suppressive functions. The data supported the potential of FliC-CAR Tregs as a promising new therapy for IBD, providing antigen-specific regulation of intestinal inflammation. The study also highlighted the potential utility of CARs targeting microbial antigens.

These studies have provided valuable insights into the complex interactions between microorganisms and the host immune system in diseases such as periodontitis and IBD. The identification of specific microbial agents and their products, such as flagellin, as triggers for immune responses underlying the pathogenesis of these inflammatory conditions opens up new avenues for therapeutic intervention. The findings not only reveal potential treatment targets but also deepen our understanding of the underlying mechanisms of these diseases. Future research will undoubtedly build on these findings to uncover more about the intricate relationship between microbiota and host health, leading to improved patient care and outcomes.

1. Kim TS, Ikeuchi T, Theofilou VI, Williams DW, Greenwell-Wild T, June A, Adade EE, Li L, Abusleme L, Dutzan N, Yuan Y, Brenchley L, Bouladoux N, Sakamachi Y; NIDCD/NIDCR Genomics and Computational Biology Core; Palmer RJ Jr, Iglesias-Bartolome R, Trinchieri G, Garantziotis S, Belkaid Y, Valm AM, Diaz PI, Holland SM, Moutsopoulos NM. Epithelial-derived interleukin-23 promotes oral mucosal immunopathology. Immunity. 2024 Apr 9;57(4):859-875.e11.

2. Boardman DA, Wong MQ, Rees WD, Wu D, Himmel ME, Orban PC, Vent-Schmidt J, Zachos NC, Steiner TS, Levings MK. Flagellin-specific human CAR Tregs for immune regulation in IBD. J Autoimmun. 2023 Jan;134:102961.