Investigations into the aetiopathogenesis of orofacial granulomatosis using multiple omics technologies reveal a potential role for B cells

Dear Editor, Orofacial granulomatosis (OFG) is a granulomatous inflammatory disease of the oral cavity, with similarities to Crohn's disease (CD). OFG affects children and young adults, a proportion of whom have coexisting CD. Indeed, childhood-onset OFG is a risk factor for CD. However, not all OFG patients develop CD.1 The aetiopathogenesis of OFG is unknown. Studies revealed that oral granulomatous diseases are genetically distinguishable from CD,2, 3 and microbial dysbiosis, including changes in abundance of Streptococcus and Neisseria, have been suggested to drive the granulomatous inflammatory response in OFG.4, 5 Alternatively, OFG has been described as an atopic condition and dietary intolerances implicated in its aetiology. However, not all patients see clinical improvements upon dietary exclusion.5 Studies into the immunopathogenesis of OFG are few and mainly histological. Tissue from patients with OFG has been found to contain a significantly higher number of CD3+ T cells, CD11c+ dendritic cells and B cells, including a novel subset of IgE-expressing B cells, than tissue from patients with concurrent CD.6 Since OFG is an uncommon condition, there is a lack of robust clinical studies investigating its aetiopathogenesis. Therefore, 32 participants with OFG and 43 healthy controls were recruited, and multiple ‘omics’ technologies were applied to interrogate the aetiopathogenic mechanisms of disease (Figure 1A and Supporting Information Materials and Methods). There were no significant differences between demographic or clinical parameters between the study groups, including those with OFG alone and concurrent CD (Table S1). RNA-seq analysis revealed that participants with OFG alone showed a heterogeneous spread in variation (Figure 1B) with 2304 differentially expressed genes (DEGs) (Figure 1C). Gene ontology enrichment analysis revealed the DEGs to be predominantly associated with adaptive immune responses (Figure 1D). STRING analysis of the top 50 up- (Table S2) and down- (Table S3) regulated genes identified upregulation of pathways associated with B cell activity and phagocytosis (Figure 1E) and downregulation of pathways associated with keratinocyte function (Figure 1F). Whole Exome Sequencing revealed the most frequent mutations associated with OFG, ranked on allele frequency (AF), were found in the KIAA2018 and TAS2R31 genes. However, based on the Combined Annotation-Dependent Depletion (CADD) score, mutations in the SRGAP2 and AK2 genes had a greater potential impact on disease (Table S4). Analysis of the salivary immunoproteome revealed that 41.79% of variance was attributed to the first component and 13.08% to the second component (Figure 2A), and 55 proteins were significantly elevated in the saliva of patients with OFG (Figure 2B and Table S5). However, no significant differences in levels were determined between participants with OFG and those with concurrent CD (data not shown). STRING analysis identified upregulated pathways associated with regulation of adaptive immune responses, TNF and cytokine production (Figure 2C,D). ELISA analysis confirmed IL-6 (Figure 2E), CXCL9 (Figure 2F) and CCL3 (Figure 2G) were elevated in all participants with OFG compared to healthy control participants (all p 0.7) between ELISA levels and Olink NPX values (Figure 2H). Faecal calprotectin (MRP8/14; S100A8/9) is used in both the diagnostic workup and monitoring of patients with IBD. In a paediatric population with early onset IBD, salivary calprotectin levels were significantly elevated in those with OFG, compared to those with IBD alone.7 Analysis of salivary calprotectin revealed levels that were significantly elevated in all participants with OFG (p < 0.001), but there was no difference between participants with OFG and those with concurrent CD (Figure 3A). In addition, salivary calprotectin levels strongly correlated with levels of IL-6 and CXCL9 but no other parameters (Figure 3B). Analysis of the salivary microbiome was performed to determine if dysbiosis contributed to the aetiopathogenesis of OFG. Using ecological principles (see Supporting Information), the OFG cohort had 185 core taxa and 724 satellite taxa. The core taxa accounted for 98.0% abundance. The healthy cohort had 216 core and 703 satellite taxa. The core taxa accounted for 99.6% abundance (Figure 4A). Diversity was significantly higher in the healthy cohort, compared to the OFG cohort, for the whole microbiota, core and satellite taxa (p < 0.05 in all instances) (Figure 4B). The compositional similarity between the OFG and healthy control cohorts was significantly different for the whole microbiota, core and satellite taxa (p < 0.0001 in all instances) (Figure 4C). SIMPER analysis suggested that Streptococcus and Neisseria were contributing most to the dissimilarity between cohorts (Table S6). In terms of clinical presentation, salivary biomarkers and the oral microbiome, there were no differences between participants with OFG alone and those with concurrent CD. Microbiome analysis, in agreement with previous studies, implicated Streptococcus and Neisseria species in the aetiopathogenesis of OFG.3, 4 Interestingly, Neisseria subflava, from patients with OFG, was demonstrated to elicit granulomatous inflammation.4 Therefore, dysbiosis may play a role in driving granulomatous inflammation in patients with OFG. However, to position organisms as specific aetiological agents requires further studies. The data show that B-cell responses may play a role in OFG immunopathogenesis. RNAseq revealed a significant role for B cell genes and signalling pathways. In addition, WES identified mutations in SRGAP2 and AK2 that may predispose to OFG. Interestingly, SRGAP2 is expressed in immune cells (https://v23.proteinatlas.org), but its function remains unknown. However, AK2 plays an important role in B cell immunometabolism, activation and antibody production.8 Furthermore, IL6 (B cell stimulatory factor 2), CXCL9 and CCL3 were elevated in the saliva of participants with OFG, all of which play a role in B cell maturation, viability, migration, differentiation into plasma cells and B cell receptor signalling.9 Adding to the evidence that B cells may play a role in OFG, a recent case study reported the use of the B-cell-depleting monoclonal antibody Rituximab as a treatment, which revealed promising results.10 Therefore, large-scale, well-controlled clinical studies using Rituximab may reveal more about the role of B cells in OFG immunopathogenesis and determine if targeting B cells is a treatment option for OFG. Christopher John Nile, John Gibson and Andrew Smith conceived and supervised the study. Maria Tumelty, Christopher John Nile, John Gibson, Gordon Ramage and Andrew Smith designed the research methodology. Maria Tumelty, David Lappin, Krupali Patel and Helen Petersen collected the data and performed the experiments. Maria Tumelty, Christopher John Nile, David Lappin, Chris Delaney and Christopher van der Gast conducted the formal analysis. Christopher John Nile, Maria Tumelty, Christopher van der Gast and Andrew Smith wrote the original draft of the manuscript. All authors contributed to data interpretation, critically revised the manuscript and approved the final version. Christopher Nile is funded by the NIHR Newcastle Biomedical Research Centre (BRC), awarded to the Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle University and Cumbria, Northumberland, Tyne and Wear Foundation Trust. Andrew Smith was funded by the National Institute for Health Research Biomedical Research Centre at NIHR UCLH BRC and UCL. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. Maria Tumelty was supported by grants from Tenovus Scotland, the Royal College of Surgeons and Physicians (Glasgow) and the Faculty of Dental Surgery, Royal College of Surgeons of England. Krupali Patel and Helen Peterson were supported by grants from The Royal College of Surgeons of England. The authors declare no conflict of interest. The authors declare that AI technologies were not used in the preparation of this work. Thirty-two participants with OFG, with and without CD, attending Glasgow Dental Hospital & School's Department of Oral Medicine were recruited into the study. Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.