Amphiregulin-producing γδ T cells are vital for safeguarding oral barrier immune homeostasis

Significance Loss of oral barrier homeostasis leads to the development of periodontitis, the most common chronic inflammatory condition of mankind. Therefore, it is important to better understand the immune mediators acting at this unique barrier to safeguard tissue integrity. Here we identify a vital role for γδ T cells in constraining pathological inflammation at the oral barrier, as the absence of γδ T cells resulted in enhanced pathology during periodontitis. We show that oral barrier γδ T cells produce the reparative cytokine Amphiregulin, administration of which rescued the elevated oral pathology of tcrδ−/− mice. Collectively, we identify a pathway controlling oral immunity mediated by barrier-resident γδ T cells, highlighting that these cells are crucial guards of oral barrier immune homeostasis.


Treatment protocols during Ligature-induced Periodontitis
For some periodontitis experiments mice were treated with 7ug/mouse Areg or PBS i.v. every other day starting from the day after ligature placement. For other experiments mice were placed on antibiotics (0.3mg/ml trimethoprim and 0.7mg/ml sulfamethoxazole) in the drinking water for 5 days prior to ligature placements and maintained on this regimen throughout the experiment. For other experiments mice were treated with 200ug/treatment of anti-TCRγδ (BioXCell) every 3 days starting from 3 days before induction of periodontitis.

Bone loss Measurements
Periodontal bone heights were assessed following defleshing and staining with Methylene blue. The distance between the Cemento-enamel junction and alveolar bone crest (CEJ-ABC distance) was measured at 6 predetermined sites and combined to give a total CEJ-ABC distance presented in mm. For ligature induced periodontitis final change in bone heights was determined by subtracting the CEJ-ABC for ligated molars from un-ligated molars of mice of the same genotype.

Oral microbiome evaluation via 16S rRNA gene sequencing and qPCR
For microbiome analyses, the murine oral cavity was sampled for 30s using sterile ultra-fine swabs. Serial dilutions of the swab extracts were plated on Luria broth (aerobic growth) and Wilkins-Chalgren (anaerobic growth) agar and colony-forming units counted. Bacterial DNA from the swabs was isolated using the DNeasy Powersoil kit (Qiagen). Total 16S rRNA copy numbers were determined using described primers(3). Species-specific primers for Aggregatibacter actinomycetemcomitans 16S were forward GGACGGGTGAGTAATGCTTG and reverse CCTTTACCCCACCAACTACC with an annealing temperature of 58 o C as previously described(4, 5).
For 16S rRNA gene sequencing amplicon libraries were generated using previously described primers that amplify the V4 region of the 16S gene(6). Primer sequences allow a secondary nested PCR process, which incorporates Illumina adapter sequences for sequencing of samples on Illumina Sequencing platforms.
Libraries were generated in triplicate before pooling. Initial processing and quality SI 3 assessment of the sequencing data was carried out at the Centre for Genomics, University of Liverpool, using an in-house pipeline. Sequences were classified using the QIIME script assign_taxonomy.py, using the RDP classifier (7)  permutations. Significantly different OTUs between wild-type and tcrδ -/microbiota were generated using analysis of compositions of microbiomes (ANCOM) with a false discovery rate < 0.05(10).

Real-time and conventional RT-PCR
Total RNA was obtained from gingival tissues using Trizol and cDNA synthesized using Superscript reverse transcription kit (Invitrogen/Life Technologies). To determine TCR Vγ usage, utilized primers were previously described(11). Products from PCR reactions were run on a 2% agarose gel and visualized with SYBR-safe (Invitrogen). Quantitative real-time PCR was done with SYBR green qPCR super mix (Invitrogen/Life Technologies) and normalized to hprt expression.

RNA-sequencing
At LC sciences, cDNAs were constructed from total RNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech/Takara). Libraries were prepared using Nextera XT DNA sample preparation kit (Illumina), pooled and sequenced on HiSeq X Ten