Paediatric obstructive sleep apnoea syndrome (OSAS) is associated with tonsil colonisation by Streptococcus pyogenes

The involvement of pathogenic bacteria in obstructive sleep apnoea syndrome (OSAS) has yet to be elucidated. We investigated the possible role of group A streptococcus (GAS) in OSAS pathogenesis. In 40 tonsillectomized patients affected by OSAS and 80 healthy controls, significant (p < 0.0001) association of GAS with paediatric OSAS was found. Supernatant from streptolysin O (SLO)-producing GAS induced production of cysteinyl leukotrienes (CysLTs) in tonsil mononuclear cells (TMCs). CysLTs-treated TMCs showed significant (p < 0.05) proliferation of CD4+ T, CD19+ and CD19+CD27+CD38+ B lymphocytes. We discovered a SLO-dependent activation of CysLTs production through a pathway involving TOLL-like receptor 4 (TLR4), TIR-domain-containing adapter-inducing interferon-β (TRIF), Myeloid differentiation primary response gene 88 (MyD88), and p38 MAP Kinase. In conclusion, we hypothesise that GAS may contribute to paediatric tonsillar hyperplasia through CysLTs production induced by SLO, and this might explain its association with OSAS.


Supplementary Methods S3
Tonsil tissue preservation and purification of TMCs. From each specimen, two portions were embedded in OCT compound, plunged in dry ice-cooled isopentane for 1 min and kept frozen at -80°C; two portions were fixed in 4% buffered formaldehyde and paraffin-embedded (FFPE). The remnant portions were single-cell mechanically dissociated, mononuclear cells were purified by Ficoll-Paque Plus (GE Healthcare) following the manufacturer's instructions, and were then treated with RBC (red blood cell) Lysis buffer 1X for 2 min on ice and washed. Cells were counted and resuspended in fetal bovine serum (FBS, Sigma-Aldrich) with 10% DMSO, pre-frozen at -80°C with a Nalgene Cryo freezing container, and stored at -150°C.
Antibody production. Recombinant SLO protein was purified as previously described 1  Overnight cultures were identified on the basis of macro-and microscopic morphology and identity was confirmed through specific biochemical tests, according to standard methods 2 . Glycerol stocks were made for each bacterial strain.
Bacterial mutant strains, media and growth conditions. The Streptococcus pyogenes M1 strain 3348 was kindly provided by Istituto Superiore di Sanità (Rome, Italy); its isogenic mutants 3348∆slo, and 3348slodm were provided by Cristina Faralla and Robert Janulczyk (GSK Vaccines, Siena, Italy). The complemented strain 3348∆slo(pAM_slo) was obtained following a previously described method 3, 4 by electro transformation of electrocompetent 3348∆slo strain cells. Briefly, the following primers were used for the slo gene insert: and incubated on ice for 5 min. Bacteria were then grown with 0.9 mL of THYE with 250 mmol/L of sucrose for 2 h at 37°C with 5% CO 2 , centrifuged for 10 min at RT at 900 x g and grown on TSA plates with 5% sheep blood and 10 µg/mL chloramphenicol overnight at 37°C with 5% CO 2. Clones were screened by PCR. All S.
pyogenes isolates were grown at 37°C with 5% CO 2 in Todd-Hewitt medium supplemented with 0.5% yeast extract (THY, Difco), or on Tryptic-soy agar (TSA) plates with 5% sheep blood, except for 3348∆slo(pAM_slo) as stated above. Western blot assay. We performed western blot with mouse polyclonal anti-SLO on cell-free S. pyogenes supernatants harvested at A 600 = 0.2, 0.4, and 0.8 from all S. pyogenes strains isolated in OSAS tonsils to verify SLO in vitro production; the supernatants were concentrated by trichloroacetic acid (TCA) protein precipitation 5 , and the samples obtained were run as previously described 6 .

Purification of human peripheral blood mononuclear cells (PBMCs). PBMCs from healthy volunteers from the
San Giuseppe General Hospital (Empoli, Italy) were purified as described with minor adaptations 7 . Briefly, heparinised blood was mixed with a double volume of PBS and separated by Ficoll-paque, to obtain mononuclear cells which were washed in HBSS and incubated on ice for 2 min with 1X red blood cell (RBC) lysis buffer. Cells were washed, filtered with 70 µm-cell strainers and counted. The cysteinyl leukotriene production assay was performed on PBMCs as described above.