title: Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness.
abstract: BACKGROUND
  Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF).
  
  
  METHODS
  We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100μg/100μl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge.
  
  
  RESULTS
  Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1β, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100μg LPS). At 100μg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10μg and 30μg LPS).
  
  
  CONCLUSIONS
  Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa.
  
  
  CONCLUSIONS
  Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1β, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS.
  
  
  BACKGROUND
  Clinical Trials.gov NCT02284074.
authors.affiliation: Centre for Respiratory Infection (CRI), National Heart and Lung Institute (NHLI) at Imperial College, St. Mary's Hospital: Mint Wing, Entrance C, Paddington, London, W2 1NY, United Kingdom.
authors.forename: Jaideep
authors.initials: J
authors.lastname: Dhariwal
authors.affiliation: GlaxoSmithKline, Stevenage, SG1 2NY, United Kingdom.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Kitson
authors.affiliation: GlaxoSmithKline, Stevenage, SG1 2NY, United Kingdom.
authors.forename: Reema E
authors.initials: RE
authors.lastname: Jones
authors.affiliation: Centre for Respiratory Infection (CRI), National Heart and Lung Institute (NHLI) at Imperial College, St. Mary's Hospital: Mint Wing, Entrance C, Paddington, London, W2 1NY, United Kingdom.
authors.forename: Grant
authors.initials: G
authors.lastname: Nicholson
authors.affiliation: Centre for Respiratory Infection (CRI), National Heart and Lung Institute (NHLI) at Imperial College, St. Mary's Hospital: Mint Wing, Entrance C, Paddington, London, W2 1NY, United Kingdom.
authors.forename: Tanushree
authors.initials: T
authors.lastname: Tunstall
authors.affiliation: Centre for Respiratory Infection (CRI), National Heart and Lung Institute (NHLI) at Imperial College, St. Mary's Hospital: Mint Wing, Entrance C, Paddington, London, W2 1NY, United Kingdom.
authors.forename: Ross P
authors.initials: RP
authors.lastname: Walton
authors.affiliation: GlaxoSmithKline, Stockley Park West, Uxbridge, UB11 1BT, United Kingdom.
authors.forename: Grace
authors.initials: G
authors.lastname: Francombe
authors.affiliation: GlaxoSmithKline, Stockley Park West, Uxbridge, UB11 1BT, United Kingdom.
authors.forename: Jane
authors.initials: J
authors.lastname: Gilbert
authors.affiliation: Centre for Respiratory Infection (CRI), National Heart and Lung Institute (NHLI) at Imperial College, St. Mary's Hospital: Mint Wing, Entrance C, Paddington, London, W2 1NY, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Tan
authors.affiliation: GlaxoSmithKline, Stevenage, SG1 2NY, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Murdoch
authors.affiliation: Centre for Respiratory Infection (CRI), National Heart and Lung Institute (NHLI) at Imperial College, St. Mary's Hospital: Mint Wing, Entrance C, Paddington, London, W2 1NY, United Kingdom.
authors.forename: Onn Min
authors.initials: OM
authors.lastname: Kon
authors.affiliation: Centre for Respiratory Infection (CRI), National Heart and Lung Institute (NHLI) at Imperial College, St. Mary's Hospital: Mint Wing, Entrance C, Paddington, London, W2 1NY, United Kingdom.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Openshaw
authors.affiliation: Centre for Respiratory Infection (CRI), National Heart and Lung Institute (NHLI) at Imperial College, St. Mary's Hospital: Mint Wing, Entrance C, Paddington, London, W2 1NY, United Kingdom.
authors.forename: Trevor T
authors.initials: TT
authors.lastname: Hansel
mesh_terms: D000293:Adolescent; D000328:Adult; D018925:Chemokines / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D007113:Immunity, Innate; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D007378:Interleukins / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D009297:Nasal Mucosa / Q000276:immunology*; D059085:Nasal Sprays; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D018925:Chemokines; D007378:Interleukins; D008070:Lipopolysaccharides; D059085:Nasal Sprays; D012333:RNA, Messenger; D018799:Intercellular Adhesion Molecule-1