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.

Key messages: 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.

Trial registration: Clinical Trials.gov NCT02284074.

Conflict of interest statement

Competing Interests: This study was partly funded by Glaxo SmithKline, the employer of JK, REJ, GF, JG and RM. In the past 5 years TTH has been Principal Investigator for respiratory clinical studies carried out at Imperial College, that have been sponsored by Pharmaceutical Companies: Novartis, Merck, GlaxoSmithKline (GSK) and Dainippon-Sumitomo. TTH has received lecture fees from Novartis, Merck, GSK and Dainippon-Sumitomo. TTH is involved in setting up a spin-off company with Imperial Innovations called Mucosal Diagnostics, and has received support to attend Respiratory Conferences from Boehringer Ingelheim. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Fig 1. Consort Flow Chart.

Fig 2. Nasal lipopolysaccharide challenge study design and procedures.

14 subjects completed the entire protocol (per protocol), while 1 subject only received nasal challenges at placebo and LPS. TNSS–total nasal symptom score. AE–adverse events

Fig 3. Median levels of chemokines and cytokines (pg/ml) in nasal mucosal lining fluid (MLF) are shown after nasal spray challenge with 4 different doses of LPS and a placebo (see key below).

Lower limits of detection (LLOD) for cytokines and chemokines were as follows: IL-1β (5.0pg/ml), IL-6 (5.0pg/ml), CXCL8/IL-8 (10.0pg/ml), CCL3/MIP-1α (5.0pg/ml), CCL2/MCP-1 (25.0pg/ml), IL-10 (5.0pg/ml). Time -0.5h refers to nasosorption performed 30 min prior to topical nasal challenge, and this varies considerably between individuals due to variations in individual’s microbial flora and mucosal immune responses. Nasal lavage was performed after nasosorption at -0.5h, but prior to nasal challenge, in order to partially wash the nose free of baseline inflammatory microbes and mediators, but causes detectable levels of inflammatory mediators at 0.5h to decrease markedly from -0.5h. There is a tendency for nasosorption levels of mediators to gradually increase from 0.5h to 10h in the placebo arm, but levels of inflammatory cytokines and chemokines after LPS are generally higher.

Fig 4. Levels of interleukins (IL) in nasosorption eluates presented as area under the curve (AUC) for times 0.5-10h in relation to topical nasal challenge (n = 14 or 15).

AUC is derived from from 0.5 since the baseline sample at -0.5h is pre-nasal lavage and is highly variable due to difference in individuals in terms of nasal microbes and inflammatory mediators. A) IL-1β as medians with quartiles; B) IL-6 as medians with quartiles; C) IL-1β AUC data for individual subjects; D) IL- AUC data for individual subjects. Note the idiosyncratic large responses of subject 2009 after nasal challenge with 10μg LPS, and subject 3101 after nasal challenge with 30μg LPS, these responses being too large to be presented on the y axis.

Fig 5. Idiosyncratic large nasal responses to lipopolysaccharide (LPS) on 2 occasions.

Raw data for levels of chemokines and cytokines (pg/ml) in nasal mucosal lining fluid (MLF). Data is presented for individual subjects: A. Subject 2009 after nasal challenge with 10μg LPS. B. Subject 3101 after nasal challenge with 30μg LPS. Note that baseline levels of cytokines and chemokines at -0.5h are markedly raised, and come down to only a small extent after nasal lavage at 0.5h. It is suggested that these individuals may have had a non-symptomatic alteration in their nasal microbial flora prior to LPS challenge on these opccasions: possibly the individuals had a subclinical viral infection or bacterial colonisation.

Fig 6. Nasal lavage leukocyte counts (medians expressed as cells/ml) following nasal LPS challenge.

Cells were counted in a modified Neubauer chamber, and a leukocyte differential performed on a stained cytospin observed under a light microscope.

Fig 7. ICAM-1 mRNA abundance within nasal curettage samples after nasal LPS challenge, Data is shown as means with 95% confidence intervals.

Relative abundance of expression with Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) using normalized data and (glyceraldehyde-3-phosphate dehydrogenase) (GAPDH) as a housekeeper gene (see Methods).