A PCR-based method for quantifying neutrophils in human nasal secretions

Abstract

Neutrophil recruitment to the nasopharynx (NP) is a central event in resolution of NP-initiated microbial infections. A vigorous neutrophil response in infected tissues is also associated with the outcome of adverse tissue pathology. Therefore, differences in infection-induced tissue neutrophil numbers may correlate with pathogenesis events. Existing methods of quantifiying neutrophils require evaluation of NP samples within hours of procurement as flow cytometry based cell quantification methods require live neutrophil cells. Therefore, we developed a novel RT-PCR method that could reliably quantify neutrophil counts in frozen NP wash samples. mRNA transcripts of the genes encoding CD16, CD18, and CD62L were identified as neutrophil-specific in NP samples and not significantly variable in response to stimulation by heat killed bacteria, and can be used to derive an accurate assessment of neutrophil content in a sample even in the presence of epithelial cells. Using flow cytometry as a comparator, the method was validated in human NP wash samples. We conclude that this PCR-based method should prove useful for providing a quantitative estimate of neutrophil recruitment to the NP during infection and pathogenesis.

Streptococcus pneumoniaeburden and nasopharyngeal inflammation during acute otitis media

“…The transcription of inflammatory genes followed similar patterns, with IL-8, TNF-a and SOD2 correlating positively with Spn bacterial burden, whereas transcription of CXCL1, a neutrophil chemoattractant, was significantly elevated during AOM. Since neutrophils are the predominant cell type present in nasal wash samples, 19 the bulk of gene transcription can be attributed to neutrophils. These measurements thus constitute a survey of the activation status, as well as the recruitment of neutrophils.…”

“…RNA was extracted from nasal wash cell pellets in TRI Reagent (Sigma) by chloroform extraction and alcohol precipitation as previously described. 19 RNA samples were resuspended in 30 µl UltraPure nuclease-free water (ThermoFisher, Waltham, MA, USA). cDNA was prepared using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions.…”

“…Neutrophil recruitment was measured according to the mean CT value of CD16, CD18 and CD62L as previously described. 19 Samples were classified as Spn-positive or Spn-negative based on microbial culture. Following this, Spn was quantified by PCR.…”

“…Since neutrophils constitute the predominant cell type present in NP samples, 19 measurements of RNA transcription in the cell pellets can be attributed largely to neutrophils. In contrast, protein secretion in the supernatants of these samples could come from any cell present in the NP at the time of sample collection, especially epithelial cells.…”

“…We recently described a novel method for measuring neutrophil content in nasal secretions by PCR. 19 By applying this method to pediatric NP samples collected during health, viral URI without progression to AOM and viral URI with progression to AOM during colonization by Spn, we were able to measure and compare the influence of NP bacterial burden and physiological state on signals of local NP inflammation, including neutrophil recruitment and transcription and secretion of various innate immune cytokines.…”