Introduction The most common pathogen in the lower airway of patients with COPD is Haemophilus influenzae. H. influenzae has been shown to be linked to inflammation and increased inflammation. Emerging evidence shows that pathogens can exist as either cell-associated or free-living (non-cell associated). We investigated whether detectable free-living H. influenzae correlates with pulmonary inflammation.

Methods Cell-free sputum supernatants samples from 29 COPD patients (24 men), with a mean (range) age of 71 (45 to 88) years were analysed. All samples were collected at stable state and bacterial DNA was extracted, using a commercial assay and then quantified using real time-PCR utilising taqman hydrolysis probes. The omp P6 gene from H. influenzae was inserted into a positive cloning vector and transformed to generate plasmids. These plasmids were used as standards within the qPCR, allowing the accurate detection of very small levels of H. influenzae within the samples. Cytokines were measured using the meso-scale multi-array platform within the same sample set.

Results Free Living H. influenzae was detected in 15/29 (52%) of cell-free samples, with a bacterial load of (geometric mean (95% CI)) of 1.23 × 10⁶ gene copies/ml (2.63 × 10⁵ to 5.75 × 10⁶). Correlations were seen between free-living H. influenzae and Interleukin-1 Beta (IL1-β) (r = 0.47, p = 0.01), MMP8 (p = 0.04, r = 0.38), CCL3 (p = 0.002 =, r = 0.57), CCL13 (p = 0.02, r = 0.54), CCL26 (p = 0.03, r = -0.31), CCL4 (p = 0.04, r = 0.38). No significant correlations were seen between free-living H. influenzae and IL8 (p = 0.11, r = -0.30), IL10 (p = 0.10, r = -0.36) or TNF-alpha (p = 0.61, r = -0.12).

Conclusion Free-living H. influenzae is associated with increased pro-inflammatory mediators in the airway. Whether this is related to the pathogenesis of COPD needs to be further investigated.