Abstract
Cystic Fibrosis (CF), caused by mutations affecting the CFTR gene, is characterised by viscid secretions in multiple organ systems. CF airways contain thick mucus, creating a gradient of hypoxia, which promotes the establishment of polymicrobial infection. Such inflammation predisposes to further infection, a self-perpetuating cycle in mediated by NF-κB. Anaerobic Gram-negative Prevotella spp. are found in sputum from healthy volunteers and CF patients and in CF lungs correlate with reduced levels of inflammation. Prevotella histicola (P. histicola) can suppress murine lung inflammation, however, no studies have examined the role of P. histicola in modulating infection and inflammation in the CF airways. We investigated innate immune signalling and NF-kB activation in CF epithelial cells CFBE41o- in response to clinical stains of P. histicola and Pseudomonas aeruginosa (P. aeruginosa). Toll-Like Receptor (TLR) expressing HEK-293 cells and siRNA assays for TLRs and IKKα were used to confirm signalling pathways. We show that P. histicola infection activated the alternative NF-kB signalling pathway in CF bronchial epithelial cells inducing HIF-1α protein. TLR5 signalling was responsible for the induction of the alternative NF-kB pathway through phosphorylation of IKKα. The induction of transcription factor HIF-1α was inversely associated with the induction of the alternative NF-kB pathway and knockdown of IKKα partially restored canonical NF-kB activation in response to P. histicola. This study demonstrates that different bacterial species in the respiratory microbiome can contribute differently to inflammation, either by activating inflammatory cascades (P. aeruginosa) or by muting the inflammatory response by modulating similar or related pathways (P. histicola). Further work is required to assess the complex interactions of the lung microbiome in response to mixed bacterial infections and their effects in people with CF.
Highlights
Cystic Fibrosis (CF), is an autosomal recessive life-limiting disease, characterised by viscid secretions in multiple organ systems due to mutations affecting the CFTR gene, which codes for a cAMP-regulated chloride channel, found on epithelial surfaces, including the airways, pancreas, and intestine [1]
To test the hypothesis that infection with P. histicola in CF airway epithelial cells may contribute to anti-inflammatory signalling through activation of the alternative NF-κB pathway we compared NF-κB signalling in CFBE41o- cells infected with P. histicola and P. aeruginosa
CFBE41o- cells infected with P. histicola did not demonstrate elevated levels of phosphorylated IκBα at any time point and there was no change in nuclear p50 or p65 in response to infection expression Beta-2-microglobulin was used as the endogenously expressed house-keeping gene for all experiments and 2-ΔΔCT was calculated for F: IL-6 mRNA, G: IL-8 mRNA and H: CXCL-1. 2-Way ANOVA with Bonferroni post-test, n = 3–5, p
Summary
Cystic Fibrosis (CF), is an autosomal recessive life-limiting disease, characterised by viscid secretions in multiple organ systems due to mutations affecting the CFTR gene, which codes for a cAMP-regulated chloride channel, found on epithelial surfaces, including the airways, pancreas, and intestine [1]. The contribution of bacterial species such as Pseudomonas aeruginosa (P. aeruginosa), Burkholderia cepacia complex, Haemophilus influenza and Staphylococcus aureus (S. aureus) to inflammation in the CF lung have been extensively studied [3,4,5], a broader picture of the microenvironment in the CF lung has recently become apparent with advances in high fidelity generation sequencing (NGS) [1]. This method has identified a diverse microbiome in the lungs of people with CF, including the presence of the Gramnegative anaerobic genus Prevotella spp. We recently showed that a strain of P. nigrescens isolated from a person with CF induced a lower proinflammatory cytokine expression in CF bronchial epithelial cells than P. aeruginosa, suggesting that the presence of certain Prevotella species in CF lungs may lower the inflammatory response and could be beneficial to the host [9]
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