Dysregulated inflammation

There is evidence that inflammation exists in cystic fibrosis even before any signs of infection or lung disease [186]. Scientists originally thought of excessive

inflammation in cystic fibrosis as a consequence of chronic and repetitive

infections. However, in a review of primary literature and clinical studies by Rao et al., the authors propose that the cause of inflammation in cystic fibrosis extends far beyond infection [186]. Rao presents inflammation as an important pathological component of cystic fibrosis. Inflammation in cystic fibrosis is exaggerated, results in lung injury, persists after pathogen clearance, and is present even before the first pulmonary infection [186]. Human sputum and bronchoalveolar lavage samples reflect an established pro-inflammatory state in cystic fibrosis lungs independent of the presence of an active infection. Several clinical studies show at least 10 fold more neutrophils in uninfected cystic fibrosis lungs compared to normal humans with or without lower respiratory tract

infections. Moreover, there is an elevated presence of pro-inflammatory cytokines including TNF-α, IL-6, IL-8, and LTB-4 in un-infected cystic fibrosis lungs as well [173, 227, 228]. There is an increased number of macrophages in un-infected cystic fibrosis lungs. These macrophages are believed to have a more profound ability to release mediators and to induce an inflammatory state compared to macrophages from non-cystic fibrosis lungs [186]. Studies show that in the absence of any infection, macrophages in cystic fibrosis lungs are polarized towards a pro-inflammatory M1 phenotype [173, 184]. Naïve human

macrophages from cystic fibrosis patients stimulated with LPS show significantly higher stimulation of the NF-κB and MAPK signaling pathways, higher levels of pro-inflammatory cytokine release, and a decreased ability to produce the anti- inflammatory cytokine IL-10 [134, 172-174, 181, 229-232]. Similarly, T

lymphocytes from cystic fibrosis lungs have an increased pro-inflammatory potential in culture when compared to T lymphocytes from healthy volunteers [231].

In addition, cells that makeup tracheal and epithelial tissues are also capable of producing dysregulated inflammation. In a study by Tirouvanziam et al., naïve tissues from newborns with cystic fibrosis were grafted into immunocompromised mice to investigate the inflammatory potential of cystic fibrosis lung tissues [233]. When fetal tracheal tissues were transplanted into severe combined

immunodeficient mice, there was an eight-fold greater increase in IL-8 secretion as compared to mice which received non-cystic fibrosis tracheal grafts. When these mice were infected with PA, there was a rapid increase in leukocyte migration and spread of PA into the lamina propria within 3 hours in mice which received grafts from diseased neonates [233]. In mice with grafts from normal neonates, the inflammatory response occurred later and at a slower rate after 6 hours of infection. The inflammation in mice grafted with tissues from cystic fibrosis neonates was associated with severe exfoliation of the lung epithelium along with damage in the mucosa which was not otherwise observed in mice with normal grafts. These pulmonary changes are believed to create a niche for PA growth and replication which is thought to resemble the chronic infection in humans [233]. This study supports the idea that CFTR mutations predispose to an inflammatory state in cystic fibrosis patients independent of infection. And that in the presence of an infection, the already primed and inflamed lungs of cystic fibrosis patients respond in an exaggerated manner to invading pathogens.

Additionally, Khan et al. analyzed lavage fluids from the lungs of 16 human newborns with cystic fibrosis whose cultures were negative for any possible

infection (bacterial, viral, or fungal) [234]. In comparison with normal infants, the lavage fluids of the diseased population had significantly higher levels of

neutrophils, neutrophil elastases, anti-protease inhibitors, and IL-8. Collectively, this study by Khan, in addition to many similar studies, confirm the inverse relation between neutrophil counts and lung function in cystic fibrosis which is again independent of the infection state [185, 234-240].

Cystic fibrosis patients also have a reduction in the typical anti-inflammatory set point of the pulmonary environment, represented by a decrease in the

concentrations of IL-10, nitric oxide, and lipoxin-A4. IL-10 acts to stop the

inflammatory response by suppressing the pro-inflammatory transcription factors, shutting down the synthesis of inflammatory mediators, and inducing the

regulated-cell death of neutrophils. The reduction in nitric oxide prevents

bacterial killing and relaxation of the airways. And finally, lipoxin-A4 is essential to limit the neutrophil-mediated inflammatory response [228, 241-250]. These changes exist along with the pre-described pro-inflammatory state. Thus, there is a disrupted balance of the inflammatory state in patients with cystic fibrosis.

Another factor that clearly relates to the inflammatory picture of cystic fibrosis which is independent of infection is the presence of reactive airway (asthma-like) signs and symptoms. In fact, smooth muscle cells express CFTR receptors. When the latter are mutated they are believed to impair the smooth muscle contractility due to impairment of ion flux [248]. Additionally, smooth muscle cells in children and adults with cystic fibrosis were observed to be affected with

hyperplasia and hypertrophy which impairs their contraction [12, 249]. The airway hyper-responsiveness and the asthma-like phenotype are also attributed to inflammatory mediators which induce smooth muscle contractions. In response to these inflammatory cytokines, smooth muscle cells can release IL-8 which further contributes to the inflammatory process. Additionally, the excessive release of IL-4 and IL-13 by T lymphocytes with mutated CFTR (Th2s) drives airway hyper-responsiveness directly and through the increased production of

IgE [68]. This presents an inflammatory pulmonary disease overlapping with cystic fibrosis pathology and independent of infection state [248-250].

Collectively, the evidence presented distinguishes the inflammatory process as a unique pathological element of cystic fibrosis. This is supported by the facts that inflammation is (1) present prior to any infection; (2) is excessive compared to the number of infective microbes; (3) contributes to lung injury and damage; (4) and is innately skewed towards a pro-inflammatory response. This further emphasizes that dysregulated inflammation is a key factor in cystic fibrosis pathology to be closely considered for therapeutic targeting and treatment.