Efficacy of melatonin treatment in a cystic fibrosis mouse model of airway infection

Abstract

Approaches to mitigate the severity of infections and of immune responses are still needed for the treatment of cystic fibrosis (CF) even with the success of highly effective modulator therapies. Previous studies identified reduced levels of melatonin in a CF mouse model related to circadian rhythm dysregulation. Melatonin is known to have immunomodulatory properties and it was hypothesized that treatment with melatonin would improve responses to bacterial infection in CF mice. Data demonstrate that CF mice (G542X/G542X) treated with melatonin (10 µg/mL) in drinking water for 10 weeks had improved responses to airway infection with a clinical isolate of Pseudomonas aeruginosa. Melatonin-treated mice exhibited improved bacterial clearance, reduced inflammatory markers. Mice treated in drinking water for 1 week had improved bacterial clearance but no improvement in inflammation. Wild type (WT) control mice showed no response to melatonin treatment suggesting melatonin is eliciting a CF-specific response in this model. The efficacy of direct melatonin (1 µM) treatment to the airways was also tested and found to be ineffective. In conclusion, long-term systemic treatment with melatonin is an effective therapy in a CF mouse model that normalizes the response to airway infection to a WT pattern.

Fig. 1

Changes in animal weight post-infection. Animals were weighed immediately prior to infection and daily until harvest. Change was calculated as a percentage of initial weight. (A) Comparison of weight retention between melatonin-treated and control CF mice. Melatonin-treated animals had a statistically significant improvement over control. (B) Comparison of weight retention between melatonin-treated and control WT mice. Significance determined by t-test at each time point: *p < 0.05.

Fig. 2

Airway clearance of P. aeruginosa post-infection. Colony-forming unit (CFU) counts per mL recovered from. (A) BAL fluid, (B) lung homogenates, and (C) total (BAL + lung homogenate) of control and post-weaning chronic melatonin-treated WT and CF mice. Significance determined by t-test, *p < 0.05, **p < 0.01, ***p, 0.001.

Fig. 3

Immune cell composition post-infection. BAL fluid was used to determine cell differentials (Giesma/Miller). (A) Comparison of % neutrophils in BAL between control and post-weaning chronic melatonin-treated WT and CF mice. Melatonin treated mice had significantly lower neutrophil counts than controls. (B) Comparison of % monocytes in BAL between control and melatonin-treated WT and CF mice. Melatonin mice had significantly higher monocytes counts than controls. Significance determined by t-test, *p < 0.05, **p < 0.01, ***p, 0.001.

Fig. 4

Pro-inflammatory cytokine levels post-infection. Cytokines were measured from BAL and lung homogenate supernatant using Luminex. Concentration (pg/mL) of CXCL1 in BAL (A) and homogenate (B), TNF-alpha in BAL (C) and homogenate (D), and CXCL2 in BAL (E) and homogenate (F) of control and post-weaning chronic melatonin-treated CF mice. Significance determined by t-test; *p < 0.05.

Fig. 5

Efficacy of 1-week melatonin treatment on responses to infection. (A) Changes in animal weight post-infection. Animals were weighed immediately prior to infection and daily until harvest. Change was calculated as a percentage of initial weight. (B) Airway clearance of P. aeruginosa post-infection. Colony-forming unit (CFU) counts per mL recovered from. BAL fluid, lung homogenates (HOM), and TOTAL (BAL + lung homogenate) of control and post-weaning chronic melatonin-treated WT and CF mice. (C) Immune cell composition post-infection. BAL fluid was used to determine cell differentials of % neutrophils and % monocytes in BAL between control and 1-week melatonin-treated WT and CF mice. Significance determined by t-test, *p < 0.05.

Fig. 6

Pro-inflammatory cytokine levels post-infection. Cytokines were measured from BAL and lung homogenate supernatant using Luminex. Concentration (pg/mL) of CXCL1 in BAL (A) and homogenate (B), TNF-alpha in BAL (C) and homogenate (D), and CXCL2 in BAL (E) and homogenate (F) of control and 1-weekmelatonin-treated CF mice.

Fig. 7

Airway clearance of P. aeruginosa post-infection. Colony-forming unit (CFU) counts per mL recovered from. (A) BAL fluid, (B) lung homogenates, and (C) total (BAL + lung homogenate) of control and acute intratracheal melatonin-treated WT and CF mice. Significance determined by t-test, **p < 0.01.

Fig. 8

Changes in weight post-infection in animals treated with intratracheal melatonin. Animals were weighed immediately prior to infection and daily until harvest. Change was calculated as a percentage of initial weight. (A) Comparison of weight retention between melatonin-treated and control CF mice. (C,D) Immune cell composition with intratracheal melatonin application. BAL fluid was used to determine (C) % neutrophils and (D) % monocytes between control and intratracheal melatonin-treated CF mice.