Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation

Abstract

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF.

Figure 1

Internalization and clearance of P. aeruginosa-O1 (PAO1) in bone marrow-derived macrophages (BMDMs) from Wild-type (WT) and Cftr^{F508del/F508del} mice. (a–g) WT or Cftr^{F508del/F508del} BMDMs infected with PAO1 for 10 min followed by 10 min (a–c) or 3 h (d–f) of culture in the presence of gentamicin. (a, d) Number of colony-forming units (CFU) of PAO1 in WT (n=10) and Cftr^{F508del/F508del} cells (n=13) BMDMs (**P<0.01, Student's t-test) and (b, e) q-PCR transcript level of total PAO1 DNA in WT (n=3) and Cftr^{F508del/F508del} (n=3) BMDMs related to proC housekeeping gene (***P<0.001, Student's t-test). (c, f) Immunofluorescence detection of viable intracellular bacteria. (g) Clearance of bacteria expressed as percentage of living bacteria after 3 h of culture respect to the total amount of internalized bacteria in WT and Cftr^{F508del/F508del} BMDMs (**P<0.01, Student's t-test). (h) WT or Cftr^{F508del/F508del} BMDMs infected with PAO1-GFP at different time points. Enumeration of intracellular PAO1-GFP by immufluorescence in WT and Cftr^{F508del/F508del} BMDMs. Means±S.D. of three independent measurements for each group of experiments. Asterisks indicate significant differences

Figure 2

Effects of cysteamine on internalization and clearance of P. aeruginosa-O1 (PAO1) in bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cftr^{F508del/F508del} mice. BMDMs from Cftr^{F508del/F508del} (a–c) or WT (d–f) mice treated with 250 μM cysteamine (n=10) or vehicle (n=10) for 10 min before infection followed by 10 min (a–b, d–e) or 3 h (c, f) of gentamicin culture. P. aeruginosa internalization expressed as number of CFUs of PAO1 (a, d) or enumerated by immunofluosescence (b, e) after 10 min of gentamicin culture (***P<0.001 versus vehicle; Student's t-test); (c, f) clearance of P. aeruginosa expressed as percentage of living bacteria after 3 h of culture respect to the total amount of internalized bacteria (***P<0.001 versus treatment with vehicle; Student's t-test). Means±S.D. of three independent measurements for each group of experiments. Asterisks indicate significant differences

Figure 3

Effects of cysteamine on autophagy markers in bone marrow-derived macrophages (BMDMs). Wild-type (WT) or Cftr^{F508del/F508del} BMDMs treated for 18 h with cysteamine or vehicle. Representative immunoblots with anti-BECN1 (a) and anti-SQSTM1/p62 (b) (top) and densitometric measurements (bottom) in BMDMs from one mice per group. Mean±S.D. of three independent measurements (**P<0.01 versus vehicle-treated mice; ANOVA). Asterisks indicate significant differences

Figure 4

Cysteamine does not restore defective killing of P. aeruginosa by bone marrow-derived macrophages (BMDMs) from Cftr^{F508del/F508del}/Becn1^+/− mice. BMDMs from wild-type (WT) (n=10) (a–c), Becn1 haplo-insufficient (Becn1^+/−) (n=3) (a–c), Cftr^{F508del/F508del} (n=10) and Cftr^{F508del/F508del}/Becn1^+/− (n=3) (d–f), mice infected with P. aeruginosa-O1 (PAO1) for 10 min followed by 10 min (a, d), or 3 h (b–c and e–f) of culture with gentamicin. Number of CFUs of PAO1 after 10 min (a, d) or 3 h (b, e); clearance of PA expressed as percentage of living bacteria after 3 h of culture respect to the total amount of internalized bacteria (c, f). Effects of the pre-treatment with cysteamine in BMDMs from Becn1^+/− (a–c) and Cftr^{F508del/F508del}/Becn1^+/− (d–f) mice. Means±S.D. of three independent measurements for each group of experiments. *P<0.05, **P<0.01, Student's t-test

Figure 5

Cysteamine reverts defective killing of P. aeruginosa through rescuing CFTR. Bone marrow-derived macrophages (BMDMs) from Cftr^{F508del/F508del} (n=3) (a–c), Cftr^−/− (n=3) (a–c), and Cftr^F508del/− (n=3) (d–f), and infected with P. aeruginosa-O1 (PAO1) for 10 min followed by 10 min (a, d) or 3 h (b–c and e–f) of culture in the presence of gentamicin. (a–b, d–e) number of CFU of PAO1; (c, f) clearance of bacteria expressed as percentage of living bacteria after 3 h of culture respect to the total amount of internalized bacteria. BMDMs from Cftr^−/− (n=3) (a–c) and Cftr^F508del/− (d–f) mice treated with 250 μM cysteamine or vehicle in the presence or absence of 3-methyl-adenine (3-MA) (3 mM) and/or CFTR_inh172 (20 μM) (d–f) and then infected with PAO1 for 10 min followed by 10 min (a, d) or 3 h (b–c and e–f) of gentamicin culture. *P<0.05, **P<0.01, Student's t-test. Means±S.D. of three independent measurements for each group of experiments. Asterisks indicate signifcant differences

Figure 6

Cysteamine controls P. aeruginosa-induced increase of TNFα and IL-1β levels in bone marrow-derived macrophages (BMDMs) from Cftr^{F508del/F508del} mice. BMDMs from Cftr^{F508del/F508del} (n=3) (a and b) and Cftr^{F508del/F508del}/Becn1^+/− (n=3) (c and d) mice treated with 250 μM cysteamine in the presence or absence of 3-methyl-adenine (3-MA) (3 mM) (a and b) and then infected with P. aeruginosa-O1 (PAO1) for 10 min followed by culture with gentamicin up to 24 h. Quantitation of TNFα (a, c) and IL-1β (b, d) levels in culture supernatants by ELISA. ***P<0.001, °°°P<0.001, Student's t-test. Means±S.D. of three independent measurements for each group of experiments. Asterisks indicate signifcant differences