Cysteamine-mediated clearance of antibiotic-resistant pathogens in human cystic fibrosis macrophages

Abstract

Members of the Burkholderia cepacia complex are virulent, multi-drug resistant pathogens that survive and replicate intracellularly in patients with cystic fibrosis (CF). We have discovered that B. cenocepacia cannot be cleared from CF macrophages due to defective autophagy, causing continued systemic inflammation and infection. Defective autophagy in CF is mediated through constitutive reactive oxygen species (ROS) activation of transglutaminase-2 (TG2), which causes the sequestration (accumulation) of essential autophagy initiating proteins. Cysteamine is a TG2 inhibitor and proteostasis regulator with the potential to restore autophagy. Therefore, we sought to examine the impact of cysteamine on CF macrophage autophagy and bacterial killing. Human peripheral blood monocyte-derived macrophages (MDMs) and alveolar macrophages were isolated from CF and non-CF donors. Macrophages were infected with clinical isolates of relevant CF pathogens. Cysteamine caused direct bacterial growth killing of live B. cenocepacia, B. multivorans, P. aeruginosa and MRSA in the absence of cells. Additionally, B. cenocepacia, B. multivorans, and P. aeruginosa invasion were significantly decreased in CF MDMs treated with cysteamine. Finally, cysteamine decreased TG2, p62, and beclin-1 accumulation in CF, leading to increased Burkholderia uptake into autophagosomes, increased macrophage CFTR expression, and decreased ROS and IL-1β production. Cysteamine has direct anti-bacterial growth killing and improves human CF macrophage autophagy resulting in increased macrophage-mediated bacterial clearance, decreased inflammation, and reduced constitutive ROS production. Thus, cysteamine may be an effective adjunct to antibiotic regimens in CF.

Fig 1. Cysteamine increases bacterial killing.

A) Summed end-point analysis of 24h bacterial killing assay of cysteamine against multi-drug resistant B. cenocepacia (Bc), P. aeruginosa (Pa), Methicillin-resistant Staphylococcus aureus (MRSA), and B. multivorans (Bm) in media devoid of human cells. NT = media alone, 3h = cysteamine added 3h after the start of culture, n = 3. B) Colony-forming units (CFU) for bacteria during 1A conditions, n = 4. C) Summed end-point analysis of 24h direct bacterial killing assays of cysteamine against enteric pathogens in media: Escherichia coli (E. coli), Salmonella typhimurium (S. typhimurium), Citrobacter rodentium (C. rodentium), Porphyromonas gingivalis (P. gingivalis), Bifidobacterium animalis (B. animalis), and Lactobacillus reuteri (L. reuteri), n = 3. “*” denotes a p value < 0.05, “**” denotes a p value < 0.01, and “***” denotes a p value < 0.001 by ANOVA for 1A and t-test for 1B, 1C. Means and standard deviation reported. D) Colony-forming units (CFU) for bacteria during 1C conditions, n = 4.

Fig 2. Cysteamine decreases bacterial loads in CF macrophages.

A) Colony forming unit (CFU) assay for human CF MDMs infected with B. cenocepacia (Bc), B. multivorans (Bm), P. aeruginosa (Pa), and MRSA for 24h with or without cysteamine, n = 3. Total and intracellular (IC) only counts are displayed. “*” denotes a p value < 0.05. “**” denotes a p value < 0.01. B) Macrophage apoptosis assay for human CF and non-CF MDMs uninfected (NT), during B. cenocepacia infection (Bc), exposure to cysteamine, and during B. cenocepacia infection and combined cysteamine exposure. Results are presented as % non-viable macrophage apoptosis as measured by flow cytometry, n = 4.

Fig 3. Cysteamine decreases TG2 and increases CFTR expression in CF.

A) Representative Western blot of TG2 and CFTR expression in human CF MDMs at baseline (NT) and after 1 or 24h infection with B. cenocepacia (Bc) with or without cysteamine. CFTR expression is representative of both class 1 and class II mutations. B) Densitometric analysis of ≥ 3 Western blots per TG2 and CFTR conditions in 3A, normalized to the loading control calreticulin. “*” denotes a p value < 0.05, “**” denotes a p value < 0.01 for 2-sample comparisons. For CFTR expression, 3 patients with a second class I mutation and 3 with a second class II mutation are presented as aggregate data. C) Representative Western blot of TG2 expression in human non-CF and CF MDMs at baseline (NT) and after 24h infection with B. cenocepacia (Bc) with or without cysteamine (cyst). D) Confocal microscopy images of CFTR expression in non-CF and CF human MDMs at baseline (NT) and infected with B. cenocepacia (Bc). CF MDMs were additionally treated with cysteamine during Bc infection (Cysteamine). The macrophage nucleus is stained blue with DAPI, B. cenocepacia (Bc) is shown in green, and CFTR expression in red.

Fig 4. Cysteamine increases CF macrophage autophagy.

A) Western blot of LC3, beclin-1, and p62 expression in human CF MDMs at baseline (NT) or following 24h infection with B. cenocepacia (Bc) with or without cysteamine. B) Densitometric analysis of ≥ 3 Western blots per condition in 4A, normalized to the loading control calreticulin. C) Confocal microscopy images of CF human alveolar macrophages infected with B. cenocepacia with or without a 24h of cysteamine and analyzed for autophagosome formation (LC3). The macrophage nucleus is stained blue with DAPI, B. cenocepacia (Bc) is shown in red, LC3 is shown in green, and bacteria co-localized with LC3 are yellow in the merged image. D) Summed scoring of bacterial co-localization from 4C (BAL, n = 1) as well as MDMs (n = 3) and THP-1 macrophages (n = 3). 100 MDMs scored per condition. BAL p value <0.0001, MDM p value = 0.02, THP-1 p value < 0.0001. “*” denotes a p value < 0.05, “**” denotes a p value < 0.01, and “***” denotes a p value < 0.001, 2-sample comparisons.

Fig 5. Cysteamine increases single-membrane autophagosome formation.

Electron microscopy (EM) images of CF and non CF MDMs during B. cenocepacia infection with or without cysteamine. A) Untreated non-CF MDMs, magnification 4500X, 2μm. White arrow indicates vacuole with few bacteria. Square indicates zoom area for 5B. B) Untreated non-CF MDM, zoom 34000X, 500nm. C) Treated non-CF MDMs, 4500X, 2μm. Black arrow indicates clearance of bacteria. Square indicates zoom area for 5D. D) Treated non-CF MDM, zoom 34000X, 500nm. E) Untreated CF MDMs, 4500X, 2μm. White arrow indicates large vacuole of bacteria. F) Untreated CF MDM, zoom 34000X, 500nm. G) Treated CF MDMs, 4500X, 2μm. Black arrow indicates clearance of bacteria, square indicates zoom area for 5H. H) Treated CF MDM, zoom 34000X, 500nm.

Fig 6. Cysteamine regulates autophagy proteostasis.

Confocal microscopy images of CF MDMs infected B. cenocepacia with or without a 24h of cysteamine and analyzed for (A) beclin-1 and (B) p62. The macrophage nucleus is stained blue with DAPI, B. cenocepacia (Bc) is shown in red, beclin-1 and p62 are shown in green in their respective sections, and bacteria co-localized with beclin-1 or p62 are yellow in the merged image. C) Summed scoring of bacterial co-localization from 6A and 6B. 100 MDMs scored per condition. “**” denotes a p value < 0.01. D) Colony forming unit (CFU) assay for human CF MDMs infected with B. cenocepacia (Bc) for 24h with or without cysteamine, n = 4. Cysteamine was removed for 24h prior to infection (washout). Intracellular (IC) only counts are displayed. “*” denotes a p value < 0.05.

Fig 7. Cysteamine reduces ROS and inflammatory cytokine production.

A) Summed end-point analysis expressed as %ROS production at 2h of CF MDMs infected with live or paraformaldehyde-killed B. cenocepacia or MRSA with or without cysteamine and assessed for ROS production using RFUs via a DCF assay. Results normalized to live B. cenocepacia and live MRSA. Representative assay of n = 3, “*” denotes a p value < 0.05, “**” denotes a p value < 0.01. B) IL-1β ELISA for CF MDM supernatants basally (NT), or following 24h B. cenocepacia infection (Bc), or Bc infection plus cysteamine, n = 4, p value = 0.007. C) Western blot of pro-IL-1β expression in human CF MDM lysates basally (NT) or following 24h infection with B. cenocepacia (Bc), with or without cysteamine. D) Densitometric analysis of ≥ 3 Western blots per condition in 7D, normalized to the loading control calreticulin. “*” denotes a p value < 0.05, “**” denotes a p value < 0.01, and “***” denotes a p value < 0.001.