Airway Epithelial Cells Differentially Adapt Their Iron Metabolism to Infection With Klebsiella pneumoniae and Escherichia coli In Vitro

Abstract

BackgroundPneumonia is often elicited by bacteria and can be associated with a severe clinical course, respiratory failure and the need for mechanical ventilation. In the alveolus, type-2-alveolar-epithelial-cells (AECII) contribute to innate immune functions. We hypothesized that AECII actively adapt cellular iron homeostasis to restrict this essential nutrient from invading pathogens – a defense strategy termed ‘nutritional immunity’, hitherto mainly demonstrated for myeloid cells.MethodsWe established an in-vitro infection model using the human AECII-like cell line A549. We infected cells with Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli), two gram-negative bacteria with different modes of infection and frequent causes of hospital-acquired pneumonia. We followed the entry and intracellular growth of these gram-negative bacteria and analyzed differential gene expression and protein levels of key inflammatory and iron metabolism molecules.ResultsBoth, K. pneumoniae and E. coli are able to invade A549 cells, whereas only K. pneumoniae is capable of proliferating intracellularly. After peak bacterial burden, the number of intracellular pathogens declines, suggesting that epithelial cells initiate antimicrobial immune effector pathways to combat bacterial proliferation. The extracellular pathogen E. coli induces an iron retention phenotype in A549 cells, mainly characterized by the downregulation of the pivotal iron exporter ferroportin, the upregulation of the iron importer transferrin-receptor-1 and corresponding induction of the iron storage protein ferritin. In contrast, cells infected with the facultative intracellular bacterium K. pneumoniae exhibit an iron export phenotype indicated by ferroportin upregulation. This differential regulation of iron homeostasis and the pathogen-specific inflammatory reaction is likely mediated by oxidative stress.ConclusionAECII-derived A549 cells show pathogen-specific innate immune functions and adapt their iron handling in response to infection. The differential regulation of iron transporters depends on the preferential intra- or extracellular localization of the pathogen and likely aims at limiting bacterial iron availability.

Proteomic analysis of meropenem-induced outer membrane vesicles released by carbapenem-resistant Klebsiella pneumoniae

“…The previous studies found that Escherichia coli induces iron retention in A549 cells and causes the corresponding induction of the protein ferritin. This regulation of iron homeostasis may be mediated by oxidative stress ( 56 , 57 ).…”

Availability of Ferritin-Bound Iron to Enterobacteriaceae

“…Nrf-2 has previously been shown to be an important regulator in nutritional immunity, depriving intracellular pathogens of iron by enhancing the egress of iron via ferroportin [ 80 ]. Furthermore, Nrf-2 activation upon infection has also been observed in non-professional immune cells, emphasizing the importance of this mechanism [ 81 , 82 ].…”

Timing of Interleukin-4 Stimulation of Macrophages Determines Their Anti-Microbial Activity during Infection with Salmonella enterica Serovar Typhimurium

“…Cells were prepared as described [ 39 ]. On the day of infection, 15 min before S .tm was added at a MOI 10, 0.5 µL of CellROX TM Deep Red (Thermo Fisher, catalog number: C10422) was pipetted to each well.…”