Quantitative Analysis of Proteome Modulations in Alveolar Epithelial Type II Cells in Response to Pulmonary Aspergillus fumigatus Infection

Abstract

The ubiquitous mold threatens immunosuppressed patients as inducer of lethal invasive aspergillosis. conidia are airborne and reach the alveoli, where they encounter alveolar epithelial cells (AEC). Previous studies reported the importance of the surfactant-producing AEC II during infection experiments using cell lines. We established a negative isolation protocol yielding untouched primary murine AEC II with a purity >90%, allowing analyses of the cells, which encountered the mold By label-free proteome analysis of AEC II isolated from mice 24h after or mock infection we quantified 2256 proteins and found 154 proteins to be significantly differentially abundant between both groups (ANOVA value ≤ 0.01, ratio of means ≥1.5 or ≤0.67, quantified with ≥2 peptides). Most of these proteins were higher abundant in the infected condition and reflected a comprehensive activation of AEC II on interaction with This was especially represented by proteins related to oxidative phosphorylation, hence energy production. However, the most strongly induced protein was the l-amino acid oxidase (LAAO) Interleukin 4 induced 1 (IL4I1) with a 42.9 fold higher abundance (ANOVA value 2.91). IL4I1 has previously been found in B cells, macrophages, dendritic cells and rare neurons. Increased IL4I1 abundance in AEC II was confirmed by qPCR, Western blot and immunohistology. Furthermore, infected lungs showed high levels of IL4I1 metabolic products. Importantly, higher IL4I1 abundance was also confirmed in lung tissue from human aspergilloma. Because LAAO are key enzymes for bactericidal product generation, AEC II might actively participate in pathogen defense. We provide insights into proteome changes of primary AEC II thereby opening new avenues to analyze the molecular changes of this central lung cell on infectious threats. Data are available ProteomeXchange with identifier PXD005834.

An Overview of l-Amino Acid Oxidase Functions from Bacteria to Mammals: Focus on the Immunoregulatory Phenylalanine Oxidase IL4I1

“…In humans, IL4I1 is expressed in monocytes/macrophages and dendritic cells present in chronic T helper type 1 (Th1) inflammation, such as sarcoidosis or tuberculosis granulomas, and tumors ( Figure 3 ). It has also been reported in alveolar type II cells during infection by the mold Aspergillus fumigatus [ 59 ]. In accordance with these observations, the induction of IL4I1 expression is mediated by inflammatory and Th1 stimuli, such as pathogen-associated molecular patterns (ligands for Toll like receptors), tumor necrosis factor (TNF) α, and interferons (IFNs), acting via NFκB and STAT1.…”

Anti-Aspergillus Activities of the Respiratory Epithelium in Health and Disease

“…Recent studies have delineated morphotype-specific epithelial responses to conidial and germinated forms of A. fumigatus . Given that germinated forms of A. fumigatus pose a major threat, both in vivo and in vitro, to epithelial integrity, and that epithelial cells can potently neutralise the vast majority of internalised spores [ 6 ], we, and others [ 72 ], hypothesise that epithelial cells, in collaboration with professional phagoctyes, provide an important, directly microbicidal, defence against A. fumigatus which can become dysfunctional in settings of respiratory disease or immune compromise. Within this context, epithelial entry would serve, in the healthy host, as a means to control everyday A. fumigatus exposure, and, in the diseased setting, as a critical gateway through which to elicit host entry and damage.…”

“…Due to the difficulties in isolating primary alveolar epithelial cells (AECs) from human patients, animal models remain, notwithstanding the significant influence of host cell isolation and handling on cellular physiology, the closest alternative for the study of host- A. fumigatus interactions. Seddigh et al recently developed a novel negative immunomagnetic protocol for isolating untouched primary AECs from murine lung for quantitative-label free in vivo proteomics [ 72 ]. By circumventing flow-cytometry-based cell sorting, which, by itself, massively influences cellular physiology, this approach permitted the targeted comparative ex vivo analysis, in nine biological replicates, of type II AECs from A. fumigatus -infected and sham-infected C57BL/6 mice at 24 h post-challenge.…”

“…By circumventing flow-cytometry-based cell sorting, which, by itself, massively influences cellular physiology, this approach permitted the targeted comparative ex vivo analysis, in nine biological replicates, of type II AECs from A. fumigatus -infected and sham-infected C57BL/6 mice at 24 h post-challenge. The study demonstrated a remarkable increase in abundance of L-amino acid oxidase (also known as interleukin 4-induced gene-1, IL4I1) mRNA and protein in primary alveolar epithelial cells from immunocompetent mice infected intratracheally with A. fumigatus spores for 24 h [ 72 ]. Via immunofluorescence-mediated histopathology it was determined that IL4I1 was often co-localized with the presence, and in some cases even germination of, A. fumigatus conidia an observation which did not apply to tissues harvested from sham-infected mice.…”

Changed Expression of Cytoskeleton Proteins During Lung Injury in a Mouse Model of Streptococcus pneumoniae Infection

“…Méndez et al employed quantitative proteomics in order to characterize the proteome of Acinetobacter baumanii under conditions that simulate those found in the airways (Méndez et al, 2015 ). Seddigh et al recently studied the effect of Aspergillus fumigatus infection on alveolar epithelial cells by means of proteomic approaches (Seddigh et al, 2017 ).…”