Alternaria alternata Accelerates Loss of Alveolar Macrophages and Promotes Lethal Influenza A Infection

Abstract

Chronic inhalation of fungi and fungal components has been linked to the development of respiratory disorders, although their role with respect to the pathogenesis of acute respiratory virus infection remains unclear. Here, we evaluate inflammatory pathology induced by repetitive administration of a filtrate of the ubiquitous fungus, Alternaria alternata, and its impact on susceptibility to infection with influenza A. We showed previously that A. alternata at the nasal mucosae resulted in increased susceptibility to an otherwise sublethal inoculum of influenza A in wild-type mice. Here we demonstrate that A. alternata-induced potentiation of influenza A infection was not dependent on fungal serine protease or ribonuclease activity. Repetitive challenge with A. alternata prior to virus infection resulted proinflammatory cytokines, neutrophil recruitment, and loss of alveolar macrophages to a degree that substantially exceeded that observed in response to influenza A infection alone. Concomitant administration of immunomodulatory Lactobacillus plantarum, a strategy shown previously to limit virus-induced inflammation in the airways, blocked the exaggerated lethal response. These observations promote an improved understanding of severe influenza infection with potential clinical relevance for individuals subjected to continuous exposure to molds and fungi.

Respiratory Epithelial Cells Respond to Lactobacillus plantarum but Provide No Cross-Protection against Virus-Induced Inflammation

“…Primary mouse tracheal epithelial cells (mTEC) cultures [ 36 ] were generated as previously described [ 37 , 38 ]. Briefly, tracheae were isolated from WT and Tlr2 −/− mice under sterile conditions and processed to yield isolated mTECs as described.…”

“…Mouse-passaged stocks were prepared from both viruses. Tissue culture infectious dose (TCID 50 ) and quantitative PCR titration were performed as previously described [ 38 , 40 ].…”

“…Samples were then reverse transcribed using the First Strand cDNA Synthesis Kit for RT-PCR (Roche) using random primers and no reverse transcriptase controls. Quantitative PCR reactions were carried out using a 7500 Real-Time PCR System (Applied Biosystems) under standard thermal cycling parameters with 25 µL of final volume including the reverse-transcribed cDNA, ABI TaqMan 2× Reagent (Thermo), and primer/probes specific for the Influenza A/FM/1/47-MA matrix protein (M1), including Forward Primer 5′-GCGAGGACTGCAGCGTAGAC-3′, Reverse Primer 5′-GGATCCCCGTTCCCATTAAG-3′, and Probe 6FAM-CTTTGTCCAAAATGCMGBNFQ, and primer/probes specific for Influenza A/HK/1/68, as previously described [ 38 ]. A VIC-labeled probe and primers that target mouse GAPDH were used for the normalization of all samples (ABI #43058313).…”

“…However, infection of mTECs with replicating Inf A resulted in the production and release of CCL5 and CXCL10 only; little to no CCL2 or CXCL1 was detected over levels observed in response to heat-inactivated virus (Figure 2D). In a previous study, we presented immunohistochemical data documenting infection of bronchial epithelial cells at day 6 after inoculation with Influenza A [38]. The findings presented here are also consistent with those published previously by Weinheimer et al [45] who presented immunohistochemical data documenting Influenza A infection in human type II pneumocytes and those of Ibricevic et al [46] who used a similar strategy to document Influenza A infection of type II alveolar epithelial cells in vivo; the latter publication also documented infection of mTECs via this method.…”

“…The findings presented here are also consistent with those published previously by Weinheimer et al [45] who presented immunohistochemical data documenting Influenza A infection in human type II pneumocytes and those of Ibricevic et al [46] who used a similar strategy to document Influenza A infection of type II alveolar epithelial cells in vivo; the latter publication also documented infection of mTECs via this method. In a previous study, we presented immunohistochemical data documenting infection of bronchial epithelial cells at day 6 after inoculation with Influenza A [38]. The findings presented here are also consistent with those published previously by Weinheimer et al [45] who presented immunohistochemical data documenting Influenza A infection in human type II pneumocytes and those of Ibricevic et al [46] who used a similar strategy to document Influenza A infection of type II alveolar epithelial cells in vivo; the latter publication also documented infection of mTECs via this method.…”