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.

Keywords: Alternaria alternata; Lactobacillus plantarum; alveolar macrophage; cytokine; fungal rhinitis; neutrophil.

Figure 1

Repetitive administration of Alternaria alternata results in increased susceptibility to lethal Inf A infection. (A) Strategy: A. alternata at the nasal mucosa (0–50 µg per inoculum; 2.5 µL per nare) on days indicated followed by Influenza A/H3N2 (Inf A; 30 tissue culture infectious dose (TCID)₅₀ units per mouse, 2.5 µL per nare) on day 0. (B) Survival in response to A. alternata followed by Inf A, as per the inoculation strategy in A.; n = 5 mice per group, *** p < 0.001, Kaplan–Meier log-rank. (C) Weight loss in response to A. alternata followed by Inf A as per the inoculation strategy in A.; n = 5 mice per group, * p < 0.05, ** p < 0.01, 1-way ANOVA; ^† no survival after this time point in response to the two highest doses of A. alternata as indicated. (D) Virus (copies virus M1 gene/µL bronchoalveolar lavage fluid [BAL]) determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on day 6 as per Figure 1A in mice treated with A. alternata (5 µg per inoculum) or diluent only prior to inoculation with Inf A; ns = no significant difference. (E) Wet-to-dry lung weight ratios at time points shown as per Figure 1A; 5 µg A. alternata per inoculum, n = 5 mice per group, * p < 0.05, 1-way ANOVA.

Figure 2

Repetitive administration of A. alternata amplifies the biochemical inflammatory response to infection with Inf A. Repetitive administration of A. alternata (5 µg per inoculum) as in Figure 1A results in amplification of (A) TNFα, (B) CXCL10, and (C) CCL2 in BAL fluid (day 6 as per Figure 1A) compared to responses observed in response to a sublethal inoculum of Inf A infection alone. By contrast, repetitive administration of A. alternata had no impact on levels of (D) IL-6 or (E) CXCL1 (day 6 as per Figure 1A) compared to those generated in response to Inf A infection alone. For all cytokines evaluated (a–e), administration of A. alternata alone (i.e., no Inf A) resulted in no detection over background levels; n = 5–8 mice per point, ** p < 0.01, *** p < 0.005, 1-way ANOVA.

Figure 3

Repetitive administration of A. alternata amplifies total neutrophil recruitment and accelerates loss of alveolar macrophages in response to infection with Inf A. (A) Total leukocytes and (B) total PMNs recruited to lung tissue in mice treated with A. alternata (5 µg per inoculum) prior to infection with a sublethal inoculum of Inf A compared to that observed in response to inoculation with Inf A alone. (C) Infection with Inf A results in a loss of AMs; repetitive exposure to A. alternata (5 µg per inoculum) prior to Inf A infection amplifies this response. (D) AMs as percent of total leukocytes in A. alternata-treated Inf A-infected mice are reduced to undetectable levels; n = 5 per group, * p < 0.05, ** p < 0.01; *** p < 0.005, 1-way ANOVA. By contrast, repetitive administration of A. alternata (5 µg per inoculum) had no impact on (E) total CD4⁺ T cells, (F) total CD8⁺ T cells, or (G) total B cells recruited to the lungs in response to infection with Inf A; n = 5 mice per group, day 6 as per Figure 1A; * p < 0.05, ** p < 0.01, 1-way ANOVA.

Figure 4

Bacterial colonization of the airways of A. alternata-challenged, Inf A-infected mice. Overnight growth at 37 °C on blood agar plates streaked with dilutions of BAL fluid from (A) a mouse infected with a sublethal inoculum of Inf A only and (B) a mouse subjected to repetitive inoculation with A. alternata (5 µg per inoculum) prior to Inf A infection. Both samples were from day 8 as per the timeline shown in Figure 1A. (C) Colony forming units (CFUs) per 50 µL BAL fluid on day 8 as per the timeline in Figure 1A. Shown are results from mice challenged with A. alternata alone, sublethal Inf A alone, A. alternata followed by Inf A, and A. alternata followed by Inf A and ampicillin (20 mg/mouse/day i.p. on days 0–8.) (D) Total body weight over time for mice in groups indicated in (C); mice treated with A. alternata alone (5 µg per inoculum) do not lose weight; n = 10 mice per group, * p < 0.05, 1-way ANOVA; ^† no survival after this time point in groups indicated.

Figure 5

Administration of immunobiotic Lactobacillus plantarum directly to the respiratory mucosa protects against the lethal response to Inf A. (A) Strategy: repetitive administration of A. alternata (days −5, −3 and −1; 2.5 µL per nare, 5 µg per inoculum) immediately preceded (10–15 min) by L. plantarum (10⁸ CFU in 50 µL phosphate-buffered saline [PBS] with 0.1% bovine serum albumin [BSA]) or diluent control. On day 0, mice were inoculated with Inf A. (B) Administration of L. plantarum protects against increased susceptibility to lethal Inf A; n = 5 mice per group, * p < 0.05; ** p < 0.01, ^† no survival after this time point in the group indicated. (C) Virus titer (copies per µL BAL fluid) on day 8 in mice challenged with A. alternata as in Figure 5A both with and without L. plantarum; ns, no significant difference. (D) CXCL10 (pg/mL BAL fluid) and (E) CCL2 (pg/mL BAL fluid); day 8 as per Figure 5A; n = 5 per group, ** p < 0.01, Mann–Whitney U-test.

Figure 6

Serine protease activity, RNase activity, and low molecular weight biomolecules are not among the critical factors promoting the amplified lethal response to Inf A. (A) Treatment with the irreversible serine protease inhibitor AEBSF reduces the protease activity in filtrates from A. alternata by >10-fold; *** p < 0.005. (B) Proteolytic inactivation of the A. alternata filtrate with AEBSF had no impact on its ability to promote weight loss and mortality when administered at 5 µg per inoculum in response to a sublethal infection with Inf A; n = 5 mice per group, ** p < 0.01, ^† no survival after this time point in groups indicated. (C) Responses of Par2 gene-deleted (Par2^-/-) mice to Inf A alone and Inf A after repetitive inoculation with A. alternata (as per Figure 1A; 5 µg per inoculum) are similar to those of wild-type mice; n = 5 mice per group, ** p < 0.01, ^† no survival after this time point in the group indicated. (D) Heat (95 °C for 10 min) and diethylpyrocarbonate (DEPC), but not human placental ribonuclease inhibitor (RI) inhibited the ribonuclease activity in A. alternata filtrates, consistent with the profile of a T2 family enzyme (see Genbank Acc. No. XM_018531878.1); *** p < 0.005. (E) Ribonucleolytic inactivation of the A. alternata filtrate with DEPC has no impact on its capacity to promote weight loss and mortality (5 µg per inoculum) in response to an otherwise sublethal Inf A infection; n = 5 mice per group, ** p < 0.01, 1-way ANOVA; ^† no survival after this time point in groups indicated. (F) Dialysis of the A. alternata filtrate to remove small biomolecules (<12 kDa) had no impact on the amplified lethal response to Inf A; ** p < 0.01, * p < 0.05, ^† no survival after this time point in the group indicated.