Abstract

Background

   Given the poor outcomes of influenza-associated pulmonary aspergillosis
   (IAPA), there is a major unmet need for innovative therapeutic
   strategies. Impairment of pathogen recognition receptor (PRR) signaling
   is a known pathomechanism and prognostic factor in IAPA patients.
   Therefore, we performed immunotherapy with nebulized PRR agonists in a
   murine IAPA model and studied its impact on infection outcomes and
   pulmonary immunopathology.

   graphic file with name ofae631_p-1865_f1.jpg
   [35]Open in a new tab

   Timeline of experimental interventions. Abbreviations: AF = Aspergillus
   fumigatus, CA = cortisone acetate (10 mg intraperitoneally), IAV =
   nebulization with influenza A virus (7.5% of the 90% lethal dose), PBS
   = phosphate-buffered saline (mock therapy), P/O = Pam2/ODN, VPS = viral
   pneumonia score (0 = healthy – 12 = moribund).

Methods

   8-week-old BALB/c mice were infected with influenza A virus (IAV,
   H3N2), immunosuppressed with cortisone acetate (CA) on days (d) 5 and 8
   after IAV infection, and infected intranasally with 50,000 Aspergillus
   fumigatus (AF) conidia on d9 (Fig. 1). Mice received nebulized Pam2+ODN
   (P/O, 4 µM/1 µM) or phosphate-buffered saline (mock therapy) before and
   after AF super-infection (d8/d12). Therapeutic outcomes were assessed
   using a combined morbidity (viral pneumonia score ≥ 7/12) and mortality
   endpoint. On d14, nCounter-based transcriptomics with Ingenuity pathway
   enrichment analysis was performed on lung tissue, along with
   histopathology and flow cytometry.

   graphic file with name ofae631_p-1865_f2.jpg
   [36]Open in a new tab

   Event-free survival (A; Mantel-Cox log-rank test) and median viral
   pneumonia scores of survivors (B) depending on the treatment arm.

Results

   All mock-treated mice with IAPA reached the morbidity/mortality
   endpoint by d13 (Fig. 2). Compared to CA-immunosuppressed mice with IAV
   infection only (100% survival), nCounter analysis revealed minimal
   incremental inflammatory response and suppression of several PRR
   pathways, interferon signaling, and neutrophil activation in the IAPA
   group. P/O therapy led to 80% event-free survival until d21 (p < 0.001
   vs. mock therapy) and full recovery of all survivors (Fig. 2).
   Histopathology revealed significantly reduced fungal burden and
   hemorrhagic lesions in P/O-treated animals. nCounter analysis showed
   induction of PRR signaling, several key effector cytokine pathways, and
   epithelial signaling after P/O therapy. Moreover, nCounter and flow
   cytometry revealed enhanced recruitment of macrophages, natural killer
   cells, and T cells in P/O-treated mice (Fig. 3).

   graphic file with name ofae631_p-1865_f3.jpg
   [37]Open in a new tab

   Summary of nCounter and flow cytometry results.

Conclusion

   Consistent with previous studies in human IAPA patients, our data
   revealed severe immune paralysis in mice with IAPA. Immunotherapy with
   nebulized P/O strongly improved infection outcomes, enhanced
   mobilization of key immune effector cells, and re-invigorated PRR and
   cytokine signaling.

Disclosures

   Sebastian Wurster, MD, MSc, Astellas Pharma: Grant/Research
   Support|Gilead Sciences: Grant/Research Support Dimitrios P.
   Kontoyiannis, MD, AbbVie: Advisor/Consultant|Astellas Pharma:
   Advisor/Consultant|Astellas Pharma: Grant/Research Support|Astellas
   Pharma: Honoraria|Cidara Therapeutics: Advisor/Consultant|Gilead
   Sciences: Advisor/Consultant|Gilead Sciences: Grant/Research
   Support|Gilead Sciences: Honoraria|Knight: Advisor/Consultant|Merck:
   Advisor/Consultant|Scynexis: Advisor/Consultant
     __________________________________________________________________

   Articles from Open Forum Infectious Diseases are provided here courtesy
   of Oxford University Press
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