Loss of virulence in the SIN kinase mutants is characterized by decreased fungal burden and host response to infection.
A) Analysis of fungal burden by qPCR at day +4 post inoculation. Mice (n = 5 / group) were immune suppressed with cyclophosphamide and triamcinolone acetonide and inoculated with 1x 106 conidia from each strain. Data are represented as nanograms of A. fumigatus specific DNA in 500 ng of total DNA. * p < 0.02. Quantitation of IL-1β (B) and TNFα (C) revealed decreased host response in SIN kinase mutant infected mice. Mice (n = 5 / group) were immune suppressed as indicated for fungal burden analysis and lung tissue was removed at day +4 post-inoculation, homogenized and analyzed by ELISA. **p = 0.0024 for (B); **p = 0.0031 for (C). An in vitro IL-1β release assay uncovered decreased induction of inflammasome activation by the SIN kinase mutants. D) Conidia from each strain were co-incubated with phorbol 12-myristate 13-acetate (PMA)-activated THP-1 cells (MOI 10:1) for 16 hrs and supernatants analyzed by ELISA for IL-1β concentration. ***p < 0.0001; **p = 0.0014. E) Inflammasome dependence of IL-1β release was established by co-culturing PMA-activated WT (THP1-null), Nlrp3−/− (THP1-KO-NLRP3), and Asc−/− (THP1-KO-ASC) THP-1 cells with CEA10 conidia (MOI 10:1) as indicated for (D). ***p < 0.0001. F) Inflammasome dependence of Aspergillus-induced IL-1β release was further confirmed by repeating this assay in the presence of the inflammasome inhibitor, MCC950 (10 μM). ***p < 0.0001. All experiments were conducted in technical replicates (n = 4) and repeated independently in triplicate. Statistical comparisons in (A), (B), (C), and (D) were made by one-way ANOVA with Dunnett’s multiple comparisons test post hoc and represent comparison of each SIN kinase mutant to the CEA10 control. Statistical comparisons in (E) were made by one-way ANOVA with Dunnett’s multiple comparisons post hoc and represent the NLRP3-/- and ASC-/- versus WT control. The statistical comparison of MCC950 versus vehicle in (F) was made by unpaired T-test.