Aspergillus fumigatus stimulates the NLRP3 inflammasome through a pathway requiring ROS production and the Syk tyrosine kinase

Abstract

Invasive aspergillosis (IA) is a life-threatening disease that occurs in immunodepressed patients when infected with Aspergillus fumigatus. This fungus is the second most-common causative agent of fungal disease after Candida albicans. Nevertheless, much remains to be learned about the mechanisms by which A. fulmigatus activates the innate immune system. We investigated the inflammatory response to conidia and hyphae of A. fumigatus and specifically, their capacity to trigger activation of an inflammasome. Our results show that in contrast to conidia, hyphal fragments induce NLRP3 inflammasome assembly, caspase-1 activation and IL-1beta release from a human monocyte cell line. The ability of Aspergillus hyphae to activate the NLRP3 inflammasome in the monocytes requires K(+) efflux and ROS production. In addition, our data show that NLRP3 inflammasome activation as well as pro-IL-1beta expression relies on the Syk tyrosine kinase, which is downstream from the pathogen recognition receptor Dectin-1, reinforcing the importance of Dectin-1 in the innate immune response against fungal infection. Furthermore, we show that treatment of monocytes with corticosteroids inhibits transcription of the gene encoding IL-1beta. Thus, our data demonstrate that the innate immune response against A. fumigatus infection involves a two step activation process, with a first signal promoting expression and synthesis of pro-IL-1beta; and a second signal, involving Syk-induced activation of the NLRP3 inflammasome and caspase-1, allowing processing and secretion of the mature cytokine.

Figure 1. A. fumigatus hyphae upregulate pro-IL-1β transcription and induce IL-1β secretion in monocytes.

One million THP-1 cells/ml were treated with 10 ng/ml of LPS with and without nigericin, spores or HFs at an MOI = 10 for 6 hours (A, B), or spores for 12 hours and HFs for 6 hours (C, D). (A) Intracellular IL-1β gene transcription was quantified by real-time PCR and compared to control. (B, C) The amount of secreted IL-1β was quantified by ELISA. (D) TNFα secretion was measured in supernatants by ELISA. Error bars represent standard deviation of at least three separate experiments. * p<0.05; ** p<0.01; *** p<0.001, compared to infected untreated cells.

Figure 2. A. fumigatus induced-caspase-1 activation depends on ROS production and K⁺ efflux.

THP-1 cells were incubated with HFs for 6 hours in the presence or absence of 130 mM KCl, 25 mM NAC, 100 µM caspase-1/caspase-5 inhibitor (Z-WEHD-FMK), or pretreated for 30 min with 1 µM of Syk kinase inhibitor (Syk I). (A inset) Caspase-1 activation was analyzed by Western blot, using an antibody against the Caspase-1 p20 cleavage product. Each band intensity was measured by NIH ImageJ software (Ctrl = 1, HF = 4.848, HF + Z-WEHD-FMK = 2.92, HF + Syk I = 1.67, and HF + NAC = 1.54). (A) Secreted Caspase-1 p20 and (B, C) mature IL-1β p17 in the supernatant of infected cells, compared to the control, was assessed by ELISA. Error bars represent the standard deviation of at least three separate experiments. * p<0.05; ** p<0.01; *** p<0.001, compared to infected untreated cells.

Figure 3. The NLRP3 inflammasome controls the anti-A. fumigatus innate immune response.

THP-1 cells were stably transfected with shRNA targeting NLRP3 or ASC in order to induce gene silencing. (A inset) Western blot analysis of wildtype (WT) cells, cells treated with non-target control (SH control), and cells treated with shNLRP3, confirming decreased expression of the NLRP3 protein after mRNA depletion. Western blot was performed with an anti-NLRP3 antibody, which detects the 118 kDa protein. (A) mRNA levels of NLRP3 and ASC were quantified by real-time PCR and compared to wild type (WT) and non-target control (SH Ctrl). Supernatants of each of the knocked down (KD) cells treated with nigericin after LPS priming, or HFs for 6 hours was analyzed by ELISA for the presence (B, C) caspase-1 p20 and (D, E) mature IL-1β. All values are representative of at least three independent experiments. The error bars represent the standard deviation of at least three separate experiments. * p<0.05; ** p<0.01; *** p<0.001, compared to infected untreated cells.

Figure 4. Syk kinase signaling provides the stimulus for both IL-1β synthesis and caspase-1 activation during A. fumigatus infection.

THP-1 cells were pretreated with 1 µM of the Syk kinase inhibitor (Syk I) for 30 min prior to challenge with HFs, and (A) mature IL-1β and (B) active caspase-1 p20 subunit were measured by ELISA. MyD88 and Syk were stably silenced by RNA interference using shRNA. (C) Transcript levels of pro-IL-1β in MyD88 KD and Syk KD cells treated with HFs was measured using real-time PCR. Representative real-time PCR values representative of three independent experiments are shown. The secretion (D) of IL-1β and (E) caspase-1 p20 into the supernatants of MyD88 KD and Syk KD cells treated with HFs was assessed by ELISA. All values are representatives of at least three independent experiments. Error bars represent standard deviation of at least three separate experiments. * p<0.05; ** p<0.01; *** p<0.001, compared to infected untreated cells.

Figure 5. The inflammatory response against A. fumigatus is impaired in immunosuppressed monocytes.

(A) THP-1 cells were stimulated for 10 min with 30 µM β-methasone prior to stimulation with HFs for 6 hours. IL-1β secretion was measured by ELISA. (B) THP-1 cells were stimulated for 10 min with 30 µM β-methasone prior to stimulation with 10 ng/ml LPS for 6 hours. IL-1β mRNA was quantified by real-time PCR.