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

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-1β 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-1β 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-1β. 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-1β; and a second signal, involving Syk-induced activation of the NLRP3 inflammasome and caspase-1, allowing processing and secretion of the mature cytokine

Aspergillus fumigatus Stimulates the NLRP3 Inflammasome through a Pathway Requiring ROS Production and the Syk Tyrosine Kinase

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-1β 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-1β 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-1β. 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-1β; and a second signal, involving Syk-induced activation of the NLRP3 inflammasome and caspase-1, allowing processing and secretion of the mature cytokine

Activation of an NLRP3 Inflammasome Restricts Mycobacterium kansasii Infection

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1β secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1β derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii

Alarmins, inflammasomes and immunity

The elaboration of an effective immune response against pathogenic microbes such as viruses, intracellular bacteria or protozoan parasites relies on the recognition of microbial products called pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Ligation of the PRRs leads to synthesis and secretion of pro-inflammatory cytokines and chemokines. Infected cells and other stressed cells also release host-cell derived molecules, called damage-associated molecular patterns (DAMPs, danger signals, or alarmins), which are generic markers for damage. DAMPs are recognized by specific receptors on both immune and nonimmune cells, which, depending on the target cell and the cellular context, can lead to cell differentiation or cell death, and either inflammation or inhibition of inflammation. Recent research has revealed that DAMPs and PAMPs synergize to permit secretion of pro-inflammatory cytokines such as interleukin-1β (IL-1β): PAMPs stimulate synthesis of pro-IL-1β, but not its secretion; while DAMPs can stimulate assembly of an inflammasome containing, usually, a Nod-like receptor (NLR) member, and activation of the protease caspase-1, which cleaves pro-IL-1β into IL-1β, allowing its secretion. Other NLR members do not participate in formation of inflammasomes but play other essential roles in regulation of the innate immune response

Alarmins, inflammasomes and immunity

The elaboration of an effective immune response against pathogenic microbes such as viruses, intracellular bacteria or protozoan parasites relies on the recognition of microbial products called pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Ligation of the PRRs leads to synthesis and secretion of pro-inflammatory cytokines and chemokines. Infected cells and other stressed cells also release host-cell derived molecules, called damage-associated molecular patterns (DAMPs, danger signals, or alarmins), which are generic markers for damage. DAMPs are recognized by specific receptors on both immune and nonimmune cells, which, depending on the target cell and the cellular context, can lead to cell differentiation or cell death, and either inflammation or inhibition of inflammation. Recent research has revealed that DAMPs and PAMPs synergize to permit secretion of pro-inflammatory cytokines such as interleukin-1β (IL-1β): PAMPs stimulate synthesis of pro-IL-1β, but not its secretion; while DAMPs can stimulate assembly of an inflammasome containing, usually, a Nod-like receptor (NLR) member, and activation of the protease caspase-1, which cleaves pro-IL-1β into IL-1β, allowing its secretion. Other NLR members do not participate in formation of inflammasomes but play other essential roles in regulation of the innate immune response

The inflammatory response against <i>A. fumigatus</i> is impaired in immunosuppressed monocytes.

<p>(<b>A</b>) 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>B</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.</p

<i>A. fumigatus</i> induced-caspase-1 activation depends on ROS production and K⁺ efflux.

<p>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). (<b>A inset</b>) 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). (<b>A</b>) Secreted Caspase-1 p20 and (<b>B, C</b>) 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. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001, compared to infected untreated cells.</p

Syk kinase signaling provides the stimulus for both IL-1β synthesis and caspase-1 activation during <i>A. fumigatus</i> infection.

<p>THP-1 cells were pretreated with 1 µM of the Syk kinase inhibitor (Syk I) for 30 min prior to challenge with HFs, and (<b>A</b>) mature IL-1β and (<b>B</b>) active caspase-1 p20 subunit were measured by ELISA. MyD88 and Syk were stably silenced by RNA interference using shRNA. (<b>C</b>) 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 (<b>D</b>) of IL-1β and (<b>E</b>) 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. * <i>p</i><0.05; ** <i>p</i><0.01; *** <i>p</i><0.001, compared to infected untreated cells.</p