Compartment-Specific and Sequential Role of MyD88 and CARD9 in Chemokine Induction and Innate Defense during Respiratory Fungal Infection

Abstract

Aspergillus fumigatus forms ubiquitous airborne conidia that humans inhale on a daily basis. Although respiratory fungal infection activates the adaptor proteins CARD9 and MyD88 via C-type lectin, Toll-like, and interleukin-1 family receptor signals, defining the temporal and spatial pattern of MyD88- and CARD9-coupled signals in immune activation and fungal clearance has been difficult to achieve. Herein, we demonstrate that MyD88 and CARD9 act in two discrete phases and in two cellular compartments to direct chemokine- and neutrophil-dependent host defense. The first phase depends on MyD88 signaling because genetic deletion of MyD88 leads to delayed induction of the neutrophil chemokines CXCL1 and CXCL5, delayed neutrophil lung trafficking, and fatal pulmonary damage at the onset of respiratory fungal infection. MyD88 expression in lung epithelial cells restores rapid chemokine induction and neutrophil recruitment via interleukin-1 receptor signaling. Exogenous CXCL1 administration reverses murine mortality in MyD88-deficient mice. The second phase depends predominately on CARD9 signaling because genetic deletion of CARD9 in radiosensitive hematopoietic cells interrupts CXCL1 and CXCL2 production and lung neutrophil recruitment beyond the initial MyD88-dependent phase. Using a CXCL2 reporter mouse, we show that lung-infiltrating neutrophils represent the major cellular source of CXCL2 during CARD9-dependent recruitment. Although neutrophil-intrinsic MyD88 and CARD9 function are dispensable for neutrophil conidial uptake and killing in the lung, global deletion of both adaptor proteins triggers rapidly progressive invasive disease when mice are challenged with an inoculum that is sub-lethal for single adapter protein knockout mice. Our findings demonstrate that distinct signal transduction pathways in the respiratory epithelium and hematopoietic compartment partially overlap to ensure optimal chemokine induction, neutrophil recruitment, and fungal clearance within the respiratory tract.

Extrapulmonary Aspergillus infection in patients with CARD9 deficiency

“…Notably, CARD9-deficient neutrophils exhibited normal capacity to internalize conidia ( Figure 5), to prevent conidial germination to hyphae (Supplemental Figure 2), and to kill opsonized or unopsonized conidia and hyphae ( Figure 5), in agreement with intact anti-Aspergillus killing of Card9 -/-mouse neutrophils (28). Furthermore, CARD9-deficient neutrophils had normal oxidative burst towards fungal and nonfungal stimuli ex vivo (Supplemental Figure 3), and exerted oxidative cytotoxicity on the B-5233/GFP Aspergillus strain that expresses GFP during oxidative stress (Supplemental Figure 3).…”

“…The absence of pulmonary Aspergillus involvement in CARD9 deficiency may be explained by lung-specific, CARD9-independent immune mechanisms that may compensate for lack of CLR/CARD9 signaling. Indeed, recent mouse studies showed that Card9 plays only a partial role in neutrophil pulmonary recruitment after Aspergillus challenge, being compensated by mononuclear phagocyte and epithelial cell IL-1α and Myd88 signaling (28,33). Thus, CARD9 appears to mediate organ-specific effects on neutrophil recruitment during aspergillosis, being largely redundant for pulmonary recruitment (28) but necessary for recruitment into extrapulmonary organs (current study), in agreement with its CNS-specific role in mediating neutrophil recruitment during systemic candidiasis (14).…”

“…Similar necrosis without neutrophil infiltration was seen in the infected mesenteric lymph node of patient 1 (Supplemental Figure 4). Because aspergillosis in nonneutropenic mice and humans is accompanied by robust neutrophilic tissue accumulation (28,30,31), the lack of tissue neutrophil infiltration in CARD9 deficiency despite normal peripheral neutrophil numbers represents a suboptimal innate immune response and is consistent with a defect in neutrophil recruitment from the blood into the Aspergillus-infected tissue.…”

Immunity against fungi

“…CARD9 is critical for neutrophil recruitment to the Candidainfected CNS but not for neutrophil recruitment to the Candida-infected kidney or Staphylococcus-infected CNS (159). This tropism is mediated by CARD9-dependent production of CXC chemokines by resident glial cells and neutrophils in the Candida-infected CNS, while neutrophil-intrinsic chemotaxis and effector functions are largely intact in CARD9-deficient humans and mice (159)(160)(161). Development of CNS and intra-abdominal aspergillosis is another example of altered tissue tropism in CARD9-deficient patients (162) and results from defective CARD9-dependent neutrophil chemoattractant production in and neutrophil mobilization to extrapulmonary infection sites.…”

“…Development of CNS and intra-abdominal aspergillosis is another example of altered tissue tropism in CARD9-deficient patients (162) and results from defective CARD9-dependent neutrophil chemoattractant production in and neutrophil mobilization to extrapulmonary infection sites. The absence of pulmonary Aspergillus involvement in CARD9 deficiency may be explained by compensatory IL-1R/MyD88-dependent neutrophil lung recruitment (161,163). Collectively, these human observations have led to our appreciation that CARD9 appears to mediate fungus-, cell type-, and organ-specific neutrophil recruitment during invasive fungal infection.…”