Innate responses of phagocytes are thought to be paramount to host defenses against A. fumigatus
. Neutrophils, monocytes, macrophages and conventional DC subsets have been shown to recognize and exert antifungal responses that promote clearance of this opportunistic fungus (Bozza et al., 2002
; Dagenais and Keller, 2009
; Hartigan et al., 2009
; Hohl et al., 2009
; Park et al., 2010
). Here we show that pDCs inhibit the growth of A. fumigatus
hyphae, produce cytokines capable of activating and recruiting other immune cells, and are critical to pulmonary and systemic host defenses against invasive aspergillosis.
Hyphae, the tissue invasive form of A. fumigatus
, rapidly grow to a size that precludes phagocytosis. However, upon incubation of pDCs with A. fumigatus
, we found that within two hr, pDCs had spread over the hyphal surface. While the repertoire of surface receptors on pDCs associated with fungal recognition is not well defined, human pDCs reportedly express dectin-2, but not dectin-1, mannose receptor and DC-SIGN (Graham and Brown, 2009
; Meyer-Wentrup et al., 2008
). Moreover, human pDCs express some complement and Fc receptors, although we found that hyphal recognition did not require opsonization. Future studies are needed to define receptor(s) and their cognate ligand(s) responsible for recognition of A. fumigatus
hyphae by pDCs.
Other cell types, including neutrophils and monocytes, spread over hyphae and cause damage to A. fumigatus by oxidative and non-oxidative mechanisms. Using two independent assays, one that assesses metabolic activity and the other that directly measures hyphal elongation, we demonstrated that human pDCs have antifungal activity against A. fumigatus. However, as opposed to the situation with neutrophils and monocytes, where both growth inhibition and killing of A. fumigatus have been demonstrated, we only found evidence for growth inhibition following incubation of hyphae with pDCs. Thus, hyphal growth proceeded, but at a significantly slower rate, in the presence of pDCs.
In the absence of activating signals, pDCs reportedly undergo spontaneous apoptosis (Grouard et al., 1997
; Lepelletier et al., 2010
). Interestingly though, after a two hr incubation of pDCs with A. fumigatus
hyphae, over half of the pDCs died. Moreover, pDC lysates had antifungal activity against A. fumigatus
and some antifungal activity was retained even if the pDCs were separated from the hyphae by a permeable insert. These observations strongly suggest that diffusible, preformed mediators were responsible for the antifungal activity. The essential role of Fe3+
as fungal growth factors along with the known presence of chelators of these cations in other leukocyte populations (Mambula et al., 2000
; Urban et al., 2009
), led us to examine whether supplemental Fe3+
would reverse the pDC-mediated growth inhibition. The observation that ZnCl2
(but not FeCl3
) partially restored fungal growth suggests a role for the Zn2+
binding protein, calprotectin (Urban et al., 2009
). Neutrophils contain large amounts of cytoplasmic calprotectin. Free and NET-associated calprotectin, released from dying neutrophils, inhibit the growth of C. albicans
and A. fumigatus
by chelating zinc (Bruns et al., 2010
; Lulloff et al., 2004
). Although it is unknown whether a process similar to NETosis occurs with pDCs, we do demonstrate that human pDCs contain calprotectin and that immunodepletion of calprotectin reduces the antifungal activity of cell lysates. It is important to emphasize that Zn2+
supplementation only partially restored hyphal growth, suggesting that the antifungal activity of pDCs is probably mediated by more than one pathway.
Two lines of evidence strongly suggest that the high rate of pDC cytotoxicity following incubation with A. fumigatus
hyphae is at least partially due to secreted factors released by the fungi. First, pDC cytotoxicity was observed (albeit at a lower level) when the pDCs and hyphae were separated by a Transwell. Second, pDC cytotoxicity was significantly (albeit not completely) reduced following incubation with hyphae from A. fumigatus
strains genetically engineered to be deficient in gliotoxin production. Moreover, purified gliotoxin, at concentrations found in the lungs of patients with invasive pulmonary aspergillosis (Lewis et al., 2005
; Stanzani et al., 2005
), killed pDCs in a dose-dependent manner.
Gliotoxin is a low molecular weight mycotoxin secreted by many fungal species including A. fumigatus
(Bok et al., 2006
; Kupfahl et al., 2008
; Sutton et al., 1994
). Induction of apoptosis by gliotoxin has been described in many cell types, including PMNs and monocytes (Stanzani et al., 2005
). To dissect the mechanism of pDC death induced by A. fumigatus
hyphae, we performed a TUNEL assay. The majority of pDCs incubated with hyphae or purified gliotoxin was TUNEL positive, suggesting that the pDCs are dying by apoptosis. However, recent studies have shown that cells undergoing pyroptosis may also exhibit degradation of DNA and a positive TUNEL reaction (Fink et al., 2008
). The finding that pDCs release cytokines when stimulated with hyphae points to their undergoing an inflammatory (rather than an apoptotic) cell death, although it is possible that the fraction of pDCs which remain viable is responsible for the cytokine secretion. Finally, while early apoptotic cells normally preserve their cell membrane integrity, apoptosis can progress to secondary necrosis and membrane leakage (Challa and Chan, 2010
), which we speculate contributes to the antifungal activity of the dying pDCs.
pDCs secrete large amounts of type I IFN in response to viral infections and certain DNA and RNA sequences (Cao and Liu, 2007
; Pietras et al., 2006
), but their role during fungal infections has received little study. While Romani et al. did not find IFNα secretion by pDCs stimulated by A. fumigatus
resting conidia (Romani et al., 2004
), we found that the tissue invasive hyphal morphotype stimulates pDCs to release IFNα. Furthermore, induction of type I IFN appears to be independent of TLR7 and TLR9 as inhibition of endosomal acidification had no effect on hyphae-stimulated IFNα production. This may not be surprising given that hyphae are extracellular and TLR7 and TLR9 are endosomally localized, although the possibility that hyphal components could eventually end up in lysosomal compartments cannot be excluded. An in vivo role for type I IFNs in invasive aspergillosis was suggested by our finding that compared to wild-type mice, IFNα/βR−/−
mice had accelerated mortality after intravenous challenge with A. fumigatus
. Similarly, IFNα/βR−/−
mice were hypersusceptible to Cryptococcus neoformans
and failed to develop protective Th1 cytokine responses (Biondo et al., 2008
). Recombinant human IFNα2a and IFNα2b did not have direct antifungal activity in vitro (unpublished studies).
Remarkably, mice treated with the pDC-depleting antibody, 120G8, were dramatically more susceptible to both pulmonary and systemic challenge with A. fumigatus
than their control counterparts suggesting that pDCs are critical for defenses against the mold. In addition, following pulmonary infection of wild-type mice with A. fumigatus
, a substantial recruitment of pDCs to the lungs was observed. These recruited cells could have antifungal activity, present antigen to T cells and secrete cytokines. While the studies with IFNα/βR−/−
implicate a contribution of pDC-derived type I IFNs, further studies are required to determine the exact mechanisms by which pDCs mediate protection. Finally, it should be noted that although widely used to deplete pDCs, the antigen recognized by 120G8 is present, albeit at a reduced density, on plasma cells and may be induced by IFNs (Asselin-Paturel et al., 2003
; Blasius et al., 2006
Thus, our data demonstrate that pDCs play a nonredundant role in host defenses against invasive aspergillosis. The host-pathogen interaction between pDCs and A. fumigatus has unusual, yet seemingly paradoxical, features. The significant antifungal activity of pDCs against A. fumigatus hyphae appears to be dependent, at least in part, on dying pDCs releasing antifungal effector molecules, such as zinc chelators. This process is enhanced by fungal release of cytotoxic molecules, including gliotoxin, which induce apoptosis or pyroptosis of the pDCs. Release of cytokines by fungal stimulated pDCs may serve to recruit and activate other immune cells, thereby boosting innate responses and helping to initiate adaptive immunity. We postulate that pDCs also make critical contributions to defenses against other fungal infections and that immunotherapies that target pDC could prove beneficial in the treatment of invasive mycoses.