Compartment-specific and sequential role of MyD88 and CARD9 in chemokine induction and innate defense during respiratory fungal infection.

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.

Role of Granulocyte-Macrophage Colony-Stimulating Factor Signaling in Regulating Neutrophil Antifungal Activity and the Oxidative Burst During Respiratory Fungal Challenge.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that plays a critical role in regulating myeloid cell host defense. In this study, we demonstrated that GM-CSF signaling plays an essential role in antifungal defense against Aspergillus fumigatus. Mice that lack the GM-CSF receptor ß chain (GM-CSFRß) developed invasive hyphal growth and exhibited impaired survival after pulmonary challenge with A. fumigatus conidia. GM-CSFRß signaling regulated the recruitment of inflammatory monocytes to infected lungs, but not the recruitment of effector neutrophils. Cell-intrinsic GM-CSFRß signaling mediated neutrophil and inflammatory monocyte antifungal activity, because lung GM-CSFRß(-/-) leukocytes exhibited impaired conidial killing compared with GM-CSFRß(+/+) counterparts in mixed bone marrow chimeric mice. GM-CSFRß(-/-) neutrophils exhibited reduced (hydrogenated) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in vivo. Conversely, administration of recombinant GM-CSF enhanced neutrophil NADPH oxidase function, conidiacidal activity, and lung fungal clearance in A. fumigatus-challenged mice. Thus, our study illustrates the functional role of GM-CSFRß signaling on lung myeloid cell responses against inhaled A. fumigatus conidia and demonstrates a benefit for systemic GM-CSF administration.

Inflammatory monocytes orchestrate innate antifungal immunity in the lung.

Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2⁺Mo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2⁺Mo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2⁺Mo and Mo-DCs exert innate antifungal activity. First, CCR2⁺Mo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2⁺Mo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2⁺Mo and their derivatives in innate antifungal immunity in the lung.

Inflammatory monocytes mediate early and organ-specific innate defense during systemic candidiasis.

Candida albicans is a commensal fungus that can cause systemic disease in patients with breaches in mucosal integrity, indwelling catheters, and defects in phagocyte function. Although circulating human and murine monocytes bind C. albicans and promote inflammation, it remains unclear whether C-C chemokine receptor 2 (CCR2)- and Ly6C-expressing inflammatory monocytes exert a protective or a deleterious function during systemic infection. During murine systemic candidiasis, interruption of CCR2-dependent inflammatory monocyte trafficking into infected kidneys impaired fungal clearance and decreased murine survival. Depletion of CCR2-expressing cells led to uncontrolled fungal growth in the kidneys and brain and demonstrated an essential antifungal role for inflammatory monocytes and their tissue-resident derivatives in the first 48 hours postinfection. Adoptive transfer of purified inflammatory monocytes in depleted hosts reversed the defect in fungal clearance to a substantial extent, indicating a compartmentally and temporally restricted protective function that can be transferred to enhance systemic innate antifungal immunity.

CD22 is required for protection against West Nile virus Infection.

West Nile virus (WNV) is a RNA virus of the family Flaviviridae and the leading cause of mosquito-borne encephalitis in the United States. Humoral immunity is essential for protection against WNV infection; however, the requirements for initiating effective antibody responses against WNV infection are still unclear. CD22 (Siglec-2) is expressed on B cells and regulates B cell receptor signaling, cell survival, proliferation, and antibody production. In this study, we investigated how CD22 contributes to protection against WNV infection and found that CD22 knockout (Cd22(-/-)) mice were highly susceptible to WNV infection and had increased viral loads in the serum and central nervous system (CNS) compared to wild-type (WT) mice. This was not due to a defect in humoral immunity, as Cd22(-/-) mice had normal WNV-specific antibody responses. However, Cd22(-/-) mice had decreased WNV-specific CD8(+) T cell responses compared to those of WT mice. These defects were not simply due to reduced cytotoxic activity or increased cell death but, rather, were associated with decreased lymphocyte migration into the draining lymph nodes (dLNs) of infected Cd22(-/-) mice. Cd22(-/-) mice had reduced production of the chemokine CCL3 in the dLNs after infection, suggesting that CD22 affects chemotaxis via controlling chemokine production. CD22 was not restricted to B cells but was also expressed on a subset of splenic DCIR2(+) dendritic cells that rapidly expand early after WNV infection. Thus, CD22 plays an essential role in controlling WNV infection by governing cell migration and CD8(+) T cell responses.

Anti-CD180 (RP105) activates B cells to rapidly produce polyclonal Ig via a T cell and MyD88-independent pathway.

CD180 is homologous to TLR4 and regulates TLR4 signaling, yet its function is unclear. We report that injection of anti-CD180 mAb into mice induced rapid Ig production of all classes and subclasses, with the exception of IgA and IgG2b, with up to 50-fold increases in serum IgG1 and IgG3. IgG production after anti-CD180 injection was not due to reactivation of memory B cells and was retained in T cell-deficient (TCR knockout [KO]), CD40 KO, IL-4 KO, and MyD88 KO mice. Anti-CD180 rapidly increased both transitional and mature B cells, with especially robust increases in transitional B cell number, marginal zone B cell proliferation, and CD86, but not CD80, expression. In contrast, anti-CD40 induced primarily follicular B cell and myeloid expansion, with increases in expression of CD80 and CD95 but not CD86. The expansion of splenic B cells was due, in part, to proliferation and occurred in wild-type and TCR KO mice, whereas T cell expansion occurred in wild-type, but not in B cell-deficient, mice, indicating a direct role for B cells in CD180 stimulation in vivo. Combination of anti-CD180 with various MyD88-dependent TLR ligands biased B cell fate because coinjection diminished Ig production, but purified B cells exhibited synergistic proliferation. Anti-CD180 had no effect on cytokine production from B cells, but it increased IL-6, IL-10, and TNF-α production in combination with LPS or CpG. Thus, CD180 stimulation induces intrinsic B cell proliferation and differentiation, causing rapid increases in IgG, and integrates MyD88-dependent TLR signals to regulate proliferation, cytokine production, and differentiation.

During Aspergillus Infection, Monocyte-Derived DCs, Neutrophils, and Plasmacytoid DCs Enhance Innate Immune Defense through CXCR3-Dependent Crosstalk.

Aspergillus fumigatus, a ubiquitous mold, is a common cause of invasive aspergillosis (IA) in immunocompromised patients. Host defense against IA relies on lung-infiltrating neutrophils and monocyte-derived dendritic cells (Mo-DCs). Here, we demonstrate that plasmacytoid dendritic cells (pDCs), which are prototypically antiviral cells, participate in innate immune crosstalk underlying mucosal antifungal immunity. Aspergillus-infected murine Mo-DCs and neutrophils recruited pDCs to the lung by releasing the CXCR3 ligands, CXCL9 and CXCL10, in a Dectin-1 and Card9- and type I and III interferon signaling-dependent manner, respectively. During aspergillosis, circulating pDCs entered the lung in response to CXCR3-dependent signals. Via targeted pDC ablation, we found that pDCs were essential for host defense in the presence of normal neutrophil and Mo-DC numbers. Although interactions between pDC and fungal cells were not detected, pDCs regulated neutrophil NADPH oxidase activity and conidial killing. Thus, pDCs act as positive feedback amplifiers of neutrophil effector activity against inhaled mold conidia.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus.

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia.

Aspergillus fumigatus is a ubiquitous fungal pathogen that forms airborne conidia. The process of restricting conidial germination into hyphae by lung leukocytes is critical in determining infectious outcomes. Tracking the outcome of conidia-host cell encounters in vivo is technically challenging and an obstacle to understanding the molecular and cellular basis of antifungal immunity in the lung. Here, we describe a method that utilizes a genetically engineered Aspergillus strain [called FLARE (Jhingran et al., 2012; Espinosa et al., 2014; Heung et al., 2015)] to monitor conidial phagocytosis and killing by leukocytes within the lung environment at single encounter resolution.

Sex-specific effects of carotenoid intake on the immunological response to allografts in guppies (Poecilia reticulata).

Rarely are the evolutionary origins of mate preferences known, but, recently, the preference of female guppies (Poecilia reticulata) for males with carotenoid-based sexual coloration has been linked to a sensory bias that may have originally evolved for detecting carotenoid-rich fruits. If carotenoids enhance the immune systems of these fishes, as has been suggested for other species, this could explain the origin of the attraction to orange fruits as well as the maintenance of the female preference for orange males. We used the classic immunological technique of tissue grafting to assay a component of the immune response of guppies raised on two different dietary levels of carotenoids. Individual scales were transplanted between pairs of unrelated fishes, creating reciprocal allografts. Transplanted scales were scored on a six-point rejection scale every day for 10 days. Five days later, the same pairs of fishes received a second set of allografts and were scored again. Compared with low-carotenoid-diet males, high-carotenoid-diet males mounted a significantly stronger rejection response to the second allograft but not to the first allograft. High-carotenoid-diet females, however, showed no improvement in graft rejection compared with low-carotenoid-diet females. To our knowledge, this is the first experimental evidence for sex-specific effects of carotenoid consumption on the immune system of a species with carotenoid-based sexual coloration. These results are consistent with the hypothesis that the mate preference for carotenoid coloration is maintained by the benefits to females of choosing healthy mates, but they cast doubt on the idea that the benefits of carotenoid consumption, per se, could account for the origin of the preference. The sex-specificity of carotenoid effects on allograft rejection in guppies provides indirect support for the general hypothesis that males pay an immunological cost for sexual ornamentation.

Enhanced antibacterial activity in Hydra polyps lacking nerve cells.

The nervous system evolved within cnidarians. When assessing antibacterial activity in the freshwater polyp Hydra, we observed a strong correlation between the number of neurons present and the antibacterial activity. Tissue lacking neurons had a drastically enhanced antibacterial activity against Gram-positive (Bacillus subtilis) and Gram-negative (E. coli) bacteria compared to control tissue. The results indicate direct and strong neural influences on immunity in the phylogenetically oldest animals having a nervous system.

Dendritic cell-associated lectin 2 (DCAL2) defines a distinct CD8α- dendritic cell subset.

CLRs on DCs play important roles in immunity and are expressed selectively on certain DC subsets. Murine DCAL2 (myeloid inhibitory C-type lectin/Clec12a) is a type-II CLR with an ITIM. Using a mouse DCAL2-specific mAb, we found that DCAL2 is expressed at relatively high levels on APCs and that DCAL2 expression can be used to divide CD8α- DCs into DCAL2+DCIR2- and DCAL2-DCIR2+ subpopulations. CD8α-DCAL2+ DC, CD8α-DCIR2+ DC, and CD8α+DCAL2+ DC subsets each express different levels of TLRs and respond to unique classes of TLR ligands by producing distinct sets of cytokines. Whereas CD8α-DCAL2+ DCs robustly produce cytokines, including IL-12, in response to CpG, CD8α-DCIR2+ DCs produce only TNF-α and IL-10 in modest amounts when stimulated with zymosan. However, CD8α-DCIR2+DCs, unlike the other DC subsets, strongly up-regulate OX40L when stimulated with bacterial flagellin. As predicted from their cytokine expression, CD8α-DCAL2+ DCs efficiently induced Th1 responses in the presence of CpG in vitro and in vivo, whereas CD8α-DCIR2+ DCs induced Th2 cells in response to flagellin. Thus, CD8α-DCAL2+ DCs comprise a distinct CD8α- DC subset capable of supporting Th1 responses. DCAL2 is a useful marker to identify a Th1-inducing CD8α- DC population.

Sex-specific effects of carotenoid intake on the immunological response to allografts in guppies (Poecilia reticulata)

Rarely are the evolutionary origins of mate preferences known, but, recently, the preference of female guppies (Poecilia reticulata) for males with carotenoid-based sexual coloration has been linked to a sensory bias that may have originally evolved for detecting carotenoid-rich fruits. If carotenoids enhance the immune systems of these fishes, as has been suggested for other species, this could explain the origin of the attraction to orange fruits as well as the maintenance of the female preference for orange males. We used the classic immunological technique of tissue grafting to assay a component of the immune response of guppies raised on two different dietary levels of carotenoids. Individual scales were transplanted between pairs of unrelated fishes, creating reciprocal allografts. Transplanted scales were scored on a six-point rejection scale every day for 10 days. Five days later, the same pairs of fishes received a second set of allografts and were scored again. Compared with low-carotenoid-diet males, high-carotenoid-diet males mounted a significantly stronger rejection response to the second allograft but not to the first allograft. High-carotenoid-diet females, however, showed no improvement in graft rejection compared with low-carotenoid-diet females. To our knowledge, this is the first experimental evidence for sex-specific effects of carotenoid consumption on the immune system of a species with carotenoid-based sexual coloration. These results are consistent with the hypothesis that the mate preference for carotenoid coloration is maintained by the benefits to females of choosing healthy mates, but they cast doubt on the idea that the benefits of carotenoid consumption, per se, could account for the origin of the preference. The sex-specificity of carotenoid effects on allograft rejection in guppies provides indirect support for the general hypothesis that males pay an immunological cost for sexual ornamentation.