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Fungal pathogens are underappreciated causes of significant morbidity and mortality worldwide. In previous studies, we determined that a heat-killed, Cryptococcus neoformans fbp1-deficient strain (HK-fbp1) is a potent vaccine candidate. We determined that vaccination with HK-fbp1 confers protective immunity against lethal Cryptococcosis in an interferon γ (IFNγ)-dependent manner. In this study, we set out to uncover cellular sources and relevant targets of the protective effects of IFNγ in response to the HK-fbp1 vaccine. We found that early IFNγ production peaks at day 3 and that monocytes and neutrophils are important sources of this cytokine after vaccination. Neutralization of IFNγ at day 3 results in impaired CCR2 + monocyte recruitment and reduced differentiation into monocyte-derived dendritic cells (Mo-DC). In turn, depletion of CCR2 + cells prior to immunization results in impaired activation of IFNγ-producing CD4 and CD8 T cells. Thus, monocytes are important targets of innate IFNγ and help promote further IFNγ production by lymphocytes. We employed monocyte-fate mapper and conditional STAT1 knockout mice to uncover that STAT1 activation in CD11c + cells, including alveolar macrophages, Mo-DCs, and monocyte-derived macrophages (Mo-Mac) is essential for HK-fbp1 vaccine-induced protection. Altogether, our aggregate findings suggest critical roles for innate cells as orchestrators of vaccine-induced protection against Cryptococcus infection.IMPORTANCEThe number of patients susceptible to invasive fungal infections across the world continues to rise at an alarming pace yet current antifungal drugs are often inadequate. Immune-based interventions and novel antifungal vaccines hold the promise of significantly improving patient outcomes. In previous studies, we identified a Cryptococcus neoformans mutant strain (Fbp1-deficient) as a potent, heat-inactivated vaccine candidate capable of inducing homologous and heterologous antifungal protection. In this study, we used a combination of methods together with a cohort of conditional knockout mouse strains to interrogate the roles of innate cells in the orchestration of vaccine-induced antifungal protection. We uncovered novel roles for neutrophils and monocytes as coordinators of a STAT1-dependent cascade of responses that mediate vaccine-induced protection against invasive cryptococcosis. This new knowledge will help guide the future development of much-needed antifungal vaccines., Competing Interests: The authors declare no conflict of interest.

The detrimental impact of fungal infections to human health has steadily increased over the past decades. In October of 2022, the World Health Organization published the first ever fungal-pathogen priority list highlighting increased awareness of this problem, and the need for more research in this area. There were four distinct fungal pathogens identified as critical priority groups with Aspergillus fumigatus (Af) being the only mold. Af is a common environmental fungus responsible for over 90% of invasive aspergillosis cases worldwide. Pulmonary protection against Af is critically dependent on innate effector cells with essential roles played by neutrophils and monocytes. In this review, we will summarize our current understanding of how monocytes help orchestrate antifungal defense against Af., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Neutrophils are critical for the direct eradication of Aspergillus fumigatus conidia, but whether they mediate antifungal defense beyond their role as effectors is unclear. In this study, we demonstrate that neutrophil depletion impairs the activation of protective antifungal CCR2 + inflammatory monocytes. In the absence of neutrophils, monocytes displayed limited differentiation into monocyte-derived dendritic cells, reduced formation of reactive oxygen species, and diminished conidiacidal activity. Upstream regulator analysis of the transcriptional response in monocytes predicted a loss of STAT1-dependent signals as the potential basis for the dysfunction seen in neutrophil-depleted mice. We find that conditional removal of STAT1 on CCR2 + cells results in diminished antifungal monocyte responses, whereas exogenous administration of IFN-γ to neutrophil-depleted mice restores monocyte-derived dendritic cell maturation and reactive oxygen species production. Altogether, our findings support a critical role for neutrophils in antifungal immunity not only as effectors but also as important contributors to antifungal monocyte activation, in part by regulating STAT1-dependent functions., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Neuroimmune interactions are crucial for regulating immunity and inflammation. Recent studies have revealed that the central nervous system (CNS) senses peripheral inflammation and responds by releasing molecules that limit immune cell activation, thereby promoting tolerance and tissue integrity. However, the extent to which this is a bidirectional process, and whether peripheral immune cells also promote tolerance mechanisms in the CNS remains poorly defined. Here we report that helminth-induced type 2 inflammation promotes monocyte responses in the brain that are required to inhibit excessive microglial activation and host death. Mechanistically, infection-induced monocytes express YM1 that is sufficient to inhibit tumor necrosis factor production from activated microglia. Importantly, neuroprotective monocytes persist in the brain, and infected mice are protected from subsequent lipopolysaccharide-induced neuroinflammation months after infection-induced inflammation has resolved. These studies demonstrate that infiltrating monocytes promote CNS homeostasis in response to inflammation in the periphery and demonstrate that a peripheral infection can alter the immunologic landscape of the host brain.

Macrophages are known to mediate anti-helminth responses, but it remains uncertain which subsets are involved or how macrophages actually kill helminths. Here, we show rapid monocyte recruitment to the lung after infection with the nematode parasite Nippostrongylus brasiliensis. In this inflamed tissue microenvironment, these monocytes differentiate into an alveolar macrophage (AM)-like phenotype, expressing both SiglecF and CD11c, surround invading parasitic larvae, and preferentially kill parasites in vitro. Monocyte-derived AMs (Mo-AMs) express type 2-associated markers and show a distinct remodeling of the chromatin landscape relative to tissue-derived AMs (TD-AMs). In particular, they express high amounts of arginase-1 (Arg1), which we demonstrate mediates helminth killing through L-arginine depletion. These studies indicate that recruited monocytes are selectively programmed in the pulmonary environment to express AM markers and an anti-helminth phenotype., Competing Interests: Declaration of interests M.C.S. is the founder and President of NemaGen Discoveries., (Copyright © 2021 Rutgers University. Published by Elsevier Inc. All rights reserved.)

The etiology of ulcerative colitis is poorly understood and is likely to involve perturbation of the complex interactions between the mucosal immune system and the commensal bacteria of the gut, with cytokines acting as important cross-regulators. Here we use IFN receptor-deficient mice in a dextran sulfate sodium (DSS) model of acute intestinal injury to study the contributions of type I and III interferons (IFN) to the initiation, progression and resolution of acute colitis. We find that mice lacking both types of IFN receptors exhibit enhanced barrier destruction, extensive loss of goblet cells and diminished proliferation of epithelial cells in the colon following DSS-induced damage. Impaired mucosal healing in double IFN receptor-deficient mice is driven by decreased amphiregulin expression, which IFN signaling can up-regulate in either the epithelial or hematopoietic compartment. Together, these data underscore the pleiotropic functions of IFNs and demonstrate that these critical antiviral cytokines also support epithelial regeneration following acute colonic injury.

RIG-I-like receptors (RLR) are cytosolic RNA sensors that signal through the MAVS adaptor to activate IFN responses against viruses. Whether the RLR family has broader effects on host immunity against other pathogen families remains to be fully explored. In this study, we demonstrate that MDA5/MAVS signaling was essential for host resistance against pulmonary Aspergillus fumigatus challenge through the regulation of antifungal leukocyte responses in mice. Activation of MDA5/MAVS signaling was driven by dsRNA from live A. fumigatus serving as a key vitality-sensing pattern recognition receptor. Interestingly, induction of type I IFNs after A. fumigatus challenge was only partially dependent on MDA5/MAVS signaling, whereas type III IFN expression was entirely dependent on MDA5/MAVS signaling. Ultimately, type I and III IFN signaling drove the expression of CXCL10. Furthermore, the MDA5/MAVS-dependent IFN response was critical for the induction of optimal antifungal neutrophil killing of A. fumigatus spores. In conclusion, our data broaden the role of the RLR family to include a role in regulating antifungal immunity against A. fumigatus ., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Pulmonary infections with Aspergillus fumigatus ( Af ) are a significant cause of invasive fungal disease and lead to high morbidity and mortality in diverse populations throughout the world. Currently available antifungal drugs are often ineffective, thus contributing to unacceptably high mortality rates in patients suffering from invasive fungal infections. The use of cytokines as adjunctive immune therapies holds the promise of significantly improving patient outcomes in the future. In recent studies, we identified an essential role for type I and III interferons as regulators of optimal antifungal responses by pulmonary neutrophils during infection with Af . Although various membrane and cytosolic nucleic acid sensors are known to regulate interferon production in response to viruses, the pathways that regulate the production of these cytokines during fungal infection remain uncovered. In the current study, we demonstrate that dectin-1-mediated recognition of β-glucan on the cell wall of the clinically relevant fungal pathogen Aspergillus fumigatus promotes the activation of a protective cascade of type I and III interferon expression. We further demonstrate that exogenous administration of type I and III interferons can rescue inadequate antifungal responses in dectin-1 -/- mice, suggesting the potential therapeutic benefit of these cytokines as activators of antifungal defense in the context of innate defects., (Copyright © 2020 Dutta, Espinosa, Wang, Avina and Rivera.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cryptococcus neoformans is the main etiologic agent of cryptococcal meningitis and causes a significant number of deadly infections per year. Although it is well appreciated that host immune responses are crucial for defense against cryptococcosis, our understanding of factors that control the development of effective immunity to this fungus remains incomplete. In previous studies, we identified the F-box protein Fbp1 as a novel determinant of C. neoformans virulence. In this study, we found that the hypovirulence of the fbp1 Δ mutant is linked to the development of a robust host immune response. Infection with the fbp1 Δ mutant induces a rapid influx of CCR2 + monocytes and their differentiation into monocyte-derived dendritic cells (mo-DCs). Depletion of CCR2 + monocytes and their derivative mo-DCs resulted in impaired activation of a protective inflammatory response and the rapid death of mice infected with the fbp1 Δ mutant. Mice lacking B and T cells also developed fungal meningitis and succumbed to infection with the fbp1 Δ mutant, demonstrating that adaptive immune responses to the fbp1 Δ mutant help to maintain the long-term survival of the host. Adaptive immune responses to the fbp1 Δ mutant were characterized by enhanced differentiation of Th1 and Th17 CD4 + T cells together with diminished Th2 responses compared to the H99 parental strain. Importantly, we found that the enhanced immunogenicity of fbp1 Δ mutant yeast cells can be harnessed to confer protection against a subsequent infection with the virulent H99 parental strain. Altogether, our findings suggest that Fbp1 functions as a novel virulence factor that shapes the immunogenicity of C. neoformans IMPORTANCE Cryptococcus neoformans is the most common cause of deadly fungal meningitis, with over 270,000 infections per year. Immune responses are critically required for the prevention of cryptococcosis, and patients with impaired immunity and low CD4 + T cell numbers are at high risk of developing these deadly infections. Although it is well appreciated that the development of protective immunity is shaped by the interactions of the host immune system with fungal cells, our understanding of fungal products that influence this process remains poor. In this study, we found that the activity of F-box protein 1 (Fbp1) in highly virulent C. neoformans clinical strain H99 shapes its immunogenicity and thus affects the development of protective immune responses in the host. The identification of this new mechanism of virulence may facilitate the future development of therapeutic interventions aimed at boosting antifungal host immunity., (Copyright © 2018 Masso-Silva et al.)

Type III interferons (IFN-λs) are the most recently found members of the IFN cytokine family and engage IFNLR1 and IL10R2 receptor subunits to activate innate responses against viruses. We have identified IFN-λs as critical instructors of antifungal neutrophil responses. Using Aspergillus fumigatus ( Af ) as a model to study antifungal immune responses, we found that depletion of CCR2 + monocytes compromised the ability of neutrophils to control invasive fungal growth. Using an unbiased approach, we identified type I and III IFNs as critical regulators of the interplay between monocytes and neutrophils responding to Af We found that CCR2 + monocytes are an important early source of type I IFNs that prime optimal expression of IFN-λ. Type III IFNs act directly on neutrophils to activate their antifungal response, and mice with neutrophil-specific deletion of IFNLR1 succumb to invasive aspergillosis. Dysfunctional neutrophil responses in CCR2-depleted mice were rescued by adoptive transfer of pulmonary CCR2 + monocytes or by exogenous administration of IFN-α and IFN-λ. Thus, CCR2 + monocytes promote optimal activation of antifungal neutrophils by initiating a coordinated IFN response. We have identified type III IFNs as critical regulators of neutrophil activation and type I IFNs as early stimulators of IFN-λ expression., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cells contribute to type 2 cytokine-mediated inflammation through their enhanced ability to develop into mast cells. In this study, we show that carbonic anhydrase (Car) enzymes are up-regulated in type 2-associated progenitor cells and demonstrate that Car enzyme inhibition is sufficient to prevent mouse mast cell responses and inflammation after Trichinella spiralis infection or the induction of food allergy-like disease. Further, we used CRISPR/Cas9 technology and illustrate that genetically editing Car1 is sufficient to selectively reduce mast cell development. Finally, we demonstrate that Car enzymes can be targeted to prevent human mast cell development. Collectively, these experiments identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that Car enzyme inhibitors may possess therapeutic potential that can be used to treat mast cell-mediated inflammation., (© 2016 Henry et al.)

Mycotic infections and their effect on the human condition have been widely overlooked and poorly surveilled by many health organizations even though mortality rates have increased in recent years. The increased usage of immunosuppressive and myeloablative therapies for the treatment of malignant as well as non-malignant diseases has contributed significantly to the increased incidence of fungal infections. Invasive fungal infections have been found to be responsible for at least 1.5 million deaths worldwide. About 90% of these deaths can be attributed to Cryptococcus, Candida, Aspergillus, and Pneumocystis. A better understanding of how the host immune system contains fungal infection is likely to facilitate the development of much needed novel antifungal therapies. Innate cells are responsible for the rapid recognition and containment of fungal infections and have been found to play essential roles in defense against multiple fungal pathogens. In this review we summarize our current understanding of host-fungi interactions with a focus on mechanisms of innate cell-mediated recognition and control of pulmonary aspergillosis.

Allergic asthma is a chronic respiratory disease that results from an exaggerated inflammatory response in the airways. Environment stimuli, such as pollen and HDM, cause activation and migration of inflammatory WBCs into the respiratory tract, where they cause lung damage. Migration of these WBCs is dependent on the active configuration of the β2 integrin LFA-1. The experimental therapeutic agent LtxA specifically targets active LFA-1 and causes cell death. We investigated the association between LFA-1 and allergic asthma and hypothesized that targeting LFA-1 with LtxA could be an attractive strategy for treatment of the condition. We examined LFA-1 (CD11a) levels on PBMCs from patients with allergic asthma compared with healthy controls. Patients exhibited a significantly higher percentage of PBMCs expressing LFA-1 than healthy controls. Furthermore, the level of LFA-1 expression on patient PBMCs was greater than on healthy PBMCs. We identified a unique cellular population in patients that consisted of CD4(-) CD11a(hi) cells. We also evaluated LtxA in a HDM extract-induced mouse model for allergic asthma. LtxA caused resolution of disease in mice, as demonstrated by a decrease in BALF WBCs, a reduction in pulmonary inflammation and tissue remodeling, and a decrease in proinflammatory cytokines IL-4, IL-5, IL-9, IL-17F, and IL-23α in lung tissue. LFA-1 may serve as an important marker in allergic asthma, and the elimination of activated WBCs by use of LtxA could be a viable therapeutic strategy for treating patients with this condition., (© Society for Leukocyte Biology.)

Aspergillus fumigatus is a mold that causes severe pulmonary infections. Our knowledge of how A. fumigatus growth is controlled in the respiratory tract is developing, but still limited. Alveolar macrophages, lung resident macrophages, and airway epithelial cells constitute the first lines of defense against inhaled A. fumigatus conidia. Subsequently, neutrophils and inflammatory CCR2+ monocytes are recruited to the respiratory tract to prevent fungal growth. However, the mechanism of neutrophil and macrophage recruitment to the respiratory tract after A. fumigatus exposure remains an area of ongoing investigation. Here we show that A. fumigatus pulmonary challenge induces expression of the inflammasome-dependent cytokines IL-1β and IL-18 within the first 12 hours, while IL-1α expression continually increases over at least the first 48 hours. Strikingly, Il1r1-deficient mice are highly susceptible to pulmonary A. fumigatus challenge exemplified by robust fungal proliferation in the lung parenchyma. Enhanced susceptibility of Il1r1-deficient mice correlated with defects in leukocyte recruitment and anti-fungal activity. Importantly, IL-1α rather than IL-1β was crucial for optimal leukocyte recruitment. IL-1α signaling enhanced the production of CXCL1. Moreover, CCR2+ monocytes are required for optimal early IL-1α and CXCL1 expression in the lungs, as selective depletion of these cells resulted in their diminished expression, which in turn regulated the early accumulation of neutrophils in the lung after A. fumigatus challenge. Enhancement of pulmonary neutrophil recruitment and anti-fungal activity by CXCL1 treatment could limit fungal growth in the absence of IL-1α signaling. In contrast to the role of IL-1α in neutrophil recruitment, the inflammasome and IL-1β were only essential for optimal activation of anti-fungal activity of macrophages. As such, Pycard-deficient mice are mildly susceptible to A. fumigatus infection. Taken together, our data reveal central, non-redundant roles for IL-1α and IL-1β in controlling A. fumigatus infection in the murine 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.

CD4 T cells play important and non-redundant roles in protection against infection with diverse fungi. Distinct CD4 T cell subsets can mediate protection against fungal disease where Th1 and Th17 CD4 T cell subsets have been found to promote fungal clearance and protective immunity against diverse fungal pathogens. The differentiation of naïve CD4 T cells into Th1 or Th17 cells is crucially controlled by their interaction with dendritic cells and instructed by cytokines. IL-12 and IFN-γ promote Th1 differentiation while TGF-β, IL-6, IL-1, IL-21 and IL-23 promote Th17 differentiation and maintenance. The production of these cytokines by DCs is in turn regulated by innate receptors triggered in response to fungal infection. In this review we will discuss the contributions of cytokines found to influence fungus-specific CD4 T cell differentiation and their role in defense against fungal disease. We will also highlight the contributions of innate receptors involved in recognition of fungi and how they shape cytokine secretion and CD4 T cell differentiation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)