Tobias M. Hohl - US grants

[unreadable] DESCRIPTION (provided by applicant): This proposal describes a five-year program for the development of a laboratory-based academic career in Infectious Diseases. The candidate, Tobias Hohl, MD, PhD, a fellow in Infectious Diseases, will conduct the program under the supervision of Eric Pamer, MD, Chief of the Infectious Diseases Service at Memorial Sloan-Kettering Cancer Center, and study innate and adaptive immune responses to Aspergillus fumigatus, the most common invasive mold infection in immune compromised patients. The candidate has enlisted the support of an advisory committee of well-known scientists to provide scientific guidance and advice in career development. The research plan is guided by the hypothesis that inflammatory monocytes, a subset of white blood cells, play a pivotal role in host defense against A. fumigatus and that deficiency or dysfunction of these cells promotes pathologic disease states. Two transgenic mouse strains have been developed to test this hypothesis. The first strain labels inflammatory monocytes with a fluorescent protein. The second strain permits toxin-induced ablation of these cells. These mouse strains provide the experimental foundation for the specific aims of this proposal, as follows: (1) to determine the mechanism of pulmonary inflammatory monocyte recruitment during A. fumigatus infection, (2) to examine the effect of inflammatory monocyte depletion on the outcome of respiratory fungal infection and host immune defense mechanisms against A. fumigatus, and (3) to analyze the protective function of inflammatory monocytes during invasive aspergillosis through cell-based adoptive transfer studies. These experiments will provide novel insight into host defenses against fungal spores and invasive disease and provide an experimental basis for adoptive cell transfer experiments to alter the outcome of invasive infection in the most vulnerable host settings. The Infectious Diseases Service and the Immunology Program at MSKCC provide an ideal setting for the candidate to accomplish these goals, develop a scientific identity, and establish the basis for a career in academic medicine. [unreadable] [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Aspergillus fumigatus is pathogenic fungus that humans inhale on a daily basis. The ability of the respiratory innate immune system to prevent the germination of inhaled conidia (vegetative spores) into tissue-invasive hyphae represents a critical immunologic checkpoint. Due to advances in medical technology, there has been a significant rise in patients that live in immune compromised conditions, leading to an estimated 200,000 cases of aspergillosis annually. In high-risk groups, mortality rates remain 25-50% despite contemporary combination antifungal therapies. These trends highlight the need for an improved understanding of the molecular and cellular basis of sterilizing immunity to advance immune-based adjunctive approaches. In the initial funding period of this project we introduced a novel fungal bioreporter, termed fluorescent Aspergillus reporter (FLARE), to trace the viability of conidia during cellular interactions with immune cells in the lung. Using this approach, we identified spleen tyrosine kinase (Syk) as essential for innate defense in the lung, and in pilot studies, show that Syk acts in a cell-extrinsic manner to regulate neutrophil fungicidal activity. Although Syk can integrate signals from C-type lectin receptors, Fc receptors, and integrins in myeloid cells, its essential cellular source and fungus-induced activation signals during respiratory fungal challenge remain poorly defined. Beyond neutrophils, our studies show that -CC- chemokine receptor 2 (CCR2)-expressing inflammatory monocytes (IMs) form a second essential innate effector cell during respiratory challenge. In the lung, IMs differentiate into monocyte-derived dendritic cells (Mo-DCs), coincident with direct conidial uptake and killing, and engage in intercellular crosstalk with lung neutrophils to boost antifungal activity in the lun. In pilot studies, transcriptional profiling of IM/Mo-DCs and lung proteomic analyses identified fiv candidate mediators of intercellular crosstalk and the respiratory burst as a candidate effector mechanism. During the next project period, we propose to gain a deeper understanding of IM/MoDC - neutrophil intercellular crosstalk, its induction and regulation by Syk activity, its relevant molecular transmitters, and its role in anticonidial defense in the lung. In Aim 1, we identify protective mechanisms of Syk signaling by identifying its essential cellular source and by determining essential upstream adaptor proteins and receptors that mediate conidial uptake and killing in the lung. In Aim 2, we will define reciprocal regulation of IM/Mo-DC and neutrophil antifungal activity by establishing the cellular and trafficking requirements, induction signals, ad directional nature of intercellular crosstalk. In Aim 3, we will define molecular transmitters and effector systems of IM/Mo-DC-neutrophil crosstalk. The proposed studies are significant and innovative because they identify innate immune crosstalk between IMs and neutrophils as a novel mechanism to regulate antifungal immunity in the lung. Understanding the induction, regulation, and participants of innate immune crosstalk addresses a critical knowledge gap that will inform immune-enhancing strategies in vulnerable patient groups.

DESCRIPTION (provided by applicant): Airborne conidia (spores) represent infectious propagules that are responsible for transmission of major human mycoses. Humans inhale ubiquitous Aspergillus fumigatus conidia on a daily basis. Conidial germination into tissue-invasive hyphae leads to invasive aspergillosis (IA), a devastating cause of infectious morbidity and mortality in patients with impaired respiratory innate immune function. Although conidial engulfment and killing are hallmarks of alveolar macrophage (AM) function during respiratory infection, antifungal effector mechanisms of these cells remain poorly defined, since assays that measure alveolar macrophage conidiacidial activity in the lung have been difficult to achieve. To dissect AM conidiacidal activity, we developed a fluorescent Aspergillus reporter (FLARE) strain that, upon conidial uptake, tags AMs with a fluorescent signature. Conidial killing induces a change in the fluorescence signature, enabling us to observe and quantify cell type-specific conidial killing in the lung and test tube. We harness the FLARE strain to demonstrate that AMs employ distinct conidiacidal mechanisms and implicate matrix metalloprotease 12 (MMP12) in this process. With this approach, we examine a model of AM function that integrates matrix metalloprotease 12 as a significant cytotoxic effector mechanism to achieve fungicidal activity. The hypothesis that underlies this proposal is that AM-specific factors - that include MMP12 - control A. fumigatus conidial germination at the earliest stages of respiratory fungal infection. The aims will (1) investigate MMP12 as a major effector of AM conidiacidal activity and (2) integrate alternate AM conidiacidal mechanisms using a functional RNAi-based approach. The experimental design incorporates both a candidate gene (MMP12) and a systematic discovery approach to dissect AM antifungal activity. These studies will define matrix metalloproteolytic activity as a potential novel antifungal effector mechanism and will identify candidate genes that regulate the full spectrum of AM conidiacidal activity.

PROJECT SUMMARY Humans inhale fungal conidia (i.e, vegetative spores) on a daily basis. The ability of the respiratory innate immune system to prevent germination of inhaled conidia into tissue-invasive hyphae represents a critical immunologic checkpoint. Using Aspergillus fumigatus, the most common etiologic agent of invasive aspergillosis, as a model system for human fungal pathogens, we discovered that conidia undergo programmed cell death with apoptosis-like features during interactions with innate immune cells. This finding was facilitated by a novel fluorescent reporter of fungal physiology that enables visualization and quantitation of fungal apoptosis markers, including histone degradation, caspase activation, and DNA fragmentation. Our work demonstrates that A. fumigatus conidia express an essential and druggable anti-apoptotic protein, termed Bir1, that counters host induction of apoptosis-like programmed cell death by the action of phagocyte NADPH oxidase. Genetic and pharmacologic studies demonstrate that Bir1 expression and activity underlie conidial susceptibility to host apoptosis-like programmed cell death, and in turn, host susceptibility to invasive aspergillosis. These findings indicate that mammalian fungal immune surveillance exploits a fungal apoptosis- like programmed cell death pathway to maintain barrier immunity in the lung. In this collaborative proposal with two co-investigators, we seek to determine the mechanism through which Bir1 regulates anti-apoptotic activity during fungal-host cell encounters. Our preliminary data support a model in which Bir1 exerts anti-apoptotic activity via two conserved BIR domains, underlies post-translational regulation in response to pro-apoptotic stress, regulates candidate fungal caspase-like enzymes as apoptosis effectors, and demonstrates functional conservation across human pathogenic fungi. Based on these observations, our model predicts that fungal apoptosis-like programmed cell death is a general feature of fungal-host cell encounters and central to the establishment of invasive fungal disease. We explore this model in the following aims: (1) define the functional domains and post-translational regulation of Bir1 critical for resistance to host induction of apoptosis-like programmed cell death, (2) define the mechanism of Bir1- mediated resistance to host induction of apoptosis-like programmed cell death, with an emphasis on regulation of a candidate fungal caspase-like activity, and (3) define the role of apoptosis-like programmed cell death and Bir1 homologs following Aspergillus nidulans and Candida albicans challenge. The proposed studies are significant and innovative because they identify a novel mechanism of immune surveillance and demonstrate that higher eukaryotes can exploit programmed cell death in lower eukaryotes for the purpose of sterilizing immunity. This work will provide a mechanistic understanding of Bir1 function in regulating host-fungal encounters. Knowledge gained from these studies will inform strategies that target fungal Bir1 homologs and exploit fungal apoptosis-like programmed cell death for therapeutic gain.

PROJECT SUMMARY Aspergillus fumigatus is the most common etiologic agent of invasive aspergillosis, a devastating infection in patients with hematologic malignancies and in hematopoietic cell transplant recipients. Classically, patients with prolonged quantitative or qualitative defects in neutrophil and/or monocyte function are at risk for invasive aspergillosis. However, recent advances in targeted lymphoma therapies have resulted in new at-risk patient groups, particularly if a novel agent targets a signal transduction pathway implicated in cancer cell proliferation as well as immune surveillance. In a clinical study, we identified ibrutinib (IBT) as a risk factor for invasive fungal infections, and invasive aspergillosis in particular. Although IBT targets Bruton?s tyrosine kinase (BTK) to interrupt tonic B cell receptor signaling in malignant cells, IBT therapy leads to a defect in fungal immune surveillance, though the underlying molecular and cellular mechanisms remain undefined. BTK knockout mice are susceptible to A. fumigatus challenge and preliminary studies in mice and in human neutrophils suggest that IBT disrupts myeloid cell antifungal activity, in part by interfering with fungal cell phagocytosis. A critical knowledge gap relates to BTK function in myeloid cells and its role in mediating sterilizing antifungal immunity. The central hypothesis that underlies this proposal is that BTK signaling in myeloid cells is essential to couple A. fumigatus recognition to innate immune activation and fungal eradication. This model predicts that IBT impairs this immune surveillance function independent of its effects on B cells. To examine this model, we harness a unique fluorescent fungal reporter strain that monitors fungal uptake and viability during cellular encounters with murine leukocytes in the lung and human leukocytes in vitro and utilize gene knockout mice and a conditional gene targeting strategy to target myeloid cells that respond to, engulf, and kill conidia. We explore this model in the following aims: (1) define the fungal immune surveillance function and essential cellular source of BTK signaling in the lung, and (2) define the fungal immune surveillance defect caused by IBT administration in the lung and in human leukocytes. The proposed studies are significant and innovative because they translate an unexpected clinical observation into a systematic approach to decipher how a novel precision pharmaceutical compound interferes with a central pathway of fungal immune surveillance. Insight into the IBT-dependent fungal immune surveillance defect is likely to inform screening and prophylactic strategies for at-risk patients.

PROJECT SUMMARY. Despite advances in allogeneic hematopoietic cell transplantation (allo-HCT), transplant-related morbidity and mortality remain substantial barriers to improving clinical outcomes. A recent body of work introduced the concept that shifts in the composition and density of the human intestinal bacterial microbiota shapes allo-HCT outcomes, including overall survival, non-relapse mortality, acute graft-versus-host disease (GvH), and invasive bacterial infections. A major knowledge gap relates to the role of non-bacterial microbiota constituents in allo-HCT outcomes. We established a high-resolution bioinformatics pipeline to analyze the role of intestinal fungi, the mycobiota, in allo-HCT outcomes. In pilot studies, we discovered that intestinal fungal pathobionts, specifically Candida species, can dominate the microbiota and translocate across the intestinal mucosa to cause invasive fungal bloodstream infections. In a murine major antigen mismatch allo-HCT model we found that Candida intestinal colonization exacerbates transplant outcomes while drug-induced elimination of intestinal fungi from the gut ameliorates transplant outcomes, respectively. However, how the composition, diversity, and magnitude of the mycobiota relates to human allo-HCT outcomes, specifically for overall and non-relapse survival, the development of GvH, and transplant complications, remains unknown. Based on these findings, the central hypothesis of this proposal is that human allo-HCT induces shifts in the intestinal mycobiota (in density, composition, and diversity) and promotes states of fungal dysbiosis that predict the risk of acute GvH, and overall and non-relapse mortality. To test this hypothesis, we have assembled a 287 patient allo-HCT study cohort (transplanted between January 1, 2017 and December 31, 2018) that will be followed for two years. This hypothesis will be explored in the following aims. Aim 1 will define the magnitude, composition, diversity, and stability of the intestinal mycobiota during human allo-HCT and identify the impact of antifungal drugs on mycobiota dynamics. Aim 2 will define the relationship between the intestinal mycobiota and a broad range of clinical outcomes in allo-HCT. The proposed study will measure the dynamics of the intestinal mycobiota during allo-HCT, define states of dysbiosis linked to transplant-related and supportive care practices, and define the relationship between shifts in the mycobiota and transplant outcomes. Insight into the role of mycobiota in allo-HCT outcomes will open opportunities to test mycobiota-based or mycobiota- perturbing interventions for clinical benefit. This exploratory study relies on a unique patient biorepository and innovative methodologies in mycobiota analyses. Beyond allo-HCT, the results of our study may have the potential to be highly informative for understanding the impact of the mycobiota in other organ transplant and immune compromised patient populations.