Multiparametric Profiling of Neutrophil Function via a High-Throughput Flow Cytometry-Based Assay.

Neutrophils are a vital component of the innate immune system and play an essential function in the recognition and clearance of bacterial and fungal pathogens. There is great interest in understanding mechanisms of neutrophil dysfunction in the setting of disease and deciphering potential side effects of immunomodulatory drugs on neutrophil function. We developed a high throughput flow cytometry-based assay for detecting changes to four canonical neutrophil functions following biological or chemical triggers. Our assay detects neutrophil phagocytosis, reactive oxygen species (ROS) generation, ectodomain shedding, and secondary granule release in a single reaction mixture. By selecting fluorescent markers with minimal spectral overlap, we merge four detection assays into one microtiter plate-based assay. We demonstrate the response to the fungal pathogen, Candida albicans and validate the assay's dynamic range using the inflammatory cytokines G-CSF, GM-CSF, TNFα, and IFNγ. All four cytokines increased ectodomain shedding and phagocytosis to a similar degree while GM-CSF and TNFα were more active in degranulation when compared to IFNγ and G-CSF. We further demonstrated the impact of small molecule inhibitors such as kinase inhibition downstream of Dectin-1, a critical lectin receptor responsible for fungal cell wall recognition. Bruton's tyrosine kinase (Btk), Spleen tyrosine kinase (Syk), and Src kinase inhibition suppressed all four measured neutrophil functions but all functions were restored with lipopolysaccharide co-stimulation. This new assay allows for multiple comparisons of effector functions and permits identification of distinct subpopulations of neutrophils with a spectrum of activity. Our assay also offers the potential for studying the intended and off-target effects of immunomodulatory drugs on neutrophil responses.

Transcellular biosynthesis of leukotriene B4 orchestrates neutrophil swarming to fungi.

Neutrophil swarming is an emergent host defense mechanism triggered by targets larger than a single neutrophil's capacity to phagocytose. Swarming synergizes neutrophil functions, including chemotaxis, phagocytosis, and reactive oxygen species (ROS) production, and coordinates their deployment by many interacting neutrophils. The potent inflammatory lipid mediator leukotriene B4 (LTB4) has been established as central to orchestrating neutrophil activities during swarming. However, the details regarding how this eicosanoid choreographs the neutrophils involved in swarming are not well explained. Here we leverage microfluidics, genetically deficient mouse cells, and targeted metabolipidomic analysis to demonstrate that transcellular biosynthesis occurs among neutrophils to generate LTB4. Furthermore, transcellular biosynthesis is an entirely sufficient means of generating LTB4 for the purposes of orchestrating neutrophil swarming. These results further our understanding of how neutrophils coordinate their activities during swarming, which will be critical in the design of eventual therapies that can harness the power of swarming behavior.

TLR Signaling Rescues Fungicidal Activity in Syk-Deficient Neutrophils.

An impaired neutrophil response to pathogenic fungi puts patients at risk for fungal infections with a high risk of morbidity and mortality. Acquired neutrophil dysfunction in the setting of iatrogenic immune modulators can include the inhibition of critical kinases such as spleen tyrosine kinase (Syk). In this study, we used an established system of conditionally immortalized mouse neutrophil progenitors to investigate the ability to augment Syk-deficient neutrophil function against Candida albicans with TLR agonist signaling. LPS, a known immunomodulatory molecule derived from Gram-negative bacteria, was capable of rescuing effector functions of Syk-deficient neutrophils, which are known to have poor fungicidal activity against Candida species. LPS priming of Syk-deficient mouse neutrophils demonstrates partial rescue of fungicidal activity, including phagocytosis, degranulation, and neutrophil swarming, but not reactive oxygen species production against C. albicans, in part due to c-Fos activation. Similarly, LPS priming of human neutrophils rescues fungicidal activity in the presence of pharmacologic inhibition of Syk and Bruton's tyrosine kinase (Btk), both critical kinases in the innate immune response to fungi. In vivo, neutropenic mice were reconstituted with wild-type or Syk-deficient neutrophils and challenged i.p. with C. albicans. In this model, LPS improved wild-type neutrophil homing to the fungal challenge, although Syk-deficient neutrophils did not persist in vivo, speaking to its crucial role on in vivo persistence. Taken together, we identify TLR signaling as an alternate activation pathway capable of partially restoring neutrophil effector function against Candida in a Syk-independent manner.

Baseline procalcitonin as a predictor of bacterial infection and clinical outcomes in COVID-19: A case-control study.

PURPOSE: Coronavirus disease-2019 (COVID-19) is associated with a wide spectrum of clinical symptoms including acute respiratory failure. Biomarkers that can predict outcomes in patients with COVID-19 can assist with patient management. The aim of this study is to evaluate whether procalcitonin (PCT) can predict clinical outcome and bacterial superinfection in patients infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). METHODS: Adult patients diagnosed with SARS-CoV-2 by nasopharyngeal PCR who were admitted to a tertiary care center in Boston, MA with SARS-CoV-2 infection between March 17 and April 30, 2020 with a baseline PCT value were studied. Patients who were presumed positive for SARS-CoV-2, who lacked PCT levels, or who had a positive urinalysis with negative cultures were excluded. Demographics, clinical and laboratory data were extracted from the electronic medical records. RESULTS: 324 patient charts were reviewed and grouped by clinical and microbiologic outcomes by day 28. Baseline PCT levels were significantly higher for patients who were treated for true bacteremia (p = 0.0005) and bacterial pneumonia (p = 0.00077) compared with the non-bacterial infection group. Baseline PCT positively correlated with the NIAID ordinal scale and survival over time. When compared to other inflammatory biomarkers, PCT showed superiority in predicting bacteremia. CONCLUSIONS: Baseline PCT levels are associated with outcome and bacterial superinfection in patients hospitalized with SARS-CoV-2.

tiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche.

Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.

Redundant Trojan horse and endothelial-circulatory mechanisms for host-mediated spread of Candida albicans yeast.

The host innate immune system has developed elegant processes for the detection and clearance of invasive fungal pathogens. These strategies may also aid in the spread of pathogens in vivo, although technical limitations have previously hindered our ability to view the host innate immune and endothelial cells to probe their roles in spreading disease. Here, we have leveraged zebrafish larvae as a model to view the interactions of these host processes with the fungal pathogen Candida albicans in vivo. We examined three potential host-mediated mechanisms of fungal spread: movement inside phagocytes in a "Trojan Horse" mechanism, inflammation-assisted spread, and endothelial barrier passage. Utilizing both chemical and genetic tools, we systematically tested the loss of neutrophils and macrophages and the loss of blood flow on yeast cell spread. Both neutrophils and macrophages respond to yeast-locked and wild type C. albicans in our model and time-lapse imaging revealed that macrophages can support yeast spread in a "Trojan Horse" mechanism. Surprisingly, loss of immune cells or inflammation does not alter dissemination dynamics. On the other hand, when blood flow is blocked, yeast can cross into blood vessels but they are limited in how far they travel. Blockade of both phagocytes and circulation reduces rates of dissemination and significantly limits the distance of fungal spread from the infection site. Together, this data suggests a redundant two-step process whereby (1) yeast cross the endothelium inside phagocytes or via direct uptake, and then (2) they utilize blood flow or phagocytes to travel to distant sites.

Candida albicans necrotizing fasciitis following elective surgery.

Necrotizing fasciitis is a potentially fatal soft tissue infection that requires prompt clinical suspicion, pharmacological and surgical interventions. Bacterial pathogens, such as beta-hemolytic streptococcus and Staphylococcus aureus, are the main etiology of necrotizing fasciitis, however, rare cases caused by fungal pathogens, such as Candida albicans, have been reported following trauma. Here, we present the first case of C. albicans necrotizing fasciitis following an elective surgical procedure in an immunocompetent adult.

Spleen Tyrosine Kinase Is a Critical Regulator of Neutrophil Responses to Candida Species.

Invasive fungal infections constitute a lethal threat, with patient mortality as high as 90%. The incidence of invasive fungal infections is increasing, especially in the setting of patients receiving immunomodulatory agents, chemotherapy, or immunosuppressive medications following solid-organ or bone marrow transplantation. In addition, inhibitors of spleen tyrosine kinase (Syk) have been recently developed for the treatment of patients with refractory autoimmune and hematologic indications. Neutrophils are the initial innate cellular responders to many types of pathogens, including invasive fungi. A central process governing neutrophil recognition of fungi is through lectin binding receptors, many of which rely on Syk for cellular activation. We previously demonstrated that Syk activation is essential for cellular activation, phagosomal maturation, and elimination of phagocytosed fungal pathogens in macrophages. Here, we used combined genetic and chemical inhibitor approaches to evaluate the importance of Syk in the response of neutrophils to Candida species. We took advantage of a Cas9-expressing neutrophil progenitor cell line to generate isogenic wild-type and Syk-deficient neutrophils. Syk-deficient neutrophils are unable to control the human pathogens Candida albicans, Candida glabrata, and Candida auris Neutrophil responses to Candida species, including the production of reactive oxygen species and of cytokines such as tumor necrosis factor alpha (TNF-α), the formation of neutrophil extracellular traps (NETs), phagocytosis, and neutrophil swarming, appear to be critically dependent on Syk. These results demonstrate an essential role for Syk in neutrophil responses to Candida species and raise concern for increased fungal infections with the development of Syk-modulating therapeutics.IMPORTANCE Neutrophils are recognized to represent significant immune cell mediators for the clearance and elimination of the human-pathogenic fungal pathogen Candida The sensing of fungi by innate cells is performed, in part, through lectin receptor recognition of cell wall components and downstream cellular activation by signaling components, including spleen tyrosine kinase (Syk). While the essential role of Syk in macrophages and dendritic cells is clear, there remains uncertainty with respect to its contribution in neutrophils. In this study, we demonstrated that Syk is critical for multiple cellular functions in neutrophils responding to major human-pathogenic Candida species. These data not only demonstrate the vital nature of Syk with respect to the control of fungi by neutrophils but also warn of the potential infectious complications arising from the recent clinical development of novel Syk inhibitors for hematologic and autoimmune disorders.

Yeast and Filaments Have Specialized, Independent Activities in a Zebrafish Model of Candida albicans Infection.

Candida albicans dimorphism is a crucial virulence factor during invasive candidiasis infections, which claim the lives of nearly one-half of those afflicted. It has long been believed that filaments drive tissue invasion and yeast mediates bloodstream dissemination, but observation of these activities during infection has been prevented by technical limitations. We used a transparent zebrafish infection model to analyze more comprehensively how C. albicans utilizes shape to disseminate and invade. This model facilitated the use of diverse, complementary strategies to manipulate shape, allowing us to monitor dissemination, invasion, and pathogenesis via intravital imaging of individual fungal cells throughout the host. To control fungal cell shape, we employed three different strategies: gene deletion (efg1Δ/Δ cph1Δ/Δ, eed1Δ/Δ), overexpression of master regulators (NRG1 or UME6), and modulation of the infection temperature (21°C, 28°C, or 33°C). The effects of these orthogonal manipulations were consistent, support the proposed specialized roles of yeast in dissemination and filaments in tissue invasion and pathogenesis, and indicate conserved mechanisms in zebrafish. To test if either morphotype changes the effectiveness of the other, we infected fish with a known mixture of shape-locked strains. Surprisingly, mixed-strain infections were associated with additive, but not synergistic, filament invasion and yeast dissemination. These findings provide the most complete view of morphotype-function relationships for C. albicans to date, revealing independent roles of yeast and filaments during disseminated candidiasis.

Glucose Homeostasis Is Important for Immune Cell Viability during Candida Challenge and Host Survival of Systemic Fungal Infection.

To fight infections, macrophages undergo a metabolic shift whereby increased glycolysis fuels antimicrobial inflammation and killing of pathogens. Here we demonstrate that the pathogen Candida albicans turns this metabolic reprogramming into an Achilles' heel for macrophages. During Candida-macrophage interactions intertwined metabolic shifts occur, with concomitant upregulation of glycolysis in both host and pathogen setting up glucose competition. Candida thrives on multiple carbon sources, but infected macrophages are metabolically trapped in glycolysis and depend on glucose for viability: Candida exploits this limitation by depleting glucose, triggering rapid macrophage death. Using pharmacological or genetic means to modulate glucose metabolism of host and/or pathogen, we show that Candida infection perturbs host glucose homeostasis in the murine candidemia model and demonstrate that glucose supplementation improves host outcomes. Our results support the importance of maintaining glucose homeostasis for immune cell survival during Candida challenge and for host survival in systemic infection.

Control of Mucosal Candidiasis in the Zebrafish Swim Bladder Depends on Neutrophils That Block Filament Invasion and Drive Extracellular-Trap Production.

Candida albicans is a ubiquitous mucosal commensal that is normally prevented from causing acute or chronic invasive disease. Neutrophils contribute to protection in oral infection but exacerbate vulvovaginal candidiasis. To dissect the role of neutrophils during mucosal candidiasis, we took advantage of a new, transparent zebrafish swim bladder infection model. Intravital microscopic tracking of individual animals revealed that the blocking of neutrophil recruitment leads to rapid mortality in this model through faster disease progression. Conversely, artificial recruitment of neutrophils during early infection reduces disease pressure. Noninvasive longitudinal tracking showed that mortality is a consequence of C. albicans breaching the epithelial barrier and invading surrounding tissues. Accordingly, we found that a hyperfilamentous C. albicans strain breaches the epithelial barrier more frequently and causes mortality in immunocompetent zebrafish. A lack of neutrophils at the infection site is associated with less fungus-associated extracellular DNA and less damage to fungal filaments, suggesting that neutrophil extracellular traps help to protect the epithelial barrier from C. albicans breach. We propose a homeostatic model where C. albicans disease pressure is balanced by neutrophil-mediated damage of fungi, maintaining this organism as a commensal while minimizing the risk of damage to host tissue. The unequaled ability to dissect infection dynamics at a high spatiotemporal resolution makes this zebrafish model a unique tool for understanding mucosal host-pathogen interactions.