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.

Cytokine Augmentation Reverses Transplant Recipient Neutrophil Dysfunction Against the Human Fungal Pathogen Candida albicans.

BACKGROUND: Solid organ transplant (SOT) and stem cell transplant (SCT) recipients are at increased risk of invasive fungal disease despite normal neutrophil counts. Here, we measure neutrophil anti-Candida activity. METHODS: Twenty-one SOT and 19 SCT recipients were enrolled 2-4 months posttransplant and compared to 23 healthy control patients (HC). Neutrophils were coincubated with Candida albicans, and percentage killing and swarming responses were measured. RESULTS: Neutrophils from transplant patients had decreased fungicidal capacity compared to HC (42%, 43%, and 72% for SCT, SOT, and HC, respectively; SCT vs HC: P < .0001; SOT vs HC: P < .0001; SOT vs SCT: P = .8), including diminished ability to control hyphal growth (HC vs SOT: 0.1455 vs 0.3894, P ≤ .001; HC vs SCT: 0.1455 vs 0.6295, P ≤ .0001, respectively). Serum from SCT, but not SOT, recipients, inhibited the ability of HC neutrophils to control C. albicans (37%, 45%, and 55% for SCT, SOT, and HC, respectively). Neutrophils' control of hyphal growth was partially restored with granulocyte colony-stimulating factor or granulocyte macrophage colony-stimulating factor. CONCLUSIONS: Despite normal circulating numbers, our data suggest that neutrophils from SOT and SCT recipients mount dysfunctional responses against C. albicans. Intrinsic neutrophil changes and extrinsic serum factors may be responsible for the dysfunction, which is partially reversed with cytokine augmentation.

Paracrine signals influence patterns of fibrocartilage differentiation in a lyophilized gelatin hydrogel for applications in rotator cuff repair.

Rotator cuff injuries present a clinical challenge for repair due to current limitations in functional regeneration of the native tendon-to-bone enthesis. A biomaterial that can regionally instruct unique tissue-specific phenotypes offers potential to promote enthesis repair. We have recently demonstrated the mechanical benefits of a stratified triphasic biomaterial made up of tendon- and bone-mimetic collagen scaffold compartments connected via a continuous hydrogel, and we now explore the potential of a biologically favorable enthesis hydrogel for this application. Here we report in vitro behavior of human mesenchymal stem cells (hMSCs) within thiolated gelatin (Gel-SH) hydrogels in response to chondrogenic stimuli as well as paracrine signals derived from MSC-seeded bone and tendon scaffold compartments. Chondrogenic differentiation media promoted upregulation of cartilage and entheseal fibrocartilage matrix markers COL2, COLX, and ACAN as well as the enthesis-associated transcription factors SCX, SOX9, and RUNX2 in hMSCs within Gel-SH. Similar effects were observed in response to TGF-ß3 and BMP-4, enthesis-associated growth factors known to play a role in entheseal development and maintenance. Conditioned media generated by hMSCs seeded in tendon- and bone-mimetic collagen scaffolds influenced patterns of gene expression regarding enthesis-relevant growth factors, matrix markers, and tendon-to-bone transcription factors for hMSCs within the material. Together, these findings demonstrate that a Gel-SH hydrogel provides a permissive environment for enthesis tissue engineering and highlights the significance of cellular crosstalk between adjacent compartments within a spatially graded biomaterial.

Evaluating osteogenic effects associated with the incorporation of ascorbic acid in mineralized collagen scaffolds.

Current treatments for craniomaxillofacial (CMF) defects motivate the design of instructive biomaterials that can promote osteogenic healing of complex bone defects. We report methods to promote in vitro osteogenesis of human mesenchymal stem cells (hMSCs) within a model mineralized collagen scaffold via the incorporation of ascorbic acid (vitamin C), a key factor in collagen biosynthesis and bone mineralization. An addition of 5 w/v% ascorbic acid into the base mineralized collagen scaffold significantly changes key morphology characteristics including porosity, macrostructure, and microstructure. This modification promotes hMSC metabolic activity, ALP activity, and hMSC-mediated deposition of calcium and phosphorous. Additionally, the incorporation of ascorbic acid influences osteogenic gene expression (BMP-2, RUNX2, COL1A2) and delays the expression of genes associated with osteoclast activity and bone resorption (OPN, CTSK), though it reduces the secretion of OPG. Together, these findings highlight ascorbic acid as a relevant component for mineralized collagen scaffold design to promote osteogenic differentiation and new bone formation for improved CMF outcomes.

Shaping the mechanical properties of a gelatin hydrogel interface via amination.

Injuries to musculoskeletal interfaces, such as the tendon-to-bone insertion of the rotator cuff, present significant physiological and clinical challenges for repair due to complex gradients of structure, composition, and cellularity. Advances in interface tissue engineering require stratified biomaterials able to both provide local instructive signals to support multiple tissue phenotypes while also reducing the risk of strain concentrations and failure at the transition between dissimilar materials. Here, we describe adaptation of a thiolated gelatin (Gel-SH) hydrogel via selective amination of carboxylic acid subunits on the gelatin backbone. The magnitude and kinetics of HRP-mediated primary crosslinking and carbodiimide-mediated secondary crosslinking reactions can be tuned through amination and thiolation of carboxylic acid subunits on the gelatin backbone. We also show that a stratified biomaterial comprised of mineralized (bone-mimetic) and non-mineralized (tendon-mimetic) collagen scaffold compartments linked by an aminated Gel-SH hydrogel demonstrate improved mechanical performance and reduced strain concentrations. Together, these results highlight significant mechanical advantages that can be derived from modifying the gelatin macromer via controlled amination and thiolation and suggest an avenue for tuning the mechanical performance of hydrogel interfaces within stratified biomaterials.

BTK inhibitor-induced defects in human neutrophil effector activity against Aspergillus fumigatus are restored by TNF-α.

Inhibition of Bruton's tyrosine kinase (BTK) through covalent modifications of its active site (e.g., ibrutinib [IBT]) is a preferred treatment for multiple B cell malignancies. However, IBT-treated patients are more susceptible to invasive fungal infections, although the mechanism is poorly understood. Neutrophils are the primary line of defense against these infections; therefore, we examined the effect of IBT on primary human neutrophil effector activity against Aspergillus fumigatus. IBT significantly impaired the ability of neutrophils to kill A. fumigatus and potently inhibited reactive oxygen species (ROS) production, chemotaxis, and phagocytosis. Importantly, exogenous TNF-α fully compensated for defects imposed by IBT and newer-generation BTK inhibitors and restored the ability of neutrophils to contain A. fumigatus hyphal growth. Blocking TNF-α did not affect ROS production in healthy neutrophils but prevented exogenous TNF-α from rescuing the phenotype of IBT-treated neutrophils. The restorative capacity of TNF-α was independent of transcription. Moreover, the addition of TNF-α immediately rescued ROS production in IBT-treated neutrophils, indicating that TNF-α worked through a BTK-independent signaling pathway. Finally, TNF-α restored effector activity of primary neutrophils from patients on IBT therapy. Altogether, our data indicate that TNF-α rescued the antifungal immunity block imposed by inhibition of BTK in primary human neutrophils.

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.

Novel Host Response-Based Diagnostics to Differentiate the Etiology of Fever in Patients Presenting to the Emergency Department.

Fever is a common presentation to urgent-care services and is linked to multiple disease processes. To rapidly determine the etiology of fever, improved diagnostic modalities are necessary. This prospective study of 100 hospitalized febrile patients included both positive (FP) and negative (FN) subjects in terms of infection status and 22 healthy controls (HC). We evaluated the performance of a novel PCR-based assay measuring five host mRNA transcripts directly from whole blood to differentiate infectious versus non-infectious febrile syndromes as compared to traditional pathogen-based microbiology results. The FP and FN groups observed a robust network structure with a significant correlation between the five genes. There were statistically significant associations between positive infection status and four of the five genes: IRF-9 (OR = 1.750, 95% CI = 1.16-2.638), ITGAM (OR = 1.533, 95% CI = 1.047-2.244), PSTPIP2 (OR = 2.191, 95% CI = 1.293-3.711), and RUNX1 (OR = 1.974, 95% CI = 1.069-3.646). We developed a classifier model to classify study participants based on these five genes and other variables of interest to assess the discriminatory power of the genes. The classifier model correctly classified more than 80% of the participants into their respective groups, i.e., FP or FN. The GeneXpert prototype holds promise for guiding rapid clinical decision-making, reducing healthcare costs, and improving outcomes in undifferentiated febrile patients presenting for urgent evaluation.

The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan.

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly among immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient serum and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2-dependent signaling, including activation of spleen tyrosine kinase, gene transcription, and tumor necrosis factor alpha (TNF-α) production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan, which leads to a proinflammatory immune response in the lung. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptors can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified Dectin-2 as a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients.

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.

Transfusable neutrophil progenitors as cellular therapy for the prevention of invasive fungal infections.

The use of mature neutrophil (granulocyte) transfusions for the treatment of neutropenic patients with invasive fungal infections (IFIs) has been the focus of multiple clinical trials. Despite these efforts, the transfusion of mature neutrophils has resulted in limited clinical benefit, likely owing to problems of insufficient numbers and the very short lifespan of these donor cells. In this report, we employed a system of conditionally immortalized murine neutrophil progenitors that are capable of continuous expansion, allowing for the generation of unlimited numbers of homogenous granulocyte-macrophage progenitors (GMPs). These GMPs were assayed in vivo to demonstrate their effect on survival in 2 models of IFI: candidemia and pulmonary aspergillosis. Mature neutrophils derived from GMPs executed all cardinal functions of neutrophils. Transfused GMPs homed to the bone marrow and spleen, where they completed normal differentiation to mature neutrophils. These neutrophils were capable of homing and extravasation in response to inflammatory stimuli using a sterile peritoneal challenge model. Furthermore, conditionally immortalized GMP transfusions significantly improved survival in models of candidemia and pulmonary aspergillosis. These data confirm the therapeutic benefit of prophylactic GMP transfusions in the setting of neutropenia and encourage development of progenitor cellular therapies for the management of fungal disease in high-risk patients.

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.