Neonatal low-density granulocytes internalize and kill bacteria but suppress monocyte function using extracellular DNA.

Low-density granulocytes (LDGs) are found abundantly in neonatal blood; however, there is limited mechanistic understanding of LDG interactions with bacteria and innate immune cells during acute infection. We aimed to determine how human neonatal LDGs may influence control of the bacterial burden at sites of infection, both individually and in the presence of mononuclear phagocytes. LDGs from human umbilical cord blood do phagocytose Escherichia coli O1:K1:H7 and traffic bacteria into acidic compartments. However, LDGs were significantly less efficient at bacterial uptake and killing compared to monocytes, and this activity was associated with a reduced inflammatory cytokine response. The presence of bacteria triggered the release of DNA (eDNA) from LDGs into the extracellular space that resembled neutrophil extracellular traps, but had limited anti-bacterial activity. Instead, eDNA significantly impaired monocyte control of bacteria during co-culture. These results suggest that LDG recruitment to sites of bacterial infection may compromise host protection in the neonate. Furthermore, our findings reveal novel insights into LDG activity during infection, clarify their inflammatory contributions relative to monocytes, and identify a novel LDG mechanism of immunosuppression.This article has an associated First Person interview with the first author of the paper.

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.

The impact of opioid exposure during pregnancy on the human neonatal immune profile.

BACKGROUND: The increasing magnitude of the opioid crisis and rising rates of neonatal abstinence syndrome (NAS) diagnoses highlight the need for increased research into how maternal substance use during pregnancy can impact the neonatal immune profile and its functionality. We hypothesized that neonates with opioid exposure would have reduced proportions of some immune cells, an anti-inflammatory cytokine profile, reduced T cell proliferation, and monocyte bacterial killing activity compared to the control population. METHODS: The present study compares immune cell populations, inflammatory and anti-inflammatory cytokine and chemokine levels in the serum, and monocyte and T cell functional activity using umbilical cord samples from neonates with known opioid exposure during gestation and from control neonates without known exposure. RESULTS: Our findings demonstrated a significant reduction in neutrophils, decreased levels of inflammatory cytokines in the serum, and reduced IL-2 production during in vitro CD4+ T cell proliferation in neonates exposed to opioids compared to controls. The neutrophil findings were supported by retrospective analysis of an extended network of deidentified patient records. CONCLUSIONS: This study is the first of its kind to evaluate differences in neonatal immunity as a result of opioid exposure in the human population that will inform continued mechanistic studies. IMPACT: The opioid epidemic has become a public health crisis in the United States, and the corresponding incidence of neonatal abstinence syndrome (NAS) have risen accordingly. New research is required to understand the short and long-term health impacts of opioid exposure to the neonate. This is the first human study to investigate the immunologic profile and functionality in neonates with known opioid exposure in utero. The abundance of neutrophils and the ratio of neutrophils to lymphocytes is significantly reduced along with inflammatory cytokines and chemokines following opioid exposure during pregnancy. The immune profile in opioid-exposed neonates may promote susceptibility to infection.

Elevated Levels of Interleukin-27 in Early Life Compromise Protective Immunity in a Mouse Model of Gram-Negative Neonatal Sepsis.

Neonates are at increased risk for bacterial sepsis. We established that the immune-suppressive cytokine interleukin-27 (IL-27) is elevated in neonatal mice. Similarly, human cord blood-derived macrophages express IL-27 genes and secrete more cytokine than macrophages from adults. In the present work, we hypothesized that increased levels of IL-27 predispose neonatal mice to more severe infection during Gram-negative sepsis. Serum IL-27 levels continued to rise during infection. Peripheral tissue analysis revealed systemic IL-27 expression, while myeloid cell profiling identified Gr-1- and F4/80-expressing cells as the most abundant producers of IL-27 during infection. Increased IL-27 levels were consistent with increased mortality that was improved in IL-27 receptor α (IL-27Rα)-/- mice that lack a functional IL-27 receptor. Infected IL-27Rα-/- pups also exhibited improved weight gain and reduced morbidity. This was consistent with reduced bacterial burdens and more efficient bacterial killing by Ly6B.2+ myeloid cells and macrophages compared to WT neonates. Live animal imaging further supported a more severe and disseminated infection in WT neonates. This is the first report to describe the impact of elevated early-life IL-27 on the host response in a neonatal infection model while also defining the cell and tissue sources of cytokine. IL-27 is frequently associated with suppressed inflammation. In contrast, our findings demonstrate that IL-27 indirectly promotes an inflammatory cytokine response during neonatal sepsis by directly compromising control of bacteria that drive the inflammatory response. Collectively, our results suggest that IL-27 represents a therapeutic target to limit susceptibility and improve infectious outcomes in neonatal sepsis.

Murine myeloid-derived suppressor cells are a source of elevated levels of interleukin-27 in early life and compromise control of bacterial infection.

Microbial infections early in life remain a major cause of infant mortality worldwide. This is consistent with immune deficiencies in this population. Interleukin (IL)-27 is suppressive toward a variety of immune cell types, and we have shown that the production of IL-27 is elevated in humans and mice early in life. We hypothesize that elevated levels of IL-27 oppose protective responses to infection during the neonatal period. In this study, we extended previous findings in neonatal mice to identify a population of IL-27 producers that express Gr-1 and were further identified as myeloid-derived suppressor cells (MDSCs) based on the expression of surface markers and functional studies. In neonates, MDSCs are more abundant and contribute to the elevated pool of IL-27 in this population. Although the ability of MDSCs to regulate T lymphocyte activation has been well-studied, sparingly few studies have investigated the influence of MDSCs on innate immune function during bacterial infection. We demonstrate that macrophages are impaired in their ability to control growth of Escherichia coli when cocultured with MDSCs. This bacterium is a significant concern for neonates as a common cause of bacterial sepsis and meningitis. The suppressive effect of MDSCs on macrophage function is mediated by IL-27; inclusion of a reagent to neutralize IL-27 promotes improved control of bacterial growth. Taken together, these results suggest that the increased abundance of MDSCs may contribute to early life susceptibility to infection and further highlight production of IL-27 as a novel MDSC mechanism to suppress immunity.

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.

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.

Candida albicans and Pseudomonas aeruginosa Interact To Enhance Virulence of Mucosal Infection in Transparent Zebrafish.

Polymicrobial infections often include both fungi and bacteria and can complicate patient treatment and resolution of infection. Cross-kingdom interactions among bacteria, fungi, and/or the immune system during infection can enhance or block virulence mechanisms and influence disease progression. The fungus Candida albicans and the bacterium Pseudomonas aeruginosa are coisolated in the context of polymicrobial infection at a variety of sites throughout the body, including mucosal tissues such as the lung. In vitro, C. albicans and P. aeruginosa have a bidirectional and largely antagonistic relationship. Their interactions in vivo remain poorly understood, specifically regarding host responses in mediating infection. In this study, we examine trikingdom interactions using a transparent juvenile zebrafish to model mucosal lung infection and show that C. albicans and P. aeruginosa are synergistically virulent. We find that high C. albicans burden, fungal epithelial invasion, swimbladder edema, and epithelial extrusion events serve as predictive factors for mortality in our infection model. Longitudinal analyses of fungal, bacterial, and immune dynamics during coinfection suggest that enhanced morbidity is associated with exacerbated C. albicans pathogenesis and elevated inflammation. The P. aeruginosa quorum-sensing-deficient ΔlasR mutant also enhances C. albicans pathogenicity in coinfection and induces extrusion of the swimbladder. Together, these observations suggest that C. albicans-P. aeruginosa cross talk in vivo can benefit both organisms to the detriment of the host.

Comparison of Dose-Response Relationships for Two Isolates of SARS-CoV-2 in a Nonhuman Primate Model of Inhalational COVID-19.

Background: As the COVID-19 pandemic has progressed, numerous variants of SARS-CoV-2 have arisen, with several displaying increased transmissibility. Methods: The present study compared dose-response relationships and disease presentation in nonhuman primates infected with aerosols containing an isolate of the Gamma variant of SARS-CoV-2 to the results of our previous study with the earlier WA-1 isolate of SARS-CoV-2. Results: Disease in Gamma-infected animals was mild, characterized by dose-dependent fever and oronasal shedding of virus. Differences were observed in shedding in the upper respiratory tract between Gamma- and WA-1-infected animals that have the potential to influence disease transmission. Specifically, the estimated median doses for shedding of viral RNA or infectious virus in nasal swabs were approximately 10-fold lower for the Gamma variant than the WA-1 isolate. Given that the median doses for fever were similar, this suggests that there is a greater difference between the median doses for viral shedding and fever for Gamma than for WA-1 and potentially an increased range of doses for Gamma over which asymptomatic shedding and disease transmission are possible. Conclusions: These results complement those of previous studies, which suggested that differences in exposure dose may help to explain the range of clinical disease presentations observed in individuals with COVID-19, highlighting the importance of public health measures designed to limit exposure dose, such as masking and social distancing. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, as well as to inform dose selection in future studies examining the efficacy of therapeutics and vaccines in animal models of inhalational COVID-19.

The Enigma of Low-Density Granulocytes in Humans: Complexities in the Characterization and Function of LDGs during Disease.

Low-density granulocytes (LDGs) have been characterized as important immune cells during healthy and disease states in humans, including microbial infections, cancer, and autoimmune dysfunction. However, the classification of this cell type is similar to other immune cells (e.g., neutrophils, myeloid-derived suppressor cells) and ambiguous functional standards have rendered LDG identification and isolation daunting. Furthermore, most research involving LDGs has mainly focused on adult cells and subjects, leaving increased uncertainty surrounding younger populations, especially in vulnerable neonatal groups where LDG numbers are elevated. This review aims to bring together the current research in the field of LDG biology in the context of immunity to disease, with a focus on infection. In addition, we propose to highlight the gaps in the field that, if filled, could improve upon isolation techniques and functional characterizations for LDGs separate from neutrophils and myeloid-derived suppressor cells (MDSCs). This will not only enhance understanding of LDGs during disease processes and how they differ from other cell types but will also aid in the interpretation of comparative studies and results with the potential to inform development of novel therapeutics to improve disease states in patients.

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Microglia and amyloid precursor protein coordinate control of transient Candida cerebritis with memory deficits.

Bloodborne infections with Candida albicans are an increasingly recognized complication of modern medicine. Here, we present a mouse model of low-grade candidemia to determine the effect of disseminated infection on cerebral function and relevant immune determinants. We show that intravenous injection of 25,000 C. albicans cells causes a highly localized cerebritis marked by the accumulation of activated microglial and astroglial cells around yeast aggregates, forming fungal-induced glial granulomas. Amyloid precursor protein accumulates within the periphery of these granulomas, while cleaved amyloid beta (Aß) peptides accumulate around the yeast cells. CNS-localized C. albicans further activate the transcription factor NF-κB and induce production of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor (TNF), and Aß peptides enhance both phagocytic and antifungal activity from BV-2 cells. Mice infected with C. albicans display mild memory impairment that resolves with fungal clearance. Our results warrant additional studies to understand the effect of chronic cerebritis on cognitive and immune function.