a-neonatal-imaging-model-of-gram-negative-bacterial-sepsis

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 ...

neonatal-mouse-bone-marrow-isolation-preparation-bone-marrow-derived

Neonatal Mouse Bone Marrow Isolation and Preparation of Bone Marrow-Derived Macrophages

Various techniques for isolating bone marrow from adult mice have been well established. However, isolating bone marrow from neonatal mice is challenging ...

West Virginia University School of Medicine

Vaccine Development Center at West Virginia University Health Sciences Center

West Virginia University Health Sciences Center

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.

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

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.

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

IL-27 is a pleiotropic cytokine capable of influencing both innate and adaptive immune responses. With anti- and pro-inflammatory activity, IL-27 exerts its opposing effects in a cell-dependent and infectious context-specific manner. Upon pathogenic stimuli, IL-27 regulates innate immune cells, such as monocytes, dendritic cells, macrophages and neutrophils. Immune responses involving these innate cells that are negatively regulated by IL-27 signaling include inflammatory cytokine production, phagolysosomal acidification following phagocytosis, oxidative burst and autophagy. IL-27 signaling is crucial in maintaining the subtle balance between Th1 and Th2 immunity, in which protective inflammation is upregulated within the early stages of infection and subsequently downregulated once microbial growth is controlled. The immunomodulatory effects of IL-27 provide promising therapeutic targets for multiple disease types.

IL-27 regulation of innate immunity and control of microbial growth.

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  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.

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

Neonates are at an increased risk of an infectious disease. This is consistent with an increased abundance of myeloid-derived suppressor cells (MDSCs) compared with older children and adults. Using a murine model of neonatal bacterial sepsis, we demonstrate that MDSCs modulate their activity during an infection to enhance immune suppressive functions. A gene expression analysis shows that MDSCs increased NOS2, Arg-1 and IL-27p28 expression in vitro and in vivo in response to  O1:K1:H7 and this is regulated at the level of the gene expression. Changes in the effector gene expression are consistent with increased enzymatic activity and cytokine secretion. The neonatal MDSCs express toll-like receptor (TLR) 2, 4 and 5 capable of recognizing pathogen-associated molecular patterns (PAMPS) on . However, a variable level of effector expression was achieved in response to LPS, peptidoglycan or flagellin. Individual bacterial PAMPs did not stimulate the expression of Arg-l and IL-27p28 equivalently to . However, the upregulation of NOS2 was achieved in response to LPS, peptidoglycan and flagella. The increased immune suppressive profile translated to an enhanced suppression of CD4+ T cell proliferation. Collectively, these findings increase our understanding of the dynamic nature of MDSC activity and suggest that these cells abundant in early life can acquire activity during an infection that suppresses protective immunity.

Myeloid-Derived Suppressor Cells Gain Suppressive Function during Neonatal Bacterial Sepsis.

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.

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

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.

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

Bacille Calmette Guérin (BCG) is a live-attenuated vaccine for protection against . Despite high disease protection in infancy and early childhood, it generates poor long-term protection against pulmonary tuberculosis. We hypothesized that the unique immune profile that includes elevated interleukin (IL)-27, contributes to insufficient protection from routine neonatal BCG administration. Using a novel method to obtain neonatal progenitors, we showed that neonatal bone marrow-derived dendritic cells (BMDCs) increase production of IL-27 following BCG stimulation. To study the effect of IL-27 on BMDCs, we utilized mice deficient for IL-27 receptor-α (KO). We observed greater BCG clearance and elevated IL-12 production in the neonatal KO BMDCs compared to WT. BMDCs from KO neonates in turn stimulated more interferon-γ production from CD4 T cells isolated from BCG-vaccinated mice than WT counterparts. To further confirm the importance of these findings, C57BL/6 mice were vaccinated as neonates in line with the approach to human vaccination in high TB burden regions. IL-27 levels progressively increased through 5 weeks and were significantly elevated in mice vaccinated with BCG compared to controls. The impact of IL-27 production on clearance of BCG was significant as KO mice cleared BCG from peripheral tissues that persisted in WT mice 5 weeks post-vaccination. These results are the first to highlight the suppressive role of IL-27 on DCs in the neonatal period and the impact on neonatal immune responses to BCG.

Interleukin-27 impairs BCG antigen clearance and T cell stimulatory potential by neonatal dendritic cells.

Human newborns exhibit increased vulnerability and risk of mortality from infection that is consistent with key differences in the innate and adaptive immune responses relative to those in adult cells. We have previously shown an increase in the immune suppressive cytokine, IL-27, in neonatal cells and tissues from mice and humans. In a murine model of neonatal sepsis, mice deficient in IL-27 signaling exhibit reduced mortality, increased weight gain, and better control of bacteria with reduced systemic inflammation. To explore a reprogramming of the host response in the absence of IL-27 signaling, we profiled the transcriptome of the neonatal spleen during -induced sepsis in wild-type (WT) and IL-27Rα-deficient (KO) mice. We identified 634 genes that were differentially expressed, and those most upregulated in WT mice were associated with inflammation, cytokine signaling, and G protein coupled receptor ligand binding and signaling. These genes failed to increase in the IL-27Rα KO mice. We further isolated an innate myeloid population enriched in macrophages from the spleens of control and infected WT neonates and observed similar changes in gene expression aligned with changes in chromatin accessibility. This supports macrophages as an innate myeloid population contributing to the inflammatory profile in septic WT pups. Collectively, our findings highlight the first report of improved pathogen clearance amidst a less inflammatory environment in IL-27Rα KO. This suggests a direct relationship between IL-27 signaling and bacterial killing. An improved response to infection that is not reliant upon heightened levels of inflammation offers new promise to the potential of antagonizing IL-27 as a host-directed therapy for neonates.

Interleukin-27-dependent transcriptome signatures during neonatal sepsis.

Efforts to control tuberculosis (TB), caused by the pathogen  (Mtb), have been hampered by the immense variability in protection from BCG vaccination. While BCG protects young children from some forms of TB disease, long-term protection against pulmonary disease is more limited, suggesting a poor memory response. New vaccines or vaccination strategies are required to have a realistic chance of eliminating TB disease. In TB endemic areas, routine immunization occurs during the neonatal period and as such, we hypothesized that inadequate protective immunity elicited by BCG vaccination could be the result of the unique early-life immune landscape. Interleukin (IL)-27 is a heterodimeric cytokine with immune suppressive activity that is elevated in the neonatal period.

Article Total : 2	Year
Publication title	2020
Publication title	2024

Article	Year
Murine myeloid-derived suppressor cells are a source of elevated levels of interleukin-27 in early life and compromise control of bacterial infection. Immunology and cell biology\| PubMed ID: 30575117	2019
Elevated Levels of Interleukin-27 in Early Life Compromise Protective Immunity in a Mouse Model of Gram-Negative Neonatal Sepsis. Infection and immunity\| PubMed ID: 31818960	2020
IL-27 regulation of innate immunity and control of microbial growth. Future science OA\| PubMed ID: 32802395	2020
Neonatal low-density granulocytes internalize and kill bacteria but suppress monocyte function using extracellular DNA. Journal of cell science\| PubMed ID: 33589502	2021
Myeloid-Derived Suppressor Cells Gain Suppressive Function during Neonatal Bacterial Sepsis. International journal of molecular sciences\| PubMed ID: 34208904	2021
The Enigma of Low-Density Granulocytes in Humans: Complexities in the Characterization and Function of LDGs during Disease. Pathogens (Basel, Switzerland)\| PubMed ID: 34578124	2021
The impact of opioid exposure during pregnancy on the human neonatal immune profile. Pediatric research\| PubMed ID: 35288639	2022
Interleukin-27 impairs BCG antigen clearance and T cell stimulatory potential by neonatal dendritic cells. Current research in microbial sciences\| PubMed ID: 36530844	2023
Interleukin-27-dependent transcriptome signatures during neonatal sepsis. Frontiers in immunology\| PubMed ID: 36891292	2023
IL-27 alters inflammatory cytokine expression and limits protective immunity against in a neonatal BCG vaccination model. Frontiers in immunology\| PubMed ID: 38390338	2024

Cory M. Robinson

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