IL-27 alters inflammatory cytokine expression and limits protective immunity against Mycobacterium tuberculosis in a neonatal BCG vaccination model.

Background: Efforts to control tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (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. Objective: We investigated the impact of IL-27 on regulation of immune responses during neonatal BCG vaccination and protection against Mtb. Methods: Here, we used a novel model of neonatal vaccination and adult aerosol challenge that models the human timeline of vaccine delivery and disease transmission. Results: Overall, we observed improved control of Mtb in mice unresponsive to IL-27 (IL-27Rα-/-) that was consistent with altered expression patterns of IFN-γ and IL-17 in the lungs. The balance of these cytokines with TNF-α expression may be key to effective bacterial clearance. Conclusions: Our findings suggest the importance of evaluating new vaccines and approaches to combat TB in the neonatal population most likely to receive them as part of global vaccination campaigns. They further indicate that temporal strategies to antagonize IL-27 during early life vaccination may improve protection.

Interleukin-27-dependent transcriptome signatures during neonatal sepsis.

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 Escherichia coli-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 impairs BCG antigen clearance and T cell stimulatory potential by neonatal dendritic cells.

Bacille Calmette Guérin (BCG) is a live-attenuated vaccine for protection against Mycobacterium tuberculosis. 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.

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.

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

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 Escherichia coli 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 E. coli. 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 E. coli. 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.

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.

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

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.

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.

Executive (dys)function after stroke: special considerations for behavioral pharmacology.

Stroke is a worldwide leading cause of death and long-term disability with concurrent secondary consequences that are largely comprised of mood dysfunction, as well as sensory, motor, and cognitive deficits. This review focuses on the cognitive deficits associated with stroke specific to executive dysfunction (including decision making, working memory, and cognitive flexibility) in humans, nonhuman primates, and additional animal models. Further, we review some of the cellular and molecular underpinnings of the individual components of executive dysfunction and their neuroanatomical substrates after stroke, with an emphasis on the changes that occur during biogenic monoamine neurotransmission. We concentrate primarily on changes in the catecholaminergic (dopaminergic and noradrenergic) and serotonergic systems at the levels of neurotransmitter synthesis, distribution, reuptake, and degradation. We also discuss potential secondary stroke-related behavioral deficits (specifically, poststroke depression as well as drug-abuse potential and addiction) and their relationship with stroke-induced deficits in executive function, an especially important consideration given that the average age of the human stroke population is decreasing. In the final sections, we address pharmacological considerations for the treatment of ischemia and the subsequent functional impairment, as well as current limitations in the field of stroke and executive function research.

Executive (dys)function after traumatic brain injury: special considerations for behavioral pharmacology.

Executive function is an umbrella term that includes cognitive processes such as decision-making, impulse control, attention, behavioral flexibility, and working memory. Each of these processes depends largely upon monoaminergic (dopaminergic, serotonergic, and noradrenergic) neurotransmission in the frontal cortex, striatum, and hippocampus, among other brain areas. Traumatic brain injury (TBI) induces disruptions in monoaminergic signaling along several steps in the neurotransmission process - synthesis, distribution, and breakdown - and in turn, produces long-lasting deficits in several executive function domains. Understanding how TBI alters monoamingeric neurotransmission and executive function will advance basic knowledge of the underlying principles that govern executive function and potentially further treatment of cognitive deficits following such injury. In this review, we examine the influence of TBI on the following measures of executive function - impulsivity, behavioral flexibility, and working memory. We also describe monoaminergic-systems changes following TBI. Given that TBI patients experience alterations in monoaminergic signaling following injury, they may represent a unique population with regard to pharmacotherapy. We conclude this review by discussing some considerations for pharmacotherapy in the field of TBI.

Gradual common carotid artery occlusion as a novel model for cerebrovascular Hypoperfusion.

Chronic cerebrovascular hypoperfusion results in vascular dementia and increases predisposition to lacunar infarcts. However, there are no suitable animal models. In this study, we developed a novel model for chronic irreversible cerebral hypoperfusion in mice. Briefly, an ameroid constrictor was placed on the right carotid artery to gradually occlude the vessel, while a microcoil was placed on the left carotid artery to prevent compensation of the blood flow. This procedure resulted in a gradual hypoperfusion developing over a period of 34 days with no cerebral blood flow recovery. Histological analysis of the brain revealed neuronal and axonal degeneration as well as necrotic lesions. The most severely affected regions were located in the hippocampus and the corpus callosum. Overall, our paradigm is a viable model to study brain pathology resulting from gradual cerebrovascular hypoperfusion.

Absolute lymphocyte and neutrophil counts in neonatal ischemic brain injury.

OBJECTIVES: This study aimed to identify differences in absolute neutrophils, lymphocytes, and neutrophil-to-lymphocyte ratio between neonates with two forms of ischemic brain injury, hypoxic-ischemic encephalopathy, and acute ischemic stroke, compared to controls. We also aimed to determine whether this neutrophil/lymphocyte response pattern is associated with disease severity or is a consequence of the effects of total-body cooling, an approved treatment for moderate-to-severe hypoxic-ischemic encephalopathy. METHODS: A retrospective chart review of 101 neonates with hypoxic-ischemic encephalopathy + total-body cooling (n = 26), hypoxic-ischemic encephalopathy (n = 12), acute ischemic stroke (n = 15), and transient tachypnea of the newborn (n = 48) was conducted; transient tachypnea of the newborn neonates were used as the control group. Absolute neutrophil count and absolute lymphocyte count at three time-intervals (0-12, 12-36, and 36-60 h after birth) were collected, and neutrophil-to-lymphocyte ratio was calculated. RESULTS: Hypoxic-ischemic encephalopathy + total-body cooling neonates demonstrated significant time-interval-dependent changes in absolute lymphocyte count and neutrophil-to-lymphocyte ratio levels compared to transient tachypnea of the newborn and acute ischemic stroke patients. Pooled analysis of absolute lymphocyte count for neonates with acute ischemic stroke and hypoxic-ischemic encephalopathy (not hypoxic-ischemic encephalopathy + total-body cooling) revealed that absolute lymphocyte count changes occurring at 0-12 h are likely due to disease progression, rather than total-body cooling treatment. CONCLUSION: These data suggest that the neutrophil/lymphocyte response is modulated following neonatal ischemic brain injury, representing a possible target for therapeutic intervention. However, initial severity of hypoxic-ischemic encephalopathy among these patients could also account for the observed changes in the immune response to injury. Thus, additional work to clarify the contributions of cooling therapy and disease severity to neutrophil/lymphocyte response following hypoxic-ischemic encephalopathy in neonates is warranted.

Locomotor effects of a low-frequency fire alarm on C57BL/6 male mice: a preliminary study.

Maintaining appropriate acoustic conditions for animal welfare and data collection are crucial in biomedical research facilities. Negative impacts of disruptive sound are known and can include auditory damage, immune function changes, and behavioral alterations. One type of disruptive sound occurring in research facilities is that of fire alarms. To ameliorate this problem, many facilities have incorporated the use of low-frequency fire alarms that emit tones outside the rodent audible range. The impact of these devices has been assumed to be negligible. However, this has yet to be evaluated with controlled behavioral experiments. Thus, our objective was to investigate the impact of low-frequency fire alarm exposure on locomotor behavior in the open field, a test sensitive to acoustic stimuli disruption. Male mice were randomized to three alarm exposure groups (No-Alarm; Alarm-During; and Alarm-After) and placed in individual photobeam-activated locomotor chambers. The Alarm-During group displayed significantly reduced horizontal locomotion, with a trend towards reduced vertical locomotion. These data suggest that a low-frequency brief alarm tone can temporarily disrupt movement, a valuable insight should an alarm be deployed. Further, findings support close collaboration between researchers and institutional facility staff to ensure appropriate acoustic conditions are maintained, whenever possible, for research animals.

Effects of an α5GABAA inverse agonist on MK-801-induced learning deficits in an incremental repeated acquisition task.

N-methyl-D-aspartate receptors (NMDARs) are essential for several kinds of synaptic plasticity and play a critical role in learning and memory. Deficits in NMDAR functioning may be partially responsible for the learning and memory deficits associated with aging and numerous diseases. Administration of MK-801, a noncompetitive NMDAR antagonist, is commonly used as a preclinical model of NMDAR dysfunction. The objective of this study was to assess the effects of α5GABAA receptor inhibition on learning deficits in the incremental repeated acquisition (IRA) task induced by acute MK-801 administration. The IRA task, commonly used to examine factors that affect learning, begins with a single response and increments to progressively longer chains throughout a single session as behavior meets preset criteria. MK-801 (0.03-0.5 mg/kg, intraperitoneally), administered 10 min pretesting, produced a significant dose-dependent decrease in measures of IRA performance at doses greater than or equal to 0.25 mg/kg. The MK-801-induced deficit was attenuated after treatment with an α5GABAA receptor inverse agonist, L-655,708 (1 mg/kg, intraperitoneally). The present study provides the focus for, and supports the feasibility of, further in-depth definitive studies examining α5GABAA receptor inhibition as a suitable candidate for the attenuation of NMDAR-related deficits.