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Daniel P Nemeth,1– 3 Xiaoyu Liu,3 Daniel B McKim,4 Damon J DiSabato,2,5 Braedan Oliver,2 Anu Herd,3 Asish Katta,1 Christina E Negray,1,2 James Floyd,3 Samantha McGovern,3 Paige S Pruden,2 Feiyang Zhutang,2 Maria Smirnova,3 Jonathan P Godbout,2,5 John Sheridan,1,2 Ning Quan3 1College of Dentistry, The Ohio State University, Columbus, OH, USA; 2Institute for Behavioral Medicine Research, The Ohio State University, Columbus, OH, USA; 3Stiles-Nicholson Brain Institute, Florida Atlantic University, Jupiter, FL, USA; 4Department of Animal Sciences, University of Illinois Urbana-Champaign, Champaign, IL, USA; 5Department of Neuroscience, The Ohio State University, Columbus, OH, USACorrespondence: Daniel P Nemeth; Ning Quan, 5353 Parkside Drive, Jupiter, FL, 33458, USA, Email nemethd@fau.edu; nquan@health.fau.eduIntroduction: Lipopolysaccharide (LPS) preconditioning involves repeated, systemic, and sub-threshold doses of LPS, which induces a neuroprotective state within the CNS, thus preventing neuronal death and functional losses. Recently, proinflammatory cytokine, Interleukin-1 (IL-1), and its primary signaling partner, interleukin-1 receptor type 1 (IL-1R1), have been associated with neuroprotection in the CNS. However, it is still unknown how IL-1/IL-1R1 signaling impacts the processes associated with neuroprotection.Methods: Using our IL-1R1 restore genetic mouse model, mouse lines were generated to restrict IL-1R1 expression either to endothelia (Tie2-Cre-Il1r1r/r) or microglia (Cx3Cr1-Cre-Il1r1r/r), in addition to either global ablation (Il1r1r/r) or global restoration of IL-1R1 (Il1r1GR/GR). The LPS preconditioning paradigm consisted of four daily i.p. injections of LPS at 1 mg/kg (4d LPS). 24 hrs following the final i.p. LPS injection, tissue was collected for qPCR analysis, immunohistochemistry, or FAC sorting.Results: Following 4d LPS, we found multiple phenotypes that are dependent on IL-1R1 signaling such as microglia morphology alterations, increased microglial M2-like gene expression, and clustering of microglia onto the brain vasculature. We determined that 4d LPS induces microglial morphological changes, clustering at the vasculature, and gene expression changes are dependent on endothelial IL-1R1, but not microglial IL-1R1. A novel observation was the induction of microglial IL-1R1 (mIL-1R1) following 4d LPS. The induced mIL-1R1 permits a unique response to central IL-1β: the mIL-1R1 dependent induction of IL-1R1 antagonist (IL-1RA) and IL-1β gene expression. Analysis of RNA sequencing datasets revealed that mIL-1R1 is also induced in neurodegenerative diseases.Discussion: Here, we have identified cell type-specific IL-1R1 mediated mechanisms, which may contribute to the neuroprotection observed in LPS preconditioning. These findings identify key cellular and molecular contributors in LPS-induced neuroprotection.Graphical Abstract: Keywords: BBB, neuroinflammation, perivascular microglia

Objective: Dilution of concentrated epinephrine prior to intravenous use during clinical emergencies can delay urgent interventions. The objective of this study was to determine whether diluted epinephrine remains stable and sterile over time in common hospital settings., Methods: Epinephrine samples were prepared by clinically relevant double dilution techniques. Samples were stored in 10-mL syringes and incubated under 1 of 4 simulated hospital environments with a mixture of lighting and temperature settings: 4°C/20°C, with or without fluorescent lighting. Samples were incubated for 0, 15, 30, 60, or 90 days. Capillary zonal electrophoresis was used to quantify the concentration of epinephrine and/or presence of any degradation products. All samples were tested for the presence of bacterial growth using blood agar cultures., Results: Diluted epinephrine samples remained stable for up to 90 days in all 4 simulated clinical storage conditions. No bacterial colony-forming units were detected in any of the environmental samples regardless of incubation duration, light, or temperature conditions., Conclusion: Diluted epinephrine for anticipated clinical emergencies may remain clinically useful for up to 90 days, thus improving patient safety, access to medications, and overhead costs by reducing waste.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a. NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

Background: Recent data suggest that myelin may be altered by physiological events occurring outside of the central nervous system, which may cause changes to cognition and behavior. Similarly, peripheral infection by non-neurotropic viruses is also known to evoke changes to cognition and behavior., Methods: Mice were inoculated with saline or influenza A virus. Bulk RNA-seq, lipidomics, RT-qPCR, flow cytometry, immunostaining, and western blots were used to determine the effect of infection on OL viability, protein expression and changes to the lipidome. To determine if microglia mediated infection-induced changes to OL homeostasis, mice were treated with GW2580, an inhibitor of microglia activation. Additionally, conditioned medium experiments using primary glial cell cultures were also used to test whether secreted factors from microglia could suppress OL gene expression., Results: Transcriptomic and RT-qPCR analyses revealed temporal downregulation of OL-specific transcripts with concurrent upregulation of markers characteristic of cellular stress. OLs isolated from infected mice had reduced cellular expression of myelin proteins compared with those from saline-inoculated controls. In contrast, the expression of these proteins within myelin was not different between groups. Similarly, histological and immunoblotting analysis performed on various brain regions indicated that infection did not alter OL viability, but increased expression of a cellular stress marker. Shot-gun lipidomic analysis revealed that infection altered the lipid profile within the prefrontal cortex as well as in purified brain myelin and that these changes persisted after recovery from infection. Treatment with GW2580 during infection suppressed the expression of genes associated with glial activation and partially restored OL-specific transcripts to baseline levels. Finally, conditioned medium from activated microglia reduced OL-gene expression in primary OLs without altering their viability., Conclusions: These findings show that peripheral respiratory viral infection with IAV is capable of altering OL homeostasis and indicate that microglia activation is likely involved in the process., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Cross sectional studies have shown that statin-users have improved odds of surviving severe sepsis. Meanwhile controlled clinical trials failed to demonstrate improved sepsis survival with acute statin administration following hospitalization. Here, a lethal murine peritoneal lipopolysaccharide (LPS) endotoxemia model was used to assess the efficacy of chronic versus acute simvastatin on survival. Mirroring clinical observations, chronic but not acute treatment with simvastatin significantly increased survival. At a pre-mortality time point in LPS-treated mice, chronic simvastatin suppressed granulocyte trafficking in to the lungs and peritoneum without otherwise suppressing emergency myelopoiesis, myeloid cells in circulation, or inflammatory cytokines. Chronic simvastatin treatment significantly downregulated inflammatory chemokine gene signature in the lungs of LPS-treated mice. Thus, it was unclear if simvastatin was inhibiting granulocyte chemotaxis in a cell intrinsic or extrinsic manner. Adoptive transfer of fluorescently labeled granulocytes from statin and vehicle treated mice into LPS-treated mice showed that simvastatin inhibited lung-granulocyte trafficking in a cell intrinsic manner. Congruent with this, chemotaxis experiments using in vitro macrophages and ex vivo granulocytes demonstrated that simvastatin inhibited chemotaxis in a cell-intrinsic manner. Collectively, chronic but not acute simvastatin treatment improved survival in murine endotoxemia, and this was associated with cell-intrinsic inhibition of granulocyte chemotaxis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier GmbH.)

Influenza is a common cause of pneumonia-induced hospitalization and death, but how host factors function to influence disease susceptibility or severity has not been fully elucidated. Cellular cholesterol levels may affect the pathogenesis of influenza infection, as cholesterol is crucial for viral entry and replication, as well as immune cell proliferation and function. However, there is still conflicting evidence on the extent to which dietary cholesterol influences cholesterol metabolism. In this study, we examined the effects of a high-cholesterol diet in modulating the immune response to influenza A virus (IAV) infection in mice. Mice were fed a standard or a high-cholesterol diet for 5 wk before inoculation with mouse-adapted human IAV (Puerto Rico/8/1934), and tissues were collected at days 0, 4, 8, and 16 postinfection. Cholesterol-fed mice exhibited dyslipidemia characterized by increased levels of total serum cholesterol prior to infection and decreased triglycerides postinfection. Cholesterol-fed mice also displayed increased morbidity compared with control-fed mice, which was neither a result of immunosuppression nor changes in viral load. Instead, transcriptomic analysis of the lungs revealed that dietary cholesterol caused upregulation of genes involved in viral-response pathways and leukocyte trafficking, which coincided with increased numbers of cytokine-producing CD4 + and CD8 + T cells and infiltrating dendritic cells. Morbidity as determined by percent weight loss was highly correlated with numbers of cytokine-producing CD4 + and CD8 + T cells as well as granulocytes. Taken together, dietary cholesterol promoted IAV morbidity via exaggerated cellular immune responses that were independent of viral load., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Microglia activation and proliferation are hallmarks of many neurodegenerative disorders and may contribute to disease pathogenesis. Neurons actively regulate microglia survival and function, in part by secreting the microglia mitogen interleukin (IL)-34. Both IL-34 and colony stimulating factor (CSF)-1 bind colony stimulating factor receptor (CSFR)1 expressed on microglia. Systemic treatment with central nervous system (CNS) penetrant, CSFR1 antagonists, results in microglia death in a dose dependent matter, while others, such as GW2580, suppress activation during disease states without altering viability. However, it is not known how treatment with non-penetrant CSF1R antagonists, such as GW2580, affect the normal physiology of microglia. To determine how GW2580 affects microglia function, C57BL/6J mice were orally gavaged with vehicle or GW2580 (80mg/kg/d) for 8 days. Body weights and burrowing behavior were measured throughout the experiment. The effects of GW2580 on circulating leukocyte populations, brain microglia morphology, and the transcriptome of magnetically isolated adult brain microglia were determined. Body weights, burrowing behavior, and circulating leukocytes were not affected by treatment. Analysis of Iba-1 stained brain microglia indicated that GW2580 treatment altered morphology, but not cell number. Analysis of RNA-sequencing data indicated that genes related to reactive oxygen species (ROS) regulation and survival were suppressed by treatment. Treatment of primary microglia cultures with GW2580 resulted in a dose-dependent reduction in viability only when the cells were concurrently treated with LPS, an inducer of ROS. Pre-treatment with the ROS inhibitor, YCG063, blocked treatment induced reductions in viability. Finally, GW2580 sensitized microglia to hydrogen peroxide induced cell death. Together, these data suggest that partial CSF1R antagonism may render microglia more susceptible to reactive oxygen and nitrogen species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Soto-Diaz, Vailati-Riboni, Louie, McKim, Gaskins, Johnson and Steelman.)

Stromal cells are critical regulators of bone marrow hematopoietic niches, but assessment of their regulatory roles has been impeded by difficult and ineffective dissociation methods. Here, we methodically address bone marrow stromal cell dissociation. Yield of bone marrow CD45 - /Ter119 - /CD31 + /CD202b + endothelial cells (ECs) and CD45 - /Ter119 - /CD44 - /PDGFR + mesenchymal stromal cells (MSCs) were determined by flow cytometry. Liberase DL, Collagenase D, and Dispase II (all supplemented with DNase) enhanced EC and MSC yields, with Dispase II + DNase proving most effective. Combinations of these enzymes did not exhibit additive benefits, nor did the addition of Elastase, TrypLE, Hyaluronidase, or Accutase. Similarly, common mechanical dissociation approaches also proved ineffective. However, the combination of gentle Dispase II + DNase dissociation with magnetic sorting dramatically enriched both ECs and MSCs. This work methodically addressed common approaches for bone marrow stromal dissociation and established an effective approach for enrichment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Traumatic brain injury (TBI) can lead to significant neuropsychiatric problems and neurodegenerative pathologies, which develop and persist years after injury. Neuroinflammatory processes evolve over this same period. Therefore, we aimed to determine the contribution of microglia to neuropathology at acute [1 d postinjury (dpi)], subacute (7 dpi), and chronic (30 dpi) time points. Microglia were depleted with PLX5622, a CSF1R antagonist, before midline fluid percussion injury (FPI) in male mice and cortical neuropathology/inflammation was assessed using a neuropathology mRNA panel. Gene expression associated with inflammation and neuropathology were robustly increased acutely after injury (1 dpi) and the majority of this expression was microglia independent. At 7 and 30 dpi, however, microglial depletion reversed TBI-related expression of genes associated with inflammation, interferon signaling, and neuropathology. Myriad suppressed genes at subacute and chronic endpoints were attributed to neurons. To understand the relationship between microglia, neurons, and other glia, single-cell RNA sequencing was completed 7 dpi, a critical time point in the evolution from acute to chronic pathogenesis. Cortical microglia exhibited distinct TBI-associated clustering with increased type-1 interferon and neurodegenerative/damage-related genes. In cortical neurons, genes associated with dopamine signaling, long-term potentiation, calcium signaling, and synaptogenesis were suppressed. Microglial depletion reversed the majority of these neuronal alterations. Furthermore, there was reduced cortical dendritic complexity 7 dpi, reduced neuronal connectively 30 dpi, and cognitive impairment 30 dpi. All of these TBI-associated functional and behavioral impairments were prevented by microglial depletion. Collectively, these studies indicate that microglia promote persistent neuropathology and long-term functional impairments in neuronal homeostasis after TBI. SIGNIFICANCE STATEMENT Millions of traumatic brain injuries (TBIs) occur in the United States alone each year. Survivors face elevated rates of cognitive and psychiatric complications long after the inciting injury. Recent studies of human brain injury link chronic neuroinflammation to adverse neurologic outcomes, suggesting that evolving inflammatory processes may be an opportunity for intervention. Here, we eliminate microglia to compare the effects of diffuse TBI on neurons in the presence and absence of microglia and microglia-mediated inflammation. In the absence of microglia, neurons do not undergo TBI-induced changes in gene transcription or structure. Microglial elimination prevented TBI-induced cognitive changes 30 d postinjury (dpi). Therefore, microglia have a critical role in disrupting neuronal homeostasis after TBI, particularly at subacute and chronic timepoints., (Copyright © 2021 the authors.)

As a major producer of the inflammatory cytokine interleukin-1 (IL-1), peripheral macrophages can augment IL-1 expression via type 1 IL-1 receptor (IL-1R1) mediated autocrine self-amplification. In the CNS, microglial cells are the major producers of inflammatory cytokines, but express negligible levels of IL-1R1. In the present study, we showed CNS IL-1 induced microglial proinflammatory cytokine expression was mediated by endothelial, not microglial, IL-1R1. This paracrine mechanism was further dissected in vitro. IL-1 was unable to stimulate inflammatory cytokine expression directly from the microglial cell line BV-2, but it stimulated the brain endothelial cell line bEnd.3 to produce a factor(s) in the culture supernatant, which was capable of inducing inflammatory cytokine expression in BV-2. We termed this factor IL-1-induced microglial activation factors (IMAF). BV-2 cytokine expression was inducible by extracellular ATP, but IL-1 did not stimulate the release of ATP from bEnd.3 cells. Filtration of IMAF by size-exclusion membranes showed IMAF activity resided in molecules larger than 50 kd and incubation of IMAF at 95 °C for 5 min did not alter its activity. Microglial inhibitor minocycline was unable to block IMAF activity, even though it blocked LPS induced cytokine expression in BV-2 cells. Adding NF-κB inhibitor to the bEnd.3 cells abolished IL-1 induced cytokine expression in this bi-cellular system, but adding NF-κB inhibitor after IMAF is already produced failed to abrogate IMAF induced cytokine expression in BV-2 cells. RNA sequencing of IL-1 stimulated endothelial cells revealed increased expression of genes involved in the production and processing of hyaluronic acid (HA), suggesting HA as a candidate of IMAF. Inhibition of hyaluronidase by ascorbyl palmitate (AP) abolished IMAF-induced cytokine expression in BV-2 cells. AP administration in vivo also inhibited ICV IL-1-induced IL-1 expression in the hippocampus and hypothalamus. In vitro, either TLR2 or TLR4 inhibitors blocked IMAF induced BV-2 cytokine expression. In vivo, however, IL-1 induced cytokine expression persisted in either TLR2 or TLR4 knockouts. These results demonstrate IL-1 induced inflammatory cytokine expression in the CNS requires a bi-cellular system and HA could be a candidate for IMAF., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Circadian rhythms influence various aspects of biology, including hormonal, immunological, and behavioral processes. These 24-hour oscillations are necessary to optimize cellular functions and to synchronize these processes with the environment. Breast cancer patients and survivors frequently report disruptions in circadian oscillations that adversely affect quality-of-life, including fragmented sleep-wake cycles and flattened cortisol rhythms, which are associated with negative behavioral comorbidities (e.g., fatigue). However, the potential causal role of tumor biology in circadian dysregulation has not been investigated. Here, we examined the extent to which sham surgery, non-metastatic mammary tumors, or mammary tumor removal in mice disrupts circadian rhythms in brain clock gene expression, locomotor behavior (free-running and entrained), and physiological rhythms that have been associated with cancer behavioral comorbidities. Tumors and tumor resection altered time-of-day differences in hypothalamic expression of eight circadian-regulated genes. The onset of activity in entrained running behavior was advanced in tumor-bearing mice, and the amplitude of free-running rhythms was increased in tumor-resected mice. Tumors flattened rhythms in circulating corticosterone and Ly6c Hi monocytes which were largely restored by surgical tumor resection. This work implies that tumors alone may directly impact central and/or peripheral circadian rhythmicity in breast cancer patients, and that these effects may persist in cancer survivors, potentially contributing to behavioral comorbidities., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Anxiety and mood disorders affect both men and women. The majority of experimental models of stress, however, are completed using only male animals. For repeated social defeat (RSD), a rodent model, this is due to the inherent difficulty in eliciting male aggression toward female mice. To address this limitation, a recent study showed that a DREADD-based activation of the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) was effective in inducing aggressive behavior in male mice towards females in a social defeat paradigm. Therefore, the goal of this study was to determine if this modified version of RSD in females elicited behavioral, physiological, and immune responses similar to those reported in males. Here, we show that female mice subjected to RSD with the male DREADD aggressor developed anxiety-like behavior and social avoidance. These behavioral alterations coincided with enhanced neuronal and microglial activation in threat-appraisal regions of the brain. Moreover, stressed female mice had an enhanced peripheral immune response characterized by increased myelopoiesis, release of myeloid cells into circulation, and monocyte accumulation in the spleen and brain. These results are consistent with previously reported findings that male mice exposed to RSD exhibited increased fear and threat appraisal responses, enhanced myelopoiesis, myeloid cell release and trafficking, and anxiety-like behavior. These findings validate that RSD is a relevant model to study stress responses in female mice., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Stress is associated with an increased prevalence of anxiety and depression. Repeated social defeat (RSD) stress in mice increases the release of monocytes from the bone marrow that are recruited to the brain by microglia. These monocytes enhance inflammatory signaling and augment anxiety. Moreover, RSD promotes stress sensitization, in which exposure to acute stress 24 days after cessation of RSD causes anxiety recurrence. The purpose of this study was to determine whether microglia were critical to stress sensitization and exhibited increased reactivity to subsequent acute stress or immune challenge., Methods: Mice were exposed to RSD, microglia were eliminated by colony-stimulating factor 1 receptor antagonism (PLX5622) and allowed to repopulate, and responses to acute stress or immune challenge (lipopolysaccharide) were determined 24 days after RSD sensitization., Results: Microglia maintained a unique messenger RNA signature 24 days after RSD. Moreover, elimination of RSD-sensitized microglia prevented monocyte accumulation in the brain and blocked anxiety recurrence following acute stress (24 days). When microglia were eliminated prior to RSD and repopulated and mice were subjected to acute stress, there was monocyte accumulation in the brain and anxiety in RSD-sensitized mice. These responses were unaffected by microglial elimination/repopulation. This may be related to neuronal sensitization that persisted 24 days after RSD. Following immune challenge, there was robust microglial reactivity in RSD-sensitized mice associated with prolonged sickness behavior. Here, microglial elimination/repopulation prevented the amplified immune reactivity ex vivo and in vivo in RSD-sensitized mice., Conclusions: Microglia and neurons remain sensitized weeks after RSD, and only the immune reactivity component of RSD-sensitized microglia was prevented by elimination/repopulation., (Copyright © 2018 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Spinal cord injury (SCI) produces a toxic inflammatory microenvironment that negatively affects plasticity and recovery. Recently, we showed glial activation and peripheral myeloid cell infiltration extending beyond the epicenter through the remote lumbar cord after thoracic SCI. The presence and role of infiltrating monocytes is important, especially in the lumbar cord where locomotor central pattern generators are housed. Therefore, we compared the inflammatory profile of resident microglia and peripheral myeloid cells after SCI. Bone marrow chimeras received midthoracic contusive SCI, and trafficking was determined 1-7 days later. Fluorescence-activated cell (FAC) sorting showed similar infiltration timing of both neutrophils and macrophages in epicenter and lumbar regions. While neutrophil numbers were attenuated by day 3, macrophages remained unchanged at day 7, suggesting that macrophages have important long-term influence on the microenvironment. Nanostring gene array identified a strong proinflammatory profile of infiltrating macrophages relative to microglia at both epicenter and lumbar sites. Macrophages had elevated expression of inflammatory cytokines (IL-1β, IFNγ), chemokines (CCL2, CXCL2), mediators (COX-1, MMP-9), and receptors (CCR2, Ly6C), and decreased expression of growth promoting genes (GDNF, BDNF). Importantly, lumbar macrophages had elevated expression of active trafficking genes (CCR2, l-selectin, MMP-9) compared with epicenter macrophages. Further, acute rehabilitation exacerbated the inflammatory profile of infiltrated macrophages in the lumbar cord. Such high inflammatory potential and negative response to rehabilitation of infiltrating macrophages within lumbar locomotor central pattern generators likely impedes activity-dependent recovery. Therefore, limiting active trafficking of macrophages into the lumbar cord identifies a novel target for SCI therapies to improve locomotion.

Clinical studies indicate that psychosocial stress contributes to adverse chronic pain outcomes in patients, but it is unclear how this is initiated or amplified by stress. Repeated social defeat (RSD) is a mouse model of psychosocial stress that activates microglia, increases neuroinflammatory signaling, and augments pain and anxiety-like behaviors. We hypothesized that activated microglia within the spinal cord facilitate increased pain sensitivity following RSD. Here we show that mechanical allodynia in male mice was increased with exposure to RSD. This stress-induced behavior corresponded with increased mRNA expression of several inflammatory genes, including IL-1β, TNF-α, CCL2, and TLR4 in the lumbar spinal cord. While there were several adhesion and chemokine-related genes increased in the lumbar spinal cord after RSD, there was no accumulation of monocytes or neutrophils. Notably, there was evidence of microglial activation selectively within the nociceptive neurocircuitry of the dorsal horn of the lumbar cord. Elimination of microglia using the colony stimulating factor 1 receptor antagonist PLX5622 from the brain and spinal cord prevented the development of mechanical allodynia in RSD-exposed mice. Microglial elimination also attenuated RSD-induced IL-1β, CCR2, and TLR4 mRNA expression in the lumbar spinal cord. Together, RSD-induced allodynia was associated with microglia-mediated inflammation within the dorsal horn of the lumbar spinal cord. SIGNIFICANCE STATEMENT Mounting evidence indicates that psychological stress contributes to the onset and progression of adverse nociceptive conditions. We show here that repeated social defeat stress causes increased pain sensitivity due to inflammatory signaling within the nociceptive circuits of the spinal cord. Studies here mechanistically tested the role of microglia in the development of pain by stress. Pharmacological ablation of microglia prevented stress-induced pain sensitivity. These findings demonstrate that microglia are critical mediators in the induction of pain conditions by stress. Moreover, these studies provide a proof of principle that microglia can be targeted as a therapeutic strategy to mitigate adverse pain conditions., (Copyright © 2019 the authors 0270-6474/19/391139-11$15.00/0.)

Microglia undergo dynamic structural and transcriptional changes during the immune response to traumatic brain injury (TBI). For example, TBI causes microglia to form rod-shaped trains in the cerebral cortex, but their contribution to inflammation and pathophysiology is unclear. The purpose of this study was to determine the origin and alignment of rod microglia and to determine the role of microglia in propagating persistent cortical inflammation. Here, diffuse TBI in mice was modeled by midline fluid percussion injury (FPI). Bone marrow chimerism and BrdU pulse-chase experiments revealed that rod microglia derived from resident microglia with limited proliferation. Novel data also show that TBI-induced rod microglia were proximal to axotomized neurons, spatially overlapped with dense astrogliosis, and aligned with apical pyramidal dendrites. Furthermore, rod microglia formed adjacent to hypertrophied microglia, which clustered among layer V pyramidal neurons. To better understand the contribution of microglia to cortical inflammation and injury, microglia were eliminated prior to TBI by CSF1R antagonism (PLX5622). Microglial elimination did not affect cortical neuron axotomy induced by TBI, but attenuated rod microglial formation and astrogliosis. Analysis of 262 immune genes revealed that TBI caused profound cortical inflammation acutely (8 hr) that progressed in nature and complexity by 7 dpi. For instance, gene expression related to complement, phagocytosis, toll-like receptor signaling, and interferon response were increased 7 dpi. Critically, these acute and chronic inflammatory responses were prevented by microglial elimination. Taken together, TBI-induced neuronal injury causes microglia to structurally associate with neurons, augment astrogliosis, and propagate diverse and persistent inflammatory/immune signaling pathways., (© 2018 Wiley Periodicals, Inc.)

Psychosocial stress accelerates myelopoietic production of monocytes and neutrophils that contributes to a variety of health complications ranging from atherosclerosis to anxiety. Here, we show that social stress in mice mobilizes hematopoietic stem progenitor cells (HSPCs) from the bone marrow that enter circulation, engraft into the spleen, and establish a persistent extramedullary hematopoietic depot. These splenic progenitors actively proliferate and differentiate into multiple cell types, including monocytes, neutrophils, and erythrocytes. Splenic erythropoiesis partially abrogates stress-induced anemia. Repeated injection with isoprenaline induces progenitor mobilization to the spleen, identifying a key role for β-adrenergic signaling. Moreover, protracted splenic production of CD11b + cells persists for at least 24 days after the cessation of social stress. Thus, chronic stress establishes a persistent extramedullary hematopoietic depot that can modify a wide range of chronic disease processes and alter homeostasis of the bi-directional regulatory axis between the nervous and immune systems., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Microglia are the resident innate immune cells of the central nervous system. Limited turnover throughout the lifespan leaves microglia susceptible to age-associated dysfunction. Indeed, we and others have reported microglia develop a pro-inflammatory or "primed" profile with age, characterized by increased expression of inflammatory mediators (e.g., MHC-II, CD68, IL-1β). Moreover, immune challenge with lipopolysaccharide (LPS) causes an exaggerated and prolonged neuroinflammatory response mediated by primed microglia in the aged brain. Recent studies show colony-stimulating factor 1 receptor (CSF1R) antagonism results in rapid depletion of microglia without significant complications. Therefore, we hypothesized that CSF1R antagonist-mediated depletion of microglia in the aged brain would result in repopulation with new and unprimed microglia. Here we provide novel evidence that microglia in the brain of adult (6-8 weeks old) and aged (16-18 months old) BALB/c mice were depleted following 3-week oral PLX5622 administration. When CSF1R antagonism was stopped, microglia repopulated equally in the adult and aged brain. Microglial depletion and repopulation reversed age-associated increases in microglial CD68 + lysosome enlargement and lipofuscin accumulation. Microglia-specific RNA sequencing revealed 511 differentially expressed genes with age. Of these, 117 genes were reversed by microglial repopulation (e.g., Apoe, Tgfb2, Socs3). Nevertheless, LPS challenge still induced an exaggerated microglial inflammatory response in the aged brain compared to adults. RNA sequencing of whole-brain tissue revealed an age-induced inflammatory signature, including reactive astrocytes, that was not restored by microglial depletion and repopulation. Furthermore, the microenvironment of the aged brain produced soluble factors that influenced developing microglia ex vivo and induced a profile primed to LPS challenge. Thus, the aged brain microenvironment promotes microglial priming despite repopulation of new microglia. Collectively, aged microglia proliferate and repopulate the brain, but these new cells still adopt a pro-inflammatory profile in the aged brain.

Psychosocial stress contributes to the development of anxiety and depression. Recent clinical studies have reported increased inflammatory leukocytes in circulation of individuals with stress-related psychiatric disorders. Parallel to this, our work in mice shows that social stress causes release of inflammatory monocytes into circulation. In addition, social stress caused the development of prolonged anxiety that was dependent on inflammatory monocytes in the brain. Therefore, we hypothesize that chronic stress drives the production of inflammatory monocytes that are actively recruited to the brain by microglia, and these monocytes augment neuroinflammatory signaling and prolong anxiety. Here we show that repeated social defeat stress in mice activated threat appraisal centers in the brain that spatially coincided with microglial activation and endothelial facilitation of monocyte recruitment. Moreover, microglial depletion with a CSF1R antagonist prior to stress prevented the recruitment of monocytes to the brain and abrogated the development of anxiety. Cell-specific transcriptional profiling revealed that microglia selectively enhanced CCL2 expression, while monocytes expressed the pro-inflammatory cytokine interleukin-1β (IL-1β). Consistent with these profiles, the recruited inflammatory monocytes with stress adhered to IL-1R1 + neurovascular endothelial cells and this interaction was blocked by microglial depletion. Furthermore, disruption of IL-1β signaling by caspase-1 KO specifically within bone marrow-derived cells revealed that monocytes promoted anxiogenesis through stimulation of neurovascular IL-1R1 by IL-1β. Collectively, the development of anxiety during stress was caused by microglial recruitment of IL-1β-producing monocytes, which stimulated brain endothelial IL-1R1. Thus, monocyte IL-1β production represents a novel mechanism that underlies behavioral complications associated with stress-related psychiatric disorders.

Background: Before primary oral tumors are treated, various prophylactic procedures that require tissue repair are often necessary (e.g. biopsies, tooth extractions, radiation, and tracheotomies). Wound healing and tumor growth harness similar immune/inflammatory mechanisms. Our previous work indicates that tumors impair wound healing, although the extent to which tissue repair conversely influences tumor growth is poorly understood. Here, we test the hypothesis that dermal wound healing exacerbates primary tumor growth and influences tumor immunobiology., Materials and Methods: Female, immunocompetent mice were inoculated subcutaneously with murine oral cancer cells (AT-84) to induce flank tumors. Half of the mice received dermal excisional wounds (4 × 3.5 mm diameter) on their dorsum 16 days later, whereas the skin of controls remained intact. Tumor and blood tissues were harvested 1 and 5 days post wounding, and tumor myeloid cell populations and inflammatory gene expression were measured. Circulating myeloid cells, cytokines, and corticosterone were also quantified., Results: Wounding increased tumor mass, early tumor infiltration of macrophages, and tumor inflammatory gene expression. While wounding attenuated tumor growth-induced increases in circulating myeloid cells, no effects of wounding on circulating cytokine/endocrine measures were observed., Conclusions: These results indicate that modest skin immune/inflammatory processes can enhance distal tumor growth and alter innate tumor immunity. The implication for this work is that, in the presence of a tumor, the benefits of tissue-damaging procedures that occur clinically must be weighed against the potential consequences for tumor biology., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tissue repair is an integral component of cancer treatment (e.g., due to surgery, chemotherapy, radiation). Previous work has emphasized the immunosuppressive effects of tumors on adaptive immunity and has shown that surgery incites cancer metastases. However, the extent to which and how tumors may alter the clinically-relevant innate immune process of wound healing remains an untapped potential area of improvement for treatment, quality of life, and ultimately, mortality of cancer patients. In this study, 3.5 mm full-thickness dermal excisional wounds were placed on the dorsum of immunocompetent female mice with and without non-malignant flank AT-84 murine oral squamous cell carcinomas. Wound closure rate, inflammatory cell number and inflammatory signaling in wounds, and circulating myeloid cell concentrations were compared between tumor-bearing and tumor-free mice. Tumors delayed wound closure, suppressed inflammatory signaling, and altered myeloid cell trafficking in wounds. An in vitro scratch "wounding" assay of adult dermal fibroblasts treated with tumor cell-conditioned media supported the in vivo findings. This study demonstrates that tumors are sufficient to disrupt fundamental and clinically-relevant innate immune functions. The understanding of these underlying mechanisms provides potential for therapeutic interventions capable of improving the treatment of cancer while reducing morbidities and mortality., Competing Interests: The authors have declared that no competing interests exist.

Background: Neuroinflammatory signaling may contribute to the pathophysiology of chronic anxiety disorders. Previous work showed that repeated social defeat (RSD) in mice promoted stress-sensitization that was characterized by the recurrence of anxiety following subthreshold stress 24 days after RSD. Furthermore, splenectomy following RSD prevented the recurrence of anxiety in stress-sensitized mice. We hypothesize that the spleen of RSD-exposed mice became a reservoir of primed monocytes that were released following neuroendocrine activation by subthreshold stress., Methods: Mice were subjected to subthreshold stress (i.e., single cycle of social defeat) 24 days after RSD, and immune and behavioral measures were taken., Results: Subthreshold stress 24 days after RSD re-established anxiety-like behavior that was associated with egress of Ly6C(hi) monocytes from the spleen. Moreover, splenectomy before RSD blocked monocyte trafficking to the brain and prevented anxiety-like behavior following subthreshold stress. Splenectomy, however, had no effect on monocyte accumulation or anxiety when determined 14 hours after RSD. In addition, splenocytes cultured 24 days after RSD exhibited a primed inflammatory phenotype. Peripheral sympathetic inhibition before subthreshold stress blocked monocyte trafficking from the spleen to the brain and prevented the re-establishment of anxiety in RSD-sensitized mice. Last, β-adrenergic antagonism also prevented splenic monocyte egress after acute stress., Conclusions: The spleen served as a unique reservoir of primed monocytes that were readily released following sympathetic activation by subthreshold stress that promoted the re-establishment of anxiety. Collectively, the long-term storage of primed monocytes in the spleen may have a profound influence on recurring anxiety disorders., (Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Repeated social defeat (RSD) is a murine stressor that recapitulates key physiological, immunological, and behavioral alterations observed in humans exposed to chronic psychosocial stress. Psychosocial stress promotes prolonged behavioral adaptations that are associated with neuroinflammatory signaling and impaired neuroplasticity. Here, we show that RSD promoted hippocampal neuroinflammatory activation that was characterized by proinflammatory gene expression and by microglia activation and monocyte trafficking that was particularly pronounced within the caudal extent of the hippocampus. Because the hippocampus is a key area involved in neuroplasticity, behavior, and cognition, we hypothesize that stress-induced neuroinflammation impairs hippocampal neurogenesis and promotes cognitive and affective behavioral deficits. We show here that RSD caused transient impairments in spatial memory recall that resolved within 28 d. In assessment of neurogenesis, the number of proliferating neural progenitor cells (NPCs) and the number of young, developing neurons were not affected initially after RSD. Nonetheless, the neuronal differentiation of NPCs that proliferated during RSD was significantly impaired when examined 10 and 28 d later. In addition, social avoidance, a measure of depressive-like behavior associated with caudal hippocampal circuitry, persisted 28 d after RSD. Treatment with minocycline during RSD prevented both microglia activation and monocyte recruitment. Inhibition of this neuroinflammatory activation in turn prevented impairments in spatial memory after RSD but did not prevent deficits in neurogenesis nor did it prevent the persistence of social avoidance behavior. These findings show that neuroinflammatory activation after psychosocial stress impairs spatial memory performance independent of deficits in neurogenesis and social avoidance., Significance Statement: Repeated exposure to stress alters the homeostatic environment of the brain, giving rise to various cognitive and mood disorders that impair everyday functioning and overall quality of life. The brain, previously thought of as an immune-privileged organ, is now known to communicate extensively with the peripheral immune system. This brain-body communication plays a significant role in various stress-induced inflammatory conditions, also characterized by psychological impairments. Findings from this study implicate neuroimmune activation rather than impaired neurogenesis in stress-induced cognitive deficits. This idea opens up possibilities for novel immune interventions in the treatment of cognitive and mood disturbances, while also adding to the complexity surrounding the functional implications of adult neurogenesis., (Copyright © 2016 the authors 0270-6474/16/362590-15$15.00/0.)

Repeated social defeat (RSD) in mice causes myeloid cell trafficking to the brain that contributes to the development of prolonged anxiety-like behavior. Myeloid cell recruitment following RSD occurs in regions where neuronal and microglia activation is observed. Thus, we hypothesized that crosstalk between neurons, microglia, and endothelial cells contributes to brain myeloid cell trafficking via chemokine signaling and vascular adhesion molecules. Here we show that social defeat caused an exposure- and brain region-dependent increase in several key adhesion molecules and chemokines involved in the recruitment of myeloid cells. For example, RSD induced distinct patterns of adhesion molecule expression that may explain brain region-dependent myeloid cell trafficking. VCAM-1 and ICAM-1 mRNA expression were increased in an exposure-dependent manner. Furthermore, RSD-induced VCAM-1 and ICAM-1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Next, mRNA expression of additional adhesion molecules (E- and P-selectin, PECAM-1) and chemokines (CXCL1, CXCL2, CXCL12, CCL2) were determined in the brain. Social defeat induced an exposure-dependent increase in mRNA levels of E-selectin, CXCL1, and CXCL2 that increased with additional days of social defeat. While CXCL12 was unaffected by RSD, CCL2 expression was increased by six days of social defeat. Last, comparison between enriched CD11b(+) cells (microglia/macrophages) and enriched GLAST-1(+)/CD11b(-) cells (astrocytes) revealed RSD increased mRNA expression of IL-1β, CCL2, and CXCL2 in microglia/macrophages but not in astrocytes. Collectively, these data indicate that key mediators of leukocyte recruitment were increased in the brain vasculature following RSD in an exposure- and brain region-dependent manner., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Psychosocial stress is associated with altered immunity, anxiety and depression. Previously we showed that repeated social defeat (RSD) promoted microglia activation and social avoidance behavior that persisted for 24days after cessation of RSD. The aim of the present study was to determine if imipramine (a tricyclic antidepressant) would reverse RSD-inducedsocial avoidance and ameliorate neuroinflammatory responses. To test this, C57BL/6 mice were divided into treatment groups. One group from RSD and controls received daily injections of imipramine for 24days, following 6 cycles of RSD. Two other groups were treated with saline. RSD mice spent significantly less time in the interaction zone when an aggressor was present in the cage. Administration of imipramine reversed social avoidance behavior, significantly increasing the interaction time, so that it was similar to that of control mice. Moreover, 24days of imipramine treatment in RSD mice significantly decreased stress-induced mRNA levels for IL-6 in brain microglia. Following ex vivo LPS stimulation, microglia from mice exposed to RSD, had higher mRNA expression of IL-6, TNF-α, and IL-1β, and this was reversed by imipramine treatment. In a second experiment, imipramine was added to drinking water confirming the reversal of social avoidant behavior and decrease in mRNA expression of IL-6 in microglia. These data suggest that the antidepressant imipramine may exert its effect, in part, by down-regulating microglial activation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The development and exacerbation of depression and anxiety are associated with exposure to repeated psychosocial stress. Stress is known to affect the bidirectional communication between the nervous and immune systems leading to elevated levels of stress mediators including glucocorticoids (GCs) and catecholamines and increased trafficking of proinflammatory immune cells. Animal models, like the repeated social defeat (RSD) paradigm, were developed to explore this connection between stress and affective disorders. RSD induces activation of the sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA) axis activation, increases bone marrow production and egress of primed, GC-insensitive monocytes, and stimulates the trafficking of these cells to tissues including the spleen, lung, and brain. Recently, the observation that these monocytes have the ability to traffic to the brain perivascular spaces and parenchyma have provided mechanisms by which these peripheral cells may contribute to the prolonged anxiety-like behavior associated with RSD. The data that have been amassed from the RSD paradigm and others recapitulate many of the behavioral and immunological phenotypes associated with human anxiety disorders and may serve to elucidate potential avenues of treatment for these disorders. Here, we will discuss novel and key data that will present an overview of the neuroendocrine, immunological and behavioral responses to social stressors., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Interleukin-1 (IL-1) mediates diverse neurophysiological and neuropathological effects in the CNS through type I IL-1 receptor (IL-1R1). However, identification of IL-1R1-expressing cell types and cell-type-specific functions of IL-1R1 remains challenging. In this study, we created a novel genetic mouse model in which IL-1R1 gene expression is disrupted by an intronic insertion of a loxP flanked disruptive sequence that can be deleted by Cre recombinase, resulting in restored IL-1R1 gene expression under its endogenous promoters. A second mutation was introduced at stop codon of the IL-1R1 gene to allow tracking of the restored IL-1R1 protein by a 3HA tag and IL-1R1 mRNA by tdTomato fluorescence. These animals were designated as IL-1R1(r/r) and exhibited an IL-1R1 knock-out phenotype. We used IL-1R1 globally restored mice (IL-1R1(GR/GR)) as an IL-1R1 reporter and observed concordant labeling of IL-1R1 mRNA and protein in brain endothelial cells. Two cell-type-specific IL-1R1 restore lines were generated: Tie2Cre-IL-1R1(r/r) and LysMCre-IL-1R1(r/r). Brain endothelial COX-2 expression, CNS leukocyte infiltration, and global microglia activation induced by intracerebroventricular injection of IL-1β were not observed in IL-1R1(r/r) or LysMCre-IL-1R1(r/r) mice, but were restored in Tie2Cre-IL-1R1(r/r) mice. These results reveal IL-1R1 expression in endothelial cells alone is sufficient to mediate these central IL-1-induced responses. In addition, ex vivo IL-1β stimulation increased IL-1β expression in bone marrow cells in wild-type, Tie2Cre-IL-1R1(r/r), and LysMCre-IL-1R1(r/r), but not IL-1R1(r/r) mice. These results demonstrate this IL-1R1 restore model is a valuable tool for studying cell-type-specific functions of IL-1R1., (Copyright © 2015 the authors 0270-6474/15/352860-11$15.00/0.)

HIGHLIGHTSPsychological stress activates neuroendocrine pathways that alter immune responses.Stress-induced alterations in microglia phenotype and monocyte priming leads to aberrant peripheral and central inflammation.Elevated pro-inflammatory cytokine levels caused by microglia activation and recruitment of monocytes to the brain contribute to development and persistent anxiety-like behavior.Mechanisms that mediate interactions between microglia, endothelial cells, and macrophages and how these contribute to changes in behavior are discussed.Sensitization of microglia and re-distribution of primed monocytes are implicated in re-establishment of anxiety-like behavior. Psychological stress causes physiological, immunological, and behavioral alterations in humans and rodents that can be maladaptive and negatively affect quality of life. Several lines of evidence indicate that psychological stress disrupts key functional interactions between the immune system and brain that ultimately affects mood and behavior. For example, activation of microglia, the resident innate immune cells of the brain, has been implicated as a key regulator of mood and behavior in the context of prolonged exposure to psychological stress. Emerging evidence implicates a novel neuroimmune circuit involving microglia activation and sympathetic outflow to the peripheral immune system that further reinforces stress-related behaviors by facilitating the recruitment of inflammatory monocytes to the brain. Evidence from various rodent models, including repeated social defeat (RSD), revealed that trafficking of monocytes to the brain promoted the establishment of anxiety-like behaviors following prolonged stress exposure. In addition, new evidence implicates monocyte trafficking from the spleen to the brain as key regulator of recurring anxiety following exposure to prolonged stress. The purpose of this review is to discuss mechanisms that cause stress-induced monocyte re-distribution in the brain and how dynamic interactions between microglia, endothelial cells, and brain macrophages lead to maladaptive behavioral responses.

Background: Persistent anxiety-like symptoms may have an inflammatory-related pathophysiology. Our previous work using repeated social defeat (RSD) in mice showed that recruitment of peripheral myeloid cells to the brain is required for the development of anxiety. Here, we aimed to determine if 1) RSD promotes prolonged anxiety through redistribution of myeloid cells and 2) prior exposure to RSD sensitizes the neuroimmune axis to secondary subthreshold stress., Methods: Mice were subjected to RSD and several immune and behavioral parameters were determined .5, 8, or 24 days later. In follow-up studies, control and RSD mice were subjected to subthreshold stress at 24 days., Results: Repeated social defeat-induced macrophage recruitment to the brain corresponded with development and maintenance of anxiety-like behavior 8 days after RSD, but neither remained at 24 days. Nonetheless, social avoidance and an elevated neuroinflammatory profile were maintained at 24 days. Subthreshold social defeat in RSD-sensitized mice increased peripheral macrophage trafficking to the brain that promoted re-establishment of anxiety. Moreover, subthreshold social defeat increased social avoidance in RSD-sensitized mice compared with naïve mice. Stress-induced monocyte trafficking was linked to redistribution of myeloid progenitor cells in the spleen. Splenectomy before subthreshold stress attenuated macrophage recruitment to the brain and prevented anxiety-like behavior in RSD-sensitized mice., Conclusions: These data indicate that monocyte trafficking from the spleen to the brain contributes re-establishment of anxiety in stress-sensitized mice. These findings show that neuroinflammatory mechanisms promote mood disturbances following stress-sensitization and outline novel neuroimmune interactions that underlie recurring anxiety disorders such as posttraumatic stress disorder., (Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Recent evidence indicates that inflammatory insults in neonates significantly influenced white matter development and caused behavioral deficits that manifest in young adulthood. The mechanisms underlying these developmental and behavioral complications, however, are not well understood. We hypothesize that acute brain inflammation caused by neonatal infection reduces the bioavailability of iron required for oligodendrocyte maturation and white matter development. Here, we confirm that peripheral Escherichia coli infection in neonates at postnatal day 3 (P3) caused acute brain inflammation that was resolved within 72 h. Nonetheless, transient early life infection (ELI) profoundly influenced behavior, white matter development, and iron homeostasis in the brain. For instance, mice exposed to E. coli as neonates had increased locomotor activity and impaired motor coordination as juveniles (P35) and young adults (P60). In addition, these behavioral deficits were associated with marked hypomyelination and a reduction of oligodendrocytes in subcortical white matter and motor cortex. Moreover, ELI altered transcripts related to cellular sequestration of iron in the brain including hepcidin, ferroportin, and L-ferritin. For example, ELI increased hepcidin mRNA and decreased ferroportin mRNA and protein in the brain at P4, which preceded increased L-ferritin mRNA at P12. Consistent with the mRNA results, L-ferritin protein was robustly increased at P12 specifically in neurons of E. coli infected mice. We interpret these data to indicate that neonatal infection causes significant neuronal sequestration of iron at a time point before myelination. Together, these data indicate a possible role for aberrant neuronal iron storage in neonatal infection-induced disturbances in myelination and behavior.