Cell-Type-Specific Interleukin 1 Receptor 1 Signaling in the Brain Regulates Distinct Neuroimmune Activities.

Interleukin-1 (IL-1) signaling is important for multiple potentially pathogenic processes in the central nervous system (CNS), but the cell-type-specific roles of IL-1 signaling are unclear. We used a genetic knockin reporter system in mice to track and reciprocally delete or express IL-1 receptor 1 (IL-1R1) in specific cell types, including endothelial cells, ventricular cells, peripheral myeloid cells, microglia, astrocytes, and neurons. We found that endothelial IL-1R1 was necessary and sufficient for mediating sickness behavior and drove leukocyte recruitment to the CNS and impaired neurogenesis, whereas ventricular IL-1R1 was critical for monocyte recruitment to the CNS. Although microglia did not express IL-1R1, IL-1 stimulation of endothelial cells led to the induction of IL-1 in microglia. Together, these findings describe the structure and functions of the brain's IL-1R1-expressing system and lay a foundation for the dissection and identification of IL-1R1 signaling pathways in the pathogenesis of CNS diseases.

Interleukin-1 receptor on hippocampal neurons drives social withdrawal and cognitive deficits after chronic social stress.

Chronic stress contributes to the development of psychiatric disorders including anxiety and depression. Several inflammatory-related effects of stress are associated with increased interleukin-1 (IL-1) signaling within the central nervous system and are mediated by IL-1 receptor 1 (IL-1R1) on several distinct cell types. Neuronal IL-1R1 is prominently expressed on the neurons of the dentate gyrus, but its role in mediating behavioral responses to stress is unknown. We hypothesize that IL-1 acts on this subset of hippocampal neurons to influence cognitive and mood alterations with stress. Here, mice subjected to psychosocial stress showed reduced social interaction and impaired working memory, and these deficits were prevented by global IL-1R1 knockout. Stress-induced monocyte trafficking to the brain was also blocked by IL-1R1 knockout. Selective deletion of IL-1R1 in glutamatergic neurons (nIL-1R1-/-) abrogated the stress-induced deficits in social interaction and working memory. In addition, viral-mediated selective IL-1R1 deletion in hippocampal neurons confirmed that IL-1 receptor in the hippocampus was critical for stress-induced behavioral deficits. Furthermore, selective restoration of IL-1R1 on glutamatergic neurons was sufficient to reestablish the impairments of social interaction and working memory after stress. RNA-sequencing of the hippocampus revealed that stress increased several canonical pathways (TREM1, NF-κB, complement, IL-6 signaling) and upstream regulators (INFγ, IL-1ß, NF-κB, MYD88) associated with inflammation. The inductions of TREM1 signaling, complement, and leukocyte extravasation with stress were reversed by nIL-1R1-/-. Collectively, stress-dependent IL-1R1 signaling in hippocampal neurons represents a novel mechanism by which inflammation is perpetuated and social interactivity and working memory are modulated.

Serial Systemic Injections of Endotoxin (LPS) Elicit Neuroprotective Spinal Cord Microglia through IL-1-Dependent Cross Talk with Endothelial Cells.

Microglia are dynamic immunosurveillance cells in the CNS. Whether microglia are protective or pathologic is context dependent; the outcome varies as a function of time relative to the stimulus, activation state of neighboring cells in the microenvironment or within progression of a particular disease. Although brain microglia can be "primed" using bacterial lipopolysaccharide (LPS)/endotoxin, it is unknown whether LPS delivered systemically can also induce neuroprotective microglia in the spinal cord. Here, we show that serial systemic injections of LPS (1 mg/kg, i.p., daily) for 4 consecutive days (LPSx4) consistently elicit a reactive spinal cord microglia response marked by dramatic morphologic changes, increased production of IL-1, and enhanced proliferation without triggering leukocyte recruitment or overt neuropathology. Following LPSx4, reactive microglia frequently contact spinal cord endothelial cells. Targeted ablation or selective expression of IL-1 and IL-1 receptor (IL-1R) in either microglia or endothelia reveal that IL-1-dependent signaling between these cells mediates microglia activation. Using a mouse model of ischemic spinal cord injury in male and female mice, we show that preoperative LPSx4 provides complete protection from ischemia-induced neuron loss and hindlimb paralysis. Neuroprotection is partly reversed by either pharmacological elimination of microglia or selective removal of IL-1R in microglia or endothelia. These data indicate that spinal cord microglia are amenable to therapeutic reprogramming via systemic manipulation and that this potential can be harnessed to protect the spinal cord from injury.SIGNIFICANCE STATEMENT Data in this report indicate that a neuroprotective spinal cord microglia response can be triggered by daily systemic injections of LPS over a period of 4 d (LPSx4). The LPSx4 regimen induces morphologic transformation and enhances proliferation of spinal cord microglia without causing neuropathology. Using advanced transgenic mouse technology, we show that IL-1-dependent microglia-endothelia cross talk is necessary for eliciting this spinal cord microglia phenotype and also for conferring optimal protection to spinal motor neurons from ischemic spinal cord injury (ISCI). Collectively, these novel data show that it is possible to consistently elicit spinal cord microglia via systemic delivery of inflammogens to achieve a therapeutically effective neuroprotective response against ISCI.

Interleukin-1 causes CNS inflammatory cytokine expression via endothelia-microglia bi-cellular signaling.

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.

Interleukin 1 type 1 receptor restore: a genetic mouse model for studying interleukin 1 receptor-mediated effects in specific cell types.

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-1ß 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-1ß stimulation increased IL-1ß 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.

Euflammation attenuates peripheral inflammation-induced neuroinflammation and mitigates immune-to-brain signaling.

Peripheral inflammation can trigger a number of neuroinflammatory events in the CNS, such as activation of microglia and increases of proinflammatory cytokines. We have previously identified an interesting phenomenon, termed "euflammation", which can be induced by repeated subthreshold infectious challenges. Euflammation causes innate immune alterations without overt neuroimmune activation. In the current study, we examined the protective effect of euflammation against peripheral inflammation-induced neuroinflammation and the underlying mechanisms. When Escherichia coli or lipopolysaccharide (LPS) was injected inside or outside the euflammation induction locus (EIL), sickness behavior, global microglial activation, proinflammatory cytokine production in the brain, expression of endothelial cyclooxygenase II and induction of c-fos expression in the paraventricular nucleus of the hypothalamus were all attenuated in the euflammatory mice compared with those in the control unprimed mice. Euflammation also modulated innate immunity outside the EIL by upregulating receptors for pathogen-associated molecular patterns in spleen cells. In addition, euflammation attenuated CNS activation in response to an intra-airpouch (outside the EIL) injection of LPS without suppressing the cytokine expression in the airpouch. Collectively, our study demonstrates that signaling of peripheral inflammation to the CNS is modulated dynamically by peripheral inflammatory kinetics. Specifically, euflammation can offer effective protection against both bacterial infection and endotoxin induced neuroinflammation.

IL-1 Receptor-1 on Vglut2 + neurons in the hippocampus is critical for neuronal and behavioral sensitization after repeated social stress.

Myriad findings connect stress and inflammation to mood disorders. Social defeat in mice promotes the convergence of neuronal, central inflammatory (microglia), and peripheral immune (monocytes) pathways causing anxiety, social avoidance, and "stress-sensitization." Stress-sensitization results in augmented inflammation and the recurrence of anxiety after re-exposure to social stress. Different cell compartments, including neurons, may be uniquely sensitized by social defeat-induced interleukin-1 (IL-1) signaling. Therefore, the aim of this study was to determine if glutamatergic neuronal IL-1 receptor signaling was essential in promoting stress-sensitization after social defeat. Here, wild-type (IL-1R1+/+) mice and mice with IL-1 receptor-1 deleted selectively in glutamatergic neurons (Vglut2-IL-1R1-/-) were stress-sensitized by social defeat (6-cycles) and then exposed to acute defeat (1-cycle) at day 30. Acute defeat-induced neuronal activation (ΔFosB and phospo-CREB) in the hippocampus of stress-sensitized mice was dependent on neuronal IL-1R1. Moreover, acute defeat-induced social withdrawal and working memory impairment in stress-sensitized mice were also dependent on neuronal IL-1R1. To address region and time dependency, an AAV2-IL-1 receptor antagonist construct was administered into the hippocampus after sensitization, but prior to acute defeat at day 30. Although stress-sensitized mice had increased hippocampal pCREB and decreased working memory after stress re-exposure, these events were not influenced by AAV2-IL-1 receptor antagonist. Hippocampal ΔFosB induction and corresponding social withdrawal in stress-sensitized mice after stress re-exposure were prevented by the AAV2-IL-1 receptor antagonist. Collectively, IL-1 signaling in glutamatergic neurons of the hippocampus was essential in neuronal-sensitization after social defeat and the recall of social withdrawal.

Dynamic Interleukin-1 Receptor Type 1 Signaling Mediates Microglia-Vasculature Interactions Following Repeated Systemic LPS.

Introduction: 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-Il1r1 r/r), in addition to either global ablation (Il1r1 r/r) or global restoration of IL-1R1 (Il1r1 GR/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-1ß: the mIL-1R1 dependent induction of IL-1R1 antagonist (IL-1RA) and IL-1ß 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.

Modulation of Neural Networks by Interleukin-1.

Interleukin-1 (IL-1) is an inflammatory cytokine that has been shown to modulate neuronal signaling in homeostasis and diseases. In homeostasis, IL-1 regulates sleep and memory formation, whereas in diseases, IL-1 impairs memory and alters affect. Interestingly, IL-1 can cause long-lasting changes in behavior, suggesting IL-1 can alter neuroplasticity. The neuroplastic effects of IL-1 are mediated via its cognate receptor, Interleukin-1 Type 1 Receptor (IL-1R1), and are dependent on the distribution and cell type(s) of IL-1R1 expression. Recent reports found that IL-1R1 expression is restricted to discrete subpopulations of neurons, astrocytes, and endothelial cells and suggest IL-1 can influence neural circuits directly through neuronal IL-1R1 or indirectly via non-neuronal IL-1R1. In this review, we analyzed multiple mechanisms by which IL-1/IL-1R1 signaling might impact neuroplasticity based upon the most up-to-date literature and provided potential explanations to clarify discrepant and confusing findings reported in the past.