Natalya Belevych

Endothelial-Specific Knockdown of Interleukin-1 (IL-1) Type 1 Receptor Differentially Alters CNS Responses to IL-1 Depending on Its Route of Administration

Interleukin-1 (IL-1) has been implicated as a critical mediator of neuroimmune communication. In the brain, the functional receptor for IL-1, type 1 IL-1 receptor (IL-1R1), is localized primarily to the endothelial cells. In this study, we created an endothelial-specific IL-1R1 knockdown model to test the role of endothelial IL-1R1 in mediating the effects of IL-1. Neuronal activation in the hypothalamus was measured by c-fos expression in the paraventricular nucleus and the ventromedial preoptic area. In addition, two specific sickness symptoms, febrile response and reduction of locomotor activity, were studied. Intracerebroventricular injection of IL-1 induced leukocyte infiltration into the CNS, activation of hypothalamic neurons, fever, and reduced locomotor activity in normal mice. Endothelial-specific knockdown of IL-1R1 abrogated all these responses. Intraperitoneal injection of IL-1 also induced neuronal activation in the hypothalamus, fever, and reduced locomotor activity, without inducing leukocyte infiltration into the brain. Endothelial-specific knockdown of IL-1R1 suppressed intraperitoneal IL-1-induced fever, but not the induction of c-fos in hypothalamus. When IL-1 was given intravenously, endothelial knockdown of IL-1R1 abolished intravenous IL-1-induced CNS activation and the two monitored sickness symptoms. In addition, endothelial-specific knockdown of IL-1R1 blocked the induction of cyclooxygenase-2 expression induced by all three routes of IL-1 administration. These results show that the effects of intravenous and intracerebroventricular IL-1 are mediated by endothelial IL-1R1, whereas the effects of intraperitoneal IL-1 are partially dependent on endothelial IL-1R1.

Interleukin-1R3 mediates interleukin-1–induced potassium current increase through fast activation of Akt kinase

Inflammatory cytokine interleukin-1 (IL-1) performs multiple functions in the central nervous system. The type 1 IL-1 receptor (IL-1R1) and the IL-1 receptor accessory protein (IL-1RAcP) form a functional IL-1 receptor complex that is thought to mediate most, if not all, IL-1–induced effects. Several recent studies, however, suggest the existence of a heretofore-unidentified receptor for IL-1. In this study, we report that the IL-1R1 gene contains an internal promoter that drives the transcription of a shortened IL-1R1 mRNA. This mRNA is the template for a unique IL-1R protein that is identical to IL-1R1 at the C terminus, but with a shorter extracellular domain at the N terminus. We have termed this molecule IL-1R3. The mRNA and protein for IL-1R3 are expressed in normal and two strains of commercially available IL-1R1 knockout mice. Western blot analysis shows IL-1R3 is preferentially expressed in neural tissues. Furthermore, IL-1β binds specifically to IL-1R3 when it is complexed with the newly discovered alternative IL-1 receptor accessory protein, IL-1RAcPb. Stimulation of neurons expressing both IL-1R3 and IL-1RAcPb with IL-1β causes fast activation of the Akt kinase, which leads to an increase in voltage-gated potassium current. These results demonstrate that IL-1R3/IL-1RAcPb complex mediates a unique subset of IL-1 activity that accounts for many previously unexplained IL-1 effects in the central nervous system.

Endothelial IL-1R1 is a critical mediator of EAE pathogenesis

Interleukin-1 (IL-1) has been implicated in the disease progression of multiple sclerosis (MS). In the animal model of MS, experimental autoimmune encephalomyelitis (EAE), the induction of disease is significantly attenuated in mice lacking the type I IL-1 receptor (IL-1R1). In this study, we created a transgenic mouse (eIL-1R1 kd) in which IL-1R1 expression is knocked down specifically in endothelial cells. Induction of EAE in eIL-1R1 kd mice results in a decrease in incidence, severity and delayed onset of EAE. In addition, eIL-1R1 kd mice show significant decrease in VCAM-1 expression and diminished CD45(+) and CD3(+) infiltrating leukocytes in the spinal cord in animals challenged with EAE. Further, IL-1 and IL-23 stimulate IL-17 production by splenocytes from both wild type and the eIL-1R1 kd animals. Similarly, IL-1 and IL-23 synergistically stimulate splenocytes proliferation in these two strains of animals. After immunization with MOG(79-96), although eIL-1R1 kd mice displayed greatly reduced clinical scores, their splenocytes produced IL-17 and proliferated in response to a second MOG challenge, similar to wild type animals. These findings indicate a critical role for endothelial IL-1R1 in mediating the pathogenesis of EAE, and describe a new model that can be used to study endothelial IL-1R1.

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-1R1r/r and exhibited an IL-1R1 knock-out phenotype. We used IL-1R1 globally restored mice (IL-1R1GR/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-1R1r/r and LysMCre-IL-1R1r/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-1R1r/r or LysMCre-IL-1R1r/r mice, but were restored in Tie2Cre-IL-1R1r/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-1R1r/r, and LysMCre-IL-1R1r/r, but not IL-1R1r/r mice. These results demonstrate this IL-1R1 restore model is a valuable tool for studying cell-type-specific functions of IL-1R1.

Location-specific activation of the paraventricular nucleus of the hypothalamus by localized inflammation.

The existence of an immunological homunculus has been proposed, but evidence for location-specific response of the central nervous system to immunological stimulation is lacking. In this study, we show that inflammation induced by injection of casein into one of the causes c-fos expression in the paraventricular nucleus of the hypothalamus (PVN) in an asymmetrical manner: much stronger activation is always induced in the contralateral PVN. Unilateral sciatic nerve transection abolished the casein-induced PVN activation if casein was injected into the hindlimb with the nerve transection, but had no effect if casein was injected into the hindlimb with intact nerve innervation. Injection of casein into one the forelimbs also caused contralateral PNV activation. Further, stronger PVN activation was found in the anterior PVN after the forelimb injection, but in the posterior PVN after the hindlimb injection. Casein-induced PVN activation is absent in IL-1R1 KO, IL-6 KO, TNFα KO, and in C3H/HeJ (TLR4 mutant) animals. In comparison, injection of LPS, a systemic inflammagen, into one hindlimb induced bilateral PVN activation but injection of live Escherichia coli into one hindlimb induced contralateral PVN activation. These results support the notion that local inflammation may activate the PVN by neural routes in a location-specific manner.

Neuronal and Nonneuronal COX-2 Expression Confers Neurotoxic and Neuroprotective Phenotypes in Response to Excitotoxin Challenge

Treating acute brain injuries with COX‐2 inhibitors can produce both neuroprotective and neurotoxic effects. This study investigated the role of COX‐2 in modulating acute brain injury induced by excitotoxic neural damage. Intrastriatal injection of excitotoxin (RS)‐(tetrazole‐5yl) glycine elicited COX‐2 expression in two distinct groups of cells. cortical neurons surrounding the lesion and vascular cells in the lesion core. The vascular COX‐2 was expressed in two cell types, endothelial cells and monocytes. Selective deletion of COX‐2 in vascular cells in Tie2Cre Cox‐2flox/flox mice did not affect the induction of COX‐2 in neurons after the excitotoxin injection but resulted in increased lesion volume, indicating a neuroprotective role for the COX‐2 expressed in the vascular cells. Selective deletion of monocyte COX‐2 in LysMCre Cox‐2flox/flox mice did not reduce COX‐2‐dependent neuroprotection, suggesting that endothelial COX‐2 is sufficient to confer neuroprotection. Pharmacological inhibition of COX‐2 activity in Tie2Cre Cox‐2flox/flox mice reduced lesion volume, indicating a neurotoxic role for the COX‐2 expressed in neurons. Furthermore, COX‐2‐dependent neurotoxicity was mediated, at least in part, via the activation of the EP1 receptor. These results show that Cox‐2 expression induced in different cell types can confer opposite effects.

Prostacyclin mediates endothelial cOX-2- dependent neuroprotective effects during excitotoxic brain injury

In a previous study, we found that intracerebral administration of excitotoxin (RS)-(tetrazole-5yl) glycine caused increased neural damage in the brain in an endothelial COX-2 deleted mouse line (Tie2Cre COX-2flox/flox). In this study, we investigated whether prostacyclin might mediate this endothelial COX-2-dependent neuroprotection. Administration of excitotoxin into the striatum induced the production of prostacyclin (PGI2) in wild type, but not in endothelial COX-2 deleted mice. Inhibition of PGI2 synthase exacerbated brain lesions induced by the excitotoxin in wild type, but not in endothelial COX-2 deleted mice. Administration of a PGI2 agonist reduced neural damage in both wild type and endothelial COX-2 deleted mice. Increased PGI2 synthase expression was found in infiltrating neutrophils. In an ex vivo assay, PGI2 reduced the excitotoxin-induced calcium influx into neurons, suggesting a cellular mechanism for PGI2 mediated neuroprotection. These results reveal that PGI2 mediates endothelial COX-2 dependent neuroprotection.

Desensitizing mouse cardiac troponin C to calcium converts slow muscle towards a fast muscle phenotype

The Ca2+‐desensitizing D73N mutation in slow skeletal/cardiac troponin C caused dilatated cardiomyopathy in mice, but the consequences of this mutation in skeletal muscle were not known. The D73N mutation led to a rightward shift in the force versus pCa (‐log [Ca]) relationship in slow‐twitch mouse fibres. The D73N mutation led to a rightward shift in the force–stimulation frequency relationship and reduced fatigue resistance of mouse soleus muscle. The D73N mutation led to reduced cross‐sectional area of slow‐twitch fibres in mouse soleus muscle without affecting fibre type composition of the muscle. The D73N mutation resulted in significantly shorter times to peak force and to relaxation during isometric twitches and tetani in mouse soleus muscle. The D73N mutation led to major changes in physiological properties of mouse soleus muscle, converting slow muscle toward a fast muscle phenotype.

Brain endothelial IL-1R1 mediates central IL-1 induced neuroinflammation and anxiety-like behavior

Interleukin-1 (IL-1) mediates diverse neuropathological effects and behavioral changes in the central nervous system (CNS) through type I IL-1 receptor (IL-1R1). Different CNS cell types are known to mediate different IL-1 effects. In this study, we investigated IL-1 induced neuroinflammation and anxiety using three cell-type specific IL-1R1 expressing mouse lines: Tie2Cre-IL-1R1r/r (IL-1R1 restricted to endothelial cells and hematopoietic cells), LysMCre-IL-1R1r/r (myeloid cells) or HipAAV2Cre-IL-1R1r/r (hippocampal neurons). Following intracerebroventricular (ICV) IL-1 injection microglial activation was induced in all three mouse lines but with distinct morphological characteristics. Microglia in Tie2Cre-IL-1R1r/r mice developed most abundant branches and longest processes length. Cytokine expression analysis showed microglia in wild-type (WT) and Tie2Cre-IL-1R1r/r mice upregulated proinflammatory cytokines IL-1 β and TNF- α , which did not occur in LysMCre-IL-1R1 and AAVCre-IL-1R1 mice which lacked endothelial IL-1R1. We then adoptively transferred SVEC4-10 cells, an IL-1R1 expressing endothelial cell line, into striatum of IL-1R1 knockout mice (IL-1R1r/r). After the transfer, microglial activation was induced by ICV IL-1 but not by saline, indicating endothelial IL-1R1 is sufficient to mediate inflammatory microglial activation. Furthermore, we found endothelial IL-1R1 dependent microglial activation was mediated via exocytosis of danger signals such as ATP. In addition, anxiety-like behavior developed in WT and Tie2Cre-IL-1R1r/r mice but not LysMCre-IL-1R1r/r or AAV2Cre-IL-1R1r/r lines. Collectively, these findings demonstrate that brain endothelial IL-1R1 mediates central IL-1 induced neuroinflammation and anxiety-like behavior.

71. Infiltrating PGI2 producing leukocytes mediate neuroprotecitive effect of cyclooxygenase-2 (COX-2)

We showed previously that COX-2 is induced in the brain by excitotoxin in two distinct cell populations, neurons and non-neuronal cells. When cox-2 gene is selectively deleted in non-neuronal cells in a conditional knockout mouse line (KO), brain injury volumes induced by excitotoxin were nearly twice as large as those in WT controls, suggesting that the COX-2 expression in non-neuronal cells plays a neuroprotective role. In this study, we investigated the cellular and molecular mechanisms underlying COX-2-dependent neuroprotection. Correlated with the smaller lesion in WT mice, PGI2-producing leukocytes were found to infiltrate into the brain parenchyma in and near the site of lesion in WT, but not the KO, mice. At the site of injury, the level of PGI2 increased significantly after excitotoxin injection in WT, but not the KO, mice. In addition, inhibition of the PGI2 synthesis in WT animals induced an increase in the lesion size, whereas injection of PGI2 agonist in the KO mice reduced the lesion size. Further, after receiving adoptive transfer of blood leukocytes from WT mice, PGI2-expressing cells were found at the site of excitotoxic lesion in the KO animals with a concomittant reduction of lesion volume. These data suggest that PGI2 produced by the infiltrating leukocytes mediates the neuroprotective effect of the non-neuronal expression of COX-2.