Tlr4 Deletion Modulates Cytokine and Extracellular Matrix Expression in Chronic Spinal Cord Injury, Leading to Improved Secondary Damage and Functional Recovery.

Toll-like receptors (TLRs) play an important role in the innate immune response after CNS injury. Although TLR4 is one of the best characterized, its role in chronic stages after spinal cord injury (SCI) is not well understood. We examined the role of TLR4 signaling in injury-induced responses at 1 d, 7 d, and 8 weeks after spinal cord contusion injury in adult female TLR4 null and wild-type mice. Analyses include secondary damage, a range of transcriptome and protein analyses of inflammatory, cell death, and extracellular matrix (ECM) molecules, as well as immune cell infiltration and changes in axonal sprouting and locomotor recovery. Lack of TLR4 signaling results in reduced neuronal and myelin loss, reduced activation of NFκB, and decreased expression of inflammatory cytokines and necroptotic cell death pathway at a late time point (8 weeks) after injury. TLR4 null mice also showed reduction of scar-related ECM molecules at 8 weeks after SCI, accompanied by increase in ECM molecules associated with perineuronal nets, increased sprouting of serotonergic fibers, and improved locomotor recovery. These findings reveal novel effects of TLR4 signaling in chronic SCI. We show that TLR4 influences inflammation, cell death, and ECM deposition at late-stage post-injury when secondary injury processes are normally considered to be over. This highlights the potential for late-stage targeting of TLR4 as a potential therapy for chronic SCI.

Annexin A1 restores cerebrovascular integrity concomitant with reduced amyloid-ß and tau pathology.

Alzheimer's disease, characterized by brain deposits of amyloid-ß plaques and neurofibrillary tangles, is also linked to neurovascular dysfunction and blood-brain barrier breakdown, affecting the passage of substances into and out of the brain. We hypothesized that treatment of neurovascular alterations could be beneficial in Alzheimer's disease. Annexin A1 (ANXA1) is a mediator of glucocorticoid anti-inflammatory action that can suppress microglial activation and reduce blood-brain barrier leakage. We have reported recently that treatment with recombinant human ANXA1 (hrANXA1) reduced amyloid-ß levels by increased degradation in neuroblastoma cells and phagocytosis by microglia. Here, we show the beneficial effects of hrANXA1 in vivo by restoring efficient blood-brain barrier function and decreasing amyloid-ß and tau pathology in 5xFAD mice and Tau-P301L mice. We demonstrate that young 5xFAD mice already suffer cerebrovascular damage, while acute pre-administration of hrANXA1 rescued the vascular defects. Interestingly, the ameliorated blood-brain barrier permeability in young 5xFAD mice by hrANXA1 correlated with reduced brain amyloid-ß load, due to increased clearance and degradation of amyloid-ß by insulin degrading enzyme (IDE). The systemic anti-inflammatory properties of hrANXA1 were also observed in 5xFAD mice, increasing IL-10 and reducing TNF-α expression. Additionally, the prolonged treatment with hrANXA1 reduced the memory deficits and increased synaptic density in young 5xFAD mice. Similarly, in Tau-P301L mice, acute hrANXA1 administration restored vascular architecture integrity, affecting the distribution of tight junctions, and reduced tau phosphorylation. The combined data support the hypothesis that blood-brain barrier breakdown early in Alzheimer's disease can be restored by hrANXA1 as a potential therapeutic approach.

The Alzheimer's Disease-Associated Protein BACE1 Modulates T Cell Activation and Th17 Function.

ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is best known for its role in Alzheimer's disease amyloid plaque formation but also contributes to neurodegenerative processes triggered by CNS injury. In this article, we report that BACE1 is expressed in murine CD4+ T cells and regulates signaling through the TCR. BACE1-deficient T cells have reduced IL-17A expression under Th17 conditions and reduced CD73 expression in Th17 and inducible T regulatory cells. However, induction of the Th17 and T regulatory transcription factors RORγt and Foxp3 was unaffected. BACE1-deficient T cells showed impaired pathogenic function in experimental autoimmune encephalomyelitis. These data identify BACE1 as a novel regulator of T cell signaling pathways that impact autoimmune inflammatory T cell function.

CD73 is expressed by inflammatory Th17 cells in experimental autoimmune encephalomyelitis but does not limit differentiation or pathogenesis.

CD73 works together with CD39 to convert extracellular ATP to immunoregulatory adenosine, thus inhibiting inflammation. TGFß-mediated CD73 expression on 'regulatory' Th17 cells limits their ability to eradicate tumors, similar to the immunosuppressive mechanism described for CD73 on Tregs. However, CD73 is also expressed on Th17 cells thought to be inflammatory in Crohn's disease. CD73 has previously been reported to contribute to inflammation in the central nervous system (CNS). In experimental autoimmune encephalomyelitis (EAE), we found that inflammatory cytokine-producing Th17 cells showed increased CD73 expression as disease progressed. We therefore hypothesized that CD73 could be important for limiting the expansion or pathogenic function of Th17 cells in autoimmune inflammation of the CNS. Surprisingly, EAE development was not enhanced or inhibited by CD73 deficiency; there was correspondingly no difference in induction of Th17-associated cytokines IL-17, IFNγ or GM-CSF or recruitment of either inflammatory or regulatory cells to the central nervous system. We confirmed that CD73 was similarly not required for differentiation of Th17 cells in vitro. These data show that while CD73 expression is regulated during EAE, this enzyme is not absolutely required to either promote or limit Th17 cell expansion or EAE severity.

CCAAT/Enhancer-binding protein ß promotes pathogenesis of EAE.

The CCAAT/Enhancer Binding Protein ß (C/EBPß) transcription factor is activated by multiple inflammatory stimuli, including IL-17 and LPS, and C/EBPß itself regulates numerous genes involved in inflammation. However, the role of C/EBPß in driving autoimmunity is not well understood. Here, we demonstrate that Cebpb-/- mice are resistant to EAE. Cebpb-/- mice exhibited reduced lymphocyte and APC infiltration into CNS following EAE induction. Furthermore, MOG-induced Th17 cytokine production was impaired in draining LN, indicating defects in Th17 cell priming. In vitro Th17 polarization studies indicated that T cell responses are not inherently defective, instead supporting the known roles for C/EBPß in myeloid lineage cell activation as the likely mechanism for defective Th17 priming in vivo. However, we did uncover an unexpected role for C/EBPß in regulating ll23r expression in APCs. ChIP assays confirmed that C/EBPß binds directly to the Il23r gene promoter in dendritic cells and Th17 cells. These data establish C/EBPß as a key driver of autoimmune inflammation in EAE, and propose a novel role for C/EBPß in regulation of IL-23R expression.

MCPIP1 Endoribonuclease Activity Negatively Regulates Interleukin-17-Mediated Signaling and Inflammation.

Interleukin-17 (IL-17) induces pathology in autoimmunity and infections; therefore, constraint of this pathway is an essential component of its regulation. We demonstrate that the signaling intermediate MCPIP1 (also termed Regnase-1, encoded by Zc3h12a) is a feedback inhibitor of IL-17 receptor signal transduction. MCPIP1 knockdown enhanced IL-17-mediated signaling, requiring MCPIP1's endoribonuclease but not deubiquitinase domain. MCPIP1 haploinsufficient mice showed enhanced resistance to disseminated Candida albicans infection, which was reversed in an Il17ra(-/-) background. Conversely, IL-17-dependent pathology in Zc3h12a(+/-) mice was exacerbated in both EAE and pulmonary inflammation. MCPIP1 degraded Il6 mRNA directly but only modestly downregulated the IL-6 promoter. However, MCPIP1 strongly inhibited the Lcn2 promoter by regulating the mRNA stability of Nfkbiz, encoding the IκBζ transcription factor. Unexpectedly, MCPIP1 degraded Il17ra and Il17rc mRNA, independently of the 3' UTR. The cumulative impact of MCPIP1 on IL-6, IκBζ, and possibly IL-17R subunits results in a biologically relevant inhibition of IL-17 signaling.

Vestibulotoxic properties of potential metabolites of allylnitrile.

This study addressed the hypothesis that epoxidation of the double bond in allylnitrile mediates its vestibular toxicity, directly or after subsequent metabolism by epoxide hydrolases. The potential metabolites 3,4-epoxybutyronitrile and 3,4-dihydroxybutyronitrile were synthesized and characterized. In aqueous solutions containing sodium or potassium ions, 3,4-epoxybutyronitrile rearranged to 4-hydroxybut-2-enenitrile, and this compound was also isolated for study. Male adult Long-Evans rats were exposed to allylnitrile or 3,4-epoxybutyronitrile by bilateral transtympanic injection, and vestibular toxicity was assessed using a behavioral test battery and scanning electron microscopy (SEM) observation of the sensory epithelia. Overt vestibular toxicity was caused by 3,4-epoxybutyronitrile at 0.125 mmol/ear and by allylnitrile in some animals at 0.25 mmol/ear. Additional rats were exposed by unilateral transtympanic injection. In these studies, behavioral evidences and SEM observations demonstrated unilateral vestibular toxicity after 0.125 mmol of 3,4-epoxybutyronitrile and bilateral vestibular toxicity after 0.50 mmol of allylnitrile. However, 0.25 mmol of allylnitrile did not cause vestibular toxicity. Unilateral administration of 0.50 mmol of 3,4-dihydroxybutyronitrile or 4-hydroxybut-2-enenitrile caused no vestibular toxicity. The four compounds were also evaluated in the mouse utricle explant culture model. In 8-h exposure experiments, hair cells completely disappeared after 3,4-epoxybutyronitrile at concentrations of 325 or 450µM but not at concentrations of 150µM or lower. In contrast, no difference from controls was recorded in utricles exposed to 450µM or 1.5mM of allylnitrile, 3,4-dihydroxybutyronitrile, or 4-hydroxybut-2-enenitrile. Taken together, the present data support the hypothesis that 3,4-epoxybutyronitrile is the active metabolite of allylnitrile for vestibular toxicity.

Vestibular toxicity of cis-2-pentenenitrile in the rat.

cis-2-Pentenenitrile, an intermediate in the synthesis of nylon and other products, causes permanent behavioral deficits in rodents. Other low molecular weight nitriles cause degeneration either of the vestibular sensory hair cells or of selected neuronal populations in the brain. Adult male Long-Evans rats were exposed to cis-2-pentenenitrile (0, 1.25, 1.50, 1.75, or 2.0mmol/kg, oral, in corn oil) and assessed for changes in open field activity and rating scores in a test battery for vestibular dysfunction. Surface preparations of the vestibular sensory epithelia were observed for hair cell loss using scanning electron microscopy. A separate experiment examined the impact of pre-treatment with the universal CYP inhibitor,1-aminobenzotriazole, on the effect of cis-2-pentenenitrile on vestibular rating scores. The occurrence of degenerating neurons in the central nervous system was assessed by Fluoro-Jade C staining. cis-2-Pentenenitrile had a dose-dependent effect on body weight. Rats receiving 1.50mmol/kg or more of cis-2-pentenenitrile displayed reduced rearing activity in the open field and increased rating scores on the vestibular dysfunction test battery. Hair cell loss was observed in the vestibular sensory epithelia and correlated well with the behavioral deficits. Pre-treatment with 1-aminobenzotriazole blocked the behavioral effect. Fluoro-Jade C staining did not reveal significant neuronal degeneration in the central nervous system apart from neurite labeling in the olfactory glomeruli. We conclude that cis-2-pentenenitrile causes vestibular toxicity in a similar way to allylnitrile, cis-crotononitrile and 3,3'-iminodipropionitrile (IDPN), and also shares other targets such as the olfactory system with these other nitriles. The present data also suggest that CYP-mediated bioactivation is involved in cis-2-pentenenitrile toxicity.

Glycoforms of human endothelial CD34 that bind L-selectin carry sulfated sialyl Lewis x capped O- and N-glycans.

Endothelial sialomucin CD34 functions as an L-selectin ligand mediating lymphocyte extravasation only when properly glycosylated to express a sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x (6-sulfo SLe(x)). It is thought that multivalent 6-sulfo SLe(x) expression promotes high-affinity binding to L-selectin by enhancing avidity. However, the reported low amount of 6-sulfo SLe(x) in total human CD34 is inconsistent with this model and prompted us to re-evaluate CD34 glycosylation. We separated CD34 into 2 glycoforms, the L-selectin-binding and nonbinding glycoforms, L-B-CD34 and L-NB-CD34, respectively, and analyzed released O- and N-glycans from both forms. L-B-CD34 is relatively minor compared with L-NB-CD34 and represented less than 10% of total tonsillar CD34. MECA-79, a mAb to sulfated core-1 O-glycans, bound exclusively to L-B-CD34 and this form contained all sulfated and fucosylated O-glycans. 6-Sulfo SLe(x) epitopes occur on core-2 and extended core-1 O-glycans with approximately 20% of total L-B-CD34 O-glycans expressing 6-sulfo SLe(x). N-glycans containing potential 6-sulfo SLe(x) epitopes were also present in L-B-CD34, but their removal did not abolish binding to L-selectin. Thus, a minor glycoform of CD34 carries relatively abundant 6-sulfo SLe(x) epitopes on O-glycans that are important for its recognition by L-selectin.