BackgroundThe migration of peripheral immune cells and splenocytes to the ischemic brain is one of the major causes of delayed neuroinflammation after permanent large vessel stroke. Other groups have demonstrated that leukemia inhibitory factor (LIF), a cytokine that promotes neural cell survival through upregulation of antioxidant enzymes, promotes an anti-inflammatory phenotype in several types of immune cells. The goal of this study was to determine whether LIF treatment modulates the peripheral immune response after stroke.MethodsYoung male (3 month) Sprague-Dawley rats underwent sham surgery or permanent middle cerebral artery occlusion (MCAO). Animals were administered LIF (125 μg/kg) or PBS at 6, 24, and 48 h prior to euthanization at 72 h. Bone marrow-derived macrophages were treated with LIF (20 ng/ml) or PBS after stimulation with interferon gamma + LPS. Western blot was used to measure protein levels of CD11b, IL-12, interferon inducible protein-10, CD3, and the LIF receptor in spleen and brain tissue. ELISA was used to measure IL-10, IL-12, and interferon gamma. Isolectin was used to label activated immune cells in brain tissue sections. Statistical analysis was performed using one-way ANOVA and Student’s t test. A Kruskal-Wallis test followed by Bonferroni-corrected Mann-Whitney tests was performed if data did not pass the D’Agostino-Pearson normality test.ResultsLIF-treated rats showed significantly lower levels of the LIF receptor and interferon gamma in the spleen and CD11b levels in the brain compared to their PBS-treated counterparts. Fluorescence from isolectin-binding immune cells was more prominent in the ipsilateral cortex and striatum after PBS treatment compared to LIF treatment. MCAO + LIF significantly decreased splenic levels of CD11b and CD3 compared to sham surgery. MCAO + PBS treatment significantly elevated splenic levels of interferon inducible protein-10 at 72 h after MCAO, while LIF treatment after MCAO returned interferon inducible protein 10 to sham levels. LIF administration with interferon gamma + LPS significantly reduced the IL-12/IL-10 production ratio compared to macrophages treated with interferon gamma + LPS alone.ConclusionsThese data demonstrate that LIF promotes anti-inflammatory signaling through alterations of the IL-12/interferon gamma/interferon inducible protein 10 pathway.

Impairment of vascular pathways of cerebral β-amyloid (Aβ) elimination contributes to Alzheimer disease (AD). Vascular damage is commonly associated with diabetes. Here we show in human tissues and AD-model rats that bloodborne islet amyloid polypeptide (amylin) secreted from the pancreas perturbs cerebral Aβ clearance. Blood amylin concentrations are higher in AD than in cognitively unaffected persons. Amyloid-forming amylin accumulates in circulating monocytes and co-deposits with Aβ within the brain microvasculature, possibly involving inflammation. In rats, pancreatic expression of amyloid-forming human amylin indeed induces cerebrovascular inflammation and amylin-Aβ co-deposits. LRP1-mediated Aβ transport across the blood-brain barrier and Aβ clearance through interstitial fluid drainage along vascular walls are impaired, as indicated by Aβ deposition in perivascular spaces. At the molecular level, cerebrovascular amylin deposits alter immune and hypoxia-related brain gene expression. These converging data from humans and laboratory animals suggest that altering bloodborne amylin could potentially reduce cerebrovascular amylin deposits and Aβ pathology.

Hypersecretion of amylin, a β-cell hormone that regulates satiation, is common in individuals with prediabetes and is associated with pancreatic amyloid deposition and type-2 diabetes. Evidence has emerged that increased circulating levels of amyloid-forming human amylin may potentially impair brain function. Because mouse amylin is non-amyloidogenic, we generated transgenic mice with conditional pancreatic expression of amyloid-forming human amylin to study how in vivo knockdown of human amylin expression influences brain function during the development of type-2 diabetes. Males and females were fed a high-fat diet starting at 3 months of age to induce amylin hypersecretion and glucose dysregulation. Males developed hyperglycemia at 5 months of age, whereas females showed glucose dysregulation 3-4 months later. At 5 months of age, human amylin-expressing male mice were randomly assigned to either amylin downregulation group (by peritoneal tamoxifen injection) or control group (maintained amylin expression) (n = 10/group). Two months later, we assessed brain function with the novel object recognition test and performed comparative non-targeted metabolomics and global RNA-seq analyses of hippocampal tissue. Mice with downregulated human amylin show enhanced recognition memory index (p < 0.001) and lower blood glucose levels (p < 0.001) compared to those that continued to express human amylin. This was associated with increased hippocampal levels of glycolysis metabolites, including lactic acid (p < 0.01), glucose-6-phosphate (p = 0.06), and fructose (p = 0.07). Hippocampal gene-expression patterns between the two mouse groups revealed extensive compensatory changes in gene expression related to glucose metabolism. In conclusion, amylin downregulation in diabetic mice improves systemic glucose homeostasis and memory. Molecular processes associated with improved memory involve increased hippocampal glycolytic fluxes and compensatory gene expression.

BackgroundEmergent large vessel occlusion (ELVO) is the deadliest form of stroke and is caused by a blockage within a major a cerebral artery, usually the middle cerebral artery. This condition triggers edema, water movement into the brain, thus pressuring the brain producing massive damage resulting in disability and death. Leukemia inhibitory factor (LIF), a neuroprotective and anti‐inflammatory cytokine, decreases neurodegeneration and increases survival in a rat model of ELVO. Previously we have reported IFNγ is the primary inflammatory mediator in this stroke model and treatment with LIF decreases its expression. This study examined the presence of IFNγ on human endothelial cells by measuring the expression of the IFNγ‐induced CXCL9 and determine LIF's ability to block IFNγ signaling.MethodsFor cells under normoxia conditions, cells were plated and allowed to mature for 7 days, then stimulated with 1 ng/ml IFNγ. At time points 6, 24, 48 and 72 hours after stimulation the supernatant was taken and used for ELISA to determine the amount of CXCL9 released. For OGD conditions, cells were allowed to grow for a week and given glucose free media and placed in oxygen free chamber for 6 hours. The cells were then perfused with media with glucose and 1 ng/ml IFNγ. The supernatant was collected the same time points previously mentioned and CXCL9 was determined as stated previously. To determine the effects inhibitors had on CXCL9 release after being treated with inhibitors to JAK2 and STAT1 under normoxic conditions, cells were treated with IFNy the same as above along coupled with 50 μM and 100 M of fludarabine and AG490.ResultsExposing cerebral microvascular endothelial cells to 1 ng/ml IFNγ significantly induced expression of CXCL9 under both normoxia and oxygen‐glucose deprivation (OGD). Inhibitors to JAK2 and STAT1 significantly blocked the induction of CXCL9. Under normoxic conditions, the addition of LIF (200 ng/ml) with IFNγ significantly augmented the increase in CXCL9 in endothelial cells exposed to OGD.ConclusionCerebral microvascular endothelial cells are a major source of CXCL9, which is upregulated by IFNγ and LIF in a JAK2/STAT1‐dependent manner. The LIF signal transduction pathway in these cells has previously been shown to be dependent upon kinases such as ERK and Akt. CXCL9 is known to break down the blood‐brain barrier, which leads to edema. One potential explanation for this induction of CXCL9 is to open the blood‐brain barrier and allow for anti‐inflammatory leukocytes to enter the ischemic brain and promote repair after stroke.Support or Funding InformationThis project was funded by NINDS Award # 5R0INS091146‐04This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.