Interleukin-10 improves stroke outcome by controlling the detrimental Interleukin-17A response.

BACKGROUND: Lymphocytes have dichotomous functions in ischemic stroke. Regulatory T cells are protective, while IL-17A from innate lymphocytes promotes the infarct growth. With recent advances of T cell-subtype specific transgenic mouse models it now has become possible to study the complex interplay of T cell subpopulations in ischemic stroke. METHODS: In a murine model of experimental stroke we analyzed the effects of IL-10 on the functional outcome for up to 14 days post-ischemia and defined the source of IL-10 in ischemic brains based on immunohistochemistry, flow cytometry, and bone-marrow chimeric mice. We used neutralizing IL-17A antibodies, intrathecal IL-10 injections, and transgenic mouse models which harbor a deletion of the IL-10R on distinct T cell subpopulations to further explore the interplay between IL-10 and IL-17A pathways in the ischemic brain. RESULTS: We demonstrate that IL-10 deficient mice exhibit significantly increased infarct sizes on days 3 and 7 and enlarged brain atrophy and impaired neurological outcome on day 14 following tMCAO. In ischemic brains IL-10 producing immune cells included regulatory T cells, macrophages, and microglia. Neutralization of IL-17A following stroke reversed the worse outcome in IL-10 deficient mice and intracerebral treatment with recombinant IL-10 revealed that IL-10 controlled IL-17A positive lymphocytes in ischemic brains. Importantly, IL-10 acted differentially on αß and γδ T cells. IL-17A producing CD4+ αß T cells were directly controlled via their IL-10-receptor (IL-10R), whereas IL-10 by itself had no direct effect on the IL-17A production in γδ T cells. The control of the IL-17A production in γδ T cells depended on an intact IL10R signaling in regulatory T cells (Tregs). CONCLUSIONS: Taken together, our data indicate a key function of IL-10 in restricting the detrimental IL-17A-signaling in stroke and further supports that IL-17A is a therapeutic opportunity for stroke treatment.

The tissue is the issue: improved methylome analysis from paraffin-embedded tissues by application of the HOPE technique.

Alterations in the DNA methylome are characteristic for numerous diseases and a typical hallmark of cancer. Therefore, DNA methylation is currently under investigation in research labs and has also entered diagnostics. Recently, protocols like the BeadChip technology have become commercially available to study DNA methylation in an array format and semiquantitative fashion. However, it is known that fixation of the sample material with formalin prior to BeadChip analysis can affect the results. In this study we compared the influence of fixation on the outcome of BeadChip analysis. From six patients each a lung cancer tissue sample and a corresponding tumor-free lung tissue sample were collected. The samples were separated into three pieces. One piece of each sample was fixed with formalin, another one by the non-cross-linking HOPE technique (Hepes-glutamic acid buffer mediated Organic solvent Protection Effect). Subsequently, both became paraffin embedded. As a reference, the remaining third piece was cryopreserved. In addition we used three adenocarcinoma cell lines (H838, A549, and H1650) to validate the results from patient tissues. We show that using the HOPE technique instead of formalin largely prevents the introduction of formalin-fixation related artifacts. An ANOVA analysis significantly separated HOPE- and cryopreserved from formalin-fixed samples (FDR<0.05), while differences in the methylation data obtained from HOPE-fixed and cryopreserved material were minor. Consequently, HOPE fixation is superior to formalin fixation if a subsequent BeadChip analysis of paraffin-embedded sample material is intended.

SNUPN deficiency causes a recessive muscular dystrophy due to RNA mis-splicing and ECM dysregulation.

SNURPORTIN-1, encoded by SNUPN, plays a central role in the nuclear import of spliceosomal small nuclear ribonucleoproteins. However, its physiological function remains unexplored. In this study, we investigate 18 children from 15 unrelated families who present with atypical muscular dystrophy and neurological defects. Nine hypomorphic SNUPN biallelic variants, predominantly clustered in the last coding exon, are ascertained to segregate with the disease. We demonstrate that mutant SPN1 failed to oligomerize leading to cytoplasmic aggregation in patients' primary fibroblasts and CRISPR/Cas9-mediated mutant cell lines. Additionally, mutant nuclei exhibit defective spliceosomal maturation and breakdown of Cajal bodies. Transcriptome analyses reveal splicing and mRNA expression dysregulation, particularly in sarcolemmal components, causing disruption of cytoskeletal organization in mutant cells and patient muscle tissues. Our findings establish SNUPN deficiency as the genetic etiology of a previously unrecognized subtype of muscular dystrophy and provide robust evidence of the role of SPN1 for muscle homeostasis.

The role of the ATP-adenosine axis in ischemic stroke.

In ischemic stroke, the primary neuronal injury caused by the disruption of energy supply is further exacerbated by secondary sterile inflammation. The inflammatory cascade is largely initiated by the purine adenosine triphosphate (ATP) which is extensively released to the interstitial space during brain ischemia and functions as an extracellular danger signaling molecule. By engaging P2 receptors, extracellular ATP activates microglia leading to cytokine and chemokine production and subsequent immune cell recruitment from the periphery which further amplifies post-stroke inflammation. The ectonucleotidases CD39 and CD73 shape and balance the inflammatory environment by stepwise degrading extracellular ATP to adenosine which itself has neuroprotective and anti-inflammatory signaling properties. The neuroprotective effects of adenosine are mainly mediated through A1 receptors and inhibition of glutamatergic excitotoxicity, while the anti-inflammatory capacities of adenosine have been primarily attributed to A2A receptor activation on infiltrating immune cells in the subacute phase after stroke. In this review, we summarize the current state of knowledge on the ATP-adenosine axis in ischemic stroke, discuss contradictory results, and point out potential pitfalls towards translating therapeutic approaches from rodent stroke models to human patients.

A preclinical randomized controlled multi-centre trial of anti-interleukin-17A treatment for acute ischaemic stroke.

Multiple consensus statements have called for preclinical randomized controlled trials to improve translation in stroke research. We investigated the efficacy of an interleukin-17A neutralizing antibody in a multi-centre preclinical randomized controlled trial using a murine ischaemia reperfusion stroke model. Twelve-week-old male C57BL/6 mice were subjected to 45 min of transient middle cerebral artery occlusion in four centres. Mice were randomly assigned (1:1) to receive either an anti-interleukin-17A (500 µg) or isotype antibody (500 µg) intravenously 1 h after reperfusion. The primary endpoint was infarct volume measured by magnetic resonance imaging three days after transient middle cerebral artery occlusion. Secondary analysis included mortality, neurological score, neutrophil infiltration and the impact of the gut microbiome on treatment effects. Out of 136 mice, 109 mice were included in the analysis of the primary endpoint. Mixed model analysis revealed that interleukin-17A neutralization significantly reduced infarct sizes (anti-interleukin-17A: 61.77 ± 31.04 mm3; IgG control: 75.66 ± 34.79 mm3; P = 0.01). Secondary outcome measures showed a decrease in mortality (hazard ratio = 3.43, 95% confidence interval = 1.157-10.18; P = 0.04) and neutrophil invasion into ischaemic cortices (anti-interleukin-17A: 7222 ± 6108 cells; IgG control: 28 153 ± 23 206 cells; P < 0.01). There was no difference in Bederson score. The analysis of the gut microbiome showed significant heterogeneity between centres (R = 0.78, P < 0.001, n = 40). Taken together, neutralization of interleukin-17A in a therapeutic time window resulted in a significant reduction of infarct sizes and mortality compared with isotype control. It suggests interleukin-17A neutralization as a potential therapeutic target in stroke.

Nt5e deficiency does not affect post-stroke inflammation and lesion size in a murine ischemia/reperfusion stroke model.

Extracellular ATP released to the ischemic brain parenchyma is quickly metabolized by ectonucleotidases. Among them, the ecto-5'-nucleotidase CD73 encoded by Nt5e generates immunosuppressive adenosine. Genetic deletion of Nt5e led to increased infarct size in the murine photothrombotic stroke model. We aimed at validating this result using the transient middle cerebral artery occlusion (tMCAO) stroke model that represents pathophysiological aspects of penumbra and reperfusion. Three days after tMACO, we did not detect a difference in stroke size between CD73-deficient (CD73-/-) and control mice. Consistent with this finding, CD73-/- and control mice showed comparable numbers and composition of brain-infiltrating leukocytes measured by flow cytometry. Using NanoString technology, we further demonstrated that CD73-/- and control mice do not differ regarding glia cell gene expression profiles. Our findings highlight the potential impact of stroke models on study outcome and the need for cross-validation of originally promising immunomodulatory candidates.

Interleukin-1 Mediates Ischemic Brain Injury via Induction of IL-17A in γδ T Cells and CXCL1 in Astrocytes.

As a prototypical proinflammatory cytokine, interleukin-1 (IL-1) exacerbates the early post-stroke inflammation, whereas its neutralization is protective. To further investigate the underlying cell-type-specific IL-1 effects, we subjected IL-1 (α/ß) knockout (Il1-/-) and wildtype (WT) littermate mice to permanent middle cerebral artery occlusion (pMCAO) and assessed immune cell infiltration and cytokine production in the ischemic hemisphere by flow cytometry 24 h and 72 h after stroke. Il1-/- mice showed smaller infarcts and reduced neutrophil infiltration into the ischemic brain. We identified γδ T cells and astrocytes as target cells of IL-1 signaling-mediated neutrophil recruitment. First, IL-1-induced IL-17A production in γδ T cells in vivo, and IL-17A enhanced the expression of the main neutrophil attracting chemokine CXCL1 by astrocytes in the presence of tumor necrosis factor (TNF) in vitro. Second, IL-1 itself was a potent activator of astrocytic CXCL1 production in vitro. By employing a novel FACS sorting strategy for the acute isolation of astrocytes from ischemic brains, we confirmed that IL-1 is pivotal for Cxcl1 upregulation in astrocytes in vivo. Our results underscore the pleiotropic effects of IL-1 on immune and non-immune cells within the CNS to mount and amplify the post-stroke inflammatory response.