Junwei Hao

Impact of an immune modulator fingolimod on acute ischemic stroke

Significance In patients with acute ischemic stroke (AIS), the abrupt and massive influx of lymphocytes from the periphery to the ischemic region orchestrates focal inflammatory responses, catalyzes tissue death, and worsens clinical outcomes. In this early phase clinical study, we reduced lymphocyte migration to the brain during the first 72 h of AIS via oral administration of three doses of fingolimod. This administration led to a significant reduction of secondary lesion enlargement, microvascular permeability, and better clinical outcomes during the acute phase and 3-mo follow-up visit. This study will provoke new investigations on the efficacy of modulation of brain inflammation in AIS. Peripheral lymphocytes entering brain ischemic regions orchestrate inflammatory responses, catalyze tissue death, and worsen clinical outcomes of acute ischemic stroke (AIS) in preclinical studies. However, it is not known whether modulating brain inflammation can impact the outcome of patients with AIS. In this open-label, evaluator-blinded, parallel-group clinical pilot trial, we recruited 22 patients matched for clinical and MRI characteristics, with anterior cerebral circulation occlusion and onset of stroke that had exceeded 4.5 h, who then received standard management alone (controls) or standard management plus fingolimod (FTY720, Gilenya, Novartis), 0.5 mg per day orally for 3 consecutive days. Compared with the 11 control patients, the 11 fingolimod recipients had lower circulating lymphocyte counts, milder neurological deficits, and better recovery of neurological functions. This difference was most profound in the first week when reduction of National Institutes of Health Stroke Scale was 4 vs. −1, respectively (P = 0.0001). Neurological rehabilitation was faster in the fingolimod-treated group. Enlargement of lesion size was more restrained between baseline and day 7 than in controls (9 vs. 27 mL, P = 0.0494). Furthermore, rT1%, an indicator of microvascular permeability, was lower in the fingolimod-treated group at 7 d (20.5 vs. 11.0; P = 0.005). No drug-related serious events occurred. We conclude that in patients with acute and anterior cerebral circulation occlusion stroke, oral fingolimod within 72 h of disease onset was safe, limited secondary tissue injury from baseline to 7 d, decreased microvascular permeability, attenuated neurological deficits, and promoted recovery.

mTORC1 pathway disruption ameliorates brain inflammation following stroke via a shift in microglia phenotype from M1 type to M2 type.

Inflammatory factors secreted by microglia play an important role in focal ischemic stroke. The mammalian target of rapamycin (mTOR) pathway is a known regulator of immune responses, but the role that mTORC1 signaling plays in poststroke neuroinflammation is not clear. To explore the relationship between microglial action in the mTORC1 pathway and the impact on stroke, we administered the mTORC1 inhibitors sirolimus and everolimus to mice. Presumably, disrupting the mTORC1 pathway after focal ischemic stroke should clarify the subsequent activity of microglia. For that purpose, we generated mice deficient in the regulatory associated protein of mTOR (Raptor) in microglia, whose mTORC1 signaling was blocked, by crossing Raptor loxed (Raptorflox/flox) mice with CX3CR1CreER mice, which express Cre recombinase under the control of the CX3C chemokine receptor 1 promoter. mTORC1 blockade reduced lesion size, improved motor function, dramatically decreased production of pro‐inflammatory cytokines and chemokines, and reduced the number of M1 type microglia. Thus, mTORC1 blockade apparently attenuated behavioral deficits and poststroke inflammation after middle cerebral artery occlusion by preventing microglia polarization toward the M1 type.—Li, D., Wang, C., Yao, Y., Chen, L., Liu, G., Zhang, R., Liu, Q., Shi, F.‐D., Hao, J. mTORC1 pathway disruption ameliorates brain inflammation following stroke via a shift in microglia phenotype from M1 type to M2 type. FASEB J. 30, 3388–3399 (2016). www.fasebj.org

IL-10-Producing Lymphocytes in Inflammatory Disease

IL-10 is an anti-inflammatory cytokine that plays a significant role in controlling inflammation and modulating adaptive immune responses that cause tissue damage. IL-10-producing lymphocytes contribute to the delicate balance between inflammation and immunoregulation, and are thus regarded as a kind of “regulatory cells.” Dysregulation of these cells is linked with susceptibility to numerous inflammatory diseases. In this review, we summarized what is known about the regulatory effects of IL-10 produced by lymphocytes, including T cells, B cells and natural killer cells, in inflammatory diseases. We hope to augment immune responses or prevent immunopathology through making some small changes in the levels of IL-10 produced by lymphocytes, or in the cellular location where it is produced.

Antisense MMP-9 RNA inhibits malignant glioma cell growth in vitro and in vivo

The matrix-degrading metalloproteinases (MMPs), particularly MMP-9, play important roles in the pathogenesis and development of malignant gliomas. In the present study, the oncogenic role of MMP-9 in malignant glioma cells was investigated via antisense RNA blockade in vitro and in vivo. TJ905 malignant glioma cells were transfected with pcDNA3.0 vector expressing antisense MMP-9 RNA (pcDNA-ASMMP9), which significantly decreased MMP-9 expression, and cell proliferation was assessed. For in vivo studies, U251 cells, a human malignant glioma cell line, were implanted subcutaneously into 4- to 6-week-old BALB/c nude mice. The mice bearing well-established U251 gliomas were treated with intratumoral pcDNA-AS-MMP9-Lipofectamine complex (AS-MMP-9-treated group), subcutaneous injection of endostatin (endostatin-treated group), or both (combined therapy group). Mice treated with pcDNA (empty vector)-Lipofectamine served as the control group. Four or eight weeks later, the volume and weight of tumor, MMP-9 expression, microvessel density and proliferative activity were assayed. We demonstrate that pcDNA-AS-MMP9 significantly decreased MMP-9 expression and inhibited glioma cell proliferation. Volume and weight of tumor, MMP-9 expression, microvessel density and proliferative activity in the antisense-MMP-9-treated and therapeutic alliance groups were significantly lower than those in the control group. The results suggest that MMP-9 not only promotes malignant glioma cell invasiveness, but also affects tumor cell proliferation. Blocking the expression of MMP-9 with antisense RNA substantially suppresses the malignant phenotype of glioma cells, and thus can be used as an effective therapeutic strategy for malignant gliomas.

Dimethyl fumarate attenuates experimental autoimmune neuritis through the nuclear factor erythroid-derived 2-related factor 2/hemoxygenase-1 pathway by altering the balance of M1/M2 macrophages

BackgroundGuillain–Barré syndrome (GBS) is an acute, post-infectious, immune-mediated, demyelinating disease of peripheral nerves and nerve roots. Dimethyl fumarate (DMF), a fumaric acid ester, exhibits various biological activities, including multiple immunomodulatory and neuroprotective effects. However, the potential mechanism underlying the effect of DMF in GBS animal model experimental autoimmune neuritis (EAN) is unclear.MethodsUsing EAN, an established GBS model, we investigated the effect of DMF by assessing clinical score, histological staining and electrophysiological studies. Then, we further explored the potential mechanism by Western blot analysis, flow cytometry, fluorescence immunohistochemistry, PCR, and ELISA analysis. The Mann–Whitney U test was used to compare differences between control group and treatment groups where appropriate.ResultsDMF treatment reduced the neurological deficits by ameliorating inflammatory cell infiltration and demyelination of sciatic nerves. In addition, DMF treatment decreased the level of pro-inflammatory M1 macrophages while increasing the number of anti-inflammatory M2 macrophages in the spleens and sciatic nerves of EAN rats. In RAW 264.7, a shift in macrophage polarization from M1 to M2 phenotype was demonstrated to be depended on DMF application. In sciatic nerves, DMF treatment elevated the level of the antioxidant transcription factor nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and its target gene hemoxygenase-1 (HO-1) which could facilitate macrophage polarization toward M2 type. Moreover, DMF improved the inflammatory milieu in spleens of EAN rats, characterized by downregulation of messenger RNA (mRNA) of IFN-γ, TNF-α, IL-6, and IL-17 and upregulation of mRNA level of IL-4 and IL-10.ConclusionsTaken together, our data demonstrate that DMF can effectively suppress EAN, and the mechanism involves altering the balance of M1/M2 macrophages and attenuating inflammation.

Integrating genome-wide association studies and gene expression data highlights dysregulated multiple sclerosis risk pathways

Background: Much effort has been expended on identifying the genetic determinants of multiple sclerosis (MS). Existing large-scale genome-wide association study (GWAS) datasets provide strong support for using pathway and network-based analysis methods to investigate the mechanisms underlying MS. However, no shared genetic pathways have been identified to date. Objective: We hypothesize that shared genetic pathways may indeed exist in different MS-GWAS datasets. Methods: Here, we report results from a three-stage analysis of GWAS and expression datasets. In stage 1, we conducted multiple pathway analyses of two MS-GWAS datasets. In stage 2, we performed a candidate pathway analysis of the large-scale MS-GWAS dataset. In stage 3, we performed a pathway analysis using the dysregulated MS gene list from seven human MS case–control expression datasets. Results: In stage 1, we identified 15 shared pathways. In stage 2, we successfully replicated 14 of these 15 significant pathways. In stage 3, we found that dysregulated MS genes were significantly enriched in 10 of 15 MS risk pathways identified in stages 1 and 2. Conclusion: We report shared genetic pathways in different MS-GWAS datasets and highlight some new MS risk pathways. Our findings provide new insights on the genetic determinants of MS.

Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival.

It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1β and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers.

Infiltration of CCR2+Ly6Chigh Proinflammatory Monocytes and Neutrophils into the Central Nervous System Is Modulated by Nicotinic Acetylcholine Receptors in a Model of Multiple Sclerosis

Myeloid cells, including proinflammatory monocytes and neutrophils, have important roles in the pathology of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). These cells infiltrate the CNS in the early stages of disease development and contribute to the inflammatory response that is associated with symptom severity. It is thus crucial to identify and understand new mechanisms that can regulate the CNS infiltration of proinflammatory myeloid cells. Nicotinic acetylcholine receptors (nAChRs) have been increasingly studied for their immune-regulatory properties. In this study, we assessed the ability of nicotine, an nAChR ligand, to modulate proinflammatory myeloid cell numbers within the bone marrow, spleen, blood, and CNS of EAE mice. We found that nicotine significantly inhibits the infiltration of proinflammatory monocytes and neutrophils into the CNS at time points where these cells are known to play critical roles in disease pathology. In contrast, nicotine does not affect the expansion of other monocytes. We also show that nicotine exerts these effects by acting on α7 and α9 nAChR subtypes. Finally, mRNA transcript levels for CCL2 and CXCL2, chemokines involved in the chemotaxis of proinflammatory monocytes and neutrophils, respectively, are reduced in the brain of nicotine-treated EAE mice before the massive infiltration of these cells. Taken together, our data provide evidence that nAChRs can regulate proinflammatory cell infiltration into the CNS, which could be of significant value for the treatment of neuroinflammatory disorders.

Astrocyte-derived interleukin-15 exacerbates ischemic brain injury via propagation of cellular immunity.

Significance Ischemic stroke is a leading cause of death and disability worldwide. Evidence indicates the detrimental effects of lymphocyte infiltration into the ischemic brain. However, a knowledge gap exists relating to the brain-specific cellular constituents and environmental factors that dictate the phenotype and function of infiltrating lymphocytes. Astrocytes bridge interactions between ischemic neurons and lymphocytes. We show that brain ischemia induces robust up-regulation of astrocytic interleukin-15 (IL-15). The present study was directed toward understanding the role of astrocyte-derived factors such as IL-15 in stroke. We discovered that astrocytic IL-15 is necessary and sufficient to amplify cell-mediated immune responses that promote ischemic brain injury. These results provide definitive evidence on the role of astrocyte-derived IL-15 in ischemic brain injury. Astrocytes are believed to bridge interactions between infiltrating lymphocytes and neurons during brain ischemia, but the mechanisms for this action are poorly understood. Here we found that interleukin-15 (IL-15) is dramatically up-regulated in astrocytes of postmortem brain tissues from patients with ischemic stroke and in a mouse model of transient focal brain ischemia. We generated a glial fibrillary acidic protein (GFAP) promoter-controlled IL-15–expressing transgenic mouse (GFAP–IL-15tg) line and found enlarged brain infarcts, exacerbated neurodeficits after the induction of brain ischemia. In addition, knockdown of IL-15 in astrocytes attenuated ischemic brain injury. Interestingly, the accumulation of CD8+ T and natural killer (NK) cells was augmented in these GFAP–IL-15tg mice after brain ischemia. Of note, depletion of CD8+ T or NK cells attenuated ischemic brain injury in GFAP–IL-15tg mice. Furthermore, knockdown of the IL-15 receptor α or blockade of cell-to-cell contact diminished the activation and effector function of CD8+ T and NK cells in GFAP–IL-15tg mice, suggesting that astrocytic IL-15 is delivered in trans to target cells. Collectively, these findings indicate that astrocytic IL-15 could aggravate postischemic brain damage via propagation of CD8+ T and NK cell-mediated immunity.

Selective Sphingosine-1-Phosphate Receptor 1 Modulation Attenuates Experimental Intracerebral Hemorrhage

Background and Purpose— Preclinical studies and a proof-of-concept clinical study have shown that sphingosine-1-phosphate receptor (S1PR) modulator, fingolimod, improves the clinical outcome of intracerebral hemorrhage (ICH). However, the specific subtype of the S1PRs through which immune modulation provides protection in ICH remains unclear. In addition, fingolimod-induced adverse effects could limit its use in patients with stroke because of interactions with other S1PR subtypes, particularly with S1PR3. RP101075 is a selective S1PR1 agonist with superior cardiovascular safety profile. In this study, we investigated the impact of RP101075 treatment in a mouse model of ICH. Methods— ICH was induced by injection of autologous blood in 294 male C57BL/6J and Rag2−/− mice. ICH mice randomly received vehicle, RP101075, or RP101075 plus S1PR1 antagonist W146 by daily oral gavage for three consecutive days, starting from 30 minutes after surgery. Neurodeficits, brain edema, brain infiltration of immune cells, blood–brain barrier integrity, and cell death were assessed after ICH. Results— RP101075 significantly attenuated neurological deficits and reduced brain edema in ICH mice. W146 blocked the effects of RP101075 on neurodeficits and brain edema. RP101075 reduced the counts of brain-infiltrating lymphocytes, neutrophils, and microglia, as well as cytokine expression after ICH. Enhanced blood–brain barrier integrity and alleviated neuronal death were also seen in ICH mice after RP101075 treatment. Conclusions— S1PR1 modulation via RP101075 provides protection in experimental ICH. Together with the advantageous pharmacological features of RP101075, these results warrant further investigations of its mechanisms of action and translational values in ICH patients.