Estrogen-induced compensatory mechanisms protect IL-10-deficient mice from developing EAE.

BACKGROUND: IL-10 knockout (KO) mice are protected from experimental autoimmune encephalomyelitis (EAE) with low-dose estrogen (E2) treatment similar to wild-type (WT) mice. Previous studies have demonstrated a decrease in tumor necrosis factor in all E2-treated groups, which led to the protection of the mice. METHODS: This study used IL-10 KO mice and WT mice treated either with E2 or sham pellets 7 days prior to induction of EAE. Mice were observed for 21 days post-immunization. The spleen, inguinal lymph nodes, and brain were evaluated by flow cytometry. Spinal cords were evaluated using a cytokine/chemokine array, RT-PCR, and histology. RESULTS: This study demonstrates that E2 treatment induced three heightened regulatory mechanisms that potentially protect IL-10 KO mice from EAE: (1) an increase in programmed death-ligands 1 and 2 on monocytes and macrophages in the periphery and within the CNS; (2) an increase in CD73 in the inflamed CNS, which can increase the production of the anti-inflammatory molecule adenosine; and (3) a decrease in CD4+CD25+FoxP3+ regulatory T cells in the spleen. Together, these factors comprise an alternative compensatory mechanism that significantly downregulates key pro-inflammatory cytokine, chemokine, and chemokine receptor genes which are enhanced in the spinal cord of IL-10 KO mice. This group of E2-treated mice remained asymptomatic after EAE challenge similar to E2-treated WT mice, despite their having more T and B lymphocytes in the brain, and modestly increased demyelination in the spinal cord. CONCLUSION: These results indicate that previously unrecognized compensatory mechanisms of EAE protection are stimulated by E2 in the absence of IL-10, which can provide disease protection comparable to the IL-10-dependent effects induced by E2 in WT mice.

Sex differences in the therapeutic effects of anti-PDL2 neutralizing antibody on stroke.

Inflammation involving migration of immune cells across the damaged blood-brain barrier (BBB), activation of resident innate microglia and production of inflammatory humoral mediators such as cytokines and chemokines play a critical role in the pathogenesis of ischemic stroke. Cell-cell signaling involved in the process also includes checkpoint interaction between programmed death receptor (PD1) and programmed death ligands, PDL1 and PDL2. Based on our previous studies showing reduced MCAO infarct volumes in PDL2 deficient mice, we evaluated the ability of anti-PDL2 mAb to treat MCAO in male and female C57BL/6 mice. We found that anti-PDL2 neutralizing antibody treatment of MCAO significantly reduced infarct volumes in male mice but had no protective effects in female mice even at a 5-fold increased dose of anti-PDL2 mAb. The protection in male mice was likely mediated by reduced percentages in the spleen of PDL2+CD19+ B cells, PDL1+CD4+ T cells and CD86+CD11b+ macrophages in concert with reduced expression of PDL1 and TNFα and continued expression of CD206, in the injured ipsilateral brain hemisphere. The lack of a therapeutic benefit of anti-PDL2 on stroke-induced infarct volumes in female mice was reflected by no detectable reduction in expressed PDL2 or PDL1 and an increased frequency of Th1 and Th17 pro-inflammatory T cell subsets in the spleen, an effect not seen in PDL2 mAb treated males. This result potentially limits the utility of anti-PDL2 mAb therapy in stroke to males but underscores the importance of meeting the STAIR requirements for development of new stroke therapies for both sexes.

Translational Evaluation of Acid/Base and Electrolyte Alterations in Rodent Model of Focal Ischemia.

BACKGROUND AND PURPOSE: Acid/base and electrolytes could provide clinically valuable information about cerebral infarct core and penumbra. We evaluated associations between acid/base and electrolyte changes and outcomes in 2 rat models of stroke, permanent, and transient middle cerebral artery occlusion. METHODS: Three-month old Sprague-Dawley rats underwent permanent or transient middle cerebral artery occlusion. Pre- and post-middle cerebral artery occlusion venous samples for permanent and transient models provided pH, carbon dioxide, oxygen, glucose, and electrolyte values of ionized calcium, potassium, and sodium. Multiple regression determined predictors of infarct volume from these values, and Kaplan-Meier curve analyzed morality between permanent and transient middle cerebral artery occlusion models. RESULTS: Analysis indicated significant differences in the blood gas and electrolytes between pre- to post-middle cerebral artery occlusion. A decrease in pH and sodium with increases in carbon dioxide, potassium, ionized calcium, and glucose changes were found in both middle cerebral artery occlusion models; while hematocrit and hemoglobin were significant in the transient model. pH and ionized calcium were predictors of infarct volume in the permanent model, as changes in pH and ionized calcium decreased, infarct volume increased. CONCLUSIONS: There are acute changes in acid/base balance and electrolytes during stroke in transient and permanent rodent models. Additionally, we found pH and ionized calcium changes predicted stroke volume in the permanent middle cerebral artery occlusion model. These preliminary findings are novel, and warrant further exploration in human conditions.

The splenic response to stroke: from rodents to stroke subjects.

BACKGROUND: Stroke is the fifth leading cause of death and the leading cause of long-term disability in the USA, costing $40.2 billion in direct and indirect costs. Globally, stroke is the second leading cause of death and has a higher prevalence in lower- and middle-income countries compared to high-income countries. The role of the spleen in stroke has been studied in rodent models of stroke and is seen as a major contributor to increased secondary neural injury after stroke. Splenectomy 2 weeks prior to ischemic and hemorrhagic stroke in mice and rats shows decreased infarct volumes. Additionally, the spleen decreases in size following stroke in rodents. Pro-inflammatory mediators are also increased in the spleen and subsequently the brain after stroke. These data in preclinical models of stroke have led stroke neurologists to look at the splenic response in stroke subjects. The outcomes of these studies suggest the spleen is responding in a similar manner in stroke subjects as it is in animal models of stroke. CONCLUSION: Animal models demonstrating the detrimental role of the spleen in stroke are providing strong evidence of how the spleen is responding during stroke in human subjects. This indicates treatments targeting the splenic immune response in animals could provide useful targets and treatments for stroke subjects.

Antibiotics protect against EAE by increasing regulatory and anti-inflammatory cells.

A seven day pretreatment course of an oral antibiotic cocktail (Ampicillin, Metronidazole, Neomycin Sulfate, and Vancomycin) was shown to induce changes in peripheral immune regulation and protect mice from signs of experimental autoimmune encephalomyelitis (EAE). To determine if a shorter course of antibiotic pretreatment could also protect the mice from EAE and induce regulatory immune cells, studies were conducted using the same oral antibiotic cocktail for three days. In addition, the CNS was examined to determine the effects of antibiotic pretreatment on EAE disease course and immune modulation within the affected tissue. The shorter three day pretreatment course was also significantly protective against severe EAE in C57BL/6 mice. Moreover, our study found increased frequencies of regulatory cells and a decrease in the frequency of anti-inflammatory macrophages in the spleen of EAE protected mice. Additionally, a chemokine and chemokine receptor array run on mRNA from spinal cords revealed that genes associated with regulatory T cells and macrophage recruitment were strongly upregulated in the antibiotic pretreated mice. Additional RT-PCR data showed genes associated with anti-inflammatory microglia/macrophages were upregulated and pro-inflammatory genes were downregulated. This suggests the macrophages recruited to the spinal cord by chemokines are subsequently polarized toward an anti-inflammatory phenotype. These results lend strong support to the conclusion that a three day course of antibiotic treatment given prior to the induction of severe EAE profoundly protected the mice by inducing regulatory lymphocytes in the periphery and an anti-inflammatory milieu in the affected spinal cord tissue.

Regulatory B cells in experimental stroke.

Current treatment options for human stroke are limited mainly to the modestly effective infusion of tissue plasminogen activator (tPA), with additional improvement of functional independence and higher rates of angiographic revascularization observed after mechanical thrombectomy. However, new therapeutic strategies that address post-stroke immune-mediated inflammatory responses are urgently needed. Recent studies in experimental stroke have firmly implicated immune mechanisms in the propagation and partial resolution of central nervous system damage after the ischaemic event. A new-found anti-inflammatory role for regulatory B (Breg) cells in autoimmune diseases sparked interest in these cells as potential immunomodulators in stroke. Subsequent studies identified interleukin-10 as a common regulatory cytokine among all five of the currently recognized Breg cell subsets, several of which can be found in the affected brain hemisphere after induction of experimental stroke in mice. Transfer of enriched Breg cell subpopulations into both B-cell-depleted and wild-type mice confirmed their potent immunosuppressive activities in vivo, including recruitment and potentiation of regulatory T cells. Moreover, Breg cell therapy strongly reduced stroke volumes and treatment outcomes in ischaemic mice even when administered 24 hr after induction of experimental stroke, a treatment window far exceeding that of tPA. These striking results suggest that transfer of enriched Breg cell populations could have therapeutic value in human stroke, although considerable clinical challenges remain.

Estrogen protection against EAE modulates the microbiota and mucosal-associated regulatory cells.

Sex hormones promote immunoregulatory effects on multiple sclerosis. In the current study we evaluated the composition of the gut microbiota and the mucosal-associated regulatory cells in estrogen or sham treated female mice before and after autoimmune encephalomyelitis (EAE) induction. Treatment with pregnancy levels of estrogen induces changes in the composition and diversity of gut microbiota. Additionally, estrogen prevents EAE-associated changes in the gut microbiota and might promote the enrichment of bacteria that are associated with immune regulation. Our results point to a possible cross-talk between the sex hormones and the gut microbiota, which could promote neuroprotection.

Estrogen protects both sexes against EAE by promoting common regulatory cell subtypes independent of endogenous estrogen.

Autoimmune diseases including multiple sclerosis predominantly affect females. Although high levels of sex hormones, particularly estrogen (E2), can reduce proinflammatory immune responses, it remains unclear if a lack of endogenous sex hormones might affect treatment with exogenous sex hormones. Pretreatment with E2 almost completely prevents intact female and male mice from developing clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) by promoting various regulatory immune cell phenotypes. To evaluate the effects of exogenous estrogen in the absence of endogenous sex hormones, the current study compared EAE severity and the emergence of different immunoregulatory cell populations after E2 pretreatment of ovariectomized (OVX) female versus male mice. We found that E2 equally protected both OVX females and males from EAE over a 21 day observation period concomitant with reduced total cell numbers in spleen and spinal cord (males only), but enhanced percentages of CD19+CD5+CD1dhi, CD19+CD138+CD44hi and CD19+Tim-1+ Breg cells, CD8+CD122+ Treg cells and CD11b+CD 206+ARG-1+ anti-inflammatory M2-like monocytes/macrophages in both groups. In contrast, E2 decreased the percentage of CD4+CD25+FoxP3+ Treg cells in OVX females but increased these Treg cells in males and intact female mice. These data suggest that with the exception of CD4+CD25+FoxP3+ Treg cells, E2 protection against EAE promotes highly overlapping immunoregulatory subsets in OVX females and males.

Sex differences in regulatory cells in experimental stroke.

Stroke is the leading cause of disability in the United States. Sex differences, including smaller infarcts in females and greater involvement of immune-mediated inflammation in males may affect the efficacy of immune-modulating interventions. To address these differences, we sought to identify distinct stroke-modifying mechanisms in female vs. male mice. The current study demonstrated smaller infarcts and increased levels of regulatory CD19+CD5+CD1dhi B10 cells as well as anti-inflammatory CD11b+CD206+ microglia/macrophages in the ipsilateral vs. contralateral hemisphere of female but not male mice undergoing 60min middle cerebral artery occlusion followed by 96h of reperfusion. Moreover, female mice with MCAO had increased total spleen cell numbers but lower B10 levels in spleens. These results elucidate differing sex-dependent regulatory mechanisms that account for diminished stroke severity in females and underscore the need to test immune-modulating therapies for stroke in both males and females.

Leukemia Inhibitory Factor Protects Neurons from Ischemic Damage via Upregulation of Superoxide Dismutase 3.

Leukemia inhibitory factor (LIF) has been shown to protect oligodendrocytes from ischemia by upregulating endogenous antioxidants. The goal of this study was to determine whether LIF protects neurons during stroke by upregulating superoxide dismutase 3 (SOD3). Animals were administered phosphate-buffered saline (PBS) or 125 µg/kg LIF at 6, 24, and 48 h after middle cerebral artery occlusion or sham surgery. Neurons were isolated from rat pups on embryonic day 18 and used between 7 and 15 days in culture. Cells were treated with LIF and/or 10 µM Akt inhibitor IV with PBS and 0.1 % DMSO acting as vehicle controls. Neurons transfected with scrambled or SOD3 small interfering RNA (siRNA) were subjected to 24-h ischemia after PBS or LIF treatment. LIF significantly increased superoxide dismutase activity and SOD3 expression in ipsilateral brain tissue compared to PBS. Following 24-h ischemia, LIF reduced cell death and increased SOD3 messenger RNA (mRNA) in vitro compared to PBS. Adding Akt inhibitor IV with LIF counteracted the decrease in cell death. Partially silencing the expression of SOD3 using siRNA prior to LIF treatment counteracted the protective effect of LIF-alone PBS treatment. These results indicate that LIF protects neurons in vivo and in vitro via upregulation of SOD3.

Pro-inflammatory interferon gamma signaling is directly associated with stroke induced neurodegeneration.

The delayed immune response to stroke is responsible for the increased neural injury that continues to occur after the initial ischemic event. This delayed immune response has been linked to the spleen, as splenectomy prior to middle cerebral artery occlusion (MCAO) is neuroprotective. Interferon gamma (IFNγ) is linked to the splenic response, which enhances neural injury following MCAO. IFNγ activates the expression of the inflammatory chemokine interferon-inducible protein 10 (IP-10). This study was designed to determine the role of IFNγ signaling in the inflammatory response following MCAO. Expression of IP-10 increased in the brain and the spleen following MCAO. Splenectomy inhibited the increase of IP-10 in the brain post-MCAO, while recombinant IFNγ administration to splenectomized rats returned IP-10 levels in the brain to levels found in rats after MCAO only. Systemic administration of an IFNγ neutralizing antibody to MCAO-treated rats reduced infarct volume and IP-10 levels in the brain. T cell infiltration was reduced in the MCAO-damaged brains of IFNγ antibody-treated animals relative to those that received isotype control antibodies. Additionally, inhibiting IFNγ signaling with splenectomy or an IFNγ neutralizing antibody blocked the induction of IP-10 expression and decreased neurodegeneration following MCAO. Targeting this pro-inflammatory pathway following stroke could be a promising stroke therapeutic.

Leukemia inhibitor factor promotes functional recovery and oligodendrocyte survival in rat models of focal ischemia.

Human umbilical cord blood (HUCB) cells have shown efficacy in rodent models of focal ischemia and in vitro systems that recapitulate stroke conditions. One potential mechanism of protection is through secretion of soluble factors that protect neurons and oligodendrocytes (OLs) from oxidative stress. To overcome practical issues with cellular therapies, identification of soluble factors released by HUCB and other stem cells may pave the way for treatment modalities that are safer for a larger percentage of stroke patients. Among these soluble factors is leukemia inhibitory factor (LIF), a cytokine that exerts pleiotropic effects on cell survival. Here, data show that LIF effectively reduced infarct volume, reduced white matter injury and improved functional outcomes when administered to rats following permanent middle cerebral artery occlusion. To further explore downstream signaling, primary oligodendrocyte cultures were exposed to oxygen-glucose deprivation to mimic stroke conditions. LIF significantly reduced lactate dehydrogenase release from OLs, reduced superoxide dismutase activity and induced peroxiredoxin 4 (Prdx4) transcript. Additionally, the protective and antioxidant capacity of LIF was negated by both Akt inhibition and co-incubation with Prdx4-neutralising antibodies, establishing a role for the Akt signaling pathway and Prdx4-mediated antioxidation in LIF protection.

Molecular and cellular immune responses to ischemic brain injury.

Despite extensive research into stroke pathology, there have not been any major recent advancements in stroke therapeutics. Animal models of cerebral ischemia and clinical data have been used to investigate the progressive neural injury that occurs after an initial ischemic insult. This has lead researchers to focus more on the peripheral immune response that is generated as a result of cerebral ischemia. The therapies that have been developed as a result of this research thus far have proven ineffective in clinical trials. The failure of these therapeutics in clinical trials is thought to be due to the broad immunosuppression elicited as a result of the treatments and the cerebral ischemia itself. Emerging evidence indicates a more selective modulation of the immune system following stroke could be beneficial. The spleen has been shown to exacerbate neural injury following experimental stroke and would provide a strong therapeutic target. Selecting facets of the immune system to target would allow the protective and regenerative properties of the immune response to remain intact while blunting the pro-inflammatory response generated towards the injured brain.

A transient decrease in spleen size following stroke corresponds to splenocyte release into systemic circulation.

The splenic response to stroke is a proinflammatory reaction to ischemic injury resulting in expanded neurodegeneration. Splenectomy reduces neural injury in rodent models of hemorrhagic and ischemic stroke, however the exact nature of this response has yet to be fully understood. This study examines the migration of splenocytes after brain ischemia utilizing carboxyfluorescein diacetate succinimidyl ester (CFSE) to label them in vivo. The spleen was found to significantly decrease in size from 24 to 48 h following middle cerebral artery occlusion (MCAO) in rats compared to sham operated controls. By 96 h post-MCAO the spleen size returned to levels not different from sham operated rats. To track splenocyte migration following MCAO, spleens were injected with CFSE to label cells. CFSE positive cell numbers were significantly reduced in the 48 h MCAO group versus 48 h sham and CFSE labeled cells were equivalent in 96 h MCAO and sham groups. A significant increase of labeled lymphocyte, monocytes, and neutrophils was detected in the blood at 48 h post-MCAO when compared to the other groups. CFSE labeled cells migrated to the brain following MCAO but appear to remain within the vasculature. These cells were identified as natural killer cells (NK) and monocytes at 48 h and at 96 h post-MCAO NK cells, T cells and monocytes. After ischemic injury, splenocytes enter into systemic circulation and migrate to the brain exacerbating neurodegeneration.

The spleen contributes to stroke induced neurodegeneration through interferon gamma signaling.

Delayed neuronal death associated with stroke has been increasingly linked to the immune response to the injury. Splenectomy prior to middle cerebral artery occlusion (MCAO) is neuroprotective and significantly reduces neuroinflammation. The present study investigated whether splenic signaling occurs through interferon gamma (IFNγ). IFNγ was elevated early in spleens but later in the brains of rats following MCAO. Splenectomy decreased the amount of IFNγ in the infarct post-MCAO. Systemic administration of recombinant IFNγ abolished the protective effects of splenectomy with a concurrent increase in INFγ expression in the brain. These results suggest a role for spleen-derived IFNγ in stroke pathology.