Sheetal Bodhankar

Estrogen-induced protection against experimental autoimmune encephalomyelitis is abrogated in the absence of B cells.

Increased remissions in multiple sclerosis (MS) during pregnancy suggest that elevated levels of sex steroids exert immunoregulatory activity. Estrogen (E2=17β‐estradiol) protects against experimental autoimmune encephalomyelitis (EAE), but the cellular basis for E2‐induced protection remains unclear. Studies demonstrate that depletion of B cells prior to induction of EAE exacerbates disease severity, implicating regulatory B cells. We thus evaluated pathogenic and E2‐induced protective mechanisms in B‐cell‐deficient (μMT−/−) mice. EAE‐protective effects of E2 were abrogated in μMT−/− mice, with no reduction in disease severity, cellular infiltration or pro‐inflammatory factors in the central nervous system compared to untreated controls. E2 treatment of WT mice selectively upregulated expression of PD‐L1 on B cells and increased the percentage of IL‐10‐producing CD1dhighCD5+ regulatory B cells. Upregulation of PD‐L1 was critical for E2‐mediated protection since E2 did not inhibit EAE in PD‐L1−/− mice. Direct treatment of B cells with E2 significantly reduced proliferation of MOG35–55‐specific T cells that required estrogen receptor‐α (ERα). These results demonstrate, for the first time, a requirement for B cells in E2‐mediated protection against EAE involving direct E2 effects on regulatory B cells mediated through ERα and the PD‐1/PD‐L1 negative co‐stimulatory pathway. E2‐primed B cells may represent an important regulatory mechanism in MS and have strong implications for women receiving current MS therapies that cause B‐cell depletion.

Oestrogen treatment of experimental autoimmune encephalomyelitis requires 17β-oestradiol-receptor-positive B cells that up-regulate PD-1 on CD4+ Foxp3+ regulatory T cells.

It is now well accepted that sex hormones have immunoregulatory activity and may prevent exacerbations in multiple sclerosis during pregnancy. Our previous studies demonstrated that oestrogen (17β‐oestradiol; E2) protection against experimental autoimmune encephalomyelitis (EAE) is mediated mainly through oestrogen receptor‐α (ERα) and the membrane receptor G‐protein‐coupled receptor 30 (GPR30) and is abrogated in the absence of B cells and the co‐inhibitory receptor, Programmed Death‐1 (PD‐1). To critically evaluate the cell source of the E2 and PD‐1 co‐inhibitory pathways in EAE regulation, we assessed the requirement for ERs on transferred B cells and downstream effects on expression of PD‐1/PD‐ligand on CD4+ Foxp3+ regulatory T (Treg) cells in B‐cell‐replenished, E2‐treated B‐cell‐deficient (μMT−/−) mice with EAE. The results clearly demonstrated involvement of ERα and GPR30 on transferred B cells that mediated the protective E2 treatment effect on EAE and further showed an E2‐mediated B‐cell‐dependent up‐regulation of PD‐1 on CD4+ Foxp3+ Treg cells. These findings identify regulatory B‐cell populations as key players in potentiating Treg‐cell activity during E2‐mediated protection against EAE.

PD-L1 enhances CNS inflammation and infarct volume following experimental stroke in mice in opposition to PD-1

BackgroundStroke severity is worsened by recruitment of inflammatory immune cells into the brain. This process depends in part on T cell activation, in which the B7 family of co-stimulatory molecules plays a pivotal role. Previous studies demonstrated more severe infarcts in mice lacking programmed death-1 (PD-1), a member of the B7 family, thus implicating PD-1 as a key factor in limiting stroke severity. The purpose of this study was to determine if this protective effect of PD-1 involves either of its ligands, PD-L1 or PD-L2.MethodsCentral nervous system (CNS) inflammation and infarct volume were evaluated in male PD-L1 and PD-L2 knockout (-/-) mice undergoing 60 minutes of middle cerebral artery occlusion (MCAO) followed by 96 hours of reperfusion and compared to wild-type (WT) C57BL/6J mice.ResultsPD-L1-/- and PD-L2-/- mice had smaller total infarct volumes compared to WT mice. The PD-L1-/- and to a lesser extent PD-L2-/- mice had reduced levels of proinflammatory activated microglia and/or infiltrating monocytes and CD4+ T cells in the ischemic hemispheres. There was a reduction in ischemia-related splenic atrophy accompanied by lower activation status of splenic T cells and monocytes in the absence of PD-L1, suggesting a pathogenic rather than a regulatory role for both PD-1 ligands (PD-Ls). Suppressor T cells (IL-10-producing CD8+CD122+ T cells) trafficked to the brain in PD-L1-/- mice and there was decreased expression of CD80 on splenic antigen-presenting cells (APCs) as compared to the WT and PD-L2-/- mice.ConclusionsOur novel observations are the first to implicate PD-L1 involvement in worsening outcome of experimental stroke. The presence of suppressor T cells in the right MCAO-inflicted hemisphere in mice lacking PD-L1 implicates these cells as possible key contributors for controlling adverse effects of ischemia. Increased expression of CD80 on APCs in WT and PD-L2-/- mice suggests an overriding interaction leading to T cell activation. Conversely, low CD80 expression by APCs, along with increased PD-1 and PD-L2 expression in PD-L1-/- mice suggests alternative T cell signaling pathways, leading to a suppressor phenotype. These results suggest that agents (for example antibodies) that can target and neutralize PD-L1/2 may have therapeutic potential for treatment of human stroke.

Intrastriatal B-cell administration limits infarct size after stroke in B-cell deficient mice

Recent evidence emphasizes B-cells as a major regulatory cell type that plays an important role in limiting the pathogenic effects of ischemic stroke. The aim of the current study was to extend this initial observation to specifically examine the infiltration of regulatory B-cells and to determine if the effect of B-cells to limit the inflammatory response to cerebral ischemia is mediated by their action centrally or peripherally. Our data demonstrate the increased presence of a regulatory B-cell subset in the affected hemisphere of wild-type mice after middle cerebral artery occlusion (MCAO). We further explored the use of a novel method of stereotaxic cell delivery to bypass the blood brain barrier (BBB) and introduce CD19+ B-cells directly into the striatum as compared to peripheral administration of B-cells. Infarct volumes after 60 minutes of MCAO and 48 hours of reperfusion were determined in B-cell deficient μMT−/− mice with and without replacement of either B-cells or medium. Infarct size was significantly decreased in cerebral cortex after intrastriatal transfer of 100,000 B-cells to μMT−/− mice vs. controls, with a comparable effect on infarct size as obtained by 50 million B-cells transferred intraperitoneally. These findings support the hypothesis that B-cells play a protective role against ischemic brain injury, and suggest that B-cells may serve as a novel therapeutic agent for modulating the immune response in central nervous system inflammation after stroke.

Estrogen induces multiple regulatory B cell subtypes and promotes M2 microglia and neuroprotection during experimental autoimmune encephalomyelitis.

Sex hormones promote immunoregulatory effects on multiple sclerosis. The current study evaluated estrogen effects on regulatory B cells and resident CNS microglia during experimental autoimmune encephalomyelitis (EAE). Herein, we demonstrate an estrogen-dependent induction of multiple regulatory B cell markers indicative of IL-10 dependent as well as IFN-γ dependent pathways. Moreover, although estrogen pretreatment of EAE mice inhibited the infiltration of pro-inflammatory cells into the CNS, it enhanced the frequency of regulatory B cells and M2 microglia. Our study suggests that estrogen has a broad effect on the development of regulatory B cells during EAE, which in turn could promote neuroprotection.

Treatment with IL-10 producing B cells in combination with E2 ameliorates EAE severity and decreases CNS inflammation in B cell-deficient mice

Clinical improvement during pregnancy in multiple sclerosis (MS) patients suggests that sex hormones exert potent regulatory effects on autoimmune function. Our previous studies demonstrated that estrogen- (17β-estradiol; E2) mediated protection against experimental autoimmune encephalomyelitis (EAE), a mouse model for MS, hinges on the B cells, leading to elevated numbers of IL-10 secreting CD1dhiCD5+ B regulatory cells (Bregs) in wild type mice. Our data show that co-administration of E2 and IL-10+ B cells ameliorates EAE disease severity and limits CNS infiltrating leukocytes in B cell deficient mice. Additionally, treatment with E2 and Bregs reduces demyelination and dramatically decreases the proportion of CD11b+CD45hi activated microglia/macrophages found in the CNS of immunized animals compared to vehicle, E2 or Breg cells alone. Furthermore, mice given E2 and Bregs exhibit increased numbers of peripheral programmed death-1 positive CD4+Foxp3+ regulatory T cells (Tregs) and up-regulation of programmed death receptor-ligand-1 and CD80 expression on monocytes. Our study suggests IL-10 producing Bregs have powerful therapeutic potential as an agent against EAE when augmented with E2 treatment.

IL-10 producing B cells partially restore E2-mediated protection against EAE in PD-L1 deficient mice

Women with multiple sclerosis (MS) often experience clinical improvement during pregnancy, indicating that sex hormones might have therapeutic effects in MS. Our previous studies have demonstrated that B cells and PD-L1 are crucial for E2 (17β-estradiol)-mediated protection against experimental autoimmune encephalomyelitis (EAE). We here demonstrate that the transfer of IL-10(+) B cells into E2-treated PD-L1(-/-) mice after EAE induction could partially restore E2-mediated protection and decrease the frequency of pro-inflammatory cells in the CNS compared to E2/saline treated PD-L1(-/-) mice. Hence, co-administration of IL-10(+) B cells and E2 might have a powerful therapeutic potential for treatment of EAE.

PD-L1 Monoclonal Antibody Treats Ischemic Stroke by Controlling Central Nervous System Inflammation

Background and Purpose— Both pathogenic and regulatory immune processes are involved in the middle cerebral artery occlusion (MCAO) model of experimental stroke, including interactions involving the programmed death 1 (PD-1) receptor and its 2 ligands, PD-L1 and PD-L2. Although PD-1 reduced stroke severity, PD-L1 and PD-L2 appeared to play pathogenic roles, suggesting the use of anti-PD-L monoclonal antibody therapy for MCAO. Methods— Male C57BL/6 mice were treated with a single dose of anti-PD-L1 monoclonal antibody 4 hours after MCAO and evaluated for clinical, histological and immunologic changes after 96 hours of reperfusion. Results— Blockade of the PD-L1 checkpoint using a single injection of 200 &mgr;g anti-PD-L1 monoclonal antibody given intravenously 4 hours after occlusion significantly reduced MCAO infarct volumes and improved neurological outcomes after 96 hours of reperfusion. Treatment partially reversed splenic atrophy and decreased central nervous system infiltrating immune cells concomitant with enhanced appearance of CD8+ regulatory T cells in the lesioned central nervous system hemisphere. Conclusions— This study demonstrates for the first time the beneficial therapeutic effects of PD-L1 checkpoint blockade on MCAO, thus validating proposed mechanisms obtained in our previous studies using PD-1- and PD-L-deficient mice. These results provide strong support for the use of available humanized anti-PD-L1 antibodies for treatment of human stroke subjects.

RECOMBINANT T-CELL RECEPTOR LIGAND RTL1000 LIMITS INFLAMMATION AND DECREASES INFARCT SIZE AFTER EXPERIMENTAL ISCHEMIC STROKE IN MIDDLE-AGED MICE

We have previously demonstrated that recombinant T-cell receptor ligand 1000 (RTL1000) reduces infarct size and improves long-term functional recovery after experimental stroke in young transgenic mice expressing human leukocyte antigen DR2 (DR2-Tg). In this study, we determined the effect of RTL1000 on infarct size in 12-month-old middle-aged DR2-Tg mice, and investigated its mechanism of action. Twelve-month-old male DR2-Tg mice underwent 60min of intraluminal reversible middle cerebral artery occlusion (MCAO). Vehicle or RTL1000 was injected 4, 24, 48 and 72h after MCAO. Cortical, striatal and total hemispheric infarcts were measured 96h after stroke. Spleen and brain tissues were collected 96h after stroke for immunological analysis. Our data showed that RTL1000 significantly reduced infarct size 96h after MCAO in middle-aged male DR2-Tg mice. RTL1000 decreased the number of activated monocytes/microglia cells (CD11b(+)CD45(hi)) and CD3(+) T cells in the ischemic hemisphere. RTL1000 also reduced the percentage of total T cells and inflammatory neutrophils in the spleen. These findings suggest that RTL1000 protects against ischemic stroke in middle-aged male mice by limiting post-ischemic inflammation.

Targeting immune co-stimulatory effects of PD-L1 and PD-L2 might represent an effective therapeutic strategy in stroke

Stroke outcome is worsened by the infiltration of inflammatory immune cells into ischemic brains. Our recent study demonstrated that PD-L1- and to a lesser extent PD-L2-deficient mice had smaller brain infarcts and fewer brain-infiltrating cells vs. wild-type (WT) mice, suggesting a pathogenic role for PD-ligands in experimental stroke. We sought to ascertain PD-L1 and PD-L2-expressing cell types that affect T-cell activation, post-stroke in the context of other known co-stimulatory molecules. Thus, cells from male WT and PD-L-deficient mice undergoing 60 min of middle cerebral artery occlusion (MCAO) followed by 96 h of reperfusion were treated with neutralizing antibodies to study co-stimulatory and co-inhibitory interactions between CD80, cytotoxic T-lymphocyte antigen-4 (CTLA-4), PD-1, and PD-Ls that regulate CD8+ and CD4+ T-cell activation. We found that antibody neutralization of PD-1 and CTLA-4 signaling post-MCAO resulted in higher proliferation in WT CD8+ and CD4+ T-cells, confirming an inhibitory role of PD-1 and CTLA-4 on T-cell activation. Also, CD80/CD28 interactions played a prominent regulatory role for the CD8+ T-cells and the PD-1/PD-L2 interactions were dominant in controlling the CD4+ T-cell responses in WT mice after stroke. A suppressive phenotype in PD-L1-deficient mice was attributed to CD80/CTLA-4 and PD-1/PD-L2 interactions. PD-L2 was crucial in modulating CD4+ T-cell responses, whereas PD-L1 regulated both CD8+ and CD4+ T-cells. To establish the contribution of PD-L1 and PD-L2 on regulatory B-cells (Bregs), infarct volumes were evaluated in male PD-L1- and PD-L2-deficient mice receiving IL-10+ B-cells 4h post-MCAO. PD-L2- but not PD-L1-deficient recipients of IL-10+ B-cells had markedly reduced infarct volumes, indicating a regulatory role of PD-L2 on Bregs. These results imply that PD-L1 and PD-L2 differentially control induction of T- and Breg-cell responses after MCAO, thus suggesting that selective targeting of PD-L1 and PD-L2 might represent a valuable therapeutic strategy in stroke.