Abstract It has been proposed that homeostatic levels of estrogen can enhance female susceptibility to autoimmunity, whereas the heightened levels of estrogen associated with pregnancy are protective. This hypothesis was tested using the mouse model of experimental autoimmune encephalomyelitis (EAE). Diestrus (<100 pg/ml in serum) levels of 17β-estradiol were found to significantly reduce the clinical manifestations of active EAE in both male and female mice. Estriol was also effective but at doses below those previously established for pregnancy. The reduction in disease severity was accompanied by a coincident reduction in the number and size of inflammatory foci in the CNS of estrogen (17β-estradiol or estriol)-treated mice. Recipients of encephalitogenic T cells from low-dose estrogen-treated mice developed less severe paralysis than mice receiving T cells from placebo-treated mice. A modest shift in Th1/Th2 balance suggested that low dose estrogen therapy could bias the immune reaction toward a protective anti-inflammatory cytokine response. However, estrogen treatment at the onset of active EAE failed to reduce disease severity, a result that is consistent with the hypothesis that naive cells are more sensitive to sex hormones than differentiated effector cells. These data suggest that treatment with low doses of estrogen can reduce the capacity of developing myelin-reactive T cells to initiate disease and challenges the idea that increased susceptibility to autoimmunity in females is dependent on homeostatic levels of estrogen.
We have previously shown profound splenic activation by 6 hours following experimental stroke, resulting in rapid and widespread increased production of inflammatory factors by basal and activated splenocytes in male mice. Our lab and others have also shown a subsequent extensive loss of lymphocytes and increased lymphocyte apoptosis in spleen 12 hours after experimental stroke. Lastly, we and others have shown that splenic immunocytes can contribute to increased brain inflammation and infarct volume. However, it is unclear which splenic immunocyte populations may contribute to the evolving ischemic brain injury. Using a mouse splenectomy model and adoptive cell transfer protocols, we evaluated the effect of different splenic immunocyte populations, specifically CD4 and CD8 T lymphocytes, on infarct volume. Male C57BL/6J mice were splenectomized 14 days before experimental stroke and treated 24 hours before experimental stroke with either vehicle (saline), CD4 T lymphocytes, or CD8 T lymphocytes obtained from GFP reporter mice. Each mouse (n=8-10 per group) then underwent 60 minutes of middle cerebral artery occlusion via intraluminal filament. All mice were euthanized and brains collected at 96 hours of reperfusion. Infarct volume (% corrected contralateral structure) was determined by image analysis of coronal brain slices stained with 2,3,5-triphenyltetrazolium chloride. Adoptive transfer of GFP positive CD4 (cortex, 44±3%; striatum, 50±2%) or CD8 (cortex, 48±2%; striatum, 53±3%) T lymphocytes in splenectomized male mice did not alter infarct volume compared to vehicle-treated (cortex, 38±4%; striatum, 42±5%) splenectomized males (cortex, p=0.140; striatum, p=0.143). Although adding back CD4 or CD8 T lymphocytes did not appear to alter infarct volume, these immunocyte populations are comprised of both inflammatory and regulatory subtypes. Thus, future studies will evaluate the effect of different CD4 and CD8 subpopulations on ischemic brain outcomes. This study was supported by National Institutes of Health grant NS076013.
Freund's incomplete adjuvant (IFA), an aqueous/oil emulsion that is widely used in combination with antigenic proteins and peptides to induce tolerance, is considered to be immunologically inert. However, sporadic reports indicate that IFA may itself have inhibitory properties on induction of adjuvant induced arthritis and spontaneous diabetes. In the current study, the effects of IFA/saline were evaluated on the induction of experimental autoimmune encephalomyelitis (EAE) in three different strains of mice. IFA/saline given i.p. in two doses of > 100 microl 10 days apart were found to inhibit EAE induction to varying degrees in all three strains of mice in a dose dependent fashion. The IFA/saline injections inhibited both mitogen and antigen-induced T cell proliferation, induced elevated secretion of IFN-gamma and IL-10 by neuroantigen specific T cells, and reduced expression of cytokines, chemokines, and chemokine receptors of CNS-infiltrating mononuclear cells. These data demonstrate for the first time a direct inhibitory effect of IFA/saline on EAE, and re-emphasize the need to properly control experiments using IFA to induce antigen-specific tolerance.
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. 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.
We report that OX40 stimulation drives all lineages of CD4 T cell development, including regulatory T cells (Tregs), and the plasticity of the response is dependant on local cytokines. In TGF-beta1-treated cultures, an OX40 agonist increased IFN-gamma and IL-4 production and diverted T cells from the Treg lineage. However, cytokine blockade in the context of OX40 stimulation promoted enhanced Treg accumulation. This observation was evident in naive mice, as OX40 engagement enhanced Treg proliferation and accumulation in vivo. Lastly, OX40 agonist administration influenced experimental autoimmune encephalomyelitis disease severity in opposing directions, depending on the timing of administration. Given during Ag priming, the OX40 agonist drove Treg expansion and inhibited disease, whereas given later it enhanced T cell effector cytokine production in the CNS and exacerbated disease. Hence, OX40 signaling can augment the accumulation of all CD4 T cell lineages; however, its accentuation of immune responses may have vastly different biologic outcomes depending upon the local cytokine milieu.
The major goal of this study was to evaluate the efficacy and mechanism of a rTCR ligand (RTL) construct (I-A(s)/proteolipid protein (PLP)-139-151 peptide = RTL401) for treatment of SJL/J mice developing passive experimental autoimmune encephalomyelitis (EAE) that did not involve coimmunization with the highly inflammatory CFA. Our results demonstrated clearly that RTL401 was highly effective in treating passive EAE, with kinetics of recovery from disease very similar to treatment of actively induced EAE. The potent RTL401 treatment effect was reflected by a partial reduction of infiltrating mononuclear cells into CNS, minimal inflammatory lesions in spinal cord, and preservation of axons injured in vehicle-treated mice during the progression of EAE. Interestingly, in the absence of CFA, RTL401 treatment strongly enhanced production of the Th2 cytokine, IL-13, in spleen, blood, and spinal cord tissue, with variable effects on other Th1 and Th2 cytokines, and no significant effect on the Th3 cytokine, TGF-beta1, or on FoxP3 that is expressed by regulatory T cells. Moreover, pretreatment of PLP-139-151-specific T cells with RTL401 in vitro induced high levels of secreted IL-13, with lesser induction of other pro- and anti-inflammatory cytokines. Given the importance of IL-13 for protection against EAE, these data strongly implicate IL-13 as a dominant regulatory cytokine induced by RTL therapy. Pronounced IL-13 levels coupled with marked reduction in IL-6 levels secreted by PLP-specific T cells from blood after treatment of mice with RTL401 indicate that IL-13 and IL-6 may be useful markers for following effects of RTL therapy in future clinical trials in multiple sclerosis.
Abstract Clinical stroke induces inflammatory processes leading to cerebral injury. IL-10 expression is elevated during major CNS diseases and limits inflammation in the brain. Recent evidence demonstrated that absence of B cells led to larger infarct volumes and increased numbers of activated T cells, monocytes and microglial cells in the brain, thus implicating a regulatory role of B cell subpopulations in limiting CNS damage from stroke. The aim of this study was to determine whether the IL-10-producing regulatory B cell subset can limit CNS inflammation and reduce infarct volume following ischemic stroke in B-cell deficient (μMT-/-) mice. Five million IL-10-producing B cells were obtained from IL-10-GFP reporter mice and transferred i.v. to μMT-/- mice. After 24 h from this transfer, recipients were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by 48 h of reperfusion. Compared to vehicle-treated controls, the IL-10+ B-cell-replenished μMT-/- mice had reduced infarct volume and fewer infiltrating activated T cells and monocytes in the affected brain hemisphere. These effects in CNS were accompanied by significant increases in regulatory T cells and expression of the co-inhibitory receptor, PD-1, in the periphery. These novel observations demonstrate both immunoregulatory and protective functions of IL-10-secreting B cells in MCAO that potentially could impart significant benefit for stroke patients in the clinic.
Background: Recent evidence emphasizes the importance of T lymphocytes of differing subtypes in ischemic cell death mechanisms. RTL, composed of partial major histocompatibility complex (MHC) ClassIImolecules covalently bound to myelin peptides, is currently under clinical study for multiple sclerosis. We have shown that RTL is effective in male mice treated with middle cerebral artery occlusion (MCAO) by inhibiting early accumulation of inflammatory cells. We now show for the first time that post-treatment improves functional outcomes and that efficacy is present in females. Methods: HLA-DR2 transgenic female mice (17-20g) were subjected to 60 min MCAO (isoflurane). Cortical laser-Doppler flowmetry (LDF) was continuously monitored and only animals with LDF of below 40% of baseline during MCAO were included in study. Mice were treated with RTL1000 RTL 1000 (HLA-DR2 moiety linked to human MOG-35-55 peptide) (0.1mL, 1mg/mL sq) (RTL) or vehicle (VEH) at 3h, 24h, 48h, 72h after reperfusion. Mice were recovered for 96h or 2 weeks post-injury for measurement of histology (TTC staining) or behavioral testing respectively. Sham operated females received either vehicle or RTL and identical testing paradigms. Results: All sham mice showed no evidence of infarction or behavioral deficit. Mortality rate and general health score were similar between MCAO groups. RTL mice sustained smaller infarcts as compared to VEH group (% of contralateral structure): Total hemisphere: 13.9 ±4.1 % RTL (n=15) vs. 28.3 ±3.0% (p < 0.05) in VEH (n=15); cortex: 19.9 ±6.3 % in RTL vs. 44.2 ±5.0% (p < 0.05) in VEH; striatum 24.2 ±6.8% in RTL vs. 52.4 ± 4.7% (p < 0.05) in VEH. Histological protection was profound; 6 out of 15 in MCAO-RTL group showed no measurable infarction at 96 hours. Using a standard cylinder test repeated at 3, 7 and 14 days post MCAO, we observed that RTL strongly improved functional recovery as compared to MCAO-VEH group (RTL vs. VEH: day 3, p=0.02, day 7 p=0.00 and day 14 p=0.00). By 7days, animal performance in the RTL group was not different from shams. In contrast, recovery in the VEH group remained poor at all time points. Conclusion: RTL1000 offers strong protection after ischemic injury in females, including prolonged improvement of functional outcomes.
