that increased Olig2 expression compensates for the Olig1 deficiency. However, our data demonstrated that Olig2 expression remained unchanged in the optic nerve and brain, and in fact, it attenuated in the spinal cord inOlig1 2/2 mice (Figure 9). It will be intriguing to investigate other candidate genes that may be responsible for the survival of this Olig1 2/2 mouse line. One explanation for the unaltered myelin formation despite the altered level of MOG, MAG and MBP might be that the extent of reduction, which is subtle compared with the large reductions observed in the mice without the PGKneo cassette , was not enough to induce a significant change in the myelin formation during development. This speculation can be supported by the observation that not all myelin protein levels were affected; we have shown in this study that the expression levels of CNPase and PLP were unchanged in non-EAE adult Olig1 2/2 mice (Figure 8). If there was in fact less myelination in these mice, it would be logical to expect that they should be more susceptible to EAE induction. However, on the contrary, our results demon- strated that these mice had reduced EAE severity in the early phase of the experiment (Figure 1B). Hence, reduced expressions of MOG, MBP and MAG did not impair myelin formation during development, but contributed to alleviate EAE signs inOlig1 2/2 mice.
The total area of inflammatory infiltrate was estimated by examining the area of staining for macrophages / activated microglia, the most numerous immune cell types present in MOG- EAE white matter lesions. Areas of dense CD11b immunoreac- tivity were traced at x40 magnification and quantified as a proportion of the total spinal cord section area (Fig. 4a,b). There was no significant difference (p.0.05) between EAE-affected wild- type and EphA4 knockout micein the mean area of CD11b+ lesions as a proportion of spinal cord area (Fig. 4c). In both genotypes, there was a significant correlation (wild-type, p = 0.024; EphA4 knockout, p = 0.004) between the proportional area of CD11b+ lesions and the highest clinical score reached by a given mouse (Fig. 4d). Within CD11b+ white matter lesions, the density of macrophages / activated microglia was quantified. There was no difference between genotypes in the density of CD11b+ cells within lesion areas (Fig. 4e). There was a significant correlation between the density of CD11b+ cells and the highest clinical grade reached in EphA4 knockout mice (p = 0.03) and in wild-type mice (p = 0.05) (Fig. 4f). Spinal cord sections were also double labelled with CD11b and the subtype marker ARG1, which we found to be differentially expressed in wildtype and EphA4 knockout spinal cord following spinal cord injury  and which has also been shown to modulate EAE clinical severity . A subset of CD11b+ cells displayed ARG1 immunoreactivity in EAE lesions in both genotypes but there were no significant differences (p.0.05) (data not shown).
Mice (N = 20) were observed daily for clinical signs of disease up to 60 days after immunization. Mice were scored according to their clinical severity as follows: grade 0, no abnormality; grade 0.5, partial loss of tail tonicity, assessed by inability to curl the distal end of the tail; grade 1, reduced tail tone of slightly clumsy; grade 2, tail atony, moderately clumsy gait, impaired righting ability, or any combination; grade 3, hind limb weakness of partial paralysis; grade 4, complete hind limb paralysis or fore limb weakness; grade 5, tetraplegia or moribund state; grade 6, death (7). The data were plot- ted as daily mean clinical score for all ani- mals in a particular treatment group. Several parameters of disease were examined to evaluate the severity of EAE and the effi- cacy of BA therapy that include mean clini- cal score, incidence, mean day ofonset, peak clinical score, and cumulative disease index (8).
endogenous Foxp3 expression, 2–3 days prior to EAE induction with MBP Ac1–9 in CFA. In both scenarios, iTreg cells delayed disease progression (Figures 1A and 1B), demonstrating that iTreg cells mediate immune regulation independent of tTreg cells. In order to examine whether antigen-specificity plays a role in disease protection, Tg4 Rag 2/2 mice, 5–8 weeks of age, received 5610 6 iTreg cells from either Tg4 or non-transgenic B10.PL mice 3 days prior to disease induction with MBP Ac1–9 in CFA. Whereas there appeared to be a minor benefit from the administration of polyclonal B10.PL iTreg cells, this only reached statistical significance when protection was offered by antigen-specific Tg4 iTreg cells (Figure 1C). This effect was further reflected inin vitro suppression assays (Figure 1D). These results are reminiscent of those achieved previously in a PLP-specific system , although polyclonal iTreg cells were reported effective in protection against induction of EAE using MOG 35–55 in a non-transgenic model .
(thymus) (Fig. S2A,B). In inducible Sgpl1-deficientmice, analyzed two weeks after induction of gene deletion, S1P in the tissues was elevated to a lesser degree, ranging from 4-fold (heart) to 100-fold (lymph nodes and spleen) (Fig. 2A,B). In both mouse strains, there was no statistically significant increase of S1P in the brain and spinal cord. Concentrations of S1P in the blood of the inducible Sgpl1-deficientmice showed a moderate elevation (factor of 1.6), while plasma concentrations were not increased at all (Fig. 2A). S1P concentrations in inducible Sgpl1-deficientmice were similar at early time points and 6 months after induction (data not shown). Sph was increased more strongly in constitutive Sgpl1-deficientmice (up to 200-fold; Fig. S2C,D) than in inducible Sgpl1-deficientmice (up to 16-fold; Fig. 2C,D). Furthermore, while C16-ceramide was elevated in the constitutive Sgpl1-deficientmice up to 9-fold in selected tissues (Fig. S2E), there was only a trend towards C16- ceramide increase in the inducible Sgpl1-deficientmice (Fig. 2E). In summary, inducible Sgpl1-deficientmice feature less pro- nounced increase of sphingolipid metabolites as compared to the constitutive KO mice.
Gallium/Aluminum/Arsenide (GaAlA) LED arrays (75 cm 2 ) of 670 nm (LED bandwidth 25–30 nm at Full Width Max Power [FWHM]) were obtained from Quantum Devices (Barneveld, WI). Unanesthetized Mice were placed individually into a polypropyl- ene restraint device (12.76967.6 cm), and the LED array was positioned directly over the animal at a distance of 2 cm, covering the entire chamber and exposing the entire dorsal surface. Treatment consisted of once daily irradiation at 670 nm for 3 min, at a power intensity of 28 mW/cm 2 (total power output: 2100 mW) and an energy density of 5 J/cm 2 (375 J daily total). To determine the energy density at the level of the spinal cord, a small incision was made at the base of the tail, and a probe was inserted underneath the skin at the base of the brain, demonstrating an energy density of 12 mJ/cm 2 (432 mJ total energy administered daily) at the level of the spinal cord. As indicated, ‘‘restraint only’’ stress was employed, in which mice were placed in the restraint device for 3 min but not exposed to light. The Suppression Protocol consisted of once daily treatment for 10 consecutive days starting 24 h post immunization, resulting in a total of 4320 J at the level of the spinal cord administered to the animal over the course of treatment. Treatment protocols were performed as the ‘‘Onset Protocol’’, consisting of once daily treatment for 7 consecutive days beginning the day ofonsetof clinical signs (clinical score = 1.0; 3024 J total energy administered), and the ‘‘Double Treatment Protocol’’, consisting of once daily treatments initiated on the day ofonsetof clinical signs for 7 days, followed by 7 days rest, and a subsequent 7 day treatment period (6048 J total energy administered). Clinical disease was followed for an additional 7 days following cessation of the second treatment period.
immunomodulatory effects elicited by the enzymatic activity of indoleamine 2,3-dioxygenase (IDO), which promotes tryptophan starvation due to its catabolism into kynurenine. In fact, it was al- ready demonstrated that IDO-expressing plasmacytoid dendritic cells (pDCs) activate the GCN2 kinase pathway in T cells suppress- ing their proliferation upon TCR stimulation (Munn et al., 2005). Moreover, IDO induced profound anergy in responding WT T cells, but GCN2-knockout cells were refractory to IDO-induced anergy (Munn et al., 2005). In murine CD8+ T cells, IDO activity seems to result in GCN2 kinase-dependent down-regulation of the TCR zeta-chain. TCR zeta down-regulation could be demonstrated in vivo and was associated with an impaired cytotoxic effector function in vitro (Fallarino et al., 2006). Tryptophan catabolism has also been shown to induce apoptosis in thymocytes and Th1 cells (Fallarino et al., 2002). Recently, GCN2-dependent activation of the amino acid starvation response was found to inhibit mouse and human Th17 differentiation (Sundrud et al., 2009). Besides dampening the response of effector T cells, IDO-induced regulatory mechanisms also involve de novo generation/expansion of Foxp3+ regulatory T cells (Baban et al., 2009; Matteoli et al., 2010). Hence, it has been demonstrated that IDO-expressing DCs can induce Foxp3 expression when co-cultivated with Foxp3 CD4+CD25 T Fig. 4. Absence of the remission phase in GCN2-deficient animals is associated with lower frequency of Foxp3+ T cells within the CNS. Relative expression of (A) Foxp3 and (B) IL-10 in total extracts of spinal cords from WT and GCN2 KO mice at the EAE remission phase (21 days post-immunization). Representative plots showing the frequency of CNS-infiltrating Foxp3 GFP+ CD4+ T (Treg) cells from (C) WT Foxp3 GFP and (D) GCN2 KO Foxp3 GFP mice at the EAE remission phase (21 days post-immunization). (E) Statistical
Heat shock protein (Hsp)70 is one of the most important stress-inducible proteins. Intracellular Hsp70 not only mediates chaperone-cytoprotective functions but can also block multiple steps in the apoptosis pathway. In addition, Hsp70 is actively released into the extracellular milieu, thereby promoting innate and adaptive immune responses. Thus, Hsp70 may be a critical molecule in multiple sclerosis (MS) pathogenesis and a potential target in this disease due to its immunological and cytoprotective functions. To investigate the role of Hsp70 in MS pathogenesis, we examined its immune and cytoprotective roles using both in vitro and in vivo experimental procedures. We found that Hsp70.1-deficientmice were more resistant to developing experimentalautoimmuneencephalomyelitis (EAE) compared with their wild-type (WT) littermates, suggesting that Hsp70.1 plays a critical role in promoting an effective myelin oligodendrocyte glycoprotein (MOG)-specific T cell response. Conversely, Hsp70.1-deficientmice that developed EAE showed an increased level of autoreactive T cells to achieve the same production of cytokines compared with the WT mice. Although a neuroprotective role of HSP70 has been suggested, Hsp70.1-deficientmice that developed EAE did not exhibit increased demyelination compared with the control mice. Accordingly, Hsp70 deficiency did not influence the vulnerability to apoptosis of oligodendrocyte precursor cells (OPCs) in culture. Thus, the immunological role of Hsp70 may be relevant in EAE, and specific therapies down-regulating Hsp70 expression may be a promising approach to reduce the early autoimmune response in MS patients.
It is of recent concern that regulatory T cells may turn into effector inflammatory cells. It was found that natural arising and periphery induced Treg cells may become Th1 and Th17 cells in vivo and in vitro [54–57]. The events that lead to this conversion are based on the stimulation of the mTOR cascade, which induces the differentiation of Th1 and Th17 cells in inflammatory and lymphopenic conditions . We did not observe this effect in the treatment of ongoing EAE. In fact, our results show that regulatory T cells raised by the CQ treatment were not converted into effector T cells, even at the 30 th day after disease onset, as seen by the augmentation of Foxp3 expression and the reduction in IFN-c production. So, the treatment with chloroquine of established EAE resulted in reduction of EAE suggesting that a long-lasting immunomodulation can be achieved with this therapy. When CQ-elicited Treg cells are transferred to mice with ongoing EAE, the disease severity was reduced. The cellular response towards neuro-antigens in the periphery was contained and the pattern secretion of cytokines was altered as well. Transfer of CQ-Treg cells also reduced the infiltration of cell into the CNS, although the frequency of IL-10-producing cells was unaltered, which is distinct from the data observed with CQ treatment. The reduced dendritic cells number after CQ therapy may favor the higher suppression profile of CQ treatment over CQ-Treg cells transfer experiments. These data indicate that the amelioration of EAE after CQ treatment is a result of Treg-dependent and - independent mechanisms.
Though protoporphyrin treatment could restore antigen-driven expansion and tissue migration of chronical-stimulated miR-155 -/- T cells, it did not improve the capacity of T cells to produce effector cytokines, such as IFN-γ . In the adoptive transfer model, the percentage of Th1 cell was similar regardless of ZnPP treatment. However, the absolute number of Th1 cells was increased in ZnPP-treated group and the increased severity of the disease in miR- 155 -/- animals treated with the HO-1 inhibitor was caused by an enhanced T cell infiltration into the brain. Thus, miR-155-dependent regulation of HO-1 function primarily would appear to control T cell proliferation and migration to sites of inflammation. Interestingly, it has been well demonstrated that mitochondrial reactive oxygen species (mROS) signaling is required in T cells for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 produc- tion . Since lack of HO-1 induces mitochondrial oxidative stress and ROS production , one could hypothesize that miR-155-mediated control of HO-1 expression in T cells would be essential for IL-2-dependent proliferation and expansion following activation by antigen. This hypothesis is reinforced by the observation that HO-1 inhibition in T cells stimulates IL-2 pro- duction [8, 20].
Although the potential role of the kinin system on leuko- cyte entry into the CNS in MS remains unclear, it has been shown that BK can interfere with the mechanism of leuko- cyte recruitment in various tissues . BK may enhance the expression of adhesion molecules on endothelium cells . BK antagonists reduce leukocyte-endothelium interactions after diverse inflammatory conditions, including global cerebral ischemia , leukocyte infiltra- tion in murine mesenteric post-capillary venules  and lung inflammation in guinea pigs . BK could poten- tially modify leukocyte recruitment by production of che- moattractant molecules, such as chemokines. For example, treatment with bradykinin receptor antagonists has been shown to reduce production of chemokines, including KC and MCP-1, after intestinal ischemia and reperfusion . Several studies, have clearly demon- strated the relevance of chemokines for the recruitment of leukocytes into the brain of EAE mice [16-18].
Funding: This work was supported financially by the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and The Academy of Sciences for the Developing World (TWAS; Grant # 05-049 RG/BIO/LA), The National Institute for Vaccine Development and Technology (CNPq-INCTV) and Rede de Malaria (CNPq). ASF was supported by a FAPESP fellowship (#2009/15620-9). RLT, YCB and ALFL received a Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) fellowship and SCPL was supported by CNPq. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
MAPK and NF-kB pathways of the host . These pathways, although initially activated by exposure to the parasite, become silenced when the infection is firmly established, rendering the cell unresponsive to further stimulation with LPS . This silencing has been attributed to different virulence factors, including surface phosphoglycans [16,62], the metalloprotease GP63 , and cysteine proteases from L. mexicana . It is possible that in the absence of Ctsb, one or more of these key signaling pathways are no longer silenced by L. major promastigotes. This would open a range of new questions regarding the involvement of Ctsb in L. major infection, e.g., whether Ctsb interacts directly with the parasites, contributing to processing or activation of one or more virulence factors, or whether Ctsb directly interferes with any intermediate of key signaling pathways, such as NF-kB and MAPK. While Ctsb 2/2 BMDC and BMM were able to up- regulate IL-12 in response to L. major and LPS, IL-6 was not regulated. IL-6 transcription has been shown to depend on NF-kB . Therefore, we hypothesize that the molecular mechanism behind the involvement of Ctsb 2/2 in the expression of IL-12 would not be shared by IL-6. Our results suggest that the regulation of IL-12 expression by Ctsb is not NF-kB-dependent, although further work is necessary to confirm this observation and to explore the interaction of Ctsb with other candidate signaling pathways. In addition, finding the location of these interactions would be a key to further understand the mechanisms underlying these processes, e.g., whether proteolytic processing of signaling intermediates takes place in the cytoplasm, or whether cleavage of transcription factors by Ctsb occurs in the nuclear space, as described in thyroid carcinoma cells .
The role of DNA repair in hematopoietic homeostasis has become increasingly clear in recent years [20–23]. Knockout models of many DNA repair enzymes are characterized by either reduced stem cell function or alterations in specific hematopoietic populations. Moreover, since stem cells are protected from apoptosis [20,21], probably because of their quiescent state , deficiency in DNA repair enzymes allows mutations to accumulate in these cells. Conversely, more committed progenitors, which cycle more rapidly, would be more sensitive to cell cycle arrest or apoptosis, thus explaining the observed reduction in the numbers of progenitor cells in Polm-deficientmice. Thus the survival of HSC comes at the price of accumulated mutations; two recent reports show that micedeficientin DNA repair maintain expanded stem cell pools, but that these pools have a reduced differentiation potential during aging [20,21].
MLN single cell suspensions were obtained by mechanical disruption through a 40 m M cell strainer (BD Biosciences). Colon LP lymphocytes were isolated as previously described . Briefly, colon tissue was cut into 0.5 cm pieces and incubated at 37 uC for 30 min. in PBS containing 0.5% BSA, 2% HEPES, 1% NaPyruvate and 10 mM EDTA to remove epithelial cells. The remaining tissue was further digested in complete IMDM medium containing 10% FCS and 1.5 mg Collagenase VIII (Sigma Aldrich) for 20 min. at 37 uC and then smashed through a cell strainer. Cells were stained with surface antibodies diluted in PBS with 0.5% BSA (Sigma Aldrich). For intracellular staining cells were fixed in BD lysis buffer (BD Biosciences), permeabilized using 0.5% Saponin (Sigma Aldrich) in 0.5% BSA/PBS and stained with intracellular antibodies in 0.5% Saponin in 0.5% BSA/PBS. For the analysis of cytokine production by intracellular staining cells were first stimulated with PMA (Sigma-Aldrich) and ionomycin (Sigma-Aldrich) for 4 h at 37 uC in IMDM medium plus 7% FCS. For the final two hours, Brefeldin A (10 m g/ml) was added to the cultures to retain cytokines in the cytoplasm. Stained cells were analyzed on FACS Calibur (BD Bioscience) or Cyan (Dako Cytomation) flow cytometers using FlowJo software (Tree Star). Fluorescently conjugated mAbs directed against CD4 (L3T4), CD25 (PC61), CD45RB (C363-16A), CD45.1 (A20), Ki- 67 (MOPC-21), IL-17A (TC11), IFNc (XMG1.2) and Foxp3 (FJK-16s) were purchased from eBiosciences.
Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterised by demyelination, T cell and macrophage infiltration and a decrease in neurological function (Adams et al. 1989). Experimentalautoimmuneencephalomyelitis (EAE) is currently considered the best available model for human MS. As such, much of our current understanding of MS originates from studies of EAE (Raine 1984, Martin & McFarland 1995). EAE is most commonly induced by the active immunisation of animals with proteins from CNS, such as myelin proteolipid protein, myelin basic protein (MBP) or myelin oligodendrocyte glycoprotein, administered in complete Freund’s adjuvant (CFA). Re- cent findings indicate that different cell types, including Th1, Th17, cytotoxic T cells, B cells and regulatory T cells are involved in the inflammatory process of both MS and EAE (Holmøy & Hestvik 2008). Experimental evidence indicates that cytokines, such as TNF-α, IL-6, IL-1β and IFN-γ, play a relevant role in the pathophysiol- ogy of both MS and EAE (Mustafa et al. 1991, Imitola et al. 2005). In the brains of MS patients and EAE rats, TNF-α and IFN-γ are expressed by astrocytes, microglia and infiltrating monocytes and macrophages (Merril & Benveniste 1996, Stanislaus et al. 1999). Moreover, anti- TNF-α antibodies provide protection against primary
The findings of the present study, therefore, tend to suggest that D. triflorum aqueous and ethanolic extracts have anticonvulsant activity might have inhibited and/or attenuated PTZ-induced seizures of the mice used by enhancing, or in some ways interfering with GABAergic neurotransmission. A dose of 400 and 800 mg/kg of EEDT significantly delayed the onsetof convulsion and significantly reduced duration of action convulsion inmice against INH induced convulsion. A dose of EEDT 800 mg/kg showed 33.33% protection inmice against INH induced convulsion. The standard anticonvulsant drug, diazepam 10 mg/kg i.p. totally abolished the effects of INH induced convulsion inmice. It is well known that one of the factor responsible for isoniazid-induced convulsions is the decrease of GABA below a critical level in some neurons. Isoniazid was shown to lower the content of brain GABA to approximately the same extent in rats and mice (Pieri & Biry, 1985). Perhaps, the decrease in the amount of GABA stored presynaptically causes a reduction in the amount of GABA released by nerve impulses. Hence, the GABA receptors are regulated at the level of maximal sensitivity in order to maximize the action of GABA. Diazepam exhibits a marked protective effect against isoniazid-induced convulsions inmice (Corda et al., 1982). INH induced convulsion inmice significantly delayed the onsetof seizure, reduced the duration of action and suggesting that D. triflorum DC extracts have a lesser protector effect inmice. The results suggested that AEDT and EEDT indicate weak anticonvulsant activity as compared to the PTZ and MES convulsion models.
Immunohistochemistry and Fluorescence Microscopy Mice were terminally anesthetized and subjected to whole body perfusion with 4% paraformaldehyde in phosphate-buffered saline (PBS). Whole eyes were enucleated and post-fixed in the same fixative overnight before embedded in Tissue-TeK Cryomold Figure 7. Schematic model integrating oxidative stress, autophagy and lysosomal function into the etiology of AMD. RPE cells are exposed to high levels of cellular stress and, when healthy, damaged proteins and organelles are removed promptly by autophagy. Under disease conditions, such as decreased antioxidant defense and lysosome inhibition, self-renewal by autophagy becomes less efficient. The resulted cellular waste products can be exported by exocytosis and contribute to sub-RPE deposit and drusen formation. NRF2 can be involved in regulating both the antioxidant responses and the autophagic activities.
he analysis of IFN- � and IL-10 levels, produced by peripheral lymphoid organs, answered some mechanistic questions. We initially asked how BCG decreased inlamma- tion in the brain. In this sense, the most interesting inding was the accentuated drop of IFN- � and IL-10 levels induced by MBP, in EAE experimental groups previously vaccinated, in comparison to the EAE control group. A possible expla- nation for this inding could be the trapping of nervous tissue-speciic T cells in peripheral BCG inlammatory sites as elegantly demonstrated by Sewell et al. in 2003 . Alter- natively, these autoreactive T cells could be in lower numbers due to an apoptotic process occurring in the periphery. his phenomenon was clearly demonstrated by O’Connor et al. in 2005 . hese authors detected high levels of apop- tosis among activated CD4+ T cells in BCG experimental infection. Interestingly and concerning to the model used by us, these authors also described that the high apoptotic degree occurred simultaneously with a milder experimentalencephalomyelitis course. Even though we did not observed improvement in clinical parameters, a signiicant drop in brain inlammation was detected. his lower IFN- � and IL-10 production could also be linked to the migration of myelin-speciic T clones to the brain, where they could exert the detected anti-inlammatory activity. IFN- � is described as able to shape the immune iniltration of the CNS by controlling chemokine expression. In addition, this cytokine accentuates apoptosis of iniltrating encephalitogenic T cell clones [52, 53]. Additionally, the production of IL-10 by Tr1 regulatory cells has been widely accepted as one of the mech- anisms responsible for MS and EAE downregulation .
Idd5.4 interval . Notably, five genes (Ugtla10, Glrp1, Ramp1, Stk25, Ralb) localize within our newly defined Idd5.4 interval (Fig 2) and were differentially expressed between activated CD4 + T cells from NOD and congenic mice. Cd55 (Daf1) was also identified as a promising candidate gene in their study using B10-derived congenic intervals for Idd5 sub-loci . In contrast, our B6-derived congenic intervals appear to eliminate Cd55 from consideration be- cause it does not localize within the congenic intervals for NOD.B6-Chr1 R2 or NOD.B6-Chr1 R7 (Fig 2). It is, however, possible that B10 and B6 mice harbor different sequence for this or other genes within the larger B10-defined Idd5.4 interval, which alters diabetes incidence when placed on the NOD genetic background. New congenic mouse strains with smaller congenic intervals, combined with a haplotype analysis approach , will be needed to further localize and refine the list of candidate genes for Idd5.4, as well as determine if B6 and B10 harbor the same diabetogenic allele for this susceptibility locus.