Interferon-beta induces major histocompatibility complex of class I (MHC-I) expression and a proinflammatory phenotype in cultivated human astrocytes.

Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.

Granulocyte-macrophage colony-stimulating factor improves mouse peripheral nerve regeneration following sciatic nerve crush.

Peripheral nerve injuries severely impair patients' quality of life as full recovery is seldom achieved. Upon axonal disruption, the distal nerve stump undergoes fragmentation, and myelin breaks down; the subsequent regeneration progression is dependent on cell debris removal. In addition to tissue clearance, macrophages release angiogenic and neurotrophic factors that contribute to axon growth. Based on the importance of macrophages for nerve regeneration, especially during the initial response to injury, we treated mice with granulocyte-macrophage colony-stimulating factor (GM-CSF) at various intervals after sciatic nerve crushing. Sciatic nerves were histologically analyzed at different time intervals after injury for the presence of macrophages and indicators of regeneration. Functional recovery was followed by an automated walking track test. We found that GM-CSF potentiated early axon growth, as indicated by the enhanced expression of growth-associated protein at 7 days postinjury. Inducible nitric oxide synthase expression increased at the beginning and at the end of the regenerative process, suggesting that nitric oxide is involved in axon growth and pruning. As expected, GM-CSF treatment stimulated macrophage infiltration, which increased at 7 and 14 days; however, it did not improve myelin clearance. Instead, GM-CSF stimulated early brain-derived neurotrophic factor (BDNF) production, which peaked at 7 days. Locomotor recovery pattern was not improved by GM-CSF treatment. The present results suggest that GM-CSF may have beneficial effects on early axonal regeneration.

Dendritic cells treated with chloroquine modulate experimental autoimmune encephalomyelitis.

Chloroquine (CQ), an antimalarial drug, has been shown to modulate the immune system and reduce the severity of experimental autoimmune encephalomyelitis (EAE). The mechanisms of disease suppression are dependent on regulatory T cell induction, although Tregs-independent mechanisms exist. We aimed to evaluate whether CQ is capable to modulate bone marrow-derived dendritic cells (DCs) both phenotypically and functionally as well as whether transfer of CQ-modulated DCs reduces EAE course. Our results show that CQ-treated DCs presented altered ultrastructure morphology and lower expression of molecules involved in antigen presentation. Consequently, T cell proliferation was diminished in coculture experiments. When transferred into EAE mice, DC-CQ was able to reduce the clinical manifestation of the disease through the modulation of the immune response against neuroantigens. The data presented herein indicate that chloroquine-mediated modulation of the immune system is achieved by a direct effect on DCs and that DC-CQ adoptive transfer may be a promising approach for avoiding drug toxicity.

Isolated Peptide from Spider Venom Modulates Dendritic Cells In Vitro: A Possible Application in Oncoimmunotherapy for Glioblastoma.

Dendritic cells (DCs) vaccine is a potential tool for oncoimmunotherapy. However, it is known that this therapeutic strategy has failed in solid tumors, making the development of immunoadjuvants highly relevant. Recently, we demonstrated that Phoneutria nigriventer spider venom (PnV) components are cytotoxic to glioblastoma (GB) and activate macrophages for an antitumor profile. However, the effects of these molecules on the adaptive immune response have not yet been evaluated. This work aimed to test PnV and its purified fractions in DCs in vitro. For this purpose, bone marrow precursors were collected from male C57BL6 mice, differentiated into DCs and treated with venom or PnV-isolated fractions (F1-molecules < 3 kDa, F2-3 to 10 kDa and F3->10 kDa), with or without costimulation with human GB lysate. The results showed that mainly F1 was able to activate DCs, increasing the activation-dependent surface marker (CD86) and cytokine release (IL-1ß, TNF-α), in addition to inducing a typical morphology of mature DCs. From the F1 purification, a molecule named LW9 was the most effective, and mass spectrometry showed it to be a peptide. The present findings suggest that this molecule could be an immunoadjuvant with possible application in DC vaccines for the treatment of GB.

The Time Course of MHC-I Expression in C57BL/6J and A/J Mice Correlates with the Degree of Retrograde Gliosis in the Spinal Cord following Sciatic Nerve Crush.

The pleiotropic role of the major histocompatibility complex class I (MHC-I) reflects the close association between the nervous and immune systems. In turn, MHC-I upregulation postinjury is associated with a better regenerative outcome in isogenic mice following peripheral nerve damage. In the present work, we compared the time course of neuronal, glial, and sensorimotor recovery (1, 3, 5, 7, and 28 days after lesion­dal) following unilateral sciatic nerve crush in A/J and C57BL/6J mice. The A/J strain showed higher expression of MHC-I (7 dal, ** p < 0.01), Iba-1 (microglial reaction, 7 dal, *** p < 0.001), and GFAP (astrogliosis, 5 dal, * p < 0.05) than the C57BL/6J counterpart. Synaptic coverage (synaptophysin) was equivalent in both strains over time. In addition, mRNA expression of microdissected spinal motoneurons revealed an increase in cytoskeleton-associated molecules (cofilin, shp2, and crmp2, * p < 0.05), but not trkB, in C57BL/6J mice. Gait recovery, studied by the sciatic functional index, was faster in the A/J strain, despite the equivalent results of C57BL/6J at 28 days after injury. A similar recovery was also seen for the nociceptive threshold (von Frey test). Interestingly, when evaluating proprioceptive recovery, C57BL/6J animals showed an enlarged base of support, indicating abnormal ambulation postinjury. Overall, the present results reinforce the role of MHC-I expression in the plasticity of the nervous system following axotomy, which in turn correlates with the variable recovery capacity among strains of mice.

Components from spider venom activate macrophages against glioblastoma cells: new potential adjuvants for anticancer immunotherapy.

Immunomodulation has been considered an important approach in the treatment of malignant tumours. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumour size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated 23 subfractions named low weight (LW-1 to LW-12) and high weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumour cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer.

Neurodegeneration and increased production of nitrotyrosine, nitric oxide synthase, IFN-gamma and S100beta protein in the spinal cord of IL-12p40-deficient mice infected with Trypanosoma cruzi.

BACKGROUND/AIM: Chagas' disease is caused by Trypanosoma cruzi and occurs in most Latin American countries. The protozoan may colonize the central nervous system (CNS) of immune-compromised human hosts, thus causing neuronal disorders. Systemic control of the intracellular forms of the parasite greatly depends on the establishment of a TH1 response and subsequent nitric oxide (NO) release. At the CNS, it is known that low concentrations of NO promote neuronal survival and growth, while high concentrations exert toxic effects and neuron death. Accounting for NO production by astrocytes is the glia-derived factor S100beta, which is overproduced in some neurodegenerative diseases. In the current work, we studied the expression of NO, interferon (IFN)-gamma and S100beta in the spinal cord tissue of IL-12p40KO mice infected with T. cruzi, a model of neurodegenerative process. METHODS: IL-12p40KO and wild-type (WT) female mice infected with T. cruzi Sylvio X10/4 (10(5) trypomastigotes, intraperitoneally) were euthanized when IL-12p40KO individuals presented limb paralysis. Spinal cord sections were submitted to immunohistochemical procedures for localization of neurofilament, laminin, nitrotyrosine, NO synthases (NOS), IFN-gamma and S100beta. The total number of neurons was estimated by stereological analysis and the area and intensity of immunoreactivities were assessed by microdensitometric/morphometric image analysis. RESULTS: No lesion was found in the spinal cord sections of WT mice, while morphological disarrangements, many inflammatory foci, enlarged vessels, amastigote nests and dying neurons were seen at various levels of IL-12p40KO spinal cord. Compared to WT mice, IL-12p40KO mice presented a decrement on total number of neurons (46.4%, p < 0.05) and showed increased values of immunoreactive area for nitrotyrosine (239%, p < 0.01) and NOS (544%, p < 0.001). Moreover, the intensity of nitrotyrosine (16%, p < 0.01), NOS (38%, p < 0.05) and S100beta (21%, p < 0.001) immunoreactivities were also augmented. No IFN-gamma-labeled cells were seen in WT spinal cord tissue, contrary to IL-12p40KO tissue that displayed inflammatory infiltrating cells and also some parenchymal cells positively labeled. CONCLUSION: We suggest that overproduction of NO may account for neuronal death at the spinal cord of T. cruzi-infected IL-12p40KO mice and that IFN-gamma and S100beta may contribute to NOS activation in the absence of IL-12.

Improved mouse sciatic nerve regeneration following lymphocyte cell therapy.

Traumatic injury to the peripheral nervous system (PNS) is the most common cause of acquired nerve damage and impairs the quality of life of patients. The success of nerve regeneration depends on distal stump degeneration, tissue clearance and remodeling, processes in which the immune system participates. We previously reported improved motor recovery in sciatic nerve crush mice following adoptive transfer of lymphocytes, which migrated to the lesion site. However, lymphocyte activity and the nerve tissue response remain unexplored. Thus, in the present study, we evaluated sciatic nerve regeneration and T cell polarization in lymphocyte recipient mice. Splenic lymphocytes were isolated from mice 14 days after sciatic nerve crush and transferred to axotomized animals three days postinjury. Immediate lymphocyte migration to the crushed nerve was confirmed by in vivo imaging. Phenotyping of T helper (Th) cells by flow cytometry revealed an increased frequency of the proinflammatory Th1 and Th17 cell subsets in recipient mice at 7 days and showed that the frequency of these cells remained unchanged for up to 21 days. Moreover, nerve regeneration was improved upon cell therapy, as shown by sustained immunolabeling of axons, Schwann cells, growth-associated protein 43 and BDNF from 14 to 28 days after lesion. Macrophage and IgG immunolabeling were also higher in cell-transferred mice at 14 and 21 days following nerve crush. Functionally, we observed better sensory recovery in the lymphocyte-treated group. Overall, our data demonstrate that enhanced inflammation early after nerve injury has beneficial effects for the regenerative process, improving tissue clearance and axonal regrowth towards the target organs.

Immunomodulation by dimethyl fumarate treatment improves mouse sciatic nerve regeneration.

Traumatic injury to the peripheral nervous system (PNS) often generates sensorimotor deficits that impair the quality of life of the patient. The success of nerve regeneration is related to tissue clearance and the formation of a microenvironment that sustains and stimulates axon growth up to the target. In this sense, macrophages are important for axon and myelin debris removal, neovascularization and the production of neurotrophic factors. Macrophage activation is improved by T helper (Th) lymphocytes, whose role remains few explored upon traumatic nerve injuries. Dimethyl fumarate (DMF) is the first-line drug for the treatment of multiple sclerosis due to its neuroprotective, anti-inflammatory and immunomodulatory properties. DMF improves nerve regeneration via antioxidant and cytoprotective cell signaling pathways. However, the direct activity on the cell immune response following nerve axotomy requires further investigation. In the present study, we evaluated DMF activity on Th cells and macrophage polarization, axonal regeneration and motor recovery following sciatic nerve crush in mice. For this aim, operated animals received DMF or vehicle once a day, starting at 3 days postinjury (dpi). Using an in vivo cell migration assay, we observed reduced lymphocyte infiltration in the nerves of DMF-treated mice at 7 dpi. Flow cytometry revealed DMF-responsive lymphocyte polarization from the pro- (Th1) to anti-inflammatory (Th2) phenotype at 7 dpi but not at 14 dpi. No effect was observed on macrophage polarization (from M1 to M2), although DMF reduced the frequency of the proinflammatory M1 subset from 7 to 14 dpi. Quantification of neurofilament (axon marker) and growth-associated protein 43 (GAP-43) immunolabeling showed improved axonal regeneration under DMF treatment at 14 dpi. Better motor recovery was observed in the DMF-treated group, as verified by an automated walking track test. Overall, our data reinforce the pro-regenerative capacity of DMF after traumatic nerve injury based on downmodulation of the proinflammatory immune response.

Inhibitory effect of red LED irradiation on fibroblasts and co-culture of adipose-derived mesenchymal stem cells.

The objective of this study was to evaluate the effects of red Light Emiting Diode (red LED) irradiation on fibroblasts in adipose-derived mesenchymal stem cells (ASC) co-culture on the scratch assay. We hypothesized that red LED irradiation could stimulate paracrine secretion of ASC, contributing to the activation of genes and molecules involved in cell migration and tissue repair. ASC were co-cultured with NIH/3T3 fibroblasts through direct contact and subjected to red LED irradiation (1.45 J/cm2/5min6s) after the scratch assay, during 4 days. Four groups were established: fibroblasts (F), fibroblasts + LED (FL), fibroblasts + ASC (FC) and fibroblasts + LED + ASC (FLC). The analyzes were based on Ctgf and Reck expression, quantification of collagen types I and III, tenomodulin, VEGF, TGF-ß1, MMP-2 and MMP-9, as well as viability analysis and cell migration. Higher Ctgf expression was observed in FC compared to F. Group FC presented higher amount of tenomodulin and VEGF in relation to the other groups. In the cell migration analysis, a higher number of cells was observed in the scratched area of the FC group on the 4th day. There were no differences between groups considering cell viability, Reck expression, amount of collagen types I and III, MMP-2 and TGF-ß1, whereas TGF-ß1 was not detected in the FC group and the MMP-9 in none of the groups. Our hypothesis was not supported by the results because the red LED irradiation decreased the healing response of ASC. An inhibitory effect of the LED irradiation associated with ASC co-culture was observed with reduction of the amount of TGF-ß1, VEGF and tenomodulin, possibly involved in the reduced cell migration. In turn, the ASC alone seem to have modulated fibroblast behavior by increasing Ctgf, VEGF and tenomodulin, leading to greater cell migration. In conclusion, red LED and ASC therapy can have independent effects on fibroblast wound healing, but the combination of both does not have a synergistic effect. Therefore, future studies with other parameters of red LED associated with ASC should be tested aiming clinical application for tissue repair.

Reactive oxygen species production triggers green tea-induced anti-leukaemic effects on acute promyelocytic leukaemia model.

Green tea (GT) has been consumed as a beverage for thousands of years because of its therapeutic properties observed over time. Because there is no sufficient evidence supporting the protective role of tea intake during the development of acute myeloid leukaemia, we herein study GT extract effects on an acute promyelocytic leukaemia model. Our results demonstrated that GT reduces leucocytosis and immature cells (blasts) in peripheral blood, bone marrow (BM), and spleen of leukaemic mice, parallel with an increase of mature cells in the BM. In addition, GT induces apoptosis of cells in the BM and spleen, confirmed by activation of caspase-3, -8 and -9; GT reduces the malignant clones CD34+ and CD117+ in the BM and reduces CD117+ and Gr1+ immature myeloid cells in the spleen; GT increases intracellular reactive oxygen species (ROS) in the BM Gr1+ cells while reducing CD34+ and CD117+ cells; GT reduces CXCR4 expression on CD34+ and CD117+ cells, and reduces the nuclear translocation of HIF-1α. GT has anti-proliferative effects in leukaemia in vivo by inhibiting malignant clone expansion, probably by modulating the intracellular production of ROS.

Importance of major histocompatibility complex of class I (MHC-I) expression for astroglial reactivity and stability of neural circuits in vitro.

MHC-I molecules are involved in the antigenic presentation of cytosol-derived peptides to CD8T lymphocytes. In the nervous system, MHC-I expression is low to absent, occurring only during certain phases of development and aging or after injuries. The involvement of MHC-I in synaptic plasticity has been reported and, following lesion, astrocytes become reactive, limiting tissue damage. Such cells also attempt to restore homeostasis by secreting cytokines and neurotrophic factors. Moreover, astrocytes modulate synapse function, by taking up and releasing neurotransmitters and by limiting the synaptic cleft. Thus, the aim of the present study was to evaluate if astrocyte activation and reactivity are related to MHC I expression and if astrogliosis can be downregulated by silencing MHC-I mRNA synthesis. Given that, we evaluated astrocyte reactivity and synaptogenesis in co-cultures of astrocytes and spinal neurons under MHC-I RNA interference. For that, the MHC-I ß2-microglobulin subunit (ß2m) was knocked-down by siRNA in co-cultures (ß2m expression <60%, p<0.001). As measured by qRT-PCR, silencing of ß2m decreased expression of the astrocytic marker GFAP (<60%, p<0.001), as well as neurotrophic factors (BDNF and GDNF) and pro-inflammatory cytokines (TNF-α, IL-1, IL-6, IL-12 and IL-17). No significant changes in synaptic stability indicate that neuron-neuron interaction was preserved after ß2m silencing. Overall, the present data reinforce the importance of MHC-I expression for generation of astrogliosis, what may, in turn, become a target for future CNS/PNS therapies following injury.

MHC-I and PirB Upregulation in the Central and Peripheral Nervous System following Sciatic Nerve Injury.

Major histocompatibility complex class one (MHC-I) antigen-presenting molecules participate in central nervous system (CNS) synaptic plasticity, as does the paired immunoglobulin-like receptor B (PirB), an MHC-I ligand that can inhibit immune-cells and bind to myelin axon growth inhibitors. Based on the dual roles of both molecules in the immune and nervous systems, we evaluated their expression in the central and peripheral nervous system (PNS) following sciatic nerve injury in mice. Increased PirB and MHC-I protein and gene expression is present in the spinal cord one week after nerve transection, PirB being mostly expressed in the neuropile region. In the crushed nerve, MHC-I protein levels increased 2 weeks after lesion (wal) and progressively decreased over the next eight weeks. The same kinetics were observed for infiltrating cytotoxic T lymphocytes (CTLs) but not for PirB expression, which continuously increased. Both MHC-I and PirB were found in macrophages and Schwann cells but rarely in axons. Interestingly, at 8 wal, PirB was mainly restricted to the myelin sheath. Our findings reinforce the participation of MHC-I and PirB in CNS plasticity events. In contrast, opposing expression levels of these molecules were found in the PNS, so that MHC-I and PirB seem to be mostly implicated in antigen presentation to CTLs and axon myelination, respectively.

Correction: MHC-I and PirB Upregulation in the Central and Peripheral Nervous System Following Sciatic Nerve Injury.

[This corrects the article DOI: 10.1371/journal.pone.0161463.].

Neuroprotective effect of tempol (4 hydroxy-tempo) on neuronal death induced by sciatic nerve transection in neonatal rats.

Peripheral nerve injury in newborn rats triggers extensive neuronal death within the spinal cord. Because most neurodegeneration is related to oxidative stress and apoptosis, the use of antioxidants may be of therapeutic interest. Tempol is promising because of its ability to chelate reactive oxygen species and to minimize or even prevent tissue damage. Here, we evaluated neuroprotective effects of tempol following neonatal sciatic nerve transection. Two-day-old pups underwent sciatic nerve axotomy followed by tempol (12, 24 and 48 mg/kg) treatment (i.p.) at 10 min, 6 h, and every 24 h up to 1 week after injury. The rats were then killed for lumbar intumescence analysis. Nissl staining, TUNEL, synaptophysin immunolabeling and qRT-PCR (Caspase 3, Bax and Bcl2) were carried out. The results indicated that tempol treatment, at 24 mg/kg, increased up to 21% spinal cord motoneuron survival (p<0.001), also preserving pre-synaptic terminals in the neuropile. Likewise, the TUNEL-positive cell number decreased in tempol-treated animals. qRT-PCR results indicated differential increase in Caspase 3 (3-fold), Bax (13-fold) and Bcl2 (28-fold) gene expression, after 12 h following axotomy and tempol treatment. In conclusion, tempol administration has proven to be neuroprotective after neonatal nerve injury, leading to improved motoneuron survival, synapse preservation and minimizing apoptosis.

Exacerbation of autoimmune neuro-inflammation in mice cured from blood-stage Plasmodium berghei infection.

The thymus plays an important role shaping the T cell repertoire in the periphery, partly, through the elimination of inflammatory auto-reactive cells. It has been shown that, during Plasmodium berghei infection, the thymus is rendered atrophic by the premature egress of CD4+CD8+ double-positive (DP) T cells to the periphery. To investigate whether autoimmune diseases are affected after Plasmodium berghei NK65 infection, we immunized C57BL/6 mice, which was previously infected with P. berghei NK65 and treated with chloroquine (CQ), with MOG35-55 peptide and the clinical course of Experimental Autoimmune Encephalomyelitis (EAE) was evaluated. Our results showed that NK65+CQ+EAE mice developed a more severe disease than control EAE mice. The same pattern of disease severity was observed in MOG35-55-immunized mice after adoptive transfer of P. berghei-elicited splenic DP-T cells. The higher frequency of IL-17+- and IFN-γ+-producing DP lymphocytes in the Central Nervous System of these mice suggests that immature lymphocytes contribute to disease worsening. To our knowledge, this is the first study to integrate the possible relationship between malaria and multiple sclerosis through the contribution of the thymus. Notwithstanding, further studies must be conducted to assert the relevance of malaria-induced thymic atrophy in the susceptibility and clinical course of other inflammatory autoimmune diseases.

Challenge of chronically infected mice with homologous trypanosoma cruzi parasites enhances the immune response but does not modify cardiopathy: implications for the design of a therapeutic vaccine.

Chagas disease is a Trypanosoma cruzi-induced zoonosis that has no natural cure. Local damage induced by the parasite and the immune response causes chronic heart and digestive lesions. Efforts to develop a therapeutic vaccine that boosts the immune response to completely clear the parasite are needed because there is no effective treatment for chronically infected patients. In an attempt to modify the host-parasite equilibrium to increase parasite destruction, we analyzed cardiopathy and the immune response in chronically infected mice that were challenged with live homologous parasites. Challenge with a single dose of parasite increased CD4(+) and CD8(+) T cell populations, gamma interferon (IFN-γ) production, and serum-specific IgG levels. However, subpatent parasitemias and cardiac tissue were not affected. Because of the short duration of the immune boost after a single challenge, we next evaluated the impact of four parasite doses, administered 3 weeks apart. At 1 to 2 months after the last dose, the numbers of CD4(+) T cells and IFN-γ-producing CD4(+) memory cells and the CD4(+) T cell proliferative response to T. cruzi antigen were increased in the spleen. The frequency of IFN-γ-producing CD8(+) memory cells in the blood was also increased. However, the sustained challenge did not favor TH1 development; rather, it induced an increase in serum-specific IgG1 levels and mixed TH1/TH2 cytokine production. Moreover, there were no significant changes in cardiac lesions and subpatent parasitemias. In conclusion, we believe that this study may help in elucidating the necessary elements for a successful therapeutic vaccine which may reduce cardiomyopathy in chronically infected human patients.

Chloroquine treatment enhances regulatory T cells and reduces the severity of experimental autoimmune encephalomyelitis.

BACKGROUND: The modulation of inflammatory processes is a necessary step, mostly orchestrated by regulatory T (Treg) cells and suppressive Dendritic Cells (DCs), to prevent the development of deleterious responses and autoimmune diseases. Therapies that focused on adoptive transfer of Treg cells or their expansion in vivo achieved great success in controlling inflammation in several experimental models. Chloroquine (CQ), an anti-malarial drug, was shown to reduce inflammation, although the mechanisms are still obscure. In this context, we aimed to access whether chloroquine treatment alters the frequency of Treg cells and DCs in normal mice. In addition, the effects of the prophylactic and therapeutic treatment with CQ on Experimental Autoimmune Encephalomyelitis (EAE), an experimental model for human Multiple Sclerosis, was investigated as well. METHODOLOGY/PRINCIPAL FINDINGS: EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein (MOG35-55) peptide. C57BL/6 mice were intraperitoneally treated with chloroquine. Results show that the CQ treatment provoked an increase in Treg cells frequency as well as a decrease in DCs. We next evaluated whether prophylactic CQ administration is capable of reducing the clinical and histopathological signs of EAE. Our results demonstrated that CQ-treated mice developed mild EAE compared to controls that was associated with lower infiltration of inflammatory cells in the central nervous system CNS) and increased frequency of Treg cells. Also, proliferation of MOG35-55-reactive T cells was significantly inhibited by chloroquine treatment. Similar results were observed when chloroquine was administrated after disease onset. CONCLUSION: We show for the first time that CQ treatment promotes the expansion of Treg cells, corroborating previous reports indicating that chloroquine has immunomodulatory properties. Our results also show that CQ treatment suppress the inflammation in the CNS of EAE-inflicted mice, both in prophylactic and therapeutic approaches. We hypothesized that the increased number of regulatory T cells induced by the CQ treatment is involved in the reduction of the clinical signs of EAE.

IL-12p40 deficiency leads to uncontrolled Trypanosoma cruzi dissemination in the spinal cord resulting in neuronal death and motor dysfunction.

Chagas' disease is a protozoosis caused by Trypanosoma cruzi that frequently shows severe chronic clinical complications of the heart or digestive system. Neurological disorders due to T. cruzi infection are also described in children and immunosuppressed hosts. We have previously reported that IL-12p40 knockout (KO) mice infected with the T. cruzi strain Sylvio X10/4 develop spinal cord neurodegenerative disease. Here, we further characterized neuropathology, parasite burden and inflammatory component associated to the fatal neurological disorder occurring in this mouse model. Forelimb paralysis in infected IL-12p40KO mice was associated with 60% (p<0.05) decrease in spinal cord neuronal density, glutamate accumulation (153%, p<0.05) and strong demyelization in lesion areas, mostly in those showing heavy protein nitrosylation, all denoting a neurotoxic degenerative profile. Quantification of T. cruzi 18S rRNA showed that parasite burden was controlled in the spinal cord of WT mice, decreasing from the fifth week after infection, but progressive parasite dissemination was observed in IL-12p40KO cords concurrent with significant accumulation of the astrocytic marker GFAP (317.0%, p<0.01) and 8-fold increase in macrophages/microglia (p<0.01), 36.3% (p<0.01) of which were infected. Similarly, mRNA levels for CD3, TNF-α, IFN-γ, iNOS, IL-10 and arginase I declined in WT spinal cords about the fourth or fifth week after infection, but kept increasing in IL-12p40KO mice. Interestingly, compared to WT tissue, lower mRNA levels for IFN-γ were observed in the IL-12p40KO spinal cords up to the fourth week of infection. Together the data suggest that impairments of parasite clearance mechanisms in IL-12p40KO mice elicit prolonged spinal cord inflammation that in turn leads to irreversible neurodegenerative lesions.