Multiple sclerosis (MS) is a complex disorder of the central nervous system that appears to be driven by a shift in immune functioning toward excess inflammation that results in demyelination and axonal loss. Beta interferons were the first class of disease-modifying therapies to be approved for patients with MS after treatment with this type I interferon improved the course of MS on both clinical and radiological measures in clinical trials. The mechanism of action of interferon-beta appears to be driven by influencing the immune system at many levels, including antigen-presenting cells, T cells, and B cells. One effect of these interactions is to shift cytokine networks in favor of an anti-inflammatory effect. The pleiotropic mechanism of action may be a critical factor in determining the efficacy of interferon-beta in MS. This review will focus on select immunological mechanisms that are influenced by this type I cytokine.
The mammalian immune system constitutively senses vast quantities of commensal bacteria and their products through pattern recognition receptors, yet excessive immune reactivity is prevented under homeostasis. Intestinal microbiome can influence host susceptibility to extra-intestine autoimmune disorders. Here we report that polysaccharide A (PSA), a symbiosis factor for human intestinal commensal Bacteroides fragilis, protects against central nervous system demyelination and inflammation during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, through toll-like receptor 2 (TLR2). TLR2 mediates tissue-specific expansion of a critical regulatory CD39+ CD4 T cell subset by PSA. Ablation of CD39 signaling abrogates PSA control of EAE manifestations and inflammatory cytokine responses. Further, CD39 confers immune-regulatory phenotypes to total CD4 T cells and Foxp3+ CD4 Tregs. Importantly, CD39-deficient CD4 T cells show an enhanced capability to drive EAE progression. Our results demonstrate the therapeutic potential and underlying mechanism by which an intestinal symbiont product modulates CNS-targeted demyelination.
There is increasing support for the importance of risk factors such as genetic makeup, obesity, smoking, vitamin D insufficiency, and antibiotic exposure contributing to the development of autoimmune diseases, including human multiple sclerosis (MS). Perhaps the greatest environmental risk factor associated with the development of immune-mediated conditions is the gut microbiome. Microbial and helminthic agents are active participants in shaping the immune systems of their hosts. This concept is continually reinforced by studies in the burgeoning area of commensal-mediated immunomodulation. The clinical importance of these findings for MS is suggested by both their participation in disease and, perhaps of greater clinical importance, attenuation of disease severity. Observations made in murine models of central nervous system demyelinating disease and a limited number of small studies in human MS suggest that immune homeostasis within the gut microbiome may be of paramount importance in maintaining a disease-free state. This review describes three immunological factors associated with the gut microbiome that are central to cytokine network activities in MS pathogenesis: T helper cell polarization, T regulatory cell function, and B cell activity. Comparisons are drawn between the regulatory mechanisms attributed to first-line therapies and those described in commensal-mediated amelioration of central nervous system demyelination.
Mammals live in a co-evolutionary association with the plethora of microorganisms that reside at a variety of tissue microenvironments. The microbiome represents the collective genomes of these co-existing microorganisms, which is shaped by host factors such as genetics and nutrients but in turn is able to influence host biology in health and disease. Niche-specific microbiome, prominently the gut microbiome, has the capacity to effect both local and distal sites within the host. The gut microbiome has played a crucial role in the bidirectional gut-brain axis that integrates the gut and central nervous system (CNS) activities, and thus the concept of microbiome-gut-brain axis is emerging. Studies are revealing how diverse forms of neuro-immune and neuro-psychiatric disorders are correlated with or modulated by variations of microbiome, microbiota-derived products and exogenous antibiotics and probiotics. The microbiome poises the peripheral immune homeostasis and predisposes host susceptibility to CNS autoimmune diseases such as multiple sclerosis. Neural, endocrine and metabolic mechanisms are also critical mediators of the microbiome-CNS signaling, which are more involved in neuro-psychiatric disorders such as autism, depression, anxiety, stress. Research on the role of microbiome in CNS disorders deepens our academic knowledge about host-microbiome commensalism in central regulation and in practicality, holds conceivable promise for developing novel prognostic and therapeutic avenues for CNS disorders.
Genetic factors determining the pathogenesis and course of ocular toxoplasmosis are poorly understood. In this study, we explored the development of experimental ocular pathogenesis in genetically dissimilar mice infected with either the RH strain, the PLK strain, or the immunodominant surface antigen 1 (SAG1 [P30])-deficient mutant of the RH strain of Toxoplasma gondii. At 11 days postinfection, ocular infection of C57BL/6 mice with all of the strains of parasites resulted in severe inflammatory lesions and high numbers of parasites in eye tissue; less severe ocular lesions at earlier histopathology and prolonged survival were observed in this mouse strain infected with either the major surface antigen 1-deficient SAG1−/− strain or the less virulent PLK strain compared with RH infection. In contrast, both BALB/c and CBA/J mice had less severe lesions and low numbers of parasites in their eye tissue, and infection developed into the chronic stage in these mice. There were significantly higher serum levels of gamma interferon and tumor necrosis factor alpha in C57BL/6 mice than in BALB/c and CBA/J mice following ocular infection. These observations confirm earlier reports on systemic immunity to these parasites that the route of Toxoplasma infection markedly influences survival of mice. Our data indicate that genetic factors of the host as well as the parasite strain are critical in determining susceptibility to experimental ocular toxoplasmosis in murine models.
The role of CD4+ T cells in the pathogenesis of ocular toxoplasmosis was investigated in murine models utilizing inbred C57BL/6 mice deficient either in CD4+, CD8+, or B cells (μMT). Severe necrosis and inflammation with replicating parasites were observed in the eyes of control mice after primary ocular infection, and near-normal histology with few tachyzoites was observed in the eyes of mice immunized intraperitoneally with the avirulent ts-4 strain followed by intraocular challenge with the RH strain of Toxoplasma gondii. In contrast, mild inflammation without evidence of necrosis associated with increased parasite burdens were observed in the eyes of CD4 knockout (KO) mice after both primary ocular infection and challenge with RH tachyzoites. CD8 KO mice, as well as μMT mice, demonstrated increased ocular necrosis in response to either primary ocular infection or challenge. The parasite burden was increased in the eyes of both CD8 KO and μMT mice in which the parasite load was even higher. As expected, there were no increases in the levels of immunoglobulin G in serum or aqueous humor in μMT mice, and there was no increase in the levels of gamma interferon and tumor necrosis factor alpha in the sera of CD4 KO mice after both infection and challenge. These results suggest that the ocular inflammatory response to the parasite is mediated primarily by the CD4+-T-cell response. CD8+ T cells and B cells may play an important role in limiting tachyzoite proliferation in the eyes. Mice deficient in CD8+ CD4+ T cells or B cells exhibit diminished vaccine-induced resistance and increased ocular parasite burden after challenge.
To understand the role of interleukin-10 (IL-10) in ocular toxoplasmosis, we compared C57BL/6 (B6) and BALB/c background mice lacking a functional IL-10 gene (IL-10−/−) and B6 transgenic mice expressing IL-10 under the control of the IL-2 promoter. Increased cellular infiltration and necrosis were observed in the eye tissue of IL-10−/− mice of both the B6 and BALB/c backgrounds with associated changes in the levels of cytokines in serum. In contrast, there was no evidence of necrosis in the eye tissue from IL-10 transgenic mice following parasite exposure. Our results demonstrate that IL-10 is important in the regulation of inflammation during acute ocular toxoplasmosis.
We have observed previously that attachment of Toxoplasma gondii to synchronized host cells is considerably increased at the mid-S phase (4 h postrelease). Synchronized CHO host cells at the mid-S phase were fractionated by molecular weight, and the antigens were used to produce a panel of polyclonal mouse antisera. The polyclonal antisera raised against fraction 4 with molecular mass ranging approximately from 18 to 40 kDa significantly reduced attachment to mid-S-phase host cells. Immunofluorescence assays demonstrated strong reactivity to mid-S-phase host cells and identified a number of potential receptors on Western blots. These data indicate that there is a specific host membrane receptor for parasite attachment that is upregulated during the mid-S phase of the host cell cycle.
The role of mast cells (MCs) in Toxoplasma gondii infection is poorly known. Kunming outbred mice were infected intraperitoneally with RH strain T. gondii, either treated with compound 48/80 (C48/80, MC activator) or disodium cromoglycate (DSCG, MC inhibitor). Compared with infected controls, infected mice treated with C48/80 exhibited significantly increased inflammation in the liver (P < 0.01), spleen (P < 0.05), and mesentery (P < 0.05) tissues, higher parasite burden in the peritoneal lavage fluids (P < 0.01), and increased levels of mRNA transcripts of T. gondii tachyzoite surface antigen 1 (SAG1) gene in the spleen and liver tissues (P < 0.01), accompanied with significantly increased Th1 cytokine (IFN-γ, IL-12p40, and TNF-α) (P < 0.01) and decreased IL-10 (P < 0.01) mRNA expressions in the liver, and increased IFN-γ (P < 0.01) and IL-12p40 (P < 0.01) but decreased TNF-α (P < 0.01) and IL-4 (P < 0.01) in the spleens of infected mice treated with C48/80 at day 9-10 p.i. Whereas mice treated with DSCG had significantly decreased tissue lesions (P < 0.01), lower parasite burden in the peritoneal lavage fluids (P < 0.01) and decreased SAG1 expressions in the spleen and liver tissues (P < 0.01), accompanied with significantly increased IFN-γ (P < 0.01) and IL-12p40 (P < 0.05) in the liver, and decreased IFN-γ (P < 0.05) and TNF-α (P < 0.01) in the spleens; IL-4 and IL-10 expressions in both the spleen and liver were significantly increased (P < 0.01) in the infected mice treated with DSCG. These findings suggest that mediators associated with the MC activation may play an important role in modulating acute inflammatory pathogenesis and parasite clearance during T. gondii infection in this strain of mice. Thus, MC activation/inhibition mechanisms are potential novel targets for the prevention and control of T. gondii infection.
We recently showed that B cells reduce CNS inflammation in mice with experimental allergic encephalomyelitis (EAE). Here, we demonstrate that adoptively transferred CD5/CD19+ B cells protect against EAE severity. Furthermore, we show that glatiramer acetate (GA), a therapeutic for relapsing multiple sclerosis treatment, amplifies this effect. Transfer of GA-conditioned B cells leads to increased production of immunoregulatory cytokines and reduced CNS inflammation, as well as decreased expression of the chemokine receptor, CXCR5, and elevated BDNF expression in the CNS. Thus B cells can protect against EAE, and GA augments this effect in maintaining immune homeostasis and controlling EAE disease progression.
Glatiramer acetate; Inflammation; B cells; CD19; CD5
In the present study we addressed the question whether Toxoplasma gondii could promote apoptosis in T lymphocytes in the acute stage of infection. Using in vivo activated T cells and then culturing them for a short time, we observed activation induced cell death in T. gondii infected mice. A higher level of activation induced cell death (AICD) was seen in susceptible C57BL/6 mice than in resistant CBA/J mice following infection with the same P strain of parasite. Apoptosis in T cells of susceptible mice was associated with altered induction of Bcl-2/Bax, loss of Mitochondrial Transmembrane Potential. Both CD4+ and CD8+ T cells were found to be susceptible to apoptosis; CD4+ T cells were sensitive to Fas mediated death whereas CD8+ T cells were insensitive to this signal. Caspase inhibitors had less effect on DNA fragmentation in CD4+ compared to CD8+ T cells. Exposure of CD4+ T cells to anti-IFNγ mAb resulted in an increase in the number of T cells that were positive for anti-apoptotic molecule Bcl-2 and DiOC6, a cationic dye that accumulates in intact mitochondria. These changes were less noticeable in CD8+ T cells following treatment with anti-IFNγ mAb. These findings provide further insight into the mechanisms of T cell apoptosis in T. gondii infection.
We have recently shown that alteration of the gut commensal microbiota with antibiotics can modify the susceptibility to autoimmune demyelinating processes of the central nervous system. Treatment of mice with a broad spectrum of antibiotics not only induced significant changes in the regulatory T cell populations of the gut associated lymphoid tissues (GALT) and peripheral lymphoid organs but reduced the susceptibility to EAE, the most widely used animal model for human multiple sclerosis. Here, we show further that oral antibiotic treatment of EAE mice induced a CD5+B cell subpopulation that conferred protection against the disease. Protection was associated with an enhanced frequency of CD5+B cells in distal lymphoid sites such as cervical LN. In vitro stimulation with LPS increased the production of IL-10 by splenic CD5+B cells. Adoptive transfer of CD5+B cells from antibiotic treated mice reduced significantly the severity of EAE by shifting the immune responses from Th1/Th17 towards anti-inflammatory Th2-type responses. Our results demonstrate that this specific B cell population appears to be involved in the immune regulation of autoimmunity, in particular this experimental demyelinating disease of the central nervous system by gut commensal microflora.
B cells; commensal bacteria; autoimmune regulation; IL-10; EAE/MS
We report that like other T cells cultured in the presence of transforming growth factor (TGF) β, Th17 cells also produce interleukin (IL) 9. Th17 cells generated in vitro with IL-6 and TGF-β as well as purified ex vivo Th17 cells both produced IL-9. To determine if IL-9 has functional consequences in Th17-mediated inflammatory disease, we evaluated the role of IL-9 in the development and progression of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. The data show that IL-9 neutralization and IL-9 receptor deficiency attenuates disease, and this correlates with decreases in Th17 cells and IL-6–producing macrophages in the central nervous system, as well as mast cell numbers in the regional lymph nodes. Collectively, these data implicate IL-9 as a Th17-derived cytokine that can contribute to inflammatory disease.
Better understanding of host-virus interaction is essential to produce effective vaccines against influenza (H5N1) viruses.
Influenza A (H5N1) viruses are strong candidates for causing the next influenza pandemic if they acquire the ability for efficient human-to-human transmission. A major public health goal is to make efficacious vaccines against these viruses by using novel approaches, including cell-culture system, reverse genetics, and adjuvant development. Important consideration for the strategy includes preparation of vaccines from a currently circulating strain to induce broad-spectrum immunity toward newly emerged human H5 strains. This strategy would be a good solution early in a pandemic until an antigenically matched and approved vaccine is produced. The concept of therapeutic vaccines (e.g., antidisease vaccine) directed at diminishing the cytokine storm frequently seen in subtype H5N1–infected persons is underscored. Better understanding of host–virus interaction is essential to identify tools to produce effective vaccines against influenza (H5N1).
Vaccine; influenza (H5N1); virus; pandemic; antibody; CTL response; broad-spectrum immunity; cytokines; therapeutic vaccines; perspective
Human neutrophils are rescued from apoptosis following incubation with once-washed, fibroblast-derived Toxoplasma gondii tachyzoites. Both infected and uninfected neutrophils are rescued, implicating a soluble mediator. In this study we investigated the origin and identity of this soluble mediator. Neutrophils were incubated either with purified tachyzoites or with conditioned medium derived from T. gondii-infected human fibroblasts. Conditioned medium was found to be a potent stimulus that delayed neutrophil apoptosis up to 72 h, whereas purified and extensively washed tachyzoites had no effect. Delayed apoptosis correlated with up-regulation of the neutrophil antiapoptotic protein, Mcl-1, and the neutrophil interleukin 3 receptor α subunit (IL-3Rα), suggesting a role for granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF and granulocyte colony-stimulating factor (G-CSF) were measurable in conditioned medium by enzyme-linked immunosorbent assay. Neutralizing antibodies to GM-CSF and G-CSF were additive in abrogating delayed neutrophil apoptosis induced by conditioned medium. Inhibitors of Src family tyrosine kinases, Gi proteins, phosphatidylinositol 3-kinase, p44erk1 and p42erk2 mitogen-activated protein kinases, and Jak2 kinases partially attenuated the effect of conditioned medium, consistent with a role for G-CSF and/or GM-CSF. Hence, delayed neutrophil apoptosis is mediated by GM-CSF and G-CSF secreted by T. gondii-infected human fibroblasts. This enhanced neutrophil survival may contribute to the robust proinflammatory response elicited in the T. gondii-infected host.
When tachyzoites were incubated with human peripheral blood leukocytes in vitro, more monocytes and dendritic cells than neutrophils or lymphocytes were infected. Although tachyzoites were able to divide in each of these cell types, monocytes and dendritic cells were more permissive to rapid tachyzoite division than neutrophils or lymphocytes.
To assess the relationship between the changes of cellular components and the production of Th1 cytokine in the immune tissue, inbred C57BL/6 mice were orally infected with 40 cysts of 76K strain of Toxoplasma gondii. The sequential change of cell differentials and IFN-γ production of splenocytes were analyzed by Diff-Quik stain and RT-PCR. There were no significant proportional changes of cellular components of splenocytes until day 4 postinfection (PI) as compared to those of day 0, and the relative percentage of macrophages and neutrophils/eosinophils increased significantly (p<0.01) thereafter. The expression of IFN-γ mRNA of CD3- cells was observed from day 1 PI at a low level. However, IFN-γ production of CD3+ cells increased significantly from day 4 PI (p<0.01) which progressively increased thereafter. These findings provide the relative percentages of granulocytes and macrophages were increased in conjunction with increase of total number of splenocytes after oral infection with T. gondii in the susceptible murine hosts, and lymphocytes were the major cellular components and the important source of IFN-γ.
Toxoplasma gondii; toxoplasmosis; splenocytes; cell differentials; IFN-γ; granulocytes
Toxoplasma gondii remains a serious cause of morbidity and mortality in individuals that are immunosuppressed, patients with AIDS in particular. The cellular immune response, especially by gamma interferon (IFN-γ)-producing CD8+ T cells, is an essential component of protective immunity against the parasite. In the present study the role of CD8+ T cells during the reactivation of Toxoplasma infection in an immunocompromised murine model was evaluated. Chronically infected mice were challenged with LP-BM5 virus, and the kinetics of CD8+ T-cell function was studied. At 10 weeks after viral infection, mice showed obvious signs of systemic illness and began to die. At this stage, CD8+ T cells were unresponsive to antigenic stimulation and unable to kill Toxoplasma-infected targets. IFN-γ production by the CD8+ T cells from dual-infected animals reached background levels, and a dramatic fall in the frequency of precursor cytotoxic T lymphocytes was observed. Histopathological analysis of the tissues demonstrated signs of disseminated toxoplasmosis as a result of reactivation of infection. However, treatment of the dual-infected animals with immune CD8+ T cells at 5 weeks post-LP-BM5 challenge prevented the reactivation of toxoplasmosis, and mice continued to live. Our study for the first time demonstrates a therapeutic role for CD8+ T cells against an opportunistic infection in an immunocompromised state.
Previous studies have demonstrated that surface antigen proteins, in particular SAG-1, of Toxoplasma gondii are important to this parasite as attachment ligands for the host cell. An in vitro assay was developed to test whether these ligands and other secretory proteins are involved in the immune response of human cells to toxoplasma. Human monocytes were infected with tachyzoites in the presence of antiparasite antibodies, and their effect on mitogen-induced lymphoproliferation was examined. The presence of antibody to either parasite-excreted proteins (MIC-1 and MIC-2) or surface proteins (SAG-1 and SAG-2) during infection neutralized the marked decrease seen in mitogen-induced lymphoproliferation in the presence of infected monocytes. Conversely, antibodies to other secreted proteins (ROP-1) and cytoplasmic molecules had no effect on parasite-induced, monocyte-mediated downregulation. Fluorescence microscope analysis detected microneme and surface antigen proteins on the monocyte cell surface during infection. These results suggest that microneme and surface antigen proteins trigger monocytes to downregulate mitogen-induced lymphoproliferation.
Ocular toxoplasmosis is a potentially blinding intraocular inflammation. The intent of this study was to investigate the role of Fas-FasL interaction in a murine model of acquired ocular toxoplasmosis induced by intracameral inoculation of Toxoplasma gondii. Intraocular inflammation, Fas and FasL expression on lymphocytes and on ocular tissues, the occurrence of apoptosis, and the frequency of CD8+ and CD4+ T cells in the infected eyes were analyzed in C57BL/6 (B6) mice. Susceptibility to parasite-induced intraocular inflammation was observed in Fas-deficient (B6-lpr) and FasL-deficient (B6-gld) mice. Inoculation of 5,000 T. gondii tachyzoites induced significant intraocular inflammation associated with increase of Fas and FasL expression in the inoculated eyes of wild-type B6 mice. Flow cytometry demonstrated a significant increase of Fas and FasL expression on the splenocytes from naive mice incubated in vitro with the parasite and on the splenocytes harvested from the infected mice at day 8 after parasite inoculation. Apoptosis of inflammatory cells and cells in ocular tissues was seen, and a greater frequency of CD8+ than CD4+ T cells was observed in the infected eyes. The intensity of intraocular inflammation was greater in B6-lpr and B6-gld mice than in wild-type B6 mice (P < 0.05). The results suggest that Fas-FasL interaction associated with apoptosis is involved in the pathogenesis of acquired ocular toxoplasmosis in mice.
Stage conversion between bradyzoites and tachyzoites was investigated in C57BL/6 mice chronically infected with the ME-49 strain of Toxoplasma gondii. In order to promote bradyzoite-tachyzoite conversion, mice were treated in vivo with neutralizing doses of anti-gamma interferon (IFN-γ) or anti-tumor necrosis factor alpha (TNF-α) antibodies. Expression of parasite-specific antigens SAG-1, SAG-2, and heat shock protein 70 (Hsp-70) was visualized in the central nervous system by immunocytochemistry and measured by photometric assay. The immunosuppressive effect of anti-IFN-γ or anti-TNF-α treatment was immediate, leading to parasite stage conversion as indicated by the increased expression of tachyzoite-specific antigens (SAG-1 and SAG-2) and by rapid parasite replication. We also observed expression of high levels of Hsp-70 during a short period of conversion of bradyzoites to tachyzoites. Our data suggest that Hsp-70 may have an important role in the process of bradyzoite-tachyzoite conversion during the reactivation of chronic toxoplasmosis.
Neospora caninum is a coccidial protozoan parasite that appears morphologically indistinguishable from Toxoplasma gondii and that infects a large range of mammals. Both inbred and outbred strains of mice exhibit a high degree of resistance to infection with N. caninum. Three inbred strains of mice (A/J, BALB/c, and C57BL/6) that were infected intraperitoneally with N. caninum were protected against a lethal challenge from T. gondii. Vaccine-induced protection was Neospora dose dependent. A rise in the CD8+ T-cell population in mice that had been vaccinated with N. caninum and challenged with T. gondii was observed. Adoptive transfer of CD8+ T-cell splenocytes from N. caninum-infected mice was protective against challenge with Toxoplasma. The CD8+ T cells from Neospora-infected mice proliferate to both Neospora and Toxoplasma antigens in vitro and secrete substantial quantities of gamma interferon when pulsed with the parasite antigen. These observations demonstrate that N. caninum protects against lethal T. gondii infection by the induction of CD8+ T cells that are immunoreactive to both parasites.