T cell immunoglobulin and mucin domain-containing-3 (Tim-3) is an inhibitory receptor expressed on exhausted T cells during HIV-1 and HCV infection. By contrast, Tim-3 expression and function are defective in multiple human autoimmune diseases. However, the molecular mechanisms governing Tim-3 function remain poorly understood. Here we show that HLA-B-associated transcript 3 (Bat3) binds to, and represses the function of Tim-3. Bat3-deficient T cells display elevated expression of exhaustion markers, and knocking down Bat3 in myelin antigen-specific CD4+ T cells dramatically inhibits the development of experimental autoimmune encephalomyelitis while promoting the expansion of a dysfunctional Tim-3hiIFNγlo CD4+ cell population. Furthermore, exhausted Tim-3+ T cells from murine tumors and HIV-1-infected individuals display substantially reduced Bat3 expression and targeted deletion of Bat3 induces an exhausted phenotype in T cells. These data indicate that Bat3 acts as a molecular safety catch that inhibits Tim-3-dependent cell death/exhaustion, suggesting that Bat3 may represent a viable therapeutic target in autoimmune disorders, chronic infections and cancers.
Amyloid forming proteins Tau, alpha B crystallin, and amyloid P protein are all found in lesions of multiple sclerosis (MS). Our previous work established that amyloidogenic peptides from the small heat shock protein, alpha B crystallin(HspB5), and from amyloid β fibrils, characteristic of Alzheimer’s disease, were therapeutic in experimental autoimmune encephalomyelitis (EAE), reflecting aspects of the pathology of MS. To understand the molecular basis for the therapeutic effect, a set of amyloidogenic peptides composed of six amino acids, including those from tau, amyloid β A4, major prion protein (PrP), HspB5, amylin, serum amyloid P (SAP), and insulin B chain were shown to be anti-inflammatory, capable of reducing serological levels of IL-6, and attenuating paralysis in EAE. The chaperone function of the fibrils correlates with the therapeutic outcome. Fibrils composed of Tau 623–628 precipitated 49 plasma proteins, including apolipoprotein B-100, clusterin, transthyretin, and complement C3, supporting the hypothesis that the fibrils are active biological agents. Amyloid fibrils thus may provide benefit in MS and other neuroinflammatory disorders.
Small heat shock proteins; chaperone; amyloid fibrils; inflammation; experimental autoimmune encephalomyelitis
Interleukin 17 (IL-17)-producing TH17 cells are often present at the sites of tissue inflammation in autoimmune diseases, which has lead to the conclusion that TH17 are main drivers of autoimmune tissue injury. However, not all TH17 cells are pathogenic, in fact TH17 generated with TGF-β1 and IL-6 produce IL-17 but do not readily induce autoimmune disease without further exposure to IL-23. Here we show that TGF-β3, produced by developing TH17 cells, is dependent on IL-23, which together with IL-6 induces highly pathogenic TH17 cells. Moreover, TGF-β3-induced TH17 cells are functionally and molecularly distinct from TGF-β1-induced TH17 cells and possess a molecular signature that defines pathogenic effector TH17 cells in autoimmune disease.
Experimental autoimmune encephalomyelitis (EAE) is a widely used model of multiple sclerosis (MS). In NOD mice, EAE develops as a relapsing-remitting disease that transitions to a chronic progressive disease, making the NOD model the only mouse model that recapitulates the full clinical disease course observed in most MS patients. We have generated a T cell receptor (TCR) transgenic mouse that expresses the alpha and beta chains of a myelin oligodendrocyte glycoprotein (MOG) 35–55-reactive TCR (1C6) on the NOD background. 1C6 TCR transgenic mice spontaneously generate both CD4+ and CD8+ T cells that recognize MOG and produce pro-inflammatory cytokines, allowing for the first time the simultaneous examination of myelin-reactive CD4+ and CD8+ T cells in the same host. 1C6 CD8+ T cells alone can induce optic neuritis and mild EAE with delayed onset; however, 1C6 CD4+ T cells alone induce severe EAE and predominate in driving disease when both cell types are present. When 1C6 mice are crossed with mice bearing an immunoglobulin heavy chain specific for MOG, the mice develop spontaneous EAE with high incidence but surprisingly the disease pattern does not resemble the Neuromyelitis optica (NMO)-like disease observed in mice bearing CD4+ T cells and B cells reactive to MOG on the C57BL/6 background. Collectively our data show that while myelin-reactive CD8+ T cells contribute to disease, disease is primarily driven by myelin-reactive CD4+ T cells and that the co-existence of myelin-reactive T and B cells does not necessarily result in a distinct pathological phenotype.
CD47 exerts different effects on disease in distinct cell types and locations and during different stages of experimental autoimmune encephalomyelitis.
Comparison of transcriptomic and proteomic data from pathologically similar multiple sclerosis (MS) lesions reveals down-regulation of CD47 at the messenger RNA level and low abundance at the protein level. Immunohistochemical studies demonstrate that CD47 is expressed in normal myelin and in foamy macrophages and reactive astrocytes within active MS lesions. We demonstrate that CD47−/− mice are refractory to experimental autoimmune encephalomyelitis (EAE), primarily as the result of failure of immune cell activation after immunization with myelin antigen. In contrast, blocking with a monoclonal antibody against CD47 in mice at the peak of paralysis worsens EAE severity and enhances immune activation in the peripheral immune system. In vitro assays demonstrate that blocking CD47 also promotes phagocytosis of myelin and that this effect is dependent on signal regulatory protein α (SIRP-α). Immune regulation and phagocytosis are mechanisms for CD47 signaling in autoimmune neuroinflammation. Depending on the cell type, location, and disease stage, CD47 has Janus-like roles, with opposing effects on EAE pathogenesis.
Multiple sclerosis is an autoimmune disease of the central nervous system characterized by neuroinflammation and demyelination. Although considered a T cell-mediated disease, multiple sclerosis involves the activation of both adaptive and innate immune cells, as well as resident cells of the central nervous system, which synergize in inducing inflammation and thereby demyelination. Differentiation, survival, and inflammatory functions of innate immune cells and of astrocytes of the central nervous system are regulated by tyrosine kinases. Here, we show that imatinib, sorafenib, and GW2580—small molecule tyrosine kinase inhibitors can each—prevent the development of disease and treat established disease in a mouse model of multiple sclerosis. In vitro, imatinib and sorafenib inhibited astrocyte proliferation mediated by the tyrosine kinase platelet-derived growth factor receptor (PDGFR), whereas GW2580 and sorafenib inhibited macrophage tumor necrosis factor (TNF) production mediated by the tyrosine kinases c-Fms and PDGFR, respectively. In vivo, amelioration of disease by GW2580 was associated with a reduction in the proportion of macrophages and T cells in the CNS infiltrate, as well as a reduction in the levels of circulating TNF. Our findings suggest that GW2580 and the FDA-approved drugs imatinib and sorafenib have potential as novel therapeutics for the treatment of autoimmune demyelinating disease.
Imatinib; sorafenib; experimental autoimmune encephalomyelitis; tyrosine kinase inhibitors; macrophages; TNF; astrocyte proliferation
Treatment with ex vivo–generated regulatory T cells (T-reg) has been regarded as a potentially attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp knock-in (Foxp3gfp.KI) mice and myelin oligodendrocyte glycoprotein (MOG)35–55/IAb (MHC class II) tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. MOG tetramer–reactive, Foxp3+ T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. Foxp3+ T cells isolated from the CNS were effective in suppressing naive MOG-specific T cells, but failed to control CNS-derived encephalitogenic T-eff that secreted interleukin (IL)-6 and tumor necrosis factor (TNF). Our data suggest that in order for CD4+Foxp3+ T-reg to effectively control autoimmune reactions in the target organ, it may also be necessary to control tissue inflammation.
Ectopic lymphoid follicles are hallmarks of chronic autoimmune inflammatory diseases such as multiple sclerosis (MS), rheumatoid arthritis, Sjögren’s syndrome, and myasthenia gravis. However, the effector cells and mechanisms that induce their development are unknown. Here we showed that in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, Th17 cells specifically induced ectopic lymphoid follicles in the central nervous system (CNS). Development of ectopic lymphoid follicles was partly dependent on the cytokine interleukin 17 (IL-17) and on the cell surface molecule Podoplanin (Pdp), which was expressed on Th17 cells, but not on other effector T cell subsets. Pdp was also crucial for the development of secondary lymphoid structures: Pdp-deficient mice lacked peripheral lymph nodes and had a defect in forming normal lymphoid follicles and germinal centers in spleen and lymph node remnants. Thus, Th17 cells are uniquely endowed to induce tissue inflammation, characterized by ectopic lymphoid follicles within the target organ.
Analyses of varicella-zoster virus (VZV) protein expression during latency have been discordant, with rare to many positive neurons detected. We show that ascites-derived murine and rabbit antibodies specific for VZV proteins in vitro contain endogenous antibodies that react with human blood type A antigens in neurons. Apparent VZV neuronal staining and blood type A were strongly associated (by a χ2 test, α = 0.0003). Adsorption of ascites-derived monoclonal antibodies or antiserum with type A erythrocytes or the use of in vitro-derived VZV monoclonal antibodies eliminated apparent VZV staining. Animal-derived antibodies must be screened for anti-blood type A reactivity to avoid misidentification of viral proteins in the neurons of the 30 to 40% of individuals who are blood type A.
Laquinimod is a novel oral drug that is currently being evaluated for the treatment of relapsing-remitting (RR) multiple sclerosis (MS). Using the animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we examined how laquinimod promotes immune modulation. Oral laquinimod treatment reversed established RR-EAE and was associated with reduced central nervous system (CNS) inflammation, decreased Th1 and Th17 responses, and an increase in regulatory T cells (Treg). In vivo laquinimod treatment inhibited donor myelin-specific T cells from transferring EAE to naive recipient mice. In vivo laquinimod treatment altered subpopulations of myeloid antigen presenting cells (APC) that included a decrease in CD11c+CD11b+CD4+ dendritic cells (DC) and an elevation of CD11bhiGr1hi monocytes. CD11b+ cells from these mice exhibited an anti-inflammatory type II phenotype characterized by reduced STAT1 phosphorylation, decreased production of IL-6, IL-12/23 and TNF, and increased IL-10. In adoptive transfer, donor type II monocytes from laquinimod-treated mice suppressed clinical and histologic disease in recipients with established EAE. As effects were observed in both APC and T cell compartments, we examined whether T cell immune modulation occurred as a direct effect of laquinimod on T cells, or as a consequence of altered APC function. Inhibition of Th1 and Th17 differentiation was observed only when type II monocytes or DC from laquinimod-treated mice were used as APC, regardless of whether myelin-specific T cells were obtained from laquinimod-treated or untreated mice. Thus, laquinimod modulates adaptive T cell immune responses via its effects on cells of the innate immune system, and may not influence T cells directly.
IFN-γ plays a central role in anti-tumor immunity. Tim-3 is expressed on IFN-γ-producing Th1 cells; upon interaction with its ligand, galectin-9, it terminates Th1 immunity. Here, we show that transgenic over-expression of Tim-3 on T cells results in an increase in CD11b+Ly-6G+ cells and inhibition of immune responses. Molecular characterization of CD11b+Ly-6G+ cells reveals a phenotype consistent with granulocytic myeloid-derived suppressor cells (MDSC). Accordingly, we find that modulation of the Tim-3/galectin-9 pathway impacts on tumor growth. Similarly, overexpression of Tim-3 ligand, galectin-9, results in an increase in CD11b+Ly-6G+ cells and inhibition of immune responses. Loss of Tim-3 restores normal levels of CD11b+Ly-6G+ cells and normal immune responses in galectin-9 transgenic mice. Our data uncover a novel mechanism by which the Tim-3/galectin-9 pathway regulates immune responses and identifies this pathway as a therapeutic target in diseases where MDSC are disadvantageous.
This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the United States National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.
Mn superoxide dismutase (MnSOD) is an important mitochondrial antioxidant enzyme, and elevated MnSOD levels have been shown to reduce tumor growth in part by suppressing cell proliferation. Studies with fibroblasts have shown that increased MnSOD expression prolongs cell cycle transition time in G1/S and favors entrance into the quiescent state. To determine if the same effect occurs during tissue regeneration in vivo, we used a transgenic mouse system with liver-specific MnSOD expression and a partial hepatectomy paradigm to induce synchronized in vivo cell proliferation during liver regeneration. We show in this experimental system that a 2.6 fold increase in MnSOD activities leads to delayed entry into S phase, as measured by reduction in bromodeoxyuridine (BrdU) incorporation, and decreased expression of proliferative cell nuclear antigen (PCNA). Thus, compared to control mice with baseline MnSOD levels, transgenic mice with increased MnSOD expression in the liver have 23% fewer BrdU positive cells and a marked attenuation of PCNA expression. The increase in MnSOD activity also leads to an increase of the mitochondrial form of thioredoxin (thioredoxin 2), but not of several other peroxidases examined, suggesting the importance of thioredoxin 2 in maintaining redox balance in mitochondria with elevated levels of MnSOD.
MnSOD; partial hepatectomy; mitochondria; thioredoxin 2; liver regeneration; cell cycle progression
Impaired mitochondrial fusion/fission plays a causal role in neuronal death. This study delineated a PKCδ-related signaling cascade in which excessive mitochondrial fission is induced during oxidative stress. Moreover, a selective peptide inhibitor of PKCδ inhibits impaired mitochondrial fission under these pathological conditions.
Neuronal cell death in a number of neurological disorders is associated with aberrant mitochondrial dynamics and mitochondrial degeneration. However, the triggers for this mitochondrial dysregulation are not known. Here we show excessive mitochondrial fission and mitochondrial structural disarray in brains of hypertensive rats with hypertension-induced brain injury (encephalopathy). We found that activation of protein kinase Cδ (PKCδ) induced aberrant mitochondrial fragmentation and impaired mitochondrial function in cultured SH-SY5Y neuronal cells and in this rat model of hypertension-induced encephalopathy. Immunoprecipitation studies indicate that PKCδ binds Drp1, a major mitochondrial fission protein, and phosphorylates Drp1 at Ser 579, thus increasing mitochondrial fragmentation. Further, we found that Drp1 Ser 579 phosphorylation by PKCδ is associated with Drp1 translocation to the mitochondria under oxidative stress. Importantly, inhibition of PKCδ, using a selective PKCδ peptide inhibitor (δV1-1), reduced mitochondrial fission and fragmentation and conferred neuronal protection in vivo and in culture. Our study suggests that PKCδ activation dysregulates the mitochondrial fission machinery and induces aberrant mitochondrial fission, thus contributing to neurological pathology.
Peroxisome proliferator–activated receptors (PPARs; PPAR-α, PPAR-δ, and PPAR-γ) comprise a family of nuclear receptors that sense fatty acid levels and translate this information into altered gene transcription. Previously, it was reported that treatment of mice with a synthetic ligand activator of PPAR-δ, GW0742, ameliorates experimental autoimmune encephalomyelitis (EAE), indicating a possible role for this nuclear receptor in the control of central nervous system (CNS) autoimmune inflammation. We show that mice deficient in PPAR-δ (PPAR-δ−/−) develop a severe inflammatory response during EAE characterized by a striking accumulation of IFN-γ+IL-17A− and IFN-γ+IL-17A+ CD4+ cells in the spinal cord. The preferential expansion of these T helper subsets in the CNS of PPAR-δ−/− mice occurred as a result of a constellation of immune system aberrations that included higher CD4+ cell proliferation, cytokine production, and T-bet expression and enhanced expression of IL-12 family cytokines by myeloid cells. We also show that the effect of PPAR-δ in inhibiting the production of IFN-γ and IL-12 family cytokines is ligand dependent and is observed in both mouse and human immune cells. Collectively, these findings suggest that PPAR-δ serves as an important molecular brake for the control of autoimmune inflammation.
Foxp3 is a key transcription factor involved in the generation and function of regulatory T (Treg) cells. Transforming growth factor β (TGF-β) induces Foxp3, which generates inducible Foxp3+ Treg cells from naïve T cells, and interleukin 6 (IL-6) inhibits the generation of inducible Treg cells and induces T helper cells that produce IL-17 (TH-17 cells). However, a role for IL-4 in the generation of TGF-β-induced Treg cells and/or the generation of effector CD4+ T helper cells has not been studied. Here, we show that IL-4 blocked the generation of TGF-β-induced Foxp3+ Treg cells. Instead, IL-4 induced a population of T helper cells that predominantly produce IL-9 and IL-10. The IL-9+IL-10+ T cells did not exhibit any regulatory properties in spite of producing large quantities of IL-10. Adoptive transfer of IL-9+IL-10+producing T cells into RAG-1-deficient mice induced colitis and peripheral neuritis. Interestingly, the severity of tissue inflammation was aggravated when IL-9+IL-10+ T cells were co-transferred with CD45RBhi CD4+ effector T cells into RAG-1-deficient mice, which indicated that IL-9+IL-10+ T cells do not display any suppressive function and therefore constitute a unique population of IL-10-producing helper-effector T cells that promote tissue inflammation.
Experimental autoimmune encephalomyelitis (EAE) is a model of human multiple sclerosis (MS) induced by auto-reactive T helper cells that mediate tissue inflammation and demyelination in the CNS. Initially, IFN-γ-producing Th1 cells and, more recently, IL-17-producing Th17 cells with specificity for myelin antigens have been implicated in EAE induction, but whether Th17 cells are encephalitogenic has been controversial. Moreover, a new effector T cell subset, Th9 cells, has been identified, however, the ability of this T cell subset to induce EAE has not been investigated.
Here, we have developed protocols to generate myelin oligodendrocyte glycoprotein (MOG)-specific Th17, Th1, Th2, and Th9 cells in vitro, so that we could directly compare and characterize the encephalitogenic activity of each of these subsets upon adoptive transfer. We show that MOG-specific Th1, Th17, and Th9 cells but not Th2 cells induce EAE upon adoptive transfer. Importantly, each T cell subset induced disease with a different pathological phenotype.
These data demonstrate that different effector T cell subsets with specificity for myelin antigens can induce CNS autoimmunity and that the pathological heterogeneity in MS lesions might in part be due to multiple distinct myelin-reactive effector T cells.
TIM (T-cell, immunoglobulin, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in antigen-presenting cells. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating monoclonal antibodies, we show that expression of Tim-4 protein is restricted to CD11c+ and CD11b+ cells and is upregulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on antigen-presenting cells, is a costimulatory molecule that promotes T cell expansion and survival by crosslinking Tim-1 on T cells.
T cells; costimulation; apoptosis; cell surface molecules
Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediate-early 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in estimates of the frequency of IE63-positive neurons, varying from a rare event to abundant expression. We examined IE63 expression in cadaver ganglia using a high-potency rabbit anti-IE63 antibody and corresponding preimmune serum. Using standard immunohistochemical techniques, we evaluated 10 ganglia that contained VZV DNA from seven individuals. These experiments showed that neuronal pigments were a confounding variable; however, by examining sections coded to prevent investigator bias and applying statistical analysis, we determined that IE63 protein, if present, is in a very small proportion of neurons (<2.8%). To refine estimates of IE63 protein abundance, we modified our protocol by incorporating a biological stain to exclude the pigment signal and evaluated 27 ganglia from 18 individuals. We identified IE63 protein in neurons within only one ganglion, in which VZV glycoprotein E and an immune cell infiltrate were also demonstrated. Antigen preservation was shown by detection of neuronal synaptophysin. These data provide evidence that the expression of IE63 protein, which has been referred to as a latency-associated protein, is rare. Refining estimates of VZV protein expression in neurons is important for developing a hypothesis about the mechanisms by which VZV latency may be maintained.
We examined the involvement of chemokine-like receptor-1 (CMKLR1) in experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis. Upon EAE induction by active immunization with myelin oligodendrocyte glycoprotein amino acids 35–55 (MOG35–55), microglial cells and CNS-infiltrating myeloid dendritic cells expressed CMKLR1, as determined by flow cytometric analysis. In addition, chemerin, a natural ligand for CMKLR1, was up-regulated in the CNS of mice with EAE. We found that CMKLR1-deficient (CMKLR1 knockout (KO)) mice develop less severe clinical and histologic disease than their wild-type (WT) counterparts. CMKLR1 KO lymphocytes proliferate and produce proinflammatory cytokines in vitro, yet MOG35–55-reactive CMKLR1 KO lymphocytes are deficient in their ability to induce EAE by adoptive transfer to WT or CMKLR1 KO recipients. Moreover, CMKLR1 KO recipients fail to fully support EAE induction by transferred MOG-reactive WT lymphocytes. The results imply involvement of CMKLR1 in both the induction and effector phases of disease. We conclude that CMKLR1 participates in the inflammatory mechanisms of EAE and represents a potential therapeutic target in multiple sclerosis.
Idd5.1 regulates T1D susceptibility in NOD mice and has two notable candidate genes, Ctla4 and Icos. Reduced expression of one of the four CTLA-4 isoforms, ligand independent CTLA-4 (liCTLA-4), which inhibits in vitro T cell activation and cytokine production similarly to full length CTLA-4 (flCTLA-4), has been hypothesized to increase T1D susceptibility. However, further support of this hypothesis is required since the Idd5.1 haplotypes of the diabetes-susceptible NOD and the resistant B10 strains differ throughout Ctla4 and Icos. Using haplotype analysis and the generation of novel Idd5.1 congenic strains that differ at the disease-associated Ctla4 exon 2 single nucleotide polymorphism (SNP), we demonstrate that increased expression of liCTLA-4 correlates with reduced T1D susceptibility. To directly assess the ability of liCTLA-4 to modulate T1D, we generated liCTLA-4 transgenic NOD mice and compared their diabetes susceptibility to non-transgenic littermates. NOD liCTLA-4 transgenic mice were protected from T1D to the same extent as NOD.B10 Idd5.1 congenic mice, demonstrating that increased liCTLA-4 expression alone can account for disease protection. To further investigate the in vivo function of liCTLA-4, specifically whether liCTLA-4 can functionally replace flCTLA-4 in vivo, we expressed the liCTLA-4 transgene in CTLA-4-/- B6 mice. CTLA-4-/- mice expressing liCTLA-4 accumulated fewer activated effector/memory CD4+ T cells than CTLA-4-/- mice and the transgenic mice were partially rescued from the multiorgan inflammation and early lethality caused by the disruption of Ctla4. These results suggest that liCTLA-4 can partially replace some functions of flCTLA-4 in vivo and that this isoform evolved to reinforce the function of flCTLA-4.
Rodent; T Cells; Autoimmunity; Cell Activation; Transgenic / Knockout Mice
In separate previous studies, we have shown that lipid-complexed amphotericin B (Abelcet [ABLC]) and liposomal amphotericin B (AmBisome [AmBi]) are efficacious against coccidioidal meningitis in rabbits. Here, we compared ABLC and AmBi directly in a coccidioidal meningitis model. Male New Zealand White rabbits were infected with 5 × 104 Coccidioides posadasii arthroconidia by direct cisternal puncture. Therapy with intravenous ABLC or AmBi at 7.5 or 15 mg/kg of body weight or sterile 5% dextrose water (D5W) began 5 days later. Clinical assessments were done daily; cerebrospinal fluid and blood samples were obtained on day 15 and upon euthanasia. Survivors to day 25 were euthanatized, the numbers of CFU in their tissues were determined, and histology analyses of the brains and spinal cords were done. Controls showed progressive disease, whereas animals treated with either dose of either drug showed few clinical signs of infection. All ABLC- or AmBi-treated rabbits survived, whereas eight of nine D5W-treated rabbits were euthanatized before day 25 (P < 0.0001). Numbers of CFU in the brains and spinal cords of ABLC- or AmBi-treated animals were 100- to 10,000-fold lower than those in the corresponding tissues of D5W-treated animals (P < 0.0006 to 0.0001). However, only two or fewer given a regimen of ABLC or AmBi were cured of infection in both tissues. Fewer ABLC-treated rabbits (four of eight treated with 7.5 mg/kg and five of eight treated with 15 mg/kg) than controls (nine of nine) had meningitis at any level of severity (P, 0.015 or 0.043 for animals treated with ABLC at 7.5 or 15 mg/kg, respectively). Although groups of rabbits treated with AmBi regimens did not have significantly fewer animals with meningitis than the control group (P > 0.05), ABLC and AmBi were not significantly different. In this model, intravenous ABLC and AmBi were similarly highly effective, with few clinical signs of infection, 100% survival, and significantly reduced fungal burdens among treated animals. There appeared to be little benefit in using the 15-mg/kg dosage of either formulation. There was no significant advantage of one drug over the other for this indication. Further studies are required to determine the lowest effective doses of these formulations.
It has been suggested that T cell immunoglobulin mucin (Tim)-1 expressed on T cells serves to positively costimulate T cell responses. However, crosslinking of Tim-1 by its ligand Tim-4 resulted in either activation or inhibition of T cell responses, thus raising the issue of whether Tim-1 can have a dual function as a costimulator. To resolve this issue, we tested a series of monoclonal antibodies specific for Tim-1 and identified two antibodies that showed opposite functional effects. One anti–Tim-1 antibody increased the frequency of antigen-specific T cells, the production of the proinflammatory cytokines IFN-γ and IL-17, and the severity of experimental autoimmune encephalomyelitis. In contrast, another anti–Tim-1 antibody inhibited the generation of antigen-specific T cells, production of IFN-γ and IL-17, and development of autoimmunity, and it caused a strong Th2 response. Both antibodies bound to closely related epitopes in the IgV domain of the Tim-1 molecule, but the activating antibody had an avidity for Tim-1 that was 17 times higher than the inhibitory antibody. Although both anti–Tim-1 antibodies induced CD3 capping, only the activating antibody caused strong cytoskeletal reorganization and motility. These data indicate that Tim-1 regulates T cell responses and that Tim-1 engagement can alter T cell function depending on the affinity/avidity with which it is engaged.
Hypertensive encephalopathy is a potentially fatal condition associated with cerebral edema and the breakdown of the blood-brain barrier (BBB). The molecular pathways leading to this condition, however, are unknown. We determined the role of δPKC, which is thought to regulate microvascular permeability, in the development of hypertensive encephalopathy using δV1-1 — a selective peptide inhibitor of δPKC. As a model of hypertensive encephalopathy, Dahl salt-sensitive rats were fed an 8% high-salt diet from 6 weeks of age and then were infused s.c. with saline, control TAT peptide, or δV1-1 using osmotic minipumps. The mortality rate and the behavioral symptoms of hypertensive encephalopathy decreased significantly in the δV1-1–treated group relative to the control-treated group, and BBB permeability was reduced by more than 60%. Treatment with δV1-1 was also associated with decreased δPKC accumulation in capillary endothelial cells and in the endfeet of capillary astrocytes, which suggests decreased microvasculature disruption. Treatment with δV1-1 prevented hypertension-induced tight junction disruption associated with BBB breakdown, which suggests that δPKC may specifically act to dysregulate tight junction components. Together, these results suggest that δPKC plays a role in the development of hypertension-induced encephalopathy and may be a therapeutic target for the prevention of BBB disruption.
Peroxisome proliferator–activated receptor (PPAR)α is a nuclear receptor that mediates gender differences in lipid metabolism. PPARα also functions to control inflammatory responses by repressing the activity of nuclear factor κB (NF-κB) and c-jun in immune cells. Because PPARα is situated at the crossroads of gender and immune regulation, we hypothesized that this gene may mediate sex differences in the development of T cell–mediated autoimmune disease. We show that PPARα is more abundant in male as compared with female CD4+ cells and that its expression is sensitive to androgen levels. Genetic ablation of this gene selectively removed the brake on NF-κB and c-jun activity in male T lymphocytes, resulting in higher production of interferon γ and tumor necrosis factor (but not interleukin 17), and lower production of T helper (Th)2 cytokines. Upon induction of experimental autoimmune encephalomyelitis, male but not female PPARα−/− mice developed more severe clinical signs that were restricted to the acute phase of disease. These results suggest that males are less prone to develop Th1-mediated autoimmunity because they have higher T cell expression of PPARα.