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Although IL-23 and downstream signal transduction play essential roles in neuroinflammation, the local impact of IL-23 in multiple sclerosis is still not fully understood. Our previous study revealed that the central nervous system (CNS)-restricted expression of IL-23 in a mouse model with astrocyte-specific expression of IL-23, called GF-IL23 mice, leads to spontaneous formation of infiltrates in the brain, especially in the cerebellum. To further investigate the impact of CNS-specific IL-23-expression on neuroinflammation, we studied the GF-IL23 model in mice expressing a myelin oligodendrocyte glycoprotein (MOG)-specific T cell receptor (GF23-2D2 mice). The GF23-2D2 mice developed a chronic progressive experimental autoimmune encephalomyelitis with myelitis and ataxia without requiring additional immunization. CNS-production of IL-23 alone induced pronounced neuroinflammation in the transgenic MOG-specific T cell receptor model. The GF23-2D2 mice spontaneously developed multilocular infiltrates with a high number of B cells, demyelination and a proinflammatory cytokine milieu indicating that the interaction of encephalitogenic T cells and B cells via co-stimulatory factors seemed to be crucial., (© 2022. The Author(s).)

Guillain-Barre syndrome (GBS) is an autoimmune disease of demyelination and inflammation of peripheral nerves. Current treatments are limited to plasma exchange and intravenous immunoglobulins. Cargo-free nanoparticles (NPs) have been evaluated here for their therapeutic benefit on the disease course of experimental autoimmune neuritis (EAN), mimicking the human GBS. NPs prepared from poly-lactic co-glycolic acid (PLGA) with variable size and surface charge (i.e., 500 nm vs. 130 nm, polyvinyl alcohol (PVA) vs. sodium cholate), were intravenously administered in before- or early-onset treatment schedules in a rat EAN model. NP treatment mitigated distinctly the clinical severity of EAN as compared to the P2-peptide control group (P2) in all treatments and reduced the trafficking of inflammatory monocytes at inflammatory loci and diverted them towards the spleen. Therapeutic treatment with NPs reduced the expression of proinflammatory markers (CD68 (P2: 34.8 ± 6.6 vs. NP: 11.9 ± 2.3), IL-1β (P2: 18.3 ± 0.8 vs. NP: 5.8 ± 2.2), TNF-α (P2: 23.5 ± 3.7 vs. NP: 8.3 ± 1.7) and elevated the expression levels of anti-inflammatory markers CD163 (P2: 19.7 ± 3.0 vs. NP: 41.1 ± 6.5; all for NP-PVA of 130 nm; relative to healthy control). These results highlight the therapeutic potential of such cargo-free NPs in treating EAN, which would be easily translatable into clinical use due to their well-known low-toxicity profile.

Interleukin (IL)-23 is one of the critical cytokines in autoimmune neuroinflammation. To further clarify the local function of IL-23 on the course of neuroinflammation, we recently established a transgenic mouse model with astrocyte-specific expression of IL-23 (GF-IL23). The GF-IL23 mice spontaneously developed a progressive ataxic phenotype with cerebellar infiltration with high amounts of B cells most prominent in the subarachnoid and perivascular space. To enlighten the B cell role in GF-IL23 mice, we generated GF-IL23 mice on a B cell knockout (k.o.) background (GF-IL23 B cell k.o.). GF-IL23 B cell k.o. mice compared with GF-IL23 mice had no infiltrates or only minor infiltration, and no antibody deposition was detected in the cerebellum. Furthermore, microglia, astrocyte activation, hypervascularization and demyelination were reduced in GF-IL23 B cell k.o. mice compared with GF-IL23 mice. Cytokines and chemokine receptors like IL-12a, cerebrospinal fluid 2 and CXCR3 were downregulated. Our study indicates that B cells are essential in IL-23-dependent neuroinflammation in the GF-IL23 model., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background & Aims: In celiac disease (CeD), gluten induces immune activation, leading to enteropathy. TAK-101, gluten protein (gliadin) encapsulated in negatively charged poly(dl-lactide-co-glycolic acid) nanoparticles, is designed to induce gluten-specific tolerance., Methods: TAK-101 was evaluated in phase 1 dose escalation safety and phase 2a double-blind, randomized, placebo-controlled studies. Primary endpoints included pharmacokinetics, safety, and tolerability of TAK-101 (phase 1) and change from baseline in circulating gliadin-specific interferon-γ-producing cells at day 6 of gluten challenge, in patients with CeD (phase 2a). Secondary endpoints in the phase 2a study included changes from baseline in enteropathy (villus height to crypt depth ratio [Vh:Cd]), and frequency of intestinal intraepithelial lymphocytes and peripheral gut-homing T cells., Results: In phase 2a, 33 randomized patients completed the 14-day gluten challenge. TAK-101 induced an 88% reduction in change from baseline in interferon-γ spot-forming units vs placebo (2.01 vs 17.58, P = .006). Vh:Cd deteriorated in the placebo group (-0.63, P = .002), but not in the TAK-101 group (-0.18, P = .110), although the intergroup change from baseline was not significant (P = .08). Intraepithelial lymphocyte numbers remained equal. TAK-101 reduced changes in circulating α4β7 + CD4 + (0.26 vs 1.05, P = .032), αEβ7 + CD8 + (0.69 vs 3.64, P = .003), and γδ (0.15 vs 1.59, P = .010) effector memory T cells. TAK-101 (up to 8 mg/kg) induced no clinically meaningful changes in vital signs or routine clinical laboratory evaluations. No serious adverse events occurred., Conclusions: TAK-101 was well tolerated and prevented gluten-induced immune activation in CeD. The findings from the present clinical trial suggest that antigen-specific tolerance was induced and represent a novel approach translatable to other immune-mediated diseases. ClinicalTrials.gov identifiers: NCT03486990 and NCT03738475., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Interleukin 23 is a critical cytokine in the pathogenesis of multiple sclerosis. But the local impact of interleukin 23 on the course of neuroinflammation is still not well defined. To further characterize the effect of interleukin 23 on CNS inflammation, we recently described a transgenic mouse model with astrocyte-specific expression of interleukin 23 (GF-IL23 mice). The GF-IL23 mice spontaneously develop a progressive ataxic phenotype with cerebellar tissue destruction and inflammatory infiltrates with high amounts of B cells most prominent in the subarachnoid and perivascular space., Methods: To further elucidate the local impact of the CNS-specific interleukin 23 synthesis in autoimmune neuroinflammation, we induced a MOG35-55 experimental autoimmune encephalomyelitis (EAE) in GF-IL23 mice and WT mice and analyzed the mice by histology, flow cytometry, and transcriptome analysis., Results: We were able to demonstrate that local interleukin 23 production in the CNS leads to aggravation and chronification of the EAE course with a severe paraparesis and an ataxic phenotype. Moreover, enhanced multilocular neuroinflammation was present not only in the spinal cord, but also in the forebrain, brainstem, and predominantly in the cerebellum accompanied by persisting demyelination. Thereby, interleukin 23 creates a pronounced proinflammatory response with accumulation of leukocytes, in particular B cells, CD4+ cells, but also γδ T cells and activated microglia/macrophages. Furthermore, transcriptome analysis revealed an enhanced proinflammatory cytokine milieu with upregulation of lymphocyte activation markers, co-stimulatory markers, chemokines, and components of the complement system., Conclusion: Taken together, the GF-IL23 model allowed a further breakdown of the different mechanisms how IL-23 drives neuroinflammation in the EAE model and proved to be a useful tool to further dissect the impact of interleukin 23 on neuroinflammatory models.

Background & Aims: Celiac disease could be treated, and potentially cured, by restoring T-cell tolerance to gliadin. We investigated the safety and efficacy of negatively charged 500-nm poly(lactide-co-glycolide) nanoparticles encapsulating gliadin protein (TIMP-GLIA) in 3 mouse models of celiac disease. Uptake of these nanoparticles by antigen-presenting cells was shown to induce immune tolerance in other animal models of autoimmune disease., Methods: We performed studies with C57BL/6; RAG1 -/- (C57BL/6); and HLA-DQ8, huCD4 transgenic Ab0 NOD mice. Mice were given 1 or 2 tail-vein injections of TIMP-GLIA or control nanoparticles. Some mice were given intradermal injections of gliadin in complete Freund's adjuvant (immunization) or of soluble gliadin or ovalbumin (ear challenge). RAG -/- mice were given intraperitoneal injections of CD4 + CD62L - CD44 hi T cells from gliadin-immunized C57BL/6 mice and were fed with an AIN-76A-based diet containing wheat gluten (oral challenge) or without gluten. Spleen or lymph node cells were analyzed in proliferation and cytokine secretion assays or by flow cytometry, RNA sequencing, or real-time quantitative polymerase chain reaction. Serum samples were analyzed by gliadin antibody enzyme-linked immunosorbent assay, and intestinal tissues were analyzed by histology. Human peripheral blood mononuclear cells, or immature dendritic cells derived from human peripheral blood mononuclear cells, were cultured in medium containing TIMP-GLIA, anti-CD3 antibody, or lipopolysaccharide (controls) and analyzed in proliferation and cytokine secretion assays or by flow cytometry. Whole blood or plasma from healthy volunteers was incubated with TIMP-GLIA, and hemolysis, platelet activation and aggregation, and complement activation or coagulation were analyzed., Results: TIMP-GLIA did not increase markers of maturation on cultured human dendritic cells or induce activation of T cells from patients with active or treated celiac disease. In the delayed-type hypersensitivity (model 1), the HLA-DQ8 transgenic (model 2), and the gliadin memory T-cell enteropathy (model 3) models of celiac disease, intravenous injections of TIMP-GLIA significantly decreased gliadin-specific T-cell proliferation (in models 1 and 2), inflammatory cytokine secretion (in models 1, 2, and 3), circulating gliadin-specific IgG/IgG2c (in models 1 and 2), ear swelling (in model 1), gluten-dependent enteropathy (in model 3), and body weight loss (in model 3). In model 1, the effects were shown to be dose dependent. Splenocytes from HLA-DQ8 transgenic mice given TIMP-GLIA nanoparticles, but not control nanoparticles, had increased levels of FOXP3 and gene expression signatures associated with tolerance induction., Conclusions: In mice with gliadin sensitivity, injection of TIMP-GLIA nanoparticles induced unresponsiveness to gliadin and reduced markers of inflammation and enteropathy. This strategy might be developed for the treatment of celiac disease., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Arthritogenic alphaviruses such as Ross River and Chikungunya viruses cause debilitating muscle and joint pain and pose significant challenges in the light of recent outbreaks. How host immune responses are orchestrated after alphaviral infections and lead to musculoskeletal inflammation remains poorly understood. Here, we show that myositis induced by Ross River virus (RRV) infection is driven by CD11b hi Ly6C hi inflammatory monocytes and followed by the establishment of a CD11b hi Ly6C lo CX 3 CR1 + macrophage population in the muscle upon recovery. Selective modulation of CD11b hi Ly6C hi monocyte migration to infected muscle using immune-modifying microparticles (IMP) reduced disease score, tissue damage, and inflammation and promoted the accumulation of CX 3 CR1 + macrophages, enhancing recovery and resolution. Here, we detail the role of immune pathology, describing a poorly characterized muscle macrophage subset as part of the dynamics of alphavirus-induced myositis and tissue recovery and identify IMP as an effective immunomodulatory approach. Given the lack of specific treatments available for alphavirus-induced pathologies, this study highlights a therapeutic potential for simple immune modulation by IMP in infected individuals in the event of large alphavirus outbreaks. IMPORTANCE Arthritogenic alphaviruses cause debilitating inflammatory disease, and current therapies are restricted to palliative approaches. Here, we show that following monocyte-driven muscle inflammation, tissue recovery is associated with the accumulation of CX 3 CR1 + macrophages in the muscle. Modulating inflammatory monocyte infiltration using immune-modifying microparticles (IMP) reduced tissue damage and inflammation and enhanced the formation of tissue repair-associated CX 3 CR1 + macrophages in the muscle. This shows that modulating key effectors of viral inflammation using microparticles can alter the outcome of disease by facilitating the accumulation of macrophage subsets associated with tissue repair., (Copyright © 2020 Zaid et al.)

Recently, two chimeric antigen receptor (CAR) T cell therapies were approved based on their remarkable efficacy in patients with hematological malignancies. By contrast, CAR-T cell therapies results in solid tumors have been less promising. To develop the next generation of T cell therapies a better understanding of T cell receptor (TCR) biology and its implication for the design of synthetic receptors is critical. Here, we review current and newly developed forms of T cell therapies and how their utilization of different components of the TCR signaling machinery and their requirement for engagement (or not) of human leukocyte antigen impacts their design, efficacy and applicability as cancer drugs. Notably, we highlight the development of human leukocyte antigen-independent T cell platforms that utilize the full TCR complex as having promise to overcome some of the limitations of existing T cell therapies.

Interleukin 23 (IL-23) is a key mediator in neuroinflammation in numerous autoimmune diseases including multiple sclerosis (MS). However, the pathophysiology of IL-23 and how it contributes to neuroinflammation is poorly defined. To further clarify the role of IL-23 in CNS inflammation, we generated a transgenic mouse model (GF-IL23) with astrocyte-targeted expression of both IL-23 subunits, IL-23p19, and IL-23p40. These GF-IL23 mice spontaneously develop a progressive ataxic phenotype, which corresponds to cerebellar tissue destruction, and inflammatory infiltrates most prominent in the subarachnoidal and perivascular space. The CNS-cytokine milieu was characterized by numerous inflammatory mediators such as IL-17a and IFNγ. However, the leukocytic infiltrates were surprisingly predominated by B cells. To further examine the impact of the CNS-specific IL-23 synthesis on an additional systemic inflammatory stimulus, we applied the LPS-induced endotoxemia model. Administration of LPS in GF-IL23 mice resulted in early and pronounced microglial activation, enhanced cytokine production and, in sharp contrast to control animals, in the formation of lymphocytic infiltrates. Our model confirms a critical role for IL-23 in the induction of inflammation in the CNS, in particular facilitating the accumulation of lymphocytes in and around the brain. Thereby, CNS-specific synthesis of IL-23 is able to induce a cascade of inflammatory cytokines leading to microglia activation, astrocytosis, and ultimately tissue damage. The presented transgenic model will be a useful tool to further dissect the role of IL-23 in neuroinflammation.

Autoimmune diseases, such as celiac disease, multiple sclerosis, and type 1 diabetes, are leading causes of morbidity and mortality in the United States. In these disease states, immune regulatory mechanisms fail that result in T and B cell-mediated destruction of self-tissues. The known role of T cells in mediating autoimmune diseases has led to the emergence of numerous therapies aimed at inactivating T cells, however successful 'tolerance-inducing' strategies have not yet emerged for approved standard-of-care clinical use. In this review, we describe relevant examples of antigen-specific tolerance approaches that have been applied in clinical trials for human diseases. Furthermore, we describe the evolution of biomaterial approaches from cell-based therapies to induce immune tolerance with a focus on the Tolerogenic Immune-Modifying nanoParticle (TIMP) platform. The TIMP platform can be designed to treat various autoimmune conditions and is currently in clinical trials testing its ability to reverse celiac disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Current treatment of severe malaria and associated cerebral malaria (CM) and respiratory distress syndromes are directed primarily at the parasite. Targeting the parasite has only partial efficacy in advanced infection, as neurological damage and respiratory distress are due to accumulation of host blood cells in the brain microvasculature and lung interstitium. Here, computational analysis identifies Ly6C lo monocytes as a major component of the immune infiltrate in both organs in a preclinical mouse model. Specifically targeting Ly6C lo monocyte precursors, identified by adoptive transfer, with immune-modifying particles (IMP) prevents experimental CM (ECM) in 50% of Plasmodium berghei ANKA-infected mice in early treatment protocols. Furthermore, treatment at onset of clinical ECM with 2 doses of a novel combination of IMP and anti-malarial drug artesunate results in 88% survival. This combination confers protection against ECM and mortality in late stage severe experimental malaria and provides a viable advance on current treatment regimens., Competing Interests: N.K. and D.G. declare the following competing interests: research support and stock option in the company Cour Pharmaceutical Development Inc. The remaining authors declare no competing interests.

Type 1 diabetes (T1D) is mediated by destruction of pancreatic β cells by autoantigen-specific CD4 + and CD8 + T cells, thus the ideal solution for T1D is the restoration of immune tolerance to β cell antigens. We demonstrate the ability of carboxylated 500 nm biodegradable poly(lactide-co-glycolide) (PLG) nanoparticles PLG nanoparticles (either surface coupled with or encapsulating the cognate diabetogenic peptides) to rapidly and efficiently restore tolerance in NOD.SCID recipients of both activated diabetogenic CD4 + BDC2.5 chromagranin A-specific and CD8 + NY8.3 islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific TCR transgenic T cells in an antigen-specific manner. Further, initiation and maintenance of Ag-PLG tolerance operates via several overlapping, but independent, pathways including regulation via negative-co-stimulatory molecules (CTLA-4 and PD-1) and the systemic induction of peptide-specific Tregs which were critical for long-term maintenance of tolerance by controlling both trafficking of effector T cells to, and their release of pro-inflammatory cytokines within the pancreas, concomitant with selective retention of effector cells in the spleens of recipient mice., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Polymeric nanoparticles (NPs) have demonstrated their potential to induce antigen (Ag)-specific immunological tolerance in multiple immune models and are at various stages of commercial development. Association of Ag with NPs is typically achieved through surface coupling or encapsulation methods. However, these methods have limitations that include high polydispersity, uncontrollable Ag loading and release, and possible immunogenicity. Here, using antigenic peptides conjugated to poly(lactide-co-glycolide), we developed Ag-polymer conjugate NPs (acNPs) with modular loading of single or multiple Ags, negligible burst release, and minimally exposed surface Ag. Tolerogenic responses of acNPs were studied in vitro to decouple the role of NP size, concentration, and Ag loading on regulatory T cell (Treg) induction. CD4 + CD25 + Foxp3 + Treg induction was dependent on NP size, but CD25 expression of CD4 + T cells was not. NP concentration and Ag loading could be modulated to achieve maximal levels of Treg induction. In relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a murine model of multiple sclerosis, acNPs were effective in inhibiting disease induced by a single peptide or multiple peptides. The acNPs provide a simple, modular, and well-defined platform, and the NP physicochemical properties offer potential to design and answer complex mechanistic questions surrounding NP-induced tolerance., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common fatal sporadic encephalitis in developed countries. There is evidence from HSE animal models that not only direct virus-mediated damage caused but also the host's immune response contributes to the high mortality of the disease. Chemokines modulate and orchestrate this immune response. Previous experimental studies in HSE models identified the chemokine receptor CXCR3 and its ligands as molecules with a high impact on the course of HSE in mouse models. In this study, the role of the chemokine receptor CXCR3 was evaluated after intranasal infection with the encephalitogenic HSV-1 strain 17 syn + using CXCR3-deficient mice (CXCR3 -/- ) and wild-type controls. We demonstrated a neurotropic viral spread into the CNS of after intranasal infection. Although viral load and histological distribution of infected neurons were independent from CXCR3 signaling early after infection, CXCR3-deficient mice cleared HSV-1 more efficiently 14 days after infection. Furthermore, CXCR3 deficiency led to a decreased weight loss in mice after HSV-1 infection. T cell infiltration and microglial activation was prominently reduced by inhibition of CXCR3 signaling. Quantitative PCR of proinflammatory cytokines and chemokines confirmed the reduced neuroinflammatory response in CXCR3-deficient mice during HSE. Our results demonstrate that the recruitment of peripheral immune cells into the CNS, induction of neuroinflammation, and consecutive weight loss during herpes encephalitis is modulated by CXCR3 signaling. Interruption of the CXCR3 pathway ameliorates the detrimental host immune response and in turn, leads paradoxically to an enhanced viral clearance after intranasal infection. Our data gives further insight into the role of CXCR3 during HSE after intranasal infection.

Specific immunotherapy (SIT) is the most widely used treatment for allergic diseases that directly targets the T helper 2 (Th2) bias underlying allergy. However, the most widespread clinical applications of SIT require a long period of dose escalation with soluble antigen (Ag) and carry a significant risk of adverse reactions, particularly in highly sensitized patients who stand to benefit most from a curative treatment. Thus, the development of safer, more efficient methods to induce Ag-specific immune tolerance is critical to advancing allergy treatment. We hypothesized that antigen-associated nanoparticles (Ag-NPs), which we have used to prevent and treat Th1/Th17-mediated autoimmune disease, would also be effective for the induction of tolerance in a murine model of Th2-mediated ovalbumin/alum-induced allergic airway inflammation. We demonstrate here that antigen-conjugated polystyrene (Ag-PS) NPs, although effective for the prophylactic induction of tolerance, induce anaphylaxis in presensitized mice. Antigen-conjugated NPs made of biodegradable poly(lactide-co-glycolide) (Ag-PLG) are similarly effective prophylactically, are well tolerated by sensitized animals, but only partially inhibit Th2 responses when administered therapeutically. PLG NPs containing encapsulated antigen [PLG(Ag)], however, were well tolerated and effectively inhibited Th2 responses and airway inflammation both prophylactically and therapeutically. Thus, we illustrate progression toward PLG(Ag) as a biodegradable Ag carrier platform for the safe and effective inhibition of allergic airway inflammation without the need for nonspecific immunosuppression in animals with established Th2 sensitization.

Unlabelled: The initiating events in autoimmune disease remain to be completely understood, but it is thought that genetic predisposition synergizes with "environmental" factors, including viral infection, leading to disease. One elegant animal model used to study the pathogenesis of multiple sclerosis that perfectly blends genetics and environmental components in the context of virus-induced autoimmunity is Theiler's murine encephalitis virus-induced demyelinating disease (TMEV-IDD). TMEV-infected disease-susceptible SJL/J mice develop a persistent central nervous system (CNS) infection and later develop autoimmune demyelination, while disease-resistant C57BL/6 (B6) mice rapidly clear the infection and develop no autoimmune pathology. Mice of the (B6 × SJL/J)F1 cross between these two mouse strains are classified as intermediately susceptible. We employed this model to investigate if rapid virus clearance in B6 versus SJL/J mice was perhaps related to differences in the innate immune response in the CNS of the two strains in the first few days following intracerebral virus inoculation. Here we show that SJL/J mice lack, in addition to NK cells, a novel innate immune subset known as natural killer dendritic cells (NKDCs), which express phenotypic markers (CD11c(int) NK1.1(+)) and functional activity of both NK cells and DCs. These NKDCs are activated in the periphery and migrate into the infected CNS in a very late antigen 4 (VLA-4)-dependent fashion. Most significantly, NKDCs are critical for CNS clearance of TMEV, as transfer of NKDCs purified from B6 mice into TMEV-IDD-susceptible (B6 × SJL/J)F1 mice promotes viral clearance. Together the findings of this work show for the first time a link between NKDCs, viral infection, and CNS autoimmunity., Importance: Viral infection is an important cofactor, along with genetic susceptibility, in the initiation of a variety of organ-specific autoimmune diseases. Thus, in-depth understanding of how virus infections trigger autoimmunity may lead to novel ways to prevent or treat these diseases. Theiler's murine encephalitis virus-induced demyelinating disease (TMEV-IDD) serves as an important model for the human T cell-mediated autoimmune demyelinating disease multiple sclerosis. Induction of TMEV-IDD is genetically controlled as SJL/J mice develop persistent central nervous system (CNS) infection leading to chronic autoimmune demyelination, while C57BL/6 mice rapidly clear virus and are disease resistant. We determined that, as opposed to resistant B6 mice, disease-susceptible SJL/J mice lacked a unique innate immune population, the natural killer dendritic cell (NKDC), which was shown to play a critical role in early CNS virus clearance via its ability to both present virus antigen to T cells and to lyse target cells., (Copyright © 2015 Chastain et al.)

Recent approaches using nanoparticles engineered for immune regulation have yielded promising results in preclinical models of disease. The number of nanoparticle therapies is growing, fueled by innovations in nanotechnology and advances in understanding of the underlying pathogenesis of immune-mediated diseases. In particular, recent mechanistic insight into the ways in which nanoparticles interact with the mononuclear phagocyte system and impact its function during homeostasis and inflammation have highlighted the potential of nanoparticle-based therapies for controlling severe inflammation while concurrently restoring peripheral immune tolerance in autoimmune disease. Here we review recent advances in nanoparticle-based approaches aimed at immune-modulation, and discuss these in the context of concepts in polymeric nanoparticle development, including particle modification, delivery and the factors associated with successful clinical deployment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

IRF8 (interferon-regulatory factor-8) plays a critical role in regulating myeloid cell differentiation. However, the role of this transcription factor in the development of Ly6C+ inflammatory monocytes and their migration to the infected brain has not been examined. We have previously shown that West Nile virus (WNV) infection of wild-type (WT) mice triggers a significant increase in numbers of Ly6C+ monocytes in the bone marrow. These cells traffic via the blood to the infected brain, where they give rise to proinflammatory macrophages. Here, we show that WNV-infected IRF8-deficient (IRF8-/-) mice had significantly reduced numbers of Ly6C+ monocytes in the periphery, with few of these cells found in the blood. Furthermore, low numbers of inflammatory monocyte-derived macrophages were observed in the brains of IRF8-/- mice throughout infection. Adoptive transfer of IRF8-/- Ly6C+ monocytes demonstrated that these cells were intrinsically unable to traffic to the inflamed brain. Low expression of the chemokine receptor CCR2 and integrin VLA-4 by IRF8-/- monocytes likely contributed to this defect, as the interactions between these proteins and their ligands are critical for monocyte egress and migration to inflammatory foci. These data highlight a critical role for IRF8 in inflammatory monocyte differentiation and migration during WNV infection., (© 2014 S. Karger AG, Basel.)

Background and Objectives: TOL101 is a highly selective murine anti-αβ T cell receptor (TCR) IgM antibody and has recently completed phase II testing in primary renal transplant patients. This study was undertaken to determine the pharmacokinetic, pharmacodynamic, and immunogenic profile of TOL101., Methods: Nine cohorts of two to six patients received at least five daily doses (of, or combination of, 0.28, 1.4, 7, 14, 28, or 42 mg) of TOL-101 administered at successively higher doses. Semi-logarithmic graphs of serum TOL101 concentration versus time supported the use of a one-compartment intravenous infusion pharmacokinetic model. The model was parameterized in terms of serum clearance (CL) and volume of distribution (V d)., Results: There was a trend toward a decrease in serum CL as the dose increased from 1.4 to 28 mg. However, the mean values for CL and V d were consistent across the cohorts that received 28, 32, and 42 mg. The mean ± standard deviation half-lives for these five cohorts ranged from 15.1 ± 7.35 to 28.6 ± 8.46 h, with an overall mean of 23.8 h, supporting both daily as well as fixed (i.e., not based on weight) dosing. Using CD3+ ≤25 cells/mm(3) as the primary pharmacodynamic marker, all non-responders were in the 0.28, 1.4, or 7 mg cohorts, suggesting that starting doses above 14 mg are required. Finally, one patient out of 36 was found to have anti-drug antibody., Conclusions: Together, the data show that while TOL101 is a highly potent anti-TCR antibody, its pharmacological profile is somewhat versatile, allowing for daily dosing without immunogenicity concerns.

TOL101 is a murine IgM mAb targeting the αβ TCR. Unlike other T cell targets, the αβ TCR has no known intracellular signaling domains and may provide a nonmitogenic target for T cell inactivation. We report the 6-month Phase 2 trial data testing TOL101 in kidney transplantation. The study was designed to identify a dose that resulted in significant CD3 T cell modulation (<25 T cell/mm(3) ), to examine the safety and tolerability of TOL101 and to obtain preliminary efficacy information. Thirty-six patients were enrolled and given 5-10 daily doses of TOL101; 33 patients completed dosing, while three discontinued after two doses due to a self-limiting urticarial rash. Infusion adjustments, antihistamines, steroids and dose escalation of TOL101 reduced the incidence of the rash. Doses of TOL101 above 28 mg resulted in prolonged CD3 modulation, with rapid recovery observed 7 days after therapy cessation. There were no cases of patient or graft loss. Few significant adverse events were reported, with one nosocomial pneumonia. There were five biopsy-confirmed acute cellular rejections (13.9%); however, no donor-specific antibodies were detected. Overall TOL101 was well-tolerated, supporting continued clinical development using the dose escalating 21-28-42-42-42 mg regimen., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Targeted immune tolerance is a coveted therapy for the treatment of a variety of autoimmune diseases, as current treatment options often involve nonspecific immunosuppression. Intravenous (iv) infusion of apoptotic syngeneic splenocytes linked with peptide or protein autoantigens using ethylene carbodiimide (ECDI) has been demonstrated to be an effective method for inducing peripheral, antigen-specific tolerance for treatment of autoimmune disease. Here, we show the ability of biodegradable poly(lactic-co-glycolic acid) (PLG) nanoparticles to function as a safe, cost-effective, and highly efficient alternative to cellular carriers for the induction of antigen-specific T cell tolerance. We describe the formulation of tolerogenic PLG particles and demonstrate that administration of myelin antigen-coupled particles both prevented and treated relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a CD4 T cell-mediated mouse model of multiple sclerosis (MS). PLG particles made on-site with surfactant modifications surpass the efficacy of commercially available particles in their ability to couple peptide and to prevent disease induction. Most importantly, myelin antigen-coupled PLG nanoparticles are able to significantly ameliorate ongoing disease and subsequent relapses when administered at onset or at peak of acute disease, and minimize epitope spreading when administered during disease remission. Therapeutic treatment results in significantly reduced CNS infiltration of encephalitogenic Th1 (IFN-γ) and Th17 (IL-17a) cells as well as inflammatory monocytes/macrophages. Together, these data describe a platform for antigen display that is safe, low-cost, and highly effective at inducing antigen-specific T cell tolerance. The development of such a platform carries broad implications for the treatment of a variety of immune-mediated diseases.

Inflammatory monocyte-derived effector cells play an important role in the pathogenesis of numerous inflammatory diseases. However, no treatment option exists that is capable of modulating these cells specifically. We show that infused negatively charged, immune-modifying microparticles (IMPs), derived from polystyrene, microdiamonds, or biodegradable poly(lactic-co-glycolic) acid, were taken up by inflammatory monocytes, in an opsonin-independent fashion, via the macrophage receptor with collagenous structure (MARCO). Subsequently, these monocytes no longer trafficked to sites of inflammation; rather, IMP infusion caused their sequestration in the spleen through apoptotic cell clearance mechanisms and, ultimately, caspase-3-mediated apoptosis. Administration of IMPs in mouse models of myocardial infarction, experimental autoimmune encephalomyelitis, dextran sodium sulfate-induced colitis, thioglycollate-induced peritonitis, and lethal flavivirus encephalitis markedly reduced monocyte accumulation at inflammatory foci, reduced disease symptoms, and promoted tissue repair. Together, these data highlight the intricate interplay between scavenger receptors, the spleen, and inflammatory monocyte function and support the translation of IMPs for therapeutic use in diseases caused or potentiated by inflammatory monocytes.

Lipocalin 2 (Lcn2) is a bacteriostatic factor produced during the innate immune response to bacterial infection. Whether Lcn2 has a function in viral infection is unknown. We investigated the regulation and function of Lcn2 in the central nervous system (CNS) of mice during West Nile virus (WNV) encephalitis. Lcn2 mRNA and protein were induced in the brain by day 5, and this induction increased further by day 7 postinfection but was delayed compared with the induction of the toll-like receptor 3 (TLR3) gene, retinoic acid-inducible gene 1 (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) gene. The Lcn2 mRNA and protein were both found at high levels in the choroid plexus, vascular endothelium, macrophage/microglia, and astrocytes. However, some neuronal subsets contained Lcn2 protein but no detectable mRNA. In Lcn2 knockout (KO) mice, with the exception of CXC motif chemokine 5 (CXCL5), which was significantly more downregulated than in wild-type (WT) mice, expression levels of a number of other host response genes were similar in the two genotypes. The brain from Lcn2 and WT mice with WNV encephalitis contained similar numbers of infiltrating macrophages, granulocytes, and T cells. Lcn2 KO and WT mice had no significant difference in tissue viral loads or survival after infection with different doses of WNV. We conclude that Lcn2 gene expression is induced to high levels in a time-dependent fashion in a variety of cells and regions of the CNS of mice with WNV encephalitis. The function of Lcn2 in the host response to WNV infection remains largely unknown, but our data indicate that it is dispensable as an antiviral or immunoregulatory factor in WNV encephalitis.

Immune tolerance remains the most promising yet elusive strategy for treating immune-mediated diseases. An experimental strategy showing promise in phase 1 clinical studies is the delivery of Ag cross-linked to apoptotic leukocytes using ethylene carbodiimide. This approach originated from demonstration of the profound tolerance-inducing ability of i.v. administered Ag-coupled splenocytes (Ag-SP) in mice, which has been demonstrated to treat T cell-mediated disorders including autoimmunity, allergy, and transplant rejection. Recent studies have defined the intricate interplay between the innate and adaptive immune systems in Ag-SP tolerance induction. Innate mechanisms include scavenger receptor-mediated uptake of Ag-SP by host APCs, Ag representation, and the required upregulation of PD-L1 expression and IL-10 production by splenic marginal zone macrophages leading to Ag-specific T cell regulation via the combined effects of cell-intrinsic anergy and regulatory T cell induction. In this paper, we discuss the history, advantages, current mechanistic understanding, and clinical potential of tolerance induction using apoptotic Ag-coupled apoptotic leukocytes.

Mosquito-borne flaviviruses are a major current and emerging threat, affecting millions of people worldwide. Global climate change, combined with increasing proximity of humans to animals and mosquito vectors by expansion into natural habitats, coupled with the increase in international travel, have resulted in significant spread and concomitant increase in the incidence of infection and severe disease. Although neuroinvasive disease has been well described for some viral infections such as Japanese Encephalitis virus (JEV) and West Nile virus (WNV), others such as dengue virus (DENV) have recently displayed an emerging pattern of neuroinvasive disease, distinct from the previously observed, systemically-induced encephalomyelopathy. In this setting, the immune response is a crucial component of host defence, in preventing viral dissemination and invasion of the central nervous system (CNS). However, subversion of the anti-viral activities of macrophages by flaviviruses can facilitate viral replication and spread, enhancing the intensity of immune responses, leading to severe immune-mediated disease which may be further exacerbated during the subsequent infection with some flaviviruses. Furthermore, in the CNS myeloid cells may be responsible for inducing specific inflammatory changes, which can lead to significant pathological damage during encephalitis. The interaction of virus and cells of the myeloid lineage is complex, and this interaction is likely responsible at least in part, for crucial differences between viral clearance and pathology. Recent studies on the role of myeloid cells in innate immunity and viral control, and the mechanisms of evasion and subversion used by flaviviruses are rapidly advancing our understanding of the immunopathological mechanisms involved in flavivirus encephalitis and will lead to the development of therapeutic strategies previously not considered.

As a group of disorders, autoimmunity ranks as the third most prevalent cause of morbidity and mortality in the Western World. However, the etiology of most autoimmune diseases remains unknown. Although genetic linkage studies support a critical underlying role for genetics, the geographic distribution of these disorders as well as the low concordance rates in monozygotic twins suggest that a combination of other factors including environmental ones are involved. Virus infection is a primary factor that has been implicated in the initiation of autoimmune disease. Infection triggers a robust and usually well-coordinated immune response that is critical for viral clearance. However, in some instances, immune regulatory mechanisms may falter, culminating in the breakdown of self-tolerance, resulting in immune-mediated attack directed against both viral and self-antigens. Traditionally, cross-reactive T-cell recognition, known as molecular mimicry, as well as bystander T-cell activation, culminating in epitope spreading, have been the predominant mechanisms elucidated through which infection may culminate in an T-cell-mediated autoimmune response. However, other hypotheses including virus-induced decoy of the immune system also warrant discussion in regard to their potential for triggering autoimmunity. In this review, we discuss the mechanisms by which virus infection and antiviral immunity contribute to the development of autoimmunity., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Monoclonal antibody (mAb) technology has revolutionized treatment options for T cell mediated diseases. However, a safe, clinically available anti-T cell antibody (ab) remains elusive. Experience with anti-T cell agents and their propensity for causing immune-mediated toxicities have hampered the development of anti-T cell mAb's. Furthermore, misunderstanding regarding mechanism(s) of action of particular antibodies can influence development and clinical prescription habits. For example, the anti-CD3 Ab OKT3 is consistently described as a depleting Ab even though original studies showed the mechanism to be non-lytic. Future anti-T cell mAbs are likely to be non-depletional and focused on the expansion of regulatory T cells. This review discusses how the properties of Abs can be exploited for manipulating pathological T cell responses in the clinic., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Monocytes are a heterogeneous population of bone marrow-derived cells that are recruited to sites of infection and inflammation in many models of human diseases, including those of the central nervous system (CNS). Ly6Chi/CCR2(hi) inflammatory monocytes have been identified as the circulating precursors of brain macrophages, dendritic cells and arguably microglia in experimental autoimmune encephalomyelitis; Alzheimer's disease; stroke; and more recently in CNS infection caused by Herpes simplex virus, murine hepatitis virus, Theiler's murine encephalomyelitis virus, Japanese encephalitis virus and West Nile virus. The precise differentiation pathways and functions of inflammatory monocyte-derived populations in the inflamed CNS remains a contentious issue, especially in regard to the existence of monocyte-derived microglia. Furthermore, the contributions of monocyte-derived subsets to viral clearance and immunopathology are not well-defined. Thus, understanding the pathways through which inflammatory monocytes migrate to the brain and their functional capacity within the CNS is critical to inform future therapeutic strategies. This review discusses some of the key aspects of inflammatory monocyte trafficking to the brain and addresses the role of these cells in viral encephalitis.

Aberrant T-cell activation underlies many autoimmune disorders, yet most attempts to induce T-cell tolerance have failed. Building on previous strategies for tolerance induction that exploited natural mechanisms for clearing apoptotic debris, we show that antigen-decorated microparticles (500-nm diameter) induce long-term T-cell tolerance in mice with relapsing experimental autoimmune encephalomyelitis. Specifically, intravenous infusion of either polystyrene or biodegradable poly(lactide-co-glycolide) microparticles bearing encephalitogenic peptides prevents the onset and modifies the course of the disease. These beneficial effects require microparticle uptake by marginal zone macrophages expressing the scavenger receptor MARCO and are mediated in part by the activity of regulatory T cells, abortive T-cell activation and T-cell anergy. Together these data highlight the potential for using microparticles to target natural apoptotic clearance pathways to inactivate pathogenic T cells and halt the disease process in autoimmunity.

Infiltration of Ly6C(hi) monocytes from the blood is a hallmark of viral encephalitis. In mice with lethal encephalitis caused by West Nile virus (WNV), an emerging neurotropic flavivirus, inhibition of Ly6C(hi) monocyte trafficking into the brain by anti-very late antigen (VLA)-4 integrin antibody blockade at the time of first weight loss and leukocyte influx resulted in long-term survival of up to 60% of infected mice, with subsequent sterilizing immunity. This treatment had no effect on viral titers but appeared to be due to inhibition of Ly6C(hi) macrophage immigration. Although macrophages isolated from the infected brain induced WNV-specific CD4(+) T-cell proliferation, T cells did not directly contribute to pathology, but are likely to be important in viral control, as antibody-mediated T-cell depletion could not reproduce the therapeutic benefit of anti-VLA-4. Instead, 70% of infiltrating inflammatory monocyte-derived macrophages were found to be making nitric oxide (NO). Furthermore, aminoguanidine-mediated inhibition of induced NO synthase activity in infiltrating macrophages significantly prolonged survival, indicating involvement of NO in the immunopathology. These data show for the first time the therapeutic effects of temporally targeting pathogenic NO-producing macrophages during neurotropic viral encephalitis.

Ag-specific tolerance is a highly desired therapy for immune-mediated diseases. Intravenous infusion of protein/peptide Ags linked to syngeneic splenic leukocytes with ethylene carbodiimide (Ag-coupled splenocytes [Ag-SP]) has been demonstrated to be a highly efficient method for inducing peripheral, Ag-specific T cell tolerance for treatment of autoimmune disease. However, little is understood about the mechanisms underlying this therapy. In this study, we show that apoptotic Ag-SP accumulate in the splenic marginal zone, where their uptake by F4/80(+) macrophages induces production of IL-10, which upregulates the expression of the immunomodulatory costimulatory molecule PD-L1 that is essential for Ag-SP tolerance induction. Ag-SP infusion also induces T regulatory cells that are dispensable for tolerance induction but required for long-term tolerance maintenance. Collectively, these results indicate that Ag-SP tolerance recapitulates how tolerance is normally maintained in the hematopoietic compartment and highlight the interplay between the innate and adaptive immune systems in the induction of Ag-SP tolerance. To our knowledge, we show for the first time that tolerance results from the synergistic effects of two distinct mechanisms, PD-L1-dependent T cell-intrinsic unresponsiveness and the activation of T regulatory cells. These findings are particularly relevant as this tolerance protocol is currently being tested in a Phase I/IIa clinical trial in new-onset relapsing-remitting multiple sclerosis.

In recent years, substantial advances in T-cell immunosuppressive strategies and their translation to routine clinical practice have revolutionized management and outcomes in autoimmune disease and solid organ transplantation. More than 80 diseases have been considered to have an autoimmune etiology, such that autoimmune-associated morbidity and mortality rank as third highest in developed countries, after cardiovascular diseases and cancer. Solid organ transplantation has become the therapy of choice for many end-stage organ diseases. Short-term outcomes such as patient and allograft survival at 1 year, acute rejection rates, as well as time course of disease progression and symptom control have steadily improved. However, despite the use of newer immunosuppressive drug combinations, improvements in long-term allograft survival and complete resolution of autoimmunity remain elusive. In addition, the chronic use of nonspecifically targeted immunosuppressive drugs is associated with significant adverse effects and increased morbidity and mortality. In this article, we discuss the current clinical tools for immune suppression and attempts to induce long-term T-cell tolerance induction as well as much-needed future approaches to produce more short-acting, antigen-specific agents, which may optimize outcomes in the clinic.

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.

IL-6 is crucial for the induction of many murine models of autoimmunity including experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. To establish the role of site-specific production of IL-6 in autoimmunity, we examined myelin oligodendrocyte glycoprotein immunization-induced EAE in transgenic mice (GFAP-IL6) with IL-6 production restricted to the cerebellum. Myelin oligodendrocyte glycoprotein-immunized (Mi-) GFAP-IL6 mice developed severe ataxia but no physical signs of spinal cord involvement, which was in sharp contrast to Mi-wild type (WT) animals that developed classical EAE with ascending paralysis. Immune pathology and demyelination were nearly absent from the spinal cord, but significantly increased in the cerebellum of Mi-GFAP-IL6 mice. Tissue damage in the cerebellum in the Mi-GFAP-IL6 mice was accompanied by increased total numbers of infiltrating leukocytes and increased proportions of both neutrophils and B-cells. With the exception of IL-17 mRNA, which was elevated in both control immunized and Mi-GFAP-IL6 cerebellum, the level of other cytokine and chemokine mRNAs were comparable with Mi-WT cerebellum whereas significantly higher levels of IFN-gamma and TNF-alpha mRNA were found in Mi-WT spinal cord. Thus, site-specific production of IL-6 in the cerebellum redirects trafficking away from the normally preferred antigenic site the spinal cord and acts as a leukocyte "sink" that markedly enhances the inflammatory cell accumulation and disease. The mechanisms underlying this process likely include the induction of specific chemokines, activation of microglia, and activation and loss of integrity of the blood-brain barrier present in the cerebellum of the GFAP-IL6 mice before the induction of EAE.

In a lethal West Nile virus (WNV) model, central nervous system infection triggered a threefold increase in CD45(int)/CD11b(+)/CD11c(-) microglia at days 6-7 postinfection (p.i.). Few microglia were proliferating, suggesting that the increased numbers were derived from a migratory precursor cell. Depletion of "circulating" (Gr1(-)(Ly6C(lo))CX3CR1(+)) and "inflammatory" (Gr1(hi)/Ly6C(hi)/CCR2(+)) classical monocytes during infection abrogated the increase in microglia. C57BL/6 chimeras reconstituted with cFMS-enhanced green fluorescent protein (EGFP) bone marrow (BM) showed large numbers of peripherally derived (GFP(+)) microglia expressing GR1(+)(Ly6C(+)) at day 7 p.i., suggesting that the inflammatory monocyte is a microglial precursor. This was confirmed by adoptive transfer of labeled BM (Ly6C(hi)/CD115(+)) or circulating inflammatory monocytes that trafficked to the WNV-infected brain and expressed a microglial phenotype. CCL2 is a chemokine that is highly expressed during WNV infection and important in inflammatory monocyte trafficking. Neutralization of CCL2 not only reduced the number of GFP(+) microglia in the brain during WNV infection but prolonged the life of infected animals. Therefore, CCL2-dependent inflammatory monocyte migration is critical for increases in microglia during WNV infection and may also play a pathogenic role during WNV encephalitis.

A major challenge for human allogeneic islet transplantation is the development of effective methods to induce donor-specific tolerance to obviate the need for life-long immunosuppression that is toxic to the insulin-producing beta cells and detrimental to the host. We developed an efficient donor-specific tolerance therapy that utilizes infusions of ethylene carbodiimide (ECDI)-treated donor splenic antigen-presenting cells that results in indefinite survival of allogeneic islet grafts in the absence of immunosuppression. Furthermore, we show that induction of tolerance is critically dependent on synergistic effects between an intact programmed death 1 receptor-programmed death ligand 1 signaling pathway and CD4(+)CD25(+)Foxp3(+) regulatory T cells. This highly efficient antigen-specific therapy with a complete avoidance of immunosuppression has significant therapeutic potential in human islet cell transplantation.

Multiple Sclerosis (MS) is an inflammatory demyelinating disorder of the central nervous system (CNS). Since current treatments are aimed at nonspecifically down-regulating inflammation and natural mechanisms of repair and remyelination within the CNS are inadequate for recovery of function, MS patients presently only have available treatments that delay symptom progression. The complex nature of this disease means that only multifaceted treatments hold the promise of a cure. Recent studies indicate that the ER stress response, a cellular pathway that allows a cell to survive and recover from a stressful event, could be elicited to help the myelin-generating and myelin-support cells of the CNS survive inflammatory insult.

Viral encephalitis affects approximately 7.5 people/100 000 and carries a high rate of morbidity and mortality. Most patients with viral encephalitis will develop some form of seizure during the infectious process, and of those who survive encephalitic disease, approximately 4-20% will develop epilepsy. Arthropod-borne (arbo)viruses are the leading cause of viral encephalitis in the world today, with between 10% and 35% of patients infected with these viruses displaying some form of seizure. Several neurotropic DNA viruses, including Herpes and cytomegalovirus also commonly cause seizures in infected patients. In the clinical setting, the cause of seizures seen during viral encephalitis is usually attributed to acute febrile responses. However, it has become apparent that the mechanisms behind seizure generation during viral encephalitis are likely to be much more complicated. For example, CD4(+) and CD8(+) T cells possibly through their secretion of interferon-gamma, appear to play an important role in determining neuronal responses when challenged with kainic acid. In addition, the ability of the human immunodeficiency virus, transactivating protein to modulate NMDA signaling possibly triggering seizures, highlights the fact that elements of the antiviral response and even virally derived proteins are capable of directly manipulating neuronal function. Understanding the complex relationships between the CNS, the immune system, and invading pathogens is a critical step in understanding the pathogenesis of seizures seen during viral infections and informing the development of novel therapies.

Seizures are a major complication of viral encephalitis. However, the mechanisms of seizure-associated neuronal dysfunction remain poorly understood. We report that intranasal inoculation with West Nile virus (WNV) (Sarafend) causes limbic seizures in C57BL/6 mice, but not in interferon (IFN)-gamma-deficient (IFN-gamma-/-) mice. Both strains showed similar levels of virus in the brain, as well as similar concentrations of the cytokines, tumor necrosis factor and interleukin-6, both of which can alter neuronal excitability. Experiments in chimeric IFN-gamma-/- mice reconstituted with IFN-gamma-producing leukocytes showed that IFN-gamma is not required during central nervous system infection for limbic seizure development, suggesting a role for IFN-gamma in the developing brain. This was supported responses to pentylenetetrazole, kainic acid (KA), and N-methyl-d-aspartate (NMDA). Both strains of mice exhibited similar behavior after pentylenetetrazole challenge. However, while NMDA and KA treatment resulted in characteristic seizures in C57BL/6 mice, these responses were diminished (NMDA treatment) or absent (KA treatment) in IFN-gamma-/- mice. Furthermore, NMDA-receptor blockade with MK-801 in WNV-infected C57BL/6 mice abrogated seizures and prolonged survival. Our data show that IFN-gamma plays an important role in the development of the excitatory seizure pathways in the brain and that these cascades become pathogenic in encephalitic WNV infection.

The chemokine receptor CXCR3 promotes the trafficking of activated T and NK cells in response to three ligands, CXCL9, CXCL10, and CXCL11. Although these chemokines are produced in the CNS in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), their role in the pathogenesis of CNS autoimmunity is unresolved. We examined the function of CXCR3 signaling in EAE using mice that were deficient for CXCR3 (CXCR3(-/-)). The time to onset and peak disease severity were similar for CXCR3(-/-) and wild-type (WT) animals; however, CXCR3(-/-) mice had more severe chronic disease with increased demyelination and axonal damage. The inflammatory lesions in WT mice consisted of well-demarcated perivascular mononuclear cell infiltrates, mainly in the spinal cord and cerebellum. In CXCR3(-/-) mice, these lesions were more widespread throughout the CNS and were diffused and poorly organized, with T cells and highly activated microglia/macrophages scattered throughout the white matter. Although the number of CD4(+) and CD8(+) T cells infiltrating the CNS were similar in CXCR3(-/-) and WT mice, Foxp3(+) regulatory T cells were significantly reduced in number and dispersed in CXCR3(-/-) mice. The expression of various chemokine and cytokine genes in the CNS was similar in CXCR3(-/-) and WT mice. The genes for the CXCR3 ligands were expressed predominantly in and/or immediately surrounding the mononuclear cell infiltrates. We conclude that in EAE, CXCR3 signaling constrains T cells to the perivascular space in the CNS and augments regulatory T cell recruitment and effector T cell interaction, thus limiting autoimmune-mediated tissue damage.

With the recent emergence of the flavivirus, West Nile virus (WNV), in particular, the New York strain of Lineage I WNV in North America in 1999, there has been a significant increase in activity in neurotropic flavivirus research. These viruses cause encephalitis that can result in permanent neurological sequelae or death. Attempts to develop vaccines have made progress, but have been variably successful, despite considerable commercial underwriting. Thus, the discovery of ways and means to combat disease is no less urgent. As such, most recent work has been directed towards dissecting and understanding the pathogenesis of disease, as a way of informing possible approaches to abrogation or amelioration of illness. Whether inherent to flaviviruses or because humans are incidental, dead-end hosts, it is clear that these viruses interact with their human hosts in extremely complex ways. This occurs from the cellular level, at which infection must be established to produce disease, to its interaction with the adaptive immune response, which may result in its eradication, with or without immunopathological and consequent neurological sequelae. As human proximity to and contact with flavivirus insect vectors and amplifying hosts cannot practically be eliminated, our understanding of the pathogenesis of flavivirus-induced diseases, especially with regard to possible targets for treatment, is imperative.

The interferon (IFN)-stimulated genes (ISGs) ISG-49, ISG-54, and ISG-56 are highly responsive to viral infection, yet the regulation and function of these genes in vivo are unknown. We examined the simultaneous regulation of these ISGs in the brains of mice during infection with either lymphocytic choriomeningitis virus (LCMV) or West Nile virus (WNV). Expression of the ISG-49 and ISG-56 genes increased significantly during LCMV infection, being widespread and localized predominantly to common as well as distinct neuronal populations. Expression of the ISG-54 gene also increased but to lower levels and with a more restricted distribution. Although expression of the ISG-49, ISG-54, and ISG-56 genes was increased in the brains of LCMV-infected STAT1 and STAT2 knockout (KO) mice, this was blunted, delayed, and restricted to the choroid plexus, meninges, and endothelium. ISG-56 protein was regulated in parallel with the corresponding RNA transcript in the brain during LCMV infection in wild-type and STAT KO mice. Similar changes in ISG-49, ISG-54, and ISG-56 RNA levels and ISG-56 protein levels were observed in the brains of wild-type mice following infection with WNV. Thus, the ISG-49, ISG-54, and ISG-56 genes are coordinately upregulated in the brain during LCMV and WNV infection; this upregulation, in the case of LCMV, was totally (neurons) or partially (non-neurons) dependent on the IFN-signaling molecules STAT1 and STAT2. These findings suggest a dominant role for the ISG-49, ISG-54, and ISG-56 genes in the host response to different viruses in the central nervous system, where, particularly in neurons, these genes may have nonredundant functions.