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Abstract Cumulative evidence has established that Interferon (IFN)-γ has both pathogenic and protective roles in Multiple Sclerosis and the animal model, Experimental Autoimmune Encephalomyelitis (EAE). However, the underlying mechanisms to the beneficial effects of IFN-γ are not well understood. In this study, we found that IFN-γ exerts therapeutic effects on chronic, relapsing-remitting, and chronic progressive EAE models. The frequency of regulatory T (Treg) cells in spinal cords from chronic EAE mice treated with IFN-γ was significantly increased with no effect on Th1 and Th17 cells. Consistently, depletion of FOXP3-expressing cells blocked the protective effects of IFN-γ, indicating that the therapeutic effect of IFN-γ depends on the presence of Treg cells. However, IFN-γ did not trigger direct in vitro differentiation of Treg cells. In vivo administration of blocking antibodies against either interleukin (IL)-10, transforming growth factor (TGF)-β or program death (PD)-1, revealed that the protective effects of IFN-γ in EAE were also dependent on TGF-β and PD-1, but not on IL-10, suggesting that IFN-γ might have an indirect role on Treg cells acting through antigen-presenting cells. Indeed, IFN-γ treatment increased the frequency of a subset of splenic CD11b+ myeloid cells expressing TGF-β-Latency Associated Peptide (LAP) and program death ligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)-1-dependent manner. Furthermore, splenic CD11b+ cells from EAE mice preconditioned in vitro with IFN-γ and myelin oligodendrocyte glycoprotein (MOG) peptide exhibited a tolerogenic phenotype with the capability to induce conversion of naïve CD4+ T cells mediated by secretion of TGF-β. Remarkably, adoptive transfer of splenic CD11b+ cells from IFN-γ-treated EAE mice into untreated recipient mice ameliorated clinical symptoms of EAE and limited central nervous system infiltration of mononuclear cells and effector helper T cells. These results reveal a novel cellular and molecular mechanism whereby IFN-γ promotes beneficial effects in EAE by endowing splenic CD11b+ myeloid cells with tolerogenic and therapeutic activities.

Summary: In response to corneal injury, an activation of corneal epithelial stem cells and their direct progeny the early transit amplifying (eTA) cells to rapidly proliferate is critical for proper re-epithelialization. Thus, it is important to understand how such stem/eTA cell activation is regulated. Angiotensin-converting enzyme 2 (ACE2) is predominantly expressed in the stem/eTA-enriched limbal epithelium but its role in the limbal epithelium was unclear. Single cell RNA sequencing (scRNA-seq) suggested that Ace2 involved the proliferation of the stem/eTA cells. Ace2 was reduced following corneal injury. Such reduction enhanced limbal epithelial proliferation and downregulated LCN2, a negative regulator of proliferation in a variety of tissues, via upregulating TGFA and consequently activating epidermal growth factor receptor (EGFR). Inhibition of EGFR or overexpression of LCN2 reversed the increased proliferation in limbal epithelial cells lacking ACE2. Our findings demonstrate that after corneal injury, ACE2 is downregulated, which activates limbal epithelial cell proliferation via a TGFA/EGFR/LCN2 pathway.

Abstract In acute skin injury, healing is impaired by the excessive release of reactive oxygen species (ROS). Melanin, an efficient scavenger of radical species in the skin, performs a key role in ROS scavenging in response to UV radiation and is upregulated in response to toxic insult. In a chemical injury model in mice, we demonstrate that the topical application of synthetic melanin particles (SMPs) significantly decreases edema, reduces eschar detachment time, and increases the rate of wound area reduction compared to vehicle controls. Furthermore, these results were replicated in a UV-injury model. Immune array analysis shows downregulated gene expression in apoptotic and inflammatory signaling pathways consistent with histological reduction in apoptosis. Mechanistically, synthetic melanin intervention increases superoxide dismutase (SOD) activity, decreases Mmp9 expression, and suppresses ERK1/2 phosphorylation. Furthermore, we observed that the application of SMPs caused increased populations of anti-inflammatory immune cells to accumulate in the skin, mirroring their decrease from splenic populations. To enhance antioxidant capacity, an engineered biomimetic High Surface Area SMP was deployed, exhibiting increased wound healing efficiency. Finally, in human skin explants, SMP intervention significantly decreased the damage caused by chemical injury. Therefore, SMPs are promising and effective candidates as topical therapies for accelerated wound healing, including via pathways validated in human skin.

Purpose: To determine whether there was a relationship between sleep position and symptomatic partial- and full-thickness rotator cuff tears. Methods: A consecutive series of patients that met the inclusion/exclusion criteria (n = 58) were in seen in clinic between July 2019 and December 2019. All of these individuals had a significant partial-thickness (> 50%) or full-thickness rotator cuff tear determined by either ultrasound, magnetic resonance imaging, or both. All patients in this series either had an insidious onset of shoulder pain or their symptoms were related to the basic wear and tear of daily activities. Traumatic rotator cuff tears (those associated with a significant traumatic event such as shoulder instability, motor vehicle accidents, sports related injuries, etc.) were excluded. Previous shoulder surgery, recurrent rotator cuff tears, and worker’s compensation cases also were excluded from this series. As part of the history-taking process, the patients were asked what was their preferred sleeping position—side sleeper, back sleeper, or stomach sleeper. A χ2 test was conducted to determine the relationship between rotator cuff pathology and sleep position. Results: Of the 58 subjects, 52 of the patients were side sleepers, 4 were stomach sleepers, 1 was a back sleeper, and 1 preferred all 3 positions. Statistical analysis, using the χ2 test (P < .0001), demonstrated that rotator cuff tears were most often seen in side sleepers. Conclusions: In our study, there appeared to be a relationship between the preference of being a side sleeper and the presence of a rotator cuff tear. Level of Evidence: Level IV, prognostic case series.

Mature oligodendrocytes (OLG) are the myelin-forming cells of the central nervous system. Recent work has shown a dynamic role for these cells in the plasticity of neural circuits, leading to a renewed interest in voltage-sensitive currents in OLG. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and their respective current (Ih) were recently identified in mature OLG and shown to play a role in regulating myelin length. Here we provide a biochemical and electrophysiological characterization of HCN channels in cells of the oligodendrocyte lineage. We observed that mice with a nonsense mutation in the Hcn2 gene (Hcn2ap/ap) have less white matter than their wild type counterparts with fewer OLG and fewer oligodendrocyte progenitor cells (OPCs). Hcn2ap/ap mice have severe motor impairments, although these deficits were not observed in mice with HCN2 conditionally eliminated only in oligodendrocytes (Cnpcre/+; Hcn2F/F). However, Cnpcre/+; Hcn2F/F mice develop motor impairments more rapidly in response to experimental autoimmune encephalomyelitis (EAE). We conclude that HCN2 channels in OLG may play a role in regulating metabolism.

A20 gene variants are linked with systemic sclerosis (SS), but the mechanisms are unclear. Here, the authors show that A20 expression is reduced in SS skin and lungs, that its ablation in mice induces SS, and that show that fibrosis can be ameliorated by induction of A20.

Compelling evidence has shown that interferon (IFN)-γ has dual effects in multiple sclerosis and in its animal model of experimental autoimmune encephalomyelitis (EAE), with results supporting both a pathogenic and beneficial function. However, the mechanisms whereby IFN-γ may promote neuroprotection in EAE and its effects on central nervous system (CNS)-resident cells have remained an enigma for more than 30 years. In this study, the impact of IFN-γ at the peak of EAE, its effects on CNS infiltrating myeloid cells (MC) and microglia (MG), and the underlying cellular and molecular mechanisms were investigated. IFN-γ administration resulted in disease amelioration and attenuation of neuroinflammation associated with significantly lower frequencies of CNS CD11b+ myeloid cells and less infiltration of inflammatory cells and demyelination. A significant reduction in activated MG and enhanced resting MG was determined by flow cytometry and immunohistrochemistry. Primary MC/MG cultures obtained from the spinal cord of IFN-γ-treated EAE mice that were ex vivo re-stimulated with a low dose (1 ng/ml) of IFN-γ and neuroantigen, promoted a significantly higher induction of CD4+ regulatory T (Treg) cells associated with increased transforming growth factor (TGF)-β secretion. Additionally, IFN-γ-treated primary MC/MG cultures produced significantly lower nitrite in response to LPS challenge than control MC/MG. IFN-γ-treated EAE mice had a significantly higher frequency of CX3CR1high MC/MG and expressed lower levels of program death ligand 1 (PD-L1) than PBS-treated mice. Most CX3CR1highPD-L1lowCD11b+Ly6G- cells expressed MG markers (Tmem119, Sall2, and P2ry12), indicating that they represented an enriched MG subset (CX3CR1highPD-L1low MG). Amelioration of clinical symptoms and induction of CX3CR1highPD-L1low MG by IFN-γ were dependent on STAT-1. RNA-seq analyses revealed that in vivo treatment with IFN-γ promoted the induction of homeostatic CX3CR1highPD-L1low MG, upregulating the expression of genes associated with tolerogenic and anti-inflammatory roles and down-regulating pro-inflammatory genes. These analyses highlight the master role that IFN-γ plays in regulating microglial activity and provide new insights into the cellular and molecular mechanisms involved in the therapeutic activity of IFN-γ in EAE.

Cancer treatment utilizing infusion therapies to enhance the patient’s own immune response against the tumor have shown significant functionality in a small subpopulation of patients. Additionally, advances have been made in the utilization of nanotechnology for the treatment of disease. We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic-co-glycolic acid) (PLGA) nanoparticles (IMPs; named ONP-302) for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.

Systemic sclerosis (SSc) is a chronic, multisystem orphan disease with a highly variable clinical course, high mortality rate, and a poorly understood complex pathogenesis. We have identified an important role for a subpopulation of monocytes and macrophages characterized by surface expression of the scavenger receptor macrophage receptor with collagenous structure (MARCO) in chronic inflammation and fibrosis in SSc and in preclinical disease models. We show that MARCO+ monocytes and macrophages accumulate in lesional skin and lung in topographic proximity to activated myofibroblasts in patients with SSc and in the bleomycin-induced mouse model of SSc. Short-term treatment of mice with a potentially novel nanoparticle, poly(lactic-co-glycolic) acid (PLG), which is composed of a carboxylated, FDA-approved, biodegradable polymer and modulates activation and trafficking of MARCO+ inflammatory monocytes, markedly attenuated bleomycin-induced skin and lung inflammation and fibrosis. Mechanistically, in isolated cells in culture, PLG nanoparticles inhibited TGF-dependent fibrotic responses in vitro. Thus, MARCO+ monocytes are potent effector cells of skin and lung fibrosis and can be therapeutically targeted in SSc using PLG nanoparticles.

Food allergy is a growing health concern worldwide. Current allergen-specific immunotherapy (AIT) approaches require frequent dosing over extended periods of time and may induce anaphylaxis due to allergen-effector cell interactions. A critical need remains to develop novel approaches that refine AIT for the treatment of food allergies. Previous studies show that poly(lactide-co-glycolide) (PLG) nanoscale particles (NP) effectively suppress Th1- and Th17-driven immune pathologies. However, their ability to suppress the distinct Th2-polarized immune responses driving food allergy are unknown. Herein, we describe the safety and efficacy of NPs containing encapsulated peanut allergen in desensitizing murine models of peanut allergy. Peanut extract encapsulation allowed for the safe intravenous delivery of allergen relative to non-encapsulated approaches. Application of 2–3 doses, without the need for dose escalation, was sufficient to achieve prophylactic and therapeutic efficacy, which correlated with suppression of Th2-mediated disease and reduced mast cell degranulation. Efficacy was associated with strong reductions in a broad panel of Th1, Th2, and Th17 cytokines. These results demonstrate the ability of PLG NPs to suppress allergen-specific immune responses to induce a more tolerogenic phenotype, conferring protection from intragastric allergen challenge. These promising studies represent a step forward in the development of improved immunotherapies for food allergy.

Monitoring changes in immune phenotype during the progression of multiple sclerosis can provide insight into disease progression and inform treatment. Here the authors develop engineered biomaterial-based immunological niches for easy access to innate immune cells in a mouse model of multiple sclerosis.

Neuromyelitis optica (NMO) is a chronic inflammatory disease of the central nervous system that primarily affects the optic nerves and spinal cord of patients, and in some instances their brainstem, diencephalon or cerebrum as spectrum disorders (NMOSD). Clinical and basic science knowledge of NMO has dramatically increased over the last two decades and it has changed the perception of the disease as being inevitably disabling or fatal. Nonetheless, there is still no cure for NMO and all the disease-modifying therapies (DMTs) are only partially effective. Furthermore, DMTs are not disease- or antigen-specific and alter all immune responses including those protective against infections and cancer and are often associated with significant adverse reactions. In this review, we discuss the pathogenic mechanisms of NMO as they pertain to its DMTs and immune tolerance. We also examine novel research therapeutic strategies focused on induction of antigen-specific immune tolerance by administrating tolerogenic immune-modifying nanoparticles (TIMP). Development and implementation of immune tolerance-based therapies in NMO is likely to be an important step toward improving the treatment outcomes of the disease. The antigen-specificity of these therapies will likely ameliorate the disease safely and effectively, and will also eliminate the clinical challenges associated with chronic immunosuppressive therapies.

Summary: Inappropriate CD4+ T helper (Th) differentiation can compromise host immunity or promote autoimmune disease. To identify disease-relevant regulators of T cell fate, we examined mutations that modify risk for multiple sclerosis (MS), a canonical organ-specific autoimmune disease. This analysis identified a role for Zinc finger E-box-binding homeobox (ZEB1). Deletion of ZEB1 protects against experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis (MS). Mechanistically, ZEB1 in CD4+ T cells is required for pathogenic Th1 and Th17 differentiation. Genomic analyses of paired human and mouse expression data elucidated an unexpected role for ZEB1 in JAK-STAT signaling. ZEB1 inhibits miR-101-3p that represses JAK2 expression, STAT3/STAT4 phosphorylation, and subsequent expression of interleukin-17 (IL-17) and interferon gamma (IFN-γ). Underscoring its clinical relevance, ZEB1 and JAK2 downregulation decreases pathogenic cytokines expression in T cells from MS patients. Moreover, a Food and Drug Administration (FDA)-approved JAK2 inhibitor is effective in EAE. Collectively, these findings identify a conserved, potentially targetable mechanism regulating disease-relevant inflammation.

Injection of donor apoptotic cells induces graft tolerance in mice. Here the authors combine this approach with short immunosuppressive therapy to achieve long-term tolerance to allogeneic islets and restoration of normoglycemia in diabetic nonhuman primates, and delineate cellular and molecular correlates of tolerance induction.

Multiple Sclerosis (MS) is an immune-mediated neurological disorder, characterized by central nervous system (CNS) inflammation, oligodendrocyte loss, demyelination, and axonal degeneration. Although autoimmunity, inflammatory demyelination and neurodegeneration underlie MS, the initiating event has yet to be clarified. Effective disease modifying therapies need to both regulate the immune system and promote restoration of neuronal function, including remyelination. The challenge in developing an effective long-lived therapy for MS requires that three disease-associated targets be addressed: (1) self-tolerance must be re-established to specifically inhibit the underlying myelin-directed autoimmune pathogenic mechanisms; (2) neurons must be protected from inflammatory injury and degeneration; (3) myelin repair must be engendered by stimulating oligodendrocyte progenitors to remyelinate CNS neuronal axons. The combined use of chronic and relapsing remitting experimental autoimmune encephalomyelitis (C-EAE, R-EAE) (“outside-in”) as well as progressive diphtheria toxin A chain (DTA) and cuprizone autoimmune encephalitis (CAE) (“inside-out”) mouse models allow for the investigation and specific targeting of all three of these MS-associated disease parameters. The “outside-in” EAE models initiated by myelin-specific autoreactive CD4+ T cells allow for the evaluation of both myelin-specific tolerance in the absence or presence of neuroprotective and/or remyelinating agents. The “inside-out” mouse models of secondary inflammatory demyelination are triggered by toxin-induced oligodendrocyte loss or subtle myelin damage, which allows evaluation of novel therapeutics that could promote remyelination and neuroprotection in the CNS. Overall, utilizing these complementary pre-clinical MS models will open new avenues for developing therapeutic interventions, tackling MS from the “outside-in” and/or “inside-out”.

Multiple Sclerosis (MS) is characterized by immune cell infiltration to the central nervous system (CNS) as well as loss of myelin. Characterization of the cells in lesions of MS patients revealed an important accumulation of myeloid cells such as macrophages and dendritic cells (DCs). Data from the experimental autoimmune encephalomyelitis (EAE) model of MS supports the importance of peripheral myeloid cells in the disease pathology. However, the majority of MS therapies focus on lymphocytes. As we will discuss in this review, multiple strategies are now in place to target myeloid cells in clinical trials. These strategies have emerged from data in both human and mouse studies. We discuss strategies targeting myeloid cell migration, growth factors and cytokines, biological functions (with a focus on miRNAs), and immunological activities (with a focus on nanoparticles).

ABSTRACT Ocular herpes simplex virus 1 (HSV-1) infection leads to an immunopathogenic disease called herpes stromal keratitis (HSK), in which CD4+ T cell-driven inflammation contributes to irreversible damage to the cornea. Herpesvirus entry mediator (HVEM) is an immune modulator that activates stimulatory and inhibitory cosignals by interacting with its binding partners, LIGHT (TNFSF14), BTLA (B and T lymphocyte attenuator), and CD160. We have previously shown that HVEM exacerbates HSK pathogenesis, but the involvement of its binding partners and its connection to the pathogenic T cell response have not been elucidated. In this study, we investigated the role of HVEM and its binding partners in mediating the T cell response using a murine model of ocular HSV-1 infection. By infecting mice lacking the binding partners, we demonstrated that multiple HVEM binding partners were required for HSK pathogenesis. Surprisingly, while LIGHT−/−, BTLA−/−, and CD160−/− mice did not show differences in disease compared to wild-type mice, BTLA−/− LIGHT−/− and CD160−/− LIGHT−/− double knockout mice showed attenuated disease characterized by decreased clinical symptoms, increased retention of corneal sensitivity, and decreased infiltrating leukocytes in the cornea. We determined that the attenuation of disease in HVEM−/−, BTLA−/− LIGHT−/−, and CD160−/− LIGHT−/− mice correlated with a decrease in gamma interferon (IFN-γ)-producing CD4+ T cells. Together, these results suggest that HVEM cosignaling through multiple binding partners induces a pathogenic Th1 response to promote HSK. This report provides new insight into the mechanism of HVEM in HSK pathogenesis and highlights the complexity of HVEM signaling in modulating the immune response following ocular HSV-1 infection. IMPORTANCE Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, is capable of causing a progressive inflammatory ocular disease called herpes stromal keratitis (HSK). HSV-1 ocular infection leads to persistent inflammation in the cornea resulting in outcomes ranging from significant visual impairment to complete blindness. Our previous work showed that herpesvirus entry mediator (HVEM) promotes the symptoms of HSK independently of viral entry and that HVEM expression on CD45+ cells correlates with increased infiltration of leukocytes into the cornea during the chronic inflammatory phase of the disease. Here, we elucidated the role of HVEM in the pathogenic Th1 response following ocular HSV-1 infection and the contribution of HVEM binding partners in HSK pathogenesis. Investigating the molecular mechanisms of HVEM in promoting corneal inflammation following HSV-1 infection improves our understanding of potential therapeutic targets for HSK.

Indoleamine 2, 3-dioxygenase 1 (IDO; IDO1; INDO) is a rate-limiting enzyme that metabolizes the essential amino acid, tryptophan, into downstream kynurenines. Canonically, the metabolic depletion of tryptophan and/or the accumulation of kynurenine is the mechanism that defines how immunosuppressive IDO inhibits immune cell effector functions and/or facilitates T cell death. Non-canonically, IDO also suppresses immunity through non-enzymic effects. Since IDO targeting compounds predominantly aim to inhibit metabolic activity as evidenced across the numerous clinical trials currently evaluating safety/efficacy in patients with cancer, in addition to the recent disappointment of IDO enzyme inhibitor therapy during the phase III ECHO-301 trial, the issue of IDO non-enzyme effects have come to the forefront of mechanistic and therapeutic consideration(s). Here, we review enzyme-dependent and -independent IDO-mediated immunosuppression as it primarily relates to glioblastoma (GBM); the most common and aggressive primary brain tumor in adults. Our group's recent discovery that IDO levels increase in the brain parenchyma during advanced age and regardless of whether GBM is present, highlights an immunosuppressive synergy between aging-increased IDO activity in cells of the central nervous system that reside outside of the brain tumor but collaborate with GBM cell IDO activity inside of the tumor. Because of their potential value for the in vivo study of IDO, we also review current transgenic animal modeling systems while highlighting three new constructs recently created by our group. This work converges on the central premise that maximal immunotherapeutic efficacy in subjects with advanced cancer requires both IDO enzyme- and non-enzyme-neutralization, which is not adequately addressed by available IDO-targeting pharmacologic approaches at this time.

Patients with locally advanced and metastatic urothelial carcinoma have a low survival rate (median 15.7 months, 13.1–17.8), with only a 23% response rate to monotherapy treatment with anti-PDL1 checkpoint immunotherapy. To identify new therapeutic targets, we profiled the immune regulatory signatures during murine cancer development using the BBN carcinogen and identified an increase in the expression of the T cell inhibitory protein B7-H4 (VTCN1, B7S1, B7X). B7-H4 expression temporally correlated with decreased lymphocyte infiltration. While the increase in B7-H4 expression within the bladder by CD11b+ monocytes is shared with human cancers, B7-H4 expression has not been previously identified in other murine cancer models. Higher expression of B7-H4 was associated with worse survival in muscle-invasive bladder cancer in humans, and increased B7-H4 expression was identified in luminal and luminal-papillary subtypes of bladder cancer. Evaluation of B7-H4 by single-cell RNA-Seq and immune mass cytometry of human bladder tumors found that B7-H4 is expressed in both the epithelium of urothelial carcinoma and CD68+ macrophages within the tumor. To investigate the function of B7-H4, treatment of human monocyte and T cell co-cultures with a B7-H4 blocking antibody resulted in enhanced IFN-γ secretion by CD4+ and CD8+ T cells. Additionally, anti-B7-H4 antibody treatment of BBN-carcinogen bladder cancers resulted in decreased tumor size, increased CD8+ T cell infiltration within the bladder, and a complimentary decrease in tumor-infiltrating T regulatory cells (Tregs). Furthermore, treatment with a combination of anti-PD-1 and anti-B7-H4 antibodies resulted in a significant reduction in tumor stage, a reduction in tumor size, and an increased level of tumor necrosis. These findings suggest that antibodies targeting B7-H4 may be a viable strategy for bladder cancers unresponsive to PD-1 checkpoint inhibitors.

We have shown that PLG nanoparticles loaded with peptide antigen can reduce disease in animal models of autoimmunity and in a phase 1/2a clinical trial in celiac patients. Clarifying the mechanisms by which antigen-loaded nanoparticles establish tolerance is key to further adapting them to clinical use. The mechanisms underlying tolerance induction include the expansion of antigen-specific CD4+ regulatory T cells and sequestration of autoreactive cells in the spleen. In this study, we employed nanoparticles loaded with two model peptides, GP33–41 (a CD8 T cell epitope derived from lymphocytic choriomeningitis virus) and OVA323–339 (a CD4 T cell epitope derived from ovalbumin), to modulate the CD8+ and CD4+ T cells from two transgenic mouse strains, P14 and DO11.10, respectively. Firstly, it was found that the injection of P14 mice with particles bearing the MHC I-restricted GP33–41 peptide resulted in the expansion of CD8+ T cells with a regulatory cell phenotype. This correlated with reduced CD4+ T cell viability in ex vivo co-cultures. Secondly, both nanoparticle types were able to sequester transgenic T cells in secondary lymphoid tissue. Flow cytometric analyses showed a reduction in the surface expression of chemokine receptors. Such an effect was more prominently observed in the CD4+ cells rather than the CD8+ cells.

Intravenously infused synthetic 500 nm nanoparticles composed of poly(lactide-co-glycolide) are taken up by blood-borne inflammatory monocytes via a macrophage scavenger receptor (macrophage receptor with collagenous structure), and the monocytes no longer traffic to sites of inflammation. Intravenous administration of the nanoparticles after experimental spinal cord injury in mice safely and selectively limited infiltration of hematogenous monocytes into the injury site. The nanoparticles did not bind to resident microglia, and did not change the number of microglia in the injured spinal cord. Nanoparticle administration reduced M1 macrophage polarization and microglia activation, reduced levels of inflammatory cytokines, and markedly reduced fibrotic scar formation without altering glial scarring. These findings thus implicate early-infiltrating hematogenous monocytes as highly selective contributors to fibrosis that do not play an indispensable role in gliosis after SCI. Further, the nanoparticle treatment reduced accumulation of chondroitin sulfate proteoglycans, increased axon density inside and caudal to the lesion site, and significantly improved functional recovery after both moderate and severe injuries to the spinal cord. These data provide further evidence that hematogenous monocytes contribute to inflammatory damage and fibrotic scar formation after spinal cord injury in mice. Further, since the nanoparticles are simple to administer intravenously, immunologically inert, stable at room temperature, composed of an FDA-approved material, and have no known toxicity, these findings suggest that the nanoparticles potentially offer a practical treatment for human spinal cord injury.

Exosomes are extracellular vesicles that can favor tumor development and metastasis. Here, the authors show that cancer exosomes may also exert a suppressive function; in fact, exosomes from non-metastatic melanoma cells can lead to the recruitment of patrolling monocytes, which clear cancer cells at the pre-metastatic niche.

NOTCH signalling stimulates oligodendrocyte progenitor cell proliferation but how this regulates demyelinating disease is unclear. Here, the authors show that an IL-17 adaptor protein, Act1, interacts with the C-terminal fragment of NOTCH1 (NICD) to activate cell proliferation and an inflammatory response.

Particle-based technologies are increasingly being used in diagnostics and therapeutics. The particles employed in these applications are usually composed of polymers such as poly(lactide-co-glycolide) (PLG) and functionalized with peptides or proteins. Peptide or protein conjugation to particles is frequently achieved using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), while dimethyl sulfoxide (DMSO) is used to retrieve surface-attached or encapsulated peptides or proteins by solubilizing the particles. We examined strategies based on bicinchoninic acid (BSA), Coomassie Plus, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays for the quantification of surface-attached or encapsulated peptides or proteins. We determined that the CBQCA assay is a highly sensitive and accurate substitute for radioactivity-based assays that is suitable for measuring multiple particle-bound or particle-encapsulated peptides or proteins in the presence of EDC or PLG in DMSO, compounds that interfere with the more commonly used BSA and Coomassie Plus assays. Our strategy enables the accurate quantification of peptides or proteins loaded onto or into particles—an essential component of particle-based platform design for diagnostics and therapeutics.

Patients with neurofibromatosis type 1 (NF1) and Costello syndrome Rasopathy have behavioral deficits. In NF1 patients, these may correlate with white matter enlargement and aberrant myelin. To model these features, we induced Nf1 loss or HRas hyperactivation in mouse oligodendrocytes. Enlarged brain white matter tracts correlated with myelin decompaction, downregulation of claudin-11, and mislocalization of connexin-32. Surprisingly, non-cell-autonomous defects in perivascular astrocytes and the blood-brain barrier (BBB) developed, implicating a soluble mediator. Nitric oxide (NO) can disrupt tight junctions and gap junctions, and NO and NO synthases (NOS1–NOS3) were upregulated in mutant white matter. Treating mice with the NOS inhibitor NG-nitro-L-arginine methyl ester or the antioxidant N-acetyl cysteine corrected cellular phenotypes. CNP-HRasG12V mice also displayed locomotor hyperactivity, which could be rescued by antioxidant treatment. We conclude that Nf1/Ras regulates oligodendrocyte NOS and that dysregulated NO signaling in oligodendrocytes can alter the surrounding vasculature. The data suggest that antioxidants may improve some behavioral deficits in Rasopathy patients.

Enhancing the ability of either endogenous or transplanted oligodendrocyte progenitors (OPs) to engage in myelination may constitute a novel therapeutic approach to demyelinating diseases of the brain. It is known that in adults neural progenitors situated in the subventricular zone of the lateral ventricle (SVZ) are capable of generating OPs which can migrate into white matter tracts such as the corpus callosum (CC). We observed that progenitor cells in the SVZ of adult mice expressed CXCR4 chemokine receptors and that the chemokine SDF-1/CXCL12 was expressed in the CC. We therefore investigated the role of chemokine signaling in regulating the migration of OPs into the CC following their transplantation into the lateral ventricle. We established OP cell cultures from Olig2-EGFP mouse brains. These cells expressed a variety of chemokine receptors, including CXCR4 receptors. Olig2-EGFP OPs differentiated into CNPase-expressing oligodendrocytes in culture. To study the migratory capacity of Olig2-EGFP OPs in vivo, we transplanted them into the lateral ventricles of mice. Donor cells migrated into the CC and differentiated into mature oligodendrocytes. This migration was enhanced in animals with Experimental Autoimmune Encephalomyelitis (EAE). Inhibition of CXCR4 receptor expression in OPs using shRNA inhibited the migration of transplanted OPs into the white matter suggesting that their directed migration is regulated by CXCR4 signaling. These findings indicate that CXCR4 mediated signaling is important in guiding the migration of transplanted OPs in the context of inflammatory demyelinating brain disease.

ABSTRACT 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 (CD11cint 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.

We have previously developed a robust regimen for tolerance induction in murine models of islet cell transplantation using pre- and posttransplant infusions of donor splenocytes (SPs) treated with a chemical cross-linker ethylcarbodiimide (ECDI). However, the requirement for large numbers of fresh donor SPs for ECDI coupling impairs its clinical feasibility, and additionally, the compatibility of this tolerance regimen with commonly used immunosuppressive drugs is largely unknown. In the current study, we demonstrate that equivalent tolerance efficacy for islet cell transplantation can be successfully achieved not only with a significantly lower dose of ECDI-SPs than originally established but also with culture-expanded donor B-cells or with soluble donor antigens in the form of donor cell lysate, which is ECDI coupled to recipient SPs. We further demonstrate that tolerance induced by donor ECDI-SPs is dependent on a favorable apoptotic-to-necrotic cell ratio post-ECDI coupling and is not affected by a transient course of conventional immunosuppressive drugs including tacrolimus and mycophenolate mofetil. While splenic antigen-presenting cells of the recipient play an important role in mediating the tolerogenic effects of donor ECDI-SPs, splenectomized recipients can be readily tolerized and appear to employ liver Kupffer cells for uptaking and processing of the ECDI-SPs. We conclude that infusion of donor ECDI-SPs is a versatile tolerance strategy that has a high potential for adaptation to clinically feasible regimens for tolerance trials for human islet cell transplantation.

Epilepsy is a chronic brain disorder that affects one percent of the human population worldwide. Immune responses are implicated in seizure induction and the development of epilepsy. Pre-clinical and clinical evidence have accumulated to suggest a positive feedback cycle between brain inflammation and epileptogenesis. Prolonged or recurrent seizures and brain injuries lead to upregulation of proinflammatory cytokines and activated immune responses to further increase seizure susceptibility, promote neuronal excitability, and induce blood-brain barrier (BBB) breakdown. This review focuses on the potential role of innate and adaptive immune responses in the pathogenesis of epilepsy. Both human studies and animal models that help delineate the contributions of brain inflammation in epileptogenesis will be discussed. We highlight the critical role of brain-resident immune mediators and emphasize the contribution of brain-infiltrating peripheral leukocytes. Additionally, we propose possible immune mechanisms that underlie epileptogenesis. Several proinflammatory pathways are discussed, including the interleukin-1 receptor/ toll-like receptor signaling cascade, the pathways activated by danger-associated molecular patterns, and the cyclooxygenase-2 / prostaglandin pathway. Finally, development of better therapies that target the key constituents and processes identified in these mechanisms are considered, for instance, engineering antagonizing agents that effectively block these pathways in an antigen-specific manner.