Saparna Pai

IL-1β Breaks Tolerance through Expansion of CD25+ Effector T Cells

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn’s disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1β drives proliferation and cytokine production by CD4+CD25+FoxP3− effector/memory T cells, attenuates CD4+CD25+FoxP3+ regulatory T cell function, and allows escape of CD4+CD25− autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1β in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.

Abnormal NF-κB function characterizes human type 1 diabetes dendritic cells and monocytes

Dendritic cell (DC) differentiation is abnormal in type 1 diabetes mellitus (T1DM). However, the nature of the relationship between this abnormality and disease pathogenesis is unknown. We studied the LPS response in monocytes and monocyte-derived DCs isolated from T1DM patients and from non-T1DM controls. In T1DM patients, late LPS-mediated nuclear DNA binding by RelA, p50, c-Rel, and RelB was impaired as compared with type 2 DM, rheumatoid arthritis, and healthy subjects, associated with impaired DC CD40 and MHC class I induction but normal cytokine production. In TIDM monocytes, RelA and RelB were constitutively activated, and the src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), a negative regulator of NF-κB, was overexpressed. Addition of sodium stibogluconate, a SHP-1 inhibitor, to DCs differentiating from monocyte precursors restored their capacity to respond to LPS in ∼60% of patients. The monocyte and DC NF-κB response to LPS is thus a novel phenotypic and likely pathogenetic marker for human T1DM. SHP-1 is at least one NF-κB regulatory mechanism which might be induced as a result of abnormal inflammatory signaling responses in T1DM monocytes.

Real-time imaging reveals the dynamics of leukocyte behaviour during experimental cerebral malaria pathogenesis.

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6Chi monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8+ T lymphocytes. A critical number of CD8+ T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8+ T lymphocytes in regulating vascular pathology in this disease.

RelB Nuclear Translocation Mediated by C-Terminal Activator Regions of Epstein-Barr Virus-Encoded Latent Membrane Protein 1 and Its Effect on Antigen-Presenting Function in B Cells

ABSTRACT Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitts lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-κB activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative IκBα S32/36A, an NF-κB inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-κB subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.

Immune deficiency or hyperactivity-Nf-κb illuminates autoimmunity

Nuclear factor (NF)-kappaB is a transcription factor family which transmits signals from the cell surface to the nucleus, resulting in transcriptional effects on genes involved in inflammation, cell differentiation and survival. The signaling of NF-kappaB and mitogen-activated protein (MAP) kinases through adapter molecules is of critical importance to survival and activation of all cells in the body, including those regulating innate and adaptive immunity. Here we review the individual and intersecting roles played by the alternate and classical NF-kappaB pathways in the pathogenesis of autoimmune disease. Understanding the differences in classical and alternate NF-kappaB function has greatly assisted the development of models of their contribution to different autoimmune diseases. To exemplify these concepts, we consider the contribution of NF-kappaB to rheumatoid arthritis and type 1 diabetes pathogenesis and approaches to immunotherapy.

Current evidence for a role of the kynurenine pathway of tryptophan metabolism in multiple sclerosis

The kynurenine pathway (KP) is the major metabolic pathway of the essential amino acid tryptophan (TRP). Stimulation by inflammatory molecules, such as interferon-γ (IFN-γ), is the trigger for induction of the KP, driving a complex cascade of production of both neuroprotective and neurotoxic metabolites, and in turn, regulation of the immune response and responses of brain cells to the KP metabolites. Consequently, substantial evidence has accumulated over the past couple of decades that dysregulation of the KP and the production of neurotoxic metabolites are associated with many neuroinflammatory and neurodegenerative diseases, including Parkinson’s disease, AIDS-related dementia, motor neurone disease, schizophrenia, Huntington’s disease, and brain cancers. In the past decade, evidence of the link between the KP and multiple sclerosis (MS) has rapidly grown and has implicated the KP in MS pathogenesis. KP enzymes, indoleamine 2,3-dioxygenase (IDO-1) and tryptophan dioxygenase (highest expression in hepatic cells), are the principal enzymes triggering activation of the KP to produce kynurenine from TRP. This is in preference to other routes such as serotonin and melatonin production. In neurological disease, degradation of the blood–brain barrier, even if transient, allows the entry of blood monocytes into the brain parenchyma. Similar to microglia and macrophages, these cells are highly responsive to IFN-γ, which upregulates the expression of enzymes, including IDO-1, producing neurotoxic KP metabolites such as quinolinic acid. These metabolites circulate systemically or are released locally in the brain and can contribute to the excitotoxic death of oligodendrocytes and neurons in neurological disease principally by virtue of their agonist activity at N-methyl-d-aspartic acid receptors. The latest evidence is presented and discussed. The enzymes that control the checkpoints in the KP represent an attractive therapeutic target, and consequently several KP inhibitors are currently in clinical trials for other neurological diseases, and hence may make suitable candidates for MS patients. Underpinning these drug discovery endeavors, in recent years, several advances have been made in how KP metabolites are assayed in various biological fluids, and tremendous advancements have been made in how specimens are imaged to determine disease progression and involvement of various cell types and molecules in MS.

Immunotherapy with Costimulatory Dendritic Cells To Control Autoimmune Inflammation

Costimulation-deficient dendritic cells (DCs) prevent autoimmune disease in mouse models. However, autoimmune-prone mice and humans fail to control expansion of peripheral autoreactive effector memory T cells (TEMs), which resist immunoregulation by costimulation-deficient DCs. In contrast, activation of DC costimulation may be coupled with regulatory capacity. To test whether costimulatory DCs control TEMs and attenuate established autoimmune disease, we used RelB-deficient mice, which have multiorgan inflammation, expanded peripheral autoreactive TEMs, and dysfunctional Foxp3+ regulatory T cells (Tregs) cells and conventional DCs. TEMs were regulated by Foxp3+ Tregs when costimulated by CD3/CD28-coated beads or wild-type DCs but not DCs deficient in RelB or CD80/CD86. After transfer, RelB and CD80/CD86-sufficient DCs restored tolerance and achieved a long-term cure of autoimmune disease through costimulation of TEM and Foxp3+ Treg IFN-γ production, as well as induction of IDO by host APCs. IDO was required for regulation of TEMs and suppression of organ inflammation. Our data challenge the paradigm that costimulation-deficient DCs are required to regulate established autoimmune disease to avoid TEM activation and demonstrate cooperative cross-talk between costimulatory DCs, IFN-γ, and IDO-dependent immune regulation. IFN-γ and IDO activity may be good surrogate biomarkers measured against clinical efficacy in trials of autoimmune disease immunoregulation.

Nasopharyngeal carcinoma-associated Epstein-Barr virus-encoded oncogene latent membrane protein 1 potentiates regulatory T-cell function.

Sequence variation in the Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) oncogene structure may affect antigen‐presenting cell (APC) function of infected B cells and immune escape by EBV‐specific T cells and thus contribute to the development of malignancy. Normal B cell‐associated LMP1 (B‐LMP1) upregulates B cell APC function through activation of the necrosis factor (NF)‐κB subunit, RelB. We examined the ability of B‐LMP1 and a nasopharyngeal carcinoma‐associated LMP1 (NPC‐LMP1) to modulate B cell APC function and T‐cell responses. B lymphoma cells transfected with NPC‐LMP1 stimulated resting T cells in mixed lymphocyte reaction less efficiently than B‐LMP1 transfectants. Unexpectedly, antigen presentation to CD4+ T helper cells was reduced owing to potentiation of regulatory T‐cell function by NPC‐LMP1 transfectants, which produce increased levels of interleukin‐10, rendering CD4+ T cells hyporesponsive. Thus, after primary EBV infection, T cells may escape activation by NPC‐LMP1. These observations have important implications for the establishment of EBV‐associated malignancy in the context of infection with tumour‐associated EBV LMP1 variants.

Monocyte-derived Dc Primed With Tlr Agonists Secrete Il-12p70 in a Cd40-dependent Manner Under Hyperthermic Conditions

Fever is an evolutionarily conserved mechanism to improve survival during infection. Previous studies have shown that feverlike temperatures directly enhance the function of murine bone marrow-derived dendritic cells (DCs). In the present study, we examined the response of human monocyte-derived DC to 39.5°C hyperthermia. When primed with toll-like receptor agonists or bacterial extract but not proinflammatory cytokines, hyperthermia specifically enhanced secretion of interleukin (IL)-12p70 by DC, without altering the secretion of IL-10, tumor necrosis factor α or IL-1β. These DC induced significantly higher levels of T-cell proliferation and interferon γ production in assays of antigen presentation and MLR. Endogenous heat-sock protein 70 colocalized with CD40 in DC exposed to hyperthermic conditions. Recombinant CD40-Fc fusion protein blocked the increase in IL-12p70 secretion by DC primed with bacterial extract and hyperthermia. Thus, DC primed with toll-like receptor-agonists respond to hyperthermia with increased IL-12p70 secretion, mediated by heat-shock protein binding and activation of CD40. The data have important applications for clinical immunotherapy and the mechanism of fever.

Visualizing leukocyte trafficking in the living brain with 2-photon intravital microscopy.

Intravital imaging of the superficial brain tissue in mice represents a powerful tool for the dissection of the cellular and molecular cues underlying inflammatory and infectious central nervous system (CNS) diseases. We present here a step-by-step protocol that will enable a non-specialist to set up a two-photon brain-imaging model. The protocol offers a two-part approach that is specifically optimized for imaging leukocytes but can be easily adapted to answer varied CNS-related biological questions. The protocol enables simultaneous visualization of fluorescently labeled immune cells, the pial microvasculature and extracellular structures such as collagen fibers at high spatial and temporal resolution. Intracranial structures are exposed through a cranial window, and physiologic conditions are maintained during extended imaging sessions via continuous superfusion of the brain surface with artificial cerebrospinal fluid (aCSF). Experiments typically require 1–2 h of preparation, which is followed by variable periods of immune cell tracking. Our methodology converges the experience of two laboratories over the past 10 years in diseased animal models such as cerebral ischemia, lupus, cerebral malaria, and toxoplasmosis. We exemplify the utility of this protocol by tracking leukocytes in transgenic mice in the pial vessels under steady-state conditions.