Oxidized Low-Density Lipoprotein Immune Complex Priming of the Nlrp3 Inflammasome Involves TLR and FcγR Cooperation and Is Dependent on CARD9.

Oxidized low-density lipoprotein (oxLDL) is known to activate inflammatory responses in a variety of cells, especially macrophages and dendritic cells. Interestingly, much of the oxLDL in circulation is complexed to Abs, and these resulting immune complexes (ICs) are a prominent feature of chronic inflammatory disease, such as atherosclerosis, type-2 diabetes, systemic lupus erythematosus, and rheumatoid arthritis. Levels of oxLDL ICs often correlate with disease severity, and studies demonstrated that oxLDL ICs elicit potent inflammatory responses in macrophages. In this article, we show that bone marrow-derived dendritic cells (BMDCs) incubated with oxLDL ICs for 24 h secrete significantly more IL-1ß compared with BMDCs treated with free oxLDL, whereas there was no difference in levels of TNF-α or IL-6. Treatment of BMDCs with oxLDL ICs increased expression of inflammasome-related genes Il1a, Il1b, and Nlrp3, and pretreatment with a caspase 1 inhibitor decreased IL-1ß secretion in response to oxLDL ICs. This inflammasome priming was due to oxLDL IC signaling via multiple receptors, because inhibition of CD36, TLR4, and FcγR significantly decreased IL-1ß secretion in response to oxLDL ICs. Signaling through these receptors converged on the adaptor protein CARD9, a component of the CARD9-Bcl10-MALT1 signalosome complex involved in NF-κB translocation. Finally, oxLDL IC-mediated IL-1ß production resulted in increased Th17 polarization and cytokine secretion. Collectively, these data demonstrate that oxLDL ICs induce inflammasome activation through a separate and more robust mechanism than oxLDL alone and that these ICs may be immunomodulatory in chronic disease and not just biomarkers of severity.

Autoimmune-mediated glucose intolerance in a mouse model of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against self-antigens such as double-stranded DNA and phospholipids. Classical comorbidities of SLE include glomerulonephritis, infection, cardiovascular disease, arthritis, skin disorders, and neurological disease. In addition to these classical comorbidities, there is emerging evidence that SLE patients are at higher risk of developing insulin resistance and other components of the metabolic syndrome. Visceral adipose tissue inflammation is a central mediator of insulin resistance in the obese setting, but the mechanism behind the pathogenesis of metabolic disease in the SLE patient population is unclear. We hypothesize that lupus-associated changes in the adaptive immune system are associated with disruption in glucose homeostasis in the context of SLE. To test this hypothesis, we assessed the metabolic and immunological phenotype of SLE-prone B6.SLE mice. B6.SLE mice fed a low-fat diet had significantly worsened glucose tolerance, increased adipose tissue insulin resistance, increased ß-cell insulin secretion, and increased adipocyte size compared with their respective B6 controls. Independently of diet, B cells isolated from the white adipose tissue of B6.SLE mice were skewed toward IgG production, and the level of IgG1 was elevated in the serum of SLE-prone mice. These data show that B6.SLE mice develop defects in glucose homeostasis even when fed a low-fat diet and suggest that B cells may play a role in this metabolic dysfunction.

Activation of invariant natural killer T cells by lipid excess promotes tissue inflammation, insulin resistance, and hepatic steatosis in obese mice.

Obesity triggers a low-grade systemic inflammation, which plays an important role in the development of obesity-associated metabolic diseases. In searching for links between lipid accumulation and chronic inflammation, we examined invariant natural killer T (iNKT) cells, a subset of T lymphocytes that react with lipids and regulate inflammatory responses. We show that iNKT cells respond to dietary lipid excess and become activated before or at the time of tissue recruitment of inflammatory leukocytes, and that these cells progressively increase proinflammatory cytokine production in obese mice. Such iNKT cells skew other leukocytes toward proinflammatory cytokine production and induce an imbalanced proinflammatory cytokine environment in multiple tissues. Further, iNKT cell deficiency ameliorates tissue inflammation and provides protection against obesity-induced insulin resistance and hepatic steatosis. Conversely, chronic iNKT cell stimulation using a canonical iNKT cell agonist exacerbates tissue inflammation and obesity-associated metabolic disease. These findings place iNKT cells into the complex network linking lipid excess to inflammation in obesity and suggest new therapeutic avenues for obesity-associated metabolic disorders.

Nonequivalence of classical MHC class I loci in ability to direct effective antiviral immunity.

Structural diversity in the peptide binding sites of the redundant classical MHC antigen presenting molecules is strongly selected in humans and mice. Although the encoded antigen presenting molecules overlap in antigen presenting function, differences in polymorphism at the MHC I A, B and C loci in humans and higher primates indicate these loci are not functionally equivalent. The structural basis of these differences is not known. We hypothesize that classical class I loci differ in their ability to direct effective immunity against intracellular pathogens. Using a picornavirus infection model and chimeric H-2 transgenes, we examined locus specific functional determinants distinguishing the ability of class I sister genes to direct effective anti viral immunity. Whereas, parental FVB and transgenic FVB mice expressing the H-2K(b) gene are highly susceptible to persisting Theiler's virus infection within the CNS and subsequent demyelination, mice expressing the D(b) transgene clear the virus and are protected from demyelination. Remarkably, animals expressing a chimeric transgene, comprised primarily of K(b) but encoding the peptide binding domain of D(b), develop a robust anti viral CTL response yet fail to clear virus and develop significant demyelination. Differences in expression of the chimeric K(b)α1α2D(b) gene (low) and D(b) (high) in the CNS of infected mice mirror expression levels of their endogenous H-2(q) counterparts in FVB mice. These findings demonstrate that locus specific elements other than those specifying peptide binding and T cell receptor interaction can determine ability to clear virus infection. This finding provides a basis for understanding locus-specific differences in MHC polymorphism, characterized best in human populations.

Mycophenolate mofetil but not atorvastatin attenuates atherosclerosis in lupus-prone LDLr(-/-) mice.

RATIONALE: Recent clinical and preclinical studies have demonstrated that systemic lupus erythematosus (SLE) is associated with an increased risk for cardiovascular disease (CVD). However, unlike in the general population, little is known regarding the efficacy of atheroprotective interventions in patients with SLE. The current study aims to determine the benefit of lymphocyte inhibition on reducing the atherosclerotic burden in SLE-susceptible LDLr-deficient mice. METHODS: Female LDLr(-/-) mice were lethally irradiated and reconstituted with bone marrow from C57Bl/6 mice (LDLr.B6) or the SLE-susceptible B6.Sle1.2.3 mice (LDLr.Sle). At 16 weeks post transplant, mice were treated with atorvastatin (10 mg/kg), mycophenolate mofetil (MMF; 40 mg/kg), or both (MMF-A) for 8 weeks, after which the extent of atherosclerosis and the presence of SLE were assessed. RESULTS: Following 8 weeks of treatment, we observed that atorvastatin-mediated reduction in cholesterol levels attenuated atherogenesis in LDLr.B6 mice but failed to significantly reduce atherosclerotic lesion size in LDLr.Sle mice, in spite of a significant reduction in serum cholesterol levels. Treatment with MMF and MMF-A attenuated atherogenesis in LDLr.B6 and LDLr.Sle mice. In addition, MMF-containing regimens inhibited recruitment of CD4+ T cells to atherosclerotic lesions in LDLr.Sle mice. In these mice, MMF also reduced the proportion of activated splenic T cells, as well as interleukin 10 secretion by T cells. With regard to lupus activity, MMF had no overt effect on anti-double-stranded DNA (dsDNA) antibody titres or kidney function and pathology. CONCLUSIONS: The current study demonstrates that reduction of cholesterol levels alone is not atheroprotective in lupus-mediated atherogenesis. This is the first study to demonstrate that MMF reduces the atherosclerotic burden in a model of lupus-accelerated atherosclerosis. Our results suggest that MMF treatment may prove beneficial in preventing CVD in patients with SLE.

Theiler's murine encephalomyelitis virus as a vaccine candidate for immunotherapy.

The induction of sterilizing T-cell responses to tumors is a major goal in the development of T-cell vaccines for treating cancer. Although specific components of anti-viral CD8+ immunity are well characterized, we still lack the ability to mimic viral CD8+ T-cell responses in therapeutic settings for treating cancers. Infection with the picornavirus Theiler's murine encephalomyelitis virus (TMEV) induces a strong sterilizing CD8+ T-cell response. In the absence of sterilizing immunity, the virus causes a persistent infection. We capitalized on the ability of TMEV to induce strong cellular immunity even under conditions of immune deficiency by modifying the virus to evaluate its potential as a T-cell vaccine. The introduction of defined CD8+ T-cell epitopes into the leader sequence of the TMEV genome generates an attenuated vaccine strain that can efficiently drive CD8+ T-cell responses to the targeted antigen. This virus activates T-cells in a manner that is capable of inducing targeted tissue damage and glucose dysregulation in an adoptive T-cell transfer model of diabetes mellitus. As a therapeutic vaccine for the treatment of established melanoma, epitope-modified TMEV can induce strong cytotoxic T-cell responses and promote infiltration of the T-cells into established tumors, ultimately leading to a delay in tumor growth and improved survival of vaccinated animals. We propose that epitope-modified TMEV is an excellent candidate for further development as a human T-cell vaccine for use in immunotherapy.

The inhibitory FcγRIIb modulates the inflammatory response and influences atherosclerosis in male apoE(-/-) mice.

BACKGROUND: Atherosclerosis is widely accepted as an inflammatory disease involving both innate and adaptive immunity. B cells and/or antibodies have previously been shown to play a protective role against atherosclerosis. Aside from their ability to bind to antigens, antibodies can influence inflammatory responses by interacting with various Fcγ receptors on the surface of antigen presenting cells. Although studies in mice have determined that stimulatory Fcγ receptors contribute to atherosclerosis, the role of the inhibitory Fcγ receptor IIb (FcγRIIb) has only recently been investigated. METHODS AND RESULTS: To determine the importance of FcγRIIb in modulating the adaptive immune response to hyperlipidemia, we generated FcγRIIb-deficient mice on the apoE-deficient background (apoE/FcγRIIb(-/-)). We report that male apoE/FcγRIIb(-/-) mice develop exacerbated atherosclerosis that is independent of lipid levels, and is characterized by increased antibody titers to modified LDL and pro-inflammatory cytokines in the aorta. CONCLUSIONS: These findings suggest that antibodies against atherosclerosis-associated antigens partially protect against atherosclerosis in male apoE(-/-) mice by conveying inhibitory signals through the FcγRIIb that downregulate pro-inflammatory signaling via other immune receptors. These data are the first to describe a significant in vivo effect for FcγRIIb in modulating the cytokine response in the aorta in male apoE(-/-) mice.

Statin therapy in lupus-mediated atherogenesis: two birds with one stone?

The atherosclerotic process is accelerated in patients with systemic lupus erythematosus (SLE). In addition to a robust lipid-lowering effect, various immunomodulatory functions have been ascribed to statins. By virtue of the latter they may be able to reduce atherosclerotic vascular disease in SLE by inhibiting immune activation within the arterial wall and by attenuating lupus activity. The effects of statins on SLE as well as on lupus-mediated atherogenesis in vivo are discussed in this viewpoint.

Sizing up stability: combination therapy with Apo-AI peptide mimetics and statins in systemic lupus erythematosus-mediated atherosclerosis.

In a study published recently in Arthritis Research & Therapy, Woo and colleagues investigated the effects of pravastatin in combination with an apolipoprotein-AI (Apo-AI) mimetic peptide in a mouse model of lupus-accelerated atherosclerosis. Combination treatment resulted in a significant decrease in systemic inflammation but increased aortic root lesion size. However, this treatment changed the phenotype of the lesion to a more stable plaque. Because plaque stability is also important for protection against the deadly manifestations of atherosclerosis, combination therapies using Apo-AI mimetics and statin might offer a good additional therapy to treat autoimmunity and cardiovascular disease in patients with lupus.

Development of spontaneous anergy in invariant natural killer T cells in a mouse model of dyslipidemia.

OBJECTIVE: In this study, we investigated whether dyslipidemia-associated perturbed invariant natural killer T (iNKT) cell function is due to intrinsic changes in iNKT cells or defects in the ability of antigen-presenting cells to activate iNKT cells. METHODS AND RESULTS: We compared iNKT cell expansion and cytokine production in C57BL/6J (B6) and apolipoprotein E-deficient (apoE(-/-)) mice. In response to in vivo stimulation with alpha-galactosylceramide, a prototypic iNKT cell glycolipid antigen, apoE(-/-) mice showed significantly decreased splenic iNKT cell expansion at 3 days after injection, a profile associated with iNKT cell anergy due to chronic stimulation. This decrease in expansion and cytokine production was accompanied by a 2-fold increase in percentage of iNKT cells expressing the inhibitory marker programmed death-1 in apoE(-/-) mice compared with controls. However, in vivo and in vitro blockade of programmed death-1 using monoclonal antibody was not able to restore functions of iNKT cells from apoE(-/-) mice to B6 levels. iNKT cells from apoE(-/-) mice also had increased intracellular T cell receptor and Ly49 expression, a phenotype associated with previous activation. Changes in iNKT cell functions were cell autonomous, because dendritic cells from apoE(-/-) mice were able to activate B6 iNKT cells, but iNKT cells from apoE(-/-) mice were not able to respond to B6 dendritic cells. CONCLUSIONS: These data suggest that chronic dyslipidemia induces an iNKT cell phenotype that is unresponsive to further simulation by exogenous glycolipid and that sustained unresponsiveness is iNKT cell intrinsic.

Thymus leukemia antigen controls intraepithelial lymphocyte function and inflammatory bowel disease.

Intestinal intraepithelial lymphocytes (IEL) bear a partially activated phenotype that permits them to rapidly respond to antigenic insults. However, this phenotype also implies that IEL must be highly controlled to prevent misdirected immune reactions. It has been suggested that IEL are regulated through the interaction of the CD8alpha alpha homodimer with the thymus leukemia (TL) antigen expressed by intestinal epithelial cells. We have generated and characterized mice genetically-deficient in TL expression. Our findings show that TL expression has a critical role in maintaining IEL effector functions. Also, TL deficiency accelerated colitis in a genetic model of inflammatory bowel disease. These findings reveal an important regulatory role of TL in controlling IEL function and intestinal inflammation.

Flexibility accompanies commitment of memory CD4 lymphocytes derived from IL-4 locus-activated precursors.

Differentiation of T helper (Th) subset 2 effector lymphocytes is thought to foreclose on IFN-gamma gene expression. Using an IL-4 locus modified to detect transcriptional induction of this effector cytokine gene in developing Th2 cells, we show here that these cells contributed effectively to a long-term memory population. A memory CD4 subset formed efficiently from an activated population after transcriptional induction of the IL-4 locus and differentiation into an IL-4-producing subset with Th2 characteristics. Memory lymphocytes derived from Th2 cells with IL-4 locus activation remained committed to transcriptional competence of Th2 cytokine genes when reactivated and cultured under strong Th1-polarizing conditions. This commitment to transcriptional competence at Th2 cytokine gene loci upon recall activation indicates that linear differentiation is a substantial component of type 2 memory. Strikingly, however, descendants of the Th2 population could turn on IFN-gamma expression when reactivated after a quiescent period, revealing an unexpected flexibility allowing activation of the forbidden IFN-gamma gene after reactivation and growth.

In vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules.

Endoplasmic reticulum (ER)-associated aminopeptidase (ERAP)1 has been implicated in the final proteolytic processing of peptides presented by major histocompatibility complex (MHC) class I molecules. To evaluate the in vivo role of ERAP1, we have generated ERAP1-deficient mice. Cell surface expression of the class Ia molecules H-2Kb and H-2Db and of the class Ib molecule Qa-2 was significantly reduced in these animals. Although cells from mutant animals exhibited reduced capacity to present several self- and foreign antigens to Kb-, Db-, or Qa-1b-restricted CD8+ cytotoxic T cells, presentation of some antigens was unaffected or significantly enhanced. Consistent with these findings, mice generated defective CD8+ T cell responses against class I-presented antigens. These findings reveal an important in vivo role of ER-associated peptidase activity in tailoring peptides for presentation by MHC class Ia and class Ib molecules.

Antigen-specific CD8+ T cells mediate a peptide-induced fatal syndrome.

Peptide immunotherapy both activates and suppresses the T cell response against known peptide Ags. Although pretreatment with VP2(121-130) peptide inhibits the development of antiviral CTL specific for the immunodominant D(b):VP2(121-130) epitope expressed during acute Theiler's murine encephalomyelitis virus infection, i.v. injection of this same peptide or MHC tetramers containing the peptide during an ongoing antiviral CTL response results in a peptide-induced fatal syndrome (PIFS) within 48 h. Susceptibility to PIFS is dependent on peptide-specific CD8(+) T cells, varies among inbred strains of mice, and is not mediated by traditionally defined mechanisms of shock. Analyses using bone marrow chimeras and mutant mice demonstrate that susceptibility to PIFS is determined by the genotype of bone marrow-derived cells and requires the expression of perforin. Animals responding to peptide treatment with PIFS develop classical stress responses in the brain. These findings raise important considerations for the development of peptide therapies for active diseases to modify immune responses involving expanded populations of T cells. In summary, treatment with peptides or MHC-tetramers during a peptide-specific immune response can result in a fatal shock-like syndrome. Susceptibility to the syndrome is genetically determined, is mediated by CD8(+) T cells, and requires expression of perforin. These findings raise concerns about the use of peptides and MHC tetramers in therapeutic schemes.

Anatomical and cellular requirements for the activation and migration of virus-specific CD8+ T cells to the brain during Theiler's virus infection.

Theiler's murine encephalomyelitis virus (TMEV) infection of the brain induces a virus-specific CD8(+) T-cell response in genetically resistant mice. The peak of the immune response to the virus occurs 7 days after infection, with an immunodominant CD8(+) T-cell response against a VP2-derived capsid peptide in the context of the D(b) molecule. The process of activation of antigen-specific T cells that migrate to the brain in the TMEV model has not been defined. The site of antigenic challenge in the TMEV model is directly into the brain parenchyma, a site that is considered immune privileged. We investigated the hypothesis that antiviral CD8(+) T-cell responses are initiated in situ upon intracranial inoculation with TMEV. To determine whether a brain parenchymal antigen-presenting cell is responsible for the activation of virus-specific CD8(+) T cells, we evaluated the CD8(+) T-cell response to the VP2 peptide in bone marrow chimeras and mutant mice lacking peripheral lymphoid organs. The generation of the anti-TMEV CD8(+) T-cell response in the brain requires priming by a bone marrow-derived antigen-presenting cell and the presence of peripheral lymphoid organs. Although our results show that activation of TMEV-specific CD8(+) T cells occurs in the peripheral lymphoid compartment, they do not exclude the possibility that the immune response to TMEV is initiated by a brain-resident, bone marrow-derived, antigen-presenting cell.

Inability of bm14 mice to respond to Theiler's murine encephalomyelitis virus is caused by defective antigen presentation, not repertoire selection.

Natural selection drives diversification of MHC class I proteins, but the mechanism by which selection for polymorphism occurs is not known. New variant class I alleles differ from parental alleles both in the nature of the CD8 T cell repertoire formed and the ability to present pathogen-derived peptides. In the current study, we examined whether T cell repertoire differences, Ag presentation differences, or both account for differential viral resistance between mice bearing variant and parental alleles. We demonstrate that nonresponsive mice have inadequate presentation of viral Ag, but have T cell repertoires capable of mounting Ag-specific responses. Although previous work suggests a correlation between the ability to present an Ag and the ability to generate a repertoire responsive to that Ag, we show that the two functions of MHC class I are independent.

Clearance of Theiler's virus infection depends on the ability to generate a CD8+ T cell response against a single immunodominant viral peptide.

Theiler's murine encephalomyelitis virus (TMEV) induces a chronic demyelinating disease in the central nervous system of susceptible mice. Resistance to persistent TMEV infection maps to he D locus of the major histocompatibility complex suggesting a prominent role of antiviral CTL in the protective immune response. Introduction of the D(b) gene into the FVB strain confers resistance to this otherwise susceptible mouse line. Infection of the FVB/D(b) mouse with TMEV provides a model where antiviral resistance is determined by a response elicited by a single class I molecule. Resistant mice of the H-2(b) haplotype mount a vigorous H-2D(b)-restricted immunodominant response to the VP2 capsid protein. To investigate the extent of the contribution of the immunodominant T cell population in resistance to TMEV, FVB/D(b) mice were depleted of VP2-specific CD8(+) T cells by peptide treatment prior to virus infection. Peptide-treated mice were not able to clear the virus and developed extensive demyelination. These findings demonstrate that the D(b)-restricted CD8(+) T cells specific for a single viral peptide can confer resistance to TMEV infection. Our ability to manipulate this cellular response provides a model for investigating the mechanisms mediating protection against virus infection by CD8(+) T cells.