noninvasive-assessment-cardiac-abnormalities-experimental-autoimmune

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

in-situ-detection-autoreactive-cd4-t-cells-brain-heart-using-major

<em>In Situ</em> Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

University of Nebraska-Lincoln

University of Nebraska, Lincoln

Reactive oxygen species (ROS) produced in macrophages is critical for microbial killing, but they also take part in inflammation and antigen presentation functions. MicroRNAs (miRNAs) are endogenous regulators of gene expression, and they can control immune responses. To dissect the complex nature of ROS-mediated effects in macrophages, we sought to characterize miRNAs that are responsive to oxidative stress-induced with hydrogen peroxide (H(2)O(2)) in the mouse macrophage cell line, RAW 264.7. We have identified a set of unique miRNAs that are differentially expressed in response to H(2)O(2). These include miR-27a*, miR-27b*, miR-29b*, miR-24-2*, and miR-21*, all of which were downregulated except for miR-21*. By using luciferase reporter vector containing nuclear factor-kB (NF-kB) response elements, we demonstrate that overexpression of miR-27b* suppresses lipopolysaccharide-induced activation of NF-kB in RAW 264.7 cells. Our data suggest that macrophage functions can be regulated by oxidative stress-responsive miRNAs by modulating the NF-kB pathway.

miR-27b*, an oxidative stress-responsive microRNA modulates nuclear factor-kB pathway in RAW 264.7 cells.

Inactivation of the Staphylococcus aureus tricarboxylic acid (TCA) cycle delays the resolution of cutaneous ulcers in a mouse soft tissue infection model. In this study, it was observed that cutaneous lesions in mice infected with wild-type or isogenic aconitase mutant S. aureus strains contained comparable inflammatory infiltrates, suggesting the delayed resolution was independent of the recruitment of immune cells. These observations led us to hypothesize that staphylococcal metabolism can modulate the host immune response. Using an in vitro model system involving RAW 264.7 cells, the authors observed that cells cultured with S. aureus aconitase mutant strains produced significantly lower amounts of nitric oxide (NO(•)) and an inducible nitric oxide synthase as compared to those cells exposed to wild-type bacteria. Despite the decrease in NO(•) synthesis, the expression of antigen-presentation and costimulatory molecules was similar in cells cultured with wild-type and those cultured with aconitase mutant bacteria. The data suggest that staphylococci can evade innate immune responses and potentially enhance their ability to survive in infected hosts by altering their metabolism. This may also explain the occurrence of TCA cycle mutants in clinical S. aureus isolates.

TCA cycle inactivation in Staphylococcus aureus alters nitric oxide production in RAW 264.7 cells.

Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 T cells, CD4 T cells - especially self-reactive cells - are challenging to detect with major histocompatibility complex (MHC) class II tetramers because of low frequencies and low affinities of their T cell receptors to MHC-peptide complexes. Here, we report the use of fluorescent multimers, designated MHC dextramers that contain a large number of peptide-MHC complexes per reagent.

Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers.

A/J mice bearing the H-2 allele IA(k) are highly susceptible to autoimmune myocarditis induced with cardiac myosin heavy chain (Myhc)-α 334-352, whereas B10.A mice carrying a similar allele IA(k) are relatively resistant, suggesting that the generation of Myhc-α-reactive T cell repertoires is influenced by genetic background. To enumerate the precursor frequencies of Myhc-α-specific CD4 T cells, we sought to create IA(k) tetramers for Myhc-α 334-352. Tetramers were created using approaches that involve covalent tethering of individual peptide sequences or exogenous loading of peptides into empty IA(k) molecules by peptide-exchange reaction. Using ribonuclease 43-56 tetramers as controls, we demonstrated that by flow cytometry (FC), Myhc-α 334-352 tetramers specifically bind myosin-reactive T cells. CD4 cells isolated from A/J mice immunized with Myhc-α 334-352 were used to optimize conditions for tetramer staining, and neuraminidase treatment prior to tetramer staining permitted the detection of Myhc-α-specific cells ex vivo. The reagents are useful tools for monitoring the frequency of Myhc-α-reactive CD4 cells and to determine their pathogenic potential at a single cell level by FC.

Detection of cardiac myosin heavy chain-α-specific CD4 cells by using MHC class II/IA(k) tetramers in A/J mice.

Myocarditis is one cause of sudden cardiac death in young adolescents, and individuals affected with myocarditis can develop dilated cardiomyopathy, a frequent reason for heart transplantation. Exposure to environmental microbes has been suspected in the initiation of heart autoimmunity, but the direct causal link is lacking. We report here identification of novel mimicry epitopes that bear sequences similar to those in cardiac myosin heavy chain (MYHC)-α 334-352. These epitopes represent Bacillus spp., Magnetospirillum gryphiswaldense, Cryptococcus neoformans and Zea mays. The mimicry peptides induced varying degrees of myocarditis in A/J mice reminiscent of the disease induced with MYHC-α 334-352. We demonstrate that the mimics induce cross-reactive T cell responses for MYHC-α 334-352 as verified by MHC class II IA(k)/tetramer staining and Th-1 and Th-17 cytokines similar to those of MYHC-α 334-352. The data suggest that exposure to environmental microbes which are otherwise innocuous can predispose to heart autoimmunity by molecular mimicry.

Identification of novel mimicry epitopes for cardiac myosin heavy chain-α that induce autoimmune myocarditis in A/J mice.

We had previously reported that Acanthamoeba castellanii (ACA) contains a mimicry epitope for proteolipid protein 139-151 capable of inducing central nervous system (CNS) autoimmunity in SJL/J mice. We now present evidence that ACA also contains a mimicry epitope for myelin basic protein (MBP) 89-101, a derivative from amoebic nicotinamide adenine dinucleotide dehydrogenase subunit 2 (NAD). The epitope, NAD 108-120, contains a discontinuous stretch of six amino acids in the core region (VVFFKNIILIGFL) sharing 46% identity with MBP 89-101 (VHFFKNIVTPRTP; identical residues are underlined). SJL mice immunized with NAD 108-120 develop encephalomyelitis similar to the disease induced by the cognate peptide. We demonstrate that NAD 108-120 induces T cells that cross-react with MBP 89-101; the antigen-sensitized T cells, which produce predominantly T helper (T(h)) 1 and T(h)17 cytokines, transfer disease in naive SJL recipients reminiscent of the disease induced with MBP 89-101. This is the first report to demonstrate that a solitary microbe can induce CNS autoimmunity by generating cross-reactive T cells for multiple myelin antigens.

Identification of a second mimicry epitope from Acanthamoeba castellanii that induces CNS autoimmunity by generating cross-reactive T cells for MBP 89-101 in SJL mice.

Enteroviruses like coxsackievirus B3 (CVB3) are common suspects in myocarditis/dilated cardiomyopathy patients. Autoimmunity has been proposed as an underlying mechanism, but direct evidence of its role is lacking. To delineate autoimmune response in CVB3 myocarditis, we used IA(k) dextramers for cardiac myosin heavy chain (Myhc)-α 334-352. We have demonstrated that myocarditis-susceptible A/J mice infected with CVB3 generate Myhc-α-reactive CD4 T cells and such a repertoire was absent in naïve mice as measured by proliferative response to Myhc-α 334-352 and IA(k) dextramer staining. We also detected Myhc-α 334-352 dextramer(+) cells in the hearts of CVB3-infected mice. The autoreactive T cell repertoire derived from infected mice contained a high frequency of interleukin-17-producing cells capable of inducing myocarditis in naïve recipients. The data suggest that CVB3, a bona fide pathogen of cardiovascular system that primarily infects the heart can lead to the secondary generation of autoreactive T cells and contribute to cardiac pathology.

Coxsackievirus B3 infection leads to the generation of cardiac myosin heavy chain-α-reactive CD4 T cells in A/J mice.

In this report, we have addressed the role of copper-zinc superoxide dismutase (SOD1) deficiency in the mediation of central nervous system autoimmunity. We demonstrate that SOD1-deficient C57Bl/6 mice develop more severe autoimmune encephalomyelitis induced with myelin oligodendrocyte glycoprotein (MOG) 35-55, compared with wild type mice. This alteration in the disease phenotype was not due to aberrant expansion of MOG-specific T cells nor their ability to produce inflammatory cytokines; rather lymphocytes generated in SOD1-deficient mice were more prone to spontaneous cell death when compared with their wild type littermate controls. The data point to a role for SOD1 in the maintenance of self-tolerance leading to the suppression of autoimmune responses.

Copper-zinc superoxide dismutase-deficient mice show increased susceptibility to experimental autoimmune encephalomyelitis induced with myelin oligodendrocyte glycoprotein 35-55.

A definitive diagnostic test for multiple sclerosis (MS) does not exist; instead physicians use a combination of medical history, magnetic resonance imaging, and cerebrospinal fluid analysis (CSF). Significant effort has been employed to identify biomarkers from CSF to facilitate MS diagnosis; however, none of the proposed biomarkers have been successful to date. Urine is a proven source of metabolite biomarkers and has the potential to be a rapid, noninvasive, inexpensive, and efficient diagnostic tool for various human diseases. Nevertheless, urinary metabolites have not been extensively explored as a source of biomarkers for MS. We demonstrate that urinary metabolites have significant promise for monitoring disease-progression, and response to treatment in MS patients. NMR analysis of urine permitted the identification of metabolites that differentiate experimental autoimmune encephalomyelitis (EAE)-mice (prototypic disease model for MS) from healthy and MS drug-treated EAE mice.

Potential of urinary metabolites for diagnosing multiple sclerosis.

We report here identification of novel mimicry epitopes for interphotoreceptor retinoid-binding protein (IRBP) 201-216, a candidate ocular antigen that causes experimental autoimmune uveoretinitis (EAU) in A/J mice. One mimicry epitope from Ehrlichia canis (EHC), designated EHC 44-59, induced cross-reactive T cells for IRBP 201-216 capable of producing T helper (Th)1 and Th17 cytokines, but failed to induce EAU in A/J mice. In addition, animals first primed with suboptimal doses of IRBP 201-216 and subsequently immunized with EHC 44-59 did not develop EAU; rather, the mimicry epitope prevented the disease induced by IRBP 201-216. However, alteration in the composition of EHC 44-59 by substituting alanine with valine at position 49, similar to the composition of IRBP 201-216, enabled the mimicry epitope to acquire uveitogenicity. The data provide new insights as to how microbes containing mimicry sequences for retinal antigens can prevent ocular inflammation by acting as naturally occurring altered peptide ligands.

Mimicry epitope from Ehrlichia canis for interphotoreceptor retinoid-binding protein 201-216 prevents autoimmune uveoretinitis by acting as altered peptide ligand.

We report here the utility of major histocompatibility complex (MHC) class II dextramers for in situ detection of self-reactive CD4 T cells in two target organs, the brain and heart. We optimized the conditions for in situ detection of antigen-specific CD4 T cells using brain sections obtained from SJL mice immunized with myelin proteolipid protein (PLP) 139-151; the sections were costained with IA(s)/PLP 139-151 (specific) or Theiler's murine encephalomyelitis virus (TMEV) 70-86 (control) dextramers and anti-CD4. Analysis of sections by laser scanning confocal microscope revealed detection of cells positive for PLP 139-151 but not for TMEV 70-86 dextramers to be colocalized with CD4-expressing T cells, indicating that the staining was specific to PLP 139-151 dextramers. Further, we devised a method to reliably enumerate the frequencies of antigen-specific T cells by counting the number of dextramer⁺ CD4⁺ T cells in the 'Z' serial images acquired sequentially. We next extended these observations to detect cardiac myosin-specific T cells in autoimmune myocarditis induced in A/J mice by immunizing with cardiac myosin heavy chain-α (Myhc) 334-352. Heart sections prepared from immunized mice were costained with Myhc 334-352 (specific) or bovine ribonuclease 43-56 (control) dextramers together with anti-CD4; the sections showed the infiltrations of Myhc-specific CD4 T cells. The data suggest that MHC class II dextramers are useful tools for enumerating the frequencies of antigen-specific CD4 T cells in situ by direct staining without having to amplify the fluorescent signals, an approach commonly employed with conventional MHC tetramers.

Direct staining with major histocompatibility complex class II dextramers permits detection of antigen-specific, autoreactive CD4 T cells in situ.

We recently reported that Acanthamoeba castellanii (ACA), an opportunistic pathogen of the central nervous system (CNS) possesses mimicry epitopes for proteolipid protein (PLP) 139-151 and myelin basic protein 89-101, and that the epitopes induce experimental autoimmune encephalomyelitis (EAE) in SJL mice reminiscent of the diseases induced with their corresponding cognate peptides. We now demonstrate that mice infected with ACA also show the generation of cross-reactive T cells, predominantly for PLP 139-151, as evaluated by T cell proliferation and IAs/dextramer staining. We verified that PLP 139-151-sensitized lymphocytes generated in infected mice contained a high proportion of T helper 1 cytokine-producing cells, and they can transfer disease to naïve animals. Likewise, the animals first primed with suboptimal dose of PLP 139-151 and later infected with ACA, developed EAE, suggesting that ACA infection can trigger CNS autoimmunity in the presence of preexisting repertoire of autoreactive T cells. Taken together, the data provide novel insights into the pathogenesis of Acanthamoeba infections, and the potential role of infectious agents with mimicry epitopes to self-antigens in the pathogenesis of CNS diseases such as multiple sclerosis.

SJL Mice Infected with Acanthamoeba castellanii Develop Central Nervous System Autoimmunity through the Generation of Cross-Reactive T Cells for Myelin Antigens.

ArticleTotal : 2	Year
Publication title Cited by 7	2014
Publication title Cited by 11	2014

Article	Year
miR-27b*, an oxidative stress-responsive microRNA modulates nuclear factor-kB pathway in RAW 264.7 cells. Molecular and cellular biochemistry\| PubMed ID: 21350856	2011
TCA cycle inactivation in Staphylococcus aureus alters nitric oxide production in RAW 264.7 cells. Molecular and cellular biochemistry\| PubMed ID: 21519920	2011
Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers. BMC immunology\| PubMed ID: 21767394	2011
Detection of cardiac myosin heavy chain-α-specific CD4 cells by using MHC class II/IA(k) tetramers in A/J mice. Journal of immunological methods\| PubMed ID: 21782819	2011
Identification of novel mimicry epitopes for cardiac myosin heavy chain-α that induce autoimmune myocarditis in A/J mice. Cellular immunology\| PubMed ID: 21939961	2011
Identification of a second mimicry epitope from Acanthamoeba castellanii that induces CNS autoimmunity by generating cross-reactive T cells for MBP 89-101 in SJL mice. International immunology\| PubMed ID: 22058327	2011
Coxsackievirus B3 infection leads to the generation of cardiac myosin heavy chain-α-reactive CD4 T cells in A/J mice. Clinical immunology (Orlando, Fla.)\| PubMed ID: 22854287	2012
Copper-zinc superoxide dismutase-deficient mice show increased susceptibility to experimental autoimmune encephalomyelitis induced with myelin oligodendrocyte glycoprotein 35-55. Journal of neuroimmunology\| PubMed ID: 23294897	2013
Potential of urinary metabolites for diagnosing multiple sclerosis. ACS chemical biology\| PubMed ID: 23369377	2013
Mimicry epitope from Ehrlichia canis for interphotoreceptor retinoid-binding protein 201-216 prevents autoimmune uveoretinitis by acting as altered peptide ligand. Journal of neuroimmunology\| PubMed ID: 24029580	2013
Direct staining with major histocompatibility complex class II dextramers permits detection of antigen-specific, autoreactive CD4 T cells in situ. PloS one\| PubMed ID: 24475302	2014
SJL Mice Infected with Acanthamoeba castellanii Develop Central Nervous System Autoimmunity through the Generation of Cross-Reactive T Cells for Myelin Antigens. PloS one\| PubMed ID: 24879066	2014

Arunakumar Gangaplara

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