Oxidative stress, danger, and immune diseases

Intro slides Spring 2013MCB 5255

Reactive Oxygen Species

• Molecules or ions formed by the incomplete one-electron reduction of oxygen

• Include singlet oxygen; superoxides; peroxides; hydroxyl radical; and hypochlorous acid

• Contribute to the microbicidal activity of phagocytes, regulation of signal transduction and gene expression, and the oxidative damage to nucleic acids; proteins; and lipids

Formation and Function• In immune function, synthesized by dedicated

enzymes in phagocytic cells– Generated for killing engulfed bacteria

• Unavoidable byproduct of cellular respiration• Interaction of ionizing radiation with biological

molecules

Oxidative Stress SIGMA-ALDRICH

Oxidative StressOxidative stress is imposed on cells as a result of one of three factors: 1) an increase in oxidant generation, 2) a decrease in antioxidant protection, or 3) a failure to repair oxidative damage. Cell damage is induced by reactive oxygen species (ROS). ROS are either free radicals, reactive anions containing oxygen atoms, or molecules containing oxygen atoms that can either produce free radicals or are chemically activated by them. Examples are hydroxyl radical, superoxide, hydrogen peroxide, and peroxynitrite. The main source of ROS in vivo is aerobic respiration, although ROS are also produced by peroxisomal ß-oxidation of fatty acids, microsomal cytochrome P450 metabolism of xenobiotic compounds, stimulation of phagocytosis by pathogens or lipopolysaccharides, arginine metabolism, and tissue specific enzymes. Under normal conditions, ROS are cleared from the cell by the action of superoxide dismutase (SOD), catalase, or glutathione (GSH) peroxidase. The main damage to cells results from the ROS-induced alteration of macromolecules such as polyunsaturated fatty acids in membrane lipids, essential proteins, and DNA. Additionally, oxidative stress and ROS have been implicated in disease states, such as Alzheimer’s disease, Parkinson’s disease, cancer, and aging.ReferencesFiers, W., et al., More than one way to die: apoptosis, necrosis and reactive oxygen damage Oncogene., 18, 7719-7730 (1999).Nicholls, D.G., and Budd, S.L., Mitochondria and neuronal survival. Physiol. Rev., 80, 315-360 (2000).Hayes, J.D., et al., Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defense against oxidative stress. Free Radic. Res., 31, 273-300 (1999).

Free Radical Production

Oxidative Stress• An imbalance between the production and

manifestation of reactive species and the ability to readily detoxify the reactive intermediates– Can cause damage to all components of the cell

including proteins, lipids, and DNA• ROS vs RNS– Highly reactive molecules containing oxygen

• Peroxides, hydroxyl radicals, superoxide– Highly reactive molecules containing nitrogen

• Nitrogen dioxide (·NO2) and dinitrogen trioxide (N2O3)

Antioxidant

• A molecule capable of inhibiting the oxidation of other molecules– Oxidation: Loss of electron(s) resulting in an increase in oxidation

state– Reduction: Gain of electron(s) resulting in a decrease in oxidation

state• Antioxidants are reducing agents• Prevent reactive species from causing damage in the body

• Both endogenous and exogenous antioxidants– Endogenous: SOD, glutathione peroxidase, CAT– Exogenous: vitamin C, vitamin E, carotenoids and

polyphenols

Defenses against ROS• Antioxidant enzymes such as

Superoxide Dismutase and Catalase (2H2O2 -> 2H2O + O2)

• Antioxidants such as glutathione GSH– Glu-cys-gly tripeptide

• Antioxidant proteins such as Metallothionein

• Autoimmunity: self recognition by the immune response– Dual recognition (self-MHC plus antigenic peptide)– Jerne network hypothesis– “don’t eat me” signaling (CD47 on erythrocytes)

• Autoimmune disease: self recognition with damaging consequences to tissue function– Tissue specific (e.g. T1D)– Systemic (SLE)

• “Danger signals”

Autoimmunity vs Autoimmune disease

• 4 main hypersensitivities (I-IV)– Type I Anaphalaxis; Immediate; IgE mediated mast cell

degranulation• Allergies, atopy

– Type II Cytotoxic (IgM and IgG mediated)• Erythroblastosis fetalis, autoimmune hemolytic anemia,

pemphigus vulgaris– Type III Immune complex

• Serum sickness, RA,– Type IV DTH/contact sensitivity

• Contact dermatitis, T1D, RA, Multiple sclerosis

Hypersensitivities

Figure 10-2

Figure 10-1

– Discrimination of self vs non-self• Central tolerance develops in thymus and bone marrow

– (negative selection to eliminate cells reactive with antigens » Present soon after cell expresses antigen receptor» Present at high concentration over long periods of time

• Peripheral tolerance/anergy– When cells encounter antigen in the absence of co-stimulatory

signals that are usually provided by inflammation• Antigen segregation

– Physical barriers to restrict immune cell access» Thyroid, pancreas, intracellular

• Regulatory cells that suppress responses• Clonal deletion post activation

Tolerance

• Organ specific– T1D– Multiple sclerosis– Grave’s disease– Autoimmune hemolytic anemia– Myasthenia gravis

• Systemic– RA– Scleroderma– SLE

Differentiation of autoimmune diseases; organ specific vs systemic

disease autoantibody Symptom

Myesthenia gravis Anti-acetylcholine receptor Muscle weakness

Graves disease Anti-thyroid stimulating hormone receptor

Hyperthyroidism

Thrombocytopenic propura Anti-platelet antibodies Bruises and hemorrhaging

Pemphigus vulgaris Anti-desmoglein Blistering rash

Examples of autoimmune disease that can be transferred across the placenta

Disease T cells B cells Antibody

SLE Pathogenic help for antibody

Present antigen to T cells

Pathogenic

T1D Pathogenic Present antigens to T cells

Present but unclear role

Myesthenia gravis Help for antibody Antibody secretion Pathogenic

Multiple sclerosis Pathogenic Present antigen to T cells

Present but unclear role

Components of immunity that are part of autoimmune disease

• Pathogens– Cross-reactive antigens/molecular mimicry

• Lyme arthritis• Rheumatic fever

– Chronic inflammation, immune dysregulation– Disruption of cell/tissue barriers

• Sympathetic ophthalmia (granulomatous uveitis)• Toxicants and other stressors• Genetic predisposition• Combinations of the above

Routes to Autoimmune Disease

http://pubs.acs.org/doi/pdf/10.1021/tx9003787(see class website for link)

Figure 10-28 part 1 of 2

Figure 10-28 part 2 of 2

• Single gene models– Fas, FasL; ALPS (defects in apoptosis, lymphoaccumulation, angergy

and SLE-like autoimmune disease)– Mev; viable motheaten, Hcph-1; SHP1 (chronic inflammation)– IPEX immune dysregulation X linked recessive mutation in

transcription factor FoxP3; severe allergic inflammation, hemolytic anemia, thrombocytopenia, etc.

– Deficiency in CD25 (IL2R); impaired peripheral tolerance– CTLA4 mutation; Graves disease, T1D, etc.– C1q mutation SLE– MHC associations with autoimmune disease (e.g. HLA-B27)

Genes involved in autoimmune disease

Mutations at the Motheaten Locus are Within the Hcph Gene

• Negative regulator of signal transduction

– growth factor receptors: c-kit, EPO– activation signaling: BCR, TCR, NK activating receptor– SHP-1 inactivates anti-apoptotic signaling molecules in

neutrophil proliferation– induces apoptosis in sympathetic neurons

Function of SHP-1

Clinical disease in viable motheaten mice

• Anemia

• Immunodeficiency

• Autoimmunity

• Death from acidophilic   macrophage pneumonia

Macrophage pneumonia in mev/mev mice

mev/mev

+/?

• GWAS genome wide associational studies• Family studies to identify SNP that track with

autoimmune disease• Animal models with mutations in candidate

genes• Meta-analysis of data to enlarge patient

populations studied for autoimmune disease

Approaches to identifying genes involved in autoimmune disease

• Biochemical events that potentiate autoimmunity– events that cause damage to membrane, etc• Reactive oxygen, chronic inflammation

• Biochemistry of damaging events associated with autoimmune disease

» Reactive oxygen, chronic inflammation

Biochemistry of autoimmune disease

Figure 1. Pathogenesis of diabetic microvascular complications. This schematic proposes that the development of microvascularcomplications begins early in the course of diabetes, well before clinical diabetes is detected. Certain genetic characteristicsor polymorphisms (Apo E4, Aldose reductase, ACE) may increase individual predisposition for development of microvascular

complications of diabetes [30,31], whereas other genetic factors, such as the toll receptor, are protective and decreasepredisposition. The various inflammatory mediators listed under the heading of inflammation cause direct cellular injury and

initiate the cycle of functional and progressive pathologic changes, which ultimately manifest as microvascular complications[13,15–18,21]. As the disease progresses, lipotoxicity [28], glucotoxicity [42,43], and epigenetic factors further contribute to the

functional and pathologic changes. Intervention with insulin or insulin sensitizers, particularly in the early stages of pathogenesis,can counteract inflammatory changes, control glycemia, prevent formation of advanced glycation end products, and ameliorateoxidative-stress-induced overactivation of poly adenosine diphosphate ribose polymerase (PARP), with the potential to changethe natural history of microvascular complications [29,37]. ApoE4 = Apolipoprotein E4; ACE = Angiotensin-converting enzyme;

PKCβ = Protein kinase C beta; IL-6 = Interleukin-6; TNFα = Tumor necrosis factor alpha; NFκ B = Nuclear factor kappa B. Adaptedwith permission from Vinik A, Mehrbyan A. Diabetic neuropathies. Med Clin North Am 2004; 88: 947–999

http://onlinelibrary.wiley.com/doi/10.1002/dmrr.530/pdfDiabetes Metab Res Rev 2005; 21: 85–90.

http://nihroadmap.nih.gov/epigenomics/epigeneticmechanisms.asp

Histone modifications

http://www.nature.com/nsmb/journal/v14/n11/images/nsmb1337-F1.gif

http://www.cellsignal.com/reference/pathway/Histone_Methylation.html

•H3K4me3 demethylases : link between histone modifications and XLMR. X-linked mental retardation (XLMR) gene SMCX (JARID1C), which encodes a JmjC-domain protein, reversed H3K4me3 to di- and mono- but not unmethylated products//Cell 2007

•The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3//Nature (2006) 442: 307-11.

•Sustained JNK1 activation is associated with altered histone H3 methylations in human liver cancer.  //J Hepatol.  2009, 50: 323-33

•Perturbation of epigenetic status by toxicants//Toxicology LettersVolume 149, Issues 1-3, 1 April 2004, Pages 51-58

Diabetes is not the only context in which histone methylation is potentially important. For example:

http://www.cdc.gov/diabetes/consumer/learn.htm

Type 1 diabetes, which was previously called insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes, may account for 5% to 10% of all diagnosed cases of diabetes.

Type 2 diabetes, which was previously called non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes, may account for about 90% to 95% of all diagnosed cases of diabetes.

Gestational diabetes is a type of diabetes that only pregnant women get. If not treated, it can cause problems for mothers and babies. Gestational diabetes develops in 2% to 5% of all pregnancies but usually disappears when a pregnancy is over.

Other specific types of diabetes resulting from specific genetic syndromes, surgery, drugs, malnutrition, infections, and other illnesses may account for 1% to 2% of all diagnosed cases of diabetes.

Rate of new cases of type 1 and type 2 diabetes among youth aged <20 years, by race/ethnicity,

2002–2003

CDC. National Diabetes Fact Sheet, 2007.Source: SEARCH for Diabetes in Youth StudyNHW=Non-Hispanic whites; AA=African Americans; H=Hispanics; API=Asians/Pacific Islanders; AI=American Indians

<10 years 10–19 years

Humanized mouse models

Humanized mouse models to study human diseases Brehm et al.

NOD/SCID/Akita mouse

• Each presentation is ~1 hour• Spend first 20 minutes or so describing the

fundamental information: what do we need to know to understand the papers you have assigned? How does this presentation fit into the course main topic?

• Divide the second 30 minutes into discussions of each of the two contemporary papers that you assigned to the class at the previous class period

Your presentations

Grantsmanship:

NIH Steps to the NIH grant application process  http://funding.niaid.nih.gov/researchfunding/grant/pages/applying.aspx

NIH electronic grant forms http://grants.nih.gov/grants/funding/424/index.htm

Examples of outstanding titles and abstacts  http://funding.niaid.nih.gov/researchfunding/grant/pages/titleabs.aspx

Search engine for currently funded grants  http://projectreporter.nih.gov/reporter.cfm

Tongue-in-cheek": how to fail in grant writing  http://chronicle.com/article/How-to-Fail-in-Grant-Writing/125620/ 

• What is the fundamental hypothesis that is being tested?

• What techniques did they use that we have to understand to evaluate the data?

• What are the most important figures/data sets that we should discuss?

• Are there alternative interpretations of their data?• What conclusions did they reach?• What new questions do they open up with their

results?

Discussion points to include

• Hypothesis and ONE specific aim are due March 5 to be discussed on March 6th

• Grant is due May 5 (first day of exam period) by 5pm (hard copy plus electronic e-mailed file please)

Grant application

• TEXT:• Hypothesis and specific aim (0.5 page)• Background and Significance (3-4 pages)

– What do we know about the system?– What makes this hypothesis tenable?– How is the approach you propose innovative?

• Research designs and Experimental approach (4-5 pages)– Rationale– Experimental design and methods– Anticipated outcomes– Potential pitfalls and alternative approaches

• We will talk about NIH forms later in the semester

Grant format:

Inflammatory Bowel Disease

• Include:– Crohn’s Disease– Ulcerative Colitis

• Autoimmune disease—idiopathic• Current treatments:– Treat symptoms, reduce frequency– Surgical resectioning

Effects of IBD

• Severe inflammation, perforation of intestinal epithelium

• Strictures, fistulae, toxic megacolon, perianal disease

• Arthritis common, may be unrelated• Increased risk of cancer, infection

Oxidative Stress in Autoimmune Disease

• Excessive oxidative stress is thought to have an important role in the pathogenesis of many autoimmune diseases– Enhances inflammation, induce apoptotic cell death,

disrupt signal pathways • Seen in diseases such as:

• RA• SLE• IBD• MS