Oxidative stress, danger, and
immune diseases
Intro slides
Spring 2013
MCB 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 Stress
Oxidative 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.
References
Fiers, 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, 315360 (2000).
Hayes, J.D., et al., Glutathione and glutathione-dependent enzymes represent a coordinately 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 vs Autoimmune disease
• 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”
Hypersensitivities
• 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
Figure 10-2
Figure 10-1
Tolerance
– 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 costimulatory 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
Differentiation of autoimmune diseases;
organ specific vs systemic
• Organ specific
–
–
–
–
–
T1D
Multiple sclerosis
Grave’s disease
Autoimmune hemolytic anemia
Myasthenia gravis
• Systemic
– RA
– Scleroderma
– SLE
Examples of autoimmune disease that can be transferred across
the placenta
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
Components of immunity that are part of
autoimmune disease
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
Routes to 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
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
Genes involved in autoimmune disease
• 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)
Mutations at the Motheaten Locus are Within the
Hcph Gene
Function of SHP-1
• 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
Clinical disease in viable motheaten mice
• Anemia
• Immunodeficiency
• Autoimmunity
• Death from acidophilic
macrophage pneumonia
Macrophage pneumonia in mev/mev mice
+/?
mev/mev
Approaches to identifying genes involved in
autoimmune disease
• 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
Biochemistry of 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
Figure 1. Pathogenesis of diabetic microvascular complications. This schematic proposes that the development of microvascular
complications begins early in the course of diabetes, well before clinical diabetes is detected. Certain genetic characteristics
or 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 decrease
predisposition. 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 ameliorate
oxidative-stress-induced overactivation of poly adenosine diphosphate ribose polymerase (PARP), with the potential to change
the 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. Adapted
with 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/pdf
Diabetes 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
Diabetes is not the only context in which histone
methylation is potentially important. For example:
•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
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.
http://www.cdc.gov/diabetes/consumer/learn.htm
Rate of new cases of type 1 and type 2 diabetes
among youth aged <20 years, by race/ethnicity,
2002–2003
<10 years
10–19 years
CDC. National Diabetes Fact Sheet, 2007.
Source: SEARCH for Diabetes in Youth Study
NHW=Non-Hispanic whites; AA=African Americans; H=Hispanics; API=Asians/Pacific Islanders; AI=A
Indians
Humanized mouse models
Humanized mouse models to study human diseases Brehm et al.
NOD/SCID/Akita mouse
Your presentations
• 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
Grantsmanship:
NIH Steps to the NIH grant application
process http://funding.niaid.nih.gov/researchfunding/grant/pages/apply
ing.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/
Discussion points to include
• 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?
Grant application
• 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 format:
• 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
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