Autoimmunity and Risk Assessment Luebke

7/30/2019 Autoimmunity and Risk Assessment Luebke

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AutoimmunityImmunotoxicity Risk Assessment

Bob LuebkeImmunotoxicology BranchExperimental Toxicology Division

NHEERL, ORD

Bob LuebkeITB/ETD/NHEERLUS EPA


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Autoimmunity and Autoimmune Disease

Autoimmunity: response to self antigens

Recognition autoimmune disease Required for normal immune function

Interaction between innate and adaptive response: Major Histocompatibility Complexmolecules

Autoantibodies are common

Autoimmune disease: Damage to organs, structures, cells or interferingwith receptors

Examples

Rheumatoid arthritis

Multiple sclerosis

Systemic lupus erythematosus

Hashimoto's thyroiditis

Myasthenia gravis



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Factors Affecting Immunocompetence.and

Autoimmunity

Age

Gender * Genotype*

Life style choices

Stress



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Age and Autoimmunity

Accumulated damage to host DNA/cells/tissues

Altered immune function related to age

Telomere shortening in T helper cells? Replicative senescence causing altered gene expression

Proinflammatory cytokines

Loss of self tolerance



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Host Factors Associated with

Autoimmunity

Gender

Incidence of autoimmunity in females > in males

Constitutive differences in immunoreactivity

Estrogen increases and androgen decreases B cellfunction

Physiologic changes in hormone levels modulate theseverity of certain autoimmune diseases

SLE worse during pregnancy RA and MS severity decreased in pregnancy

Males with RA tend to have lower Te

Physiological shift in Th1-Th2 balance



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Autoimmunity Genetics

Animal modes: incidence approaching 100%

Humans: concordance rates in monozygotic twins: 30-

50% Susceptibility gene(s) suspected, but most not identified

Environmental factors provide triggers

HLA and autoimmunity

Strongest association with HLA-B27 and gram negative infections

Strong associations with other MHC haplotypes

Differences in toxicant metabolism or elimination

SLE associated with gene polymorphisms in drug metabolism and clearance

Polymorphism in various cytokine genes

Associations with autoimmune disease unproven



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Autoimmunity

Lifestyle

Smoking

Pulmonary damage and Goodpastures syndrome

Increased susceptibility to RA due to polyclonal

lymphocyte activation

Alcohol abuse

Elevated Ig levels, particularly IgA

Formation of autoimmunogenic protein adducts by

EtOH metabolites



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Autoimmunity

Lifestyle

Stress

Acute stress associated with the onset of RA

Chronic stress associated with RA flare-ups

Exacerbation of SLE by acute and chronic stress

Occupation

Exposure to solvents, pesticides, metals



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Potential Causes of

Autoimmunity

Failure to remove potentially autoreactive

cells

Altered self Molecular mimicry

Unmaksing of cryptic antigens

Altered regulatory protein production (e.g.,cytokines) or gene expression



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Potential Causes of

Autoimmunity

Failure to remove potentially autoreactive cells


Positive selection: optimalbinding to self Ag preventsapoptosis

Negative selection:superoptimal binding toself Ag induces apoptosis


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Potential Causes of

Autoimmunity

Failure to remove potentially autoreactive cells



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Potential Causes of

Autoimmunity

Defects in peripheral tolerance: T regulatory cells CD4+CD25+FOXP3+ (Treg) subpopulation

Develop in the periphery from naive CD4+ T cells

FOXP3 transcription factor represses IL-2 production byCD4 cells Mutation in FOXP3 associated with type I diabetes and

hypothyroidism

Reduced numbers of circulating Treg

Juvenile idiopathic arthritis Psoriatic arthritis

Autoimmune liver disease

SLE

Correlate with a higher disease activity or poorer prognosis

Hu et al., 2007. Trends Immunol. 28, 329-332 for review



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Potential Causes of

Autoimmunity

Altered self Conformational change in host protein: autoimmune hemolytic anemia

or platelet destruction

Molecular mimicry Structure of (typically) pathogen component and host protein similar

Antibodies to bacterial or viral Ag cross-react with host tissues Rheumatic heart disease

Unmasking of cryptic antigens Damage to host tissues or cells , release of normally hidden proteins

(antigens)

Ab recognizes extracellular matrix Goodpastures syndrome

Altered regulatory protein production (e.g., cytokines) or geneexpression



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Immunopathology of Autoimmune

Disease

Ab interaction with receptors

Agonist: Graves disease

Inhibitor: Myasthenia gravis

Immune complex damage

Antibody-antigen complexes

Chronic infections

Complexes localized in small vessels, joints, organs

Activation of macrophages and complement

system upregulates inflammation



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Immunopathology of Autoimmune

Disease T cell mediated

Organ-specific

Type 1 diabetes mellitus: antigenic mimicry?

Multiple sclerosis: antigenic mimicry

Epstein Barr virus, Herpes virus-6, milk proteins

Components of nerve sheath

Hashimotos thyroiditis: antigenic mimicry?

Mediated by direct T cell cytotoxicity (CD8+)

Mediated by proinflammatory products from T cells,M

Cytokines: IL-1beta, TNFalpha

Reactive nitrogen or oxygen intermediates



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Agents Associated with

Autoimmunity

Metals

Solvents

Infectious agents

Pesticides

Particles (asbestos, silica)

Ultraviolet light



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Autoimmunity

Mercury

Human exposure (amalgam, occupation, drugs)

T cell activation, ANA, elevated IgE, proteinuria, glomerulonephritis

in humans

Th2-dominated response

Upregulated IL-4 stimulates IgG1/IgE antibodies and polyclonal B cell

expansion

Possible altered-self mechanism

Possible alteration of antigen processing mechanism,leading to presentation of cryptic nuclear antigens



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Autoimmunity Lead

Proximal tubule damage associated withoccupational lead exposure

Typically associated with enhanced Ab synthesis,

rather than autoimmunity in lab animals Pb activates Th2 cells in vitro and in vivo

Stimulates production of IL-4, IL-8 and IL-12

Autoimmune disease possibly due to increased

regulatory gene transcription



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Autoimmunity

Hydrocarbons Occupational exposure to trichloroethylene: ~400,000 workers in US

TCE detected in 35% of tested US water supplies Increased symptoms of SLE in a population drinking TCE-contaminated water in Tucson,

AZ

Systemic sclerosis (scleroderma) Fibrosis, collagen deposition

Suggested link to various MHC haplotypes

Aromatic hydrocarbons (benzene, toluene, xylene) associated withlocalized scleroderma

Aliphatic HCs (naphtha, hexachloroethane, n-hexane), vinyl chloridemonomer associated with systemic form



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Autoimmunity

Infection

Enteric bacteria, HLA-B27 and inflammatory disease of

joints, connective tissues

Molecular mimicry Sequence homology in bacterial proteins and B27 molecule

Bacterial or viral superantigens

Polyclonal activation of T cells by Ag-presenting cells

Stimulated T cells produce huge quantities of cytokines Drive development of autoreactive T cells?

Flare-up or exacerbation of existing disease



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Autoimmunity

Pesticides

Occupational chlorpyrifos exposure associatedwith autoantibodies 1- 4.5 yr after exposure

(n=12) Most patients had pre-existing allergic conditions,

although 3 developed allergies after exposure

Increased incidence of antinuclear antibodies in a

rural population (n=322). Associated with lifetimeexposure to: carbamate, OCs (aldrin, dieldrin,endrin, chlordane, heptachlor, lindane),pyrethroids, and phenoxyacetic acid herbicides

Reviews: Holsapple, 2002. Toxicol Lett. 127, 101-109; Luebke, 2002. Human

Ecol. Risk Assess. 8, 293-303; Voccia et al., 1999. Toxicol. Ind. Health. 15, 119-132.



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Autoimmunity

Asbestos, Libby, Montana

Systemic autoimmune diseases

Odds ratio for systemic autoimmune disease = 2.14

Odds ratio for rheumatoid arthritis = 3.23

Occupational and military exposure to asbestos

increased risk

Noonan et al., 2006. Environ. Health Perspect. 114,1243-1247

Markers


Pfau et al., 2005. Environ. Health Perspect. 113, 25-30


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Summary

Genotype: significant, but not exclusive

predisposing factor in development of

autoimmune disease

Probable contribution by other host factors

Environment has definite role

Autoimmunity (or at least autoantibodyproduction) may be as common or more

common than xenobiotic-induced

immunosuppression



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Additional Resources

Autoimmunity And Toxicology - Immune

Disregulation Induced By Drugs And

Chemicals, M.E. Kammller, N. Bloksma, W.

Seinen, eds. Elsevier, 2006

WHO, 2006. Environmental Health Criteria

Series, No. 236 Principles and Methods for

Assessing Autoimmunity Associated withExposure to Chemicals



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Risk Assessment



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Xenobiotic Exposure and Immunocompetence

Most likely outcome: mild to moderatesuppression

Reduced resistance to infection

Recovery expected when exposure ends

Detection in humans at the population level is difficult

Animal models: must distinguish overt toxicity

from immunotoxicity Dose response

Effects at doses that do not alter body weight or foodconsumption

Young healthy well-fed animals are usedBob LuebkeITB/ETD/NHEERLUS EPA


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Immunotoxicology Hazard ID

Nomination for testing by NTP

Research in government and academic labs

Observations in routine toxicity testing

Mass, cellularity, architecture of spleen, thymus orlymph nodes

Abnormal hematology

Tiered Testing (NTP, OECD407)

Tier 1. Organ weights and cellularity, phenotypicanalysis, hematology, enhanced histopathology

Tier 2. Functional parameters, host resistance



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Testing Schemes

In vitro immunotoxicology

Little experience with cell culture for hazard ID

Better for mode or mechanism studies

Microarray analysis Functional vs observational endpoints

Model choice

Species, strain and gender

Visit http://www.inchem.org/documents/ehc/ehc/ehc180.htmfor WHOguidelines

http://www.inchem.org/documents/ehc/ehc/ehc180.htmhttp://www.inchem.org/documents/ehc/ehc/ehc180.htm


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Upstream Markers, Downstream

Consequences


Upstream (observational) Downstream (consequential)

Indicator Assessment Function Interpretation

Lymphoidorgan data

Weights, cellularityviability, architecture

Maturation,differentiation

Suggestive

Cell surfacemarkers

Flow cytometry Relative distribution byfunction/type

Suggestive/confirmatory

Innateimmunity

Activation, phagocytosis

killing, mediatorproduction

Resistance to infection Intracellular & extracellularinfection, viral infections,tumors

Antibodysynthesis

TDAR: PFC or ELISA

responseResistance to infectionToxin neutralization

Risk of extracellular infection,viral infections, tetanus

Cellmediatedimmunity

Delayed hypersensitivity

Cytotoxicity

Cytokine production

Resistance to infection,neoplasia

Help for Ig response

Risk of intracellular infection,viral infections, tumors

Up

Down


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Equivalent Markers


Marker Rodent Assay Human Equivalent Observational Functional

Cellularity Organs, CBC CBC x

CellPopulations Organs, PBMC PBMC x

Mediators Organs, PBMC PBMC x x

CellProliferation

Organs, PBMC PBMC x xAntibody

Production

TDAR Vaccination

xCellularImmunity

DTH DTH xHostResistance Challenge Epidemiology/Challenge x


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Immunotoxicology Hazard ID

EPA Health Effects Test Guidelines: OPPTS 870.7800Immunotoxicity (TSCA, FIFRA), Published 10/26/07


Immunize(SRBC)

Assayon d29

Adult Mice and/or Rats

Exposure28 Days

Suppression Phenotypic analysisoptional

Case by case NK cell assay


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Immunotoxicity Hazard IDEPA Health Effects Test Guidelines: OPPTS 870.7800

Immunotoxicity (TSCA, FIFRA)


Inject SRBC

Measure relative or absolute

antibody concentration by ELISA

Serum samples

Single cell

suspension

Count relative (per million)or absolute (per spleen)number of cells producingantibody
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Regulatory Interest in Life Stage and

Susceptibility

Pesticides in the diets of infants and children(NRC, 1993) Need for data on the effects of pesticides on the

developing reproductive, immune, and centralnervous systems.

Food Quality Protection Act (EPA, 1996) Separate assessment for infants and children to

establish pesticide residue levels

Safe Drinking Water Act (EPA, 1996)

EPA to conduct studies to identify sensitive subgroups Executive Order # 13045, 1997

Identification of potential health risks to kids a highpriority



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Critical Windows of Exposure:

Rodents vs. Humans

Event Mouse (days)(% of term)

Human (weeks)(% of term)

Appearance of T cells in fetal liver 14 (67%) 6-8 (15-20%)

Organogenesis of thymus begins 11 (52%) 6 (15%)

Lymph nodes evident 10.5 (50%) 8-12 (20-30%)

Spleen develops 13 (62%) 10-14 (25-35%)

B cell lymphopoiesis begins in bone marrow 17 (81%) 12 (30%)

B lymphocytes detectable in blood 13 (62%) 12 (30%)

CD4+ and CD8+ T cells detectable in spleen 19 (91%) 14 (35%)

Thymus development completed 13 (62%) 15-16 (37-40%)

Bone marrow becomes the major site ofhematopoiesis

17.5 (83%) 22 (55%)

T cell receptor expression in periphery Early post-natal 23 (58%)

Bob Luebke

ITB/ETD/NHEERLUS EPA


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Implications for Risk Assessment

In the young

Late gestational exposure in rodents mimics early-

to-mid gestation in humans

Does not address the aged population

Stress affects resistance to infection

Chemicals? Critical data gap

Bob Luebke



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Advanced Age, Chemical Exposure and

Host Resistance

Bob Luebke


Luebke et al. 1999. Toxicology 136, 1526.


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Hazard Identification:

Developmental vs. Adult Exposure

Developmental vs. adult exposure to xenobiotics (DES, DZP, Pb, TCDD,

TBTO) (Luebke et al., 2006. J. Toxicol. Environ. Health B Crit. Rev. 9, 1-26)

Can cause long-lasting ( lifetime) effects at doses that cause short-term

immunotoxicity in adults (e.g., DES, DZP)

Testing adults will detect effects, but grossly underestimate the relative risk of

gestational/neonatal exposure

Can cause immunotoxicity at lower doses in the young than in mature

animals, and developmental effects are more persistent (DZP, Pb, TBTO)

Application of an uncertainty factor may provide protection, but would not predict persistence

Can cause long-lasting ( lifetime) effects in offspring at doses that do not

affect immune function in adults (e.g., TCDD in rats) Testing adults only would fail to detect immunotoxicity

Bob Luebke



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Hazard ID: Identifying Potential

Autoimmunogenic Compounds

Autoimmune-prone animal models

NZBXNZW-F1 mice: lupus like disease, esp. in

females

MRL/Ipr: uncontrolled lymphoproliferation

More rapid onset of disease following HgCl2 or

TCE exposure

Female Fisher rats: defect in CRH production

Bob Luebke



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Interpreting Evidence of

Immunosuppression Clear Evidence of Toxicity to the Immune System

Dose-related changes in function (e.g., Ab synthesis or resistance to disease)

Effects in multiple endpoints that suggest biological plausibility (i.e. alterationsin NK cell cytotoxicity and NK cell numbers) would also provide clear evidenceof immune toxicity

Some Evidence of Toxicity to the Immune System

No change in functional parameters but statistically significant, dose-relatedadverse changes in lymphoid tissue histopathology or WBC counts

Equivocal Evidence of Toxicity to the Immune System Marginal deficits in immune parameters that may be chemically-related

Example: statistically significant changes in one or more parameters at middleor low doses, but not at high doses, in the absence of other supportive data

Bob Luebke


Courtesy of Dori Germolec, NIEHS


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Weight of Evidence for

Immunosuppression

No Evidence of Toxicity to the Immune System

Inadequate study of immunotoxicity

Major qualitative or quantitative limitations

cannot be interpreted as valid for showing the

presence or absence of immunotoxicity

Bob Luebke


Courtesy of Dori Germolec, NIEHS


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Next Steps

Setting safe exposure levels

Is immunotoxicity the most sensitive endpoint?

Are developmental exposure data available?

What is the level of confidence in the data?

EPA guidelines for immunosuppression risk

assessment in progress

Bob Luebke

ITB/ETD/NHEERL

Documents

Autoimmunity and Risk Assessment Luebke