MICR 304 Immunology &

Serology

MICR 304 Immunology &

Serology

Lecture 5Epithelial Cells

Chapter 2.3, Primary Literature

Lecture 5Epithelial Cells

Chapter 2.3, Primary Literature

Overview of Today’s Lecture

• Epithelial cell types • Pattern recognition receptors (TLR,

NOD)• Contributions of Epithelial cells to

host defense• Closer look at selected epithelial

cells

Key Players in Immunology

Innate Adaptive

Cells PhagocytesEpithelial Cells

NK Cells

Lymphocytes(B-Ly, T-Ly)

Effector Molecules

ComplementAntimicrobial (Poly)PeptidesAntimicrobial

Lipids?

Antibodies

Epithelial Cells Line All Body Surfaces

Epithelial Cell Types

• Keratinocytes– Keratinizing squamous epithelial cells

• Non-keratinizing squamous epithelial cells

• Columnar epithelial cells– Ciliated– Non-ciliated– Pseudostratified

• Transitional epithelial cells– Urinary bladder

Epithelial Cell Surfaces are Covered

• Skin– Lipids– Sweat

• Mucosa– Mucus

Challenges for Epithelial Cells

• Lungs– Large surface area– Exposure to vascular system

• Intestine– Nutrient rich– Large surface area– Heavy colonization– Food resorption

• Genital tract– Protection of offspring– Direct connection to peritoneum in the female tract

Epithelial Cell Surfaces are Port of Entry for Microbes• Skin

– Rarely direct– Insect bite– Wounds

• Airways– Inhalation

• Gastrointestinal tract– Contaminated

water or food

• Reproductive tract:– Direct contact

Epithelial Cell Surfaces are Port of Entry for Microbes

Salmonella entering intestinal epithelial cellsSchistosoma entering skin

Epithelial Cells Contribute to Host Defense in Many Ways

• Mechanical– Physical barrier (keratin)– Removal by ciliar transport– Removal by apoptotic cell shedding

• Biochemical– Production and secretion of antimicrobials– Proinflammatory cytokine production and

secretion– Immunoglobulin transport to the surface

• Microbial– Maintain normal flora

, keratin

General Defense Mechanisms Preventing Microbial Invasion

Mechanical Removal

• Epithelial cell shedding– Constant proliferation of basal cell

layer• Ciliar transport

– Respiratory tract– Mucociliary escalator

• Peristaltic– All tubular organs– Occlusion leads to infection

Mucociliary Escalator

Low pH

• Stomach– Gastric acid

• Vagina– Lactic acid

• Urine

Mucus Covering all Internal Surfaces

• Produced by Goblet cells and submucosal glands

• Viscous• Mucin (negatively charged carbohydrate

polymers) has high binding capacities• Glycoproteins• Penetration requires special microbial

pathogenic factors– Axial filament in T. pallidum– E. histolytica via proteinase (Moncada et al

2003)

Normal Microbiota• Increasingly recognized as important• NIH Roadmap: The Human Microbiome

ProjectSkin Tongue Esophagus

Normal Microbiota• Bacteria >>> fungi >>

protozoa• Mucosal surfaces more

populated than skin• Sites close to body

entrances are more populated

• Combined weight ~ 1 kg• Outnumber host by 1014 : 1,

host cells by > 10:1

Loss of Normal Microbiota Triggers Infectious Diseases

Pseudomembranous enterocolitis caused by C. difficile

Contributions of Normal Microbiota to Health

• Competition for space and nutrients• Production of antimicrobial factors

– Short chained fatty acids• Lactic acid by Lactobacilli

– Bacteriocins– H2O2 production

• Metabolic function– Vitamin production (K, some B)– Degradation of nutrients

• Enhance development of intestinal architecture, capillary network, epithelial cell differentiation

• Modulation of immune response– Induction of enzymes required for antimicrobial peptide processing

(Lopez-Boado et al 2000)– Induction of cytokine production (Klebanoff et al 1999)– Induction of IgA– Development of immune system

(reviewed in Salzman et al, Seminars in Immunology, 2007)

Probiotics

• Uptake of viable bacteria to the benefit of human health– Lactobacilli, Streptococci,

Bifidobacteria

• Withstand HCl, bile salts• Adhere to host

intestinal mucosa• Produce useful enzymes

or physiological end products

• Therapeutic applications – Infectious diseases

• Chronic UTI with E. coli

– Lactose intolerance– Inflammatory

disorders– Anti-carcinogenic

Prebiotics

• Food ingredients that alter normal microbiota to the benefit of the health

• Oligosaccharides stimulating the growth of bifidobacteria and lactobacilli

Epithelial Cell Defense

TLR

Microbial Products(LPS, PG, etc)

Antimicrobial Peptides

Cytokines

TLR: Toll-like receptor (pattern recognition)LPS: lipopolysaccharidePG: peptidoglycan

Antimicrobial Lipids?

Pattern recognition Receptors (PRP)

• Receptors that recognize pathogen associated microbial patterns (PAMP)

• Transmembrane receptors– Toll and Toll-like receptors (TLRs)

• Cytosolic receptors– Nod-like receptors (NLRs)

Toll and Toll-Like Receptors

• Transmembrane Receptor• Extracellular leucine-rich repeats• Intracytoplasmic conserved region

– TIR domain (Toll-like/ IL1 Receptor domain)

– Cystine-rich motifs that flank the leucine rich repeats

– Toll: in insects– Toll-like receptors: in other organisms

• Role in innate immunity

Toll-Like Receptors• TLR1 -10 in humans, TLR1-13 in

mice• Structural homology to Toll• All involved in Immune defense

– Intracellular region with homology to IL1 receptor

• Activated directly by microbial products not normally found in host

A relative small # of receptorscan recognize a relative large # of different

microbes.

Ligands of TLRs: Microbial Products

TLRs and Their Ligands

Intracellular

Extracellular

Cytoplasmic membrane

PeptidoglycanTLR2

[ ]

, LTA

Effects of TLR Activation• Cytokine up-regulation and secretion

– Pro-inflammatory cytokines– Chemokines

• Reactive oxygen and nitrogen metabolites

• Antimicrobial peptide production– HBD2

• Up-regulation of surface molecules enhancing adaptive immune responses– Co-stimulatory signals– MHC-II

• Apoptosis

InnateImmunity

AdaptiveImmunity

Epithelial Cells Recognize Microbial Products via TLR and NODs

• NOD: nucleotide-binding oligomerization domain proteins– NOD1: muramyl tripeptide containing

diaminopimelic acid (gram-negative bacteria)

– NOD2: muramyl dipeptide (most bacteria)

• TLR and NOD activation leads to NFB activation

Production of pro-inflammatory cytokines, chemokines, and antimicrobial

peptides

* Chronic NOD2 stimulation down regulates immune response (Hedl et al. 2007; Canto et al., 2009)

Epithelial Cell derived Cytokines

• Interleukins– IL-1, IL6 (Interleukin 1,6)

• Pro-inflammatory: fever, T-cell activation, macrophage activation, epithelial cell activation

• Chemokines– CXCL8 (IL-8, Interleukin 8): neutrophil and T-

cells– CCL2-5: monocytes, eosinophils, T cells

• Attracts and stimulates neutrophils, macrophages, T-cells

– CCL20: dendritic cells– CCL27: memory T-cells, B-cells

Production and Secretion of Antimicrobials

• Continuous secretion of human beta defensin HBD1

• Upregulation of human beta defensin HBD2, HBD3 and human defensin HD5 during infection and in response to cytokines

• Release of preformed antimicrobials– HD5 and HD6 in Paneth cells in the

intestine

Up-Regulation of HBD2 in Keratinocytes

CM: conditioned medium from

monocytes stimulated with LPS

Liu et al, The Journal of Immunology, 2003

Immunohistochemistry, organotypic cultures

IgA Transport Through Epithelial Cells

The Respiratory Tract Epithelium is Diverse

Alveoloar Duct

Clara CellsGloblet Cells

Ciliated Cells

Pneumocyte I

Pneumocyte II

Gas exchange

Surfactant

Anti-inflammatoryRepair

Mucus

TransportAntimicrobials

Pneumocytes II Secrete Surfactant

• Surfactant composition:– 90% Lipids– 10% Protein – Some carbohydrates

• Secreted as lamellar bodies with densely arranged phospholipid membranes

Surfactant: A Multitask Performers

• Surfactant important to reduce surface tension

• Opsonization

• Antimicrobial activity (Surfactant protein A-D)

Phagocytozing lung macrophage

Mucosa in Small Intestine

Schumick, Cleveland Clinic

Small Intestinal Epithelial Cells

• Intestinal stem cell• Four epithelial cell

lineages– Enterocytes– Goblet cells– Enteroendocrine cells– Paneth cells

Potten et al. ,2001

Columnar base cellLgr5+ (Clever’s Group)

Paneth Cells• Located in the base of crypts• Pyramidal shaped• Numerous secretory granules

– Lysozyme– Defensins

• Respond to microbial products– TLR9 +– NOD2+

• Respond to cholinergic agonists• Pivotal role in maintaining

normal flora • Protection of intestinal stem

cell• Paracrine signaling

– Chloride channel– IL8

(Salzman, Underwood and Bevins, Seminars in Immunology, 2007)

M-Cells in the Intestine

M-Cells in the Intestine

Today’s Take Home Message• Epithelial cells contribute in many ways to host

defense: mechanically (keratin, ciliar transport, apoptosis), biochemically (antimicrobial peptides, cytokines), and through maintaining the microbiota.

• TLR and NOD are pattern recognition receptors, whereby TLRs are membrane bound and NODs cytosolic. Their activation leads to AMP and cytokin production.

• Contributions of normal microbiota to host defense include competition for space and nutrients, production of antimicrobial factors,, metabolic function, development of intestinal architecture, modulation of immune response.

Additional Resources• http://education.vetmed.vt.edu/curriculum/vm8054/Labs

/Lab6/IMAGES/MONOCYTE%20IN%20SMEAR.JPG• Cantó E, Moga E, Ricart E, Garcia-Bosch O, Garcia-

Planella E, Juarez C, Vidal S. MDP-Induced selective tolerance to TLR4 ligands: impairment in NOD2 mutant Crohn's disease patients. Inflamm Bowel Dis. 2009 Nov;15(11):1686-96. PubMed PMID: 19572373.

• Hedl M, Li J, Cho JH, Abraham C. Chronic stimulation of Nod2 mediates tolerance to bacterial products. Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19440-5. Epub 2007 Nov 21. PubMed PMID: 18032608; PubMed Central PMCID: PMC2148308.