micr 304 s2010 lecture 5_epithelial.ppt
TRANSCRIPT
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.