9/24/2012. The Skin as a Barrier. Dermal Barriers to Infection-How Normal Flora Become Opportunistic, Invasive and Pathogenic

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1 Dermal Barriers to Infection-How Normal Flora Become Opportunistic, Invasive and Pathogenic PCPC Conference, October 2012 Lipo Chemicals Inc. Nava Dayan PhD All content included on this presentation, is the property of Nava Dayan and is protected by International copyright laws. This presentation and its contents may not be duplicated, reproduced or used in any way other than for your personal use, unless permission is obtained in writing. In no event shall Nava Dayanor her sources be liable for lost profits or any other special, incidental or consequential damages arising out of or in connection with this presentation Outline The skin as a barrier Innate immunity Anti-microbial peptides Anti-microbial lipids Commensals contribution to skin s immunity Skin normal flora Opportunistic bacteria Strategies bacteria uses to invade the skin Susceptible population- what is an immuno-compromised skin? Summary The Skin as a Barrier 1

2 Stratum Corneum Structure and Function The outmost layer composed of dead cells but live tissue An assembly of 15 to 20 layers of flattened cells that are filed with filaments of keratin Surface cells continuously shed Brick and mortar model where corneocytes are bricks, and intercellular lipids and corneo-desmosomes are mortar The stratuncorneum consists of less then 10% of the skin, but contributes over 80% of the skin s permeability barrier function Lipophilic in nature Skin Structure Human skin is composed of three main layers: the epidermis, dermis, and hypodermis The epidermis is the topmost layer and can be further divided into the stratum corneum and the viable epidermis picture from Courtesy of Prof. Michniak-Kohn, Center for Dermal Research The Stratum Corneum Barrier for Protection The desquamation process is a protective mechanism to eliminate: Microorganisms Damaged cells- protection against cancer Harmful agents Slow down penetration In human forearm, for example, about 1300 cells/cm 2 /hr are shed and commonly accumulate as house dust 2

3 Stratum Corneum- Skin Penetration Most compounds will penetrate the skin via the intracellular pathway (between the corneocytes) This is a lipophilic, sinuous route for penetration Size threshold is around MW of 500 Desmosomes Plasma membrane Intercellular space Connecting fibers Keratin fibers associated with cytplasmic plague 1mm Tightly connect adjacent cells but permit compounds to move around them in the intercellular space Follicles A route into the dermal layer Constant flow of sebum that is controlled by blood hormone levels Sebum secreted by glands is composed of free fatty acids, triglycerides and waxes 3

4 Location of the Epidermal Barrier Mackee et al 1945 upper viable epidermis Berenson & Burch 1951 inner SC Blank 1953 inner SC Blank 1953 all layers of the SC??? Kligman 1960 all layers of the SC Courtesy of Prof. P. Wertz, Iowa University Courtesy of Prof. Wertz, University of Iowa Intercellular Lipids Generated from lamellar granules that are synthesized by keratinocytes Composed of fatty acids, cholesterol and ceramides Matrix arranged in lamellar sheets along with intercellular adhesion complexes,corneo-desmosomes Provides barrier for water loss, controls skin hydration The intercellular lipids comprise only 1-5% of the total volume of the stratum corneum, but is the major permeation route for most molecules, both hydrophilic and lipophilic Stratum corneum lipids include chains much longer than those of most biological membranes Wertz P.W. SeminDermatol. 11 (1992) Lipids Composition in Pig s Epidermis Courtesy From: of Prof. Prof. Wertz P. UOI Wertz, Iowa University G.M. Gray & H. Yardley 4

5 Cosmetics is Generally Safe when Applied to Healthy Intact Skin Serious problems from cosmetic use are rare when applied to healthy intact skin and used in an appropriate manner Misuse of cosmetics is the #1 reason concerning the safety issues For example: Adding liquids to the product Mixing two different products Sharing makeup Unhygienic practices (e.g. applying with dirty hands) Inappropriate storage (e.g. excessive heat, open container/closure etc.) Applying it on an impaired barrier (irritated skin or infected eye) Using the product after it is expired Cases of Compromised Barrier The integrity of natural skin barrier may be compromised when exposed to detergents/ organic solvents/ sensitizers during normal household or occupational settings A slightly damaged skin may allow for faster rate, shorter lag time and higher total penetration of chemicals/ pathogens Immune system, if compromised (e.g. due to treatment of cancer or other infections), the resident microflora may become an opportunistic infectious pathogen (sepsis) Skin s Innate Immunity 5

6 Skin s Innate Immunity, Definition Innate immunity is composed of a multitude of antigen-nonspecific defense mechanisms that a host uses immediately or within several hours after exposure to almost any insult This is the immunity one is born with and is the initial response by the body to eliminate insult such as microbial contamination and prevent infection Unlike adaptive immunity, innate immunity is designed to recognize molecules shared by groups of related insulting agents that are essential for the survival of those organisms and are not found associated with mammalian cells The innate immune responses do not improve with repeated exposure Skin s Innate Immunity- Pathways for Defense Examples of innate immunity mechanisms include: Anatomical Barriers / Mechanical Removal Bacterial Antagonism Pattern-recognition Receptors Antigen-nonspecific Defense Chemicals Complement Pathways Phagocytosis Inflammation Fever Skin Innate Immunity Components of Skin Innate Immunity: Keratin and physical shield Lipids Peptides Sebum (organic acids such as Undecylinicacid) Sweat Resident vs. Transient microflora Acidic ph Low water content Phagocytic cells (neutrophils, monocytes, and macrophages) Cells that release inflammatory mediators (basophils, mast cells, and eosinophils) Natural killer cells (NK cells) Molecules such as complement proteins, acute phase proteins, and cytokines 6

7 Lipids Lipids at the skin surface are thought to be part of the anti-microbial barrier Synthesized in the epidermis and travel to the surface Secreted by sebaceous glands Free sphingoid bases, maybe generated by ceramides hydrolysis Exhibits broad anti-microbial activity Sapienic acid, derived from sebum triglycerides Showed anti microbial activity against MRSA Drake D. R. et al. J. Lipid Res. 49 (2008) 4-11 Peptides Peptides on skin and mucosa display an anti-microbial activity against bacteria and viruses Newly isolated dermicidins where found to be produced by eccrine sweat glands In addition to their anti-microbial activity, these peptides were also found to regulate the adaptive immune system through cytokines release (IL-17, IL- 22) A certain type of dermicidin (DCD-IL) was shown to stimulate keratinocytes to generate cytokines and chemokines Kolls J.K. et al Nat. Rev. Immunol. 8 (2008) Niyonsaba M. et al. Br. J. Dermatol. Oct., 2008 Innate Immune System Opportunistic pathogens may penetrate the skin via the extra cellular matrix Some permeability barrier lipids exhibit anti-microbial properties Anti-microbial peptides (AMP) localized in lamellar bodies are co-delivered to intercellular matrix Both AMP expression and lipid synthesis are accelerated after barrier insult 7

8 Possible Mechanism of Action of Anti Microbial Lipids Morphological Changes in C. Striatum Control Normal morphology characterized by club shape rod Scanning electron microscopy Epidermal synthesized lipid Bacteria clump into aggregates with distortion of the membrane surface Epidermal synthesized lipid in combination with sebum generated fatty acid Disruption of bacteria cell wall and membrane integrity Studies conducted by Carol L Bratt; Dow Institute; The University of Iowa Possible Mechanism of Action of Anti Microbial Lipids Morphological Changes in C. Striatum Control Normal diffuse granular cytoplasm Epidermal synthesized lipid Cytoplasm components flocculated Apparent disruption of membrane and cell wall and leakage of cellular contents Epidermal synthesized lipid in combination with sebum generated fatty acid Flocculation of the cellular contents and detached membrane Transmission Elecrton Microscopy (TEM; Uranyl acetate staining) Studies conducted by Carol L Bratt; Dow Institute; The University of Iowa Possible Mechanism of Action of Anti Microbial Lipids Morphological Changes in C. Striatum Epidermal synthesized lipid in combination with sebum generated fatty acid, TEM Studies conducted by Carol L Bratt; Dow Institute; The University of Iowa 8

9 Skin Microflora: Source, Nature, Roles The topical normal bacterial flora that inhabits the skin is related to its barrier function Normal flora interferes with pathogenic microorganism growth Examples of normal flora include: Staphylococcus, Micrococcus, Neisseria, Peptococcus, Corynebacterium, Propionobacterium, Streptococcus and Acinetobacter Not all species are found on one person, but most people carry at least five of them Skin Microflora: Source, Nature, Roles Microflora include: bacteria, fungi, algae, protozoa and viruses that live in harmless association with our skin The microflora increases the acidic nature of the skin, therefore making it inhospitable for pathogens Different parts of our body have different populations of microflora This balance can change as a result of variations in diet, use of antibiotics or personal hygiene products and physical injury Skin Microflora: Source, Nature, Roles Some of the flora that are harmless on the surface of the skin can become harmful when the surface is broken and they can pave their way to the blood stream Staphylococcus aureusfor example can cause serious disease if penetrates the circulation Propionobacterium acnesresides in hair follicles and is usually harmless, however when the follicle pore is clogged, the bacteria may infect the surrounding tissue causing inflammation The ideal skin cleanser will remove unwanted microorganisms while maintaining the skin barrier function 9

10 Opportunistic Bacteria and Strategies for Skin Invasion Bacteria Associated Disease By bacteria that is not part of the normal flora (example M. tuberculosis) Bacteria which is part of the normal flora that acquires extra virulence factors that makes it pathogenic (example: E. coli) Bacteria that is part of the normal flora that can gain access to deep tissues by trauma(example: S. epidermidis) Immuno-compromised patients are prone to disease by free living bacteria and components of the normal flora, especially if introduced to deep tissues (example: Acinetobacter) Conditions When Resident Flora Becomes a Threat Deep wounds and/or large areas of burn-common among war burn victims Conditions when immune system is compromised -HIV, cancer treatment Aged population- the immune system activity is diminished Neonates and young children 10

11 Conditions for Colonization Sufficiently large inoculum Reaching and adhering specific target cells Existence of conducive environment for pathogen survival In skin Damaged skin (e.g. cuts) Sebaceous gland Hair follicles Age related change in skin resistance (due to environmental factors, chemical exposure or disease) Strategies for Skin Invasion Take Advantage of Breaks in Skin Compromised barrier- shaving, irritation, allergic reaction, sunburn, applied heat, humidity Cuts allow penetration of opportunistic pathogens Catheters- allow direct access to already immuno-challenged individual Burns- pseudomonas infections 11

12 General Mechanisms of Action Composition of skin flora of the body varies from site to site and depends on factors including: Amount and composition of sebum and sweat Location of sweat glands Moisture content Age Ethnicity Predominant flora: Micrococci Saprophytic corynebacterium Propioniobacterium S. aureus-exists on average on 30% of the population Population in Higher Risk for Skin Infections Weakened general immunity ( systemic disease, prior disease or hormone imbalance) Weakened skin immunity (skin chronic disorder) Skin allergy Genetic pre-disposition (?) Life style (malnutrition), environment (extremes of temperature, humidity) Age- young and old Work related exposure hospitals, military, athletes Emotional stress and fatigue Summary The skin is a dynamic organ that provides more then just a physical shield It naturally hosts a variety of organisms that, if healthy and balance, assist maintain its integrity In recent years we advanced significantly in the research and understanding of the role of skin s innate immunity and identified key lipids and peptides that participate in it When the individual is immuno-compromised it can lead to imbalance in normal flora and the initiation of a disease 12

13 August 13, 2012 Bacteria on Skin Boost Immune Cell Function The harmless bacteria that thrive on the skin can help immune cells fight diseasecausing microbes, according to a new study in mice. The finding gives new insight into skin health. By Dr. Yasmine Belkaid, NIH Additional Reading Peter M. Elias and Kenneth Feingold Acknowledgments Special thanks go to my collaborators at the Dow Institute at the University of Iowa: Prof. Philip Wertz Prof. Kim Brogden Prof. David Drake Carol Bratt; graduate student Thank you Nava Dayan Ph. D. 13