Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D.

MUCOSAL IMMUNITY

LEARNING GOAL You will be able to describe the mucosal .

OBJECTIVES To attain the goal for this lecture you will be able to:

 Describe the components of the mucosal immune system.  Draw the structure of secretory IgA.  Explain the mechanism of IgA transport across mucosal surfaces.  Explain how a response to antigen is generated in the mucosal system.  Identify the differences in tolerogenic versus immunogenic responses to mucosal antigen administration.  Delineate the functions of the mucosal immune system, including M cells.  Describe how the mucosal immune system might be used for immunization.  Describe the characteristics of selective IgA deficiency.  Describe how intestinal commensal bacteria interact with the host to promote a healthy environment

READING ASSIGNMENT Janeway’s Immunobiology 8th Edition (2012), Chapter 12 and Article “Mucosal Immunity and Vaccines” by Robyn Seipp attached at end of lecture notes.

LECTURER Katherine L. Knight, Ph.D.

Page 1 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D.

CONTENT SUMMARY

I. INTRODUCTION TO MUCOSAL IMMUNITY

II. ORGANIZATION OF THE MUCOSAL IMMUNE SYSTEM A. Components of the Mucosal Immune System B. Induction of a Response C. Features of Mucosal Immunity D. Intraepithelial lymphocytes (IEL)

III. IgA SYNTHESIS, STRUCTURE AND TRANSPORT

IV. FUNCTIONS OF IgA AT MUCOSAL SURFACES A. Barrier Functions B. Intraepithelial Viral Neutralization C. Excretory Immunity D. Passive Immunity E. IgA Deficiency State

V. MUCOSAL IMMUNIZATION

VI. MUCOSAL TOLERANCE A. The Induction of Tolerance via Mucosal Sites B. The interaction Between Gut Bacteria and the Intestine

I. INTRODUCTION TO MUCOSAL IMMUNITY

Mucosal surfaces are continually exposed to external infectious agents, and consequently, immunologic defense against pathogens is paramount at these surfaces. Specific immunologic defense at mucosal surfaces is mediated by a specialized arm of the immune system that is termed the mucosal immune system. The mucosal immune system includes lymphoid tissues of the , respiratory tract, salivary glands, lacrimal glands, mammary glands, and genito-urinary tract. The mucosal, or secretory, branch of the immune system is quite extensive, as the mucosal surfaces of the human body represent an area 100 times greater that of the skin. The importance of this system is underscored by the fact that 70 to 80% of all immunoglobulin producing cells in the body are physically located within the tissues of the mucosal immune system. Worldwide, over 12 million (1.2 x 107) deaths result from mucosal

Page 2 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D. infections.

Mucosal tissues are exposed to a large number of both potentially harmful and benign antigens from the environment, food, and microorganisms. For example, the intestine is host to hundreds/thousands of different bacteria. The mucosal immune system must therefore continually control responsiveness and unresponsiveness. Unlike many other components of the immune system, our understanding of the regulation of mucosal immunity remains somewhat incomplete.

II. ORGANIZATION OF THE MUCOSAL IMMUNE SYSTEM

A. Components of the Mucosal Immune System Mucosal immunity is triggered by the coordinated interaction of multiple cell types within the mucosal tissues. The process involves the initiation of the response at an inductive site, leading to an immune response at multiple effector sites.

Components of the mucosal immune system (MALT) include:  Gastrointestinal tract – gut associated lymphoid tissue (GALT); includes and  Respiratory tract – bronchial associated lymphoid tissue (BALT)  Nasal associated lymphoid tissue (NALT)  Genitourinary tract  Lacrimal glands  Salivary glands  Mammary glands

Cells of the mucosal immune system are located in anatomically defined compartments (e.g., Peyer’s patches and isolated lymphoid follicles) as well as throughout mucosal tissues.

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B. Induction of a Response The inductive process has been best described for the GALT, which can be used as a prototype to explain the generation of mucosal immunity. Another inductive site that is gaining attention is the NALT, as inductive sites that are similar to those found in the GI tract are also present in nasal mucosa. Evidence for induction through BALT is also available.

Lymphocytes reside in defined compartment of MALT (GALT is best defined example).

Mechanistically, the induction process can be divided into the following steps:

 Antigens entering the digestive tract can be taken up

Page 4 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D. - By nonspecific transport across the epithelium or by specialized mucosal cells called M cells which internalize the antigen and transport it across the epithelium where antigen can be taken up by APCs such as dendritic cells (DC). - By FcRn transport of Ag/Ab complexes - By apoptosis of infected enterocytes - By DC that have dendrites extending through the epithelial tight junction into the lumen

 Antigens are then presented to lymphocytes (in the intestine, these are located in Peyer’s patches).

 Lymphocytes (both B and T cells) leave the mucosal site and travel to the mesenteric lymph nodes, then into the lymph.

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 Via the thoracic duct, the lymphocytes exit the lymph and enter the circulation.

 Circulating lymphocytes “home” to positions within the mucosal lamina propria throughout the body, including sites distant from the original antigenic encounter. The homing of lymphocytes to mucosal sites involves specific interactions of both adhesion molecules and chemokines.

 B Lymphocytes within the peripheral tissues proliferate and differentiate into IgA secreting plasma cells at effector sites.

C. Features of Mucosal Immunity

1. The administration of antigen at one mucosal site results in specific antibody production at distant mucosal sites. Some regional preference seems to occur, however. For example, induction via NALT leads to a more robust response in the respiratory sites than in gastrointestinal sites.

2. B cells in the mucosa are selectively induced to produce dimeric IgA rather than other isotypes. The selective switch of B cells to IgA is believed to be mediated by specific cytokines produced by T cells in the inductive sites.

3. Conventional T cells, particularly CTLs, are also an important component of the mucosal immune response. The induction and homing requirements for these cells are not as well described as those for mucosal B cells.

Page 6 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D. 4. Induction of a response via a mucosal site generally elicits a systemic immune response as well, such that serum antibodies can be detected. This indicates that a mucosal encounter with antigen generates subsets of T and B cells that home to mucosal sites and also to and regional nodes.

D. Intraepithelial Lymphocytes (IEL)

A distinct population of lymphocytes, mostly CD8+ T cells are found in the gut epithelium. There are 10-15 IEL for every 100 epithelial cells and because the mucosa has a huge surface area, IELs are one of the largest population of lymphocytes in the body. The function of these cells is still not clear but they may readily kill infected epithelial cells.

III. IgA SYNTHESIS, STRUCTURE AND TRANSPORT

The predominant immunoglobulin in mucosal secretions is IgA. Serum Ig – 12% IgA class, primarily monomeric Secreted Ig at mucosal sites – 96% IgA, primarily dimeric IgA in mucous secretions is called secretory IgA, or sIgA.

The production of secretory IgA (sIgA) requires both plasma cells in the lamina propria and epithelial cells of the mucosa.

 Dimeric IgA (2 monomeric IgA units covalently joined a J chain) is produced by plasma cells within the mucosal lamina propria.  Dimeric IgA binds to the polymeric immunoglobulin receptor (pIgR) on the basal surface of mucosal epithelial cells.  The IgA-pIgR complex is endocytosed and transported through the epithelial cell to the lumenal surface for release.

Page 7 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D.  During this transport, the pIgR is cleaved and a small fragment is lost.  The remaining large component, secretory component, is covalently bound to the dimeric IgA.  IgA is secreted at the mucosal surface as dimeric IgA covalently bound to secretory component.

 Secretory IgA production requires two different cell types.  Only polymeric immunoglobulins (dimeric IgA or pentameric IgM) are capable of binding and being transported by pIgR.  Mice that are genetically deficient for pIgR exhibit the expected decreases in IgA transport. PIgR deficiency also leads to an increased mucosal leakiness.

IV. FUNCTIONS OF IgA AT MUCOSAL SURFACES

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A. Barrier Functions

Secretory IgA can bind to bacteria and viruses and prevent their adherence and invasion into mucosal tissues. Secretory IgA can neutralize many viruses in this way, including polio, herpesvirus, coxsackie virus, and rotaviruses. Secretory IgA can also neutralize bacterial toxins at mucosal surfaces

B. Intraepithelial Viral Neutralization

IgA that is internalized by mucosal epithelial cells (via the pIgR) may contribute to intracellular viral inactivation.

C. Excretory Immunity

Viral particles that complex with dimeric IgA in the lamina propria may be endocytosed and transported out by the pIgR pathway.

D. Passive Immunity sIgA in breast milk provides passive immunity to the infant.

E. IgA Deficiency

Selective IgA deficiency is the most common primary immune deficiency, with an estimated incidence of 1 per every 500 to 1000 persons. The precise characteristics of the deficiency are variable, as some patients have complete IgA deficiency but others have decreased but detectable levels of IgA.

Patients present with low or no levels of serum IgA, but have normal cell mediated immunity and serum antibody responses. Not all patients exhibit increased susceptibility to infection. Reasons to suspect selective IgA deficiency include 1) a family history of IgA deficiency of agammaglobulinemia, 2) a high incidence of oral infections, 3) frequent respiratory infections, and 4) chronic diarrhea.

Autoimmune diseases, including SLE, juvenile rheumatoid arthritis, and thyroiditis, are often associated with selective IgA deficiency. Immunoglobulin therapy is generally not indicated, as the patient’s normal antibody response can produce anti-IgA antibody in response to IgA treatment. People with a complete absence of IgA may develop or even anaphylactic shock if given gammaglobulin.

Page 9 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D. V. MUCOSAL IMMUNIZATION

Mucosal surfaces are portals of entry for many pathogens (e.g. cholera, HIV, influenza).

The development of immunization strategies that would produce a robust mucosal immune response is a high priority.

When compared to systemic immunization by intramuscular, intraperitoneal, or intradermal routes, immunization with mucosally administered antigens has both advantages and disadvantages.

ORAL IMMUNIZATION

Advantages Disadvantages  Ease of administration (oral)  Difficulty in eliciting robust response  Generates both mucosal and systemic  Response may not be long-lasting immunity

An example of an effective oral immunization is the polio vaccine. Effective nasal spray vaccines for influenza have recently been developed.

New strategies for oral immunization include the use of cholera toxin chimeric molecules as well as recombinant avirulent bacteria (e.g. avirulent salmonella expressing S. pneumoniae proteins). Can also target M cells using bacteria and viruses that preferentially bind M cells or antigen encased in biodegradable particles such as latex. These strategies attempt to boost the uptake of foreign antigen at mucosal induction sites.

VI. MUCOSAL TOLERANCE

A. The Induction of Tolerance via Mucosal Sites

 The mucosa is exposed to many environmental antigens such as food that are not infectious. To operate in an effective manner, the mucosal immune system must distinguish between pathogenic antigens, which require a response, and non-dangerous antigens, such as those in food and in the commensal bacteria that make the gut their home. The response to most antigens is tolerance, and the type of antigen is critical to eliciting the appropriate response. The key feature that appears to distinguish between the induction of a response and the induction of tolerance is inflammation. Antigen encounters that occur alongside inflammation generally illicit an immune response. Antigen encounterd in the absence of inflammation generally induces tolerance. Thus:  Food antigens generally induce tolerance.

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 Microbes (bacteria and viruses) that cause inflammation generally evoke a mucosal immune response.  Peptides generally induce tolerance, unless attached to a mucosal adjuvant, such as cholera toxin.  Tolerance can occur by anergy (unresponsiveness) or regulatory T cells (Treg)  The induction of tolerance might be exploited therapeutically in autoimmune diseases, or to limit transplant rejection.

B. Interaction Between Gut Bacteria and the Intestine

 Commensal bacterial species coinhabit the gut; 10X more bacterial cells than human cells  The mechanism by which the mucosal administration of some antigens induces tolerance, rather than immunity, is incompletely understood. Recent studies suggest that mucosal tolerance is mediated by mucosal dendritic cells.

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 Regulatory T cells are a prominent feature at mucosal sites, and may synergize with suppressive dendritic cells. Regulatory populations have been isolated from draining lymph nodes of mucosal sites.

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MUCOSAL IMMUNITY AND VACCINES By Robyn Seipp The mucous membranes are one of the largest organs of the body (Figure 1). Collectively, they cover a surface area of more than 400m2 (equivalent to one and a half tennis courts) and comprise the linings of the gastrointestinal, urogenital and respiratory tracts [15,18].

Figure 1: Schematic diagram of the mucous membranes of the body and surfaces with which they are in contact. These mucosal surfaces, while located inside the body, are actually a physical barrier between the outside and the sterile interior cavity of the body known as the “systemic” environment. Critical nutrients, oxygen and other molecules are constantly taken up across these mucosal barriers; however, another important function of the mucous is to keep invading pathogens out [15]. Daily these mucous membranes are bombarded by outside elements and it is up to the unique immune system of the mucous to determine what is potentially harmful and what is beneficial [15,18].

Why mucus matters

Interest in mucosal tissues stems from their importance in immunity, with the vast majority of human pathogens initiating infections at mucosal surfaces, making the gastrointestinal, urogenital and respiratory tracts major routes of entry into the body [14,18]. In fact, virtually the only way to contract an infection other than mucosally is through blood-borne routes such as injections, transfusions and bites. Examples of mucosally-infecting agents include cold viruses, influenza, food poisoning agents, tuberculosis, sexually transmitted diseases, cholera, diphtheria and plague [15]. Despite its important role, only a handful of vaccines specifically target this area of

Page 13 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D. the immune system despite strong evidence that a good mucosal response can effectively prevent systemic infections [15].

Components of a mucosal immune response

The systemic and mucosal immune systems have an arsenal of molecules specifically designed to eliminate invading pathogens. One of the first lines of defense are antibodies, made and secreted by plasma B cells. Antibodies are large protein molecules capable of binding and neutralizing an invading organism7. A picture of a typical antibody is shown in Figure 2.

Figure 2: Schematic diagram of the prototypical IgG antibody. It shows the two antigen binding sites of the variable regions of light and heavy chains. The constant region determines the type of antibody and its effector function. It is composed of heavy chains only.

The mucous membranes produce a special type of antibody called secretory IgA or sIgA. In the mucous, this antibody is secreted as a dimer, joined at the non-antigen binding end by a protein known as the J chain as shown in Figure 3.

Figure 3. Structure of secretory IgA. It consists of at least two IgA molecules covalently linked by a J chain and the secretory component, which is added as the antibody crossed the mucosal epithelial cells into the lumen.

This form of the antibody is more stable, less resistant to proteolysis by the digestive enzymes of the gut, and has higher avidity for mucosal surfaces [13,15,18]. The mucous membranes are bathed in huge quantities of sIgA, which act as a first line of

Page 14 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D. defense to neutralize invading pathogens [15,15]. Experimental evidence shows that the presence of sIgA correlates with resistance to infection by various pathogens, including bacteria, viruses, parasites and fungi [13,15,18]. It has also been shown to neutralize viruses and prevent their adherence to the epithelial cells lining the mucous (thereby preventing infection) as well as mediating excretion of pathogens and preventing the assembly of mature virus particles [13,15,18,21]. Another important component of mucosal immunity is the T cell-mediated immune response. T cells that specifically recognize pathogens can help antibodies to clear the infection or directly kill the invader themselves. T cells produced in the mucous are capable of traveling throughout the mucosal tissues through special “homing” receptors on their membranes [13,15,18]. This means that if an immune response is generated in the gastrointestinal lining, T cells produced there can travel to other mucosal sites, for example, the lungs or nasal cavity, providing protection over a large surface area [13,15,21].

The importance of mucosal immunology lies in the interplay between the mucosal response and the systemic immune response. Several studies have demonstrated that stimulating the immune system systemically (i.e. via injection or blood-borne routes) results in production of protective antibody and T cells only within the sterile, internal environment of the body—no mucosal response is generated. On the other hand, stimulation of the mucosal immune response can result in production of protective B and T cells in both mucosal and systemic environments so that infections are stopped before they get into the body [13,15,18].

Mucosal vaccinations

The goal of current mucosal vaccination strategies is to prevent both initial stages of disease (colonization and infection by pathogens) and block its development [14]. Currently, the vast majority of vaccines available only block disease development once the pathogen has crossed the mucosal barrier into the normally sterile systemic environment [13]. Mucosal vaccines have several advantages over traditional systemic vaccines. They can be administered orally or nasally rather than via injection. This is more widely accepted by the public, as well as making the vaccine simpler to administer and distribute. In addition, there is less risk of needle stick injury or cross-contamination [13,15,18]. Inoculation could be accomplished using recombinant bacteria to express pieces of proteins from the pathogen (known as antigens) that the immune system will respond to and “remember” for the next infection. Another possibility is the creation of transgenic plants expressing pathogen antigens that could be eaten to administer the vaccine. Certain types of transgenic plants such as banana trees and potatoes have also been modified to overexpress protein antigens from pathogens such as hepatitis B and rotavirus; however, to date the level of protein expressed in the plant is variable and often too low for effective immunization [13,14,15].

Page 15 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D. Spotlight on a mucosal AIDS vaccine Studies of populations with high incidences of HIV infection have shed some light on possible approaches to the development of an effective vaccine. Analysis of several groups of HIV-exposed individuals has consistently revealed the existence of a small subgroup that is resistant to infection despite multiple, long-term exposures [11,12]. In about 30% of cases it appears to be the presence of high levels of HIV- neutralizing sIgA in the genital tract and HIV-reactive T cells in the cervix that confers resistance to infection [10-12]. Because 70-80% of people contract the disease mucosally, and because the gut is the major reservoir for HIV replication, there is interest in the potential of the mucosal immune response in preventing and/or clearing infection [2,10]. Among the newer vaccines designed to induce a protective immune response to HIV is one designed to mimic the mucosal responses seen in these resistant individuals [2]. In 2001, a US group tested the first mucosal HIV vaccine in rhesus macaques [2]. While the vaccine showed promising results, it failed to prevent infection.

Autoimmunity

Autoimmunity occurs when the immune system begins attacking “self” components of the body aberrantly. The reasons why this occurs remain largely unknown, but the consequences can be devastating as parts of the body are slowly destroyed by the very system designed to protect them. Examples of autoimmune diseases include multiple sclerosis, rheumatoid arthritis, lupus and type I diabetes [15]. Mucosal immunity offers some possibilities for alleviating or reversing symptoms of autoimmune diseases. The basis of treatment involves a phenomenon known as mucosal tolerance. In the 1940’s, M.W. Chase noticed that repeated feeding of a substance to mice reduced their immune response when the same substance was injected systemically [15]. His experiment is outlined in Figure 4.

Figure 4. Chase’s experiment that demonstrated mucosal tolerance. a) Injection of picryl chloride into mice led to a strong systemic immune response. b) When mice

Page 16 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D. were first fed picryl chloride for several weeks, then injected with picryl chloride, a significant loss in the systemic immune response was observed. This phenomenon, known as mucosal tolerance, has been demonstrated not only with picryl chloride, but also with heavy metals, proteins, blood cells, mites, inactivated viruses, bacteria and many other substances [15]. The exact mechanisms of how the body adapts its immune response in this way are still not well understood [15]. Nevertheless it is believed to be a normal body function to prevent hypersensitivity to food proteins, normal bacterial flora that inhabit the gastrointestinal tracts and other mucosal surfaces of healthy individuals, and other environmental macromolecules [15].

Mucosal tolerance is being exploited for the development of autoimmune disease therapies. Because autoimmunity is caused by the immune system reacting to “self” antigens, the theory is that administration of the self antigen to which the body is aberrantly reacting by oral or nasal routes could induce mucosal tolerance and thus lower the harmful systemic immune response [15]. For example, in multiple sclerosis (MS), myelin (a fatty substance surrounding nerve cells) is slowly degraded by the immune system, resulting in gradual loss of nerve function (see Figure 5 and http://www.mssociety.ca/).

Figure 5. Electron micrograph of a cross-section of a nerve cell showing the myelin sheath. Mice suffering from EAE (acute experimental autoimmune encephalomegalitis) have very similar symptoms and are used as experimental models for human MS. Myelin deterioration was suppressed in these mice by oral or nasal administration of very high doses of MBP (myelin basic protein) [6,9,15]. Clinical trials in humans using bovine MBP have had controversial results: MBP reappeared on nerves of patients in a pilot study but with a larger group the positive observations of the initial study failed to be confirmed [6,9,15].

Immunocontraceptives

Page 17 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D. The idea of harnessing the immune response to control fertility was first proposed in the 1930’s [3]. However, only recently has our comprehension of the immune system progressed to the level where the idea may finally be put into practice. Over the past 30 years, the World Health Organization Task Force on Vaccines for Fertility Regulation has supported basic and clinical research on the development of birth control “vaccines” [8]. Studies have shown that approximately 30% of human infertility is associated with the presence of anti-sperm antibodies in mucous secretions of men and/or women and sperm-reactive T cells in men only [3,16]. These individuals show no other health problems other than immune-mediated infertility: therefore, inducing an immune response against critical components of reproduction might be a safe and effective way to control conception [3,16]. One approach being considered is the design of a “vaccine” capable of inducing an immune response against sperm antigens in either partner. Immunization of the male partner would result in inviable sperm production, while immunization of the female partner would result in reduced sperm motility or neutralization in the cervix and/or impaired penetration of sperm into the oocyte [3]. Another target for immunocontraceptive vaccines are reproductive hormones such as follicle stimulating hormone and human gonadotropin. Interfering with the functioning of these hormones can also reduce fertility.

References and Additional Reading 1) Barber B. Introduction: Emerging vaccine strategies. Sem. Immunol. 1997 9: 269- 270. 2) Belyakov IM, Hel Z, Kelsall B, Kuznetsov VA, Ahlers JD, Nacsa J, Watkins DI, Allen TM, Sette A, Altman J, Woodward R, Markham PD, Clements JD, Franchini G, Strober W, Berzofsky JA. Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques. Nat Med. 2001 Dec;7(12):1320-6. 3) Delves PJ, Lund T, Roitt IM. Antifertility vaccines. Trends Immunol. 2002 Apr;23(4):213-9. Review. 4) Delves PJ, Lund T, Roitt IM. Future prospects for vaccines to control fertility.Trends Immunol. 2002 Apr;23(4):220-1. 5) Fujihashi K, Koga T, van Ginkel FW, Hagiwara Y, McGhee JR. A dilemma for mucosal vaccination: efficacy versus toxicity using enterotoxin-based adjuvants.Vaccine. 2002 Jun 7;20(19-20):2431-8. Review. 6) Fukaura H, Kent SC, Pietrusewicz MJ, Khoury SJ, Weiner HL, Hafler DA. Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-beta1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J Clin Invest. 1996 Jul 1;98(1):70-7. 7) Goldsby RA, Kindt TJ and Osborne BA. “Kuby Immunology” 4th ed. W.H. Freeman & Co: New York. 2000. 8) Griffin PD. The WHO Task Force on Vaccines for Fertility Regulation. Its formation, objectives and research activities. Hum Reprod. 1991 Jan;6(1):166-72. Review.

Page 18 Host Defense 2013 Mucosal Immunity April 16, 2013 Katherine L.Knight, Ph.D. 9) Hafler DA, Kent SC, Pietrusewicz MJ, Khoury SJ, Weiner HL, Fukaura H. Oral administration of myelin induces antigen-specific TGF-beta 1 secreting T cells in patients with multiple sclerosis. Ann N Y Acad Sci. 1997 Dec 19;835:120-31. 10) Kaul R, Plummer FA, Kimani J, Dong T, Kiama P, Rostron T, Njagi E, MacDonald KS, Bwayo JJ, McMichael AJ, Rowland-Jones SL. HIV-1-specific mucosal CD8+ lymphocyte responses in the cervix of HIV-1-resistant prostitutes in Nairobi. J Immunol. 2000 Feb 1;164(3):1602-11. 11) Kaul R, Trabattoni D, Bwayo JJ, Arienti D, Zagliani A, Mwangi FM, Kariuki C, Ngugi EN, MacDonald KS, Ball TB, Clerici M, Plummer FA.HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan sex workers. AIDS. 1999 Jan 14;13(1):23- 9. 12) Mazzoli S, Lopalco L, Salvi A, Trabattoni D, Lo Caputo S, Semplici F, Biasin M, Bl C, Cosma A, Pastori C, Meacci F, Mazzotta F, Villa ML, Siccardi AG, Clerici M.Human immunodeficiency virus (HIV)-specific IgA and HIV neutralizing activity in the serum of exposed seronegative partners of HIV-seropositive persons. J Infect Dis. 1999 Sep;180(3):871-5. 13) McCluskie MJ, Davis HL. Mucosal immunization with DNA vaccines. Microbes Infect. 1999 Jul;1(9):685-98. Review. 14) Medina E, Guzman CA. Modulation of immune responses following antigen administration by mucosal route. FEMS Immunol Med Microbiol. 2000 Apr;27(4):305-11. Review. 15) Ogra PL, Faden H, Welliver RC.Vaccination strategies for mucosal immune responses. Clin Microbiol Rev. 2001 Apr;14(2):430-45. Review. 16) Rajalakshmi M, Griffin PD. Male Contraception: present and future. New Age International (P) Ltd: New Delhi. 1999. pp.309-328 17) Raychaudhuri S, Rock KL. Fully mobilizing host defense: building better vaccines.Nat Biotechnol. 1998 Nov;16(11):1025-31. Review. 18) Rosenthal KL, Gallichan WS Challenges for vaccination against sexually- transmitted diseases: induction and long-term maintenance of mucosal immune responses in the female genital tract. Semin Immunol. 1997 Oct;9(5):303-14. Review. 19) Talwar GP. Vaccines and passive immunological approaches for the control of fertility and hormone-dependent cancers. Immunol Rev. 1999 Oct;171:173-92. Review. 20) United Nations AIDS Information. 21) van Ginkel FW, Nguyen HH, McGhee JR Vaccines for mucosal immunity to combat emerging infectious diseases. Emerg Infect Dis. 2000 Mar-Apr;6(2):123-32. Review. (Art by Jane Wang - note that high res versions of these image files are available)

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Multiple Immune Compartments • Peripheral lymph nodes and spleen • Adaptive immune response to antigen in blood • Peritoneum • Skin • Mucosal immune system (MALT) • Mucosa is exposed to billions of foreign antigens • Must protect against invading pathogens Lymphocytes have tissue-specific (compartment-specific) homing receptors

How to maintain homeostatic relationship with the microbiota?

• Minimize direct contact between intestinal bacteria and epithelial cells (mucin and 1 layer of epithelial cells) • Keep penetrant bacteria in intestinal sites (limit exposure to systemic immune compartment)

Looking Inside-out: Immune system control of microbiota

Hooper et al (2012) Science 336:1268

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Challenge for the Mucosal Immune System:

• Distinguish between foreign antigens such as food (no response) and pathogenic organisms (respond)

• In the context of 1014 commensal bacteria

Pathogens/toxins often enter our bodies across mucosal surfaces

Surface Areas: Skin – 2 m2 Lung – 140 m2 G.I. – 200 m2 Basketball court = 400 m2 Thus, a very large commitment of lymphocytes is needed to protect these surfaces

Nature Reviews Immunology Nagler-Anderson Vol 1: 59-67 (2003)

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Mucosal Tissues (MALT) • Gastrointestinal tract (GALT) • Respiratory tract (BALT) • Nasal mucosa (NALT) • Salivary glands • Lacrimal glands • Mammary glands • Genito-urinary tract

Intestinal Lymphoid Tissues

Isolated lymphoid follicle

Mucosal Immunology(2008) 1, 31–37 10.1038/mi.2007.9

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Intestinal IgA, CD4 T and CD8 T cells

Mucosal Immunology (2008) 1, 31–37 10.1038/mi.2007.9

70% to 80% of all Ig producing cells are in mucosal areas

…and most of these are IgA plasma cells

Epithelium and Lamina Propria

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Inductive vs Effector Sites

Induction of Mucosal Immune Response: Antigen taken-up by M cells

Induction of Mucosal Immune Response: Antigen uptake

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T cells in Peyer’s patches Become Activated and Migrate to Lamina Propria of Small Intestine

Lymphocytes Activated in Mucosa Home to Other Mucosal Sites

Induction of Mucosal Immunity

Mucosal Immunology (2008) 1, 31–37 10.1038/mi.2007.9

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Features of Mucosal Immunity:

• Ag at one mucosal sites Ab at a distant mucosal site • B cells in MALT selectively produce dimeric IgA • T cells, especially CTLs important • Mucosal immunity also results in systemic immunity

Intraepithelial Lymphocytes (IEL) (kill infected epithelial cells)

IgA Deficiency

• 1 in 500 to 1000 persons

• Often no clinical symptoms

• Some with increased susceptibility to infection

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STRUCTURE OF SECRETORY IgA

IgA1

Secretory Component

IgA2 Secretory Component

Transport of IgA across the epithelium Figure 9-20

IgA Function

sIgA in breast milk provides passive immmunity

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The Potential of Oral (Mucosal) Immunization

Advantages: • Ease of Administration • Generate both mucosal and systemic immunity Disadvantages: • Response may be short-lived • Difficult to elicit robust immune response - because of tolerance

Tolerance vs Immunity

• Food antigens result in tolerance • Pathogenic microorganisms lead to inflammation and immunity • Commensals – local induction of IgA antibodies that bind the microorganisms, thereby confining them to the lumen

Protective Immunity vs Oral Tolerance

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Mucosal (oral) Tolerance

Tolerance

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Vaccination

Systemic provides no mucosal immunity

Mucosal provides both mucosal and systemic immunity

But, how to induce mucosal immunity in the tolerogenic environment? • Need to induce inflammation

Mucosal Tolerance Mediated by Dendritic Cells

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Commensal bacteria can prevent inflammatory responses in intestine

The Danger of Antibiotics

Infection by Clostridium difficile.

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Fig. 12.26 Commensal bacteria can prevent inflammatory responses in the intestine.

13 Host Defense 2013 Commensals and Immunity April 16, 2013 Katherine L.Knight, Ph.D.

COMMENSAL MICROBIOTA AND THE IMMUNE SYSTEM

LEARNING GOAL You will be able to describe how commensal microbiota affect the immune system and how the intestinal microbiota contributes to disease.

OBJECTIVES To attain the goal for this lecture you will be able to:

 Compare the total number of human cells to mucosal bacterial cells  Describe 3 ways in which intestinal commensal bacteria interact with the host to promote a healthy environment  Identify immune and non-immune diseases resulting from changes in the intestinal microbiota  Describe causes of intestinal dysbiosis  Discuss how intestinal microbiota may protect from allergic inflammation  State the evidence that intestinal microbiota can promote inflammatory bowel diseases  Describe the mechanism by which intestinal microbiota can protect from inflammatory bowel diseases  Describe how commensal microbiota contributes to the balance between pro- inflammatory and anti-inflammatory immune mechanisms  State how intestinal microbiota can affect systemic immunity as well as mucosal immunity

READING ASSIGNMENT Janeway’s Immunobiology 8th Edition (2012), Chapter 12 p486-503 and attached article: Round, J.L. and Mazmanian, S.K. (2009) The shapes intestinal immune responses during health and disease. Nature Reviews Immunology 9:S13

LECTURER Katherine L. Knight, Ph.D.

Page 1 Host Defense 2013 Commensals and Immunity April 16, 2013 Katherine L.Knight, Ph.D.

CONTENT SUMMARY

I. INTRODUCTION TO COMMENSAL MICROBIOTA AT MUCOSAL SURFACES

II. CONTRIBUTION OF COMMENSAL MICROBIOTA TO GOOD HEALTH A. Development of secondary lymphoid organs (germ-free vs. normal) B. Protection from infectious agents (C. difficile) C. Balance pro- and anti-inflammatory immune reactions (TH17 vs Treg cells) D. Protection from intestinal inflammation (inflammatory bowel diseases)

III. DISEASES RESULTING FROM ALTERED GUT MICROBIOTA (DYSBIOSIS) A. Gut microbes affect tissues far beyond the intestine B. C. Autoimmunity D. Metabolic syndrome (obesity)

IV. CAUSES OF DYSBIOSIS A. Antibiotics B. Diet C. Early childhood exposure

V. SUMMARY

VI. KEY QUESTIONS TO BE ANSWERED

I. COMMENSAL MICROBIOTA AT DIFFERENT MUCOSAL SURFACES

The large intestine is home to ~1000 different bacterial species and the total number of bacterial cells outnumbers human cells by 10:1. That means that only 10% of cells in our bodies are human cells. The vast majority of these bacteria are not pathogenic and are referred to as beneficial microbes because they:  Are required for development of the immune system  Provide energy by metabolizing dietary polysaccharides  Provide vitamins

Page 2 Host Defense 2013 Commensals and Immunity April 16, 2013 Katherine L.Knight, Ph.D.  Protect from pathogenic bacteria

Commensal bacteria are present at all mucosal surfaces and the composition is different at the different sites. Commensal Microbiota at Mucosal Surfaces

II. CONTRIBUTION OF COMMENSAL MICROBIOTA TO GOOD HEALTH

A. Development of secondary lymphoid organs (germ-free vs. normal)

Intestinal bacteria responsible for development of immune system; germfree animals have almost no secondary lymphoid tissues including mucosal tissues. Some but not all commensal bacteria can promote development of lymphoid tissues

B. Protection from infectious disease agents

i. Clostridium difficile disease. Treatment with antibiotics kill most commensals, allowing toxin-producing C. difficile to become dominant

Page 3 Host Defense 2013 Commensals and Immunity April 16, 2013 Katherine L.Knight, Ph.D.

ii. Enteropathogenic E. coli (traveler’s diarrhea) In mouse model using enteric pathogen Citrobacter rodentium, mice are protected from enteric disease by a single oral dose of the commensal Bacillus subtilis C. Balance pro- and anti-inflammatory immune reactions

Commensals balance pro- and anti-inflammatory immune mechanisms; some bacteria induce an inflammatory response while others induce anti-inflammatory response

TH17 cells are needed for protection from pathogenic bacteria, but overproduction of IL-17 can lead to intestinal inflammation; TH17 cells are regulated by Treg cells; a “healthy” balance of TH17 and Treg cells is generated by a “healthy” intestinal microbiota. Dysbiosis can favor pro- inflammatory status.

Changes in the commensals can tip the balance between inflammatory (TH1 and TH17) and anti-inflammatory (Treg) responses.

D. Protection from intestinal inflammation How can commensals protect from Intestinal Inflammation? a. Inflammatory bowel diseases likely result from dysbiosis (altered microbiota) b. It is not clear if all commensals, or a subset of them can promote IBD c. Disease can be transferred between individuals with intestinal microbiota d. IBD can be treated by Bacteroides fragilis which induces an anti- inflammatory response

III. DISEASES RESULTING FROM ALTERED GUT MICROBIOTA (DYSBIOSIS)

Page 4 Host Defense 2013 Commensals and Immunity April 16, 2013 Katherine L.Knight, Ph.D. A. Gut microbes affect tissues far beyond the intestine

B. Allergy- allergy is on the rise, especially in industrialized and urban parts of the world. What is the nature of Western lifestyles that leads to increased allergy?

Hygiene hypothesis: Since Koch and Pasteur, microbes are known to be the cause of infectious diseases and emphasis has been placed on eradicating bacteria. But that’s like “throwing the baby out with the bathwater.” Strachan found epidemiologic evidence that the prevalence of hay fever in a family was inversely related to the number of older children in the household leading to the speculation that early childhood infections protect from allergy later in life. He suggested that “declining family size, … and higher standards of personal cleanliness have reduced the opportunity for cross infection in young families.” The important issue is not cleanliness, but that early life exposure to some microbes can alter susceptibility to disease, particularly allergy.

After respiratory exposure to house dust mite allergen, antibiotic-treated mice have increased allergic response. Decreased intestinal microbiota alters MyD88 stimulation of B cells which results in increased IgE level which causes expansion of basophils, mediators of allergic response.

Khosravi & Mazmanian (2012) Nature Medicine 18:492

C. Autoimmunity In mouse models of autoimmunity:  Intestinal bacteria can protect from experimental autoimmune encephalomyelitis (EAE). B. fragilis induces anti-inflammatory Treg cells which provide EAE protection

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Willing et al., (2011) Nature Reviews Microbiology 9:279

 Type 1 diabetes (T1D) caused by T-cell mediated destruction of insulin-producing - cells; in mouse model, T1D-prone mice do not develop T1D in the absence of TLR signaling by the microbiota

D. Metabolic Syndrome (obesity)

Does the microbiota contribute to obesity, type 2 diabetes and cardiovascular disease (metabolic syndrome)? Mice lacking TLR5 are obese and have many characteristics of metabolic syndrome including obesity, insulin resistance and cardiovascular disease

Sandoval and Seeley 2010 Science 328:179

Page 6 Host Defense 2013 Commensals and Immunity April 16, 2013 Katherine L.Knight, Ph.D. IV. CAUSES OF DYSBIOSIS Gut microbiota is altered with numerous diseases.

A. Antibiotics can induce EAE (autoimmunity), Type 1 and Type 2 diabetes and asthma/allergy B. Diet can affect intestinal lymphoid cells and increase production of IL-22 which alters the microbiota composition; also, quantity of fat and polysaccharides in diet affects composition of microbial community, e.g., high fat and carbyhydrate diet leads to increase in Firmicutes and decrease in Bacteroidetes C. Early childhood experiences- see hygiene hypothesis (above)

V. SUMMARY- TAKE HOME MESSAGE  Commensals can suppress Inflammatory responses and protect from diseases both in intestine and other organs  Dysbiosis of gut commensals likely contributes to chronic inflammatory diseases, e.g., IBD, autoimmunity, allergy and metabolic syndrome (obesity)  Protection is mediated by balancing TH (TH1 & TH17, pro-inflammatory) and Treg (anti-inflammatory) cells

Page 7 Host Defense 2013 Commensals and Immunity April 16, 2013 Katherine L.Knight, Ph.D.  Dysbiosis caused by diet, antibiotics, stress, and possibly by early childhood exposure to commensals

VI. KEY QUESTIONS TO BE ANSWERED

• How much of immune dysfunction is due to alterations in Microbiota? • How does the microbiota influence differentiation to generate TH17 vs Treg cells? • What microbial molecules drive the differentiation to Treg instead of TH17? • Do viruses and fungi have similar protective or disease-ionducing effects on immunity? • How does the immune system regulate the microbiota • How does the microbiota regulate the immune system? • How is it that TLR signaling from pathjogens results in pro-inflammatory responses, but TLR signals from commensals induce anti-inflammatory responses?

An Alternate view for Treating Chronic Immune-mediated Diseases: Identify strategies that microbes use to keep our immune system healthy rather than on trying to correct the immune imbalances caused by dysbiosis.

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Tipping the balance between sickness and health

Science (2005) 307:1914

The Human-Bacterial Superorganism • >90% of the cells in our bodies are not ours; they are microbial cells

• Most bacteria (>7000 kinds) reside in the large intestine

• Gut microbiota has metabolic activity equal to a virtual organ

Commensal Gut Bacteria:

• Required for development of the immune system

• Provide energy by metabolizing dietary polysaccharides

• Provide vitamins

• Protect from pathogenic bacteria

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Protective Power of Intestinal Bacteria

Jones and Megarrity (1986) Aust Vet J. 63:259

Commensal Bacteria Protect from Infection by Pathogenic Bacteria

Limited Normal Microbiota Microbiota Antibiotics

Toxin-producing Toxin-producing Clostridium Clostridium Colitis No Disease

Gut Microbes Affect Systems Far Beyond The Gut:

Obesity Allergy

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Commensal Bacteria May Contribute to Obesity

Germ-free

Normal Normal

&

20%Obese more fat

Intestinal Bacteria Can Promote Allergy

Antibiotics

Normal mouse Limited Microbiota

Introduce Candida albicans

Allergic mouse

The Human:Microbial Superorganism

• Changes due e.g., to stress and diet, may alter the microbiome and accordingly, the functional balance between host and microbes

• Such imbalances may contribute to: inflammatory bowel disease, allergy, and GI stress.

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The Intestinal Microbiota Protects Against Allergic Inflammation

Khosravi & Mazmanian (2012) 18:492

Crosstalk between an Organism and it gut commens Has both potentiating and det On the immune response

Goldszmid & Trinchieri Nature Immunol 13:932 (2012)

The lymphotoxin‐IL‐23‐IL‐22 pathway guides diet‐induced Changes in the microbiota to promote obesity

Ubeda & Pamer (2012) Nature Immunology 13:940

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