Barrier immunity: gut, skin and lungs.

Question 1

Name the five barrier organs of the human body.

Answer 1

Our mucosal tissues are highly connected to the lymphatic system and include:
- Gastrointestinal tract (mouth and intestine)
- Respiratory tract (airway and lungs)
- Reproductive tract
- Urinary tract

Non mucosal tissue:
- Skin (~2 m2 out of 400 m2 in total)

Question 2

All of our barrier surfaces harbors communities of microorganisms, which include bacteria, viruses, protozoa, fungi, and even worms. What are these collectively called?

Answer 2

Microbes that live in relative harmony at our barrier surfaces are collectively called commensal organisms. Collectively, these microorganisms are referred to as our commensal microbiome.

Question 3

What does MALT stand for?

Answer 3

MALT stands for mucosal-associated lymphoid tissue - the immune systems associated specifically with mucosal tissues. Their surfaces are moist, mucus-rich, and are often covered by a single epithelial cell layer (but some have more layers), the skin is not considered a MALT.

Question 4

Describe the term “physiological inflammation”.

Answer 4

Since homeostasis is an active and dynamic state involving immune cells, microbiome homeostasis is sometimes referred to as “physiological inflammation”. It is maintained by a combination of mechanisms that inhibit inflammation and promote tolerance.

Question 5

Describe two “common themes” of barrier immunity.

Answer 5

• There are two “sides”: homeostasis and inflammation, dynamic process between a tolerogenic-mode and immune challenge response-mode. Balance is key, “lagom”.
• There are many microbes associated with the barrier surfaces that are commensal microbes, benign and should be there for optimal function.

Question 6

What is the common denominator for all barrier surfaces?

Answer 6

Each of our barrier organs is separated from the external environment by at least one layer of epithelial cells, which not only provide a physical barrier but are active participants in our response to the microbiome.

Question 7

Out of all the barrier organs, which have several vs one epithelial layer?

Answer 7

The epidermis of our skin, mouth, and reproductive and urinary tracts is made up of multiple layers of epithelial cells, while our intestinal and respiratory tracts
are each separated from the outside world by a single layer of epithelial cells.

Question 8

There are several specialized lymphoid tissues connected to the mucosal barrier tissues, which?

Answer 8

• the lung is served by bronchus-associated lymphoid tissue (BALT)
• The upper airways are served by nasal-associated lymphoid tissue (NALT)
• Gut-associated lymphoid tissue (GALT)
• the skin is served by less organized skin associated lymphoid tissue (SALT).

Question 9

Gut Associated Lymphoid Tissue (GALT) include Peyer’s patches and ILFs, what are these?

Answer 9

Peyer’s patches are small clusters of highly organized and stable lymphatic tissue found in the lamina propria in the wall of the small intestine. They share many anatomical and functional features with traditional lymph nodes.

ILFs, or isolated lymphoid follicles are more loosely organized clusters of lymphatic tissue, which are associated with epithelial surfaces along the entire intestinal tract. These are much smaller than peyer’s patches but are the primary source of IgA producing
cells.

Both of these contain a lot of immune cells, are highly plastic and respond quickly to environmental changes. These structures are placed just under the layer of epithelial cells and are drained by the lymphatic system via lymphatic vessels.

Question 10

Where in the GI tract is the microbial load the highest?

Answer 10

In the large intestine. The number and diversity of microbes increase the further down we go.

Starting from the stomach which have a low pH not habitable for many microorganisms, we have a microbial load of about 10^2-10^3 (very few), in the small intestine we have less than 10^5 microorganisms in the jejunum and somewhere between 10^3-10^7 in the ileum. In the colon we have a microbial load of between 10^9-10^12 microbes, much higher than in the rest of the GI tract.

Question 11

How does the morphology of the small and large intestine differ?

Answer 11

The small intestine is longer, and have taller and more dense villi than the large intestine, specialized for nutrient absorption.

The large intestine is shorter and have shorter and less dense villi, the mucus layer is a lot thicker in the large intestine which lubricates and provides protection against the high microbial load. Specialized for uptake of water and some vitamins and delivers waste to the rectum and anus.

Question 12

The epithelial layer of the intestine is highly heterogenic, explain the function of four different cell types found in the intestinal epithelium.

Answer 12

• Regular enterocytes make up the bulk of the epithelium and transport nutrients. Express a variety of PRRs.
• Paneth cells maintain the stem cell niche in the clefts between villi, also secrete antimicrobial peptides (AMPs).
• Goblet cells produce mucus and secrete AMPs, that are also able to secrete cytokines.
• Immune cells like IELs, Intraepithelial lymphocytes, long-lived resistant effector cells that release cytokines without priming, only need to encounter antigen.
• Microfold (M) cells are highly specialized for the transcytosis of antigen across the epithelium.

Question 13

What is the core function of mucous?

Answer 13

The mucous layer form a barrier to have distance between the epithelial layer and microorganisms, it also inhibit bacterial mobility.

Question 14

Are microbiotas universal?

Answer 14

No! The microbime is both individual and species dependent. When studying the human microbiome its often inserted into sterile mice.

Question 15

What is the lamina propria?

Answer 15

The lamina propria is a layer of connective tissue located r just under the gut epithelial layer populated by resident and migrating immune cells as well as a network of capillaries and lymph vessels. It is the site of most immune cell activity in the GI tract.

Question 16

The intestinal immune system employs three major strategies to prevent microbes from penetrating the gut epithelium without permission, which?

Answer 16

• Mucous that hinders movement and functions as a barrier
• Cells in the intestinal epithelium secrete antimicrobial peptides like defensins into the mucus that can kill bacteria. All very important to keep the enormous load of bacteria and other microorganisms at bay
• Plasma cells in the healthy lamina propria secrete large amounts of IgA that inhibits microbes from penetrating the epithelial cells, and at the same time discourages inflammatory responses to any of the antigens it binds to.

Secretory IgA also plays a role in transporting antigen from the lumen to the subepithelial space and delivering it to antigen-presenting cells in a manner that promotes tolerance.

Question 17

There are four ways in which antigen can be delivered from the lumen to antigen-presenting cells in the gastrointestinal tract, which?

Answer 17

1. M cell dependent antigen delivery: M cells are specialized for transcytosis of antigens and microbes non-specifically and carry them to DCs in the lamina propria. They also express receptors that bind to specific classes of whole pathogens which can be transported to the lamina propria where APCs can take up the antigen.
2. FcR dependent: Antigen bound to IgA in immune complexes can be shuttled into the lamina propria by Fc receptors (FcR), such as the polyIg receptor, to then be shown to APCs.
3. Goblet cell dependent: Goblet cells are able to convey small, soluble antigens from lumen to lamina propria. (similar to M cell dependent antigen delivery, but work through transcytosis but goblet cells only transport soluble small antigens).
4. Direct APC contact: e.g. dendritic cells can extend their processes through the tight junctions, between epithelial cells, into the lumen to get in direct contact with the pathogen and internalize it.

Question 18

What trio of molecules maintains a tolerogenic environment in the intestinal lumen under homeostatic conditions?

Answer 18

Under healthy conditions, epithelial cells are stimulated by commensal microbes that interact with PRRs, often Toll-like receptors (TLRs). These interactions result in the production of TGF-β, the vitamin A metabolite retinoic acid (RA), and thymic stromal lymphopoietin (TSLP). This trio helps polarize T cell differentiation into Tregs (induced/peripheral Tregs) that secrete IL-10, an anti-inflammatory cytokine, which is particularly important in maintaining immune tolerance in the gut.

Individuals with low IL-10 levels are susceptible to inflammatory bowel disease (IBD).

Note: resident macrophages and dendritic cells also seem to contribute to the IL-10 production.

Question 19

How is IgA transported into the intestinal lumen?

Answer 19

The IgA dimers are shuttled over the epithelial barrier from the basolateral to apical side via the polymeric Ig receptor (polyIgR), an FcR expressed by intestinal epithelial cells. (also in the bronchial/respiratory tract).

Question 20

IgA class switching occurs in both T-dependent and T-independent manners, explain T-dependent briefly.

Answer 20

The T-dependent manner is the usual route of class switching, activated T cells differentiate into follicular helper T cells, that go into the germinal center (mostly in the peyer’s patches) and activate B cells with high affinity receptors to the specific antigen and induces class switching to IgA by providing the combinations of signals that favors class switching to IgA, mainly CD40L and the cytokine TGF-β. The IgA+ B cells then leave the GC and differentiate into plasma cells that secrete IgA.

Question 21

Explain the process of T-independent IgA class switching.

Answer 21

T-independent class switching is induced by the cytokines BAFF and APRIL, which are expressed by TLR-antigen stimulated epithelial cells, as well as mucosal APCs (mainly DCs) which stimulate lamina propria B cells to class switch to IgA. Mostly IgA with broad specificity.

Remember: T-dependent and T-independent class switching happens simultaneously.

Question 22

Commensal microbes help maintain a tolerogenic “tone” in the intestine, name two bacteria that are included in a healthy human microbiome.

Answer 22

Microbes that colonize our guts in early life (“old friends”) tune and tolerize our immune system by inducing the development of regulatory T cells and the production of IgA specific for commensal bacteria.

• Bacteria in the phylum Firmicutes, e.g clostridium produce short-chain fatty acids (SCFAs) by fermenting dietary fiber which help directly influence dendritic cells
  in the gut and enhance the development of regulatory T cells.
• Another firmicutes bacteria member, segmented filamentous bacteria (SFB) enhance IgA production and Th17 development.
• Bacteroides fragilis produce polysaccharide A (PSA) that help to maintain the Treg pool.

In the absence of microorganisms, the intestine does not develop normally and the immune system is underdeveloped both locally and systemically.

Question 23

There are two major problematic outcomes when a challenge to the gut homeostasis occur, which?

Answer 23

• GI-infections: an unwelcome bug eaten, leading to an immune response.
• Non-infectious exposures causing dysbiosis: common after antibiotic treatment which kills many commensal bacteria, leaving room for bacteria that are only beneficial in small amounts to expand for example.

Question 24

What is a clear disadvantage for pathogens invading through the intestine vs a wound for example?

Answer 24

In the intestine, the pathogen need to compete for nutrients and growth factors with the numerous commensal bacteria, so they have a harder time than pathogens entering a wound for example.

Question 25

The intestinal immune system can mount two different responses, the type 1 or type 2 response, depending on the pathogen. Give an example of the type 1 response in the gut.

Answer 25

An example of a pathogen triggering the type 1 response is the single-cell bacterium Salmonella. Salmonella first need to compete with the commensal microbiome and evade AMPs that can kill them, if they succeed, they can breach the epithelium through transcytosis by M cells, enter epithelial cells or be engulfed by resident macrophages. Many innate immune cells like APCs and ILCs (Innate lymphoid cells, e.g. NKT cells) and epithelial cells sense their presence by PRRs like TLRs and NLRs and cooperate in developing a cytokine environment that encourages a type 1 response - this makes up the inductive response. The PRR engagement leads to expression of IL-23, a very potent cytokine that is strongly inflammatory and up-regulates the production of antimicrobial proteins. It also enhances the production of IL-22 and IL-17 by Th17 (and ILC3) cells which mounts a protective response (healing and proliferation of damaged epithelium to restore its integrity) and recruits neutrophils that help clear the salmonella. NLR signalling induce inflammasome activity and IFN-γ secretion by T cell and NKT subsets, T cells are also primed to induce class switch to IgG in B cells to combat the infection.

Question 26

Give an example of the type 2 response in the gut.

Answer 26

A typical example of a pathogen that mounts the type 2 response are intestinal worms. Tuft cells in the intestinal epithelium "taste" products after some parasites, and when they detect one, they produce and secrete the alarmin IL-25, which stimulates secretion of key type 2 cytokine IL-13 by ILC2 cells in the lamina propria. IL-13 enhances goblet cells to produce mucus, which help clear worms from the lumen. Worms can also be detected by PRRs/damage by epithelial cells or damage induced responses which lead to production of alarmins and cytokines. The alarmins and cytokines recruit basophils but more importantly eosinophils, which produce IL-4 that polarizes CD4+ T cells to differentiate into Th2 cells, that enhance B cell class switching to IgE. Worm-specific IgE antibodies bind via their antigen-binding sites to worms and via their constant regions to granulocytes such as eosinophils, and induce the release of granule mediators; these granule mediators, like histamine, have potent toxic effects that can be fatal to worms.

Question 27

Name the key players in a type 1 vs a type 2 response.

Answer 27

Pattern recognition receptors and NLRs expressed by epithelial cells, APCs, and ILCs trigger a type 1 response to the gram-negative bacterium Salmonella, which is coordinated by several cytokines, including IL-23 and IL-17, and several cell types, including Th1 cells and ILCs. It culminates in the recruitment of phagocytic neutrophils. Worms are sensed by tuft cells in the epithelium, which generate alarmins and cytokines, including IL-25, that trigger a type 2 effector response coordinated by ILC2 and Th2 cells, which produce the trio of type 2 cytokines, IL-4, IL-5, and IL-13. The response culminates in the production of IgE and the degranulation of eosinophils, which kills the worms directly.

Question 28

What does the term "pathobionts" mean?

Answer 28

Pathobionts means potentially pathogenic microbes. Pathobionts are chill when present in small numbers but can become pathogenic if they are allowed to increase in number => dysbiosis (antibiotics - major driver of this) that can lead to patholigical inflammation.

Question 29

Where in the world is Inflammatory bowel disease, IBD, the most common?

Answer 29

In westernized societies. Inflammatory bowel disease includes two diseases: Crohn’s disease and ulcerative colitis. They are characterized by both a decrease in tolerogenic and increase in inflammatory (type 1 and type 2) immune responses in the intestine. Causes are not fully understood but may be associated with diet, the microbiome, and a failure to induce homeostasis conditions, which also may have a genetic component. It is typically treated with immunosuppressive drugs and anti-inflammatory therapies.

Question 30

Can the gut immune system influence other parts of the body?

Answer 30

Yes, there's much evidence supporting that the gut immunity can affect the brain and vice versa. Stress hormones are known to influence the gut immune system. Gut immunity also seem top have a connection to the respiratory system. Overall it makes sense that soluble immune factors produced in the gut can travel through the circulation to other organs and impact them. It's all connected.

Question 31

When is the gut microbiota established?

Answer 31

We are sterile during gestation, but as soon as we are born we start to be invaded by microbes. Vaginal birth helps with establishing a solid microbiome, so C-sections can lead to dysbiosis unless it gets established early also early antibiotic treatments can lead to dysbiosis. It is very important that the microbiome gets established early, as it helps form the immune cell repertoire. Problems can lead to allergy or autoimmune diseases.

Question 32

Which tissue in the respiratory system is functionally comparable to peyer's patches in the intestine?

Answer 32

Tonsils and adenoids are nasal associated lymphoid tissue (NALT) and are comparable in function to Peyer’s patches.

Question 33

The epithelium in the respiratory tract is only one layer, as in the intestine, how does it differ?

Answer 33

- The epithelial cells in the respiratory tract decrease in thinness from the trachea, where its quite thick with mucous and get gradually thinner in the bronci and broncioles all the way down to the alveoli, where its extremely thin to allow for gas exchange with the blood vessels underlying the epithelial layer. - The epithelial cells of the respiratory tract have cilia on their apical end to sweep microbes upwards and out.

Question 34

The alveoli are associated with a unique type of immune cell, which?

Answer 34

Alveoli are are associated with alveolar macrophages (dust cells) that monitor the lower airways and alveoli for infection and work with respiratory epithelial cells to regulate the balance between tolerogenic and inflammatory responses in this very delicate site. The epithelial cells secrete IL-10 and ligands for TGF-β receptors, e.g. integrins, both anti-inflammatory signals and the dust cells express a lot of IL-10 receptors to keep the tolerogenic tune and decrease inflammation.

Question 35

Most of the intestine is home to commensal bacteria, how is it for the respiratory tract?

Answer 35

The upper respiratory tract is home to many commensal microbes and the lamina propria here harbors many of the same immune cells as the intestine. A healthy lower respiratory tract (from the trachea down) does not support a community of commensal bacteria and, instead, focuses on ridding itself of microbe visitors

Question 36

Which receptor is often expressed by ag-specific lymphocytes generated in lymph nodes that drain the lung? why?

Answer 36

Ag-specific lymphocytes in respiratory tissues often express the CCR4 receptor, a chemokine receptor. The CCR4 functions as a honing signal to direct immune cells back to the lung, which expresses the CCR4 ligands CCL17 and CCL22. Other tissues like the skin can also attract CCR4+ cells, which could explain how antigen exposure in the respiratory system can have systemic effects.

Question 37

The overall responses (type 1 and 2) of the respiratory system is similar to that of the intestine, although type 1 responses are more common. Give an example of a common pathogen for the respiratory system and describe the response.

Answer 37

The response elicited depend on the type of antigen, diff antigens are bound by certain PRRs which initiate and dictate an appropriate response. Viruses are common in the respiratory tract, and influenza virus is one of the most common invaders of the respiratory epithelium. The virus express hemagglutinin that binds to sialic acid expressed on the surface of epithelial cells and other innate immune cells in the epithelium.This interaction induces endocytosis of the virus, releasing its single-stranded RNA genome into the cytoplasm of the infected cell. The SS RNA gets recognized by the PRRs TLR7 and RIG-1 (and NLRs) which sets off production of pro-inflammatory cytokines like IL-17, IL-23 and IL-1β, which recruit NK cells (ILC1) to the area, capable of killing virus infected cells without antigen specificity, which limits the initial spread of the virus. The activated APCs migrate to the NALT and iBALT where they activate T cells. The pro-inflammatory cytokines program the antigen-presenting cells for polarizing cytokine production that induce differentiation into the Th1 subset, producing IFN-γ. The Th1 cells go on to activate CD8+ T cells that become cytotoxic effector cells that go on to kill the virus infected cells and B cells to produce antibodies. The IFN-γ recruit macrophages that clear the virus. The CD4+ Th cells increase a lot in number, leading to a lot of and CD8+ Tc cells keeping the virus in check.

Question 38

Briefly describe the asthma induced immune response.

Answer 38

Asthma is an inappropriate respiratory immune response to non-pathogenic antigens and conditions. When inspired antigens causing asthma are recognized, a type 2 response is triggered very similar to that triggered by worms in the intestine. Epithelial cells produce alarmins (endogenous danger signals, DAMPs example heat shock proteins, ATP, HMGB1) and IL-25 and IL-33 that trigger a type 2 response. Alarmins activate ILC2 and Th2 cells (and Th9) to secrete attractants for eosinophils (IL-4/5), basophils and mast cells (IL-9), class switching to IgE (IL-4) and mucus production (IL-13). Antigen bound IgE and cytokines trigger degranulation of the granulocytes, eg histamine, which would kill pathogen but when inappropriate it triggers unwarranted inflammation in the airways. Chronic exposure to these stimuli induces more permanent changes in the bronchial tissue, resulting in asthma.

Question 39

How does intranasal vaccines work in short?

Answer 39

Intranasal vaccines are promising because of easy delivery (spray into the nose) and its ability to induce both local and systemic immune responses. When the vaccine (attenuated virus for example) is delivered, it permeates the nasal mucosa and APCs can internalize it, migrate to lymph nodes and stimulate the generation of mucosal tissue–homing T cells and B cells.

Question 40

The skin is a unique barrier immune system, briefly describe the skin as a barrier and include immune cells that are specific to the skin.

Answer 40

The skin consists of three major layers, the upper epithelial layers or epidermis, the area directly underneath the epithelium or dermis, and the hypodermis, the deepest layer of our skin. Unlike other barriers, the skin does not produce mucus. - The epidermis consists of keratinocytes (skin epithelial cells) in layers that move outwards as they are generated at the border between the dermis and epidermis. The outermost part consists of dead keratinocytes and lipids form the stratum corneum, a waterproof barrier to the external environment. Keratinocytes are active members of the innate immune system and produce antimicrobial molecules like psoriasin and express several PRRs, like TLR3. - In the epidermis we also find langerhans cells, a unique, specialized type of dendritic cells (pAPC) that browse the epidermis and communicate with immune cells in the dermis. They also carry skin antigens to the draining lymph nodes to activate the adaptive immune system if needed. - There is also a large population of resident memory CD8 T cells (CD8+ Trm cells) in the epidermis.

Question 41

As in the other barrier tissues, Tregs are also found in the skin (dermis) and contribute to tolerance of commensal microbes. But how do the Tregs of the skin differ from those in the intestine and respiratory system?

Answer 41

The generation of skin-specific Treg cells is age dependent and occurs when newborns (neonates) are first colonized by microorganisms. Skin T cells may arise in one wave early in development and then take up long-term residence in the skin. Recent studies show that tolerance to commensal bacteria arises in a narrow window of time during neonatal development. In fact, mice were not able to generate tolerizing Tregs if they were colonized with commensal bacteria when mature. This observation underscores the possibility that exposure to microbes in infants has a profound influence on our immune health as adults.

Question 42

Upon a lesion in the skin followed by pathogen invasion, which cell type are the first responders?

Answer 42

Neutrophils and resident memory T cells are the first responders after a skin lesion. Neutrophils are so abundant in the blood and thus have a shorter way to the affected area than activated lymphocytes, which travel from the lymph nodes. Resident memory T cells are early responders to infection in the skin as they are already there. They release inflammatory cytokines, including IFN-γ, upon Ag engagement. They are the first responders to herpes viruses foir example.

Question 43

There are three different routes of antigen delivery to lymphocytes after skin lesion, which?

Answer 43

1. APCs internalize and process antigen and migrate to the lymph nodes and present aag-peptides to naive T cells to activate them 2. whole pathogens can be opsonized and delivered to the lymph nodes where B cells with non-antigen specific BCRs take them up through binding complement proteins that is then grabbed by follicular dendritic cells that present it to naive B cells and activate them 3. Whole pathogens like Sporozoites delivered from infected mosquito bite can travel directly to a draining lymph node, where Lymph node APCs, and not skin DCs, process and present parasite Ag.

Question 44

Resident memory T cells have a unique shape, why?Describe their shape.

Answer 44

Resident memory T cells are very branched (normally round) because they are squeezed in between keratinocytes which are tightly packed in the epidermis.

Question 45

The immune system is not only involved in the inflammatory response after skin lesions, but have another very important role, which?

Answer 45

The immune system is also involved in the healing process! The inflammatory response recruits monocytes which secrete compounds involved in tissue healing, which stops the entry of more pathogen, providing the opportunity to clear all pathogen that did enter.

Question 46

Atopic dermatitis is a common problem involving the skin. what is the cause and pathogenesis of atopic dermatitis?

Answer 46

Atopic dermatitis can develop after disruption of the skin's barrier function (genetic factors, dysbiosis or injury) which allows entry of pathogens --> mast cell and DC activation causes activation of both type one and 2 response and IL-3/4 secretion witch causes itching in the area --> more damage to the keratinocytes --> more pathogen can enter --> more itch, more damage in a spiral that can turn chronic if not treated.

Question 47

Mast cells express many receptors that can cause non-immunologic activation, give an example.

Answer 47

Mas-related G-protein coupled receptor member X2 (MRGPRX2) is a Mast cell-specific receptor for basic secretagogues, including inflammatory peptides and drugs associated with allergic-type reactions. This receptor is involved in antibiotics allergy and itchiness in response to opioids for example.