ANSC 4650

Describe the Hygiene Hypothesis

- For humans, it is hypothesized that lack of exposure to certain microbes (i.e. GIT parasites) during early life development can affect immune development and increase risk of atopic diseases (includes = asthma, food allergy and atopic dermatitis) o These atopic diseases are caused by antibody-mediated hypersensitivity reactions (i.e. IgE)

What is the structure of an LPS monomer?

2 parts to LPS → 1. Lipid A portion (toxic) 2. Long acyl side chain

Describe C-Type lectin membrane receptors

C-Type lectin membrane receptors: important for recognizing polysaccharides like fungal glucans and mannose - Examples → o Mannose receptor: recognizes mannose o Dectin-1: recognizes fungal glucans (like beta-glucan)

Give examples of how mutations in the DNA can cause DNA polymorphisms

Gene 1 (Before/ original codon)→ o TAT = tyrosine Gene 2 (After; mutated codon)→ o TAC = tyrosine • Because the same AA is encoded before and after the mutation, it is called a synonymous single nucleotide polymorphism (SNP) o TAA and TAG = Stop codons • Results in the formation of a pseudogene (non functional) or truncated mRNA (eg that functions as miRNA) • remember that were dealing with the DNA gene sequence; the real stop codons are mRNA and these translate to the UAA and UAG stop codons when transcribed o TCT = serine • Because the AA before and after the mutation are different, this is called a non-synonymous SNP • Non-synonymous SNPs could modify gene product, or result in novel gene product • Eg- If this in a rc region that binds an antigen, this could affect the rc's ability to bind o TGT = cysteine • Another non-synonymous SNP example Note - when you undergo genome duplication, you increase the risk that these random mutations occur over time causing DNA polymorphisms

What are uropygial glands?

glands that produces lipids to condition feathers to repel water - found within the skin of chickens

Describe sweat glands in pigs, dogs and cats

• Sweat glands are concentrated in the footpads and snout

Describe sweat glands in hoofed animals (i.e. horses, cattle, sheep)

• Sweat glands are concentrated under the axilla (armpits)

Describe the Antimicrobial proteins and peptides released by sentinel cells

• can be produced by • sentinel cells • granulocytes

Describe Sweat glands in primates

• sweat glands are distributed all over the body allowing for → • very efficient thermoregulation • potentially facilitating ideal environment for skin microbiome • interestingly, environmental temperatures experienced during early life epigenetically determine the number of functional sweat glands on our body • In addition to thermoregulation, sweat of primates also contains different antimicrobial peptides • Dermcidin: peptide with antimicrobial properties • Lactoferrin: peptide with antimicrobial properties and binds iron • These antimicrobial peptides that help to control microbial invaders and commensal microbes

There were 4 whole genome duplication events that that affected immune system evolution. Describe them

#1 = 794-652 MYA (1R arrow) • This had a huge impact on MHC and BCR and TCR and other genes regulated to the immune system • Profoundly affected all species related to this course #2 = 652-525 MYA (2R arrow) • also profoundly affected species related to this course #3 = 326- 300 MYA (3R arrow) • affected bony fish (teleostei) #4 = 85- 100 MYA (arrow not added) • this event was partial genome duplication • affected bony fish (salmonids, ie. trout and salmon)

Once activated, sentinel cells release 4 types of molecules. List them

1. Vasoactive compounds 2. Pro-inflammatory cytokines 3. Chemokines 4. Antimicrobial proteins and peptides

Describe GALT

- Epithelial cells lining the GIT also act as innate immune cells that protect against pathogen invasion and induce tolerance to commensal microflora by communicating with the gut-associated lymphoid tissues (GALT) o Intestinal M- cells for example, can sample antigens from the lumen and pass them along to underlying tissue macrophages and DCs for antigen-presentation in secondary lymphoid tissues (ie underlying lymphoid follicles within the Peyer's patches)

Changes in the composition of the GIT microflora throughout life (eg antibiotic use, infections etc) have also been recently implicated in the development a number of human diseases. List 5 of them

1. atopic diseases 2. obesity 3. diabetes 4. inflammatory bowel disease 5. various types of cancer

Describe resistance of chickens to LPS

- Chickens are also more resistant to endotoxemia than mammals - Chickens possess orthologs (compared to mammals) of = o TLR4 o MD2 - Chickens lack orthologs of= o LPS-BP o possibly CD14 - Also, the TRIF pathway leading to IFN production is not responsive to LPS, possibly because they don't possess IRF3 o however, chickens do have the capacity to produce IFN-b in response to TLR3 agonists

Describe the neuroendocrine-immune axis

- During microbial invasion for example, immune cells recognize the pathogen then release immune signaling molecules called cytokines, as well as various neuropeptides that bind to their respective receptors on neural cells within the gut, skin, liver/spleen, respiratory and urogenital tracts and brain o These cells become activated and subsequently alert the CNS to danger o These danger signals are perceived within the brain, and the CNS responds with neural (i.e. SAM) and endocrine signals (i.e. HPA) that help regulate the immune response o Bidirectional communication among these systems occurs within what is referred to as the neuroendocrine-immune axis

Most PRRs are cell- associated receptors including which 3 locations?

1. cell membrane receptors 2. cytoplasmic receptors 3. vesicle/endosome/exosome receptors

Describe Janeway's "Stranger Hypothesis"

- In 1989, Charles Janeway proposed the "Stranger hypothesis" to explain how the host recognizes microbial invaders o He proposed that PRRs on sentinel cells were able to recognize MAMPs as "danger signals", thus providing an innate level of non-self-recognition o MAMPs trigger what is called microbial inflammation - Since then, many conserved MAMPs have been identified across pathogen species.

Describe Matzinger's "Danger Hypothesis"

- In 1994, Polly Matzinger therefore, came up with the "Danger hypothesis" to explain the role of alarmins in danger signaling - Alarmins are "danger signals" that are released by stressed, damaged or dying cells, and by activated sentinel cells. The identification of alarmins is ongoing o Normally, these alarmins are just normally found in the cell but if cell is damaged it gets released into the blood o Alarmins trigger what is called sterile inflammation

Describe the role of the immune system in tissue repair

- In addition to confronting and eliminating invading pathogens, the immune system (i.e. macrophages) is also involved in the repair and remodeling of tissues that were damaged during the infection or tissue trauma o This phase of the immune response (ie repairing damaged tissue) is referred to as the resolution phase

Describe the rationale for the hypothesis that Chicken TLR9 was lost during evolution

- In birds (chickens) → o They don't possess TLR9 but they do have TLR21 o recall that TLR9 recognizes bacterial CpG DNA as well as mtDNA o TLR21 is very similar in structure to TLR9 and it ALSO recognizes unmethylated bacterial CpG DNA - In zebrafish→ o They do have TLR9, but it also has TLR21 - in humans→ o we have TLR9, but not TLR21 - Thus, avian TLR21 is believed to be orthologous to fish and amphibian TLR21.

What is endotoxemia?

- In mammals, LPS-TLR4 ligation leads to a potent inflammatory reaction referred to as endotoxemia: LPS-induced SIRS and septic shock due to a high dose of gram -ve LPS binding to TLR4 o Researchers use LPS (also called bacterial endotoxin) a lot to elicit immune responses, but you need to be careful with the dose • If you give a high dose, may get endotoxemia • If you give a low dose, may get acute phase response (flu like symptoms)

Describe the rationale for the hypothesis that Chicken TLR15 arose from duplication of TLR2

- TLR15 is found in birds (chickens), but not in other species - TLR15 is phylogenetically related to mammalian TLR2 (Gene sequence of TLR15 is very similar to TLR2) o Thus it is hypothesized that TLR15 arose from gene duplication of TLR2 o Note that birds possess two isoforms of TLR2 (TLR2A and TLR2B) which also supports the hypothesis that TLR15 arose from TLR2 gene duplication

The inflammatory response typically remains localized to site(s) of infection. What happens if it becomes systemic?

- The inflammatory response typically remains localized to site(s) of infection o However, if local defenses are breached, the infection can become systemic and a potentially damaging condition referred to as Systemic Inflammatory Response Syndrome (SIRS) can occur, which can lead to sepsis resulting in organ failure and death

Describe the resistance of fish to LPS

- Unlike mammals, fish and amphibians are highly resistant to endotoxemia - Why is this the case? o In the case of fish, this is likely because fish species lack orthologs for CD14, MD2, LPS-BP, and most fish lack a TLR4 ortholog o They can still respond to LPS, but are much more resistant to it - Interestingly, LPS is toxic to the cyprinid species zebrafish o Zebrafish express tlr4a and tlr4b paralogue gene products (compared to each other and/or mammalian TLR4) o However, neither of these genes recognize LPS, and tlr4 signaling in the zebrafish elicits an anti-inflammatory rather than inflammatory response o Thus, the activation pathways remains to be determined

Describe the case of contact dermatitis in dairy cattle caused by calcium cyanamide

- farmer was using a new product with antimicrobial properties to reduce incidence of mastitis in his cows o but there was a compound in this new product (calcium cyanamide) that once bound to skin proteins became a hapten, and over time the cows became sensitized to this chemical o the cows developed contact dermatitis. Symptoms → • alopecia • erythema • crust formation on the femoral region, abdomen and mammary gland • lichenification (thickening of the skin) on the cow that died

Describe the structure of the epithelial layer

- joined by tight junctions and adhesion molecules - EC's are constantly replaced by underlying stem cells that undergo proliferation and differentiation (and migrate upward) - Epithelial cells have a polarized morphology → o apical side = exposed to commensal microbes and the environment o basolateral side = sterile, closest side to the blood and protected from microbial exposure - express PRRs but only on their basolateral side - Since PRRs are concentrated on the basolateral side, EC's are able to → o 1. Tolerate commensal microbes on their apical side, and • If we had PRRs on the apical side, we'd constantly be recognizing commensal microbes as foreign and that would be rly bad o 2. Alert the underlying immune system (eg macrophages) of microbial invaders and/or tissue damage • if the EC becomes damaged, now the PRR can recognize the pathogen and will alert the immune cells underneath

Describe the epithelial cells lining the nasal and oral cavities, trachea, and bronchi

- these are all polarized, and express PRRs o Thus, they protect against pathogen invasion and facilitate tolerance to commensal microflora by communicating with the sub-epithelial immune system, referred to as the→ • nasal- associated lymphoid tissues (NALT) • bronchi-associated lymphoid tissues (BALT)

Describe the kinetics of the innate and acquired immune response

- x axis = time - barrier o immediate protection - innate IR o happens right after barrier = broken o don't want it to be activated for too long bc will cause tissue damage if excessive o every subsequent booster doesn't yield any faster response, or any better protection - acquired IR o primary • first vaccination (for eg) • goes down over time o secondary • after booster vaccination (for eg) • secondary response occurs faster, and has greater protection compared to first vaccination o tertiary • another booster • faster response, more protection o subsequent • another booster • threshold reached, not any diff from response in tertiary response (same protection and just as fast) - arrows o any time youre exposed to the antigen (either naturally or via a vaccine/booster) - The kinetics of immune response varies between the innate and acquired immune systems. Immediate protection provided by the innate immune system is often sufficient to control and eliminate the invading pathogen - However, when this is not the case, a primary acquired immune response, which provides enhanced protection, will occur over a period of days-to-weeks to control and eliminate the invading pathogen

What are the 2 types of danger signals

1. microbe-associated molecular patterns (MAMPs) 2. Alarmins

The stratum corneum protects the underlying epidermis from which 5 things?

1. Abrasion 2. Water loss 3. UV radiation 4. Toxic chemicals 5. Microbial invasion THINK! AWTUM

Describe the 3 (progressive) layers that protect you from invading microorganisms

1. Barriers • skin = The sloughing of non-viable epithelial cells making up the stratum corneum of the skin of birds, reptiles and mammals • Mucous= Mucous flow from the skin of many fish species and amphibians, and from the urogenital, respiratory and gastrointestinal tracts • pH= Low pH of the stomach and skin • Oxygen= Anaerobic gastrointestinal tract • Temperature= Increased body temperature in homeothermic species (aka warm blooded species like humans. As opposed to cold blooded species that cant regulate their temperature and their internal temp depends on the external environment they live in) • commensal bacteria= Colonization of commensal microbes along the host epithelium • Physical processes = eg vomiting, diarrhea, and intestinal peristalsis 2. Innate immunity (immediate protection if pathogen gets past the first barriers) • Natural antibodies • Enzymes • Antimicrobial peptides • Epithelial cells • NK cells • Gt cells • Dendritic cells • Mast cells • Macrophages • granulocytes 3. Acquired immunity • cell mediated immunity = T cells • antibody/humoral mediated immunity= B cells

Give the steps of HMGB1 recognition when its fully reduced

1. Binds neighbouring cells through a receptor called Receptor for Advanced Glycation End products (RAGE) • this induces expression and release of 2 things → o 1. CXCL12: a chemokine that binds the receptor CXCR4 o 2. adhesion molecules • both of these things support immune cell trafficking to damaged tissues 2. Binds and forms heterocomplexes with CXCL12 • This increases CXCL12 affinity to CXCR4 • Increased binding of CXCL12-HMGB1 complex to CXCR4 promotes immune cell trafficking (i.e. bringing neutrophils and monocytes from the bone marrow out into blood and then into damaged tissues) o So note that technically HMGB1 DOES bind to CXCR4, but only when its complexed to CXCL12

List the 5 PRRs/PRR families that work in concert with pathogen recognition TLRs, and to steer the acquired immune response (aka these ones are TLRs that have non-PRR functions, but can work with PRR type TLRs)

1. C-Type lectin membrane receptor family 2. CD14 3. Peptidoglycan-recognition protein (PGRP) 4. NOD-like receptors (NLRs) 5. retinoic acid-inducible gene 1-like receptor (RLR) family

When B and T cells become activated (after recognizing antigen), they proliferate into different cells. Describe/list them

1. Effector T cell = cytotoxic T cells • Important for recognizing pathogen infected cell and gives it a death signal that kick starts apoptosis for that cell • Cytotoxic T cells can secrete cytokines that activate macrophages (one of the ways that the innate and acquired immune system work together) 2. Memory T cell 3. Plasma cell • Produces antibodies that can act as opsonins (act on extracellular pathogens) • Opsonins are secreted into the blood 4. Memory B cell

Give the steps of HMGB1 recognition when its partially oxidized

1. The partially oxidized HMGB1 isoform occurs when C23 and C45 become disulphide bonded 2. When this happens, HMGB1 interacts binds to TLR4 on a neighbouring cell, which is also bound to MD2 3. This binding upregulates expression of NF-kappa b: a transcription factor that increases the release of pro-inflammatory cytokines and chemokines

Gene duplication has contributed to 4 important developments. Describe them

1. Gene copy number variation • function = affecting mRNA expression • we have more than 2 copies of some of the genes in our body. These vary from person to person (ie, one person may have more of a specific cytokine than another person) 2. The development of pseudogenes (non-functional genes) • function = utilized to create somatic mutations during BCR and TCR gene recombination • For eg gene recombination with RAG1 and 2, these pseudogenes are an a easy place for nucleotides to insert into the genome, to change the DNA sequence (ie to create the somatic mutations that occur during BCR and TCR gene recombination) 3. The development of non-coding RNA (i.e. miRNA) and miRNA copy number variants • function = involved in gene regulation 4. The development of genes with slightly different or completely novel functions • these occur through DNA mutations that become fixed within a population, referred to as a DNA polymorphisms • essentially, the first 3 developments can happen randomly, but if they are mutations that end up becoming fixed within the population, now they have become DNA polymorphisms

The nasal and oral cavities, trachea, bronchi and bronchioles contain a variety of epithelial cell types. Describe the 3 types

1. Goblet cells = secrete mucus and surfactant proteins 2. Club cells = secrete antimicrobial proteins and peptides 3. Microfold cells (M-cells): non-motile, specialized epithelial cells that are found in the bronchi • these cells sample antigens from the mucus matrix and pass them along to underlying tissue macrophages and dendritic cells (DC) for antigen- presentation in secondary lymph tissue (ie lymph nodes)

List the 2 arms of the immune system

1. Innate immune system 2. Acquired/adaptive immune system - 2 parts = cell mediated immunity (T cells), humoral/antibody immunity (B cells)

The skin fulfills many different physiological functions across species. List 5 functions

1. Its an important peripheral sensory organ 2. It's involved in nutrient synthesis (i.e. vitamin D) 3. Needed for thermoregulation 4. Needed for osmoregulation 5. Acts as a protective barrier against environmental "stressors" such as UV radiation, chemical exposure and microbial invaders THINK! 'POP TN'

Give the steps of Signaling pathway 1 (MyD88)

1. MyD88 recruits IL-1 receptor-associated kinases (IRAK): group of kinases that cause early-phase activation of 2 proteins by phosphorylation = • 1. Mitogen-activated protein kinases (MAPK) • once activated, will activate AP-1: a transcription factor • 2. NF-κB: a transcription factor 2. These activated transcription factors initiate the production of various immunoregulatory and antimicrobial compounds including--> - 3 enzymes = o pro-caspase-1 o inducible nitric oxide synthase (iNOS)-2: produces NO o cyclooxygenase (COX)-2 - 6 pro-inflammatory cytokines = o tumor necrosis factor (TNF)-α o interferon (IFN)-γ o interleukin (IL)-6 o pro-IL-1 o pro-IL-18 o pro-IL-33 3. Pro- caspase-1 then gets bioactivated to caspase-1 within the NLRP3 inflammasome: a newly assembled cytoplasmic multiprotein complex 4. caspase-1 bioactivates 3 inflammatory cytokines for secretion • pro-IL-1 → IL-1 • pro-IL18 → IL-18 • pro- IL33 → IL-33 5. All of the pro-inflammatory cytokines collectively → • trigger fever and sickness responses • trigger hepatic acute-phase protein production • trigger Type 2 interferon (IFN)-γ production • act as chemoattractants for granulocytes

What are 5 essential features/steps of the acquired immune response

1. Phagocytosis of the microbe. 2. Process microbial proteins into peptides. 3. Migrate from the site(s) of infection to secondary lymphoid tissues (i.e. lymph nodes, spleen etc.) 4. Up-regulate membrane antigen receptors called major histocompatibility complex (MHC) molecules that are loaded with microbial peptides. 5. Present microbial antigenic peptides on MHC molecules to TCR on antigen- specific T cells, and the BCR on antigen- specific B cells; this is referred to as antigen presentation. • These antigen-specific lymphocytes become activated to proliferate and differentiate into the effector cells of the acquired immune system (i.e. T cells and antibody-secreting plasma cells)

What are the 2 main functions of keratinocytes?

1. Producing keratin to make up avian feathers and mammalian hair • Feathers and hair have 2 important functions= o 1. thermoregulation o 2. protective barrier for the underlying epidermis 2. Keratinocytes are also important innate immune cells (they are sentinel cells) that protect against pathogen invasion and maintain tolerance to commensal microflora • they do this by communicating with the sub-epithelial immune system, referred to as the skin- associated lymphoid tissue (SALT)

Give the steps of LPS recognition by TLR-4 (until divergence into the 2 pathways)

1. Serum LPS- binding protein (LPS-BP) binds to, and facilitates the extraction of bacterial LPS monomers 2. These LPS monomers are transferred to CD14: a membrane protein 3. LPS is then transferred to Myeloid Differentiation factor 2 protein (MD2) • binds MD2 in a specific way • The toxic lipid A portion of LPS occupies a hydrophobic pocket within MD2 • the LPS acyl chain outside the pocket hangs over and interacts with neighboring TLR4 4. Simultaneous binding of LPS to MD2 and TLR4 alters the shape of TLR4 and causes TLR4 to dimerize 5. TLR4 dimerization causes recruitment of 2 adaptor proteins→ • 1. MyD88 • recruited with the help of TIRAP: a type of sorting protein • 2. TIR domain-containing adaptor molecule 1 (TRIF) • also called TICAM1 • recruited with the help of TRAM: a type of sorting protein • note that in order for TRIF to be recruited, LPS-TLR4 must first be internalized and transported to an endosomal compartment • this process is dependent on CD14, which appears help stabilize the TLR4-MD2 complex on the plasma membrane 6. MyD88 and TRIF initiate two different intracellular signaling pathways (one each) • a) signaling pathway 1 (MyD88) = activated first • b) Signaling pathway 2 (TRIF) = activated second

Give the steps of signalling pathway 2 (TRIF)

1. TRIF recruitment leads to the induction of transcription factor Interferon Regulator Factor 3 (IRF3) 2. Activation of IRF3 results in → o Induced expression of antiviral and immunoregulatory Type-1 interferons= • IFN-α • IFN-β o Late phase activation of = • NF-κB • MAPK o Induced expression of negative regulators of inflammation = • IL-10 • suppressor of cytokine synthesis 1 (SOCS1) • Suppressor of cytokine synthesis 3 (SOCS3) Note- This TRIF pathway is also activated by TLRs 3, 7, 8, and 9.

Give the steps of HMGB1 recognition when its fully oxidized

1. The "fully oxidized" HMGB1 isoform occurs when all 3 cysteines are terminally oxidized to sulfonates • in this state, HMGB1 has no chemoattractant or cytokine-inducing properties 2. HMGB1 gets degraded and recycled

When is tolerance supposed develop?

At an early age

Describe CD14

CD14: already discussed in the context of LPS - However, it can also recognize other ligands including→ o PTG o LAM

Describe the differences in stratum corneum in humans of different skin colour

Compared to those of lighter race, people of dark race have a stratum corneum that is = • thicker • comprised of a higher lipid content • replaced more rapidly This enables dark-skinned people to = • more effectively retain water • be more resistant to chemical and microbial exposure

During the initial phase of the influx of effector cells, do granulocytes or MDMs act first

During the initial phase of the influx of effector cells, granulocytes are immediately recruited from the circulation to infected and damaged tissues - Within hours, this granulocyte population is replaced by MDM that participate as effector cells during the inflammatory response, and repair cells during the resolution phase.

What are pneumocytes

Epithelial cells that produce surfactant proteins that reduce surface tension preventing lung collapse, and have antimicrobial and immunomodulatory properties - these are the thin layer of epithelial cells that make alveolar sacs

Define paralogues

Genes that can have different functions but have arisen out of gene duplication from an ancestor. The can be within or across species (but regardless will have the same ancestor)

What needs to happen for long-term immunity to occur?

In order for long-term immunity to occur, the host must mount an acquired immune response that is highly specific. This acquired immune response typically targets microbial proteins (antigens) in the form of o immunoglobulins/Ig's (BCRs on B cells and antibodies secreted by differentiated B cells called plasma cells) o TCRs on T- lymphocytes (T cells)

What was the main flaw with the Stranger Hypothesis?

It did not explain how inflammation is triggered in sterile environments such as during tissue trauma o eg getting punched doesn't trigger inflammation because of microbes, its just due to getting punched

What is the reason for variable skin pigmentation in humans?

It is hypothesized that variable skin pigmentation in humans is a nutritional adaptive feature that has allowed humans to survive in environments with variable UV radiation o 2 observations support this → • 1. light-skinned people are more susceptible to spina bifida caused by UV radiation folic acid depletion during pregnancy • 2. the offspring of people of dark skin are more susceptible to Rickets disease and immunodeficiency resulting from insufficient vitamin D production during pregnancy and early life

What is immunological memory?

Long- term enhanced protection against pathogens - note that the innate immune response does not provide this

Describe the mucous gradient of the trachea, bronchi and bronchioles

Loose outer layer= • matrix for commensal microbes to live on • they don't penetrate the dense matrix tho Dense inner layer = • antimicrobial compounds (eg defensins) • antibodies (IgA) • enzymes (eg lysozyme) • cytokines • hormones

Describe the composition and 7 functions of mucous

Mucus is a gel gradient comprised of mucin proteins and lipids that provide a number of important functions→ 1) Act as a physical barrier preventing the attachment of pathogenic microbes and commensals to underlying epithelial cells 2) Lubricates the epithelium reducing potential abrasion 3) Traps and removes pathogenic microbes, environmental antigens, and irritating chemicals (i.e. atmospheric ammonia) • In the case of the respiratory tract, particulate trapping (> 5 μm) by mucus is facilitated by turbulent airflow through the trachea and bronchi • If they're smaller than 5 μm, they wont get trapped and will travel all the way to the alveoli 4) Maintains hydration of the epithelium supporting epithelial barrier function and the exclusion of hydrophilic chemicals (i.e. irritants) 5) The outer loose mucus layer acts as an adhesive surface for commensal microbial populations, especially in the large intestine 6) The inner dense mucus layer provides a matrix to concentrate → - antimicrobial proteins = • IgA • Lysozyme • defensins - immunomodulatory proteins = • cytokines • hormones 7) Facilitates the removal of motile immune cells (i.e. granulocytes and macrophages) that have engulfed (phagocytized) foreign particles • in the resp tract, you cough up phlegm (which is mucous) • under a microscope, youd find immune cells in there too (like granulocytes)

Are the innate and acquired immune systems mutually exclusive? Explain

No - the innate immune response provides an immediate level of protection that buys time for the more effective acquired immune response to occur, and is also required for an effective acquired immune response to occur. - and there are many examples of cells and humoral proteins that are involved in both the innate and acquired immune responses. During the AbMIR for example, innate immune cells act as antigen-presenting cells (APC), as described above, that present microbial peptides from extracellular pathogens to antigen- specific T cells - These T cells then work with the APC to activate antigen-specific B cells, which proliferate and differentiate into effector plasma cells that secrete antibodies with various effector functions (i.e. opsonins) - Likewise, during the CMIR to intracellular pathogens, APC present microbial peptides to antigen-specific T cells, which proliferate and differentiate into effector T cells (i.e. cytotoxic T cells) - These effector T cells, along with innate immune cells (i.e. monocyte-derived macrophages) that are recruited to the site of infection during the acquired immune response, target pathogen-infected cells for killing.

Differentiate between non-self and modified self

Non-self = pathogens Modified self= one of your own cells, but is mutated (for eg and grows rapidly out of control like a tumour cell)

Compare the innate vs acquired immune system in a) history b) onset c) specificity d) potency e) memory f) effectiveness g) cell types h) effector humoral proteins

Note - the innate immune system is always on, the acquired immune system needs to be induced by an antigen a) - Innate = ancient - acquired = recent b) - Innate = min-hours - Acquired = days-weeks c) - Innate = conserved molecular patterns (MAMPs) - Acquired = specific antigens d) - Innate = can be overwhelmed (can become excessive and cause damage/kill you) - Acquired = rarely overwhelmed e) - Innate = limited - Acquired = long-term memory f) - Innate = does not improve - Acquired = Improves to a threshold with subsequent exposure g) - Innate = macrophages, DCs, granulocytes, NK cells, gamma delta T cells, mast cells, epithelial cells - Acquired = T and B cells, recruited macrophages, mast cells, eosinophils h) - Innate = antimicrobial peptides, enzymes, natural antibodies - Acquired = Antibodies

Describe Peptidoglycan-recognition protein (PGRP)

Peptidoglycan-recognition protein (PGRP): a PRR that is highly conserved across species - this PRR can either be membrane-bound or secreted - Four genes have been identified coding PGRPs across species (fish to chickens to mice to mammals) - PGRP binds to = o Muramyldipeptide: a bioactive fragment of PTG that causes inflammation (very similar concept to the toxin lipid A portion that causes damage in LPS) o LPS o LTA - PGRP functions= o has bacteriostatic properties (can stop growth of bacteria) o is concentrated within granulocyte granules, meaning it can be released during degranulation o has amidase activity, which enables it to bind to and degrade PTG produced by commensal microbes • this suggests that it is involved in maintaining host tolerance to commensals

Describe the pro-inflammatory cytokines released by sentinel cells

Pro-inflammatory cytokines activate cells surrounding sentinel cells to enhance "danger" signaling and effector function (i.e. macrophage phagocytosis) - 3 types→ 1. TNF-alpha 2. IL-1 3. IL-6 • At high enough concentrations, these cytokines can spillover into the circulation and elicit a systemic inflammatory response that triggers → • acute phase response: when cytokines spill over and get into other tissues (like brain, bone marrow etc) and cause an immune response there • maybe also pathological inflammation (i.e. SIRS)

Describe the skin of bony fish (osteichthyes - zebrafish and salmonids)

Scales • By contrast to cartilaginous fish, bony fish have scales that are covered with a mucus-coated multilayered epidermis (instead of having the scales act as a barrier for the epidermis like in cartilaginous fish, now the mucous covered epidermis acts as a barrier for the scales) Multilayered epidermis • On top of scales is a multilayered epidermis, which include → • Goblet cells • Melanophores: aka called melanocytes in mammals; cells that provide skin pigmentation and help to sequester reactive oxygen species (ROS) generated during oxidative stress • Neural sensory cells: cells that are highly concentrated along the lateral line NOTE- From inside out we have scales → multilayered epidermis (includes goblet cells, melanophores, and neural sensory cells)→ mucosal layer

Describe the skin of cartilaginous fish (chondrychthyes- sharks and rays)

Scales • They have placoid scales: tooth-like structures that rise out of a mucus-coated epidermis (protects the epidermis) Epidermis • Epidermis is found underneath the placoid scales, and includes → • Goblet cells: Specialized epithelial cells that secrete mucous. Protects the viable epidermis from the surrounding aquatic environment and contains antimicrobial proteins and peptides NOTE - from inside out we have epithelial cells (includes goblet cells)→ mucosal layer → placoid scales

Describe the chemokines released by sentinel cells

Specialized cytokines called chemokines that direct the influx of effector immune cells from the circulation to infected and damaged tissues. Chemokines: specialized cytokines that direct immune trafficking. Immune cells have receptors to recognize these different chemokines

Describe the promiscuous (can recognize more than 1 MAMP) TLRs

TLR2 = recognizes the following MAMPs - bacterial PTGs - mycobacterial LAM - fungal phospholipomannan - parasite tGPI- mucin - LPS - lipoproteins - microbial proteins Note that TLR9 and TLR4 can recognize more than 1 ligand, however they recognize both MAMPs and alarmins

Describe the 2 types of TLRs complexes that interact for ligand recognition

TLR2/6 heterodimer = recognizes the following MAMPs • bacterial lipopeptides • bacterial LTA (gram positive) • zymosan: a type of fungal glucan TLR1/2 heterodimer = recognizes the following MAMPs • mycobacterial lipopeptides

Describe the 4 types of endosomal membrane TLRs

TLR3 = recognize intracellular pathogens (viruses) by binding to viral nucleic acids TLR7 = recognize intracellular pathogens (viruses) by binding to viral nucleic acids TLR8 = recognize intracellular pathogens (viruses) by binding to viral nucleic acids TLR9 = recognize intracellular pathogens (bacteria) by binding to bacterial CpG DNA: regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide; these regions are highly unmethylated in comparison to the highly methylated host DNA

Describe the 2 types of TLRs that can also recognize host alarmins

TLR4 = recognizes the following alarmins • HSP 67/70 • Fibrinogen • heparin sulfate • HMGB1 TLR9 = recognizes the following alarmins (TLR21 is the same) • mtDNA (this makes sense because mtDNA is very similar to bacterial DNA and TLR9 also recognizes bacterial CpG DNA)

Describe how the neuroendocrine system contributes to immunity

The innate and acquired immune responses are tightly self- regulated and are also regulated by signals from the neuroendocrine system. 2 aspects = 1. Neural = sympathetic-adrenal medullary (SAM) axis 2. Endocrine = hypothalamic-pituitary- adrenal (HPA) axis Collectively, these 2 axes form the 'stress axis' and ensures that these immune responses respond effectively, but not excessively, to restore homeostasis.

Describe the origin of the RAG1 and 2 genes

The origin of RAG1 and RAG2 genes is uncertain, but they may have arisen from a mutated retroviral insert that was incorporated into the vertebrate genome much earlier

What is the resolution phase

The phase of the immune response that involves repairing damaged tissue that were damaged during the infection or tissue trauma

Describe the types of epithelial cells (other characteristics) found in the small intestines

The small intestine epithelium is comprised of finger-like villi and gland- like invaginated crypts: contain pluripotent stem cells that proliferate, and as they migrate upwards they differentiate into various epithelial cell types → o Goblet cells o enteroendocrine cells: epithelial cell that can produce hormones o M-cells o Paneth cells: secrete antimicrobial peptides into the dense mucous layer that kill pathogens and help control commensal bacteria o Absorptive enterocytes: epithelial cells that facilitate nutrient uptake - Peyer's patches = important for immunity. Tissue that is dense with lymphoid follicles

List the 5 types of sentinel cells

These are the innate immune cells that express PRRs 1. epithelial cells (EC) 2. tissue macrophages (Mf) 3. dendritic cells (DC) 4. mast cells 5. mucosal eosinophils

What are 2 main the differences between avian skin and mammalian skin?

Unlike mammalian skin, avian skin lacks = 1. sweat glands 2. lipid-secreting sebaceous glands • instead of sebaceous glands they have uropygial glands: glands that produces lipids to condition the feathers to repel water

Describe the Vasoactive compounds released by sentinel cells

Vasoactive compounds (that slow down blood flow) = promote the influx and concentration of certain cells from the circulation to infected/damaged tissues 1. effector cells (i.e. granulocytes and MDM) • avian granulocytes are called = heterophils • mammalian and fish granulocytes are called = neutrophils (there are others, but main one we'll focus on) 2. humoral proteins • natural antibodies • complement proteins • antimicrobial peptides

Describe how mucous is removed from the respiratory tract

Via ciliated epithelial cells of the respiratory tract o important because mucous is removed from the bronchioles, bronchi, and trachea by the beating of ciliated epithelial cells and by coughing o The mucus is then expectorated, or swallowed and degraded at a low pH in the stomach

Where is mucous found in a) fish species b) birds and mammals

a) - covering the skin (for bony fish) and under the placoid scales for cartilaginous fish - also found bathing epithelial cells lining the respiratory tract, GIT, and urogenital tract b) - bathing epithelial cells lining the respiratory tract, GIT, and urogenital tract

Give examples alarmins of the following locations a) nuclear alarmins b) mitochondrial alarmins c) cytosolic alarmins d) ER aarmins e) alarmins found in the blood 9nor originally from a cell)

a) 1. High mobility group box protein (HMGB1) 2. DNA histones 3. Genomic DNA itself 4. IL-33 5. Pro-inflammatory cytokines (IL-1α) b) 1. Mitochondrial DNA (mtDNA) c) 1 . ATP 2. Uric acid crystals 3. Heat-shock proteins d) 1. Calreticulin e) 1. Heparin-sulfate 2. Fibrinogen 3. Serum amyloid A 4. S100A8/9 (Calprotectin)

Give examples of MAMPs on the following locations a) MAMPs on the membrane of Gram-positive bacteria (eg Staphylococcus aureus) b) MAMPs on the membrane of Gram- negative bacteria (eg Escherichia coli) c) MAMPs on acid-fast bacteria (eg Mycobacterium avium subsp. Paratuberculosis) d) MAMPs on fungal cell wall e) MAMPs on parasites f) MAMPs on viruses

a) 1. peptidoglycans (PTG) • thick peptidoglycan cell membrane 2. lipoteichoic acids (LTA) b) 1. peptidoglycan covered by a layer of lipopolysaccharide (LPS) • thin peptidoglycan cell membrane c) 1. glycolipids 2. lipoarabinomannan (LAM) 3. peptidoglycan d) 1. Mannoprotein (as well as things that bind mannoprotein like phospholipomannan) 2. beta-glucan (a polysaccharide) 3. chitin (a polysaccharide) e) 1. tGP1-mucin f) 1. unique DNA and RNA sequences (recall that MAMPs aren't always on the cell surface, could be something within cell too)

a) List 6 types of cell membrane-bound TLRs b) which MAMPs do they recognize

a) o TLR1 o TLR2 o TLR4 o TLR5 o TLR6 o TLR11 b) MAMPs on extracellular pathogens including --> o microbial proteins o lipoproteins o LPS

Describe a) High mobility group box protein (HMGB1) b) Mitochondrial DNA (mtDNA)

a) • In the nucleus, HMGB1 is involved in DNA folding, recombination and transcription • When released into the cytoplasm, HMGB1 initiates cell death pathways (apoptosis). But if the cell membrane is damaged, can be released into the blood to cause diff things to happen - HMGB1 is released from dying and stressed cells - Its recognition by different receptors depends on the redox state of its three cysteine residues → 1. C23 = can form a disulfide bond 2. C45 = can form a disulfide bond 3. C106 - the redox state of these 3 Cys residues determines which HMGB1 isoform is present. It is always fully reduced when it is first released from the cell, but it starts to become oxidized once in the blood b) • Highly unmethylated DNA and believed to be of bacterial origin • Because it is highly unmethylated and looks like bacterial DNA, the immune system recognizes this as non-self • This supports the hypothesis that mitochondrial DNA originally came from bacteria. Acquired during early eukaryote evolution by phagocytosis of a bacterium

The immune system can be out of balance. Describe what can happen if the immune system is a) attenuated b) excessive

a) • Pathogenic disease • Cancer b) • Inflammatory disease • Cancer • Allergy (due to sensitization to environmental antigens) • Autoimmune disease (due to sensitization to self-antigens)

Describe the evolution of the a) innate immune system b) acquired immune system

a) • The innate immune system is ancient and highly conserved across species • Arose early in evolution b) • The acquired immune system became functional sometime between 794-652 million years ago following the activation of recombination activation genes (RAG1 and RAG2) whose gene products are enzymes that mediate the first steps of B cell receptor (BCR on B-lymphocytes) and T cell receptor (TCR on T- lymphocytes) gene recombination (occurring only in developing lymphocytes during the early stages of T and B cell maturation. This process generates somatic mutations that facilitate adaptive changes within these antigen receptors that allow lymphocytes to recognize constantly mutating pathogens (referred to as experiential learning) • results in the highly diverse repertoire of antibodies/immunoglobulins (Igs) and T cell receptors (TCRs) found on B cells and T cells, respectively

Between the innate and acquired immune systems, a) which is more effective in controlling/eliminating pathogens? b) which one is faster?

a) Acquired b) Innate - it takes the acquired immune system considerably longer. Takes longer for the host to mount (day-to-weeks as opposed to minutes-to- hours) because a higher level of immune system orchestration is required to elicit an acquired immune response that also involves the innate inflammatory response. - In order for an acquired immune response to occur for example, there has to be sufficient inflammation to activate surveillance (sentinel) immune cells to carry out the essential features of acquired immune response

a) define orthologues b) relate this back to TLR21 and TLR9

a) Orthologues: Genes in different species with similar functions that evolved from a common ancestor gene before speciation. b) - Interestingly in fish, both TLR9 and TLR21 recognize bacterial CpG DNA; however, TLR21 is lacking in mammals - So we think that there was a common ancestor to fish, chickens and humans that had both TLR21 and 9, and after genome duplication, this expression varied between the species however which ever one was present in the species (9 or 21), the function was the same regardless

Within mammalian species, describe differences in a) hair thickness b) hair types c) stratum corneum thickness d) amount of melanocyte pigmentation e) sebaceous gland secretion

a) Vary greatly across mammalian species - this influences → • skin conditioning • potential damage caused by skin abrasion • attachment of parasites such as ticks that can be disease vectors b) - Some species have almost no hair (i.e. pigs and humans) - others species have combinations of guard and awn hairs (i.e. cattle, horses) - other species have down or wool (i.e. dog, cats, mink, domestic sheep) c) highly variable within and across mammalian species - influences → • skin conditioning • potential damage caused by skin abrasions • chemical exposure • nutritional health d) highly variable within and across mammalian species - influences → • skin conditioning • potential damage caused by skin abrasions • chemical exposure • nutritional health e) highly variable within and across mammalian species - influences → • skin conditioning • potential damage caused by skin abrasions • chemical exposure • nutritional health

a) What are melanophores? b) what are neural sensory cells

a) aka called melanocytes in mammals; cells that provide skin pigmentation and help to sequester reactive oxygen species (ROS) generated during oxidative stress - found within the multilayered epidermis of bony fish b) Neural sensory cells: sensory cells that are highly concentrated along the lateral line - found within the multilayered epidermis of bony fish

What are acid-fast bacteria?

bacteria that have cell membranes that contain mycolic acid

From a health perspective, why are sweat glands located in moist regions?

moist regions are conducive to fungal growth, bacterial invasion, and to immune sensitization to haptens

What are the differences in large intestine epithelium vs small intestine epithelium

o 1. not lined with villi o 2. Paneth cells are not usually found within the crypts • this likely explains why bacterial populations are much more concentrated here than in the small intestine o 3. Lots of Goblet cells within the large intestine crypts • produce large amounts of mucus to protect against the highly concentrated and diverse commensal population in this part of the GIT • The mucus flows through the GIT and away from the body o 4. M-cells can also be found over lymphoid follicles along the colon and rectum

What are haptens?

o Haptens: small molecular weight reactive chemicals (<1000 Da) that bind to specific proteins and together, the complex can be recognized by your immune systems • can be found in cosmetics, jewelry, cleaning agents, paints, and some antimicrobials • Repeated exposure to haptens can lead to the development of an immune disorder called allergic contact dermatitis, which is a cell- mediated hypersensitivity reaction to the hapten

What is the main difference in epidermis between fish and birds/mammals?

o In contrast to fish, the epidermis of birds and mammals is covered with a non-viable (essentially dead) layer of cells called the stratum corneum, which is constantly replaced by epithelial cells called keratinocytes that differentiate from underlying pluripotent stem cells

What is the receptor complex that binds LPS?

o TLR4, which is bound to MD2, which is also bound to CD14 (TLR4-MD2-CD14) • CD14 = binds LPS first • MD2 = binds LPS second, links CD14 to TLR4 • TLR4 = associates with MD2, and is the only component that completely spans the membrane so this is responsible for activating cell signaling in response

Describe the epithelial cell types found in the alveoli as well as a description of the environment

o The lower bronchioles and alveolar sacs are considered a sterile environment, except in cases of pneumonia, when microbes have invaded these epithelial tissues. o The epithelium making up the alveolar sacs is comprised of a very thin layer of epithelial cells called pneumocytes: cells that produce surfactant proteins that reduce surface tension preventing lung collapse, and have antimicrobial and immunomodulatory properties

Describe the stratum corneum in livestock species (cattle, sheep, pigs) aka ruminants

o cattle have a very thick stratum corneum compared to sheep and pigs o sheep secrete long chain waxy esters called lanolin rather than oils from their sebaceous glands to form a moisture barrier (oils lubricate the stratum corneum)

Describe initial Identification of TLRs

o first identified in Drosophila in the early 90s (i.e. 18 wheeler) o Homologous sequences and gene products were later identified in numerous fish, bird and mammalian species

The morphology of the epidermis varies along the mammalian GIT. What is this determined by? What does this result in?

o largely determined by colonization of unique commensal microbial populations (microbiome) after birth o These microbes ensure normal immune system maturation, which establishes life-long immune tolerance to commensal microbes and immune responsiveness to pathogens

Describe whole genome duplication

o this can also happen during meiosis if theres not separation during first stage. Results in polyploidy o when WGD occurred in evolution, this was very unstable, and genomes tried to return back to their diploid state o this process of reorganization unstable polyploidy genomes to become diploid resulted in new speciation events • This reorganization has had a profound impact on the evolution of the immune system across species

What accounts for the enhanced response of the acquired immune system with subsequent exposure whereas with the innate IR the response is the same?

the acquired immune response is equipped 2 important factors → • 1. antigen specificity • 2. immunological memory - thus, subsequent encounters with the invading pathogen will result in a higher level of protection that is much more efficient than the primary acquired immune response