MMI 39- Innate Immunity

When are eosinophils involved?

-Crucial in defense against parasitic worms (AKA helminths) -Involved in allergic responses -Rarely, eosinophilia may be due to an underlying immunodeficiency or immune dysregulation.*

Describe eosinophils:

-Histological appearance: 2-3 lobed nuclei and pink-staining from eosin color (acidic dye) -Highly cationic granule contents Major basic protein, eosinophil peroxidase, eosinophil cationic protein, eosinophil-derived neurotoxin Function extracellularly; are especially toxic to the surfaces of cells & pathogens

What is the phagocyte respiratory burst??

-Neutrophils, monocytes, and MΦ can phagocytose or use receptor-mediated endocytosis to internalize antigen & break it down through the phagosomal pathway. -Monocytes and MΦ (not PMNs) present co-stimulatory molecules & antigenic peptide epitopes in MHC II molecules. -NADPH oxidase is esp. important for defense against catalase + organisms. Recurrent infections with these organisms occur in its absence (Chronic Granulomatous Disease, CGD). Usually X-linked. -In addition to oxidative burst, phagocytes produce RNS & an abundance of antimicrobial peptides & proteolytic enzymes that may be used in microbial defense.

What happens if a person has a mutation in NADPH oxidase?

-Non-functional mutations in components of NADPH oxidase cause chronic granulomatous disease (CGD). You may have learned about G6PD deficiencies that can result in hemolytic anemia. Another consequence of G6PD deficiency is defective ROS formation (as seen with CGD) due to impaired G6PD-dependent formation of NADPH, the substrate for NADPH oxidase. -H2O2 is a waste product of cellular respiration in many organisms. Patients with CGD are especially susceptible to pathogens that produce catalase, but not catalase-negative pathogens. This is because pathogen-derived H2O2 can be converted by myeloperoxidase in phagocytes to produce hypochlorite, a powerful antimicrobial agent. Microorganisms that produce catalase can eliminate their own respiratory waste product before it can be seized/used by phagocytes.

What are PAMPs?

-PAMPs are molecules present on/in microbes, but not healthy host cells. -PAMPs are regularly found on several different microbial species & even unrelated organisms (e.g. CpG DNA in viruses & protozoa). -PAMPs are typically integral to an organisms viability.

PAMP recognition by a PRR may initiate:

-Receptor-mediated endocytosis (phagocytosis); -Respiratory burst & phago-lysosome fusion (antimicrobial digestion with proteases, reactive oxygen & nitrogen species); -Antigen processing & presentation (displaying MHC molecules with peptide antigens on the cell surface); -Inflammasome activation (secretion of pro-inflammatory alarmins—already translated cytokines, but perhaps retained within the cell in an inactive form requiring cleavage); -Transcription of various immunological genes, including integrins, cytokines, chemokines; -Induction of apoptosis, autophagy, etc.

What happens in PRR signaling pathways?

-Signaling outcomes depend on the combination of PRR pathways activated and the cell type in which intracellular signaling occurs. -Commonly triggered transcription factors include IRFs, NFκB, & AP-1. -Inflammasome action leads to the release of IL-1 & IL-18 (both highly pro-inflammatory cytokines). -Pro-inflammatory responses tend to promote leukocytosis (often neutrophilia) and the signs you'd recognize as inflammation (redness, swelling, etc). (NFkB) -Interferon responses tend to promote intracellular defenses and cell mediated immunity (less inflammation). (IRFs)

What percentage of WBCs are eosinophils? What do they look like?

1-3% of WBCs basic granules so they will take acidic dyes like eosin and will look pink.

What are neutrophils mechanism of killing?

1. Phagocytosis—engulfment & destruction using ROS, RNS, AMPs & proteolytic enzymes 2. Neutrophil extracellular traps (NETs) -expulsion of chromatin that literally trap pathogens & facilitate their digestion by toxic molecules released from granules

What do neutrophils look like?

3-5 lobed nuclei Cytoplasmic neutral-staining granules packed with proteolytic enzymes, e.g. lysozyme, & antimicrobial peptides (AMPs), e.g. defensins, lactoferrin

What percentage of WBCs are monocytes? What do they look like?

3-8% of WBCs washed away. cytoplasm is grey and dirty. They will have a banded nucleus.

What percentage of WBCs are neutrophils? What do they look like?

54-62% of WBCs aka polymorphonuclear neutrophils aka segmented neutrophil multi-segmented (3-5 lobes) bold dark nucleus

What do adaptive antigen receptors (BCRs and TCRs) result from?

Adaptive antigen receptors (i.e. BCRs & TCRs) result from somatic cell gene rearrangements, so a nearly infinite number of antigen specificities is possible. Furthermore, the receptors are extremely specific & are unable to differentiate threating from innocuous antigen without innate help.

Overview of the complement system:

Anaphylatoxins (C3A, C5A). The classical pathway of complement activation is activated following binding of the recognition molecule C1q to ligands such as immune complexes. The lectin pathway is activated following binding of recognition molecules, such as mannose-binding lectin (MBL), collectins or ficolins, to their ligands, which include carbohydrate structures. Although the alternative pathway is initiated spontaneously, properdin (not shown) might also serve as a recognition molecule for directing activation of this pathway. Following activation via the initiating molecules a cascade of proteolytic activation steps leads to the formation of C3‑convertases that cleave C3 into the anaphylatoxin C3a and the opsonin C3b. Next, C5‑convertases generate the potent pro-inflammatory anaphylatoxins C5a and C5b, the latter of which, together with C6-C9, forms the membrane attack complex (MAC).

The lines represent infectious microbe replication in a normal mammalian host, a host lacking adaptive immunity, and a host lacking innate immunity. Which of the curves depicts deficient innate immunity in the host? A. Red line B. Gray line C. Yellow line

Answer: A. Red Line Red= Host lacking innate Gray= host lacking adaptive Yellow= normal

Why is innate immunity considered conserved?

Antigens recognized by the innate immune system are "CONSERVED" as the receptors that recognize them (PRRs) are also conserved—changed little throughout evolutionary history and have likely evolved together. Because of the use of these invariant (unchanging) germline-encoded pattern recognition receptors (PRRs), the innate immune system has a limited number of specificities for broadly shared microbial patterns. Thus, the innate immune system is not very specific. Measures used to detect infections early include things like elevated C-reactive protein or erythrocyte sedimentation rate. Neither one is very helpful in identifying the cause of inflammation or infection.

What is ferritin's inflammatory action?

Binds and sequesters iron to inhibit microbial iron scavenging; can mask iron deficiency

Where is the complement pathway convergence and most abundant serum opsonin?

C3

Describe the complement pathways.

Classical- activated by antibodies (IgM the best, IgG). Lectin- activated by sugars (mannose on pathogens). Alternative- activated by spontaneous hydrolysis. The classical pathway of complement activation is activated following binding of the recognition molecule C1q to ligands such as immune complexes. The lectin pathway is activated following binding of recognition molecules, such as mannose-binding lectin (MBL), collectins or ficolins, to their ligands, which include carbohydrate structures. Although the alternative pathway is initiated spontaneously, properdin (not shown) might also serve as a recognition molecule for directing activation of this pathway. Following activation via the initiating molecules a cascade of proteolytic activation steps leads to the formation of C3‑convertases that cleave C3 into the anaphylatoxin C3a and the opsonin C3b. Next, C5‑convertases generate the potent pro-inflammatory anaphylatoxins C5a and C5b, the latter of which, together with C6-C9, forms the membrane attack complex (MAC).

What is fibrinogen's inflammatory action? NEED TO KNOW

Coagulation factor; promotes endothelial repair; correlates with ESR

What are neutrophils cubical to?

Crucial in defense against extracellular [pyogenic] bacteria & fungi (major component of pus, i.e. purulent drainage) Pus formed w/PMN responses due to complete enzymatic digestion of cells & tissues.

If you have a cytoplasm PRR, what might this signal?

Cytoplasm PRR signaling induces interferon response=good against intracellular infections like viruses

Which of the following is most likely to trigger sterile inflammation? A. Bacterial peptidoglycan B. Unmethylated CpG DNA C. Flagellin protein D. Endogenous HSP60 E. Endogenous insulin F. Nuclear phosphatidylcholine

D. Endogenous HSP60

What is serum amyloid A's inflammatory action?

DAMP; prolonged elevation can lead to amyloidosis

In the absence of infection, what triggers sterile inflammation and healing?

DAMPs

What are DAMPs?

DAMPs are released from host cells or extracellular matrix following cell death, stress, or injury.

What are macrophages role in antigen presentation?

Display co-stimulatory molecules & peptide antigens in both MHC I & II molecules to CTL (CD8) & Th (CD4) cells, respectively

What will eosinophilia indicate?

Eosinophilia (elevated eosinophils in blood) may indicate parasite infection (esp. helminths, e.g. Ascaris, Strongyloides, Schistosoma, Trichinella).

How are the innate and adaptive immune systems intimately linked?

Even though we try to lump things into separate buckets, it is important to recognize that the innate & adaptive arms of the immune system function cooperatively. The innate system alerts & informs the adaptive while the adaptive recruits & amplifies aspects of the innate system.

What are some PAMPs?

Flagellin Peptidoglycan Lipopolysaccharide (LPS) dsRNA

What are some DAMPs?

HMGB1 (high mobility group box) HSPs (heat shock proteins)

What are some characteristics of PRRs?

Invariant. Germ-line encoded. Ever-present and expressed. Recognize general structures that share certain characteristics. Protein subunits may oligomerize at varying levels & oligomerization can initiate downstream cell signaling.

What are the consequences of PRR deficiences?

Last note on PRRs is their importance for health. Remember that these receptors are conserved within the population—we all have [for the most part] these same receptors on all the same cells. When they don't function sufficiently, for instance to alert the host of potential threats, the host may become more susceptible to infections. Exacerbated function may be linked to inflammatory-associated diseases, but depending on the cellular expression of particular PRRs, it's also possible that regulatory mechanisms get circumvented by their dysfunction.

How are macrophages and neutrophils alike?

Like neutrophils, macrophages are part of the innate immune system and recognize their targets via repeated patterns, i.e. pattern recognition receptors (PRRs) bind pathogen-/damage-associated molecular patterns (PAMPs/DAMPs). And, like neutrophils, macrophages also have several mechanisms by which to kill engulfed pathogens, although those are slightly different than those used by neutrophils. One big difference is what happens after a pathogen is engulfed and killed. When neutrophils do their job, the pathogen is gone. Macrophages like to show off their kill, interacting and presenting detritus from their prey to cells from the adaptive immune systems. So, in that respect they're maybe a little less primitive than neutrophils, perhaps.

What are macrophages role in tattoos?

Long lived tissue-resident macrophages take up injected dye early in the tattoo process. When they die, new macrophages engulf the dead macrophages along with their contents. The relative permanence of tattoos is due to the limited mobility of skin macrophages. When they die, another will engulf the dead mac as well as the ink it's left behind. Isn't that "AWSOME"?

Are mast cells and macrophages found in blood?

Mast cells and MΦ will not be found in blood, but their monocyte precursors will. MΦ can present antigen to T cells (adaptive arm). Monocytosis (elevated monocytes in blood) and lymphocytosis (elevated blood lymphocytes—esp. NK & T lymphocytes) may indicate intracellular infection (e.g. viral, mycobacterial)

What do mast cells, eosinophils, and basophils participate in?

Mast cells, eosinophils, and basophils participate in allergic responses and anti-parasite immunity.

Monocytes can differentiate into...

Monocytes can differentiate into macrophages within tissues (not in blood). Both monocytes and macrophages can phagocytose antigen and present peptide epitopes of antigen within MHC molecules to T cells.

How do you create macrophages?

Monocytes that have left circulation & entered tissues may mature into long-lived MΦ

What are the acute phase reactants? Where are the produced?

More FFiSH in the C. (Ferritin, Fibrinogen, Serum amyloid A, Hepcidin, C-reactive protein). All increase. Produced by liver in response to pro-inflammatory cytokines (esp. IL-6

General overview of Neutrophils:

One-hit wonders! Neutrophils are the most abundant leukocyte in the peripheral blood. Stunningly short lived, they last only a couple of HOURS in the blood (longer if they make it into tissues). They can accumulate at sites of infection in the form of pus. Neutrophils are remarkable in the way they can sneak out of the blood and into inflamed tissues by working their way between other cells. These phagocytes are most effective against bacterial infections, especially extracellular bacteria. Synonym: polymorphonuclear cell (PMN) or polys because of their hypersegmented nucleus (usually 3-5 lobes). On a WBC differential, a "left shift" refers to the release of pre-mature neutrophils from bone-marrow with not-yet segmented nuclei (see top figure).

What is c-reactive protein's inflammatory action?

Opsonin; fixes/activates complement and facilitates phagocytosis; measured clinically as a nonspecific sign of ongoing inflammation

What do PAMPs and DAMPs act as?

PAMPs and DAMPs act as danger signals to spur the innate immune system into action.

What does neutrophilic indicate?

PMNs (aka neutrophils) are very effective phagocytes. Neutrophilia (elevated PMNs in blood) may indicate extracellular bacterial or fungal infection (e.g. Staphylococcus, Streptococcus, Candida, Aspergillus).

How do macrophages kill?

Phagolysosome killing & degradation: -Lysosomes are highly acidified (pH 3.5-4.0) -Respiratory burst (ROS & RNS) -AMPs and enzymes (cathelicidin, lysozyme, hydrolases)

How longs do neutrophils live? What recruits neutrophils?

Professional phagocytes w/distinctive 3-5 lobed nuclei Short-lived (hours to days)—One hit wonders! Most abundant leukocyte in blood -Recruited to inflamed tissues from blood stream via IL-8 (aka CXCL8) -↑ bone marrow production in response to G-CSF

What are the general characteristics of innate immunity?

Rapid response (hours) Limited # of specificities -Conserved, germ-line encoded [pattern recognition] receptors -Target PAMPs & DAMPs -All the same cells express all the same receptors Fixed response—constant in magnitude (no memory) No immunological memory

What are the general characteristics of adaptive immunity?

Slower to respond (> 7 days) Numerous highly selective specificities -Antigen receptors generated via somatic DNA recombination -Clones of lymphocytes w/distinct specificities express different receptors Response improves in magnitude Memory

If you have a surface PRR, what might this signal?

Surface PRR signaling induces endocytosis=good against extracellular infections like C. albicans, E. coli, & P. aeruginosa

Why is adaptive immunity slow to respond?

The adaptive immune system is slow to respond because new antigens are not recognized as threats, they have no context. However, once the adaptive arm has been primed (triggered), the response improves in magnitude and becomes increasingly effective over its duration. This holds true for subsequent encounters with the same antigen, i.e. the adaptive arm will respond faster, stronger and be more effective with each encounter with a particular antigen—this means the adaptive arm remembers past antigens, i.e. it has memory.

What are the earliest usable tests to identify infectious agents detecting?

The earliest usable tests to identify infectious agents should detect antigen, not aspects of the immune system because the earliest immune response will not be specific. In general, specific antibodies can be detected to confirm a specific cause of infection about a week after exposure.

Macrophages are the big eaters...

These are tissue resident cells, that, when activated engulf and destroy pathogens. Every tissue has a population of resident macrophages, in most cases they come from circulating precursors in the blood called monocytes. Monocytes are also known as mononuclear phagocytes (to contrast them from granulocytic/neutrophilic phagocytes which have multi-lobed nuclei).

What do macrophages look like?

agranulocytic THIS IS THE ROLE IN TATTO PERMANENCE

What do macrophages secrete?

cytokines to affect immune responses

What is an adjuvant?

immune stimulants that initiate immune responses. (e.g. In vaccines, if you put a PAMP with cytoplasm PRR= better anti-viral immunity). Helps to stimulate better immune response.

When do eosinophil numbers increase?

in response to IL-5

What are the major phagocyte population resident in normal tissues?

macrophages

Where are PRRs expressed?

our immune cells

What is the PRR that recognizes LPS? NEED TO KNOW.

toll-like receptor (TLR) 4 is the PRR that recognizes lipopolysaccharide (LPS), which is found in the outer membrane of Gram negative bacteria.

What is hepcidin's inflammatory action?

↓ exogenous iron absorption (by degrading ferroportin) and inhibits iron release (from macrophages) anemia of chronic disease