Section 1: Imin

what are the origins of phagocytosis?

did not start as an immune defense; was used for nutrient acquisition by unicellular organisms

what are 3 major events in the evolution of phagocytosis and when did they occur?

1.) bacterial apoptosis-like cell death was seen in multicellular biofilms (3.25 BYA in prokaryota era)

2.) sponges had the capacity for internalization and differentiation (600 MYA in porifera era)

3.) phagocyte pro-inflammatory response and homeostatic mechanisms seen in ancestral myeloid phagocytes (500 MYA in agnatha era)

what are 3 survival strategies used by intracellular pathogens?, give an example of pathogens that use these?

1.) prevent lysosome and phagosome fusion (L. pneumophilia, T. gondii, Salmonella, Yersinia, M. tuberculosis)

2.) escape phagosome before fusion occurs (T. cruzi, L. monocytogenes)

3.) survive in the phagolysosome (Leishmania, Mycobacterium)

what is phagocytosis defined as?

internalizing particles greater than 1 micron; receptor mediated; clathrin independent; involved in immunity, tissue turnover and repair

what is endocytosis defined as?

internalizing particles smaller than 1 micron; receptor mediated; clathrin dependent; involved in down regulating surface receptors, nutrient uptake and synaptic vesicle recycling

what is pinocytosis defined as?

cell drinking; non-receptor mediated; clathrin dependent; involved in fluid and soluble molecule uptake

what are professional phagocytes?

myeloid cells such as monocytes, macrophages, immature DCs and neutrophils

what is the major difference between professional and non-professional phagocytes?

phagocytic efficiency and capacity differs due to an array of phagocytic receptors that increase particle range and phagocytic rate

what are the 7 key steps in the phagocytosis process?

1.) chemotaxis

2.) adherence

3.) receptor activation

4.) inducing phagocytic signalling cascades

5.) pseudopod formation and internalization

6.) initiate intracellular degradative mechanisms, killing responses and release of pro-inflammatory mediators

7.) digest and release products

what are 4 examples of direct recognition of microbes?

1.) M pi mannose receptor

2.) DEC 205

3.) Dectin-1

4.) scavenger receptors

what is the role of the M pi mannose receptor?

recognizes branched alpha-linked mannose oligosaccharide chains on surfaces of bacteria, fungi, and protozoa, but not mammals

what is the role of DEC 205?

receptor that recognizes mannans

what is the role of Dectin-1?

C-type lectin receptor that recognizes beta-glucan polysaccharides

what is the role of scavenger receptors?

recognize both components on apoptotic cells and bacterial cell walls

what are 2 examples of scavenger receptors and their jobs?

1.) SRA: binds intact gram (+) and gram (-) bacteria, lipotechoic acid and LPS

2.) MARCO: binds intact bacteria

what are 4 examples of indirect recognition of microbes via opsonins?

1.) collectins

2.) ficolins

3.) complement

4.) antibodies

what are collectins/ficolins?

activate innate immunity at mucosal surfaces and in the bloodstream

they’re soluble proteins that recognize distinct carbohydrate moiteties on microbes but not on host

what is affinity?

strength of noncovalent binding interaction; higher affinity means higher likelihood 2 partners will exist in a complex

what is avidity?

increased “apparent” affinity” due to the presence of multiple binding sites on both partners

how do signalling pathways get activated following FcgammaR binding?

1.) receptor is activated

2.) protein tyrosine kinase if Src family mediates phosphorylation of FcR ITAM domain, creating SH2 binding site

3.) Syk, a protein tyrosine kinase, is recruited to ITAM SH2 binding site

4.) Syk gets activated

5.) recruitment and activation of PI3 kinase

6.) many pathways get activated

how is the phagolysosome formed?

requires actin rearrangements, myosin interaction with actin (entry), and dynamin (contracts and pinches off membrane)

phagosome fuses with granules and lysosomes

what are the contents of a primary granule in neutrophils?

myeloperoxidase, elastase and bacterial permeability increasing protein

what are the contents of a secondary granules in neutrophils?

lactoferrin and metalloproteinsases

what is the content if a tertiary granule in neutophils?

gelatinase

what is the role of calcium signalling in phagosome maturation?

calcium concentration is greatest near the phagosome and phagocytic cups

phagocytic receptors direct calcium in waves arounf the phagosome and is important for phagolysosome formation; Ca2+ waves branch so that 1 branch goes around the cell and the other goes around the phagosome

what is the purpose of reactive oxygen and nitrogen species?

kills internalized microorganisms

highly corrosive and toxic

what are features of reactive oxygen intermediates

produced by NADPH oxidase in phagolysosome

rapid, but limited

NADPH oxidase consists of membrane and cytosolic components which assemble in response to phagocytic signals

what are features of reactive nitrogen intermediates?

produced in cytoplasm by inducible nitric oxide synthase (iNOS) and diffuses into phagosome

requires transcriptional induction of iNOS gene expression

slower, but longer lasting

How is cell turnover related to phagocytosis?

CD47 is expressed on healthy erythrocytes, but not on dead ones; CD47 binds SIRP-1alpha receptor on kupffer cell and splenic macrophage surface, binding increases threshold of activation need for phagocytosis; therefor, live erythrocytes aren’t removed from blood prematurely

with respect to receptor and surrounding molecules, what is the look of FCgammaR initiating a phagocytic response?

large immune complexes present, high [PI3K] compared to [Cbl]; TLR activation can cause PI3K expression, among other things

with respect to receptor and surrounding molecules, what if the look of FCgammaR initiating an endocytic response?

less cross talk due to loss of large immune complexes; [Cbl] higher than [PI3K; presence of apoptotic bodies can lead to Cbl expression

Cbl leads to Syl ubiquitinaion and proteosomal degredation

how do different types of phagocytes rank with respect to speed of degradation of pathogen?

macrophages degrade pathogens the fastest, DCs do the slowest, possibly to get association with MHCII

what is SLE thought to be caused by?

formation of autoantibodies to self antigens (mostly nuclei acid)

immune complex deposition occurs around small blood vessels leading to a variety of symptoms

what is a stem cell?

an early progenitor cell with the capacity to differentiate into a broad range of mature cells

what does totipotent mean?

ability of stem cells to give rise to all embryonic and extra-embryonic tissues

what does pluripotent mean?

ability of stem cells to give rise to all embryonic tissues, but not extra-embryonic tissues

what does multipotent mean?

ability of stem cells to give rise to diverse cell types of one or a few tissues

what are hematopoietic stem cells?

they supply the entire repertoire of mature blood cells for the lifetime of an organism

what are the 2 reasons HSCs are lifelong?

1.) capacity to self renew

2.) have multi-lineage differentiation potential

what types of cells do HSCs give rise to?

B/T cells, NK cells, erythrocytes, megakaryocytes, granuloytes, monocytes

what is the relationship between differentiation and self-renewal capacity in stem cells?

increasing differentiation decreases ability for self-renewal

provide 5 genes and 2 pathways associated with self-renewal and expression of multi-lineage specific transcripts?

Hoxb4, bmi1, cdkn1a, shh, bmp4

notch pathway, wnt pathway

provide 5 genes associated with hematopoietic cell function and expression of individual lineage specific transcripts?

globin genes, mpo, gata 1, epor, mpl

what are the 2 types of progenitor cells a HSC can become?

1.) common myeloid progenitor

2.) common lymphoid progenitor

what 3 cell types can a CLP become?

B cell, T cell or NK cell

what 2 progenitor cell types can a CMP become?

1.) megakeryocyte/erythrocyte progenitor

2.) granulocyte/monocyte pregenitor

what 2 cells can a MEP become?

megakaryocyte or erythrocyte

what 2 cells can a GMP become?

granulocyte or monocyte

what is the phenotype based definition of HSCs in mice?

high MHC 1 expression, low Thy-1 positivity, positive for Sca-1, AA4.1, and c-kit, positive for adhesion molecule PGP-1, negative for Lin, negative for B220, Mac-1, Gr-1 and CD-8

how can mature blood subsets be identified?

can use surface marker expression and do flow cytometry and get stained with those surface markers

what are the 2 types of bone marrow

yellow: dormant

red: actively generates blood cells

*all bone marrow is red at birth

where does bone marrow remain in adults?

axial skeleton and epiphysis of humerus and femur

what does the bone marrow contain?

capillaries, reticular fibers of connective tissues, fibroblasts, immature blood cells

what 3 ways do cells provide a nurturing environment to stem cells in the bone marrow?

1.) cell derived components

2.) systemic control factors

3.) physiological conditions

what is the purpose of osteoblastic cells within the bone marrow?

they can sustain slow-cycling/dormant (quiescent) stem cells

this can protect stem cells from damage

what is the purpose of SCF produced by stromal cells?

associates with the extracellular matrix and acts on primitive stem cells through c-kit (CD117) *c-kit is a tyrosine kinase receptor

why is it important for a species to be heterozygous w/sI for stem cell differentiation?

will undergo impaired hematopoiesis

sI/sI mutants have normal stem cells but defective stromal production of SCF- lack the environment; can be corrected by giving them a spleen fragment which contains stromal cells

w/w mutants lack c-kit (surface receptor for SCF)- lacks normal stem cells; can be corrected by giving them bone marrow to provide c-kit

what is ontogeny?

the developmental life history of an organism

what is primitive/embryonic hematopoiesis?

occurs extra-embryonically in the yolk sac, very rapid, ensures sufficient oxygen is supplied to developing embryo and dying cells, resulting from tissue turnover, are cleared out (by macrophages)

what is definitive hematopoiesis?

occurs once embryonic hematopoiesis is beginning to decline

it sustains production of all blood cell lineages for fetus and adult

originates in aorta-gonad-mesonephros (AGM) region of the para-aortic splanchnopleura (P-Sp), where de novo formation of HSC occurs followed by migration from AGM to fetal liver, spleen, and bone marrow where more maturation will take place

what organ is a constant among species for a location of hematopoiesis?

the spleen

what are colony stimulating factors?

cytokines central to hematopoiesis, functional responses of resulting blood cells and maintenance of homeostasis

production and degradation of these are strictly controlled under steady state and stress periods

what are the functions of M-CSF and G-CSF?

colony stimulating factors that have roles in proliferation, differentiation, and survival of macrophages, neutrophils, and their precursors

what are the functions of GM-CSF and multi-CSF (IL-3)?

colony stimulating factors that function at early stages of lineage commitment regulating the expansion and maturation of primitive hematopoietic progenitors

where does integration of signals promoting hematopoiesis occur?

occurs at the level of gene expression

transcription factors integrate all incoming signalling pathways and modulation of transcription machinery

interactions between transcription factors, co-regulatory molecules, and specific binding sequences in the DNA

how is hematopoiesis regulated through changes in concentration of PU.1?

uniformly high during early stages of hematopoietic development (concentration changes do not occur during differentiation of HSCs into CMPs or CLPs)

PU.1 concentration reduction is required for normal megakaryocyte-erythroid progenitor, B cell progenitor and T cell progenitor development

PU.1 concentration increase is required for development of granulocyte-macrophage progenitors

what is PU.1?

a transcription factor that has a positive effect on myeloid differentiation and negative effect on lymphoid differentation

what happens when PU.1 concentration decreases in myeloid lineages?

failed differentiation, abnormal proliferation, and leukemia

how does GATA-1 and GATA-2 affect PU.1?

inhibit PU.1 function

in stem cells: GATA-2 blocks PU.1 and c-Jun interaction, inhibiting PU.1 activation of ita downstream target genes

in erythroblasts: up-regulating GATA-1 blocks coactivation of PU.1 by c-Jun

in developing myeloid progenitors: decreasing GATA-1 and GATA-2 results in PU.1 and c-Jun synergistically activating PU.1 target genes like the M-CSF receptor

what are SAMPs?

self associated molecular patterns

what are ACAMPs?

apoptotic cell associated molecular patterns

what are DAMPs?

danger/associated molecular patterns

how does the skin act as a physical barrier from pathogens?

in the dermis, there are structures such as hair follicles, sweat glands and sebaceous glands which prevent pathogen entry to the body

what times of immune cells are always present in the epidermis and dermis?

epidermis: langerhans cells

dermis: DCs, macrophages and mast cells

what are HAMPs?

homeostasis altering molecular process

how were toll receptors discovered in drosophilia?

a mutation in toll signalling affected the production of drosomyosin which resulted in severe aspergillus fumigatus infection

what is the structure of TLRs?

contain a leucine rich repeat region which is for PAMP binding that form a horseshoe shape; internal domain called TIR which is the trigger for downstream signalling

where are TLRs located?

on the cell membrane (usually recognize structural features of pathogens) and the endosomal membrane (recognize nucleic acids)

how must TLRs present to be active?

they must dimerize (homo or hetero)

what is the only TLR dimer that can migrate from the cell membrane to the endosomal membrane?

TLR4/TLR4

how does LPS activate TLRs?

LPS binds LBP which activates CD14, CD14 activates MD2 (myeloid differentiation factor) and bind TLR4

what is the equivalent in drosophilia in the following TLR signalling structures in mammals?

MyD88, IRAK, IKK, IKB, NFKB

MyD88= dMyD88

IRAK= Pelle

IKK= cactus kinase

IKB= cactus

NFKB= DIF

why do drosophilia have indirect PAMP-Toll interactions?

PAMPs first encounter a protease for breakdown, then activate spatzle which activates Toll

how does an antibacterial response occur through TLRs?

typically begin on the cell membrane

MyD88 as an adaptor which activates NFKB

Proinflammatory receptors get transcribed (IL-1 beta and TNF-alpha)

macrophages and neutrophils get recruited and activated

antimicrobial proteins get synthesized

how does a viral response occur through TLRs?

typically begin on endosomal membrane

MyD88 and TRIF are adaptors

adaptors activate IRF7 transcription factor

transcription factors transcribe type 1 IFNs which inhibit viral replication

what are the 2 receptors of SARS-COV2?

ACE2 and TMPRSS2

what PRRs are activated by SARS-COV2?

TLRs are triggered in the endosome, resulting in NFKB activation

RLRs are triggered in cytosol, resulting in IRF3/IRF7 activation

what are 3 DNA sensing PRRs? what do they sense?

TLR9 (senses CpGDNA)

AIM2 (senses dsDNA)

cGAS (senses dsDNA)

what is the pathway following TLR9 activation?

activates MyD88, which activates IRAK1/IRAK4, which activates IKK, which activates TNF and IFN alpha

what is the pathway following cGAS activation?

activates 2’3’-cGAMP, which activates STING, which activates TBK1, which activates NFKB or IRF3, which produces TNF or IFN beta

what are ALRs?

bind cytosolic DNA and form inflammasomes

AIM2 binds dsDNA; binding multiple ALRs to same dsDNA strand allows AIM2 PYDs to associate, driving long filament assembly with ASC and pro-caspase 1, forms the inflammasome

what are NLRs?

NOD like receptors

bind PAMPs from cytosolic pathogens

what are the 3 major functional domains of NLRs?

1.) LRR domain (ligand binding at C terminus)

2.) central domain (NBD domain)

3.) N-terminal domain

what is an inflammasome?

multiprotein complex that promotes inflammation by processing inactive precursors of pro-inflammatory cytokines like IL-1-beta and IL-18

what structures are present in inflammasomes?

NLRs, AIM2, and pyrin act as cytosolic sensors detecting PAMPs or DAMPs

ASC (Pyd and CARD) bind NLRP3 via Pyd-Pyd interactions

caspase-1 binds ASC via CARD-CARD interactions

what is signal 1 in the inflammasome?

PAMP/DAMP binding to PRR

signalling pathway activation to transcribe and synthesize pro-IL-1-beta, pro-IL-18 and NLRP3

what is signal 2 in the inflammasome?

sterile and pathogen-derived activators initiate changes to produce signal 2

NEK7 binds to NLRP3 which triggers NLRP3 assembly with ASC and procaspase-1

procaspase 1 cleaved into caspase 1

caspase 1 cleaves pro-IL-1-beta and pro-IL-18 into IL-1-beta and IL-18

also generates gasdermin D to form pores for release of these cytokines

what is pyropoptosis?

causes membrane disruption and nuclei celling, associated with cytokines IL-1 and IL-18, pro-inflammatory

what are some activators of signal 2 of the inflammasome?

K+ efflux and ROS generation

what signals do viable cells put out?

no ACAMP or alarmin expression

express SAMPs to prevent phagocytic recognition