PEPITEM Flashcards
what is adiponectin?
Most abundant circulating adipokine
Adiponectin – cytokine generated by adipose tissue (adipokine)
what are the functions of adiponectin?
Metabolic function
immune system
what is the metabolic function of adiponectin?
- Insulin sensitising
- Low levels associate with T2D, CVD, atherosclerosis – metabolic diseases
what is the function of adiponectin in the immune system?
Immune system → adiponectin is anti-inflammatory
- Decreases T cell proliferation, so decreases inflammation
- Decreases APC function (MHC II, CD86)
- Modulates neutrophil function (chemotaxis, apoptosis)
how does adiponectin (adi) deficiency affect leukocyte recruitment?
Mouse model K/O of adi gene:
- Looked via intravital microscopy at peri-intestinal venules in vivo
- WT mouse: normal leukocyte rolling, adhesion, E-sel, VCAM1
- Adi K/O = more leukocyte adhesion, more E-sel and VCAM1 - more rolling and adhesion
Adi dampens down leukocyte recruitment by reducing adhesion molecules
what is a static adhesion assay?
in vitro model of T cell trafficking
- HUVEC (human umbilical vein endothelial cells) cultured in dish
- stimulate HUVEC for 24 hours with TNF and IFNy
- add healthy peripheral blood lymphocytes (PBLs) purified from donor blood - within 20 mins can see some cells have adhered and others migrated below HUVECs
Phase dark (red circles) = migrated through endothelium
phase light = on surface of endothelium
what is a flow-based adhesion assay?
Lower throughput than static
- model what occurs in blood using microscope by pumping to pull leukocytes across HUVECs at flow rate similar to circulation
- can see capture of leukocytes on endothelium via selectins in real time
how do adiponectin levels in diabetes patients affect leukocyte recruitment?
low adiponectin in T2D, so more leukocyte recruitment - more inflammatory T cells into pancreas
what happens when adi is added to static adhesion assay?
When adi is added, it inhibits PBL migration
- We expect circulating adi levels to be 5-15ug/ml in plasma
- Adi gives tonic inhibition of T cell trafficking
same result shown in flow based assay
what is the mechanism of action of adiponectin/PEPITEM?
Stimulated endothelial cells:
- these recruit B cells to endothelial surface during inflammation
- B cells don’t migrate across, the just bind to endothelium via integrins
- Adi activates B cells via adiR1/2, leading to pepitem release
- Pepitem regulates T cell trafficking indirectly
- Cadherin 15 on endothelial cells binds peptiem
- Leads to SPHK1 activation to generate S1P which is released from endothelium
- S1P turns off function of b2 integrins on T cell to prevent trafficking
how was PEPITEM identified?
PEPITEM was found in B cell supernatant
- Isolated B cells from healthy donors, give adi or not (control)
- Took supernatant and used column-based purification to isolate protein and used mass spec
- In silico subtrative analysis of control and adi-stimulated sample
- PEPITEM was the only unique product in adi-stimulated supernatant
what is PEPITEM?
Product of the 14-3-3z protein, which is a product of YWHAZ gene
- It is 14 amino acids in size
very potent
- IC50 = 45pM
- not much needed to have effects
how is PEPITEM processed?
PEPITEM is secreted by B cells as the whole 14-3-3z protein
- And then 14-3-3z is cleaved/proteolytically processed extracellularly by proteases to form activate PEPITEM
- enzymes on surface of different cells can process 14-3-3z, e.g. endothelial cells, B cells
- MMP2 and MMP9 can cleave 14-3-3 to generate PEPITEM
what has PEPITEM been shown to do in mouse models?
Regulate T cell trafficking in disease models in vivo
- Salmonella model with infected foci in liver and T cell infiltration – PEPITEM given to mice reduces T cells in foci
- Uveitis model – inject mouse eye with LPS to cause inflammation, PEPITEM reduces T cell infiltrate
- Ischaemia injury in liver – tie off blood supply to liver for 30 mins, release to induce reperfusion injury, which drives inflammation and T cell recruitment to injured liver – PEPITEM prevents T cell engraftment in liver so less inflammation
- Sjorgen’s syndrome – inject viruses into salivary glands of mouse – PEPITEM reduces T cell infiltrate
what are the overall effects of PEPITEM in preclinical models?
PEPITEM reduces inflammation and T cell recruitment into the environment
how is PEPITEM pathway affected in T1D and RA?
- adiR2 expression on B cells of patients is reduced compared to healthy controls – same for RA patients
- Downstream signalling of adi receptor also compromised – less PEPITEM generation by adiponectin signalling
- PEPTIEM release from isolated B cells: healthy controls = more pepitem release, T1D patients = no PEPITEM release from B cells
- PEPITEM in patient serum circulation is reduced compared to healthy controls
how is T cell migration affected in T1D and RA? can it be resolved?
In healthy controls = high T cell migration, add adi can reduce migration
In T1D, same level of basal recruitment of T cells, but addition of adi doesn’t reduce migration, addition of synthetic pepitem regains control of migration
T1D AdiRs are non functional, so adi itself doesn’t work, hence why synthetic PEPITEM is needed as it mimics native PEPTIEM
how does PEPITEM change in healthy people?
PEPITEM always circulating in healthy people to control inflammation and avoid chronic inflammatory disease
As we age, we lose PEPITEM – increased risk of inflammatory disease
PEPITEM is a homeostatic agent that is always circulating in health
what happens when PEPITEM pathway is dysregulated or lost?
chronic and exaggerated inflammation
- occurs in ageing and disease
- immune-mediated inflammatory diseases are age-related, and related to reduced PEPITEM
how was the active amino acid sequence of PEPITEM for drug development identified?
Take 14 amino acids and one by one substitute with alanine to see which are essential for function
- Amino acid 7 in PEPITEM is naturally alanine = control
- K/O each amino acid in turn = no effect
- showed that there are 2 active amino acids, not just one - these are called small pharmacophores
what pharmacophores were shown to be important for PEPITEM?
Can K/O pharmacophores e.g. SVT K/O still works due to redundancy of QGA
- Means point mutations alone in PEPITEM doesn’t ablate function
SVT and QGA are two independently functional tripeptides – found from studying many truncated tripeptides
- they are chemically distinct
- Have similar IC50 to PEPITEM itself in vitro and in vivo
- These peptides are as effective as PEPITEM
what is the zymosan-induced peritonitis model?
Zymosan-induced peritonitis is a simple and reproducible model of self-resolving inflammation
- Zymosan is a yeast cell wall extract ofSaccharomyces cerevisiaeand is recognized by TLR2 and Dectin-1(dendritic cell-associated C-type lectin 1) on innate immune cells
- inject zymosan into peritoneum of mice to induce inflammation and T cell recruitment
- can see kinetics of leukocyte recruitment, cytokine release and pro-resolution e.g. macrophage efferocytosis
what is the normal kinetics of zymosan-induced peritonitis?
At 4 hours = many neutrophils, some monocytes
over the 2 weeks = monocytes increase
at 3 days = T cells and B cells
how do the tripeptides affect the zymosan-induced perionitis model and leukocyte trafficking?
Put zymosan into mice to induce inflammation and recruit leukocytes:
- Co-administer PEPITEM or tripeptides
- PEPITEM and tripeptides inhibit T cell recruitment to peritoneum e.g. CD45+, CD3+
- PEPITEM better in inhibiting neutrophils and monocytes
- QGA less effective in eosinophils
- SVT less effective in B cells
