Lecture 17 - T1D Research

Question 1

What are the limitations of studying T1D in humans

Answer 1

Not possible to take pancreatic biopsies from at-risk individuals prior to or once diagnosed with T1D (whatever functional beta cells they have left - not a viable option to do surgery on the pancreas)

• Difficult to determine what’s happening during disease development
• Need access to key tissues and cells, but only have access to:
  o Peripheral blood of living individuals
  o Cadaveric donor pancreatic specimens
• Difficult to identify and confirm genetic and environmental factors:
  o Need controlled breeding  allows us to test the effect of different genes
  o Need controlled isolation  allows us to test the effect of different environmental factors

Question 2

What is the NOD mouse?

Answer 2

Nonobese diabetic (NOD) mouse
Spontaneously develop diabetes

Higher rates in females than males (humans about same)

Has to do with sex hormones in mice, if you castrate males then they get higher rates similar to females, giving testosterone to females will lower rates

> lymphocytes mediate specific destruction of insulin-producing Beta cells

Question 3

How does T1D arise in the NOD mouse?

Answer 3

Very consistent development of diabetes in NOD mouse

> day 20: immune cell invasion of pancreas
day 40-120: increasing insulitis and beta cell destruction, progressive loss of insulin
day 120-death (200): clinical diagnosis (hyperglycemia), residual to no insulin secretion

Question 4

What is peri-insulitis?

Answer 4

When insulin is in the peripheral/outside of the cell

Question 5

What are the genetic factor similarities between mouse and human?

Answer 5

Mouse genetic studies identified > 30 T1D susceptibility loci

Humans >40 alleles that increase risk of T1D

Many of the genetic susceptibility loci overlap between mouse and humans

Mouse T1D loci termed Idd, Human T1D loci termed IDDM

We find more genes in heterogenous humans, because we could have different combinations of loci, mouse are all basically clones

Question 6

What are the immunological abnormalities in NOD mouse: thymus?

Answer 6

• Thymus:
  o Abnormality is the impaired deletion of autoreactive T cells
  o Causes beta-cell specific T cells to escape into the periphery

Question 7

What are the immunological abnormalities in NOD mouse: macrophages?

Answer 7

o Less efficient at clearing dying cells/beta cells
o Produces higher levels of inflammatory cytokines
o This causes an increase in inflammation in islet and exposure of beta cell antigens

Question 8

What are the immunological abnormalities in NOD mouse: dendritic cells?

Answer 8

o Defective maturation an responses to certain stimuli
o Fails to eliminate autoreactive T cells in the periphery
o Causes enhanced activation of beta-cell specific T cells

Question 9

What are the immunological abnormalities in NOD mouse: T cells?

Answer 9

o Enhanced propensity to proliferative and produce pro-inflammatory cytokines (esp. IFN-gamma)
o Regulatory T cells are less effective at suppressing autoreactive CD4+ and C8+ T cells in the periphery
o Causes increased numbers and enhanced activation of beta-cell specific T cells

Question 10

What are the genetic factors: Idd1 and IDDM1?

Answer 10

• A similar amino acid change for the MHC/HLA class II molecule associated with T1D
  High risk allele genotype for beta chain
  >human HLA (DQ2 or DQ8) = DQB1 *0201 or *0302
  >Mouse MHC = NOD H2-Ag7
• Beta chain position 57 (peptide cleft where beta cell is presented with B-cell receptor)
  >human = code for alanine
  >mouse = code for serine
  >both associated with increased T1D risk

Question 11

What are the salt bridge changes in NOD mice and humans during the amino acid change for MHC/HLA class II molecule

Answer 11

Salt bridge DISRUPTED when aspartic acid at position 57 is changed to serine or alanine
>associated with susceptibility to T1DM

Salt bridge FORMED between aspartic acid at position 57 in beta chain and arginine on alpha chain
>associated with resistance to T1DM

Question 12

How can aspartic acid changes be proven to affect T1D development?

Answer 12

Use gene editing in NOD mouse
>CRISPR-Cas9 mutagenesis to change MHC amino acid

> show that changing 1 aa from serine to aspartic acid is protective

NOD MICE WITH ASPARTIC ACID ARE PROTECTED

Question 13

What are the environmental factors that are similar between human and mouse?

Answer 13

• Positive correlation between equatorial distance and incidence of T1D – decreased temperature = increased diabetes incidence
• Correlation does not equal causation
  Different diabetes incidence rates for different countries - definitely has environmental factors that affect incidence

Question 14

What effect does the environmental factor of temperature have on T1D?

Answer 14

Higher temperature reduces incidence of diabetes in NOD mice

what does this mean for humans? temp does it affect? prob not cause weather is seasonal and doesnt always stay 24 degrees but** (add in)

Question 15

What effect does the environmental factor of microbial pathogens have on T1D?

Answer 15

• Can move NOD mice from a facility that has microbial pathogens to one that doesn’t
• Mice in conventional dirty breeding facilities have lower incidence of diabetes
  >T1D incidence in NOD mice is affected by microbial pathogens –> link to the theory that early exposure of pathogens tempers the immune system and reduces incidence of T1D

Question 16

What effect does the environmental factor of gut microbiota have on T1D?

Answer 16

Observed:
NOD males < NOD females for T1D incidence
NOD males have different gut microbiomes to NOD females

Hypothesis
>NOD females fed gut-derived microbes isolated from NOD males should reduce incidence of T1D

> > > Microbes in gut of NOD males provide protection against development of T1D (not complete protection)

Question 17

What is the immune cell infiltration and insulin autoantibodies like in NOD mice?

Answer 17

Same types of immune cells that invade humans also invade NOD mice
>macrophges, dendritic cells, CD8+ T cells (cytotoxic), CD4+ T cells (proinflammatory cytokines), B cells (autoantibodies)

> insulin autoantibodies can be detected in peripheral blood and precede T1D in NOD mice

NOD mice exhibit more extensive insulitis than human with T1D

Question 18

What is the role of autoreactive CD8+ T cells in NOD mice?

Answer 18

• Perforin causes pore formation within the beta cell membrane
  o Enables granzymes, which are proteases, to enter the cell and initiate perforin

Question 19

How is gene editing used to delete the perforin gene in NOD mice?

Answer 19

Design and synthesis of guide RNAs: short RNA molecules that can be designed to target specific regions of the DNA sequence - to induce double-strand breaks.

Introduction of gRNAs and Cas9 protein: can cut DNA at specific locations, are introduced into the fertilized eggs of NOD mice by microinjection.

Homology-directed repair: To delete the perforin gene, a repair template is also introduced into the fertilized eggs. This repair template contains a modified sequence that replaces the perforin gene with a non-functional sequence.

Selection and breeding of edited mice: The fertilized eggs are implanted into surrogate mothers, and the resulting pups are screened for the presence of the perforin gene deletion using PCR and other molecular biology techniques. Mice with the desired gene edit are bred to produce a colony of perforin-deficient NOD mice.

• Insulitis still occurs, but development of T1D is significantly delayed and reduced

Question 20

What is the effect of depleting specific immune cells in NOD mice?

Answer 20

Deplete macrophages by antibody treatment: Reduces T1D incidence

Deplete dendritic cells by gene editing: Prevents T1D

Deplete B cells by gene editing or antibody treatment: Prevents T1D

Do not produce antibodies, but can present antigen to T cells: Still develops T1D

Do not produce antibodies, but do not present antigen to T cells: Reduces T1D incidence
Getting rid of autoantibodies alone has not affect on T1D incidence

Deplete both CD4+ and CD8+ T cells by gene editing or treatment with antibody: Prevents T1D

Transfer both T cells from diabetic mouse to non-diabetic mouse: Causes T1D onset

Transfer only CD4+ or only CD8+ T cells from diabetic mouse to non-diabetic mouse” Does not cause T1D

Need both T cells to develop pathogenesis that leads to T1D

Question 21

What are 3 classical signs of diabetes mellites?

Answer 21

Polydipsia: This refers to excessive thirst and an increased desire to drink fluids. People with diabetes mellitus often experience polydipsia due to high blood glucose levels that cause the kidneys to produce more urine, leading to dehydration.

Polyuria: This refers to excessive urination, which is often associated with polydipsia. People with diabetes mellitus often experience polyuria because their kidneys are working to remove excess glucose from the blood, which leads to increased urine production.

Polyphagia: This refers to increased hunger and an increased desire to eat. People with diabetes mellitus may experience polyphagia because their body’s cells are not able to use glucose effectively, which leads to a lack of energy and increased hunger.