Characterization of the TLR/IL1 beta signaling pathway in type 1 diabetes
- Children’s Medical Center (Dallas, Plano, Southlake)
- UT Southwestern-Other
Ellen Grishman, M.D.
aim 1. (a). To determine if there are differences in mRna and protein expression of TLR1/2/4/6, naLP3/aSC/Caspase1, the activity of the inflammasome and iL1[Beta] production between PBMC obtained from newly diagnosed T1D versus healthy controls. (b). To determine the changes in expression of these molecules when PBMC from healthy controls are incubated in sera from patients with newly diagnosed T1D.
Stimulation of TLR2/4 results in production of pro-iL1[Beta] (measured as iL1[Beta] mRna) and is processed to mature iL1[Beta] by the activation of Caspase1 which requires the adaptor molecules naLP3 and aSC. Patients with T1D have increased mRna expression of TLR2/4 and iL1[Beta] in PBMC, but we do not know if iL1[MiCRo-SYMBoL] is increased as a result of TLR or another pathway. Because iL1[MiCRo-SYMBoL] likely plays a role in T1D pathophysiology, it is important to understand the mechanisms by which it is increased. This aim will allow us to connect the observations that TLR2/4 and iL1[MiCRo-SYMBoL] expression are increased in newly diagnosed T1D and that T1D sera can induce iL1[MiCRo-SYMBoL] expression. Better understanding of the innate immune system in the development of T1D may identify novel therapeutic targets to improve therapeutic options for patients with evolving T1D.
Patient population: approximately 200 children are diagnosed with T1D each year at Children's Medical Center Dallas. We are the only center caring for children with diabetes in a catchment area of ~2 million people. The large number of patients makes recruitment straightforward. We require 20 patients with T1D and 20 healthy controls for this aim. However, we will recruit 28 patients from each group since we will not be able to use some samples in the analysis due to too few peripheral blood mononuclear cells, experimental error, inability of the cells to respond to positive control, etc. Healthy controls can be either children or young adults since we are looking for basal expression and the response of healthy PBMC incubated with sera from patients with T1D, so age matching is not necessary. We cannot enroll young children due to blood volume requirements for the study.
aim 2. To determine if TLR2/4 blockade (using antibodies and specific siRna) in healthy PBMC exposed to sera from newly diagnosed T1D will abrogate the production of pro-iL1[Beta], the activation of Caspase1, and production of mature iL1[Beta].
Rationale: in order to mechanistically implicate TLR2/4 in the induction of iL1[Beta], we will need to inhibit each TLR or possibly both, and determine if inflammasome activation and iL1[Beta] induction are blocked. These initial experiments will allow further dissection of the signaling pathways involved in induction of pro-iL1[Beta] and the activation of Caspase1 in PBMC from newly diagnosed T1D patients. understanding the mechanisms leading to increased iL1[Beta] expression will identify potential novel therapeutic targets in the treatment of evolving T1D.
Patient population: identical to aim .
Study Procedures: Methods for aim 1.
Blood collection and PBMC extraction
We will collect 1 ml/kg (minimum 30 ml and maximum 40 ml) of blood from T1D patients and healthy controls in eDTa tubes and extract plasma by centrifugation. We will add 10% (v/v) thrombin to the plasma and incubate the samples at 37[Degrees]C for 20 minutes before discarding the clot. Sera will be stored at -80[Degrees]C, if not used immediately. PBMC will be obtained by Ficoll-Histopaque gradient centrifugation and used immediately.
Basal expression of the members of the TLR/inflammasome/iL1[Beta] pathway will be determined by quantitative RT-PCR, western blotting, and flow cytometry.
Methods for aim 2.
Blood collection is the same as aim 1. We will then perform antibody blocking studies on PBMC cultures, and we will use siRna to decrease expression of TLR 2 and/or 4 to mechanistically determine the impact of these TLR on iL1 [Beta] production.
See attached for full Project Summary.
Inclusion Criterion for Patients with T1D
1) Diagnosed with T1D (as defined by the American Diabetes Association) within 1 week of enrollment
2) Ages 7-18 years inclusive
3) Weight >= 30 kg
4) Males and females
Inclusion Criterion for Healthy Controls
1) Healthy people ages 7-40 years inclusive
2) Weight >= 30 kg
3) Males and females