Induction of TIMP1 in Human Monocytes

Polytraumatic injury has been known to lead to severe systemic inflammation in select patients, where the specific mechanism behind this outcome is still not known. What is witnessed is when patients receive blood transfusions during hemorrhaging associated with polytraumatic injuries some patients have a beneficial reaction, while others have an adverse reaction in relation to systemic inflammation. TIMP 1 is a protein that regulates MMPs, which are proteins that play a vital role in wound healing and in the composition of the extracellular matrix. TIMP 1 specifically inhibits MMP 9, which plays a crucial role in the inflammatory response during polytraumatic injury. MMP 9 is involved in the degradation of the extracellular matrix and activates cytokines and chemokines to regulate tissue remodeling. This study aims to understand the mechanisms of TIMP1 induction in human monocytes. This will be done by selecting TIMP1 primers for RT-PCR amplification. In a previous experiment, cDNA was prepared from human monocyte cells (U937) treated or not treated with endotoxin. A specific immune response gene TIMP1 associated with inflammation will be amplified from the cDNA preparations using PCR, which will be compared with a housekeeping gene GADPH. Gel electrophoresis will be used to separate these RT-PCR products to determine whether endotoxin enhances TIMP1 expression without affecting GADPH expression levels. The expected outcome is endotoxin will induce higher TIMP1 expression. Understanding the mechanisms behind induction of inflammatory factors will allow for the possibility of developing predicative models to determine a patients’susceptibility to systemic inflammation.

Hello everyone. My name is Mahadin Ahmed and I’m here to share with you all my and my mentor Dr. Caroline Hoemann and Julia Leonard’s project involving induction of TIMP1 in human monocytes.
So, let’s begin discussing what started the initial interest into this topic. It starts with understanding polytrauma, which involves individuals sustaining injuries to multiple body parts and organs. These kinds of traumatic injuries have been known to lead to severe systemic inflammation affecting the entire body and slowing recovery in selected patients. These types of injuries are typically associated with severe blood loss requiring administration of a blood transfusion from donors. These transfusion products are known to have both beneficial and adverse effects on systemic inflammation. The reason behind why certain patients experience this severe form of inflammation is still unclear and is an area intense interest termed ‘trauma immunology’.

What me and my mentor Dr. Caroline Hoemann believe to be the cause is related to a key enzyme called MMP-9 which is one of several factors involved in the inflammatory response and its inhibitory molecule TIMP1. MMP-9 is involved in the degradation of the extracellular matrix and activates cytokines and chemokines to regulate tissue remodeling. The extracellular matrix or, ECM for short, helps binds cells and tissues, which acts almost like as a foundation would for a building. The MMP9 breaks down this damaged foundation (extracellular) to make way for new tissue. As you can imagine one would not want this MMP9 to be constantly active otherwise there would only be constant degradation of the tissue and no opportunity for the new ECM to form. The way MMP9 is inhibited or shut off is through a regulatory molecule TIMP1. TIMP1 binds to the MMP9 enzyme to deactivate MMP9 and thus prevents constant degradation of the ECM.

This study aims to understand the mechanisms of TIMP1 induction in human monocytes. This will be done by selecting TIMP1 primers for RT-PCR amplification. In a previous experiment, cDNA was prepared from human monocyte cells (U937) treated or not treated with endotoxin. A specific immune response gene TIMP1 associated with inflammation will be amplified from the cDNA preparations using PCR, which will be compared with a housekeeping gene GADPH. PCR involves the amplification of the cDNA by heating our sample cDNA to separate the two strands of DNA from each other. After which the temperature is lowered to allow these primers to anneal to each strand. These primers act as guides so that a thermophilic polymerase can add nucleotides to the cDNA strands to make two copies of our cDNA. This goes on for multiple cycles turning 1 cDNA to 2 to 4 to 8 and so on and so forth. We carry out this amplification in a piece of equipment called a thermocycler.

Once amplification is done gel electrophoresis will be used to separate these RT-PCR products to determine whether endotoxin enhances TIMP1 expression without affecting GADPH expression levels. The samples will be loaded into the wells of the agarose gel and submerged in a buffer solution. A current will be run through the solution, allowing the cDNA to move towards the positive terminal due to electrostatic attraction. The separation occurs due to a difference in size as larger cDNA moves slower through the gel than smaller cDNA. The sizes of these samples can be compared with that of a ladder, where each rung represents different known sizes. The image on the right uses a 1000 base pair ladder, so the first rung is 100 then 200 then 300, and so on until the last rung of 1000 base pairs is reached. What we expect to find is that the endotoxin will induce higher TIMP1 expression, which may provide some insight into mechanisms involved in TIMP1 induction.

Me and my mentor Dr. Caroline Hoemann’s research aims to broaden the understanding on the mechanisms behind systemic inflammation brought on by polytrauma, which can act as a possible mechanism people can manipulate to prevent patients from experiencing severe systemic inflammation or even be the basis for future therapeutics.

I want to give a special thank you to both Dr. Caroline Hoemann and Julia Leonard for making this project possible. Thank you all for your time and attention.