Can combined cellular and 11beta-hydroxysteroid dehydrogenase-1 gene therapy attenuate inflammation in acute respiratory distress syndrome?

When some people have a severe infection, their body's defence systems can over-react and cause damage to their own organs through a process called inflammation. When the lungs are damaged in this way, it is called acute respiratory distress syndrome (ARDS). This can occur due to a variety of insults including smoke inhalation, physical injury, as well as bacterial and viral infection. ARDS causes the lungs to fill up with water, making it very difficult to breathe. These patients therefore need to be looked after in the intensive care unit, where a machine can help support their breathing. The death rate associated with this is 35-45%. Even those who survive ARDS have considerable recuperation periods and reduced quality of life.

The body produces natural steroids which can help reduce inflammation. These steroids are activated by a protein called HSD-1 (11beta-hydroxysteroid dehydrogenase type 1). Our previous work showed that mice which have been genetically modified to not make HSD-1 at all, develop more severe and persistent lung damage following infection, compared to normal mice. We also found that patients with ARDS have lower than normal levels of HSD-1 in their lungs. This suggests that less steroid is being activated in the lungs of these patients, and that the lung inflammation and damage is continuing unchecked. If we were able to give a treatment which increased the amount of HSD-1 specifically in the lungs of patients with ARDS, this could be effective in reducing the inflammation and damage caused by the disease.

Other work has shown that a type of stem cell found in the bone marrow of adults has the potential to be used as a treatment for ARDS. These cells are called "mesenchymal" stem cells (MSCs). It has been shown that in mice with lung
damage, injection of these stem cells can help reduce lung inflammation, fight infection, and repair damaged lung. These stem cells are known to travel to damaged organs specifically, after being injected. Since these stem cells have been taken safely from the bone marrow of adult volunteers, we therefore avoid the ethical problems associated with using foetal stem cells. The anti-inflammatory, protective quality of MSCs, mean they are also a potential treatment for ARDS.

So far we have discussed two separate potential treatments for ARDS: 1) injection of MSCs and 2) increasing HSD-1 levels in the lungs. However, we think that by combining these two ideas, a more effective treatment could be developed. We think that MSCs which are genetically modified to produce high levels of HSD-1 protein, could potentially be used as a treatment for ARDS in the future. In theory, the genetically modified MSCs would travel to the damaged lung in ARDS patients; once there, the anti-inflammatory effects of the MSCs and high levels of HSD-1 would reduce damage caused to the lungs. We have already successfully genetically modified the MSCs so that they produce high levels of HSD-1 protein, and shown that these cells can activate natural steroids in the lab. The aims of our research are to investigate whether genetically modified MSCs will have the ability to reduce lung injury in ARDS.

We will then inject the genetically modified MSCs into mice with lung injury, to mimic what happens in humans when they get ARDS. We will use genetically modified mice which do not make their own HSD-1 at all. We will then monitor the mice to see if those who receive the treatment have less lung damage than the untreated mice. We will also test the genetically modified stem cells on macrophages (one of the body's defender cells) taken from the lung washings of human ARDS patients, and see if the ability of the macrophages to reduce inflammation by clearing away dead cells and bacteria is improved. We will also use the genetically modified stem cells on human lungs in the lab which have been rejected for transplantation, to see if they can reduce inflammation in human lung tissue.

1) Is the phagocytic function (including efferocytosis) of Alveolar Macrophages (AMs) impaired in ARDS patients compared to normal volunteers?
ARDS patients will be recruited & AMs isolated from broncho-alveolar lavage fluid. Flow cytometry will be used to determine uptake of fluorescently labelled apoptotic neutrophils, E. coli & S. aureus bioparticles by AMs.

2) Do HSD-1 overexpressing tMSCs enhance the function of human AMs from ARDS patients more effectively than transgene-inactive tMSCs?
AMs from ARDS patients will be cultured with transgene-activated & transgene-inactivated tMSCs, in the presence of cortisone. After 24hrs, basal cytokine release will be assessed using ELISA. Phagocytosis and efferocytosis assays will
also be performed as described above.

3) Will HSD-1 overexpressing tMSCs attenuate inflammation in murine models of lung injury more effectively than transgene-inactive tMSCs?
I will use 2 different models of lung injury in HSD-1 knockout mice: intra-tracheal LPS to model direct lung injury, and caecal ligation & puncture to model indirect lung injury. We will utilise standard markers of lung injury to assess the impact of transgene-activated vs transgene-inactivated tMSCs.

4) Determine if HSD-1 over-expressing tMSCs can modify alveolar steroid metabolism in a human ex-vivo lung perfused (EVLP) model injured with LPS.
I will work with Prof Matthay's group at UCSF who have experience using the EVLP model. All lung lobes will receive LPS & cortisone. For each lung, one lobe will receive transgene-activated tMSCs intra-bronchially and the other saline. After 4 hours, BAL would be performed, & mass spectrometry performed on the BALF to determine cortisol and cortisone concentrations.

Opportunities: This translational work will help determine if local up-regulation of HSD-1 by tMSCs will attenuate inflammation in ARDS by restoring AM function.
Exploitation: Data generated will be published in high citation peer reviewed journals

This research will benefit investigators examining the pathophysiology of ARDS, pneumonia and sepsis. Together these diseases represent a very significant healthcare burden and there has been limited novel pharmacotherapy for these
conditions for many years. This research will also be of interest to researchers in other fields who are studying the role of local HSD-1 activity and MSCs as regulators of inflammation and fibrosis.

This novel translational work will help determine if transgenic mesenchymal stem cells over-expressing HSD-1 can be administered systemically, locally upregulate cortisol production in inflamed lung tissues, and promote resolution of
inflammation. If successful, the implications for the potential use of such tMSCs in human ARDS and in other inflammatory conditions are profound. The appeal of this concept lies in the fact that the tMSCs could be administered systemically, however the effect would be targeted to specific sites of organ inflammation/damage, due to the ability of MSCs to migrate to these areas. In this way, the high HSD-1 expression of tMSCs would not be systemic, but would be localised exactly where required - in tissues being damaged by inflammation. The potential scope for this treatment application extends beyond that of ARDS, and could be used to treat a number of inflammatory conditions and also severe sepsis. If successful, this work may influence national health research policy makers to invest further funds towards research into combined cellular and gene therapy for inflammatory conditions.

ARDS occurs in response to a variety of insults, such as trauma and severe sepsis. It affects all age groups; has a high mortality of up to 30-50% and causes a long-term reduction in quality of life for survivors. ARDS has significant resource implications, prolonging intensive care unit (ICU) and hospital stay, and requiring rehabilitation in the community. The cost per ICU bed-day in the UK exceeds £1800 and delivery of critical care to patients with ARDS accounts for a significant proportion of ICU capacity. Therefore, it is of considerable public health importance. There are no current readily available therapeutic interventions proven to prevent or treat ARDS. Clearly this represents a major unmet health need. Thus this research will not only potentially impact on the health and quality of life of patients but also the NHS and health care providing organisations. Cell based therapy to promote local HSD-1 activity as a novel anti-inflammatory would likely take 10 years to deliver.

This research will also contribute to the reputation of the University of Birmingham as a centre for cutting edge translational research. The research will take place in the Centre for Translational Inflammation Research which was opened in June 2011. This is a purpose built £8 million facility located within the new £450 million Queen Elizabeth Hospital Birmingham. The centre has all the major core facilities (FACS, cell culture facilities) necessary for this project and has laboratory and office accommodation for 100 clinical and biomedical researchers. It acts as a hub for researchers in critical care and respiratory medicine who have an extensive collaborative history. The success of the centre has attracted academic industry contacts with both big pharma (GSK, AZ) as well as SMEs (e.g. Creabilis, Biopta). Further MRC funding should therefore enhance the reputation of the unit and draw more collaborators.

During the tenure of this grant, I will learn new techniques such as mouse lung injury models and lentiviral transfection. Through my involvement in project design, analysis and writing papers for publication as well as presentation of my work on national and international conferences will enable my successful entry into the scientific community as an independent researcher. The development of these skills will be integral for my future career as an independent research leader.