MICA: Suppressing inflammation to enhance antigen-specific immunity in older humans using p38MAPK inhibitors and vitaminD3

Older humans are more susceptible to infections, even those to which they were immune to in their youth. Vaccination against many different infectious agents is also sub-optimal in these individuals. To investigate the mechanisms involved in the decline in immunity during ageing we inject safe derivatives of microorganisms into the skin of old and young volunteers. After injection of the agent, we can extract the white cells (leucocytes) that accumulate at the site of the immune response to test for their function. One of the responses we have been investigating is that to the varicella zoster virus (VZV) that induces chickenpox mainly in young individuals. Infected subjects control this persistent virus until an older age where reduced immunity leads to viral re-activation to cause shingles. Older humans are also susceptible to re-activation of TB and we can also test their response to tuberculosis (TB) proteins. Elderly subjects have decreased capacity to respond to VZV in the skin that may be an underlying reason for their increased susceptibility to shingles. These individuals also have decreased cutaneous responses to TB related proteins and the immune defect in the skin may reflect the general defect in immunity in these individuals. We have found that this defective response is associated with increased baseline inflammation in this tissue. Although inflammatory responses are required to clear infections, excessive inflammation has previously been shown to interfere with the generation of specific immunity. We will therefore investigate ways of enhancing the immune response of older humans by blocking basal inflammation.

To extend our initial observations of high background inflammation in the skin, we will investigate which cells in this tissue are responsible for the production of inflammatory mediators by using a second human experimental system that tests the response to a skin irritant known as cantharadin. Next we will block inflammation in the skin to determine whether this can lead to improved responses upon challenge with antigens. This will be achieved by pre-treating old subjects with a drug that has already been developed by GSK (Losmapimod, p38MAPkinase inhibitor) and another that is available off the shelf (vitamin D3). The GSK p38 inhibitors are currently being tested to prevent unwanted inflammation. A surprising observation we made was that p38 inhibition could also rejuvenate human T lymphocytes and enhance their ability to proliferate in vitro. Therefore the inhibition of p38 may block unwanted inflammatory response as well as enhance T lymphocyte reactivity. Vitamin D3 has also been shown to have anti-inflammatory effects and works in part by inhibiting p38MAPkinase activation. The use of 2 separate interventions in this project increases the likelihood of success in this study. While Losmapimod may be more specific in its anti-inflammatory action, the use of vitamin D3 is more cost effective as it is a cheap compound that is readily available.

The desirable outcome of this study is that either one or other of these compounds, that older volunteers will be treated with, will boost their response to microbial antigen challenge in the skin. This will provide proof of concept data that will lead to the exciting possibility of enhancing immunity by inhibiting the increased baseline inflammatory responses that are found during ageing. This may be a strategy to enhance immune responses in general and specifically to enhance vaccination efficacy to various diseases that is less effective in older subjects.

We will use two human experimental models to clarify mechanisms behind decreased immune responses during ageing. This involves injecting clinically approved antigens (varicella zoster skin test antigens, tuberculin PPD) into the upper forearm in young (<25 yrs) and elderly individuals (>70 years), followed by skin biopsy or suction blister induction to isolate cells from the site of the response. We will also inject saline or apply the irritant cantharadin to the skin to study the propensity for non-specific inflammation during ageing. Preliminary data suggest that non-specific inflammation is increased in older individuals. We will perform global gene expression analyses of these biopsies taken from old subjects who have been pre-treated with either a p38 inhibitor or vitamin D3 to determine whether the inhibition of baseline inflammation can enhance the response to antigen challenge. The global gene expression profiles will be interrogated by signalling pathway modelling and by analysis with a cell specific gene expression atlas developed by the Co-PIs at the Roslin Institute. This will identify the resident leukocytes are responsible for the high baseline inflammation and these cells will be further investigated in vitro. We will also investigate the phenotype and function of antigen specific T cells that will be isolated from the site of antigen challenge in the skin before and after p38 inhibitor or vitamin D3 treatment.
Zostavax and BCG provide live attenuated vaccination strategies against VZV and TB that are less effective in the elsderly. It is not known whether this may reflect altered viral replication kinetics in ageing skin resulting in reduced antigenicity. Each vaccine will be administered by intradermal injection into young and elderly individuals. Biopsies of the injection sites will be subjected to RNAseq analysis (Illumina HiSeq platform) to obtain quantitative host and pathogen transcriptomic data.

The UK is shifting in demography towards a more aged society. Ill health in old individuals is often associated with the increase in the incidence of infections that is related to impaired immunity. These problems reduce our working life and generate a significant cost-burden on the NHS and on social care provision. This study will provide new information on the nature and the possibility for manipulation of defective cutaneous immunity in the elderly.

Pharmaceutical Companies: 1. The identification of potential genes that are defective will indicate putative targets that can be manipulated to boost immunity during ageing. Each altered gene represents a potential new target for therapy and would be of great interest to scientists in the pharmaceutical industry. This information provide direct targets that may be manipulated to boost immunity during ageing e.g. during vaccination.
2.One of our hypotheses is that blocking the background inflammation in older humans with either p38 inhibitors or vitamin D3 can enhance the generation of an antigen-specific T cell response in vivo. GSK will provide the p38 inhibitor (Losmapimod) and the background information related to its use in humans in vivo. This study has the potential to provide the data that underpins a subsequent clinical trial on the effects of p38 blockade on boosting the immune system of healthy old humans, especially in the context of vaccination.
3. A better understanding of effects of inflammation on the development cutaneous immunity will also provide new data on which vaccine strategies and the use of adjuvants in older humans can be based. The data we will generate in humans will have more relevance to understanding immunity, dysfunction and the potential for enhancement than similar studies performed in animal models.
4. Most responses to immune stimulation in older humans are monitored by observing immune changes in the blood. We will be able to investigate immune changes in the skin as well as blood compartments.

Health Care Policy: If this study identifies ways to improve the immune response in elderly patients, this will lead to significant changes in our ability to immunise the UK population, maintaining their health during ageing.

Other Clinical Areas: Clarification of the defective immunity to VZV and tuberculin PPD in the skin of older humans will extend to the defective immunity that lead to skin cancer. Thus the identification of potential targets that can be manipulated to boost immunity to VZV and mTB may also be beneficial to enhance immunity to malignancy. Many pharmaceutical companies including GSK and Pfizer have already produced clinical grade p38 inhibitors that have been used in phase II and III trials for blocking inflammatory disease. However our preliminary data suggest that these inhibitors may enhance immunity by reversing certain aspects of T cell senescence. If our studies show that p38 blockade or vitamin D3 supplementation can boost immune responses in older humans in the skin, the possibility of using these reagents in vivo e.g. during vaccination would be eminently feasible and is the next step towards translation.
The Ageing Public: The data from our study will be disseminated directly to the general public during recruitment drives as well as during seminars in the latter parts of this grant to inform our volunteers on outcomes of the research. We aim to establish a close dialogue with the older community who are the key stakeholders in this work. This will ensure our volunteers and wider communities are aware of the effects of aging on immunity. Our results will provide proof in principle that it is possible to enhance the responsiveness of older humans to vaccination.

Hub for training: If this application is successful, UCL will allocate 4 PhD students to this project to be trained in the use of molecular, cellular and systems biology techniques to investigate the ageing human immune system in vivo.