How does blocking inflammation enhance human cutaneous immunity during ageing in vivo?

Although animal models have been used to investigate mechanisms for immune decline during ageing, differences in lifespan and species specific differences prelude direct comparison between animals and humans. Furthermore, most human studies are conducted on white cells isolated from the blood that may not reflect on events that take place during an immune response in the tissues, akin to saying that people travelling on a motorway are representative of the people living in big cities served by the motorway. We developed a new way of investigating white cells in human tissues during an immune response. With these new methods we are trying to understand why older humans respond badly to pathogens in the skin that they should be immune to, like the chickenpox virus. This may also explain why older individuals get more skin cancer and also more skin infections.

In a previous study we made a rather surprising finding that the reduced response to the chickenpox virus in old people was not due to a decrease in the number of white cells that recognize this virus as compared to young subjects in either the blood or the skin. Instead this decreased response was due to changes in the skin environment itself, where we identified more background inflammation. What we did in this previous study was to reduce the inflammation in old volunteers, using an anti-inflammatory drug from Glaxo-Smith-Kline (GSK) to see if this could enhance their response in the skin to the chickenpox virus. In a nutshell, we were able to do so and we have published this work. However key questions remained unanswered including identifying where the inflammation was coming from in the first place i.e. which cells in the skin were making it? Which cells secreted proteins to recruit inflammatory white blood cells, called monocytes, to the skin? Finally how do these monocytes contribute to the inhibition of the immune response during ageing? Do they work alone or together with other cell types?

We will take individual cells out of the skin and look at their gene expression to see which cell type makes the inflammatory proteins. We will do this part of the study with Professor Menna Clatworthy in Cambridge University who has already performed extremely important studies on human tissue using these techniques. We will also determine which cells secrete the molecules that attract inflammatory monocytes from the blood into the skin of older subjects. In collaboration with Professor Muzlifah Haniffa in Newcastle we will be able to begin to generate a genomic map of different skin cells from old people for comparison with young individuals that she has already analysed. Furthermore in collaboration with Prof. Chris Buckley who is an expert on inflammation in joints in patient with Rheumatoid Arthritis, we will investigate if cells called fibroblasts in the skin are involved, like they are in the joints. This possibility is suggested by our preliminary results and we envisage possible interactions between different cell types to induce the inflammation we observe in the skin of older humans. This will be addressed with the single cell RNAseq analyses that we will perform with Prof. Clatworthy.

The next question is how the inflammation blocks the function of the resident white cells in the skin. We will test if it is due to a direct inhibitory effect of secreted factors from the culprit cell, if the inflammation makes other cells turn on the brakes to inhibit immunity or if the inflammation calls in the "military police" known as regulatory cells that calms everything down in the skin. Finally we will used bio-banked samples that we generated in a previous study to probe how inhibiting inflammation in the old works -does it prevent recruitment of inflammatory monocytes? Prevent the generation of inhibitory cells or stop the interaction between different cell types, breaking the synergy between them and thus puts out the fire of inflammation.

Leukocyte populations will be isolated from 5mm punch biopsies taken from normal the skin of young (<35 years) and older humans (>65 years) and also from the site of injury after the injection of 20ul of air into the skin. These samples will be used for immunohistology coupled to confocal microscopy or will be digested and sorted by multiparametric flow cytometry. In addition we will perform single cell RNAseq analyses on cells isolated from skin biopsies with Prof Clatworthy in Cambridge. This data will be analysed further in collaboration with Muzlifah Haniffa's skin atlas to determine how different skin cell populations change with age.

We will also use an experimental system where senescent and non-senescent dermal fibroblasts will be co cultured with autologous CD4+ T cells to determine if the inflammatory cytokines secreted can induce FoxP3 expression and regulatory function. We will test if the interaction between senescent fibroblasts and different autologous monocyte subsets from the blood can amplify the secretion of inflammatory mediators by either cell type.

Another inhibitory mechanism identified in our preliminary RNAseq studies of skin biopsies from young and old subjects is PTGS2 which encodes the protein COX2, which is upregulated under inflammatory conditions and results in the production of the lipid mediator Prostaglandin E2 (PGE2). Preliminary analysis of skin sections has identified that COX2 is elevated in old saline-injected skin compared to normal skin taken from the same subjects. We will Investigate prostaglandin E2 (PGE2; COX2 pathway) production by infiltrating monocytes and whether the PGE2 receptors (EP4 and EP2) is increased on T resident memory (Trm) cells (identified by CD69 co-staining).

Biobanked samples of normal and air/saline injected skin from old individuals before and after p38 inhibition in vivo will be interrogated by immunohistology and confocal microscopy for the cellular interactions described above.

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. We previously showed that excessive inflammation during ageing, "inflammageing" inhibits human antigen-specific cutaneous immunity. The current programme is designed to clarify where the inflammation in the skin comes from, how monocytes are recruited in and how these monocytes specifically inhibit antigen-specific immunity.

Scientific excellence:
A paradigm shifting experimental medicine paper was published recently by the Akbar group demonstrating that the short term blockade of inflammation in old humans in vivo can enhance immunity in the skin (Vukmanovic-Stejic M et al 2017). This has considerable implications for immune enhancement during ageing. The work on immune ageing that has been performed by the Akbar Group has been highlighted on the BBSRC Business Magazine, The MRC website and in the Daily Express. The UCL Media Department produced a video podcast of this work that has been posted on Youtube (http://youtu.be/oQ-unC7D9i4) that has featured on the BBSRC, MRC and UCL websites. This media coverage highlights the scientific impact of our studies.

Pharmaceutical Companies:
We showed that blocking of p38 MAP kinase signalling in humans with a GSK drug called Losmapimod, that has been through phase III trials, can enhance immunity in the skin of older subjects (MRC MICA Experimental Medicine Grant to Akbar). It is of note that p38 inhibitors that have been through phase II and phase III trials have been developed by many different pharmaceutical companies and our investigations may build a case for drug repurposing. The current study is designed to understand the mechanism behind how inhibitory process that are induced by inflammation inhibit immunity. This will identify potential targets for future therapy to boost immunity during ageing and will be of interest to many pharmaceutical companies.

Health Care Policy:
If this study identifies ways to improve the immune response this may ultimately lead to significant changes in the way we immunize the UK population potentially utilising a short course of anti-inflammatory drugs to enhance vaccine response and subsequently maintain their health during ageing.

Public Engagement:
We already have a close relationship with the older community through previous public engagement initiatives. We have hosted 5 events to date, aimed specifically at older adults. There are ~50-100 attendees at each meeting. These events are organized to contact and engage with older adults from within the local community to inform past, present and future volunteers of the research that is taking place on ageing immunity in UCL. The ambition is to make our research understandable and accessible to all. Members of the Akbar Group also give talks to publicize our work to lay audiences at the Cheltenham and Edinburgh Science Festivals and at University of the Third Age meetings (2013 and 2015, 2016, 2017, 2018). I was interviewed for a programme on BBC Radio 4 hosted by Ms. Vivienne Parry that discussed ageing and immunity (http://www.bbc.co.uk/programmes/b012qtw0) and by Dr. Mark Porter where I discussed blocking inflammation to enhance human immunity (https://www.bbc.co.uk/programmes/b09rybgf). We also made a recruitment video that highlights our work on immune ageing (https://youtu.be/P-7nPwaW6tU).

Staff training: Medical science is progressing toward generating and handling large data sets. We are collaborating with 3 highly successful labs who have used this technology. The staff to be employed on this grant will have the opportunity to observe and learn this technology from the experts while reciprocally, members of our collaborator labs will be able to observe cutting edge technology in the Akbar Lab. for the study of human immunity in vivo.