Are long intergenic non-coding RNAs central regulators of inflammation and the innate immune response?

Upon completion of the human genome project, the biggest surprise was that humans have far fewer genes than previously expected (~ 21,000). Additionally, less than 2% of the available DNA directly codes for these genes. To address this paradox, recent studies have suggested that much of the remaining 98% of DNA is turned into 'non-coding RNA' and that these are important regulators of gene expression. For convenience, we commonly divide this non-coding RNA into small non-coding RNAs and long non-coding RNAs. At the present time, we have little idea about the role of the majority of this non-coding RNA although recent studies have indicated that specific non-coding RNAs may regulate the immune response.

In order to fight infection by bacteria, fungi and viruses, the body has a complex defense system called the immune response. This involves the recognition of these micro-organisms and release of a range of chemicals that recruit and activate immune cells (inflammation) that are involved in removing these un-wanted invaders. Under normal conditions, this inflammatory response is then switched off. However, under certain circumstances, prolonged activation can be life-threatening or lead to the development of common conditions such as asthma, diabetes, cancer and cardiovascular disease. For this reason it is important to understand the mechanisms that regulate both the activation and inhibition of this immune response.

Previous studies have shown that this immune response is altered by a family of small non-coding RNAs, called microRNAs. However, whether long non-coding RNAs might also be important is unknown. In preliminary studies, we have shown that the levels of a number of long non-coding is increased in cells following stimulation with a product made from bacteria. We also found that one of these long non-coding RNAs regulates the release of the chemicals that are used to kill the invading micro-organisms. In this project, we will extend these studies to examine what other long non-coding RNAs do and how they might exert their actions. Overall, we believe that these investigations will show that long non-coding RNAs are important regulators of inflammation and the immune response to invading pathogens.

The innate immune response, which is commonly mediated by immune cells of myeloid origin (i.e. monocytes and macrophages) and provides the initial defence against infection. This involves the recognition of invading pathogens, induction of an inflammatory response and the subsequent elimination of the relevant micro-organism. To prevent unwanted inflammation, activation and resolution of the innate immune response is tightly controlled through multiple mechanisms.

We have preliminary evidence showing that changes in the expression of long intergenic non-coding RNAs (lincRNA) might provide a novel mechanism that regulates the innate immune response. Thus, we have shown that bacterial cell wall lipopolysaccharide (LPS) induced activation of human monocytes, monocyte-derived macrophages (MDM) and the monocytic THP-1 cell line changed expression of 17 lincRNAs (12 up-regulated and 5 down-regulated). Increased expression of two of these lincRNAs (TCONS_00008694 and TCONS_00015565) was mediated by the pro-inflammatory transcription factor, nuclear factor-kB (NF-kB) whilst siRNA-mediated knockdown of TCONS_00015565 showed that this regulated the release of the inflammatory mediator, interleukin-8. Given these observations we hypothesise that long intergenic non-coding RNAs are novel regulators of inflammation and the innate immune response.

To test this hypothesis, we propose to use knockdown and over-expression to examine the functional role of the remaining 16 lincRNAs. Having identified functional lincRNAs, we shall proceed to investigate their mechanism(s) of action. As a result of the requirement for large cell numbers, these investigations will be performed in LPS-stimulated monocytic THP-1 cells although important observations will be confirmed in primary human monocytes. Overall, we believe that these investigations will provide groundbreaking information on the role of these novel lincRNAs in inflammation and the innate immune response.

Academic Impact

The work outlined in this proposal will examine the role of a novel family of non-coding RNAs in inflammation and the innate immune response. From an academic point of view, the results of these studies will benefit scientists working in the area of immunology, RNA biology and those engaged clinical work related to inflammation.

Economic and Societal Impact

Inappropriate activation of the inflammatory response is associated with multiple diseases including asthma, rhinitis, eczema, rheumatoid arthritis, coeliac disease and systemic lupus. For this reason, there is considerable interest amongst biotechnology and pharmaceutical companies in the identification of novel anti-inflammatory drug targets. A number of companies including Santaris, Regulus, Alynlam and ISIS pharmaceuticals are currently undertaking clinical trials (Phase 1-3) with oligonucleotide based therapeutics (antisense and siRNA) that target mRNAs and miRNAs implicated in a range of diseases including cancer, asthma, viral infections and high cholesterol. Importantly, an identical approach could be used to target the activity and/or expression of lincRNAs and thereby permit develop of new drugs for the treatment of inflammation. Indeed, a Boston based start-up company called RaNA therapeutics has recently raised $21M to develop a locked nucleic based antisense approach to modulating the activity of a lincRNAs. Clearly, the development of novel anti-inflammatory drugs will be of great benefit to the patients suffering from these diseases.