MICA: Towards targeted treatment for complex regional pain syndrome through determination of the underlying molecular mechanisms

Complex regional pain syndrome (CRPS) is a chronic pain condition that can occur after an injury, such as a fracture and results in localised pain and swelling. CRPS spontaneously resolves within a year in 20-30% of cases, but after this time it becomes chronic and rarely improves. Treatment options for CRPS patients, as with many other forms of chronic pain, are very limited, untargeted, and often have significant side effects. It is, therefore, important to understand why CRPS develops and the underlying mechanisms involved in order to try and develop more effective drugs targeting pain and inflammation and improve lives of those living with CRPS.

We can often gain insight into why a disease develops by looking at changes found in genes (mutations), which in turn helps design new drugs, such as pain killers (analgesics). For example, changes in a gene called nerve growth factor, were found in a family unable to feel pain (inherited as a very rare disease), and this led to the discovery of a new class of analgesics that is particularly helpful against arthritic pain. We wanted to see if patients with CRPS had mutations in their genes, compared to non-sufferers, which could alter their susceptibility to the condition. We found that CRPS patients were more likely to have mutations in four genes compared to people without CRPS. Each of these mutations was hypothesised to cause alterations of protein function. We, and others, have shown these four genes are expressed in monocytes and/or macrophages (cells that engulf foreign particles and cause inflammation). Importantly, all four genes are linked to the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammatory pathway that senses foreign particles and removes them by causing release of inflammatory molecules and cell death. These data suggest that the NLRP3 inflammasome pathway is likely to be important in the pathology of CRPS.

In this proposal we will investigate how the CRPS-mutated genes affect the activity of the NLRP3 inflammasome. In order to do this, we will introduce the gene mutations into a human monocyte cell line (THP-1 cells). Next, we will determine if these mutations alter the activity of the NLRP3 inflammasome by measuring the production of inflammatory molecules and cell death in THP-1 cells. We will validate the THP-1 model by measuring inflammasome activity in cells from healthy individuals with the CRPS-associated mutations. Our next step will be to determine how these changes in NLRP3 inflammasome activity influence pain neuron activation as a method to model the excessive pain seen in CRPS. Lastly, we will test how NLRP3 modulators affect pain and inflammation in order to translate our findings towards the development of new drugs for CRPS.

We anticipate our research will lead to the identification of a new class of non-addictive pain-killers that will reduce inflammation and pain to stop the chronic stage of CRPS from developing. This would have very significant effects on pain management, inflammation and the depression, morbidity and mortality that are a consequence of prolonged severe pain.

In order to understand the genetic basis for complex regional pain syndrome (CRPS), we recruited two patient cohorts and discovered that four SNPs, each in a different gene, were enriched in CRPS cohorts compared to healthy controls. All four genes are linked to the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome pathway. We therefore hypothesise that the SNPs identified cause aberrant activation of NLRP3 to promote the development of the chronic inflammatory pain seen in CRPS. In order to study CRPS, we will initially generate a cellular model by the introduction of each SNP allele into a monocyte cell line THP-1 using CRISPR/Cas9 gene editing (already achieved for one SNP).

Macrophages are highly plastic cells and exist in at least three different states based on environmental cues: naïve M0, pro-inflammatory M1 and anti-inflammatory M2. The transition between M1 and M2 cells is important in wound resolution and healing. We will identify whether the CRPS mutations alter inflammasome activity in macrophages in different activity states by differentiating THP-1 cells harbouring the CRPS SNPs into M0, M1, M2 and M1>M2 states. Inflammasome activity will be determined by measuring pro-inflammatory cytokine secretion (interleukin IL-1B, IL-18) and gasdermin D-driven cell death following activation of NLRP3. These results will be validated in primary macrophages taken from healthy controls with the CRPS-associated SNPs. We will investigate whether the CRPS mutations alter the cross talk between pain and inflammation by measuring the effect of inflammatory mediators released by macrophages on nociceptor sensitisation. We will develop a co-culture system with macrophages and nociceptors differentiated from embryonic stem cells. Nociceptor activity will be measured using electrophysiology. Lastly, we will investigate the effect of inflammasome modulators, provided by NodThera, on the pain and inflammatory responses in the co-culture model.