Treatment for rheumatoid arthritis (RA) has come a long way in recent years, and a battery of antirheumatic medications can in many cases successfully stymy the inflammatory cells that cause swelling and pain when they infiltrate tissues around the joints. Yet for some reason, about 20% of patients with painful, visibly swollen joints consistently get no relief from multiple rounds of even the strongest of these anti-inflammatory drugs.

Surgical interventions intended to remove inflamed tissue have revealed why: “In some cases, their joints aren’t actually inflamed,” noted co-senior author Dana Orange, MD, an associate professor of clinical investigation in Rockefeller’s Laboratory of Molecular Neuro-oncology. “With these patients, if you press on the joint, it feels mushy and thick to the touch, but it’s not caused by the infiltrating immune cells. They have excessive tissue growth, but without inflammation. So why are they experiencing pain?”

Newly reported research by Orange and colleagues now suggests an explanation. Using a machine-learning approach they developed called graph-based gene expression module identification (GbGMI), the team identified a suite of 815 genes that activate abnormal growth of sensory neurons in tissues that cushion the affected joints. “These 815 genes are rewiring the sensory nerves, which explains why anti-inflammatory drugs don’t work to alleviate pain for these patients,” said Orange. The findings, they suggest, may lead to new treatments for these outliers.

Reporting on their results in Science Translational MedicineSynovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis,” co-senior author Orange, et al., concluded, “Together, this work identifies a group of genes associated with patient report of pain in low inflammatory synovium in both early untreated and established RA … These findings have the potential to be leveraged to develop next-generation therapeutic approaches to alleviating pain in RA, particularly in the low inflammatory pathotype that may be less responsive to current therapeutics that target adaptive immune inflammation.”

Rheumatoid arthritis is a tricky chronic disease characterized by inflammation in the synovium, the tissue that lines the joint cavity. Its symptoms—stiffness, tenderness, swelling, limited motion, and pain—slowly emerge in the hands, wrists, feet, and other joints. It occurs symmetrically (not just in one hand but in both, for instance) and sporadically, with irregular flare-ups. Extreme fatigue and depression are also common.

Most cases of RA are caused by products of immune cells such as cytokines, bradykinins, or prostanoids invading the synovium—a soft tissue lining the joints—where they bind to damage-sensing pain receptors. Drugs that target immune mediators have made RA a far more tolerable condition for most, the team wrote. “Remarkable progress has been made in developing an array of conventional synthetic, targeted synthetic, and biologic disease-modifying antirheumatic drugs (csDMARDs, tsDMARDs, and bDMARDs, respectively), which target relevant immune mediators.”  However, RA patients suffering from the disconnection between inflammation and aches haven’t benefitted. “… up to 20% of patients with RA are ‘difficult to treat;’ that is, they do not improve despite treatment with at least two bDMARDs or tsDMARDs, with different mechanisms of action, after failing a csDMARD,” the investigators pointed out.

It has been assumed that synovial inflammation is the cause of RA joint pain. However, recent studies have shown that pain can be dissociated from inflammation in RA. “Patients with RA and limited synovial inflammation, also known as ‘fibroid,’ ‘low inflammatory,’ ‘pauci-immune,’ or ‘fibroblast cell type abundance phenotype’ synovium, have as much pain as those with extreme inflammation,” the scientists noted. Doctors often prescribe these patients drug after anti-inflammatory drug in an ultimately fruitless attempt to give relief. However, the scientist stated, “Patients with low synovial inflammation tend to receive less benefit from treatment with anti-inflammatory drugs…”.

Orange added, “We are subjecting some patients to a lot of medications that cause immunosuppression and yet have little chance of making their symptoms better.” For their newly reported research, Orange and colleagues sought answers in the genes expressed in the joint tissue samples of these patients. The authors commented, “… we hypothesized that a focused analysis of low inflammatory synovium might identify factors beyond inflammation that relate to joint pain.”

The team looked at tissue samples and self-reported pain reports from 39 patients with RA who had pain but little inflammation. They also developed the graph-based gene expression module identification (GbGMI) machine-learning tool, which tests every possible combination of genes in a dataset to determine the optimal set of genes that together associate with a targeted clinical feature—in this case, pain.

Using RNA sequencing, the researchers found that of the 15,000 genes expressed in the tissue samples, about 2,200 had increased expression in the 39 patients. “To uncover genes associated with pain but not inflammation, we focused our analysis on 2,227 genes that exhibited increased expression in low inflammatory synovium relative to high inflammatory synovium and on pain scores that document the extent of pain in the joint that was sampled [Hip Osteoarthritis Outcome Score/Knee Osteoarthritis Outcome Score (HOOS/KOOS)],” they stated.

In this image, abnormal synovial tissue is shot through with excessive tissue growth, including blood vessels (in magenta). Synovium should be thin and smooth.
In this image, abnormal synovial tissue is shot through with excessive tissue growth, including blood vessels (in magenta). Synovium should be thin and smooth. [Bai et al]

Using GbGMI, the team identified 815 genes that together were associated with patient reports of pain. “We then tested which number of top-ranked genes collectively best correlated with pain among patients with RA with low synovial inflammation and identified an 815-gene module, which we refer to as the GbGMI-identified pain-associated genes.”

Co-senior author Fei Wang, PhD, professor of population health sciences and founding director of the Institute of Artificial Intelligence for Digital Health at Weill Cornell Medicine, added, “This is a challenging problem because we have a large number of genes but a limited number of patients. The graph-based approach we used effectively explored the collective associations between a gene set and patient-reported pain.” The team validated the findings in a second, independent dataset of synovial biopsy samples from patients with early untreated RA.“

Single-cell sequencing analysis found that of the four types of fibroblasts in synovial tissue, CD55+ fibroblasts exhibited the highest expression of pain-associated genes. “Gene expression analysis among fibroblast subsets indicated that compared with the other fibroblast subsets, lining CD55+ fibroblasts (SC-F4) exhibited the highest expression of GbGMI-identified pain-associated genes,” the authors wrote. Located in the outer synovial lining, CD55+ cells secrete synovial fluid, allowing for frictionless joint movement.

They also expressed the NTN4 gene, which codes for a protein called Netrin-4. Proteins in the netrin family guide axon growth paths and promote new vascular growth. These genes, it turned out, were enriched in pathways that are important for neuron axon growth, the researchers discovered. The keys to sensation, sensory neurons receive and transmit information to the central nervous system. Axons are the tendrils that branch out from them into tissues.

“That led us to hypothesize that perhaps the fibroblasts are producing things that alter the growth of sensory nerves,” Orange stated. But what role was the protein playing in the sensation of pain? To find out, the researchers grew neurons in vitro and then doused them with Netrin-4. This sparked the sprouting and branching of CGRP+ (gene-related peptide) pain receptors. The results represent the first time that Netrin-4 has been shown to alter the growth of pain-sensitive neurons, Orange noted.

Imaging of RA synovial tissue also revealed an overabundance of blood vessels, which feed and nurture new cells. These vessels were encased by CGRP+ sensory nerve fibers and were growing towards the lining fibroblasts in areas of excessive tissue growth, or hyperplasia. This process likely leads to the squishy swelling that many rheumatologists and surgeons have mistaken for inflammation. Reporting on the cell-based studies, the team stated, “We conclude that neoangiogenesis into abnormal papillary processes toward lining fibroblasts in the low inflammatory RA synovium was accompanied by neoneurogenesis of CGRP+ nociceptive axons.”

In the future, the researchers aim to hone in on other products that fibroblasts may be producing that can affect the growth of pain-sensitive neurons. “… these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA,” they wrote. “Future studies are needed to further unravel how genes identified here relate to neuron growth and patient experience of pain either independently or in conjunction with other genes.” Additional work will also be needed to explore the effects of fibroblast products on human neurons, they added. “There are also many other genes associated with patient report of pain in this dataset that warrant additional study.”

The investigators, in addition, aim to look at the other types of sensory nerves that might be affected. “We studied one type, but there are about a dozen,” Orange pointed out. “We don’t know if all nerves are affected equally. And we don’t want to block all sensation. Sensory nerves are important for knowing that you should avoid certain movements and the position of your joint in space, for instance … We want to drill down on those details so that hopefully we can come up with other treatments for patients who don’t have a lot of inflammation. Right now, they’re taking medications that can cost $70,000 a year but have no chance of working. We must do a better job of getting the right drug to the right patient.”

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