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A newly identified circulating cell type may be a reliable marker for impending RA flares. The discovery and description of the cells, which bear a “striking” similarity to synovial fibroblasts, provide important clues to the origins of RA and progressive joint inflammation, investigators say.
By studying longitudinally collected blood samples from four patients with RA over 4 years, Dana E. Orange, MD and colleagues at Rockefeller University, New York, identified a pattern of B-cell activation and expansion of circulating cells that are negative for CD45 and CD31 expression, and positive for PDPN, dubbed preinflammatory mesenchymal or “PRIME” cells.
Expansion of PRIME cells in circulation increased dramatically in the weeks leading up to a flare and decreased during a flare, suggesting the possibility of a serum assay for predicting flares and allowing for early intervention to ameliorate or prevent disabling consequences, the investigators wrote in a study published in the New England Journal of Medicine.
“Our hope is that this will be a diagnostic in the future, but we need to study it in more patients to see how it will perform,” Dr. Orange said in an interview, adding that the cells, if shown to be pathogenic, could also be targets for new therapeutic strategies.
RNA sequencing
Dr. Orange and colleagues discovered the PRIME cells through a novel clinical and technical protocol involving home collection of blood by patients and longitudinal RNA sequencing to study gene expression profiles during times of both disease quiescence and flares, and noticed a distinct pattern of PRIME cell expansion, depletion, and gene expression.
“Looking at their gene expression profiles, they overlapped with fibroblasts that reside in inflamed rheumatoid arthritis synovium, and in an animal model those types of fibroblasts were important for allowing entry of inflammatory infiltrates around the joint,” she said.
PRIME cells may be a precursor of synovial fibroblasts, which have been implicated by some researchers in the spread of RA between joints, Dr. Orange added.
Patients do homework
The investigators began by enrolling four patients, followed for 1-4 years, who met 2010 American College of Rheumatology–European League Against Rheumatism criteria and who were seropositive for anti–cyclic citrullinated peptide antibodies.
They assessed disease activity from patient homes weekly or during escalation of flares up to four times daily, with the Routine Assessment of Patient Index Data 3 (RAPID3) questionnaire, as well as monthly clinic visits. At clinic visits during flares, disease activity was assessed using both the RAPID3 and 28-joint Disease Activity Score.
The patients performed fingerstick blood collection and mailed the samples overnight each week to Rockefeller University, where RNA was extracted and sequenced. The investigators identified gene transcripts that were differentially expressed in blood prior to flares, and compared them with data profiles derived from synovial single-cell RNA sequencing.
To validate the findings, the researchers used flow cytometry and sorted blood-cell RNA sequencing of samples from an additional 19 patients with RA.
They found that a total of 2,613 genes were differentially expressed during a flare, compared with baseline, and that expression of 1,437 of these genes was increased during a flare, with the remaining 1,176 decreased during flares.
Before the storm
Focusing on two flare-antecedent clusters of genes, they identified one cluster of transcripts that increased 2 weeks before a flare, enriched with genes coding for developmental pathways for naive B cells (that is, not yet exposed to antigens) and leukocytes.
The second cluster included gene transcripts that increased during the week before a flare, then decreased over the duration of the flare. Genes in this cluster were enriched for pathways that were unexpected in typical blood specimens, including genes involved in cartilage morphogenesis, endochondral bone growth, and extracellular matrix organization. The gene activity suggested the presence of a mesenchymal cell, they wrote.
The RNA expression profiles of these newly identified PRIME cells were very similar to those of synovial fibroblasts, and the investigators speculated that PRIME cells may be synovial fibroblast precursors.
They proposed a model of RA exacerbation in which PRIME cells become activated by B cells in the weeks immediately preceding a flare, and then migrate out of blood into the synovium.
The investigators are currently investigating “how reproducible this signature is in different flares in patients on different types of background therapy, and then we’re very interested in looking at the upstream triggers of the B cell and the PRIME cell,” Dr. Orange said.
“One of the reasons this is very exciting is that there are these signatures that can be found when patients are clearly asymptomatic but about to flare, and if we can intervene at that time, then the patients won’t have to live through a flare, they won’t have to have that experience,” she said.
Pros and cons
In an editorial accompanying the study, Ellen M. Gravallese, MD, from Brigham and Women’s Hospital in Boston and William H. Robinson, MD, PhD, from Stanford (Calif.) University and the Veterans Affairs Palo Alto (Calif.) Health Care System wrote that the study demonstrates an important method for identifying genetic contributions to many different types of disease.
“Orange and colleagues show that intensively collected longitudinal data from a small sample of patients can be used to identify dysregulated transcriptional signatures that are not recognized by classical cross-sectional studies. This study illustrates the exciting potential of longitudinal genomics to identify key antecedents of disease flares in an approach that may be applicable to the investigation of pathogenic and protective immune responses in a wide range of human diseases,” they wrote.
Rheumatology researcher Christopher D. Buckley, MBBS, DPhil, from the University of Birmingham (England), who was not involved in the study, said that the use of blood samples is both a strength and a weakness of the study.
“Blood is much easier to get than synovial tissue, but synovial tissue is important. If I’m trying to look at the blood and trying to make an inference about what’s going on in the synovium, if I don’t look at the synovium I don’t know what the link between the blood and synovium is,” he said in an interview.
On the plus side, “the big advantage about looking at blood is that you do multiple time points, which is really cool,” he said.
Dr. Buckley is a coauthor of a recent paper in Nature Medicine – published after the study by Dr. Orange and colleagues was accepted by the New England Journal of Medicine – showing that a population of macrophages in synovium was highly predictive of remission in patients with RA, and that therapeutic modulation of these macrophages has the potential as a treatment strategy for RA.
“We are very keen to understand the cellular basis of disease. We’re very good at understanding genes, but genes have to work in cells, and cells make organs, so the cells are critical,” he said.
The paper adds fuel to a controversy that has been raging among rheumatology researchers for more than a decade: the “flying fibroblast” hypothesis, which suggests that fibroblasts can migrate from one joint to another, hence spreading the disease in a manner akin to cancer metastases.
“It’s been quite controversial whether these cells like fibroblasts can exist in the blood, or whether they’re found in sufficient number in the blood,” Dr. Buckley said. “The fact that they have identified these PRIME cells is fascinating, because that’s going to cause us to go back and reinvestigate the whole flying fibroblast story.”
His colleague John Isaacs, MBBS, PhD, from Newcastle (England) University, is principal investigator for the BIO-FLARE study, in which participants with RA stop taking their disease-modifying antirheumatic drugs under close supervision of researchers. The investigators then study the patients looking for flare signals as well as the biology of flares themselves.
“As it happens, our protocols would not pick up this particular cell, because we have not been focusing on the stroma, at least not in peripheral blood. We’ve all been looking at synovium as part of BIO-FLARE,” he said in an interview. “We will be looking for this cell now that we have seen this research, and certainly we would want to replicate.”
He agreed with Dr. Buckley’s observation that the PRIME cell data may revive the flying fibroblast hypothesis. “This is a great paper in a top clinical journal. What isn’t there is mechanism. That’s the thing we’re all going to want to understand now: Where do the cells come from, how do they actually trigger flares, and how do they go down as flare starts?”
Both Dr. Buckley and Dr. Isaacs agreed that the study findings point to important new avenues of research, but also noted that the study was small, involving a total of only 23 patients, and that replication of the findings and elucidation of the mechanism of PRIME cell generation and disposition will be required.
The study was supported by grants from the National Institutes of Health, Simons Foundation, Robertson Foundation, Rheumatology Research Foundation, Bernard and Irene Schwartz Foundation, the Iris and Junming Le Foundation, and Rockefeller University. Dr. Orange disclosed a provisional patent for the discovery of the PRIME cells. Dr. Buckley and Dr. Isaacs reported no relevant conflicts of interest.
SOURCE: Orange DE et al. N Engl J Med. 2020 Jul 15. doi: 10.1056/NEJMoa2004114.
A newly identified circulating cell type may be a reliable marker for impending RA flares. The discovery and description of the cells, which bear a “striking” similarity to synovial fibroblasts, provide important clues to the origins of RA and progressive joint inflammation, investigators say.
By studying longitudinally collected blood samples from four patients with RA over 4 years, Dana E. Orange, MD and colleagues at Rockefeller University, New York, identified a pattern of B-cell activation and expansion of circulating cells that are negative for CD45 and CD31 expression, and positive for PDPN, dubbed preinflammatory mesenchymal or “PRIME” cells.
Expansion of PRIME cells in circulation increased dramatically in the weeks leading up to a flare and decreased during a flare, suggesting the possibility of a serum assay for predicting flares and allowing for early intervention to ameliorate or prevent disabling consequences, the investigators wrote in a study published in the New England Journal of Medicine.
“Our hope is that this will be a diagnostic in the future, but we need to study it in more patients to see how it will perform,” Dr. Orange said in an interview, adding that the cells, if shown to be pathogenic, could also be targets for new therapeutic strategies.
RNA sequencing
Dr. Orange and colleagues discovered the PRIME cells through a novel clinical and technical protocol involving home collection of blood by patients and longitudinal RNA sequencing to study gene expression profiles during times of both disease quiescence and flares, and noticed a distinct pattern of PRIME cell expansion, depletion, and gene expression.
“Looking at their gene expression profiles, they overlapped with fibroblasts that reside in inflamed rheumatoid arthritis synovium, and in an animal model those types of fibroblasts were important for allowing entry of inflammatory infiltrates around the joint,” she said.
PRIME cells may be a precursor of synovial fibroblasts, which have been implicated by some researchers in the spread of RA between joints, Dr. Orange added.
Patients do homework
The investigators began by enrolling four patients, followed for 1-4 years, who met 2010 American College of Rheumatology–European League Against Rheumatism criteria and who were seropositive for anti–cyclic citrullinated peptide antibodies.
They assessed disease activity from patient homes weekly or during escalation of flares up to four times daily, with the Routine Assessment of Patient Index Data 3 (RAPID3) questionnaire, as well as monthly clinic visits. At clinic visits during flares, disease activity was assessed using both the RAPID3 and 28-joint Disease Activity Score.
The patients performed fingerstick blood collection and mailed the samples overnight each week to Rockefeller University, where RNA was extracted and sequenced. The investigators identified gene transcripts that were differentially expressed in blood prior to flares, and compared them with data profiles derived from synovial single-cell RNA sequencing.
To validate the findings, the researchers used flow cytometry and sorted blood-cell RNA sequencing of samples from an additional 19 patients with RA.
They found that a total of 2,613 genes were differentially expressed during a flare, compared with baseline, and that expression of 1,437 of these genes was increased during a flare, with the remaining 1,176 decreased during flares.
Before the storm
Focusing on two flare-antecedent clusters of genes, they identified one cluster of transcripts that increased 2 weeks before a flare, enriched with genes coding for developmental pathways for naive B cells (that is, not yet exposed to antigens) and leukocytes.
The second cluster included gene transcripts that increased during the week before a flare, then decreased over the duration of the flare. Genes in this cluster were enriched for pathways that were unexpected in typical blood specimens, including genes involved in cartilage morphogenesis, endochondral bone growth, and extracellular matrix organization. The gene activity suggested the presence of a mesenchymal cell, they wrote.
The RNA expression profiles of these newly identified PRIME cells were very similar to those of synovial fibroblasts, and the investigators speculated that PRIME cells may be synovial fibroblast precursors.
They proposed a model of RA exacerbation in which PRIME cells become activated by B cells in the weeks immediately preceding a flare, and then migrate out of blood into the synovium.
The investigators are currently investigating “how reproducible this signature is in different flares in patients on different types of background therapy, and then we’re very interested in looking at the upstream triggers of the B cell and the PRIME cell,” Dr. Orange said.
“One of the reasons this is very exciting is that there are these signatures that can be found when patients are clearly asymptomatic but about to flare, and if we can intervene at that time, then the patients won’t have to live through a flare, they won’t have to have that experience,” she said.
Pros and cons
In an editorial accompanying the study, Ellen M. Gravallese, MD, from Brigham and Women’s Hospital in Boston and William H. Robinson, MD, PhD, from Stanford (Calif.) University and the Veterans Affairs Palo Alto (Calif.) Health Care System wrote that the study demonstrates an important method for identifying genetic contributions to many different types of disease.
“Orange and colleagues show that intensively collected longitudinal data from a small sample of patients can be used to identify dysregulated transcriptional signatures that are not recognized by classical cross-sectional studies. This study illustrates the exciting potential of longitudinal genomics to identify key antecedents of disease flares in an approach that may be applicable to the investigation of pathogenic and protective immune responses in a wide range of human diseases,” they wrote.
Rheumatology researcher Christopher D. Buckley, MBBS, DPhil, from the University of Birmingham (England), who was not involved in the study, said that the use of blood samples is both a strength and a weakness of the study.
“Blood is much easier to get than synovial tissue, but synovial tissue is important. If I’m trying to look at the blood and trying to make an inference about what’s going on in the synovium, if I don’t look at the synovium I don’t know what the link between the blood and synovium is,” he said in an interview.
On the plus side, “the big advantage about looking at blood is that you do multiple time points, which is really cool,” he said.
Dr. Buckley is a coauthor of a recent paper in Nature Medicine – published after the study by Dr. Orange and colleagues was accepted by the New England Journal of Medicine – showing that a population of macrophages in synovium was highly predictive of remission in patients with RA, and that therapeutic modulation of these macrophages has the potential as a treatment strategy for RA.
“We are very keen to understand the cellular basis of disease. We’re very good at understanding genes, but genes have to work in cells, and cells make organs, so the cells are critical,” he said.
The paper adds fuel to a controversy that has been raging among rheumatology researchers for more than a decade: the “flying fibroblast” hypothesis, which suggests that fibroblasts can migrate from one joint to another, hence spreading the disease in a manner akin to cancer metastases.
“It’s been quite controversial whether these cells like fibroblasts can exist in the blood, or whether they’re found in sufficient number in the blood,” Dr. Buckley said. “The fact that they have identified these PRIME cells is fascinating, because that’s going to cause us to go back and reinvestigate the whole flying fibroblast story.”
His colleague John Isaacs, MBBS, PhD, from Newcastle (England) University, is principal investigator for the BIO-FLARE study, in which participants with RA stop taking their disease-modifying antirheumatic drugs under close supervision of researchers. The investigators then study the patients looking for flare signals as well as the biology of flares themselves.
“As it happens, our protocols would not pick up this particular cell, because we have not been focusing on the stroma, at least not in peripheral blood. We’ve all been looking at synovium as part of BIO-FLARE,” he said in an interview. “We will be looking for this cell now that we have seen this research, and certainly we would want to replicate.”
He agreed with Dr. Buckley’s observation that the PRIME cell data may revive the flying fibroblast hypothesis. “This is a great paper in a top clinical journal. What isn’t there is mechanism. That’s the thing we’re all going to want to understand now: Where do the cells come from, how do they actually trigger flares, and how do they go down as flare starts?”
Both Dr. Buckley and Dr. Isaacs agreed that the study findings point to important new avenues of research, but also noted that the study was small, involving a total of only 23 patients, and that replication of the findings and elucidation of the mechanism of PRIME cell generation and disposition will be required.
The study was supported by grants from the National Institutes of Health, Simons Foundation, Robertson Foundation, Rheumatology Research Foundation, Bernard and Irene Schwartz Foundation, the Iris and Junming Le Foundation, and Rockefeller University. Dr. Orange disclosed a provisional patent for the discovery of the PRIME cells. Dr. Buckley and Dr. Isaacs reported no relevant conflicts of interest.
SOURCE: Orange DE et al. N Engl J Med. 2020 Jul 15. doi: 10.1056/NEJMoa2004114.
A newly identified circulating cell type may be a reliable marker for impending RA flares. The discovery and description of the cells, which bear a “striking” similarity to synovial fibroblasts, provide important clues to the origins of RA and progressive joint inflammation, investigators say.
By studying longitudinally collected blood samples from four patients with RA over 4 years, Dana E. Orange, MD and colleagues at Rockefeller University, New York, identified a pattern of B-cell activation and expansion of circulating cells that are negative for CD45 and CD31 expression, and positive for PDPN, dubbed preinflammatory mesenchymal or “PRIME” cells.
Expansion of PRIME cells in circulation increased dramatically in the weeks leading up to a flare and decreased during a flare, suggesting the possibility of a serum assay for predicting flares and allowing for early intervention to ameliorate or prevent disabling consequences, the investigators wrote in a study published in the New England Journal of Medicine.
“Our hope is that this will be a diagnostic in the future, but we need to study it in more patients to see how it will perform,” Dr. Orange said in an interview, adding that the cells, if shown to be pathogenic, could also be targets for new therapeutic strategies.
RNA sequencing
Dr. Orange and colleagues discovered the PRIME cells through a novel clinical and technical protocol involving home collection of blood by patients and longitudinal RNA sequencing to study gene expression profiles during times of both disease quiescence and flares, and noticed a distinct pattern of PRIME cell expansion, depletion, and gene expression.
“Looking at their gene expression profiles, they overlapped with fibroblasts that reside in inflamed rheumatoid arthritis synovium, and in an animal model those types of fibroblasts were important for allowing entry of inflammatory infiltrates around the joint,” she said.
PRIME cells may be a precursor of synovial fibroblasts, which have been implicated by some researchers in the spread of RA between joints, Dr. Orange added.
Patients do homework
The investigators began by enrolling four patients, followed for 1-4 years, who met 2010 American College of Rheumatology–European League Against Rheumatism criteria and who were seropositive for anti–cyclic citrullinated peptide antibodies.
They assessed disease activity from patient homes weekly or during escalation of flares up to four times daily, with the Routine Assessment of Patient Index Data 3 (RAPID3) questionnaire, as well as monthly clinic visits. At clinic visits during flares, disease activity was assessed using both the RAPID3 and 28-joint Disease Activity Score.
The patients performed fingerstick blood collection and mailed the samples overnight each week to Rockefeller University, where RNA was extracted and sequenced. The investigators identified gene transcripts that were differentially expressed in blood prior to flares, and compared them with data profiles derived from synovial single-cell RNA sequencing.
To validate the findings, the researchers used flow cytometry and sorted blood-cell RNA sequencing of samples from an additional 19 patients with RA.
They found that a total of 2,613 genes were differentially expressed during a flare, compared with baseline, and that expression of 1,437 of these genes was increased during a flare, with the remaining 1,176 decreased during flares.
Before the storm
Focusing on two flare-antecedent clusters of genes, they identified one cluster of transcripts that increased 2 weeks before a flare, enriched with genes coding for developmental pathways for naive B cells (that is, not yet exposed to antigens) and leukocytes.
The second cluster included gene transcripts that increased during the week before a flare, then decreased over the duration of the flare. Genes in this cluster were enriched for pathways that were unexpected in typical blood specimens, including genes involved in cartilage morphogenesis, endochondral bone growth, and extracellular matrix organization. The gene activity suggested the presence of a mesenchymal cell, they wrote.
The RNA expression profiles of these newly identified PRIME cells were very similar to those of synovial fibroblasts, and the investigators speculated that PRIME cells may be synovial fibroblast precursors.
They proposed a model of RA exacerbation in which PRIME cells become activated by B cells in the weeks immediately preceding a flare, and then migrate out of blood into the synovium.
The investigators are currently investigating “how reproducible this signature is in different flares in patients on different types of background therapy, and then we’re very interested in looking at the upstream triggers of the B cell and the PRIME cell,” Dr. Orange said.
“One of the reasons this is very exciting is that there are these signatures that can be found when patients are clearly asymptomatic but about to flare, and if we can intervene at that time, then the patients won’t have to live through a flare, they won’t have to have that experience,” she said.
Pros and cons
In an editorial accompanying the study, Ellen M. Gravallese, MD, from Brigham and Women’s Hospital in Boston and William H. Robinson, MD, PhD, from Stanford (Calif.) University and the Veterans Affairs Palo Alto (Calif.) Health Care System wrote that the study demonstrates an important method for identifying genetic contributions to many different types of disease.
“Orange and colleagues show that intensively collected longitudinal data from a small sample of patients can be used to identify dysregulated transcriptional signatures that are not recognized by classical cross-sectional studies. This study illustrates the exciting potential of longitudinal genomics to identify key antecedents of disease flares in an approach that may be applicable to the investigation of pathogenic and protective immune responses in a wide range of human diseases,” they wrote.
Rheumatology researcher Christopher D. Buckley, MBBS, DPhil, from the University of Birmingham (England), who was not involved in the study, said that the use of blood samples is both a strength and a weakness of the study.
“Blood is much easier to get than synovial tissue, but synovial tissue is important. If I’m trying to look at the blood and trying to make an inference about what’s going on in the synovium, if I don’t look at the synovium I don’t know what the link between the blood and synovium is,” he said in an interview.
On the plus side, “the big advantage about looking at blood is that you do multiple time points, which is really cool,” he said.
Dr. Buckley is a coauthor of a recent paper in Nature Medicine – published after the study by Dr. Orange and colleagues was accepted by the New England Journal of Medicine – showing that a population of macrophages in synovium was highly predictive of remission in patients with RA, and that therapeutic modulation of these macrophages has the potential as a treatment strategy for RA.
“We are very keen to understand the cellular basis of disease. We’re very good at understanding genes, but genes have to work in cells, and cells make organs, so the cells are critical,” he said.
The paper adds fuel to a controversy that has been raging among rheumatology researchers for more than a decade: the “flying fibroblast” hypothesis, which suggests that fibroblasts can migrate from one joint to another, hence spreading the disease in a manner akin to cancer metastases.
“It’s been quite controversial whether these cells like fibroblasts can exist in the blood, or whether they’re found in sufficient number in the blood,” Dr. Buckley said. “The fact that they have identified these PRIME cells is fascinating, because that’s going to cause us to go back and reinvestigate the whole flying fibroblast story.”
His colleague John Isaacs, MBBS, PhD, from Newcastle (England) University, is principal investigator for the BIO-FLARE study, in which participants with RA stop taking their disease-modifying antirheumatic drugs under close supervision of researchers. The investigators then study the patients looking for flare signals as well as the biology of flares themselves.
“As it happens, our protocols would not pick up this particular cell, because we have not been focusing on the stroma, at least not in peripheral blood. We’ve all been looking at synovium as part of BIO-FLARE,” he said in an interview. “We will be looking for this cell now that we have seen this research, and certainly we would want to replicate.”
He agreed with Dr. Buckley’s observation that the PRIME cell data may revive the flying fibroblast hypothesis. “This is a great paper in a top clinical journal. What isn’t there is mechanism. That’s the thing we’re all going to want to understand now: Where do the cells come from, how do they actually trigger flares, and how do they go down as flare starts?”
Both Dr. Buckley and Dr. Isaacs agreed that the study findings point to important new avenues of research, but also noted that the study was small, involving a total of only 23 patients, and that replication of the findings and elucidation of the mechanism of PRIME cell generation and disposition will be required.
The study was supported by grants from the National Institutes of Health, Simons Foundation, Robertson Foundation, Rheumatology Research Foundation, Bernard and Irene Schwartz Foundation, the Iris and Junming Le Foundation, and Rockefeller University. Dr. Orange disclosed a provisional patent for the discovery of the PRIME cells. Dr. Buckley and Dr. Isaacs reported no relevant conflicts of interest.
SOURCE: Orange DE et al. N Engl J Med. 2020 Jul 15. doi: 10.1056/NEJMoa2004114.
FROM THE NEW ENGLAND JOURNAL OF MEDICINE
Key clinical point: A newly identified cell type may be predictive of RA flares.
Major finding: Preinflammatory mesenchymal cells were expanded in the week preceding a flare and decreased during the flare.
Study details: A longitudinal observational study of 23 patients with RA.
Disclosures: The study was supported by grants from the National Institutes of Health, Simons Foundation, Robertson Foundation, Rheumatology Research Foundation, Bernard and Irene Schwartz Foundation, the Iris and Junming Le Foundation, and Rockefeller University. Dr. Orange disclosed a provisional patent for the discovery of the preinflammatory mesenchymal cells.
Source: Orange DE et al. N Engl J Med. 2020 Jul 15. doi: 10.1056/NEJMoa2004114.