Biomarkers in Connective Tissue Diseases

Abstract

Autoimmune connective tissue diseases are clinically variable, making biomarkers desirable for assessing future disease risk, supporting early and accurate diagnosis, monitoring disease activity and progression, selecting therapeutics, and assessing treatment response. Because of their correlations with specific clinical characteristics and often with disease progression, autoantibodies and other soluble mediators are considered potential biomarkers. Additional biomarkers may reflect downstream pathological processes or may appear due to ongoing inflammation and damage. Because of overlap between diseases, some biomarkers have limited specificity for a single autoimmune connective tissue disease. This review describes select current biomarkers that aid in diagnosis and treatment of several major systemic autoimmune connective tissue disorders: systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, and ANCA-associated vasculitides. Newly proposed biomarkers that target various stages in disease onset or progression are also discussed. Newer approaches to overcome the diversity observed in these diseases and to facilitate personalized disease monitoring and treatment are also addressed.

Introduction

Autoimmune connective tissue disorders are a heterogenous group of diseases that affect connective tissue in various organs resulting from poorly controlled autoimmune responses, complement activation, interferon dysregulation, and associated inflammation. Although their clinical presentations vary, these diseases share significant genetic risk factors as demonstrated by cross-analysis of genome-wide association studies¹ and common regulatory mechanisms of autoimmune diseases.² Environmental and female-associated factors also play critical roles in development of autoimmune diseases.^3–7 In nearly all systemic autoimmune rheumatic diseases evaluated to date, autoantibody production and immune dysregulation precede clinical onset,^8–15 although a significant amount of this information is not yet integrated to standard clinical care. Ongoing research is focused on improving biomarkers for diagnosis, prognosis, treatment selection and optimized therapy. This review describes current and new emerging biomarkers for major connective tissue diseases: systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, and ANCA-associated vasculitides.

Systemic lupus erythematosus (SLE)

SLE is a systemic autoimmune disease characterized by production of anti-nuclear autoantibodies (ANA). Early, accurate diagnosis and disease monitoring are hindered by its heterogenous presentation and clinical course. Serological and urinary biomarkers are either in use or are emerging as potential biomarkers for SLE. These autoantibodies, complement products, and cytokines/chemokines/soluble mediators have the potential to facilitate diagnosis, identify individuals at greater risk for developing SLE, and monitor disease activity or specific organ involvement.

Autoantibodies

ANA are a hallmark of SLE. Nearly all SLE patients exhibit ANA at diagnosis, with a 1:80 immunofluorescent titer showing up to 98% sensitivity but 75% specificity for SLE classification.¹⁶ ANA are also found in individuals with many other autoimmune diseases, malignancies, hepatic diseases, unaffected family members of lupus patients, and even up to 14% of healthy individuals,¹⁷ especially with increasing age. Therefore, a positive ANA serves as a necessary but insufficient criterion for SLE classification or diagnosis, but not as a definitive test.¹⁸ Individuals with a negative ANA are extremely unlikely to have any lupus-specific autoantibodies. Therefore, through the Choosing Wisely campaign, the American College of Rheumatology (ACR) recommends testing for specific autoantibodies only when a positive ANA and clinical suspicion are present.¹⁹ Repeat testing is not indicated in ANA-positive individuals, as changes in ANA titer alone show no clinical correlation with increased disease activity or worsening prognosis. Testing of ANA and other autoantibodies in preclinical disease states or to identify individuals for potential preventive interventions will require additional studies and guidelines.²⁰

Anti-double stranded DNA (dsDNA) antibody responses have high specificity (92–96%) and moderate sensitivity (57–67%) for SLE.²¹ They constitute a criterion for SLE classification by ACR criteria (requiring 4 of 11 criteria for classification) and by the Systemic Lupus International Collaborating Clinics (SLICC) criteria (requiring 4 of 17 criteria, or dsDNA plus biopsy-proven lupus nephritis [LN]).^22–24 Anti-dsDNA forms immune complexes with nucleosomes observed in SLE patients, leading to immune complex deposition in the kidney.²⁵ Furthermore, anti-dsDNA antibodies show cross-reactivity to alpha actinin and can bind to mesangial cells in the kidney.²⁶ Immune complexes formed by anti-dsDNA antibodies in the kidney can activate the complement cascade, leading to damage in glomerulonephritis.²⁷ Patients with proliferative LN have elevated anti-dsDNA as early as four years before diagnosis, and an increase of >1 IU/mL/year was specific for LN.²⁸ Anti-dsDNA with low complement also associates with mucocutaneous, renal, and hematological flare within one year.²⁹ In clinically stable SLE patients with rising anti-dsDNA (≥25%) and rising C3a, the free released product of complement activation, (≥50%), treatment with moderate prednisone may avert severe clinical flares.³⁰

Although less common (sensitivity 26–31%) antibodies against the Sm antigen are highly specific (95–99%) for SLE and may associate with early mortality.³¹ About 30–70% of SLE patients have anti-Ro/SSA (Sjögren’s syndrome type A antigen), and Ro/SSA is associated with subacute lupus erythematosus, sicca symptoms and secondary Sjögren’s. Anti-Ro/SSA antibodies may bind to either of two antigenic proteins, 52 kD Ro and 60 kD Ro. Antibodies to 60 kD Ro/SSA are more frequently observed in SLE, and correlate with photosensitivity, cutaneous vasculitis, and hematological disorders.³² Antibodies to a related antigen, La/SSB, are present in ~10% of SLE patients and are associated with lower prevalence of renal disease.³² Anti-ribosomal P antibodies, similar to anti-Sm, are very specific for SLE, but occur in only ~20% of Caucasian SLE patients. Anti-ribosomal P is enriched in neuropsychiatric³³ and pediatric-onset disease.³⁴ A number of other autoreactivities have been reported in SLE.²¹ Of interest are anti-nucleosome responses, which correlate with disease activity in clinically quiescent patients,³⁵ and anti-cardiolipin responses, which are implicated in thrombosis and recurrent fetal loss and are associated with a complex clinical outcome, with patients meeting a higher number of ACR criteria.¹⁰

Since no single autospecificity is sufficient for SLE diagnosis, recent efforts have focused on detecting autoantibody signatures encompassing combinations of autoreactivities. One autoantigen array covers antigens related to eight distinct autoimmune diseases.³⁶ Another microarray-based test has a reported sensitivity of 94% and specificity of 75%. The test is validated as a clinical test to exclude a diagnosis of SLE if no compelling clinical evidence exists or to support a low likelihood of SLE.³⁷

Autoantibodies typically are detectable before diagnosis; 63 to 88% of individuals have autoantibodies before disease classification (0.1 to 9 years).^9,8 Anti-Ro/SSA is among the earliest detectable specificities, while anti-ds DNA antibodies appear closer to classification (~ 3 years before).⁸ Therefore, autoantibodies may serve as a biomarker of disease risk prior to SLE onset. In individuals meeting <4 ACR classification criteria for SLE, those who later reached SLE classification showed higher baseline ANA and increased IgG autoreactivity in IgG profiling of over 80 autoantigens.³⁸ In a follow-up study of previously unaffected relatives of SLE patients, 89% of individuals who later reached SLE classification were ANA-positive at baseline, compared to 48% of those who remained unaffected.¹³ Therefore ANA positivity alone is not a definitive marker for increased risk of future disease classification, and additional predictive markers are needed.

Complement

Levels of complement C3 and C4 are used to monitor SLE disease activity. Reduced C3 and C4 levels are associated with more severe disease, and reduced C1q, C3, and C4 levels can precede disease flare.³⁹ Complement is activated in SLE by immune complex deposition. Therefore, cell bound complement C4 activation products (CBCAP) on erythrocytes are increased in SLE and have 22% higher sensitivity for SLE than reduced C3/C4. ANA positivity, anti-MCV negativity, anti-dsDNA positivity, elevated erythrocyte C4d and elevated B cell C4d demonstrated 80% sensitivity for SLE and 87% specificity against other rheumatic diseases.⁴⁰ These findings have been confirmed and refined to a commercially available, weighted SLE risk score with a two tier design.⁴¹

Emerging Biomarkers

Soluble mediators

Recent studies have suggested specific soluble mediators as potential biomarkers for SLE onset and SLE disease flare. In a survey of nearly 30 immune or inflammation-based soluble mediators, several soluble mediators were elevated >3.5 years before SLE classification, including interleukin (IL)-5, IL-6, and IFNγ. Some cytokines (BLyS, APRIL) became elevated closer to diagnosis. In this preclinical period, a combination of IFNγ, IL-4, IL-6, ANA, and anti-Ro distinguished patients from controls with 84% accuracy, compared to 58% accuracy with ANA positivity alone. Thus, evaluating immune pathway dysregulation in conjunction with ANA positivity may help identify individuals at higher risk for SLE.¹¹

Flares are a significant risk factor for end organ damage in SLE, and soluble mediators are promising biomarkers of imminent SLE flare. Levels of BLyS in SLE patients are associated with anti-dsDNA levels and disease activity.⁴² Baseline BLyS concentration ≥2ng/mL predicted SLE flare at week 52 in a combined analysis of data from phase II, world-wide clinical trials.⁴³ In a longitudinal study of SLE patients, reduced levels of regulatory cytokines such as IL1 receptor antagonist, tumor growth factor (TGF)-β and IL10, preceded disease flares. A combined soluble mediator score incorporating 52 analytes was increased in patients with impending flare compared to either matched stable patients or the same patients during a clinically stable period.^{44, 45} This score accurately predicted flare in both European American and African American study groups. Accurate prediction of SLE flare may allow early treatment or prevention of flares.

Urinary biomarkers

Glomerulonephritis, a major cause of morbidity and mortality in SLE, currently requires renal biopsy for definitive diagnosis. Urinary biomarkers would be useful for identifying LN patients at the highest risk of end stage renal disease or distinguishing between LN and other forms of renal disease in lupus patients. Urinary levels of TNF-like weak inducer of apoptosis (TWEAK) correlated with disease activity in a large multicenter longitudinal study.⁴⁶ Other possible urinary biomarkers include monocyte chemoattractant protein (MCP-1), high mobility group box-1 protein (HMGB-1), vascular cell adhesion molecule (VCAM-1), and angiostatin.^47–50 The levels of these markers correlate with histological changes in the renal tissue and can distinguish between active and inactive LN.

Interferons

Interferons (IFN) are consistently associated with SLE. Serum IFNα activity associates with autoantibodies as well as BLyS.^{51, 52} IFNα levels correlated with the number of autoantibody specificities in preclinical samples obtained from individuals later classified as SLE, suggesting a role for IFNα in autoantibody accrual.¹² Interestingly, IFNγ activity was increased before autoantibody positivity, and autoantibody positivity preceded increases in IFNα. IFNγ regulates both IFNα and B cell differentiation and possibly drives B cell maturation and class switching in early stages of SLE pathogenesis. However, only a subset of adult SLE patients show IFN activity in serum.⁵¹

IFN response is most often measured indirectly as an ‘IFN signature’ defined by upregulation of sets of IFN-regulated genes. In a large, longitudinal monitoring study, 84.8% of pediatric lupus patients demonstrated an IFN signature.⁵³ A similar IFN signature has been reported in about half of adult SLE patients, and IFN activity levels have been associated with autoantibody production and disease activity.^54–56 Longitudinal monitoring of whole blood gene expression profiles and parallel disease activity in a large pediatric cohort demonstrates seven different clusters of reactivity and association with various gene expression modules.⁵³ Although these data associate IFNs and IFN-associated gene regulation with SLE, some aspects of the IFN signature may be relatively stable over the course of the disease, based on paired analyses of longitudinal data.^{57, 58} Indeed, high serum IFNα activity appears to be a complex heritable trait.⁵¹ Therefore, IFN may influence SLE predisposition, while together with dysregulation of other immune pathways, such as BLyS, T helper, and inflammatory mediators, may influence disease pathogenesis.

Due to the heterogeneity among SLE patients, personalized treatment/monitoring based on molecular mechanisms involved may be beneficial. Using an unbiased approach with modular gene expression panels that include IFN signature genes, Bancheareau et al. stratified pediatric SLE patients into separate groups that were supported by patient genotypes.⁵³ Such patient stratification may enable studying effectiveness of biomarkers for disease activity or treatment response in a particular subset of patients with a relevant molecular mechanism of disease pathogenesis.

Rheumatoid Arthritis (RA)

Rheumatoid arthritis (RA), a systemic autoimmune rheumatic disease afflicting up to 0.8% of the population, is characterized by synovitis leading to irreversible joint destruction. Effective management of RA requires initiation of therapy with disease modifying anti-rheumatic drugs (DMARDs) within months following disease onset to maximize outcomes.⁵⁹ Even a brief delay can have a significant impact on disease progression. New autoantibody specificities can develop over time, so periodic monitoring may be warranted, especially in arthralgia-positive patients who do not yet have an RA diagnosis. However, once autoantibodies are present in clinical RA serial monitoring is not necessary, as fluctuations in titers over time are not associated with disease activity or further prognosis.

Autoantibodies

Two of the most common autoantibodies in RA are rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA).

Rheumatoid factor

Rheumatoid factor is directed against the Fc portion of IgG class of antibodies. Although RF occurs in 70–80% of RA patients, it also occurs in patients with SLE, Sjögren’s syndrome, or systemic infections, as well as ~10% of healthy individuals.⁶⁰ For RA disease classification, RF has a sensitivity of 69% and specificity of 85%.⁶¹ Higher levels of RF are associated with more severe disease marked by disease progression, rheumatoid nodules, and various extra-articular manifestations.⁶² Based upon the higher sensitivity and clinical utility of ACPA, RF has become more of a historical test and is usually now only tested in combination with anti-CCP.

Antibodies against anti-citrullinated protein antigens (ACPAs)

ACPAs target proteins or peptides where arginine residues have been converted to citrulline. HLA-DRB1 alleles are the strongest genetic association for seropositive RA. HLA-DRB1 interacts with cigarette smoking to increase the risk of ACPA-positive RA, but not seronegative RA⁶³, and IgA ACPA responses have been found in the sputum of preclinical or early clinical RA patients.⁶⁴ ACPAs also appear before the onset of clinical disease, making them valuable markers for diagnosis and prognosis. Please see⁶⁵ for a more extensive review of ACPA pathogenesis in RA.

Clinically, antibodies against cyclic citrullinated peptides (CCP) are used in RA diagnosis. In a meta-analysis, anti CCP had a pooled sensitivity of 67% and 95% specificity for RA, and anti-CCP positivity was associated with increased risk of radiographic progression.⁶¹

Anti-CCP positivity predicted progression to RA in a 3-year follow-up study of undifferentiated arthritis patients.⁶⁶ Anti-CCP and RF were strongly associated with extra-articular manifestations.⁶² Anti-CCP positivity and initial DAS28 were associated with EULAR response to abatacept in analyses to predict factors of efficacy using data from the Orencia and Rheumatoid Arthritis registry.⁶⁷ High anti-CCP titer was an independent predictor of decrease in DAS28 and EULAR good response to rituximab.⁶⁸ In recent onset RA, IgA anti-CCP was observed in 29% of patients, along with IgG. Patients positive for IgA anti-CCP had a more severe disease course over 3 years compared with IgA anti-CCP-negative cases.⁶⁹ A higher number of anti-CCP isotypes was associated with significantly more radiographic damage during the disease course over 10 years of followup.⁷⁰ Even though anti-CCP isotypes may not provide significant improvement in diagnosis compared to anti-CCP IgG, the isotypes may have possible prognostic implications.

Anti-perinuclear factor (APF) and anti-keratin antibodies (AKA) antibodies recognize citrullinated epitopes on the same autoantigen, filaggrin or pro-filaggrin, and may serve as early diagnostic markers. Anti-Sa recognizes citrullinated vimentin⁷¹ and shows high specificity of 92–100% and a moderate sensitivity of 32–43%.⁷² Several other citrullinated antigens have been identified in RA including fibronectin, filaggrin, fibrinogen, alpha enolase, and collagen.

Autoantibodies with other specificities

Antibodies against carbamylated proteins (anti-CarP) are detected in 45% of RA patients, including anti-CCP–negative patients. The targets of anti-CarP in RA include vimentin, fibrinogen, and albumin.⁷³ Anti-CarP responses have been associated with mortality in seropositive RA, and specifically with respiratory causes of death in a Spanish cohort.⁷⁴ Anti-A2/anti-RA33 antibodies occur in >60% of RA patients, and are also seen in SLE. If diagnoses of SLE or mixed connective tissue disease can be excluded, the specificity of anti-A2/anti-RA33 antibodies for RA can be as high as 96%. The specificity of anti-BiP antibodies for RA has been reported as 96%, making these antibodies promising additional candidates for the classification or diagnosis of RA.

Acute phase reactants

Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are elevated in RA patients compared to controls. ESR and swollen joint count were associated with radiographic disease progression in a systemic literature review including 57 studies with disease activity measurements in 13 to 1433 patients.⁷⁵ ESR and CRP levels are also measured as components of RA disease activity indices, such as the DAS28, which are used in trials for clinical disease monitoring.

Cytokines

Many cytokines and chemokines are active in RA patient joints, and these cytokines are critical in inflammation, joint damage and RA-associated comorbidities.⁷⁶ Indeed, a number of different cytokines, such as TNFα, IL6, and IL1RA, are successfully targeted in RA treatment as are small molecule inhibitors, such as JAK inhibitors that regulate cytokine secretion pathways. In addition, in serial samples preceding RA classification in a military cohort, the number of elevated cytokines and chemokines predicted time to RA diagnosis/classification.⁷⁷ A commercially available blood test monitors RA disease activity with a score calculated from the concentrations of 12 serum biomarkers: VCAM-1, epidermal growth factor (EGF), vascular endothelial growth factor-A (VEGF-A), IL-6, TNF-receptor type 1 (TNF-RI), matrix metalloproteinase (MMP)-1, MMP-3, cartilage glycoprotein 39 (YKL-40), leptin, resistin, serum amyloid A, and CRP. Changes in the score correlate with changes in other indicators of RA disease activity, including the DAS28 index, ESR and CRP. In addition, this score declined significantly in patients who responded to TNF inhibitors by EULAR criteria, while patients with a higher score showed greater radiographic progression over 52 weeks of TNF inhibitor treatment.⁷⁸

Emerging biomarkers

Biologics such as TNF inhibitors, anti-IL-6 receptor antibodies, anti-CD20 antibodies, and CTLA-4-Ig have shown efficacy in RA. Several new biomarkers have been proposed to identify patients who may respond to these therapies. Response to rituximab is associated with RF positivity and with normal levels of CD19⁺ B cells together with increased CD19⁺ CD27⁻ IgD⁻ B cells.⁷⁹ Treatment with infliximab leads to decreases in the chemokines CXCL10/IP-10, CCL2/MCP-1, and CCL4/MIP-1β.⁸⁰ Serum concentrations of myeloid related protein (MRP) 8/14 protein complex at baseline were proposed predictors of response to biological therapy (adalimumab, infliximab or rituximab)⁸¹ and methotrexate⁸² in active RA patients, and may be useful for monitoring response to treatment across different mechanisms of action. Additional expanded biomarker studies are needed to help select the ideal therapy at the ideal time and dose for an individual patient.

Systemic Sclerosis (SSc, Scleroderma)

SSc is a systemic autoimmune disease characterized by extensive fibrosis in skin and internal organs. Patients with limited cutaneous SSc (lcSSc) show restricted distal skin sclerosis, long history of Raynaud’s phenomenon, and better prognosis. Diffuse cutaneous SSc (dcSSc) has much more extensive skin involvement, and earlier and more severe organ manifestations.⁸³ Major complications of SSc include skin and musculoskeletal complications, pulmonary arterial hypertension (PAH), interstitial lung disease (ILD), digital vasculopathy, renal crisis, and cardiac and gastrointestinal manifestations. Autoantibodies and other serologic markers, when present, can be very useful in ascertaining potential organ involvement, monitoring needs, or overall prognosis. However, as with other systemic autoimmune rheumatic diseases, monitoring levels once detected is usually unwarranted.

Autoantibodies

SSc is diagnosed based on clinical features, which are supplemented by the ANA profile. Anti-centromere antibodies (ACA) occur in ~20–42% pts in North America, mostly in lcSSc.^{84, 85} Anti-centromere is 97% specific for lcSSc against other connective tissue diseases, with a positive predictive value of 89.5%.⁸⁵ The most likely severe complication in ACA positive patients is PAH, while digital ulcers, myocardial and kidney involvement are rare. Anti-topoisomerase I (Scl70) antibodies are also highly specific (99.5%) and predictive (98%) for SSc.⁸⁵ They occur in 14–42% of patients in North America ⁸⁵, with the vast majority having dcSSc. These autoantibodies associate with progressive pulmonary fibrosis, digital ulcers and hand disability. Anti-RNA polymerase III (RNAP III) has 98–100% specificity for SSc and occurs in 16–20% of patients, mostly in the dcSSc subset.^{84, 85} Anti-RNAP III is associated with hand disability and renal involvement, and rarely with pulmonary fibrosis.⁸⁴ Similar to Scl70, anti-RNAP III is associated with higher rates of SSc-related mortality. Patients with anti-RNAP antibodies are about twice as likely as ACA positive and four-fold as likely at anti-Topo1 positive individuals to develop cancer within 3 years of SSc onset. ⁸⁶

Anti-Th/To and U3-RNP antibodies target nucleolar antigens. Th/To autoantibodies are directed against subunits of RNase P and RNase MRP. They occur in 2–5 % of patients with SSc, 8.4% with lcSSc, and 0.6% with dcSSC. Anti-Th/To may be a marker for PAH.⁸⁷ Anti-U3 RNP antibodies target fibrillarin and are found in 18.5% of African American patients. These patients had a younger age of onset, higher frequency of digital ulcers and pericarditis, but lower lung severity scores and no difference in survival.⁸⁸ Anti-U3 RNP is most frequent in males and African Americans with SSc and is associated with muscle involvement and increased risk of PAH.⁸⁴ Anti-U11/U12 RNP antibodies have been reported in 3.2% of SSc patients who had no other SSc-associated autoantibodies. The presence of anti-U11/U12 RNP antibodies was associated with pulmonary fibrosis (79% of antibody positive vs 37% of antibody negative) and a 2.25 greater risk of death.⁸⁹ In a recent study, anti-U11/U12 RNP antibodies were associated with myopathy as well as severe gastrointestinal disease and severe Raynaud’s phenomenon, in patients with SSc and cancer.⁹⁰

Emerging biomarkers

Because the major complications of SSc include pulmonary dysfunction and ILD, lung proteins have been studied as potential biomarkers for SSc. Serum levels of both Krebs von den Lungen protein [KL-6] and surfactant protein-D are elevated in SSc and associate with decreased forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DLCO). Elevated levels are more frequent in Scl70 positive patients than ACA positive patients.⁹¹ One of the more extensively studied biomarkers in SSc is the N-terminal prohormone of brain natriuretic peptide (NT-proBNP). Higher circulating levels of NT-proBNP are associated with more severe PAH and greater risk of mortality.⁹² However, NT-proBNP is not specific to PAH and may result from cardiac dysfunction.

Fibrosis is a major player in SSc pathogenesis. TGFβ is a key regulator of fibrosis, but its utility as a biomarker is limited by technical difficulties in measuring its free, circulating form. Mediators regulated by or related to the TGFβ family have been studied in SSc. TGFβ regulates cartilage oligomeric matrix protein (COMP; also known as thrombospondin-5), and sera from SSc patients have increased COMP levels, which correlate with the extent of skin involvement.⁹³ Growth differentiation factor 15 (GDF-15), a distant member of the TGFβ family, is elevated in sera from SSc patients. GDF-15 levels strongly correlate with a modified Rodnan severity score for skin involvement and negatively correlate with FVC and DLCO.^{94, 95} Levels of GDF-15 are increased in SSc patients with ILD compared to other SSc patients, and higher levels of GDF-15 at baseline were predictive of worse lung disease severity scores at 30 months of follow-up. Therefore, GDF-15 may be a prognostic marker for lung function. Interestingly, GDF-15 levels correlate with NT-proBNP and identified PAH with higher specificity and sensitivity compared to NT-proBNP.⁹⁶

Along with TGF family proteins, certain pro-inflammatory proteins are increased in SSc and associated with fibrosis. The pro-inflammatory cytokine IL-6 is elevated in sera of SSc patients with anti-Scl70 or anti-RNAP III, but not in patients with anti-centromere.⁹⁷ Elevated IL-6 is associated with skin fibrosis, lung fibrosis, and increased mortality,^{98, 99} but has not been conclusively correlated to disease activity. Levels of CXCL4, a pro-inflammatory chemokine that regulates several immune and non-immune cells, were higher in SSc patients compared to controls. Elevated CXCL4 associated with faster progression of skin fibrosis, PAH, and faster decline of DLCO.¹⁰⁰ S100A8 and a dimer of S100A8/A9 are calcium binding proteins involved in inflammatory processes. Their levels are higher in sera and bronchoalveolar lavage fluid from SSc patients.^{101, 102} An independent study found increased levels in lcSSc patients with lung fibrosis and Scl70 positivity, but observed no correlation to pulmonary function tests.¹⁰³ Further studies are required to identify and validate specific biomarkers and their roles in pathogenesis, and to enable early prediction of patients who will need a lung transplant.

ANCA Associated Vasculitides (AAV)

ANCA associated vasculitides (AAV) are a group of autoimmune diseases characterized by the presence of anti-neutrophil cytoplasmic antibodies (ANCA). AAV include eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss Syndrome), microscopic polyangiitis (MPA), and granulomatosis with polyangiitis (GPA, Wegener’s granulomatosis).

Eosinophilic Granulomatosis with Polyangiitis (EGPA, Churg-Strauss Syndrome)

EGPA is an AAV distinguished by a history of allergic disease in the majority of patients, as well as the presence of eosinophilic infiltration in extravascular granulomas. EGPA often starts as chronic rhinitis followed by eosinophilia, which progresses to small vessel vasculitis with associated symptoms. EGPA shares several features with asthma and hypereosinophilic syndrome (HES), making early diagnosis challenging. Currently, EGPA is often a diagnosis of exclusion and based upon associated organ damage. Therefore, identifying biomarkers for early diagnosis is important. ANCA are detected in a third of patients (mostly p-ANCA directed against myeloperoxidase [MPO]). ANCA-positive patients are less likely to develop heart and non-hemorrhagic lung involvement compared to ANCA-negative patients.

EGPA is characterized by increased numbers of circulating eosinophils (>1500 cells/μL). Glucocorticoid treatment drastically lowers eosinophil numbers, and eosinophilia is usually not an adequate biomarker for EGPA once a patient has started treatment.¹⁰⁴ Chemokine eotaxin-3 (CCL26) is one of the most widely studied biomarkers of pathological significance in EGPA. Eotaxin-3 is secreted by epithelial cells and acts as a chemoattractant for eosinophils. Higher serum levels of eotaxin-3 were associated with active EGPA, although the levels dropped significantly upon treatment.¹⁰⁵ Interestingly, eotaxin-3 levels were lower in hypereosinophilic syndrome, thereby making it potentially useful in diagnosis.¹⁰⁵ At a cut-off of 80 pg/mL, eotaxin-3 has 87.5% sensitivity and 98.6% specificity for EGPA,¹⁰⁵ suggesting that eotaxin-3 may be a highly sensitive and specific marker for distinguishing active EGPA from eosinophilic (asthma, HES), rheumatic (SLE, SSc) and other AAV diseases. Serum IgG4 levels are also increased in active EGPA, and correlate with the number of organ manifestations and Birmingham vasculitis activity score.¹⁰⁶ CCL17/TARC is secreted by peripheral blood mononuclear cells and is a chemoattractant for Th-2 type cells that are important in EGPA pathogenesis. Levels of CCL17 were elevated in patients with active EGPA compared to controls and to patients with inactive disease.¹⁰⁷ CCL17 levels decreased drastically after initiation of glucocorticoid therapy, but increased prior to clinical relapse.¹⁰⁷ A study of eicosanoid levels in excreted breath condensate found increased levels of arachidonic acid metabolite 12-hydroxy-eicosatetraenoic acid (12-HETE) in active EGPA compared to inactive EGPA, HES, asthmatic, and healthy controls.¹⁰⁸ Although progress is being made, EGPA management would benefit from biomarkers to aid with initial, early diagnosis and longitudinal information about associations of emerging biomarkers with disease outcomes.

Microscopic Polyangiitis (MPA)

MPA is another AAV affecting the arterioles, capillaries, and venules, and thereby involving the skin, nerves, gastrointestinal tract, lungs, kidneys and joints. ANCA in MPA are often directed against myeloperoxidase.¹⁰⁹ However, MPO-ANCA are not specific for MPA, as they occur in EGPA, necrotizing crescentic glomerulonephritis, sarcoidosis, IgA nephropathy, and infections.¹¹⁰ MPA flares are often accompanied by increases in anti-MPO titers, elevated ESR and/or CRP. A C-terminal fragment of apolipoprotein A1, AC-13, was elevated in MPA patients compared to EGPA, GPA, and RA patients and healthy individuals, with reduced levels following treatment. Therefore, AC-13 may serve as a specific marker for MPA disease activity.¹¹¹ Necrotizing glomerulonephritis is common in MPA patients, and anti-MPO associated glomerulonephritis is less responsive to standard of care treatments and has worse renal survival.¹¹² A serum creatinine level above 4.6 mg/dL at initial MPA diagnosis was shown to be a good predictive factor for development of end stage renal failure, with 92.3% sensitivity and 84.6%specificity.¹¹³ A recent study identified differential expression of genes associated with toll-like receptor signaling in peripheral neutrophils in MPA, however, evaluating the diagnostic potential of these patterns requires further research. ¹¹⁴

Granulomatosis with Polyangiitis (GPA, Wegener’s granulomatosis)

The hallmarks of GPA include necrotizing granulomatous inflammation in the respiratory tract and pauci-immune vasculitis, primarily in lung and kidneys. ANCA in GPA are directed mostly against proteinase-3 (PR3), and much less frequently against MPO.¹¹⁵ PR3-ANCA has a high sensitivity and specificity for the diagnosis of active GPA (>90%), although ANCA-negative cases do occur.

Even with improved therapies, up to 50% of GPA patients may face relapse within 5 years. Although an increase in ANCA is generally believed to precede relapse, many ANCA rises are not followed by a clinical relapse, and relapses may occur without a preceding ANCA rise.¹¹⁶ However, ANCA rises correlated with clinical relapses in patients with renal involvement, and the avidity of anti-PR3 antibodies increased during the period preceding clinical relapse, but not during the preceding ANCA rise.¹¹⁷ Patients with severe GPA exhibit lower levels of Fc glycosylation in PR3-ANCA,¹¹⁸ and the glycosylation profile of total IgG at the time of an ANCA rise predicted a clinical relapse in patients with severe disease.¹¹⁹

Several cytokines have been proposed to contribute to the systemic inflammation in GPA and could potentially serve as biomarkers of disease activity or upcoming flare. Serum levels of S100A8/A9 are elevated in patients with active AAV compared to those in remission or to healthy controls, and serum S100A8/A9 may identify PR3-positive patients at risk of relapse.¹²⁰ Serum levels of high mobility group box-1 protein (HMGB1) are significantly higher in patients with GPA than controls.¹²¹ Recently, a reduction in the number of Tregs was reported during disease flare in GPA, whereas expansion of both Treg and Th2 compartments was observed during remission.¹²² The utility of immunophenotyping in assisting disease monitoring needs further evaluation.

Summary/conclusions

Autoimmune connective tissue diseases are complex disorders driven by environmental, genetic, and immunological mechanisms. Antibodies are used clinically for diagnosis, but fall short due to low specificity and limited understanding of their role in pathogenicity. Moreover, autoantibodies alone are often not sufficient to identify individuals at risk of developing disease. Implementing preventive measures will require biomarkers that can predict disease progression within a specific time frame or in patients with milder disease. Newer studies have proposed several new biomarkers that could serve this purpose, but several areas still need extensive research. Despite overlapping mechanisms of pathogenesis between autoimmune connective tissue diseases, these diseases are diverse, with varied clinical presentations and therapeutic efficacies. Therefore, the underlying immunological mechanisms may vary, and it is unlikely that a single biomarker will be suitable in all diseases. Indeed, even within a single disease, multiple biomarkers may be required to account for mechanistic and clinical heterogeneity between patients. A multivariate approach accounting for several aspects of the disease, as has been developed for SLE and RA, may prove useful to support diagnosis, monitor disease progression/prognosis, and to select appropriate therapy for individual patients. Confirming the clinical validity of these approaches will require longitudinal analyses with sufficient power on well characterized clinical cohorts.

Figure 1. Biomarkers in SLE.

The initial stage in SLE pathogenesis is loss of tolerance to self-antigens. Genetic predisposition and environmental factors such as viral infections can lead to anti-nuclear antibody generation by epitope spreading. Disease onset or diagnosis occurs following the amplification of autoimmune response through the interactions between innate and adaptive immune cells (dendritic cells, T cells, and B cells), differentiation of T cells into Th1, Th2, Th17 subsets, and maturation and class switching of B cells to secrete autoantibodies. These autoantibodies form immune complexes, fix complement, and activate both classical and non-classical complement. This results in decreased complement C4 and C3, and an increase in cell bound C4d (CBCAP). The increased levels of cytokines, autoantibodies, and CBCAP, and decreased levels of C3 and C4 in circulation are the most useful biomarkers at this stage. Passive deposition of immune complexes or in situ immune complex formation in end organs such as kidneys, together with complement activation and high levels of secreted pro-inflammatory cytokines causes further organ damage. Urinary biomarkers are a convenient and effective way to monitor renal disease progression in SLE. DC; dendritic cells, BLyS: B lymphocyte stimulator, IFN: interferon, Th: T helper, ANA: antinuclear antibodies, dsDNA; anti double stranded DNA antibodies, nRNP; nuclear ribonuclear protein, TWEAK: TNF like weak inducer of apoptosis, MCP1: monocyte chemoattractant protein 1, VCAM-1: vascular adhesion molecule 1, HMGB1: high mobility group box 1 protein.

Figure 2. Biomarkers in RA.

The initial stage in RA pathogenesis is loss of tolerance to self-antigens. Genetic predisposition and environmental factors such as viral infections can lead to loss of tolerance and autoantibody generation by post-translational modification of self-proteins. Disease onset or diagnosis occurs following the amplification of autoimmune response through the interactions between innate and adaptive immune cells (dendritic cells, T cells, and B cells), leading to secretion of pro-inflammatory cytokines (IL6, TNFα) that activate macrophages and neutrophils. Both macrophages and neutrophils contribute to the adaptive immune response. Antigen-exposed T cells differentiate into Th1, Th2, Th17 subsets and maturation and class switching of B cells leads to secretion of autoantibodies. The increased levels of cytokines and autoantibodies are the most useful markers at this stage. The inflammatory cytokines secreted by macrophages, T cells and reactive oxygen species secreted by neutrophils activate synovial fibroblasts and induce osteoclast maturation from pro-osteoclasts. The ensuing inflammation causes joint damage. The soluble mediators secreted by macrophages, T cells, synovial fibroblasts and osteoclasts are the major biomarkers at this stage. TNF: tumor necrosis factor, IL6: interleukin 6, IFN: interferon, DC: dendritic cells, Th: T helper, ACPA: anti-citrullinated protein antibodies, Anti-CarP: anti-carbamylated protein antibodies, RF: rheumatoid factor, MMP: matrix metalloproteinase, RANKL: receptor activator of nuclear factor-kappa B ligand.

Table 1.

Autoantibody specificities in connective tissue diseases and their association with disease phenotype

Antibody	Associated disease phenotype	Other associated diseases	Reference
Systemic lupus erythematosus
Anti-dsDNA	Lupus nephritis, Neuropsychiatric lupus erythematosus		21
Anti-Sm	Lupus nephritis		31
Anti-Ro/SSA (Ro60)	Lupus nephritis Neonatal lupus erythematosus Subacute cutaneous lupus erythematosus	Sjögren’s syndrome	32
Anti-La/SSB	Lupus nephritis Neonatal lupus erythematosus Subacute cutaneous lupus erythematosus	Sjögren’s syndrome	32
Anti- Ribosomal P	Lupus nephritis Neuropsychiatric lupus erythematosus Pediatric-onset SLE		33,34
Anti-RNP	SLE	Systemic sclerosis	21,31
Anti- Cardiolipin	SLE, Anti-phospholipid syndrome		10
Rheumatoid arthritis
RF	Rheumatoid arthritis	Systemic lupus erythematosus, Sjögren’s syndrome	60,61
ACPA	Rheumatoid arthritis		61,65
Anti-CarP	Rheumatoid arthritis		73,74
RA33	Rheumatoid arthritis		60
Systemic sclerosis
Anti- centromere	Limited cutaneous systemic sclerosis, Pulmonary arterial hypertension		83,84,85
Scl70	Diffuse cutaneous systemic sclerosis, Pulmonary fibrosis, renal crisis		83,84,85
Anti-RNAP III	Diffuse cutaneous systemic sclerosis, renal crisis		83,86
Anti-Th/To	Limited cutaneous systemic sclerosis, Pulmonary arterial hypertension, Pulmonary fibrosis		87
ANCA associated vasculitis
Anti-PR3 ANCA	Granulomatosis with polyangiitis	Microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis	109,110
Anti-MPO ANCA	Microscopic polyangiitis, eosinophilic granulomatosis with polyangiitis	Granulomatosis with polyangiitis	109,110

ANCA: anti-neutrophil cytoplasmic antibodies, dsDNA: double stranded DNA, RNP: ribonuclear protein, RF: rheumatoid factor, ACPA: anti-citrullinated protein antibodies, anti-CarP: anti-carbamylated protein antibodies, anti-RNAP III: anti-RNA Polymerase III antibodies, ANCA: anti-neutrophil cytoplasmic antibodies, PR3: Proteinase 3, MPO: Myeloperoxidase.

What do we know?

• The spectrum of autoimmune connective tissue disorders show a large degree of overlap in disease pathogenesis.

• Autoantibodies are one of most useful biomarkers to support diagnosis, and in some disease states can be used to monitor progression or response to treatment.

• Antibodies to dsDNA are more specific for SLE, and along with several soluble mediators may assist in identifying individuals at increased risk of developing SLE and in predicting disease flares.

• ACPA are more specific for RA and precede disease onset. A circulating soluble mediator score has been proved to be useful in monitoring disease progression.

• ACA, Scl70, RNAP III, and Th/To autoantibodies are observed in SSc and although their role in pathogenic mechanisms are unknown, they may allow patients to be divided into specific subsets based on clinical features.

• Patients with EGPA show eosinophilia, but this cannot distinguish between EGPA and other rheumatic or eosinophilic pathologies once treatment is initiated. A third of patients with EGPA have MPO-ANCA.

• GPA is characterized by presence of PR3-ANCA. ANCA levels may be used to monitor disease progression in patients with renal involvement.

• A multivariate approach that encompasses different measures of pathologic processes, such as autoantibodies and soluble mediators, may prove to be most informative for early accurate diagnosis, monitoring and predicting disease progress.

What is still unknown?

• A precise understanding of disease pathogenesis, and which mechanisms are disease-specific or shared among autoimmune rheumatic diseases.

• Which cytokines, chemokines and soluble mediators will be the most informative in predicting flares in SLE, RA and AAV patients.

• Which biomarkers can monitor response to treatment across diseases or within a specific autoimmune connective tissue disease

• Which biomarkers can be used to monitor disease progression in SLE, SSc, GPA, and EGPA

• Biomarkers to aid early EGPA diagnosis

• Whether biomarkers can be used for early diagnosis of EGPA or to identify high-risk AAV patients for prevention trials

Abbreviations

AAV

ANCA associated vasculitides

ACA

Anti-centromere antibodies

ACPA

anti-citrullinated protein antibodies

ACR

American College of Rheumatology

ANA

anti-nuclear autoantibodies

ANCA

anti-neutrophil cytoplasmic antibodies

anti-CarP

antibodies against carbamylated proteins

CBCAP

cell bound complement C4 activation products

CCP

cyclic citrullinated peptides

COMP

cartilage oligomeric matrix protein also known as thrombospondin-5

CRP

C-reactive protein

dcSSc

diffuse cutaneous systemic sclerosis

DLCO

diffusing capacity of the lungs for carbon monoxide

DMARDs

disease modifying anti-rheumatic drugs

EGF

epidermal growth factor

ESR

Erythrocyte sedimentation rate

FVC

forced vital capacity

GDF-15

Growth differentiation factor 15

GPA

granulomatosis with polyangiitis also known as Wegener’s granulomatosis

HMGB-1

high mobility group box-1 protein

IFN

interferons

IL

interleukin

ILD

interstitial lung disease

lcSSc

limited cutaneous systemic sclerosis

LN

proliferative lupus nephritis

MCP

monocyte chemoattractant protein

MMP

matrix metalloproteinase

MPA

microscopic polyangiitis

MPO

myeloperoxidase

MRP

myeloid related protein

NT-proBNP

brain natriuretic peptide

PAH

pulmonary arterial hypertension

PR3

proteinase-3

RA

Rheumatoid arthritis

RF

rheumatoid factor

RNAP III

Anti-RNA polymerase III

SSA

Sjögren’s syndrome type A antigen

SLE

systemic lupus erythematosus

SLICC

Systemic Lupus International Collaborating Clinics

SSc

systemic sclerosis

TGF

tumor growth factor

TNF-RI

TNF-receptor type 1

TWEAK

TNF-like weak inducer of apoptosis

VCAM-1

vascular cell adhesion molecule

VEGF-A

vascular endothelial growth factor-A

YKL-40

cartilage glycoprotein 39.