Abstract
Knowledge on gastrointestinal manifestations in early systemic sclerosis (SSc) is limited. We have investigated gastrointestinal inflammation in SSc at the time of diagnosis using the inflammatory biomarker Fecal calprotectin (F-cal). Consecutive patients with suspected SSc were characterized in relation to the 2013 classification criteria for SSc and classified as SSc or SSc-like disease. F-cal levels were measured with a polyclonal ELISA (Calpro A/S, Lysaker, Norway) and levels above 50 µg/g were considered elevated. F-cal levels were compared to those of control subjects without rheumatic disease. Of 137 patients with suspected SSc, 92 were classified as SSc and 45 as SSc-like disease. Median (interquartile range) disease duration among the SSc participants was 2.5 (1.2, 4.6) years. A substantial proportion of participants classified as SSc (35/92, 38%) and SSc-like disease (14/45, 31%) exhibited elevated F-cal compared to the control group (3/41, 7.3%; p < 0.001 and p = 0.007, respectively). Elevated F-cal was associated with proton pump inhibitor usage (OR 7.14; 95% CI 2.56–29.93; p < 0.001). We conclude that elevated F-cal is present in a subgroup of patients with SSc at the time of diagnosis, suggesting that that GI inflammation may be present in this patient group early in the disease course. F-cal did not exhibit potential to differentiate SSc from SSc-like disease.
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Introduction
Systemic sclerosis (SSc) is a systemic autoimmune disease characterized by a complex interaction of vasculopathy, immune dysregulation, and fibrosis [1]. Gastrointestinal (GI) manifestations are common, with prevalence estimates ranging up to 90%. GI manifestations have been attributed to a combination of smooth muscle atrophy and neural dysfunction, but the underlying pathology is still not well understood [2]. Yet, histopathological studies show that mural fibrosis and mucosal inflammation of the GI tract is present in this patient group [3].
GI manifestations have a profound and negative impact on health-related quality of life in the SSc population, and may fluctuate over time [4]. In addition to somatic discomfort, such as fecal incontinence, nausea, malnutrition and pain, GI morbidity may also indirectly affect quality of life negatively, causing social limitation or even depression [2, 5]. GI morbidity may develop during early stages of the disease course, causing irreversible tissue damage [6]. GI manifestations during early SSc have also been associated with increased mortality, partly because of malnutrition but also as a consequence of severe complications such as pseudo-obstruction [7].
Diagnosis of early SSc can be challenging. Both the 2013 American College of Rheumatology-European League Against Rheumatism (ACR-EULAR) classification criteria and the Very Early Diagnosis of Systemic Sclerosis (VEDOSS) criteria were partly defined to facilitate early diagnosis of SSc [8, 9]. GI items are not included in either set of criteria.
Calprotectin is a dimeric protein found in the cytoplasm of immune cells, especially neutrophils. It may be actively secreted or passively released during cellular turnover, and serves as a regulator of the innate immune response through the Toll-like receptor signaling pathway [10]. Calprotectin is a stable protein that can be measured in feces (F-cal). F-cal is an established sensitive and non-invasive marker of ongoing mucosal GI inflammation available for clinical use [10, 11]. F-cal has been reported to be elevated in patients with SSc when compared to both healthy controls and patients with other rheumatic diseases such as Sjögren’s syndrome and rheumatoid arthritis, and has also been associated with GI morbidity [12, 13]. However, F-cal has not been explored in early SSc and its diagnostic potential in the classification of SSc has not been studied.
The primary objective of this study was to investigate F-cal levels in recently diagnosed SSc. A secondary aim was to investigate the diagnostic potential of F-cal in the classification of SSc. An exploratory aim was to study F-cal in relation to GI features of early SSc.
Methods
In this cross-sectional, observational study, all patients referred to the Department of Rheumatology at Skåne University Hospital, for in-house evaluation of newly onset SSc between January 1st, 2015, and December 31st, 2020, were considered for inclusion. All patients were referred to the SSc center by a senior rheumatologist because of suspected SSc. Subjects were invited to participate in the current study if they were able to produce a stool sample within ≤ 12 months of their initial evaluation at the clinic. Subjects were excluded from the study if they had previously been diagnosed with a non-SSc condition that is associated with elevation of F-cal. Conditions included in this definition were inflammatory bowel disease (IBD), GI malignancy, concurrent GI infection, recent GI surgery (≤ 4 weeks), or usage of permanent ostomy pouch.
Clinical characteristics were assessed at the first patient visit and collected retrospectively for all patients from the electronic hospital record charts. In addition, the following data were collected: age, sex, height, weight, disease subtype, serology, tobacco smoking, and disease duration defined as years since onset of first non-Raynaud’s Phenomenon (RP) disease manifestation. Gastrointestinal morbidity defined as the presence of reflux symptoms, dysphagia fecal incontinence and involuntary weight loss was collected by the physician through an in-house standardized protocol. Recent (within 1 week) use of proton pump inhibitors (PPI) or non-steroidal anti-inflammatory drugs (NSAIDs) and history of GI symptoms were noted. Esophageal dilation observed during high-resolution computed tomography (HRCT) was evaluated. Esophageal function was further investigated by barium cineradiography and classified on a scale from 0 (no involvement) to 3 (severe dysfunction/aperistalsis). All participants were examined in reference to the 2013 ACR-EULAR classification criteria, and those who fulfilled criteria were classified as “SSc patients”. Participants investigated for suspected SSc, but who did not fulfill the 2013 ACR-EULAR criteria, were classified as having “SSc-like disease”. Consequently, the group with SSc-like disease consisted of patients with new-onset symptoms indicative of SSc according to a senior rheumatologist, but who did not meet the 2013 classification criteria. These patients were further classified in reference to the VEDOSS criteria. Age- and sex-matched controls without rheumatic disease or IBD were recruited among hospital staff and their relatives or friends.
Stool samples were analyzed within 4 days of production, using a polyclonal Enzyme-Linked Immunosorbent Assay (ELISA) for detection of calprotectin (Calpro A/S, Lysaker, Norway). Laboratory analysis was performed according to the manufacturer’s instructions [14]. Briefly, stool samples were added to an extraction buffer 1:50 and subject to physical agitation. The extract was further diluted 1:100 in a dilution buffer, and 100 µl was then added to ELISA plate coated with affinity-purified monoclonal mouse antibodies specific for calprotectin. Calprotectin levels were measured by means of alkaline phosphatase-labeled rabbit antibodies against calprotectin. The ELISA had a lower detection limit of 30 µg/g. Calprotectin was defined as elevated if above 50 µg/g according to the manufacturer’s instructions and previous reports [15, 16].
For group comparison of categorical and dichotomous data, the χ2 or Fisher’s exact test was used, as appropriate. Receiver operating characteristic (ROC) curve analysis was performed and area under curve (AUC) was calculated. Odds ratios with 95% confidence intervals were calculated to estimate effect size. For group comparison of continuous and ordinal data, the Mann–Whitney U test was used. A significance level of α = 0.05 was used. All analyses were made using IBM SPSS Statistics version 27.0.0.0 and R version 4.1.1.
Ethical approval was obtained from the Regional Ethics Review Board, Lund, Sweden (reference number 2011/596) and written consent was provided by all patients and controls according to the Declaration of Helsinki.
Results
In total, 178 participants were included in this study. Of these, 137 were patients with suspect newly onset SSc and 41 were healthy controls. Of the 137 patients, 92 were classified as SSc and 45 were classified as SSc-like disease. In all three groups, participants were of similar age and a similar proportion was female. Patient characteristics of these groups are shown in Table 1.
Among participants with confirmed SSc, median disease duration was 1.5 years. Diffuse cutaneous (dcSSc) subtype was diagnosed in 14/92 (15%) of these. Of patients with SSc-like disease, 30/45 (67%) exhibited RP, 14/43 (33%) exhibited pathological capillaroscopy, and 3/45 (6.7%) exhibited sclerodactyly. The VEDOSS criteria were met by 19/45 (42%) of these patients. Stool collection was made within 1 week of the initial clinical examination in 92% (127/138) of the study subjects.
Of the SSc patients, 35/92 exhibited an elevated F-cal measurement. This was more common compared to the healthy control group, 3/41 (38 vs 7.3%; p < 0.001). Elevated F-cal was also more common in patients with SSc-like disease compared to the control group, 14/45 (31 vs 7.3%; p = 0.007; Fig. 1). The ROC analysis resulted in a maximum AUC of 0.728 (CI: 0.657–0.800) with a suggested optimal F-cal cut-off of 31.5 to differentiate SSc patients from healthy controls, yielding a specificity of 0.644 and a sensitivity of 0.533.
Similar proportions of patients with SSc and patients with SSc-like disease exhibited elevated F-cal (38 vs 31%; p = 0.427). Median F-cal did not significantly differ between these groups (35 vs < 30 µg/g; p = 0.248), indicating that F-cal was not able to discriminate between patients fulfilling the 2013 classification criteria and patients with SSc-like disease (Fig. 1). This was supported by ROC analysis, with an AUC of 0.557 (CI: 0.459–0.655) and a suggested optimal F-cal cut-off of 23.5 to differentiate SSc from SSc-like, yielding a specificity of 0.939 and sensitivity of 0.533. Elevated F-cal levels were seen in 2/19 (11%) of the subgroup fulfilling the VEDOSS criteria.
Among the SSc patients, elevated F-cal did not associate with clinical features of disease including esophageal dilatation, disease duration or the investigated GI symptoms (reflux, dysphagia, fecal incontinence, and involuntary weight loss). Also, we did not find any association between esophageal dysmotility and F-cal (p = 0.444). PPI use was significantly more common in SSc patients who had an elevated F-cal measurement compared to those who did not (OR 7.14; 95% CI 2.56–29.93; p < 0.001, Fig. 2).
Discussion
In this cross-sectional study, we show that calprotectin levels in feces are elevated in a substantial proportion of SSc patients compared to healthy controls already at the time of diagnosis. These results are in agreement with the hypothesis that GI inflammation may be an early manifestation of SSc [6] Calprotectin has been established as a marker of inflammation and neutrophil activation in several diseases, including SSc [17]. Recently, calprotectin dependent neutrophil mediated inflammation has been described in relation to disease pathogenesis in SSc, further promoting the rationale of exploring calprotectin as biomarker in SSc [18]. Our study are in line with these reports, as 35 out of 92 patients exhibited elevated levels of F-cal.
In this study, we also show that F-cal lacks discriminatory power to separate SSc, as defined by the 2013 ACR-EULAR criteria, from SSc-like disorders in a diagnostic tertiary referral setting. One reason for this may be that patients with SSc-like disorders, including VEDOSS, experience GI inflammation at similar rate as patients classified as SSc. GI symptoms were not associated with elevated F-cal in this study. This is partly in agreement with previous studies suggesting that GI inflammation does not necessarily correlate with self-reported GI morbidity in SSc [19]. However, these results differ from studies assessing SSc GI symptoms using the University of California Gastrointestinal Rating Scale [13]. Unfortunately, this questionnaire, developed specifically for SSc, was not used in this study as we were not aware of a Swedish translation at the study inception.
In this study, F-cal elevation was not associated with radiological findings of esophageal disease, which differs from previous studies [12, 13]. It is possible that the short disease duration of patients investigated here may account for these results, especially if GI inflammation is thought to precede GI dysmotility over the disease course. It should also be noted that calprotectin in stool may originate from both the upper and the lower GI tract [20].
PPI use was significantly associated with elevated F-cal in SSc patients. This association has also been reported in other cohorts [21]. One possible explanation is that patients with GI inflammation are more likely to use PPIs. Another possible explanation is that PPI use may cause increased levels of F-cal, although such a mechanism remains unclear [22].
Early diagnosis of SSc remains a clinical challenge, and GI abnormalities have previously been reported in patients with suspect SSc not fulfilling the 2013 ACR-EULAR criteria [23]. In addition, GI pathologies have been presented as independent risk factors for VEDOSS patients to develop SSc [24]. It remains to be explored to what degree pathological F-cal testing can predict future development of SSc.
One limitation of our study is that patients with elevated F-cal levels were not investigated endoscopically, and we were consequently unable to evaluate if the F-cal originated from inflamed mucosa. Another limitation is the cross-sectional study design, wherein we do not know if patients with increased F-cal later developed GI symptoms, or vice versa. Selection bias may also pose an issue, as patients experiencing GI discomfort may be less inclined to provide voluntary stool samples. A strength of this study is the relatively large group of well-characterized participants with new-onset SSc and SSc-like disease.
In conclusion, we report that a substantial proportion of patients with SSc exhibit elevated F-cal levels already at the time of diagnosis, suggesting that GI inflammation is present in early stages of this disease. F-cal could not discriminate SSc from SSc-like diseases in a tertiary diagnostic setting. The pathophysiology and time course of GI manifestations of SSc need further exploration.
Previous publication
Some of the results reported in this manuscript has previously been published as a congress abstract for the EULAR Congress 2022 [25].
Data availability
Unidentified patient data are available upon reasonable request at: viggo.hamberg@gmail.com.
References
Denton CP, Khanna D (2017) Systemic sclerosis. Lancet 390(10103):1685–1699. https://doi.org/10.1016/s0140-6736(17)30933-9
Volkmann ER, McMahan Z (2022) Gastrointestinal involvement in systemic sclerosis: pathogenesis, assessment and treatment. Curr Opin Rheumatol 34(6):328–336. https://doi.org/10.1097/BOR.0000000000000899
Sallam H, McNearney TA, Chen JD (2006) Systematic review: pathophysiology and management of gastrointestinal dysmotility in systemic sclerosis (scleroderma). Aliment Pharmacol Ther 23(6):691–712. https://doi.org/10.1111/j.1365-2036.2006.02804.x
Bering J, Griffing WL, Crowell M, Umar SB (2021) Progression of gastrointestinal symptoms over time in patients with systemic sclerosis. Rheumatol Int 41(7):1281–1287. https://doi.org/10.1007/s00296-021-04806-6
Frantz C, Avouac J, Distler O, Amrouche F, Godard D, Kennedy AT, Connolly K, Varga J, Matucci-Cerinic M, Allanore Y (2016) Impaired quality of life in systemic sclerosis and patient perception of the disease: a large international survey. Semin Arthritis Rheum 46(1):115–123. https://doi.org/10.1016/j.semarthrit.2016.02.005
Sjogren RW (1996) Gastrointestinal features of scleroderma. Curr Opin Rheumatol 8(6):569–575. https://doi.org/10.1097/00002281-199611000-00012
Richard N, Hudson M, Wang M, Gyger G, Proudman S, Stevens W, Nikpour M, Baron M, Group CSR, Group ASI (2019) Severe gastrointestinal disease in very early systemic sclerosis is associated with early mortality. Rheumatology 58(4):636–644. https://doi.org/10.1093/rheumatology/key350
van den Hoogen F, Khanna D, Fransen J, Johnson SR, Baron M, Tyndall A, Matucci-Cerinic M, Naden RP, Medsger TA Jr, Carreira PE, Riemekasten G, Clements PJ, Denton CP, Distler O, Allanore Y, Furst DE, Gabrielli A, Mayes MD, van Laar JM, Seibold JR, Czirjak L, Steen VD, Inanc M, Kowal-Bielecka O, Muller-Ladner U, Valentini G, Veale DJ, Vonk MC, Walker UA, Chung L, Collier DH, Csuka ME, Fessler BJ, Guiducci S, Herrick A, Hsu VM, Jimenez S, Kahaleh B, Merkel PA, Sierakowski S, Silver RM, Simms RW, Varga J, Pope JE (2013) 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. Arthritis Rheum 65(11):2737–2747. https://doi.org/10.1002/art.38098
Avouac J, Fransen J, Walker UA, Riccieri V, Smith V, Muller C, Miniati I, Tarner IH, Randone SB, Cutolo M, Allanore Y, Distler O, Valentini G, Czirjak L, Muller-Ladner U, Furst DE, Tyndall A, Matucci-Cerinic M, Group E (2011) Preliminary criteria for the very early diagnosis of systemic sclerosis: results of a Delphi Consensus Study from EULAR Scleroderma Trials and Research Group. Ann Rheum Dis 70(3):476–481. https://doi.org/10.1136/ard.2010.136929
Pruenster M, Vogl T, Roth J, Sperandio M (2016) S100A8/A9: from basic science to clinical application. Pharmacol Ther 167:120–131. https://doi.org/10.1016/j.pharmthera.2016.07.015
Lehmann FS, Burri E, Beglinger C (2015) The role and utility of faecal markers in inflammatory bowel disease. Therap Adv Gastroenterol 8(1):23–36. https://doi.org/10.1177/1756283X14553384
Andréasson K, Saxne T, Scheja A, Bartosik I, Mandl T, Hesselstrand R (2014) Faecal levels of calprotectin in systemic sclerosis are stable over time and are higher compared to primary Sjögren’s syndrome and rheumatoid arthritis. Arthritis Res Ther 16(1):R46. https://doi.org/10.1186/ar4475
Marie I, Leroi AM, Menard JF, Levesque H, Quillard M, Ducrotte P (2015) Fecal calprotectin in systemic sclerosis and review of the literature. Autoimmun Rev 14(6):547–554. https://doi.org/10.1016/j.autrev.2015.01.018
CALPROLAB (2015) CALPROLAB™ Calprotectin ELISA (ALP). In: A/S C (ed) Version 08 ed., Lysaker, Norway
Andreasson K, Alrawi Z, Persson A, Jonsson G, Marsal J (2016) Intestinal dysbiosis is common in systemic sclerosis and associated with gastrointestinal and extraintestinal features of disease. Arthritis Res Ther 18(1):278. https://doi.org/10.1186/s13075-016-1182-z
Burri E, Manz M, Rothen C, Rossi L, Beglinger C, Lehmann FS (2013) Monoclonal antibody testing for fecal calprotectin is superior to polyclonal testing of fecal calprotectin and lactoferrin to identify organic intestinal disease in patients with abdominal discomfort. Clin Chim Acta 416:41–47. https://doi.org/10.1016/j.cca.2012.11.008
Ebrahimi B, Nazarinia M, Molayem M (2021) Calprotectin, an available prognostic biomarker in systemic sclerosis: a systematic review. Clin Rheumatol 40(5):1709–1715. https://doi.org/10.1007/s10067-020-05446-0
Kuley R, Stultz RD, Duvvuri B, Wang T, Fritzler MJ, Hesselstrand R, Nelson JL, Lood C (2021) N-formyl Methionine peptide-mediated neutrophil activation in systemic sclerosis. Front Immunol 12:785275. https://doi.org/10.3389/fimmu.2021.785275
Thonhofer R, Siegel C, Trummer M, Graninger W (2012) Early endoscopy in systemic sclerosis without gastrointestinal symptoms. Rheumatol Int 32(1):165–168. https://doi.org/10.1007/s00296-010-1595-y
Manz M, Burri E, Rothen C, Tchanguizi N, Niederberger C, Rossi L, Beglinger C, Lehmann FS (2012) Value of fecal calprotectin in the evaluation of patients with abdominal discomfort: an observational study. BMC Gastroenterol 12(1):5. https://doi.org/10.1186/1471-230X-12-5
Andreasson K, Scheja A, Saxne T, Ohlsson B, Hesselstrand R (2011) Faecal calprotectin: a biomarker of gastrointestinal disease in systemic sclerosis. J Intern Med 270(1):50–57. https://doi.org/10.1111/j.1365-2796.2010.02340.x
Lundgren D, Eklof V, Palmqvist R, Hultdin J, Karling P (2019) Proton pump inhibitor use is associated with elevated faecal calprotectin levels. A cross-sectional study on subjects referred for colonoscopy. Scand J Gastroenterol 54(2):152–157. https://doi.org/10.1080/00365521.2019.1566493
Lepri G, Guiducci S, Bellando-Randone S, Giani I, Bruni C, Blagojevic J, Carnesecchi G, Radicati A, Pucciani F, Marco M-C (2015) Evidence for oesophageal and anorectal involvement in very early systemic sclerosis (VEDOSS): report from a single VEDOSS/EUSTAR centre. Ann Rheum Dis 74(1):124–128. https://doi.org/10.1136/annrheumdis-2013-203889
Trapiella-Martínez L, Díaz-López JB, Caminal-Montero L, Tolosa-Vilella C, Guillén-Del Castillo A, Colunga-Argüelles D, Rubio-Rivas M, Iniesta-Arandia N, Castillo-Palma MJ, Sáez-Comet L, Egurbide-Arberas MV, Ortego-Centeno N, Freire M, Vargas-Hitos JA, Ríos-Blanco JJ, Todolí-Parra JA, Rodríguez-Carballeira M, Marín-Ballvé A, Chamorro-Fernández AJ, Pla-Salas X, Madroñero-Vuelta AB, Ruiz-Muñóz M, Fonollosa-Pla V, Simeón-Aznar CP (2017) Very early and early systemic sclerosis in the Spanish scleroderma registry (RESCLE) cohort. Autoimmun Rev 16(8):796–802. https://doi.org/10.1016/j.autrev.2017.05.013
Hamberg V, Wallman JK, Mogard E, Lindqvist E, Olofsson T, Andréasson K (2022) AB0662 Elevated fecal calprotectin in early systemic sclerosis. Ann Rheum Dis 81(Suppl 1):1459–1460. https://doi.org/10.1136/annrheumdis-2022-eular.1071
Acknowledgements
This study was funded by the Anna-Greta Crafoord Foundation, the Swedish Rheumatism Association, Stiftelsen Ulla och Roland Gustafssons Donationsfond.
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Open access funding provided by Lund University.
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Conceptualization: VH and KA. Collection of data: JKW, EM, EL, TO, KA. Literature search and data analysis: VH. Draft writing: VH. Critical revision: VH, JKW, EM, EL, TO, KA. All authors accept responsibility for the accuracy and integrity of the final manuscript as submitted.
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VH: none. JKW: consultant of AbbVie, Amgen, Celgene, Eli Lilly, Novartis (unrelated to the current study). EM: consultant of Novartis (unrelated to the current study). EL: none. To has performed consulting tasks for Merck Sharp and Dohme and for Eli Lilly (unrelated to the current study). KA: none.
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Hamberg, V., Wallman, J.K., Mogard, E. et al. Elevated fecal levels of the inflammatory biomarker calprotectin in early systemic sclerosis. Rheumatol Int 43, 961–967 (2023). https://doi.org/10.1007/s00296-022-05264-4
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DOI: https://doi.org/10.1007/s00296-022-05264-4