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α9β1 integrin & its ligands as new potential biomarkers in FMF
For correspondence: Dr Sinan Çavun, Department of Medical Pharmacology, Bursa Uludag University Faculty of Medicine, Nilufer-Bursa 16059, Turkey e-mail: scavun@uludag.edu.tr
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Received: ,
Abstract
Background & objectives
Familial Mediterranean Fever (FMF) manifests as a hereditary condition characterized by repeated bouts of fever, abdominal, chest, and joint discomfort, and swelling. Colchicine is the most common form of treatment, but it does not eliminate the disease. The underlying causes of the inflammatory mechanism are still not fully known.
Methods
A total of 20 healthy controls, 16 individuals with FMF in the attack period, and 14 in the remission period participated in the study. ITGA9, ITGB1, OPN, TNC, VEGF, VCAM-1, TGM2, TSP-1, Emilin-1, and vWF levels were measured by ELISA by obtaining serum from blood samples of individuals. In addition, gene expressions of α9β1 (ITGA9, ITGB1) and its best known ligands (TNC, SPP1) were analyzed by quantitative real-time PCR (qPCR).
Results
The findings of this study showed that serum levels of α9β1 and its ligands were higher in individuals with FMF in the attack period than in the healthy controls and the FMF group in the remission period (P<0.05). The marker levels of the healthy group were also higher than those in the remission period (p<0.05). In addition, when the gene expressions were compared between the healthy controls and FMF group, no significant difference was found for ITGA9, ITGB1, TNC, and SPP1 genes.
Interpretation & conclusions
The function of α9β1 and its ligands in FMF disease was investigated for the first time in this study as per our knowledge. Serum levels of these biomarkers may help identify potential new targets for FMF disease diagnosis and treatment approaches.
Keywords
α9β1
attack
FMF
ligands
remission
Familial Mediterranean Fever (FMF) is an ethnicity-related disease transmitted hereditarily and progresses with acute fever attack and serosal inflammation1. FMF is mainly seen in individuals in the geography close to the Mediterranean region and occurs due to point mutations in the MEFV gene2.This gene encodes the pyrin molecule, a regulatory protein with limited tissue expression, especially in neutrophils, and suppresses inflammation due to neutrophil activation. The pyrin protein, formed due to a mutation in FMF, disrupts the order in neutrophil activation3. Neutrophil activation is a defining phenomenon in the inflammatory response. The interactions between the integrins released from the surface of neutrophils and their ligands, which have crucial tasks in the development of inflammation, take an active role in critical situations such as cell activation, adhesion, and migration4.
The integrin protein family consists of alpha and beta subtypes that form transmembrane heterodimers5,6. α9β1integrin is one of the members of the integrin family that has been emphasized in recent years. Different studies have shown that α9β1 integrin plays an essential role in refractory diseases (cancer, autoimmune diseases, nerve damage, thrombosis, etc.), and it highlights the possibility of α9β1 which is considered as a potential therapeutic target7. In addition, ligands for α9β1integrin, including tenascin-C (TNC) and osteopontin (OPN), are well established as critical regulators of inflammatory conditions. However, further studies are needed on other α9β1 integrin ligands such as vascular endothelial growth factor (VEGF), vascular cell adhesion molecule (VCAM-1), thrombospondin (TSP), transglutaminase (TGM), elastin microfibril interfacer (Emilin-1), von Willebrand factor (vWF). These ligands have multiple functions, and the cell types that release these vary widely. Therefore, these contribute significantly to the functioning of the biological and chemical pathways in different body parts during the inflammatory process.
FMF is an inflammatory disease that affects all systems in the body, so it is believed that these neutrophil-related markers could potentially impact the disorder’s progression. Hence, this study was undertaken to investigate the efficacy of α9β1 integrin and its ligands in FMF disease.
Material & Methods
The study was undertaken at the department of Medical Pharmacology, Faculty of Medicine, Bursa Uludag University. The study was initiated after obtaining the approval from the Ethics Committee. and patient’s data and blood samples were provided by the department of Rheumatology of the Faculty of Medicine, Bursa Uludag University.
Twenty healthy controls (5 male, 15 female) and 30 (10 male, 20 female) individuals with FMF presenting to Rheumatology outpatient clinic, Bursa Uludag University Hospital, between March 16, 2022, and December 20, 2022, were included in this study. An informed, and a signed consent form was obtained from all those willing to participate in this study. The determination of FMF was conducted using Tel Hashomer criteria7. Accordingly, it was considered suitable for two major or one major and two minor criteria for a definitive diagnosis. The number of FMF attacks the participants had in the last six months was also considered. FMF attacks and remission period distinctions were determined by biological parameters (such as sedimentation, haemogram, and CRP values) with genetic and clinical findings. The distribution of the MEFV genotype interims of numbers of study participants as follows: M694V/-11; M680I/-4; E148Q/ -2; M694V/R761H-1; R202Q/M694V- 1; M694V/E148Q -1; M694V/V726A-1; M680I/R761H-1; E148Q/R202Q-1; no mutation-7s .All FMF patients received colchicine treatment with 0.5 to 2 mg in one or two (maximum 4) divided doses daily. Individuals in the healthy group did not receive any treatment. The study excluded people with organ failure, anemia, leukopenia, acute infection, or autoimmune/inflammatory disease. Table I shows the diagnosis criteria of FMF patients according to Tel Hashomer7.
| Total (n) | % | |
|---|---|---|
| Major criteria | ||
| Recurrent febrile episodes with serositis | 16 | 53.3 |
| Amyloidosis of AA type without a predisposing disease | 18 | 60 |
| Favorable response to regular colchicine treatment | 27 | 90 |
| Minor criteria | ||
| Recurrent febrile episodes | 20 | 66.7 |
| Erysipelas-like erythema | 8 | 26.7 |
| FMF-in a first-degree relative | 22 | 73.3 |
Measurement of markers
Blood samples (⁓20 ml) from healthy individuals and individuals with FMF were centrifuged at 3000xg for 10 min. The obtained sera were separated for each sample, moved to Eppendorf tubes, and kept at -80°C.
Concentrations of α9β1 and its ligands were measured using commercial ELISA kits (BT Lab, Zhejiang, China) as per prescribed manufacturer’s protocol. Each assay was duplicated, and the results were derived using the standard curve. The sensitivity of the assay for markers was, 0.14 ng/ml for ITGΑ9; 14.49 ng/L for ITB1;10.57 ng/L for TNC; 0.15 ng/ml for OPN; 10.42 ng/L for VEGF; 0.23 ng/ml for VCAM-1; 2.39 ng/ml for TSP1; 2.74 ng/L for TGM2; 0.048 ng/ml for Emilin-1; 0.23 ng/ml for vWF, respectively.
Quantitative real-time PCR
RNAeasy®Mini Kit (Qiagen, Germany) and RT2 First Strand Kit (Qiagen, Germany) were used for isolating RNA from the serum samples and cDNA synthesis, respectively, as previously described9. qRT-PCR assays were conducted in duplicate using RT2 SYBR Green Mastermix (Qiagen, Germany). The primer sets from commercial sources that were used in this study are detailed in Supplementary Table. Actin-β (ACTB) was utilized as the housekeeping gene. As part of the data standardization process, all Ct values were adjusted against the mean Ct values of a housekeeping gene (ACTB) present on the array using the 2−ΔΔCt method7.
Statistical analysis
The ITGA9 variable exhibited an effect size d=50. G power software (V 3.1.9.7; Heinrich-Heine-Universität Düsseldorf, Germany) was employed to derive α=0.005 and ß=0.20 values for sample size determination31. It was concluded that each group should consist of at least 14 individuals, totaling at least 42 participants for the study.
The student’s t-test was employed for comparing parameters between the groups. The mean±standard deviation was computed for the variables, and the mRNA expression analysis was conducted using RT2 Profiles PCR Array Data Analysis version 3.5 (Qiagen, Germany). ΔCt values of the difference between test and control groups was determined using One-way ANOVA. A P value below 0.05 was considered statistically significance. Statistical analysis was conducted utilizing Graph Pad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA).
Results
No significant difference was observed for the mean age value (P=0.937) between the control group (38±4.5) and FMF group (37.9±11.05). In terms of gender, the number of females was significantly (P<0.05) higher than males in each group.
Serum levels of α9β1 and its ligands
The expression of ITGA9 and ITGB1, markers of α9β1, were higher in individuals with FMF in the attack period than in the healthy controls and remission groups (P<0.05). In addition, the ITGA9 and ITGB1 levels of the healthy group were higher than the FMF group in remission (P<0.05) (Supplementary Figure). Serum levels of α9β1 ligands were also similar to α9β1. TNC, OPN, VEGF, VCAM-1, TSP-1, TGM2, Emilin-1, and vWF concentrations were highest in individuals with FMF, with an attack period. Their levels in the healthy group were higher than in the FMF group in remission (P<0.05) (Table II; Supplementary Figure).
| Study parameters | Healthy control | Attack period in participants with FMF | Remission period of participants with FMF |
|---|---|---|---|
| ITGA9 (ng/ml) | 5.6±1.1 | 7.9±1 | 3.2±1.4 |
| ITGB1 (ng/l) | 1242.6±286.4 | 2367.5±1064.2 | 506.8±254.6 |
| TNC (ng/l) | 1176.1±338.4 | 2265.5±1394.4 | 709.8±186.3 |
| OPN (ng/ml) | 10.1±3.3 | 17.8±7.1 | 5±2 |
| VEGF (ng/l) | 992.6±507 | 1367.2±441.3 | 536.6±229.3 |
| VCAM-1 (ng/ml) | 47.2±13 | 84.9±41.9 | 24.2±6.8 |
| TSP-1 (ng/ml) | 119.2±45.6 | 218.3±110.9 | 61.5±29.3 |
| TGM2 (ng/l) | 167±92.3 | 342.3±199.3 | 53.1±16.6 |
| Emilin-1 (ng/ml) | 9.6±2.9 | 18.7±10.3 | 5.5±2.5 |
| vWF (ng/ml) | 30.2±8 | 59.5±20.4 | 11.6±4.1 |
Gene expressions of ITGA9, ITGB1 and TNC, SPP1
ITGA9 and ITGB1, as the α9β1 subunits with TNC and OPN (SPP1) genes, which are the best-defined α9β1 ligands, were analyzed on the mRNA expression profile. Supplementary Table provides details regarding the primers used in this study. According to the results, it was concluded that there was no disparity in gene expression levels between the groups. However, the fold change values of the individuals with FMF in the remission period were higher for the ITGB1 (0.68 fold) and SPP1 (0.64 fold) genes compared to the healthy control. When participants in the attack and remission periods were compared, the fold change values of ITGA9 (1.57 fold) and TNC (1.47 fold) were higher in the attack group than in remission, but this was not statistically significant. The mRNA expression profile for ITGA9, ITGB1, OPN, and SPP1 is shown in Table III and IV.
| Genes | Healthy control | Attack period in participants with FMF | Remission period in participants with FMF | ||||
|---|---|---|---|---|---|---|---|
| 2^(-Avg.(∆Ct)) | 2^(-Avg.(∆Ct)) | Fold change | P value | 2^(-Avg.(∆Ct)) | Fold change | P value | |
| ACTB | 1 | 1 | 1 | 0 | 1 | 1 | 0 |
| ITGA9 | 1.03 | 0.09 | 0.09 | 0.13 | 0.08 | 0.06 | 0.15 |
| ITGB1 | 0.08 | 0.01 | 0.2 | 0.09 | 0.02 | 0.68 | 0.46 |
| TNC | 0.48 | 0.08 | 0.18 | 0.22 | 0.09 | 0.12 | 0.25 |
| SPP1 | 0.09 | 0.01 | 0.17 | 0.08 | 0.04 | 0.64 | 0.15 |
P*<0.05
| Genes | 2^(-Avg.(∆Ct)) | Attack period vs remission period in participants with FMF | P value |
|---|---|---|---|
| Fold change | |||
| ACTB | 1 | 1 | 0 |
| ITGA9 | 0.09 | 1.57 | 0.25 |
| ITGB1 | 0.01 | 0.29 | 0.35 |
| TNC | 0.08 | 1.47 | 0.36 |
| SPP1 | 0.01 | 0.29 | 0.38 |
P*<0.05
Discussion
FMF is an autoinflammatory disease (AID) characterized by recurrent fever attacks and polyserositis. It develops depending on different mechanisms, including genetic and geographical conditions. In addition to the existing information about the disease, there are current approaches to diagnosis and treatment7.This study proposed to figure out the function of α9β1, one of the frequently emphasized markers and its ligands in FMF disease. Furthermore, this study identified notable distinctions in the release of α9β1 and its ligands between the healthy controls and individuals with FMF.
OPN and TNC are two common ligands with similar structures and properties for α9β1 integrin, and binding of α9β1 integrin is primarily induced by inflammation. In the RA study 2006, it was reported that α9β1 integrin is released from osteoclasts and participates in the functionality of these cells10. Expression of α9 integrin mRNA is elevated in mouse bone marrow cells during osteoclast differentiation. In addition, impaired bone resorption has also been observed in osteoclasts from α9 integrin-deficient mice10. Like mouse RA models, α9 integrin is released from human synovial fibroblasts and macrophages11. Besides, there are essential differences between the arthritic joint regions of mice and humans. While OPN production was observed to be higher than TNC production in mouse synovial tissues during inflammatory arthritis, TNC expression in individuals with RA was also found to be considerably higher than OPN11. In addition, TNC levels in synovial cells and serum were reported to have increased in individuals with RA, and TNC levels were correlated with joint erosion. Based on these findings, it has been suggested that the interaction between α9β1 integrin and TNC may be a functional target for RA, and OPN blockade may also benefit individuals RA12,13. Besides, it has been reported that ECM proteins TNC and OPN are upregulated in various pathological foci in diseases such as autoimmune arthritis, experimental allergic encephalomyelitis (EAE), and inflammatory bowel disease (IBD). In a collagen antibody-induced arthritis model of mice, it was observed that macrophages and fibroblasts in pathological foci primarily expressed α9 integrin, and inhibition of the interaction between α9 and TNC/OPN decreased the production of inflammatory cytokines in the arthritic region14. Chabas et al 15 demonstrated overexpression of OPN transcripts in the spinal cord of rats with EAE and brain tissues of individuals with MS in microarray analysis. In addition, another study found that the induction of EAE in TNC-deficient mice was impaired through T cell down-regulation that produces IFN-γ and IL-1715.
α9β1 integrin expression is upregulated in various cancers, influencing tumor progression through different mechanisms. ITGA9 has been identified as a potential biomarker for prostate tumors and linked to drug-resistant colon cancer, as well as a diagnostic marker for hepatocellular carcinoma. Additionally, studies show that while the α9 subunit is absent in adult neurons, forced expression of α9 in certain cells promotes axonal regeneration, suggesting a significant role for the α9β1-TNC interaction in this process16,17,18.
VCAM-1, an α9β1 ligand released by activated endothelium in response to inflammation, binds to α9β1 integrin and plays a crucial role in neutrophil survival and function. Research indicates that this interaction impacts neutrophil pathophysiology and helps regulate inflammation. Additionally, studies suggest that the α9β1/VCAM-1 interaction may facilitate neutrophil migration during acute inflammation19,20,21.
A study on VEGF reported that α9β1 directly binds to VEGF-A and supports VEGF-A-induced angiogenesis22. However, previous experiments of the same study group proved that VEGF-C and D also support lymphangiogenesis by directly binding to α9β1 integrin23. Studies evaluating the relationship between VEGF and α9β1 are limited, so more studies are needed.
It has been suggested that many integrins may be associated with TGM2. The experiment conducted in collagen-induced mice determined that TGM2 activity was associated with cartilage degradation in the joints24. Different studies related to TGM2, such as cancer, vascular inflammation, tissue fibrosis, and autoimmunity, demonstrated that this marker can control the level of integrin on the cell surface. Nevertheless, the precise molecular mechanism governing this regulation remains unidentified25.
TSP-1 was previously identified as an angiogenesis modulator25. This protein was shown to directly inhibit endothelial cell functions such as proliferation and migration, but some N-module fragments stimulate similar endothelial responses. In Stanizewska et al25 study, the N-terminal domain [NoC1] binding to cells expressing α9β1activated the signaling of proteins such as Erk1/2 and paxillin. Subsequently, the proangiogenic activity of NoC1 was inhibited by α9β1 inhibitors. As a result, it was revealed that α9β1released from the microvascular endothelial surface interacts with TSP-1, which is associated with the modulation of angiogenesis26.
In a 2013 study26 on Emilin-1 protein, another α9β1ligand, it was suggested that the Emilin1/α9β1integrin interaction may play an essential role in lymphatic valve formation and maintenance. Donussi et al26 reported that Emilin1-a4/a9 integrin binding restrains dermal fibroblast and keratinocyte production. Accordingly, it was thought that Emilin1 and keratinocyte α9β1 might play a protective role in skin homeostasis. Findings regarding the involvement of homeostatic molecules in the inflammatory process highlight the link between inflammation and homeostasis27.
In addition, the link between vWF and inflammation has also taken its place among current issues. Reportedly due to the acute release of vWF from the activated endothelium, it was defined as an inflammation marker only in some pathologies. However, vWF was later found to be directly related to inflammation, suggesting it has a more extensive assignment. The ability of vWF to recruit leukocytes either by direct binding to leukocytes or via platelets has been attributed as the most crucial evidence in this regard28.There are various studies in the literature on whether the vWF level is a remarkable parameter in evaluating the course of the disease in cases of acute systemic inflammation. Still, the findings need to be more consistent. Despite some studies reporting that individuals with high vWF levels are more likely to survive than others, studies have not confirmed it29,30. Mourik et al30 showed that both vWF and propeptide levels increase during the acute response of the endothelium (septicemia). Conversely, vWFpp concentrations were only slightly elevated throughout the chronic stimulation (eg., diabetes mellitus), although vWF levels were elevated31.
Despite many studies in which systemic inflammatory diseases and various immune and genetic parameters were conducted, any study showing the relationship between FMF and α9β1 integrin molecule was not found in the literature. In this direction, we obtained significant differences in ITGA9 and ITGB1 levels of α9β1 integrin markers between healthy individuals and those with FMF. α9β1 release was highest in individuals with FMF during the attack period and more than a healthy control group in those with remission. Similarly, α9β1ligands TCN, OPN, VCAM-1, VEGF, TGM2, TSP-1, Emilin-1, and vWF levels varied significantly between groups. Based on the overall results, the expression of these parameters in individuals with FMF may provide significant benefits regarding the detection and follow up of the disease. The fact that the levels of α9β1 and ligands showed a relation with each other suggests that these factors should be handled together in the pathogenesis of FMF. In addition, the α9β1 integrin subunits ITGA9 and ITGB1 levels the gene expression of their the best known ligands, TNC and SPP1 vary between groups in terms of two genes. ITGA9 and ITGB1’s expression levels in the individuals with FMF were higher than in the healthy individuals. Accordingly, the higher expression of the α9β1 subunit ITGA9 and the SPP1 ligand in FMF individuals compared to the healthy group may provide an alternative to familiar genetic markers. It can also be evaluated genetically as an inflammation marker that can be used to diagnose FMF disease.
The main limitation of this study is the low sample size in both the study groups. In addition, some of the α9β1 ligands were examined, and the mRNA profile was studied only for the best known ligands. Examining the mRNA expression of all markers will be beneficial by adding more ligands and individuals to future studies. Additionally, further elaborate studies undertaken at the molecular level, encompassing different methods such as protein analysis in the upcoming stages, will be beneficial.
In conclusion, the present study aimed to identify the relationship between the α9β1 and FMF disease. However, the utility of α9β1 integrin as a therapeutic agent for treating inflammatory disorders needs to be further explored. A better understanding of how α9β1 integrin interacts in autoimmune/autoinflammatory diseases will contribute to developing integrin-targeted therapy approaches in this area.
Financial support & sponsorship
The study was funded by the Bursa Uludag University Scientific Research Projects Foundation (Project number:TGA-2022-1025).
Conflicts of Interest
None.
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.
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