α₉β₁ integrin & its ligands as new potential biomarkers in FMF

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.

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.

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 RA^12,13. 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 process^16,17,18.

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 inflammation^19,20,21.

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 it^29,30. 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.