Regulatory T cells in rheumatoid arthritis: functions, development, regulation, and therapeutic potential.

Rheumatoid arthritis (RA) is an autoimmune disease that mainly affects the joints but also leads to systemic inflammation. Auto-reactivity and dysregulation of self-tolerance are thought to play a vital role in disease onset. In the pathogenesis of autoimmune diseases, disturbed immunosuppressive properties of regulatory T cells contribute to the dysregulation of immune homeostasis. In RA patients, the functions of Treg cells and their frequency are reduced. Therefore, focusing on the re-establishment of self-tolerance by increasing Treg cell frequencies and preventing a loss of function is a promising strategy for the treatment of RA. This approach could be especially beneficial for those patients who do not respond well to current therapies. In this review, we summarize and discuss the current knowledge about the function, differentiation and regulation of Treg cells in RA patients and in animal models of autoimmune arthritis. In addition, we highlight the therapeutic potential as well as the challenges of Treg cell targeting treatment strategies.

Th1 and Th17 cells are resistant towards T cell activation-induced downregulation of CD6.

BACKGROUND: The cell surface molecule CD6 is a modulator of T cell receptor (TCR) signaling. Recently, it has been reported that CD6 is downregulated on CD4+ T cells following T cell activation. This mechanism could limit the efficacy of anti-CD6 therapeutical antibodies. METHODS: We analyzed CD6 expression on activated and non-activated Th1 cells and Th17 cells by flow cytometry. RESULTS: Our experiments confirmed a significant downregulation of CD6 on IFNγ- and IL17-negative CD4+ T cells from healthy individuals and from patients with rheumatoid arthritis following T cell activation with anti-CD3 and anti-CD28 antibodies. In contrast, CD6 expression remained stable on activated Th17 cells and Th1 cells. CONCLUSIONS: Th1 and Th17 cells are resistant towards T cell activation-induced downregulation of CD6. These findings are relevant for the future development of CD6 targeting therapies and show that CD6 expression is differentially regulated in CD4+ T cell subsets.

Membrane-bound IL-6R is upregulated on Th17 cells and inhibits Treg cell migration by regulating post-translational modification of VASP in autoimmune arthritis.

Autoimmune arthritis is characterized by impaired regulatory T (Treg) cell migration into inflamed joint tissue and by dysregulation of the balance between Treg cells and Th17 cells. Interleukin-6 (IL-6) is known to contribute to this dysregulation, but the molecular mechanisms behind impaired Treg cell migration remain largely unknown. In this study, we assessed dynamic changes in membrane-bound IL-6 receptor (IL6R) expression levels on Th17 cells by flow cytometry during the development of collagen-induced arthritis (CIA). In a next step, bioinformatics analysis based on proteomics was performed to evaluate potential pathways affected by altered IL-6R signaling in autoimmune arthritis. Our analysis shows that membrane-bound IL-6R is upregulated on Th17 cells and is inversely correlated with IL-6 serum levels in experimental autoimmune arthritis. Moreover, IL-6R expression is significantly increased on Th17 cells from untreated patients with rheumatoid arthritis (RA). Interestingly, CD4+ T cells from CIA mice and RA patients show reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Bioinformatics analysis based on proteomics of CD4+ T cells with low or high phosphorylation levels of VASP revealed that integrin signaling and related pathways are significantly enriched in cells with low phosphorylation of VASP. Specific inhibition of p-VASP reduces the migratory function of Treg cells but has no influence on effector CD4+ T cells. Importantly, IL-6R blockade restores the phosphorylation level of VASP, thereby improving the migratory function of Treg cells from RA patients. Thus, our results establish a link between IL6R signaling and phosphorylation of VASP, which controls Treg cell migration in autoimmune arthritis.

Kinase activity profiling reveals contribution of G-protein signaling modulator 2 deficiency to impaired regulatory T cell migration in rheumatoid arthritis.

The ability of regulatory T (Treg) cells to migrate into inflammatory sites is reduced in autoimmune diseases, including rheumatoid arthritis (RA). The reasons for impaired Treg cell migration remain largely unknown. We performed multiplex human kinase activity arrays to explore possible differences in the post-translational phosphorylation status of kinase related proteins that could account for altered Treg cell migration in RA. Results were verified by migration assays and Western blot analysis of CD4+ T cells from RA patients and from mice with collagen type II induced arthritis. Kinome profiling of CD4+ T cells from RA patients revealed significantly altered post-translational phosphorylation of kinase related proteins, including G-protein-signaling modulator 2 (GPSM2), protein tyrosine kinase 6 (PTK6) and vitronectin precursor (VTNC). These proteins have not been associated with RA until now. We found that GPSM2 expression is reduced in CD4+ T cells from RA patients and is significantly downregulated in experimental autoimmune arthritis following immunization of mice with collagen type II. Interestingly, GPSM2 acts as a promoter of Treg cell migration in healthy individuals. Treatment of RA patients with interleukin-6 receptor (IL-6R) blocking antibodies restores GPSM2 expression, thereby improving Treg cell migration. Our study highlights the potential of multiplex kinase activity arrays as a tool for the identification of RA-related proteins which could serve as targets for novel treatments.

3D Computed Tomography Mapping of Thoracolumbar Vertebrae Fractures.

BACKGROUND Fractures of the thoracolumbar (TL) spine represent 90% of all spinal fractures, followed by cervical and lumbar spine fractures. This study aimed to create fracture maps of the traumatic thoracolumbar (TL) fracture vertebral body (T12-L2) through the use of CT mapping as a big data visualization method to reveal recurrent patterns and characteristics of traumatic TL fractures. MATERIAL AND METHODS A consecutive series of 174 fractured vertebrae (T12-L2) was used to create three-dimensional (3D) reconstruction images, which were superimposed and oriented to fit a model vertebral template by aligning specific bio-landmarks and reducing reconstructed fracture fragments. Fracture lines were found and traced to create a fracture map of the vertebral body. RESULTS Our study consisted of 165 patients with an average age of 47 years. A total of 174 fractured vertebrae were collected, consisting of 59 T12 vertebral fractures, 60 L1 vertebral fractures, and 55 L2 vertebral fractures. Two-dimensional (2D) maps, 3D maps, and heat maps showed that the fracture lines tended to be concentrated in the upper third and anterior third of the vertebral body, as well as being distributed in annular wedges along the anterior and lateral sides of the vertebral body. When compared with T12, the distribution of fracture lines in L1 and especially in L2 was more scattered and disorganized. CONCLUSIONS Fracture maps revealed recurrent patterns and characteristics of the traumatic TL fracture vertebral body, which improves understanding of TL fractures, as well as helping to increase opportunities for follow-up research and aid clinical decision-making.

Treatment of rheumatoid arthritis with baricitinib or upadacitinib is associated with reduced scaffold protein NEDD9 levels in CD4+ T cells.

The JAK/STAT pathway plays a crucial role in the pathogenesis of rheumatoid arthritis (RA) and JAK inhibitors have emerged as a new group of effective drugs for RA treatment. Recently, high STAT3 levels have been associated with the upregulation of the scaffold protein NEDD9, which is a regulator of T-cell trafficking and promotes collagen-induced arthritis (CIA). In this study, we aimed to reveal how treatment with JAK inhibitors affects NEDD9 in CD4+ T cells from RA patients. We analyzed NEDD9 expression in CD4+ T cells from 50 patients treated with either baricitinib, tofacitinib, or upadacitinib and performed cell migration assays to assess the potential influence of JAK inhibitor treatment on CD4+ T-cell migration. We observed that treatment with baricitinib and upadacitinib is associated with reduced NEDD9 expression in CD4+ T cells. In contrast, NEDD9 levels were not altered during treatment with tofacitinib. Moreover, treatment with baricitinib was associated with a significantly reduced migratory capacity of effector CD4+ T cells but not with impaired migration of Treg cells. This study reveals previously unknown associations between JAK inhibitor treatment and NEDD9 expression and indicates that JAK inhibitors could reduce effector T-cell migration.

Migration and homeostasis of regulatory T cells in rheumatoid arthritis.

Regulatory T (Treg) cells are garnering increased attention in research related to autoimmune diseases, including rheumatoid arthritis (RA). They play an essential role in the maintenance of immune homeostasis by restricting effector T cell activity. Reduced functions and frequencies of Treg cells contribute to the pathogenesis of RA, a common autoimmune disease which leads to systemic inflammation and erosive joint destruction. Treg cells from patients with RA are characterized by impaired functions and by an altered phenotype. They show increased plasticity towards Th17 cells and a reduced suppressive capacity. Besides the suppressive function of Treg cells, their effectiveness is determined by their ability to migrate into inflamed tissues. In the past years, new mechanisms involved in Treg cell migration have been identified. One example of such a mechanism is the phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Efficient migration of Treg cells requires the presence of VASP. IL-6, a cytokine which is abundantly present in the peripheral blood and in the synovial tissue of RA patients, induces posttranslational modifications of VASP. Recently, it has been shown in mice with collagen-induced arthritis (CIA) that this IL-6 mediated posttranslational modification leads to reduced Treg cell trafficking. Another protein which facilitates Treg cell migration is G-protein-signaling modulator 2 (GPSM2). It modulates G-protein coupled receptor functioning, thereby altering the cellular activity initiated by cell surface receptors in response to extracellular signals. The almost complete lack of GPSM2 in Treg cells from RA patients contributes to their reduced ability to migrate towards inflammatory sites. In this review article, we highlight the newly identified mechanisms of Treg cell migration and review the current knowledge about impaired Treg cell homeostasis in RA.

Regulatory T Cells from Patients with Rheumatoid Arthritis Are Characterized by Reduced Expression of Ikaros Zinc Finger Transcription Factors.

Regulatory T (Treg) cells play an important role in immune tolerance and contribute to the prevention of autoimmune diseases, including rheumatoid arthritis (RA). The differentiation, function and stability of Treg cells is controlled by members of the Ikaros zinc finger transcription factor family. In this study, we aimed to reveal how the expression of Ikaros transcription factors is affected by disease activity in RA. Therefore, we analyzed the ex vivo expression of Ikaros, Helios, Aiolos and Eos in Treg cells, Th17 cells and Th1 cells from RA patients by flow cytometry. We found significantly reduced expression of Helios, Aiolos and Eos in Treg cells from RA patients as compared to healthy controls. Moreover, Helios and Aiolos levels correlated with disease activity, as assessed by DAS28-CRP. In addition, Ikaros, Helios and Aiolos were significantly downregulated in Th1 cells from RA patients, while no difference between healthy individuals and RA was observed in Th17 cells. In summary, Helios and Aiolos expression in Treg cells correlates with disease activity and the expression levels of Ikaros transcription factors are diminished in Treg cells from RA patients. This observation could explain the reduced stability of Treg cells in RA.

Truncated isoforms of GPSM2 containing the GoLoco motif region promote CD4+ T-cell migration in SLE.

OBJECTIVE: SLE is an autoimmune disease with a complex pathogenesis. T-cell infiltration into organs contributes to inflammation and organ damage in SLE. Recently, G-protein signalling modulator 2 (GPSM2) has been shown to be implicated in T-cell migration. METHODS: We analysed the expression levels of GPSM2 and of a truncated isoform of GPSM2 containing the GoLoco motif region in CD4+ T cells from patients with SLE and from healthy individuals by western blot. In a next step, we studied the role of the truncated GPSM2 isoform using a CD4+ T-cell migration assay. RESULTS: Our experiments revealed comparable levels of GPSM2 in CD4+ T cells from patients with SLE and healthy controls. In contrast, the truncated 35 kDa isoform of GPSM2 was significantly more highly expressed in CD4+ T cells from patients with SLE as compared with healthy subjects. Antibody-mediated blockade of the 35 kDa GPSM2 isoform reduced the in vitro capacity of CD4+ T cells to migrate towards the chemokine CCL20. CONCLUSIONS: A truncated GPSM2 isoform containing the GoLoco motif region is upregulated in CD4+ T cells from patients with SLE and promotes CD4+ T-cell migration. Targeting this isoform with specific antibodies might be a promising approach to reduce CD4+ T-cell infiltration into inflamed tissues and to prevent organ damage in SLE.

Morphological changes of contralateral intervertebral foramen induced by cage insertion orientation after unilateral transforaminal lumbar interbody fusion.

BACKGROUND: This study was performed to investigate the morphological changes of contralateral intervertebral foramen (IVF) based on computed tomography images of patients with lumbar spinal stenosis after unilateral transforaminal lumbar interbody fusion (TLIF) and to compare the influence of different orientation of cage insertion on these changes. METHODS: This is a retrospective cohort study. Sixty-nine patients with lumbar spinal stenosis who had undergone single-level unilateral TLIF were retrospectively analyzed. The patients were divided into two groups according to the cage insertion orientation: the oblique group (o-group, 39 cases) and the transverse group (t-group, 30 cases). The morphological parameters of contralateral IVF were measured before and 6 months after the operation. Changes in these parameters were compared and analyzed between the two groups. The 6-month clinical outcomes of the two groups were also collected and analyzed. RESULTS: There was a significant difference in the rate of increase in the segmental angle (p < 0.01) between the two groups, the mean value of segmental angle increased by an average of 29.08% ± 14.93% in the o-group and 48.63% ± 12.01% in the t-group. Overall, the posterior disc height had a significant positive correlation with the foraminal height and area. In the o-group, however, an increase in the segmental angle resulted in a decrease in the foraminal area. No significant difference in clinical outcomes was found between the two groups. CONCLUSIONS: Compared with oblique cage insertion, transverse cage insertion could achieve greater restoration of segmental lumbar lordosis without decreasing contralateral foraminal dimensions.

Changes in L4/5 Intervertebral Foramen Bony Morphology with Age.

The purpose of this study was to explore the morphological changes in L4/5 intervertebral foramen with age using a digital method. The closed boundaries of the intervertebral foramen (IGES) in different sagittal slices (inside, middle and outside) were obtained from Mimics, and then imported to a custom-written program, which provided quantitative distance between the nerve root and the closed curves. The quantitative information of each age group was used to produce radar chart and line chart for morphological and statistical analyses. Overall, the intervertebral foramen changes mainly occurred in the inner part from middle age to old age. The foraminal height decreased with age in the inside sagittal slice, while no significant difference was found in the middle sagittal slice or the outside sagittal slice. The foraminal width showed no decrease in each age group or each sagittal plane. The present study described foraminal geometry of asymptomatic males in different sagittal slices with age. This information enhances the knowledge of anatomical changes in intervertebral foramen with age, which provides better understanding of the pathology of intervertebral foramen diseases.

Three-Dimensional Morphological Characteristics of Lower Lumbar Intervertebral Foramen with Age.

Intervertebral foramen is the doorway of nerve root and it plays an important role of radiculopathy and surgical treatment of intervertebral foramen diseases. The purpose of the study is to obtain three-dimensional (3D) morphological characteristics of lumbar intervertebral foramen and their relationship with age. Pedicle-superior articular process (P-SAP), disc height between adjacent vertebra (DH), pedicle-inferior vertebrae (P-IV), inferior posterior vertebrae-superior articular process (IPV-SAP), and bony boundary area (BBA) were measured in entrance, middle slice, and exit of lumbar intervertebral foramen for 25 males of different age groups. Spinous process to intervertebral foramen entrance (SP-IFE) was measured for 25 males of different age groups. Overall, P-SAP and P-IV decreased and IPV-SAP increased from the entrance to the exit of intervertebral foramen for L3/4-L5S1. DH decreased at entrance slice, middle slice, and exit slice for L3/4-L5S1 with age. Significant difference with aging was found only at the middle slice of L3/4 and L4/5 for P-SAP. And the significant decrease of IPV-SAP was observed at middle slice of L3/4, entrance slice of L4/5 and L5S1, and exit slice of L5S1. SP-IFE is not consistent for all subjects. In addition, the decrease of BBA at L3/4 and L4/5 was observed earlier than at L5S1. The present study described detailed information of intervertebral foramen, which may be of benefit for better understanding of the pathology and surgical planning for intervertebral foramen diseases.