Artemisinin Analogue SM934 Ameliorates Murine Experimental Autoimmune Encephalomyelitis through Enhancing the Expansion and Functions of Regulatory T Cell

Background

Artemisinin analogue SM934 was previously reported to possess immunosuppressive properties. The aim of this study was to determine the effects and the underlying mechanisms of SM934 in murine experimental autoimmune encephalomyelitis (EAE).

Methods

Female C57BL/6 mice immunized with MOG_35–55 were treated with or without SM934, then the clinical scores and other relevant parameters were assessed. Th1, Th17 and regulatory T (Treg) cell profiles were determined through ELISA, qRT-PCR, flow cytometry and BrdU incorporation assay. The effects of SM934 on Th1, Th17 and Treg cells differentiation were explored through intracellular staining and flow cytometry examination.

Results

In vivo, administration of SM934 significantly inhibited the development of EAE and suppressed the elevation of serum IL-17. Ex vivo, upon antigen-recall stimulation, IL-2, IFN-γ, IL-17 and IL-6 production were decreased, whereas IL-10 and TGF-β production were increased from the splenocytes isolated from SM934-treated mice. Consistently, both flow cytometry and qRT-PCR results showed that SM934 treatment significantly increased the Treg, while strongly suppressed the Th17 and Th1, responses in the peripheral. Furthermore, in the spinal lesion, SM934 treatment dramatically decreased the infiltration of CD4⁺ T cells, within which the Treg cells percentage was enlarged, whereas the Th17, but not Th1 percentage, was significantly decreased comparing with the vehicle-treated groups. Finally, both BrdU incorporation and in vitro Treg differentiation assays revealed that SM934 treatment could directly promote the expansion of Treg cells in vivo and in vitro.

Conclusion

Taken together, this study demonstrated that SM934 treatment could ameliorate the murine EAE disease, which might be mediated by inducing Treg differentiation and expansion.     

PD-L1 Blockade Differentially Impacts Regulatory T Cells from HIV-Infected Individuals Depending on Plasma Viremia

Blocking the PD-1/PD-L1 pathway has emerged as a potential therapy to restore impaired immune responses in human immunodeficiency virus (HIV)-infected individuals. Most reports have studied the impact of the PD-L1 blockade on effector cells and neglected possible effects on regulatory T cells (Treg cells), which play an essential role in balancing immunopathology and antiviral effector responses. The aim of this study was to define the consequences of ex vivo PD-L1 blockade on Treg cells from HIV-infected individuals. We observed that HIV infection led to an increase in PD-1+ and PD-L1+ Treg cells. This upregulation correlated with disease progression and decreased under antiretroviral treatment. Treg cells from viremic individuals had a particularly high PD-1 expression and impaired proliferative capacity in comparison with Treg cells from individuals under antiretroviral treatment. PD-L1 blockade restored the proliferative capacity of Treg cells from viremic individuals but had no effect on its suppressive capacity. Moreover, it increased the viral production in cell cultures from viremic individuals. This increase in viral production correlated with an increase in Treg cell percentage and a reduction in the CD4/Treg and CD8/Treg cell ratios. In contrast to the effect of the PD-L1 blockade on Treg cells from viremic individuals, we did not observe a significant effect on the proliferative capacity of Treg cells from individuals in whom viremia was controlled (either spontaneously or by antiretroviral treatment). However, PD-L1 blockade resulted in an increased proliferative capacity of HIV-specific-CD8 T cells in all subjects. Taken together, our findings suggest that manipulating PD-L1 in vivo can be expected to influence the net gain of effector function depending on the subject’s plasma viremia.     

Nanotechnology as a New Therapeutic Approach to Prevent the HIV-Infection of Treg Cells

Background

HIV-1 has proved to infect regulatory T cells (Treg) modifying their phenotype and impairing their suppressive capacity. As Treg cells are a crucial component in the preservation of the immune homeostasis, we researched that the antiviral capacity of carboxilan dendrimers prevents the HIV-1 infection of Treg and their effects. The phenotype and suppressive capacity of Treg treated or non-treated with carbosilane dendrimers were studied by flow cytometry. Treated and non-treated Treg from healthy donors were infected with HIV-1_NL4.3. The infection of Treg cells by HIV-1, and protective effect of two dendrimers were determined by measuring antigen p24^gag in the supernatant of the culture and intracellular.

Results

The Treg cells were treated with cationic and anionic carbosilane dendrimers. The results showed that both dendrimers did not modify the phenotype and functionality of Treg cells compared with non- treated Treg cells. Anionic dendrimers showed high biocompatibility with normal activity of the Treg cells and in antiviral assays. These dendrimers were highly active against HIV-1 preventing the infection of Treg, and were able to protect the Treg from the Foxp3 downregulation induced by the HIV-1 infection.

Conclusions

This is the first work showing that the in vitro use of anionic dendrimers prevent the HIV-1 replication and the infection of expanded Treg cells in culture, which raises the possibility to use Treg cells therapeutically in HIV-1-infected subjects.     

Investigating the efficacy of amnion-derived compared with bone marrow–derived mesenchymal stromal cells in equine tendon and ligament injuries

Background aimsThis is the first study to compare the treatment of horse tendon and ligament injuries with the use of mesenchymal stromal cells (MSCs) obtained from two different sources: amniotic membrane (AMSCs) and bone marrow (BM-MSCs). The objective was to prove the ability of AMSCs to exert beneficial effects in vivo.MethodsFive million allogeneic frozen-thawed AMSCs or autologous fresh BM-MSCs were injected intralesionally in horses belonging to group A (51 horses) and group B (44 horses). The interval lesion/implantation was of 6–15 days for the AMSCs and 16–35 days for the BM-MSCs. Healing was assessed clinically and ultrasonographically. Follow-up was monitored for 2 further years from return to full work.ResultsNo significant adverse effects after MSCs treatment were seen in any of the horses studied, independent of the type of stromal cell implanted. All animals belonging to group A resumed their activities between 4–5 months after treatment, whereas animals of group B resumed their activities after 4–12 months. The rate of re-injury in horses treated with AMSCs is lower (4.00%) compared with the average observed when horses were treated with BM-MSCs (23.08%).ConclusionsThe possibility to inject allogeneic AMSCs in real time, before any ultrasonographic change occurs within the injured tendon and ligament, together with the higher plasticity and proliferative capacity of these cells compared with BM-MSCs, represents the main features of interest for this novel approach for the treatment of equine tendon diseases. An obvious active proliferative healing in the area injected with AMSCs makes these cells more effective than BM-MSCs.     

Human Amnion Mesenchymal Cells Negative Co-stimulatory Molecules PD-L1 Expression and Its Capacity of Modulating Microglial Activation of CNS

The objective of this study is to investigate the negative immunomodulatory capacity of human amniotic mesenchymal cells (AMSCs) and their possible intrinsic mechanism, by which we can confirm that they modulate microglial activation of central nervous system from multiple perspectives at the molecular level. The identification of the immune phenotype of AMSCs and microglial cells was executed by immunohistochemical methods and flow cytometry. Meanwhile, the influence and mechanism of amniotic mesenchymal cells in vitro on proliferation, cell cycle, and cytokine release of activated microglia (MI) would be detected by ELISA, β-liquid scintillation counting method, and flow cytometry. Human amnion mesenchymal cells highly expressed negative co-stimulatory molecules PD-L1, while its ligand PD1 was expressed with high level by activated MI. When adding the PD-L1mAb to the mixed culture system composed of AMSCs and activated MI, the proliferation inhibitory effect and the cycle-blocking effect produced by the former on the latter would be partially reversed; at the same time, the impact of the latter cytokine secretion would be adjusted. As a conclusion, AMSCs play inhibitory effects on microglial activation, proliferation, and immune effects partially through the PD-L1–PD1 signaling pathways.     

CD4+CD25hiFOXP3+ Regulatory T Cells and Cytokine Responses in Human Schistosomiasis before and after Treatment with Praziquantel

Background

Chronic schistosomiasis is associated with T cell hypo-responsiveness and immunoregulatory mechanisms, including induction of regulatory T cells (Tregs). However, little is known about Treg functional capacity during human Schistosoma haematobium infection.

Methodology

CD4⁺CD25^hiFOXP3⁺ cells were characterized by flow cytometry and their function assessed by analysing total and Treg-depleted PBMC responses to schistosomal adult worm antigen (AWA), soluable egg antigen (SEA) and Bacillus Calmette-Guérin (BCG) in S. haematobium-infected Gabonese children before and 6 weeks after anthelmintic treatment. Cytokines responses (IFN-γ, IL-5, IL-10, IL-13, IL-17 and TNF) were integrated using Principal Component Analysis (PCA). Proliferation was measured by CFSE.

Principal Findings

S. haematobium infection was associated with increased Treg frequencies, which decreased post-treatment. Cytokine responses clustered into two principal components reflecting regulatory and Th2-polarized (PC1) and pro-inflammatory and Th1-polarized (PC2) cytokine responses; both components increased post-treatment. Treg depletion resulted in increased PC1 and PC2 at both time-points. Proliferation on the other hand, showed no significant difference from pre- to post-treatment. Treg depletion resulted mostly in increased proliferative responses at the pre-treatment time-point only.

Conclusions

Schistosoma-associated CD4⁺CD25^hiFOXP3⁺Tregs exert a suppressive effect on both proliferation and cytokine production. Although Treg frequency decreases after praziquantel treatment, their suppressive capacity remains unaltered when considering cytokine production whereas their influence on proliferation weakens with treatment.     

Expression of Treg Subsets on Intestinal T Cell Immunity and Endotoxin Translocation in Porcine Sepsis After Severe Burns

In the present study, the changes of the regulatory T cells (Treg) expression, endotoxin translocation, and the relationships in intestinal lymph nodes were investigated in porcine sepsis induced by severe burns. Flow cytometry, western blot, and Tachypleus amebocyte lysate were applied to study after the burn injury model was built. We found that the upregulated Treg expression was negatively related to the CD3⁺CD4⁺/CD3⁺CD8⁺ ratio (r = ?0.832, P < 0.05) after burn injury-induced sepsis. While Treg expression and portal venous plasma endotoxin translocation levels were positively correlated (r = 0.876, P < 0.05) when compared with the control group. Moreover, we detected a transforming of T cell subsets from T helper 1 cells to T helper 2 cells. Therefore, intestinal Treg cells expression exerts immunosuppressive effects on other intestinal T lymphocytes and was closely related to endotoxin translocation in porcine sepsis after severe burns injuries. Above all, the intestinal Treg cells may play an important role in the intestinal immune barrier system after severe burns injuries.     

Quantification of Regulatory T Cells in Septic Patients by Real-Time PCR–Based Methylation Assay and Flow Cytometry

During sepsis, a relative increase of regulatory T (Treg) cells has been reported. Its persistence is associated with lymphocyte anergy, immunoparalysis and a poor prognosis. Currently, an exact quantification of human Treg cells based on protein expression of marker molecules is ambiguous, as these molecules are expressed also by activated non-regulatory T cells. Furthermore, no firm criteria for flow cytometer gate settings exist so far. Recently, a specific DNA methylation pattern within FOXP3-TSDR has been reported that allows distinguishing Treg and non-regulatory T cells, independent of their activation status. Using this epigenetic marker, we established a single-tube real-time PCR based methylation assay (QAMA) for relative quantification of Treg cells. Validation was performed on defined ratios of methylated and unmethylated target sequence and on mixtures of Treg and non-regulatory T cells. DNA-methylation was measured in CD4⁺ T cells isolated from blood samples of 30 septic patients and 30 healthy subjects and compared with results of Treg cell quantification by flow cytometry based on CD4⁺ CD25^hiCD127^low measurement. In septic patients both methods showed an increased ratio of Treg cells to all CD4⁺ T cells. In healthy individuals, the results obtained by both methods were clearly positively correlated. However, the correlation between both methods in septic patients was only weak. We showed that quantification of Treg cells by QAMA detects CD4⁺ T cells with unmethylated FOXP3-TSDR, hidden in the CD25^med/low fraction of flow cytometry. Given that unmethylated FOXP3-TSDR is the most specific feature of Treg cells to date, our assay precisely quantifies Treg cells, as it additionally detects those committed Treg cells, hidden in the CD25^med/low fraction of CD4⁺ cells. Furthermore, QAMA is a reliable method, which is easier to standardize among laboratories and can thus improve reproducibility of Treg cell quantification.     

Cell size and RNA content correlate with cell differentiation and proliferative capacity of rat keratinocytes.

Keratinocytes from rat skin were separated according to their size in a specially designed unit-gravity sedimentation chamber. The fractions obtained with this technique showed clear morphological differences, and analysis of size distribution confirmed that size was the criterion for separation. Simultaneous DNA and RNA staining of the fractions with acridine orange and subsequent flow cytometric analysis enabled one to classify cells into resting, proliferating, and differentiating stages. Cell size was not directly correlated with proliferation in situ as determined with acridine orange flow cytometry, nor with proliferative capacity in culture as assayed by BrdU/Hoechst flow cytometry. The smallest cells, exhibiting low DNA and RNA content, which do not proliferate in vivo, required a prolonged period of serum stimulation in vitro to initiate RNA and DNA synthesis. Cells of intermediate size exhibited early RNA synthesis and maximal proliferative capacity, whereas the largest cell population displayed no RNA synthesis in culture and the least proliferative capacity. In conclusion, these results suggest that RNA synthesis early after serum stimulation, in addition to a specific, optimal cell size, correlates with the proliferative capacity of keratinocytes in cell culture.     

全反式维甲酸抑制猪脂肪间充质干细胞的成脂分化

分离培养猪脂肪间充质干细胞（adipose mesenchymal stem cells, AMSCs）,流式细胞仪鉴定其表面标记.利用MTT比色检测不同浓度的全反式维甲酸（all trans retinoic acid, ATRA）对猪AMSCs增殖的影响;光学显微镜下观察猪AMSCs向脂肪细胞分化的形态学变化;油红O染色提取法分析不同浓度ATRA对猪AMSCs成脂分化的影响;RT PCR检测脂肪细胞分化标志基因LPL和aP2 mRNA的变化.MTT比色结果显示,生理浓度（1×10^－9～1×10^－8 mol/L）和药理浓度（1×10^－7～1×10^－5 mol/L）ATRA对猪AMSCs增殖均没有影响.油红O染色提取法结果表明,除1×10^－7　mol/L ATRA对猪AMSCs成脂分化没有影响外,生理浓度（1×10^－9～1×10^－8 mol/L）和其它药理浓度（1×10^－6～1×10^－5 mol/L）ATRA均显著抑制猪AMSCs成脂分化（P<0.05）.RT-PCR检测显示,ATRA显著抑制脂肪细胞分化标志基因LPL和aP2 mRNA表达（P<0.05）.     

Reduced immunomodulation potential of bone marrow-derived mesenchymal stem cells induced CCR4+CCR6+ Th/Treg cell subset imbalance in ankylosing spondylitis

Introduction

Ankylosing spondylitis (AS) is a chronic autoimmune disease, and the precise pathogenesis is largely unknown at present. Bone marrow-derived mesenchymal stem cells (BMSCs) with immunosuppressive and anti-inflammatory potential and Th17/Treg cells with a reciprocal relationship regulated by BMSCs have been reported to be involved in some autoimmune disorders. Here we studied the biological and immunological characteristics of BMSCs, the frequency and phenotype of CCR4⁺CCR6⁺ Th/Treg cells and their interaction in vitro in AS.

Methods

The biological and immunomodulation characteristics of BMSCs were examined by induced multiple-differentiation and two-way mixed peripheral blood mononuclear cell (PBMC) reactions or after stimulation with phytohemagglutinin, respectively. The interactions of BMSCs and PBMCs were detected with a direct-contact co-culturing system. CCR4⁺CCR6⁺ Th/Treg cells and surface markers of BMSCs were assayed using flow cytometry.

Results

The AS-BMSCs at active stage showed normal proliferation, cell viability, surface markers and multiple differentiation characteristics, but significantly reduced immunomodulation potential (decreased 68 ± 14%); the frequencies of Treg and Fox-P3⁺ cells in AS-PBMCs decreased, while CCR4⁺CCR6⁺ Th cells increased, compared with healthy donors. Moreover, the AS-BMSCs induced imbalance in the ratio of CCR4⁺CCR6⁺ Th/Treg cells by reducing Treg/PBMCs and increasing CCR4⁺CCR6⁺ Th/PBMCs, and also reduced Fox-P3⁺ cells when co-cultured with PBMCs. Correlation analysis showed that the immunomodulation potential of BMSCs has significant negative correlations with the ratio of CCR4⁺CCR6⁺ Th to Treg cells in peripheral blood.

Conclusions

The immunomodulation potential of BMSCs is reduced and the ratio of CCR4⁺CCR6⁺ Th/Treg cells is imbalanced in AS. The BMSCs with reduced immunomodulation potential may play a novel role in AS pathogenesis by inducing CCR4⁺CCR6⁺ Th/Treg cell imbalance.     

Foxp3+ Regulatory T Cells among Tuberculosis Patients: Impact on Prognosis and Restoration of Antigen Specific IFN-γ Producing T Cells

CD4⁺CD25⁺Foxp3⁺ Regulatory T cells (Treg) and programmed death-1 (PD-1) molecules have emerged as pivotal players in immune suppression of chronic diseases. However, their impact on the disease severity, therapeutic response and restoration of immune response in human tuberculosis remains unclear. Here, we describe the possible role of Treg cells, their M. tuberculosis driven expansion and contribution of PD-1 pathway to the suppressive function of Treg cells among pulmonary tuberculosis (PTB) patients. Multicolor flow cytometry, cell culture, cells sorting and ELISA were employed to execute the study. Our results showed significant increase in frequency of antigen-reactive Treg cells, which gradually declined during successful therapy and paralleled with decline of M. tuberculosis–specific IL-10 along with elevation of IFN-γ production, and raising the IFN-γ/IL-4 ratio. Interestingly, persistence of Treg cells tightly correlated with MDR tuberculosis. Also, we show that blocking PD-1/PD-L1 pathway abrogates Treg-mediated suppression, suggesting that the PD-1/PD-L1 pathway is required for Treg-mediated suppression of the antigen-specific T cells. Treg cells possibly play a role in dampening the effector immune response and abrogating PD-1 pathway on Treg cells significantly rescued protective T cell response, suggesting its importance in immune restoration among tuberculosis patients.     

MCL-1 is essential for survival but dispensable for metabolic fitness of FOXP3+ regulatory T cells

FOXP3⁺ regulatory T (Treg) cells are essential for maintaining immunological tolerance. Given their importance in immune-related diseases, cancer and obesity, there is increasing interest in targeting the Treg cell compartment therapeutically. New pharmacological inhibitors that specifically target the prosurvival protein MCL-1 may provide this opportunity, as Treg cells are particularly reliant upon this protein. However, there are two distinct isoforms of MCL-1; one located at the outer mitochondrial membrane (OMM) that is required to antagonize apoptosis, and another at the inner mitochondrial membrane (IMM) that is reported to maintain IMM structure and metabolism via ATP production during oxidative phosphorylation. We set out to elucidate the relative importance of these distinct biological functions of MCL-1 in Treg cells to assess whether MCL-1 inhibition might impact upon the metabolism of cells able to resist apoptosis. Conditional deletion of Mcl1 in FOXP3⁺ Treg cells resulted in a lethal multiorgan autoimmunity due to the depletion of the Treg cell compartment. This striking phenotype was completely rescued by concomitant deletion of the apoptotic effector proteins BAK and BAX, indicating that apoptosis plays a pivotal role in the homeostasis of Treg cells. Notably, MCL-1-deficient Treg cells rescued from apoptosis displayed normal metabolic capacity. Moreover, pharmacological inhibition of MCL-1 in Treg cells resistant to apoptosis did not perturb their metabolic function. We conclude that Treg cells require MCL-1 only to antagonize apoptosis and not for metabolism. Therefore, MCL-1 inhibition could be used to manipulate Treg cell survival for clinical benefit without affecting the metabolic fitness of cells resisting apoptosis.Subject terms: Disease genetics, Immune cell death     

Fingolimod Increases CD39-Expressing Regulatory T Cells in Multiple Sclerosis Patients

Background

Multiple sclerosis (MS) likely results from an imbalance between regulatory and inflammatory immune processes. CD39 is an ectoenzyme that cleaves ATP to AMP and has been suggested as a novel regulatory T cells (Treg) marker. As ATP has numerous proinflammatory effects, its degradation by CD39 has anti-inflammatory influence. The purpose of this study was to explore regulatory and inflammatory mechanisms activated in fingolimod treated MS patients.

Methods and Findings

Peripheral blood mononuclear cells (PBMCs) were isolated from relapsing-remitting MS patients before starting fingolimod and three months after therapy start. mRNA expression was assessed in ex vivo PBMCs. The proportions of CD8, B cells, CD4 and CD39-expressing cells were analysed by flow cytometry. Treg proportion was quantified by flow cytometry and methylation-specific qPCR. Fingolimod treatment increased mRNA levels of CD39, AHR and CYP1B1 but decreased mRNA expression of IL-17, IL-22 and FOXP3 mRNA in PBMCs. B cells, CD4⁺ cells and Treg proportions were significantly reduced by this treatment, but remaining CD4⁺ T cells were enriched in FOXP3⁺ cells and in CD39-expressing Tregs.

Conclusions

In addition to the decrease in circulating CD4⁺ T cells and CD19⁺ B cells, our findings highlight additional immunoregulatory mechanisms induced by fingolimod.     

Human umbilical cord blood-derived stromal cells prevent graft-versus-host disease in mice following haplo-identical stem cell transplantation

Background aimsHuman umbilical cord blood-derived stromal cells (hUCBDSC) comprise a novel population of CD34⁺ cells that has been isolated in our laboratory. They have been shown previously not only to be non-immunogenic but also to exert immunosuppressive effects on xenogenic T cells in vitro. This study investigated the role of hUCBDSC in immunomodulation in an acute graft-versus-host disease (GvHD) mouse model after haplo-identical stem cell transplantationMethodsAcute GvHD was induced in recipient (B6 × BALB/c)F₁ mice by irradiation (750 cGy) followed by infusion of bone marrow cells and splenocytes from donor C57BL/6 mice. hUCBDSC were co-transplanted in the experimental group. The survival time, body weight and clinical and histopathologic scores were recorded after transplantation. The expression of surface markers [major histocompatibility complex (MHC) I, MHC II, CD80 and CD86] on CD11c⁺ dendritic cells (DC), and the percentage of CD4⁺ regulatory T cells (Treg), in the spleens of recipient mice were examined by flow cytometryResultsThe survival time was significantly prolonged, and the clinical and histopathologic scores were reduced in mice co-transplanted with hUCBDSC. The expression levels of the surface markers on DC were significantly lower in mice transplanted with hUCBDSC compared with those without. The proportion of CD4⁺ Treg in the spleen was also increased in mice transplanted with hUCBDSCConclusionsThese results from a GvHD mouse model are in agreement with previous in vitro findings, suggesting that hUCBDSC possess immunosuppressive properties and may act via influencing DC and CD4⁺ Treg.     

Frequency and functional activity of Th17, Tc17 and other T-cell subsets in Systemic Lupus Erythematosus

To compare frequency and functional activity of peripheral blood (PB) Th(c)17, Th(c)1 and Treg cells and the amount of type 2 cytokines mRNA we recruited SLE patients in active (n = 15) and inactive disease (n = 19) and healthy age- and gender-matched controls (n = 15). The study of Th(c)17, Th(c)1 and Treg cells was done by flow cytometry and cytokine mRNA by real-time PCR. Compared to NC, SLE patients present an increased proportion of Th(c)17 cells, but with lower amounts of IL-17 per cell and also a decreased frequency of Treg, but with increased production of TGF-β and FoxP3 mRNA. Ιn active compared to inactive SLE, there is a marked decreased in frequency of Th(c)1 cells, an increased production of type 2 cytokines mRNA and a distinct functional profile of Th(c)17 cells. Our findings suggest a functional disequilibrium of T-cell subsets in SLE which may contribute to the inflammatory process and disease pathogenesis.     

Lkb1 Deficiency Alters Goblet and Paneth Cell Differentiation in the Small Intestine

The Lkb1 tumour suppressor is a multitasking kinase participating in a range of physiological processes. We have determined the impact of Lkb1 deficiency on intestinal homeostasis, particularly focussing on secretory cell differentiation and development since we observe strong expression of Lkb1 in normal small intestine Paneth and goblet cells. We crossed mice bearing an Lkb1 allele flanked with LoxP sites with those carrying a Cyp1a1-specific inducible Cre recombinase. Lkb1 was efficiently deleted from the epithelial cells of the mouse intestine after intraperitoneal injection of the inducing agent β-naphthoflavone. Bi-allelic loss of Lkb1 led to the perturbed development of Paneth and goblet cell lineages. These changes were characterised by the lack of Delta ligand expression in Lkb1-deficient secretory cells and a significant increase in the levels of the downstream Notch signalling effector Hes5 but not Hes1. Our data show that Lkb1 is required for the normal differentiation of secretory cell lineages within the intestine, and that Lkb1 deficiency modulates Notch signalling modulation in post-mitotic cells.     

Regulatory T cells (CD4CD25FoxP3) expansion in systemic sclerosis correlates with disease activity and severity

Background

The role and function of T regulatory (Treg) cells have not been fully investigated in patients with systemic sclerosis (SSc).

Methods

Ten patients with SSc donated 20 ml of peripheral blood. Activity (Valentini) and severity (Medsger) scores for SSc were calculated for all patients. Healthy volunteers (controls) were matched to each patient by gender and age. CD4⁺ cells were separated using the MACS system. The numbers of Treg cells were estimated by flow cytometry after staining for CD4, CD25, and FoxP3 and calculated as patient-to-control ratio separately for each experiment. Correlations with activity and severity indices of the disease were performed. Twenty-four-hour production of TGF-β and IL-10 by activated CD4⁺ cells was measured by ELISA in culture supernatants.

Results

The numbers of Treg cells, expressed as patient-to-control ratio, correlated significantly with both activity and severity indices (r = 0.71, p = 0.034 and r = 0.67, p = 0.044, respectively). ELISA-measured production of TGF-β and IL-10 by CD4⁺ cells was similar in patients and controls.

Conclusions

Increased numbers of Treg cells are present in patients with SSc, correlating with activity and severity of the disease. This expansion of Treg cells was not accompanied, however, by heightened TGF-β or IL-10 production. Further studies to elaborate the causes and functional significance of Treg cell expansion in SSc are needed.     

CD4+CD25+/highCD127low/- regulatory T cells are enriched in rheumatoid arthritis and osteoarthritis joints—analysis of frequency and phenotype in synovial membrane,synovial fluid and peripheral blood

Introduction

CD4⁺CD25^+/highCD127^low/- regulatory T cells (Tregs) play a crucial role in maintaining peripheral tolerance. Data about the frequency of Tregs in rheumatoid arthritis (RA) are contradictory and based on the analysis of peripheral blood (PB) and synovial fluid (SF). Because Tregs exert their anti-inflammatory activity in a contact-dependent manner, the analysis of synovial membrane (SM) is crucial. Published reports regarding this matter are lacking, so we investigated the distribution and phenotype of Tregs in concurrent samples of SM, SF and PB of RA patients in comparison to those of osteoarthritis (OA) patients.

Methods

Treg frequency in a total of 40 patients (18 RA and 22 OA) matched for age and sex was assessed by flow cytometry. Functional status was assessed by analysis of cell surface markers representative of activation, memory and regulation.

Results

CD4⁺ T cells infiltrate the SM to higher frequencies in RA joints than in OA joints (P = 0.0336). In both groups, Tregs accumulate more within the SF and SM than concurrently in PB (P < 0.0001). Relative Treg frequencies were comparable in all compartments of RA and OA, but Treg concentration was significantly higher in the SM of RA patients (P = 0.025). Both PB and SM Tregs displayed a memory phenotype (CD45RO⁺RA^-), but significantly differed in activation status (CD69 and CD62L) and markers associated with Treg function (CD152, CD154, CD274, CD279 and GITR) with only minor differences between RA and OA.

Conclusions

Treg enrichment into the joint compartment is not specific to inflammatory arthritis, as we found that it was similarly enriched in OA. RA pathophysiology might not be due to a Treg deficiency, because Treg concentration in SM was significantly higher in RA. Synovial Tregs represent a distinct phenotype and are activated effector memory cells (CD62L^-CD69⁺), whereas peripheral Tregs are resting central memory cells (CD62L⁺CD69^-).