Central tolerance is impaired in the middle‐aged thymic environment

One of the earliest hallmarks of immune aging is thymus involution, which not only reduces the number of newly generated and exported T cells, but also alters the composition and organization of the thymus microenvironment. Thymic T‐cell export continues into adulthood, yet the impact of thymus involution on the quality of newly generated T‐cell clones is not well established. Notably, the number and proportion of medullary thymic epithelial cells (mTECs) and expression of tissue‐restricted antigens (TRAs) decline with age, suggesting the involuting thymus may not promote efficient central tolerance. Here, we demonstrate that the middle‐aged thymic environment does not support rapid motility of medullary thymocytes, potentially diminishing their ability to scan antigen presenting cells (APCs) that display the diverse self‐antigens that induce central tolerance. Consistent with this possibility, thymic slice assays reveal that the middle‐aged thymic environment does not support efficient negative selection or regulatory T‐cell (Treg) induction of thymocytes responsive to either TRAs or ubiquitous self‐antigens. This decline in central tolerance is not universal, but instead impacts lower‐avidity self‐antigens that are either less abundant or bind to TCRs with moderate affinities. Additionally, the decline in thymic tolerance by middle age is accompanied by both a reduction in mTECs and hematopoietic APC subsets that cooperate to drive central tolerance. Thus, age‐associated changes in the thymic environment result in impaired central tolerance against moderate‐avidity self‐antigens, potentially resulting in export of increasingly autoreactive naive T cells, with a deficit of Treg counterparts by middle age.     

Nuclear export of histone deacetylase 7 during thymic selection is required for immune self‐tolerance

Histone deacetylase 7 (HDAC7) is a T‐cell receptor (TCR) signal‐dependent regulator of differentiation that is highly expressed in CD4/CD8 double‐positive (DP) thymocytes. Here, we examine the effect of blocking TCR‐dependent nuclear export of HDAC7 during thymic selection, through expression of a signal‐resistant mutant of HDAC7 (HDAC7‐ΔP) in thymocytes. We find that HDAC7‐ΔP transgenic thymocytes exhibit a profound block in negative thymic selection, but can still undergo positive selection, resulting in the escape of autoreactive T cells into the periphery. Gene expression profiling reveals a comprehensive suppression of the negative selection‐associated gene expression programme in DP thymocytes, associated with a defect in the activation of MAP kinase pathways by TCR signals. The consequence of this block in vivo is a lethal autoimmune syndrome involving the exocrine pancreas and other abdominal organs. These experiments establish a novel molecular model of autoimmunity and cast new light on the relationship between thymic selection and immune self‐tolerance.     

Anti‐inflammatory and immune‐modulatory impacts of berberine on activation of autoreactive T cells in autoimmune inflammation

Autoreactive inflammatory CD4⁺ T cells, such as T helper (Th)1 and Th17 subtypes, have been found to associate with the pathogenesis of autoimmune disorders. On the other hand, CD4⁺ Foxp3⁺ T regulatory (Treg) cells are crucial for the immune tolerance and have a critical role in the suppression of the excessive immune and inflammatory response promoted by these Th cells. In contrast, dendritic cells (DCs) and macrophages are immune cells that through their inflammatory functions promote autoreactive T‐cell responses in autoimmune conditions. In recent years, there has been increasing attention to exploring effective immunomodulatory or anti‐inflammatory agents from the herbal collection of traditional medicine. Berberine, an isoquinoline alkaloid, is one of the main active ingredients extracted from medicinal herbs and has been shown to exert various biological and pharmacological effects that are suggested to be mainly attributed to its anti‐inflammatory and immunomodulatory properties. Several lines of experimental study have recently investigated the therapeutic potential of berberine for treating autoimmune conditions in animal models of human autoimmune diseases. Here, we aimed to seek mechanisms underlying immunomodulatory and anti‐inflammatory effects of berberine on autoreactive inflammatory responses in autoimmune conditions. Reported data reveal that berberine can directly suppress functions and differentiation of pro‐inflammatory Th1 and Th17 cells, and indirectly decrease Th cell‐mediated inflammation through modulating or suppressing other cells assisting autoreactive inflammation, such as Tregs, DCs and macrophages.     

The immune regulatory effects of tetrahedral framework nucleic acid on human T cells via the mitogen‐activated protein kinase pathway

ObjectivesAutoimmune diseases are a heterogeneous group of diseases which lose the immunological tolerance to self‐antigens. It is well recognized that irregularly provoked T cells participate in the pathological immune responses. As a novel nanomaterial with promising applications, tetrahedral framework nucleic acid (TFNA) nanostructure was found to have immune regulatory effects on T cells in this study.Materials and MethodsTo verify the successful fabrication of TFNA, the morphology of TFNA was observed by atomic force microscopy (AFM) and dynamic light scattering. The regulatory effect of TFNA was evaluated by flow cytometry after cocultured with CD3+ T cells isolated from healthy donors. Moreover, the associated signaling pathways were investigated. Finally, we verified our results on the T cells from patients with neuromyelitis optica spectrum disorder (NMOSD), which is a typical autoimmune disease induced by T cells.ResultsWe revealed the alternative regulatory functions of TFNA in human primary T cells with steady status via the JNK signaling pathway. Moreover, by inhibiting both JNK and ERK phosphorylation, TFNA exhibited significant suppressive effects on IFNγ secretion from provoking T cells without affecting TNF secretion. Similar immune regulatory effects of TFNA were also observed in autoreactive T cells from patients with NMOSD.ConclusionsOverall, our results revealed a potential application of TFNA in regulating the adaptive immune system, as well as shed a light on the treatment of T cell–mediated autoimmune diseases.     

The effect of soluble LAG-3 (CD223) treatment in fetal thymic organ culture

Lymphocyte activation gene-3 (LAG-3; CD223) is structurally similar to CD4 and binds to MHC class II with a 100-fold higher affinity than that of CD4. Soluble LAG-3 (sLAG-3Ig) might be useful for immunotherapy by inducing MHC class II-mediated cell activation. A new form of sLAG-3Ig was constructed containing a critical binding site (D1 and D2 region) to MHC class II, combined with a Fc portion of an immunoglobulin gamma1. After treatment of sLAG-3Ig in fetal thymic organ culture from DO11.10 transgenic mouse, CD4(+) T cell precursors were increased in the positive selection but not affected in the negative selection. Further analysis by treating sLAG-3Ig on thymic epithelial cells revealed that CD40 and MHC class II were up-regulated. These results may demonstrate that the treatment of sLAG-3Ig increases the precursor frequency of CD4(+) T cells by activation of thymic epithelial cells.     

Atopic dermatitis induces the expansion of thymus‐derived regulatory T cells exhibiting a Th2‐like phenotype in mice

Atopic dermatitis (AD) is a widespread inflammatory skin disease with an early onset, characterized by pruritus, eczematous lesions and skin dryness. This chronic relapsing disease is believed to be primarily a result of a defective epidermal barrier function associated with genetic susceptibility, immune hyper‐responsiveness of the skin and environmental factors. Although the important role of abnormal immune reactivity in the pathogenesis of AD is widely accepted, the role of regulatory T cells (T_regs) remains elusive. We found that the T_reg population is expanded in a mouse model of AD, i.e. mice topically treated with vitamin D3 (VitD). Moreover, mice with AD‐like symptoms exhibit increased inducible T‐cell costimulator (ICOS)‐, cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4)‐ and Glycoprotein‐A repetitions predominant receptor (GARP)‐expressing T_regs in skin‐draining lymph nodes. Importantly, the differentiation of T_regs into thymus‐derived T_regs is favoured in our mouse model of AD. Emigrated skin‐derived dendritic cells are required for T_reg induction and Langerhans cells are responsible for the biased expansion of thymus‐derived T_regs. Intriguingly, thymus‐derived T_regs isolated from mice with AD‐like symptoms exhibit a Th2 cytokine profile. Thus, AD might favour the expansion of pathogenic T_regs able to produce Th2 cytokines and to promote the disease instead of alleviating symptoms.     

Attenuated accumulation of regulatory T cells and reduced production of interleukin 10 lead to the exacerbation of tissue injury in a mouse model of acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a pathological condition that involves diffuse lung injury and severe hypoxemia caused by pulmonary and systemic diseases. We have established a mouse model of severe ARDS, developed by intratracheal injection of α‐galactosylceramide (α‐GalCer), an activator of natural killer T (NKT) cells, followed by LPS. In the present study, we used this model to investigate the regulatory mechanism in the early inflammatory response during acute lung injury. In α‐GalCer/LPS‐treated mice, the number of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and the expression of a Treg cell‐tropic chemokine, secondary lymphoid‐tissue chemokine (SLC), in the lungs was significantly lower than in mice treated with LPS alone. Giving recombinant (r)SLC increased the number of Treg cells in α‐GalCer/LPS‐treated mice. Treatment with anti‐IFN‐γ mAb enhanced the expression of SLC and the accumulation of Treg cells in the lungs of α‐GalCer/LPS‐treated mice, whereas giving recombinant (r)IFN‐γ reduced the number of Treg cells in mice treated with LPS alone. IL‐10 production was significantly lower in α‐GalCer/LPS‐treated mice than in mice treated with LPS alone. Giving rIL‐10 prolonged survival and attenuated lung injury as a result of reduced production of inflammatory cytokines (such as IL‐1β, IL‐6, TNF‐α, and IFN‐γ) and chemokines (including MCP‐1, RANTES, IP‐10, Mig, MIP‐2, and KC) in α‐GalCer/LPS‐treated mice. Treatment with anti‐IFN‐γ mAb enhanced IL‐10 production in α‐GalCer/LPS‐treated mice. These results suggest that the attenuated accumulation of Treg cells may be involved in the development of severe ARDS through a reduction in the synthesis of IL‐10.

     

Attenuation of migration properties of CD4+ T cells from aged mice correlates with decrease in chemokine receptor expression,response to retinoic acid,and RALDH expression compared to young mice

Aging results in attenuation of abilities to mount appropriate immune responses. The influence of aging on CD4⁺ T cell migration ability toward chemokines was investigated with young and aged mice. We found functional decline in migration ability toward CCL19 and also decreased CCR7 expression level in antigen-stimulated CD4⁺ T cells from aged mice compared with those from young mice. Upon addition of retinoic acid (RA), CD4⁺ T cells from aged mice showed decreased CCR9 expression level compared to young mice and the migration ability of CD4⁺ T cells from aged mice toward CCL25 was attenuated compared to young mice. We also observed that the expression of RALDH2 mRNA was decreased in mesenteric lymph node dendritic cells from aged mice compared to those from young mice. These results demonstrate that attenuated migration abilities of CD4⁺ T cells were observed in aged mice, which correlated with decreased chemokine receptor expression. Furthermore, the reduced production and response to RA by aging may be one of the causes of such attenuated migration abilities in the intestinal immune system.     

Infusion of hESC derived Immunity‐and‐matrix regulatory cells improves cognitive ability in early‐stage AD mice

ObjectivesIn this study, we administered immunity‐and‐matrix regulatory cells (IMRCs) via tail vein (IV) and intracerebroventricular (ICV) injection to 3‐month‐old 5×FAD transgenic mice to assess the effects of IMRC transplantation on the behaviour and pathology of early‐stage Alzheimer''s disease (AD).Materials and methodsClinical‐grade human embryonic stem cell (hESC)‐derived IMRCs were produced under good manufacturing practice (GMP) conditions. Three‐month‐old 5×FAD mice were administered IMRCs via IV and ICV injection. After 3 months, the mice were subjected to behavioural tests and electrophysiological analysis to evaluate their cognitive function, memory ability and synaptic plasticity. The effect of IMRCs on amyloid‐beta (Aβ)‐related pathology was detected by thioflavin‐S staining and Western blot. Quantitative real‐time PCR, ELISA and immunostaining were used to confirm that IMRCs inhibit neuroinflammation. RNA‐seq analysis was performed to measure changes in gene expression and perform a pathway analysis in response to IMRC treatment.ResultsIMRC administration via tail vein injection significantly ameliorated cognitive deficits in early‐stage AD (5×FAD) mice. However, no significant change was observed in the characteristic pathology of AD in the ICV group. Plaque analysis revealed that IMRCs did not influence either plaque deposition or BACE1 expression. In addition, IMRCs inhibited inflammatory responses and reduced microglial activation in vivo.ConclusionsWe have shown that peripheral administration of IMRCs can ameliorate AD pathology and associated cognitive deficits.     

I kappa B kinase alpha (IKKα) activity is required for functional maturation of dendritic cells and acquired immunity to infection

Dendritic cells (DC) are required for priming antigen‐specific T cells and acquired immunity to many important human pathogens, including Mycobacteriuim tuberculosis (TB) and influenza. However, inappropriate priming of auto‐reactive T cells is linked with autoimmune disease. Understanding the molecular mechanisms that regulate the priming and activation of naïve T cells is critical for development of new improved vaccines and understanding the pathogenesis of autoimmune diseases. The serine/threonine kinase IKKα (CHUK) has previously been shown to have anti‐inflammatory activity and inhibit innate immunity. Here, we show that IKKα is required in DC for priming antigen‐specific T cells and acquired immunity to the human pathogen Listeria monocytogenes. We describe a new role for IKKα in regulation of IRF3 activity and the functional maturation of DC. This presents a unique role for IKKα in dampening inflammation while simultaneously promoting adaptive immunity that could have important implications for the development of new vaccine adjuvants and treatment of autoimmune diseases.     

Effects of indoleamine 2, 3-dioxygenase (IDO) silencing on immunomodulatory function and cancer-promoting characteristic of adipose-derived mesenchymal stem cells (ASCs)

Indoleamine 2, 3-dioxygenase (IDO) catabolizes tryptophan, mediates immunomodulatory functions, and is released by stromal cells such as mesenchymal stem cells. The aims of this study were to investigate the effects of IDO silencing on immunosuppressive function of adipose-derived mesenchymal stem cells (ASCs), T cells phenotype, and the proliferation/migration of tumor cells. ASCs isolated from adipose tissues of healthy women were transfected with IDO-siRNA. Galectin-3, transforming growth factor-β1, hepatocyte growth factor, and interleukin-10 as immunomodulators were measured in ASCs using qRT-PCR. T cells phenotype, interferon-γ, and interleukin-17 expression were evaluated in peripheral blood lymphocytes (PBLs) cocultured with IDO silenced-ASCs by flow cytometry and qRT-PCR, respectively. Scratch assay was applied to assess the proliferation/migration of MDA-MB-231 cell line. Galectin-3 was upregulated (p ˂ 0.05) while hepatocyte growth factor was downregulated (p ˂ 0.05) in IDO-silenced ASCs compared to control groups. Regulatory T cells were inhibited in PBLs cocultured with IDO-silenced ASCs; also T helper2 was decreased in PBLs cocultured with IDO-silenced ASCs relative to the scramble group. IDO-silenced ASCs caused interferon-γ overexpression but interleukin-17 downregulation in PBLs. The proliferation/migration of MDA-MB-231 was suppressed after exposing to condition media of IDO-silenced ASCs compared with condition media of untransfected (p < 0.01) and scramble-transfected ASCs (p < 0.05). The results exhibited the weakened capacity of IDO-silenced ASCs for suppressing the immune cells and promoting the tumor cells' proliferation/migration. IDO suppression may be utilized as a strategy for cancer treatment. Simultaneous blocking of immunomodulators along with IDO inhibitors may show more effects on boosting the efficiency of immune-based cancer therapies.     

Amelioration of regulatory T cells by Lactobacillus delbrueckii and Lactobacillus rhamnosus in pristane-induced lupus mice model

Regulatory T cells (Tregs) play an indispensable role in the control of immune responses and induction of peripheral tolerance. Dysregulation of Tregs is involved in the pathogenesis of systemic lupus erythematosus (SLE). Tolerogenic probiotics have shown beneficial effects in the control of autoimmune diseases. We evaluated the prophylactic and therapeutic effects of Lactobacillus delbrueckii and Lactobacillus rhamnosus on Tregs and their related molecules in pristane-induced lupus mice model. Fifty-four female BALB/c mice (3–5 weeks) were randomly divided into nine groups. Lupus was induced in all groups using pristane. Prophylactic groups were treated from Day 0 (at the time of pristane injection) and treatment groups were treated 2 months later with L. rhamnosus, L. delbrueckii, mix of both probiotics, and prednisolone. One group was considered as SLE-induced control group without any treatment. Presence of antinuclear antibodies (ANA), antidouble-stranded DNA (anti-dsDNA), antiribonucleoprotein (anti-RNP), proteinuria, and serum level of creatinine, urea, the expression of forkhead box P3 (Foxp3), interleukin 6 (IL-6), IL-10, transforming growth factor β, and the number of Tregs were determined. SLE induction by pristane led to the formation of lipogranuloma, presence of ANA, anti-dsDNA, and anti-RNP. Probiotics consumption decreased the level of lipogranuloma, ANA, and anti-dsDNA. In addition, in probiotics receiving groups, Tregs and the expression level of Foxp3 increased, while IL-6 decreased. The effect of probiotics in the prophylactic group was more prominent. The results may indicate the effectiveness of L. delbrueckii and L. rhamnosus in the enhancement of Tregs and the decrease of inflammatory cytokines and disease severity in SLE-induced mice.     

Phytohemagglutinin-enhanced hybrid colony formation by cells from aged mice: Expression of th 4mod-1 mouse locus in human-mouse hybrids

Summary Cells from a continuous human line and freshly isolated cells from old adult mice heterozygous at theMod-1 locus were fused in the presence of polyethylene glycol (PEG). The production of hybrid cells, as a function of PEG concentration in the presence and absence of phytohemagglutining (PHA), was measured by cell survival and proliferation on selective medium. The incorporation of PHA into the fusion mixture allowed cell fusion to take place at nontoxic concentrations of PEG. PHA increased the frequency of cell fusion and increased the production of viable hybrid cells from 138- to over 2800-fold depending on cell type. The results suggest that the procedure may have broad application in promoting the fusion of cells sensitive to PEG. Clones were analyzed for isozymes of malic enzyme and glucose-6-phosphate dehydrogenase. The expression of the gene encoding X-linked mouse glucose-6-phosphate dehydrogenase confirmed that the cells were hybrids. These cells lost other mouse isozymes rapidly. In those clones in which the mouse malic enzyme gene was expressed, the product ofMod-1 ^α was detected significantly more frequently than that ofMod-1 ^b.     

Decreased regulatory T‐cells and CD4+/CD8+ ratio correlate with disease onset and progression in patients with generalized vitiligo

The aim of present study was to evaluate CD4⁺/CD8⁺ ratio and CD4⁺CD25^hiFoxP3⁺ Tregs in GV patients with reference to their effect on disease onset and progression. Flow cytometry was used for determination of CD4⁺/CD8⁺ ratio and Tregs in 82 patients and 50 controls. CD8⁺ T‐cell counts were significantly higher in GV patients as compared with controls (p = 0.003). Active GV patients showed higher CD8⁺ T‐cell counts compared with stable GV patients (p = 0.001). The CD4⁺/CD8⁺ ratio decreased significantly in patients as compared with controls (p = 0.001). Moreover, the ratio in active GV patients significantly lowered as compared with stable GV patients (p = 0.002). Significant decrease in Treg cell percentage and counts in GV patients was observed compared with controls (p = 0.009, p = 0.008) with significant reduction in FoxP3 expression (p = 0.024). Treg cell percentage and counts were significantly decreased in active GV patients compared with stable GV patients (p = 0.007, p = 0.002). Our results suggest that an imbalance of CD4⁺/CD8⁺ ratio and natural Tregs in frequency and function might be involved in the T‐cell mediated pathogenesis of GV and its progression.