Th17 cell promotes apoptosis of IL-23R+ neurons in experimental autoimmune encephalomyelitis.

Myelin antigen-reactive Th1 and Th17 cells are critical drivers of central nervous system (CNS) autoimmune inflammation. Transcription factors T-bet and RORγt play a crucial role in the differentiation and function of Th1 and Th17 cells, and impart them a pathogenic role in CNS autoimmune inflammation. Mice deficient in these two factors do not develop experimental autoimmune encephalomyelitis (EAE). While T-bet and RORγt are known to regulate the expression of several cell adhesion and migratory molecules in T cells, their role in supporting Th1 and Th17 trafficking to the CNS is not completely understood. More importantly, once Th1 and Th17 cells reach the CNS, how the function of these transcription factors modulates the local inflammatory response during EAE is unclear. In the present study, we showed that myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55)-specific Th1 cells deficient in RORγt could cross the blood-brain barrier (BBB) but failed to induce demyelination, apoptosis of neurons, and EAE. Pathogenic Th17 cell-derived cytokines GM-CSF, TNF-α, IL-17A, and IL-21 significantly increased the surface expression of IL-23R on neuronal cells. Furthermore, we showed that, in EAE, neurons in the brain and spinal cord express IL-23R. IL-23-IL-23R signaling in neuronal cells caused phosphorylation of STAT3 (Ser727 and Tyr705) and induced cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1) molecules in an IL-23R-dependent manner and caused apoptosis. Thus, we provided a mechanism showing that T-bet is required to recruit pathogenic Th17 cells to the CNS and RORγt-mediated inflammatory response to drive the apoptosis of IL-23R+ neurons in the CNS and cause EAE. Understanding detailed molecular mechanisms will help to design better strategies to control neuroinflammation and autoimmunity. ONE SENTENCE SUMMARY: IL-23-IL-23R signaling promotes apoptosis of CNS neurons.

Disorganization of secondary lymphoid organs and dyscoordination of chemokine secretion as key contributors to immune aging.

Aging is characterized by progressive loss of organ and tissue function, and the immune system is no exception to that inevitable principle. Of all the age-related changes in the body, reduction of the size of, and naïve T (Tn) cell output from, the thymus occurs earliest, being prominent already before or by the time of puberty. Therefore, to preserve immunity against new infections, over much of their lives, vertebrates dominantly rely on peripheral maintenance of the Tn cell pool in the secondary lymphoid organs (SLO). However, SLO structure and function subsequently also deteriorate with aging. Several recent studies have made a convincing case that this deterioration is of major importance to the erosion of protective immunity in the last third of life. Specifically, the SLO were found to accumulate multiple degenerative changes with aging. Importantly, the results from adoptive transfer and parabiosis studies teach us that the old microenvironment is the limiting factor for protective immunity in old mice. In this review, we discuss the extent, mechanisms, and potential role of stromal cell aging in the age-related alteration of T cell homeostatic maintenance and immune function decline. We use that discussion to frame the potential strategies to correct the SLO stromal aging defects - in the context of other immune rejuvenation approaches, - to improve functional immune responses and protective immunity in older adults.

In vitro-induced Foxp3+ CD8+ regulatory T cells suppress allergic IgE response in the gut.

Several subsets of CD8+ T cells are known to have a suppressive function in different tissues and diseases in mice and humans. Due to the lack of a consensus on the phenotype of regulatory CD8+ T cells and very low frequency in the body, its clinical use as adoptive cellular therapy has not advanced much. In the present work, using DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (Aza), we efficiently and stably differentiated naïve CD8+ T cells (CD8+ CD25- CD44- cells) into the CD8+ Foxp3+ regulatory CD8+ T cells (CD8 Tregs). We also generated OVA peptide257-264 -specific CD8+ Foxp3+ Tregs. Compared with activated CD8 T cells, Aza plus TGF-ß-induced CD8+ Foxp3+ Tregs showed significantly increased surface expression of CD39, CD73, CD122, CD62L, and CD103, and secreted TGF-ß and suppressed the proliferation of effector CD4+ T cells. Interestingly, CD8+ Foxp3+ Tregs exhibited low expression of perforin and granzyme required for cytotoxic function. Analysis of chemokine receptors showed that TGF-ß + Aza induced CD8+ Foxp3+ Tregs expressed gut-tropic chemokine receptors CCR6 and CCR9, and chemokine receptors CCR7 and CXCR3 required for mobilization into the spleen, lymph nodes, and gut-associated lymphoid tissues. Adoptive transfer of induced CD8+ Foxp3+ Tregs restored cholera toxin-induced breakdown of oral tolerance to OVA by regulating OVA-specific IgE and IgG1. Altogether, we showed an efficient method to generate antigen-specific CD8+ Foxp3+ Tregs, and the adoptive transfer of these cells induces oral tolerance by suppressing allergic response and maintaining intestinal homeostasis.

Early age-related atrophy of cutaneous lymph nodes precipitates an early functional decline in skin immunity in mice with aging.

Secondary lymphoid organs (SLOs) (including the spleen and lymph nodes [LNs]) are critical both for the maintenance of naive T (TN) lymphocytes and for the initiation and coordination of immune responses. How they age, including the exact timing, extent, physiological relevance, and the nature of age-related changes, remains incompletely understood. We used "time stamping" to indelibly mark newly generated naive T cells (also known as recent thymic emigrants) (RTEs) in mice, and followed their presence, phenotype, and retention in SLOs. We found that SLOs involute asynchronously. Skin-draining LNs atrophied by 6 to 9 mo in life, whereas deeper tissue-draining LNs atrophied by 18 to 20 mo, as measured by the loss of both TN numbers and the fibroblastic reticular cell (FRC) network. Time-stamped RTEs at all ages entered SLOs and successfully completed postthymic differentiation, but the capacity of older SLOs to maintain TN numbers was reduced with aging, and that trait did not depend on the age of TNs. However, in SLOs of older mice, these cells exhibited an emigration phenotype (CCR7loS1P1hi), which correlated with an increase of the cells of the same phenotype in the blood. Finally, upon intradermal immunization, RTEs generated in mice barely participated in de novo immune responses and failed to produce well-armed effector cells detectable in blood as early as by 7 to 8 mo of age. These results highlight changes in structure and function of superficial secondary lymphoid organs in laboratory mice that are earlier than expected and are consistent with the long-appreciated reduction of cutaneous immunity with aging.

IL-6 can singlehandedly drive many features of frailty in mice.

Frailty is a geriatric syndrome characterized by age-related declines in function and reserve resulting in increased vulnerability to stressors. The most consistent laboratory finding in frail subjects is elevation of serum IL-6, but it is unclear whether IL-6 is a causal driver of frailty. Here, we characterize a new mouse model of inducible IL-6 expression (IL-6TET-ON/+ mice) following administration of doxycycline (Dox) in food. In this model, IL-6 induction was Dox dose-dependent. The Dox dose that increased IL-6 levels to those observed in frail old mice directly led to an increase in frailty index, decrease in grip strength, and disrupted muscle mitochondrial homeostasis. Littermate mice lacking the knock-in construct failed to exhibit frailty after Dox feeding. Both naturally old mice and young Dox-induced IL-6TET-ON/+ mice exhibited increased IL-6 levels in sera and spleen homogenates but not in other tissues. Moreover, Dox-induced IL-6TET-ON/+ mice exhibited selective elevation in IL-6 but not in other cytokines. Finally, bone marrow chimera and splenectomy experiments demonstrated that non-hematopoietic cells are the key source of IL-6 in our model. We conclude that elevated IL-6 serum levels directly drive age-related frailty, possibly via mitochondrial mechanisms.

The iNOS Activity During an Immune Response Controls the CNS Pathology in Experimental Autoimmune Encephalomyelitis.

Inducible nitric oxide synthase (iNOS) plays a critical role in the regulation of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Previous studies have shown that iNOS plays pathogenic as well as regulatory roles in MS and EAE. However, how does iNOS alters the pathophysiology of the central nervous system (CNS) in neuronal autoimmunity is not clearly understood. In the present work, we show that treatment of mice with L-NAME, an iNOS inhibitor, during the antigen-priming phase primarily alters brain pathology, while in the subsequent effector phase of the immune response, the spinal cord is involved. Inhibition of iNOS during the priming phase of the immune response promotes the infiltration of pathogenic CD11b+F4/80-Gr-1+ cells, but there is low recruitment of regulatory CD11b+F4/80+ cells in the brain. Inhibition of iNOS during the effector phase shows similar pathogenic alterations in the spinal cord, instead of in the brain. Treatment of wild-type mice with L-NAME or mice having genetic deficiency of iNOS show lower MHC-II expression on the dendritic cells, but not on macrophages. Our data suggest that iNOS has a critical regulatory role during antigen-priming as well as in the effector phase of EAE, and inhibition iNOS at different stages of the immune response can differentially alter either the brain or spinal cord pathology. Understanding the cellular and molecular mechanisms through which iNOS functions could help to design a better strategies for the clinical management of neuroinflammation and neuronal autoimmunity.

Blood-brain barrier and its function during inflammation and autoimmunity.

The blood-brain barrier (BBB) is an important physiologic barrier that separates CNS from soluble inflammatory mediators and effector immune cells from peripheral circulation. The optimum function of the BBB is necessary for the homeostasis, maintenance, and proper neuronal function. The clinical and experimental findings have shown that BBB dysfunction is an early hallmark of various neurologic disorders ranging from inflammatory autoimmune, neurodegenerative, and traumatic diseases to neuroinvasive infections. Significant progress has been made in the understanding of the regulation of BBB function under homeostatic and neuroinflammatory conditions. Several neurologic disease-modifying drugs have shown to improve the BBB function. However, they have a broad-acting immunomodulatory function and can increase the risk of life-threatening infections. The recent development of in vitro multicomponent 3-dimensional BBB models coupled with fluidics chamber as well as a cell-type specific reporter and knockout mice gave a new boost to our understanding of the dynamics of the BBB. In the review, we discuss the current understanding of BBB composition and recent findings that illustrate the critical regulatory elements of the BBB function under physiologic and inflammatory conditions, and also suggested the strategies to control BBB structure and function.

CCR6 signaling inhibits suppressor function of induced-Treg during gut inflammation.

CCR6 is a G protein-coupled receptor (GPCR) that binds to a specific chemokine, CCL20. The role of CCR6-CCL20 is very well studied in the migration of immune cells, but the non-chemotaxis functions of CCR6 signaling were not known. Here, we show that during gut inflammation, the frequency of Foxp3+CD4+ T cells (Tregs) reduced in the secondary lymphoid tissues and CCR6+ Tregs enhanced the expression of RORγt. The peripheral blood mononuclear cells (PBMCs) of ulcerative colitis (UC) patients showed lower percentages of Foxp3+CD4+ T cells, as compared to healthy individuals, with CCR6+ Tregs showing higher RORγt expression as compared to CCR6-Tregs. CCL20 inhibited the TGF-ß1-induced Treg (iTreg) differentiation and directed them towards the pathogenic Th17-lineage in a CCR6-dependent manner. The iTreg that differentiated in the presence of CCL20 showed lower surface expression of suppressor molecules such as CD39, CD73 and FasL, and had impaired suppressive function. Furthermore, CCR6 signaling induced phosphorylation of Akt, mTOR, and STAT3 molecules in T cells. In conclusion, we have identified a new role of CCR6 signaling in the differentiation of iTregs during inflammation and gut autoimmunity.

Differentiation and Transmigration of CD4 T Cells in Neuroinflammation and Autoimmunity.

CD4+ T cells play a central role in orchestrating protective immunity and autoimmunity. The activation and differentiation of myelin-reactive CD4+ T cells into effector (Th1 and Th17) and regulatory (Tregs) subsets at the peripheral tissues, and their subsequent transmigration across the blood-brain barrier (BBB) into the central nervous system (CNS) parenchyma are decisive events in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis. How the Th1, Th17, and regulatory Tregs transmigrate across the BBB into the CNS and cause CNS inflammation is not clearly understood. Studies with transgenic and gene knockout mice have unraveled that Th1, Th17, and Tregs play a critical role in the induction and resolution of neuroinflammation. However, the plasticity of these lineages and functional dichotomy of their cytokine products makes it difficult to understand what role CD4+ T cells in the peripheral lymphoid organs, endothelial BBB, and the CNS parenchyma play in the CNS autoimmune response. In this review, we describe some of the recent findings that shed light on the mechanisms behind the differentiation and transmigration of CD4+ T cells across the BBB into the CNS parenchyma and also highlight how these two processes are interconnected, which is crucial for the outcome of CNS inflammation and autoimmunity.

IFN-γ promotes transendothelial migration of CD4+ T cells across the blood-brain barrier.

Transendothelial migration (TEM) of Th1 and Th17 cells across the blood-brain barrier (BBB) has a critical role in the development of experimental autoimmune encephalomyelitis (EAE). How cytokines produced by inflammatory Th1 and Th17 cells damage the endothelial BBB and promote transendothelial migration of immune cells into the central nervous system (CNS) during autoimmunity is not understood. We therefore investigated the effect of various cytokines on brain endothelial cells. Among the various cytokines tested, such as Th1 (IFN-γ, IL-1α, IL-1ß, TNF-α, IL-12), Th2 (IL-3, IL-4, IL-6 and IL-13), Th17 (IL-17A, IL-17F, IL-21, IL-22, IL-23, GM-CSF) and Treg-specific cytokines (IL-10 and TGF-ß), IFN-γ predominantly showed increased expression of ICAM-1, VCAM-1, MAdCAM-1, H2-Kb and I-Ab molecules on brain endothelial cells. Furthermore, IFN-γ induced transendothelial migration of CD4+ T cells from the apical (luminal side) to the basal side (abluminal side) of the endothelial monolayer to chemokine CCL21 in a STAT-1-dependent manner. IFN-γ also favored the transcellular route of TEM of CD4+ T cells. Multicolor immunofluorescence and confocal microscopic analysis showed that IFN-γ induced relocalization of ICAM-1, PECAM-1, ZO-1 and VE-cadherin in the endothelial cells, which affected the migration of CD4+ T cells. These findings reveal that the IFN-γ produced during inflammation could contribute towards disrupting the BBB and promoting TEM of CD4+ T cells. Our findings also indicate that strategies that interfere with the activation of CNS endothelial cells may help in controlling neuroinflammation and autoimmunity.