PPARβ/δ selectively regulates phenotypic features of age-related macular degeneration

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) is a nuclear receptor that regulates differentiation, inflammation, lipid metabolism, extracellular matrix remodeling, and angiogenesis in multiple tissues. These pathways are also central to the pathogenesis of age-related macular degeneration (AMD), the leading cause of vision loss globally. With the goal of identifying signaling pathways that may be important in the development of AMD, we investigated the impact of PPARβ/δ activation on ocular tissues affected in the disease. PPARβ/δ is expressed and can be activated in AMD vulnerable cells, including retinal pigment epithelial (RPE) and choroidal endothelial cells. Further, PPARβ/δ knockdown modulates AMD-related pathways selectively. Specifically, genetic ablation of Pparβ/δ in aged mice resulted in exacerbation of several phenotypic features of early dry AMD, but attenuation of experimentally induced choroidal neovascular (CNV) lesions. Antagonizing PPARβ/δ in both in vitro angiogenesis assays and in the in vivo experimentally induced CNV model, inhibited angiogenesis and angiogenic pathways, while ligand activation of PPARβ/δ, in vitro, decreased RPE lipid accumulation, characteristic of dry AMD. This study demonstrates for the first time, selective regulation of a nuclear receptor in the eye and establishes that selective targeting of PPARβ/δ may be a suitable strategy for treatment of different clinical sub-types of AMD.     

The Time Course of Cytokine Expressions Plays a Determining Role in Faster Healing of Intestinal and Colonic Anastomatic Wounds

Objectives:Inflammation is critical in the early phases of wound healing. It has been reported previously that small intestinal and colonic wounds display a more rapid healing than those of other organs. However, the underlying mechanism has not yet been elucidated. Here we examined whether differences in the time course of specified cytokine expression, in colonic and small intestinal anastomotic lesions, might play a major role in this observation in comparison to lesions effecting skin and muscle tissue.Results:The characteristics of TNF-α, IL-6, and IFN-α expression during the healing process for intestinal and colonic lesions were comparable. However, data differed significantly with that observed during healing of skin and muscle lesions. Intestinal and colonic lesions exhibited a significant and sustained increase in specified cytokine levels on day 5 to day 14 as compared with day 1 and 3. Skin and muscle lesions had random or unaltered cytokine levels throughout the study period.Conclusion:Differences in expression of cytokines TNF-α, IL-6, and IFN-α indicate that these play an important role underlying the more rapid healing processes observed in small intestinal and colonic lesions.     

Relevance of α-defensins (HNP1-3) and defensin β-1 in diabetes

AIM: To investigate the genetic background of human defensin expression in type 1 and 2 diabetes.METHODS: Associations between DEFA1/DEFA3 gene copy number polymorphism and diabetes as well as between the promoter polymorphisms of DEFB1 and diabetes were studied. The copy number variation of the DEFA1/DEFA3 genes was determined in 257 diabetic patients (117 patients with type 1 and 140 with type 2 diabetes). The control group consisted of 221 age- and gender-matched healthy blood donors. The cumulative copy numbers of the DEFA1/DEFA3 genes were detected by using quantitative PCR analysis. To evaluate the HNP 1-3 (human neutrophil peptide 1-3 or α-defensin) levels in the circulation, plasma HNP 1-3 concentrations were measured by ELISA. The expression of DEFA1/A3 in peripheral leukocytes of the diabetic patients was measured by quantitative RT PCR analysis. Three SNPs of the human DEFB1 (human defensin β-1) gene: DEFB1 G-20A (rs11362), DEFB1 C-44G (rs1800972) and DEFB1 G-52A (rs1799946) were genotyped by Custom TaqMan^® Real Time PCR assay.RESULTS: Significant differences were observed in HNP1-3 levels between the healthy subjects and both groups of diabetic patients. The mean ± SE was 28.78 ± 4.2 ng/mL in type 1 diabetes, and 29.82 ± 5.36 ng/mL in type 2 diabetes, vs 11.94 ± 2.96 ng/mL in controls; P < 0.01 respectively. There was no significant difference between patients with type 1 and type 2 diabetes in the high plasma concentrations of HNP1-3. The highest concentrations of α-defensin were found in diabetic patients with nephropathy (49.4 ± 4.8 ng/mL), neuropathy (38.7 ± 4.8 ng/mL) or cardiovascular complications (45.6 ± 1.45 ng/L). There was no significant difference in the cumulative copy numbers of DEFA1/DEFA3 genes between controls and patients, or between patients with the two types of diabetes. Comparisons of HNP 1-3 plasma level and DEFA1/A3 copy number of the same patient did not reveal significant relationship between defensin-α levels and the gene copy numbers (r² = 0.01). Similarly, no positive correlation was observed between the copy numbers and the mRNA expression levels of DEFA1/A3. Regarding the C-44G polymorphism of DEFB1, the GG “protective” genotype was much less frequent (1%-2%) among both groups of patients than among controls (9%).CONCLUSION: Elevated HNP1-3 levels in diabetes are independent of DEFA1/DEFA3 copy numbers, but GG genotype of C-44G SNP in DEFB1 gene may result in decreased defensin β-1 production.     

Enforcement of γδ-lineage commitment by the pre–T-cell receptor in precursors with weak γδ-TCR signals

Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2^−/− DN cells via retroviral transduction. Our results showed that Rag2^−/− DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.T cells can differentiate along distinct αβ- or γδ-cell lineages, but bifurcate from a common bipotent precursor (1, 2). In mice, the earliest subset of T cells contains CD4⁻ CD8⁻ or double-negative (DN) thymocytes, and this can further be divided into four subgroups (DN1–4) based on the expression of CD25 and CD44 (3, 4). Single-cell progenitor analyses have identified the DN3 stage as the point of T-lineage commitment, and also the final stage at which a DN cell specifies its lineage fate as αβ or γδ (1, 5). The αβ- or γδ-lineage choice decision is governed by several factors. Two competing models have been proposed for this process: the stochastic and instructional models (2). Although evidence exists to support either model, a version of the instructional model posits that the strength of signal transduced by the T-cell receptor (TCR) expressed by the DN3 cell dictates its lineage specification (6, 7).The apparent connection between lineage choice and the TCR expressed by the cell can be severed by manipulations of TCR signal strength. We previously noted that stimulating stronger signals via expression of the ERK/MAPK-induced Inhibitor of DNA-binding 3 (Id3) appears to promote the γδ-lineage fate in developing DN3 cells in the absence of TCR expression (8), suggesting a critical role for Id3 in mediating αβ- versus γδ-lineage decisions at this developmental checkpoint. Nevertheless, absence of Id3 also appears to favor the emergence of innate-like Vγ1.1/Vδ6.3 γδ-TCR–bearing T cells from the thymus over other γδ-TCR subsets (9, 10).Several studies have shown that ligand engagement highly influences the αβ- versus γδ-lineage decision because of its effects on γδ-TCR signal strength (6, 7, 9, 11, 12). γδ-TCR–expressing DN3 cells develop along the αβ-lineage and become CD4⁺ CD8⁺ (double-positive, DP) cells in the absence of ligand engagement (7), whereas provision of the ligand, or the use of antibodies to mimic ligand engagement (11), allows these cells to adopt the γδ-lineage fate, remain DN, and down-regulate expression of CD24. Additional signals, such as those mediated by Notch, can also influence αβ- versus γδ-lineage fate outcomes (1, 13–16). We showed that γδ-TCR–bearing thymocytes adopting the γδ-lineage do not require concurrent signals from Notch to mature past the DN3 stage, whereas their pre-TCR–expressing counterparts are completely dependent upon Notch signaling to facilitate their pre-TCR–dependent differentiation to the DP stage (1, 17).Considering the common origin of αβ- and γδ-lineage cells, it is possible for a bipotent DN3 cell to simultaneously express and transmit signals through a functional pre-TCR and a functional γδ-TCR, especially considering that TCR-β, -γ, and -δ genes complete their rearrangements at the DN3 stage. Additionally, γδ-T cells have been shown to contain TCR-β rearrangements (18) and αβ-lineage cells show evidence of both TCR-γ and -δ rearrangements (19–21). In a previous study looking to address the consequences of simultaneously expressing a TCR-β and γδ-TCR in vivo using transgenic (Tg) mice, the numbers of αβ- and γδ-lineage cells in TCR-β/γδ–expressing cells were both high, and comparable to TCR-β- and γδ-TCR-Tg mice, respectively (22). In this case, however, the TCR chains were expressed earlier than physiological for T-cell development, and premature expression of αβ-TCR transgene can lead to aberrant developmental progression (23, 24).Here, we attempt to definitively answer the question of lineage choice by simultaneously expressing TCR-β and γδ-TCR in Rag2^−/− DN3 cells via retroviral transduction followed by in vitro coculture, including limiting dilution and clonal analyses. We now find that Rag2^−/− DN3 cells expressing both pre-TCR and γδ-TCR mature along the γδ-lineage into functionally competent cells that produce IFN-γ in response to stimulation. However, in the absence of Id3 expression and strong γδ-TCR ligand, γδ-expressing cells show a lower propensity to differentiate along the γδ-lineage, but when expressing both pre- and γδ-TCRs, these cells showed increased γδ-lineage differentiation and recover functional competence to produce IFN-γ, indicating that the pre-TCR can serve to enforce to a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells dictates its lineage choice.     

IL-15 receptor α signaling constrains the development of IL-17–producing γδ T cells

The development and homeostasis of γδ T cells is highly dependent on distinct cytokine networks. Here we examine the role of IL-15 and its unique receptor, IL-15Rα, in the development of IL-17–producing γδ (γδ-17) T cells. Phenotypic analysis has shown that CD44^high γδ-17 cells express IL-15Rα and the common gamma chain (CD132), yet lack the IL-2/15Rβ chain (CD122). Surprisingly, we found an enlarged population of γδ-17 cells in the peripheral and mesenteric lymph nodes of adult IL-15Rα KO mice, but not of IL-15 KO mice. The generation of mixed chimeras from neonatal thymocytes indicated that cell-intrinsic IL-15Rα expression was required to limit IL-17 production by γδ T cells. γδ-17 cells also were increased in the peripheral lymph nodes of transgenic knock-in mice, where the IL-15Rα intracellular signaling domain was replaced with the intracellular portion of the IL-2Rα chain (that lacks signaling capacity). Finally, an analysis of neonatal thymi revealed that the CD44^lo/int precursors of γδ-17 cells, which also expressed IL-15Rα, were increased in newborn mice deficient in IL-15Rα signaling, but not in IL-15 itself. Thus, these findings demonstrate that signaling through IL-15Rα regulates the development of γδ-17 cells early in ontogeny, with long-term effects on their peripheral homeostasis in the adult.Both αβ and γδ T cells rely heavily on cytokine signaling for their development and survival. Many of these cytokines belong to the IL-2 cytokine family, whose receptors all share a common γ receptor chain (γ_c; CD132). Among these, IL-15 has a unique, nonredundant role in both T-cell and natural killer (NK)-cell biology, such that CD8⁺ memory T cells and NK cells are absent in IL-15–deficient environments (1, 2). The predominant mechanism through which IL-15 functions is termed transpresentation, whereby IL-15 is preassociated with its specific α-chain (IL-15Rα) inside the cell and presented at the cell surface in trans to a responding cell expressing γ_c and IL-2/15Rβ (CD122) (3–6). IL-15 is unique among its family members owing to its ability to act either in cis or in trans. Whether or not direct signaling (in cis) via IL-15Rα plays a significant biological role in immunobiology has not been resolved (7–11).Certain populations of γδ T cells are known to be sensitive to the availability of IL-15 for their development and/or survival. A population of specialized γδ T cells called dendritic epidermal T cells are absent from the skin of IL-15 knockout (KO) and IL-15Rα KO mice (12, 13). The CD8αα⁺ γδ T-cell receptor (TCR) intraepithelial lymphocytes are also decreased in these two KO mouse strains (1, 2). In addition, IL-15 KO mice have a reduced population of IFN-γ⁺ γδ T cells in the peritoneum (14). All of these populations express CD122, suggesting they can receive IL-15–dependent signals via transpresentation.Recently, γδ T cells have emerged as important contributors to the generation of immune responses. The innate-like γδ T cells that produce IL-17 (γδ-17 cells) have been implicated in immune responses generated during bacterial and fungal infections, experimental autoimmune encephalomyelytois (EAE), psoriasis, and anticancer immunity (reviewed in ref. 15). The γδ-17 subset of γδ T cells has a restricted TCR use that is largely limited to Vγ2 and Vγ4 expression (Garmin nomenclature; Vγ4 and Vγ6 in Tonegawa nomenclature) and a molecularly distinct gene expression profile (16). Considering the strictly regulated developmental progression of γδ T-cell subsets (17), the vast majority of γδ-17 cells are known to emerge during a limited window early in ontogeny, ∼E16.5 up to shortly after birth (18). The exogenous signals that impact γδ-17–cell development during this window remain unclear. Here we identify the IL-15Rα chain as a critical determinant through which γδ-17–cell development is regulated. Furthermore, in contrast to IL-15Rα’s predominant role in the immune system via IL-15 transpresentation, we found IL-15Rα–dependent changes in both neonatal thymic development and peripheral homeostasis in adulthood, suggesting that γδ-17 cells are dependent on cell-intrinsic signals received through IL-15Rα in cis.     

Disruption of TAK1 in hepatocytes causes hepatic injury,inflammation, fibrosis,and carcinogenesis

TGF-β–activated kinase 1 (TAK1) is a MAP3K family member that activates NF-κB and JNK via Toll-like receptors and the receptors for IL-1, TNF-α, and TGF-β. Because the TAK1 downstream molecules NF-κB and JNK have opposite effects on cell death and carcinogenesis, the role of TAK1 in the liver is unpredictable. To address this issue, we generated hepatocyte-specific Tak1-deficient (Tak1ΔHEP) mice. The Tak1ΔHEP mice displayed spontaneous hepatocyte death, compensatory proliferation, inflammatory cell infiltration, and perisinusoidal fibrosis at age 1 month. Older Tak1ΔHEP mice developed multiple cancer nodules characterized by increased expression of fetal liver genes including α-fetoprotein. Cultures of primary hepatocytes deficient in Tak1 exhibited spontaneous cell death that was further increased in response to TNF-α. TNF-α increased caspase-3 activity but activated neither NF-κB nor JNK in Tak1-deficient hepatocytes. Genetic abrogation of TNF receptor type I (TNFRI) in Tak1ΔHEP mice reduced liver damage, inflammation, and fibrosis compared with unmodified Tak1ΔHEP mice. In conclusion, hepatocyte-specific deletion of TAK1 in mice resulted in spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis that was partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver.     

Polarizing the T helper 17 response in Citrobacter rodentium infection via expression of resistin-like molecule α

Citrobacter rodentium infection is a murine model of pathogenic Escherichia coli infection that allows investigation of the cellular and molecular mechanisms involved in host-protective immunity and bacterial-induced intestinal inflammation. We recently demonstrated that following C. rodentium infection, the absence of Resistin-Like Molecule (RELM) α resulted in attenuated Th17 cell responses and reduced intestinal inflammation with minimal effects on bacterial clearance. In this addendum, we investigated the cytokine modulatory effects of RELMα and RELMα expression in the intestinal mucosa following C. rodentium infection. We show that in addition to promoting Th17 cytokine responses, RELMα inhibits Th2 cytokine expression and Th2-cytokine effector macrophage responses in the C. rodentium-infected colons. Second, utilizing reporter C. rodentium, we examined RELMα expression and macrophage recruitment at the host pathogen interface. We observed infection-induced macrophage infiltration and RELMα expression by intestinal epithelial cells. The influence of infection-induced RELMα on macrophage recruitment in the intestine is discussed.     

Getting ‘Smad' about obesity and diabetes

Recent findings on the role of transforming growth factor (TGF)-β/Smad3 signaling in the pathogenesis of obesity and type 2 diabetes have underscored its importance in metabolism and adiposity. Indeed, elevated TGF-β has been previously reported in human adipose tissue during morbid obesity and diabetic neuropathy. In this review, we discuss the pleiotropic effects of TGF-β/Smad3 signaling on metabolism and energy homeostasis, all of which has an important part in the etiology and progression of obesity-linked diabetes; these include adipocyte differentiation, white to brown fat phenotypic transition, glucose and lipid metabolism, pancreatic function, insulin signaling, adipocytokine secretion, inflammation and reactive oxygen species production. We summarize the recent in vivo findings on the role of TGF-β/Smad3 signaling in metabolism based on the studies using Smad3^−/− mice. Based on the presence of a dual regulatory effect of Smad3 on peroxisome proliferator-activated receptor (PPAR)β/δ and PPARγ2 promoters, we propose a unifying mechanism by which this signaling pathway contributes to obesity and its associated diabetes. We also discuss how the inhibition of this signaling pathway has been implicated in the amelioration of many facets of metabolic syndromes, thereby offering novel therapeutic avenues for these metabolic conditions.     

From the Cover: The CD5 ectodomain interacts with conserved fungal cell wall components and protects from zymosan-induced septic shock-like syndrome

The CD5 lymphocyte surface receptor is a group B member of the ancient and highly conserved scavenger receptor cysteine-rich superfamily. CD5 is expressed on mature T and B1a cells, where it is known to modulate lymphocyte activation and/or differentiation processes. Recently, the interaction of a few group B SRCR members (CD6, Spα, and DMBT1) with conserved microbial structures has been reported. Protein binding assays presented herein indicate that the CD5 ectodomain binds to and aggregates fungal cells (Schizosaccharomyces pombe, Candida albicans, and Cryptococcus neoformans) but not to Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus) bacteria. Accordingly, the CD5 ectodomain binds to zymosan but not to purified bacterial cell wall constituents (LPS, lipotheicoic acid, or peptidoglycan), and such binding is specifically competed by β-glucan but not by mannan. The K_d of the rshCD5/(1→3)-β-d-glucan phosphate interaction is 3.7 ± 0.2 nM as calculated from tryptophan fluorescence data analysis of free and bound rshCD5. Moreover, zymosan binds to membrane-bound CD5, and this induces both MAPK activation and cytokine release. In vivo validation of the fungal binding properties of the CD5 ectodomain is deduced from its protective effect in a mouse model of zymosan-induced septic shock-like syndrome. In conclusion, the present results indicate that the CD5 lymphocyte receptor may sense the presence of conserved fungal components [namely, (1→3)-β-d-glucans] and support the therapeutic potential of soluble CD5 forms in fungal sepsis.     

ALK7 Acts as a Positive Regulator of Macrophage Activation through Down-Regulation of PPARγ Expression

Aim: Activin receptor-like kinase 7 (ALK7) acts as a key receptor for TGF-β family members, which play important roles in regulating cardiovascular activity. However, ALK7''s potential role, and underlying mechanism, in the macrophage activation involved in atherogenesis remain unexplored.Methods: ALK7 expression in macrophages was tested by RT-PCR, western blot, and immunofluorescence co-staining. The loss-of-function strategy using AdshALK7 was performed for functional study. Oil Red O staining was used to observe the foam cell formation, while inflammatory mediators and genes related to cholesterol efflux and influx were determined by RT-PCR and western blot. A PPARγ inhibitor (G3335) was used to reveal whether PPARγ was required for ALK7 to affect macrophage activation.Results: The results exhibited upregulated ALK7 expression in oxidized low-density lipoprotein (Ox-LDL) induced bone marrow derived macrophages (BMDMs) and mouse peritoneal macrophages (MPMs), isolated from ApoE-deficient mice, while ALK7''s strong immunoreactivity in BMDMs was observed. ALK7 knockdown significantly attenuated pro-inflammatory, but promoted anti-inflammatory, macrophage markers expression. Additionally, ALK7 silencing decreased foam cell formation, accompanied by the up-regulation of ABCA1 and ABCG1 involved in cholesterol efflux but the down-regulation of CD36 and SR-A implicated in cholesterol influx. Mechanistically, ALK7 knockdown upregulated PPARγ expression, which was required for the ameliorated effect of ALK7 silencing macrophage activation.Conclusions: Our study demonstrated that ALK7 was a positive regulator for macrophage activation, partially through down-regulation of PPARγ expression, which suggested that neutralizing ALK7 might be promising therapeutic strategy for treating atherosclerosis.     

Pilot Study on Interferon-γ-producing T Cell Subsets after the Protective Vaccination with Radiation-attenuated Cercaria of Schistosoma japonicum in the Miniature Pig Model

CLAWN miniature pig has been shown to serve as a suitable host for the experimental infection of Schistosoma japonicum. In this study, we found that radiation-attenuated cercaria (RAC) vaccine gave CLAWN miniature pigs protective immunity against subsequent challenge infection with S. japonicum cercaria. To characterize the protective immune response of the pig model vaccinated by attenuated cercaria, flow cytometric analysis of the reactive T cell subsets was performed. The intracellular interferon (IFN)-γ and the cell surface markers revealed the peripheral blood CD3+ T-lymphocytes produced significant amounts of IFN-γ during the immunization period and after the challenge infection. CD4+ αβ-T cells as well as CD4+/CD8α^mid double positive and/or CD8α^high αβ-T cells were the major IFN-γ-producing CD3+ T cells. On the contrary, γδ T cells did not produce intracellular IFN-γ. Our results suggested that RAC-vaccinated miniature pigs showed effective protective immunity through the activation of αβ T cells bearing antigen specific T-cell receptors but not through the activation of γδ T cells.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Shifting the feeding of mice to the rest phase creates metabolic alterations,which, on their own,shift the peripheral circadian clocks by 12 hours

The molecular mechanisms underlying the events through which alterations in diurnal activities impinge on peripheral circadian clocks (PCCs), and reciprocally how the PCCs affect metabolism, thereby generating pathologies, are still poorly understood. Here, we deciphered how switching the diurnal feeding from the active to the rest phase, i.e., restricted feeding (RF), immediately creates a hypoinsulinemia during the active phase, which initiates a metabolic reprogramming by increasing FFA and glucagon levels. In turn, peroxisome proliferator-activated receptor alpha (PPARα) activation by free fatty acid (FFA), and cAMP response element-binding protein (CREB) activation by glucagon, lead to further metabolic alterations during the circadian active phase, as well as to aberrant activation of expression of the PCC components nuclear receptor subfamily 1, group D, member 1 (Nr1d1/RevErbα), Period (Per1 and Per2). Moreover, hypoinsulinemia leads to an increase in glycogen synthase kinase 3β (GSK3β) activity that, through phosphorylation, stabilizes and increases the level of the RevErbα protein during the active phase. This increase then leads to an untimely repression of expression of the genes containing a RORE DNA binding sequence (DBS), including the Bmal1 gene, thereby initiating in RF mice a 12-h PCC shift to which the CREB-mediated activation of Per1, Per2 by glucagon modestly contributes. We also show that the reported corticosterone extraproduction during the RF active phase reflects an adrenal aberrant activation of CREB signaling, which selectively delays the activation of the PPARα–RevErbα axis in muscle and heart and accounts for the retarded shift of their PCCs.Pioneering studies (1, 2) have established that switching the feeding time in mice from the “active” phase [dark period of the light-dark (L/D) cycle] to the “rest” phase (light period), i.e., restricted feeding (RF), overrides the suprachiasmatic nucleus (SCN) circadian clock (CC)-derived signals and acts as a “zeitgeber” for peripheral CCs (PCCs), leading to a 12-h shift in the time at which components of PCCs are expressed. Numerous studies have shown that under homeostatic conditions, the functions of PCCs and metabolism are tightly linked and that perturbations in their interactions leads to pathologies, e.g., obesity and metabolic syndrome (3–5).The identity of some of the molecular pathways that couple PCCs to metabolism are known (3–5), but it is largely unknown how environmental cues, e.g., altered feeding schedules, may directly perturb the expression of individual CC components (5–7), thereby leading to obesity and a metabolic syndrome-like pathology (5). Assuming that specific metabolic perturbations generated by switching the feeding time could selectively affect the time of expression of some of the CC core components, we looked for both metabolic and PCC alterations at early RF times. We report here a comprehensive temporal analysis, and we found that the shift of PCCs on RF is directly linked, at the molecular level, to metabolic reprogramming that directly impinges on CC component expression.