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We report here a mAb, 14-2, reactive with TCRs that include V beta 14. The frequency of V beta 14+ T cells varies with CD4 and CD8 subset and is controlled by the H-2 genes. Thus CD8+ T cells from H-2b mice include approximately 2.3% V beta 14+ T cells while CD8+ T cells from mice expressing K kappa include greater than 8% V beta 14+ T cells. In all strains examined, 7-8% of CD4+ T cells express V beta 14. The frequent usage of V beta 14 in CD8+ T cells of K kappa-expressing mice is a result of preferential positive selection of V beta 14+ CD8+ T cells as demonstrated by analysis of radiation chimeras. These studies demonstrate that H-2-dependent positive selection occurs in unmanipulated mice. Furthermore, the results imply that positive selection, and possibly H-2 restriction, can be strongly influenced by a V beta domain, with some independence from the beta-junctional sequence and alpha chain.

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1 K74R/K74R or Klf1 (K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1 (K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1 (K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1 (K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging., Competing Interests: JW, CH, YL, CL, KY, KW, ZL, BC, TH, YS, PH, LC, TC, CY, NL, CS No competing interests declared, TL is an employee of Pro-Clintech Co. Ltd, (© 2023, Wang, Hung et al.)

The nucleotide-binding and oligomerization domain, leucine-rich repeats, and pyrin domain-containing protein 3 (NLRP3) inflammasome plays a crucial role in innate immunity and is involved in the pathogenesis of autoinflammatory diseases. Glycolysis regulates NLRP3 inflammasome activation in macrophages. However, how lactic acid fermentation and pyruvate oxidation controlled by the mitochondrial pyruvate carrier (MPC) affect NLRP3 inflammasome activation and autoinflammatory disease remains elusive. We found that the inactivation of MPC with genetic depletion or pharmacological inhibitors, MSDC-0160 or pioglitazone, increased NLRP3 inflammasome activation and IL-1β secretion in macrophages. Glycolytic reprogramming induced by MPC inhibition skewed mitochondrial ATP-associated oxygen consumption into cytosolic lactate production, which enhanced NLRP3 inflammasome activation in response to monosodium urate (MSU) crystals. As pioglitazone is an insulin sens MSDC-itizer used for diabetes, its MPC inhibitory effect in diabetic individuals was investigated. The results showed that MPC inhibition exacerbated MSU-induced peritonitis in diabetic mice and increased the risk of gout in patients with diabetes. Altogether, we found that glycolysis controlled by MPC regulated NLRP3 inflammasome activation and gout development. Accordingly, prescriptions for medications targeting MPC should consider the increased risk of NLRP3-related autoinflammatory diseases., (© 2023 John Wiley & Sons Ltd.)

The molecular mechanisms contributing to the regulation of Th17-mediated inflammation remain underexplored. We here report a SUMO-specific protease (SENP)2-mediated pathway induced in pathogenic Th17 cells that restricts the pathogenesis of inflammatory colitis. SENP2 regulates the maturation of small ubiquitin-like modifiers (SUMO) and recycles SUMO from the substrate proteins. We find higher levels of SENP2 in pathogenic Th17 cells. By deleting Senp2 in T-cell lineages in mice, we demonstrate that the lack of Senp2 exacerbates the severity of experimental colitis, which is linked to elevated levels of GM-CSF + IL-17A + pathogenic Th17 cells and more severe dysbiosis of the intestinal microbiome. Adoptive transfer experiments demonstrate the cell-autonomous effect of Senp2 in restraining Th17 differentiation and colitis. The enzymatic activity of SENP2 is important for deSUMOylation of Smad4, which reduces Smad4 nuclear entry and Rorc expression. Our findings reveal a SENP2-mediated regulatory axis in the pathogenicity of Th17 cells., (© 2023. The Author(s).)

Importance: Long-term exposure to fine particulate air pollution (PM2.5) is a known risk factor for cardiovascular events, but controversy remains as to whether the current National Ambient Air Quality Standard (12 μg/m3 for 1-year mean PM2.5) is sufficiently protective., Objective: To evaluate the associations between long-term fine particulate air pollution and cardiovascular events using electronic health record and geocoded address data., Design, Setting, and Participants: This retrospective cohort study included adults in the Kaiser Permanente Northern California integrated health care system during 2007 to 2016 and followed for up to 10 years. Study participants had no prior stroke or acute myocardial infarction (AMI), and lived in Northern California for at least 1 year. Analyses were conducted January 2020 to December 2022., Exposure: Long-term exposure to PM2.5. Individual-level time-varying 1-year mean PM2.5 exposures for every study participant were updated monthly from baseline through the end of follow-up, accounting for address changes., Main Outcomes and Measures: Incident AMI, ischemic heart disease (IHD) mortality, and cardiovascular disease (CVD) mortality. Cox proportional hazards models were fit with age as time scale, adjusted for sex, race and ethnicity, socioeconomic status, smoking, body mass index, baseline comorbidities, and baseline medication use. Associations below the current regulation limit were also examined., Results: The study cohort included 3.7 million adults (mean [SD] age: 41.1 [17.2] years; 1 992 058 [52.5%] female, 20 205 [0.5%] American Indian or Alaskan Native, 714 043 [18.8%] Asian, 287 980 [7.6%] Black, 696 796 [18.4%] Hispanic, 174 261 [4.6%] multiracial, 1 904 793 [50.2%] White). There was a 12% (95% CI, 7%-18%) increased risk of incident AMI, a 21% (95% CI, 13%-30%) increased risk of IHD mortality, and an 8% (95% CI, 3%-13%) increased risk of CVD mortality associated with a 10 μg/m3 increase in 1-year mean PM2.5. PM2.5 exposure at moderate concentrations (10.0 to 11.9 μg/m3) was associated with increased risks of incident AMI (6% [95% CI, 3%-10%]) and IHD mortality (7% [95% CI, 2%-12%]) compared with low concentrations (less than 8 μg/m3)., Conclusions and Relevance: In this study, long-term PM2.5 exposure at moderate concentrations was associated with increased risks of incident AMI, IHD mortality, and CVD mortality. This study's findings add to the evidence that the current regulatory standard is not sufficiently protective.

Bacteriophage TaidaOne was isolated from soil collected in Taipei, Taiwan, using the host Streptomyces griseus. It is a siphovirus with a 56,183-bp genome that contains 86 protein-coding genes. Based on gene content similarity, it was assigned to actinobacteriophage subcluster BI1, within which only TaidaOne and GirlPower genomes contain an acetyltransferase homolog gene.

Fine particulate matter (PM 2.5 ) is a known risk factor for cardiovascular disease (CVD). Neighborhood walkability and greenness may also be associated with CVD, but there is limited evidence on their joint or interacting effects with PM 2.5 ., Methods: Cox proportional hazard models were used to estimate the risk of CVD mortality among adults with a history of acute myocardial infarction and/or stroke living in Northern California. We assessed the independent and joint effects of walkability, greenness (Normalized Differentiated Vegetation Index [NDVI]), and PM 2.5 at residential addresses, controlling for age, sex, race/ethnicity, comorbidities, BMI, smoking, revascularization, medications, and socioeconomic status., Results: Greenness had a nonlinear association with CVD mortality ( P = 0.038), with notably protective effects (HR = 0.87 [95% confidence interval {CI} = 0.78, 0.97]) at higher greenness levels (NDVI ≥ 0.3) and moderate attenuation after adjusting for PM 2.5 (HR = 0.92 [95% CI = 0.82, 1.03]) per 0.1 increase in NDVI. Walkability had no independent effect on CVD mortality. PM 2.5 had a strong independent effect in models adjusted for greenness and walkability (HR = 1.20 [95% CI = 1.08, 1.33)) per 10 μg/m 3 increase in PM 2.5 . There was an interaction between walkability and PM 2.5 ( P = 0.037), where PM 2.5 had slightly stronger associations in more walkable than less walkable neighborhoods (HR = 1.23 [95% CI = 1.06, 1.42] vs. 1.17 [95% CI = 1.04, 1.32]) per 10 μg/m 3 increase in PM 2.5 . Greenness had no interaction with PM 2.5 ( P = 0.768) nor walkability ( P = 0.385)., Conclusions: High greenness may be protective of CVD mortality among adults with CVD history. PM 2.5 associated CVD mortality risk varies slightly by level of neighborhood walkability, though these small differences may not be clinically meaningful., Competing Interests: The authors declare that they have no conflicts of interest with regard to the content of this report., (Copyright © 2022 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of The Environmental Epidemiology. All rights reserved.)

Rationale: People with chronic obstructive pulmonary disease (COPD) have an increased risk of cardiovascular disease and may be more susceptible to air pollution exposure. However, no study has examined the association between long-term fine particulate matter exposure (≤2.5 μm in aerodynamic diameter) and risk of cardiovascular events in this potentially vulnerable population. Objectives: To estimate the association between long-term fine particulate matter and risk of cardiovascular events among adults with COPD. Methods: This retrospective cohort study included 169,714 adults with COPD who were members of the Kaiser Permanente Northern California health plan during 2007-2016. Electronic health record data were linked to 1 km modeled particulate matter ≤2.5 μm in aerodynamic diameter exposure estimates. We fit Cox proportional hazard models, adjusting for age, sex, race/ethnicity, calendar year, smoking, body mass index, comorbidities, medications, and socioeconomic status. In low exposure analyses, we examined effects below the current regulation limit (12 μg/m 3 ). Measurements and Main Results: Among adults with COPD, a 10-μg/m 3 increase in 1-year mean fine particulate matter exposure was associated with an elevated risk of cardiovascular mortality (hazard ratio, 1.10; 95% confidence interval [CI], 1.01-1.20). Effects were stronger in low exposure analyses (hazard ratio, 1.88; 95% CI, 1.56-2.27). Fine particulate matter exposure was not associated with acute myocardial infarction or stroke in overall analyses. Conclusions: Long-term fine particulate matter exposure was associated with an increased risk of cardiovascular mortality among adults with COPD. Current regulations may not sufficiently protect those with COPD.

The homeostasis of CD8 + CD122 + T cell requires IL-15 trans-presentation. We use Il15ra mutant mice and bone marrow chimeras to assess the role of IL-15 trans-presentation level in CD8 + CD122 + T cells homeostasis. We demonstrate that CD8 + CD122 + T cells require different levels of IL-15 trans-presentation to support their homeostasis., Competing Interests: Declaration of Competing Interest All contributing authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

Background Previous studies have found associations between fine particulate matter <2.5 µm in diameter (PM 2.5 ) and increased risk of cardiovascular disease (CVD) among populations with no CVD history. Less is understood about susceptibility of adults with a history of CVD and subsequent PM 2.5 -related CVD events and whether current regulation levels for PM 2.5 are protective for this population. Methods and Results This retrospective cohort study included 96 582 Kaiser Permanente Northern California adults with a history of stroke or acute myocardial infarction. Outcome, covariate, and address data obtained from electronic health records were linked to time-varying 1-year mean PM 2.5 exposure estimates based on residential locations. Cox proportional hazard models estimated risks of stroke, acute myocardial infarction, and cardiovascular mortality associated with PM 2.5 exposure, adjusting for multiple covariates. Secondary analyses estimated risks below federal and state regulation levels (12 µg/m 3 for 1-year mean PM 2.5 ). A 10-µg/m 3 increase in 1-year mean PM 2.5 exposure was associated with an increase in risk of cardiovascular mortality (hazard ratio [HR], 1.20; 95% CI, 1.11-1.30), but no increase in risk of stroke or acute myocardial infarction. Analyses of <12 µg/m 3 showed increased risk for CVD mortality (HR, 2.31; 95% CI, 1.96-2.71), stroke (HR, 1.41; 95% CI, 1.09-1.83]), and acute myocardial infarction (HR, 1.51; 95% CI, 1.21-1.89) per 10-µg/m 3 increase in 1-year mean PM 2.5 . Conclusions Adults with a history of CVD are susceptible to the effects of PM 2.5 exposure, particularly on CVD mortality. Increased risks observed at exposure levels <12 µg/m 3 highlight that current PM 2.5 regulation levels may not be protective for this susceptible population.

Expression of tissue-restricted antigens (TRAs) in thymic epithelial cells (TECs) ensures negative selection of highly self-reactive T cells to establish central tolerance. Whether some of these TRAs could exert their canonical biological functions to shape thymic environment to regulate T cell development is unclear. Analyses of publicly available databases have revealed expression of transcripts at various levels of many cytokines and cytokine receptors such as IL-15, IL-15Rα, IL-13, and IL-23a in both human and mouse TECs. Ablation of either IL-15 or IL-15Rα in TECs selectively impairs type 1 innate like T cell, such as i NKT1 and γδT1 cell, development in the thymus, indicating that TECs not only serve as an important source of IL-15 but also trans-present IL-15 to ensure type 1 innate like T cell development. Because type 1 innate like T cells are proinflammatory, our data suggest the possibility that TEC may intrinsically control thymic inflammatory innate like T cells to influence thymic environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tao, Li, Liao, Schluns, Luckhart, Sleasman and Zhong.)

Background Fine particulate matter <2.5 µm in diameter (PM 2.5 ) has known effects on cardiovascular morbidity and mortality. However, no study has quantified and compared the risks of incident myocardial infarction, incident stroke, ischemic heart disease (IHD) mortality, and cerebrovascular mortality in relation to long-term PM 2.5 exposure. Methods and Results We sought to quantitatively summarize studies of long-term PM 2.5 exposure and risk of IHD and stroke events by conducting a review and meta-analysis of studies published by December 31, 2019. The main outcomes were myocardial infarction, stroke, IHD mortality, and cerebrovascular mortality. Random effects meta-analyses were used to estimate the combined risk of each outcome among studies. We reviewed 69 studies and included 42 studies in the meta-analyses. In meta-analyses, we found that a 10-µg/m 3 increase in long-term PM 2.5 exposure was associated with an increased risk of 23% for IHD mortality (95% CI, 15%-31%), 24% for cerebrovascular mortality (95% CI, 13%-36%), 13% for incident stroke (95% CI, 11%-15%), and 8% for incident myocardial infarction (95% CI, -1% to 18%). There were an insufficient number of studies of recurrent stroke and recurrent myocardial infarction to conduct meta-analyses. Conclusions Long-term PM 2.5 exposure is associated with increased risks of IHD mortality, cerebrovascular mortality, and incident stroke. The relationship with incident myocardial infarction is suggestive of increased risk but not conclusive. More research is needed to understand the relationship with recurrent events.

Background/purpose: Sjögren's syndrome (SS) is an autoimmune disease and its conventional treatment has exhibited limited therapeutic efficacy. Traditional Chinese medicine has been demonstrated to ameliorate the sicca symptoms of SS by decreasing the level of T H 1 and T H 2 cytokines and increasing salivary flow rate. A newly designed traditional Chinese medicine, SS-1, showed improved efficacy in alleviating the dryness symptoms of SS patients in the National Taiwan SS cohort investigation. Here, we investigated the effect of SS-1 on T cell responses., Methods: SS-1 was authenticated and its major compounds were verified by high-performance liquid chromatography. We examined the effects of SS-1 on the activation and T H 1, T H 2, and T H 17 polarization of murine T cells. We also determined the level of T H 1, T H 2, and T H 17 cytokine RNA in peripheral blood mononuclear cells of SS patients before and after SS-1 treatment., Results: SS-1 treatment inhibits the activation and T H 1, T H 2, and IL-17A + IFNγ + T H polarization of murine T cells. SS-1 treatment also significantly reduces IFN-γ, IL-4, and IL-13 expression, and moderately reduces IL-17A expression in peripheral blood mononuclear cells of SS patients., Conclusion: Our results suggest that SS-1 inhibits T cell activation and diminishes T H 1, T H 2, and IL-17 + IFN-γ + T H responses in SS patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020. Published by Elsevier B.V.)

Objective: Surgical site infection (SSI) is the most common infectious complication after emergency abdominal surgery (EAS). To a large extent, most SSI can be prevented, but there are few relevant studies in China. This study mainly investigated the current situation of SSI occurrence after EAS in China, and further explored risk factors for SSI occurrence. Methods: Multi-center cross-sectional study was conducted. Clinical data of patients undergoing EAS in 33 hospitals across China between May 1, 2019 and June 7, 2019 were prospectively collected, including perioperative data and microbial culture results from infected incisions. The primary outcome was the incidence of SSI after EAS, while the secondary outcomes were postoperative hospital stay, ICU occupancy rate, length of ICU stay, hospitalization cost, and mortality within postoperative 30 days. Univariate and multivariate logistic regression models were used to analyze the risk factors of SSI after EAS. Results: A total of 660 EAS patients aged (47.9±18.3) years were enrolled in this study, including 56.5% of males (373/660). Forty-nine (7.4%) patients developed postoperative SSI. The main pathogen of SSI was Escherichia coli [culture positive rate was 32.7% (16/49)]. As compared to patients without SSI, those with SSI were more likely to be older (median 56 years vs. 46 years, U =19 973.5, P <0.001), male [71.4% (35/49) vs. 56.1% (343/611), χ(2)=4.334, P =0.037] and diabetes [14.3% (7/49) vs. 5.1% (31/611), χ(2)=5.498, P =0.015]; with-lower preoperative hemoglobin (median: 122.0 g/L vs. 143.5 g/L, U =11 471.5, P =0.006) and albumin (median: 35.5 g/L vs. 40.8 g/L, U =9452.0, P <0.001), with higher blood glucose (median: 6.9 mmol/L vs. 6.0 mmol/L, U =17 754.5, P <0.001); with intestinal obstruction [32.7% (16/49) vs. 9.2% (56/611), χ(2)=25.749, P <0.001], with ASA score 3-4 [42.9% (21/49) vs. 13.9% (85/611), χ(2)=25.563, P <0.001] and with high surgical risk [49.0% (24/49) vs. 7.0% (43/611), χ(2)=105.301, P <0.001]. The main operative procedure resulting in SSI was laparotomy [81.6%(40/49) vs. 35.7%(218/611), χ(2)=40.232, P <0.001]. Patients with SSI experienced significantly longer operation time (median: 150 minutes vs. 75 minutes, U =25 183.5, P <0.001). In terms of clinical outcome, higher ICU occupancy rate [51.0% (25/49) vs. 19.5% (119/611), χ(2)=26.461, P <0.001], more hospitalization costs (median: 44 000 yuan vs. 15 000 yuan, U =24 660.0, P <0.001), longer postoperative hospital stay (median: 10 days vs. 5 days, U =23 100.0, P <0.001) and longer ICU occupancy time (median: 0 days vs. 0 days, U =19 541.5, P <0.001) were found in the SSI group. Multivariate logistic regression analysis showed that the elderly (OR=3.253, 95% CI: 1.178-8.985, P =0.023), colorectal surgery (OR=9.156, 95% CI: 3.655-22.937, P <0.001) and longer operation time (OR=15.912, 95% CI:6.858-36.916, P <0.001) were independent risk factors of SSI, while the laparoscopic surgery (OR=0.288, 95% CI: 0.119-0.694, P =0.006) was an independent protective factor for SSI. Conclusions: For patients undergoing EAS, attention should be paid to middle-aged and elderly patients and those of colorectal surgery. Laparoscopic surgery should be adopted when feasible and the operation time should be minimized, so as to reduce the incidence of SSI and to reduce the burden on patients and medical institutions.

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2 - CD34 - Lin - Sca1 + c-Kit + (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor β subunit (CSF2RB). As a result, Flk2 - CD34 - LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2 - CD34 - LSK-HSCs.

Metabolic pathways and inflammatory processes are under circadian regulation. Rhythmic immune cell recruitment is known to impact infection outcomes, but whether the circadian clock modulates immunometabolism remains unclear. We find that the molecular clock Bmal1 is induced by inflammatory stimulants, including Ifn-γ/lipopolysaccharide (M1) and tumor-conditioned medium, to maintain mitochondrial metabolism under metabolically stressed conditions in mouse macrophages. Upon M1 stimulation, myeloid-specific Bmal1 knockout (M-BKO) renders macrophages unable to sustain mitochondrial function, enhancing succinate dehydrogenase (SDH)-mediated mitochondrial production of reactive oxygen species as well as Hif-1α-dependent metabolic reprogramming and inflammatory damage. In tumor-associated macrophages, aberrant Hif-1α activation and metabolic dysregulation by M-BKO contribute to an immunosuppressive tumor microenvironment. Consequently, M-BKO increases melanoma tumor burden, whereas administering the SDH inhibitor dimethyl malonate suppresses tumor growth. Therefore, Bmal1 functions as a metabolic checkpoint that integrates macrophage mitochondrial metabolism, redox homeostasis and effector functions. This Bmal1-Hif-1α regulatory loop may provide therapeutic opportunities for inflammatory diseases and immunotherapy., Competing Interests: RA, YL, NK, KS, CX, AH, SL, DJ, NL, CL No competing interests declared, (© 2020, Alexander et al.)

Acute ischemic stroke is followed by a complex interplay between the brain and the immune system in which ischemia-reperfusion leads to a detrimental inflammatory response that causes brain injury. In the brain, IL-15 is expressed by astrocytes, neurons and microglia. Previous study showed that ischemia-reperfusion induces expression of IL-15 by astrocytes. Transgenic over-expression of IL-15 in astrocytes aggravates ischemia-reperfusion brain damage by increasing the levels and promoting the effector functions of CD8 + T and NK cells. Treatment of neonatal rats with IL-15 neutralizing antibody before hypoxia-ischemia induction reduces the infarct volume. However, as stroke-induced inflammatory responses differ between neonate and adult brain, the effects of IL-15 blockade on the injury and immune response arising from stroke in adult animals has remained unclear. In this study, we examined the effect of post-ischemia/reperfusion IL-15 blockade on the pathophysiology of cerebral ischemia-reperfusion in adult mice. Using a cerebral ischemia-reperfusion model, we compared infarct size and the infiltrating immune cells in the brain of wild type (WT) mice and Il15 -/- mice lacking NK and memory CD8 + T cells. We also evaluated the effects of IL-15 neutralizing antibody treatment on brain infarct volume, motor function, and the status of brain-infiltrating immune cells in WT mice. Il15 -/- mice show a smaller infarct volume and lower numbers of activated brain-infiltrating NK, CD8 + T, and CD4 + T cells compared to WT mice after cerebral ischemia-reperfusion. Post-ischemia/reperfusion IL-15 blockade reduces infarct size and improves motor and locomotor activity. Furthermore, IL-15 blockade reduces the effector function of NK, CD8 + T, and CD4 + T cells in the ischemia-reperfusion brain of WT mice. Ablation of IL-15 responses after cerebral ischemia-reperfusion ameliorates brain injury in adult mice. Therefore, targeting IL-15 is a potential effective therapy for ischemic stroke., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) causes death mainly by disseminated metastasis progression from the organ being confined. Different metastatic stages are closely related to cellular metabolic profiles. Normal hepatocyte and HepG2 cell line from low metastatic HCC were studied by NMR-based metabolomic techniques. Multivariate and univariate statistical analyses were utilized to identify characteristic metabolites from cells and cultured media. Elevated levels of acetate, creatine, isoleucine, leucine, and phenylalanine were observed in HepG2 cells, suggesting more active in gathering nutrient components along with altered amino acid metabolisms and enhanced lipid metabolism. High glucose consumption was significantly different in low metastatic cells. A series of characteristic metabolites were identified and served as biomarkers. Relative metabolic pathway analysis shows that low metastatic HepG2 cell line exhibits active behaviors in metabolisms and biosynthesis of specific amino acids and energy metabolism. Moreover, characteristic metabolites-based classification models executed by support vector machines algorithm perform robustly to classify normal hepatocyte and HepG2 cell line. It is concluded that NMR-based metabolomic analyses of cell lines can provide a powerful approach to understand metastasis-related biological alterations. The present study also provides a basis for metabolic markers determination of hepatic carcinoma in the future clinical study., (© 2017 International Federation for Cell Biology.)

Astrocytes play a pivotal role in neuronal survival under the condition of post-ischemic brain inflammation, but the relevant astrocyte-derived mediators of ischemic brain injury remain to be defined. IL-15 supports survival of multiple lymphocyte lineages in the peripheral immune system, but the role of IL-15 in inflammatory disease of the central nervous system is not well defined. Recent research has shown an increase of IL-15-expressing astrocytes in the ischemic brain. Since astrocytes promote neuron survival under cerebral ischemia by buffering excess extracellular glutamate and producing growth factors, recovery of astrocyte function could be of benefit for stroke therapy. Here, we report that IL-15 is the pro-survival cytokine that prevents astrocyte death from oxygen glucose deprivation (OGD)-induced damage. Astrocytes up-regulate expression of the IL-15/IL-15Rα complex under OGD, whereas OGD down-regulates the levels of pSTAT5 and pAkt in astrocytes. IL-15 treatment ameliorates the decline of pAkt, decreases the percentage of annexin V + cells, inhibits the activation of caspase-3, and activates the Akt pathway to promote astrocyte survival in response to OGD. We further identified that activation of Akt, but not PKCα/βI, is essential for astrocyte survival under OGD. Taken together, this study reveals the function of IL-15 in astrocyte survival via Akt phosphorylation in response to OGD-induced damage., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

PPARγ modulates energy metabolism and inflammation. However, its specific functions in the balance of immunity in vivo have been explored incompletely. In this study, by the age of 14 mo, Pparg(C/-) mice with PPARγ expression at 25% of the normal level exhibited high autoantibody levels and developed mesangial proliferative glomerulonephritis, which resembled systemic lupus erythematosus (SLE)-like autoimmune disease. These symptoms were preceded by splenomegaly at an early age, which was associated with increases in splenocyte accumulation and B-cell activation but not with relocation of hematopoiesis to the spleen. The mechanism of splenic lymphocyte accumulation involved reduced sphingosine-1-phosphate receptor 1 (S1P1) expression and diminished migration toward S1P in the Pparg(C/-) splenocytes, which impeded lymphocyte egression. Mechanistically, increased Th17 polarization and IL-17 signaling in the Pparg(C/-) CD4(+) T cells contributed to B-cell hyperactivation in the spleen. Finally, the activation of the remaining PPARγ in Pparg(C/-) mice by pioglitazone increased S1P1 levels, reduced the Th17 population in the spleen, and ameliorated splenomegaly. Taken together, our data demonstrated that reduction of Pparg expression in T-helper cells is critical for spontaneous SLE-like autoimmune disease development; we also revealed a novel function of PPARγ in lymphocyte trafficking and cross talk between Th17 and B cells.

Background: Interleukin 15 (IL-15) is thought to be abundant in the skeletal muscle under steady state conditions based on RNA expression; however, the IL-15 RNA level may not reflect the protein level due to post-transcriptional regulation. Although exogenous protein treatment and overexpression studies indicated IL-15 functions in the skeletal muscle, how the skeletal muscle cell uses IL-15 remains unclear. In myositis patients, IL-15 protein is up-regulated in the skeletal muscle. Given the supporting role of IL-15 in CD8(+) T-cell survival and activation and the pathogenic role of cytotoxic CD8(+) T cells in polymyositis and inclusion-body myositis, we hypothesize that IL-15 produced by the inflamed skeletal muscle promotes myositis via CD8(+) T cells., Methods: Expression of IL-15 and IL-15 receptors at the protein level by skeletal muscle cells were examined under steady state and cytokine stimulation conditions. The functions of IL-15 in the skeletal muscle were investigated using Il15 knockout (Il15 (-/-) ) mice. The immune regulatory role of skeletal muscle IL-15 was determined by co-culturing cytokine-stimulated muscle cells and memory-like CD8(+) T cells in vitro and by inducing autoimmune myositis in skeletal-muscle-specific Il15 (-/-) mice., Results: We found that the IL-15 protein was not expressed by skeletal muscle cells under steady state condition but induced by tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) stimulation and expressed as IL-15/IL-15 receptor alpha (IL-15Rα) complex. Skeletal muscle cells expressed a scanty amount of IL-15 receptor beta (IL-15Rβ) under either conditions and only responded to a high concentration of IL-15 hyperagonist, but not IL-15. Consistently, deficiency of endogenous IL-15 affected neither skeletal muscle growth nor its responses to TNF-α and IFN-γ. On the other hand, the cytokine-stimulated skeletal muscle cells presented antigen and provided IL-15 to promote the effector function of memory-like CD8(+) T cells. Genetic ablation of Il15 in skeletal muscle cells greatly ameliorated autoimmune myositis in mice., Conclusions: These findings together indicate that skeletal muscle IL-15 directly regulates immune effector cells but not muscle cells and thus presents a potential therapeutic target for myositis.

The interleukin-15 (IL-15) system is important for regulating both innate and adaptive immune responses, however, its role in autoimmune disease remained unclear. Here we found that Il15(-/-) and Il15ra(-/-) mice spontaneously developed late-onset autoimmune phenotypes. CD4(+) T cells of the knockout mice showed elevated autoreactivity as demonstrated by the induction of lymphocyte infiltration in the lacrimal and salivary glands when transferred into nude mice. The antigen-presenting cells in the thymic medullary regions expressed IL-15 and IL-15Rα, whose deficiency resulted in insufficient negative selection and elevated number of natural IL-17A-producing CD4(+) thymocytes. These findings reveal previously unknown functions of the IL-15 system in thymocyte development, and thus a new layer of regulation in T cell-mediated autoimmunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Hypoxic-ischemia (HI) and inflammation are the two major pathogenic mechanisms of brain injury in very preterm infants. The neurovascular unit is the major target of HI injury in the immature brain. Systemic inflammation may worsen HI by up-regulating neuroinflammation and disrupting the blood-brain barrier (BBB). Since neurons and oligodendrocytes, microvascular endothelial cells, and microglia may closely interact with each other, there may be a common signaling pathway leading to neuroinflammation and neurovascular damage after injury in the immature brain. TNF-α is a key pro-inflammatory cytokine that acts through the TNF receptor (TNFR), and c-Jun N-terminal kinases (JNK) are important stress-responsive kinases., Objective: To determine if TNFR1-JNK signaling is a shared pathway underlying neuroinflammation and neurovascular injury after lipopolysaccharide (LPS)-sensitized HI in the immature brain., Methods: Postpartum (P) day-5 mice received LPS or normal saline (NS) injection before HI. Immunohistochemistry, immunoblotting and TNFR1- and TNFR2-knockout mouse pups were used to determine neuroinflammation, BBB damage, TNF-α expression, JNK activation, and cell apoptosis. The cellular distribution of p-JNK, TNFR1/TNFR2 and cleaved caspase-3 were examined using immunofluorescent staining., Results: The LPS + HI group had significantly greater up-regulation of activated microglia, TNF-α and TNFR1 expression, and increases of BBB disruption and cleaved caspase-3 levels at 24 hours post-insult, and showed more cortical and white matter injury on P17 than the control and NS + HI groups. Cleaved caspase-3 was highly expressed in microvascular endothelial cells, neurons, and oligodendroglial precursor cells. LPS-sensitized HI also induced JNK activation and up-regulation of TNFR1 but not TNFR2 expression in the microglia, endothelial cells, neurons, and oligodendrocyte progenitors, and most of the TNFR1-positive cells co-expressed p-JNK. Etanercept (a TNF-α inhibitor) and AS601245 (a JNK inhibitor) protected against LPS-sensitized HI brain injury. The TNFR1-knockout but not TNFR2-knockout pups had significant reduction in JNK activation, attenuation of microglial activation, BBB breakdown and cleaved caspase-3 expression, and showed markedly less cortical and white matter injury than the wild-type pups after LPS-sensitized HI., Conclusion: TNFR1-JNK signaling is the shared pathway leading to neuroinflammation and neurovascular damage after LPS-sensitized HI in the immature brain.

T-cell receptor (TCR)-transduced signaling is critical to thymocyte development at the CD4/CD8 double-positive stage, but the molecules involved in this process are not yet fully characterized. We previously demonstrated that GM-CSF/IL-3/IL-5 receptor common β-chain-associated protein (CBAP) modulates ZAP70-mediated T-cell migration and adhesion. On the basis of the high expression of CBAP during thymocyte development, we investigated the function of CBAP in thymocyte development using a CBAP knockout mouse. CBAP-deficient mice showed normal early thymocyte development and positive selection. In contrast, several negative selection models (including TCR transgene, superantigen staphylococcal enterotoxin B, and anti-CD3 antibody treatment) revealed an attenuation of TCR-induced thymocyte deletion in CBAP knockout mice. This phenotype correlated with a reduced accumulation of BIM upon TCR crosslinking in CBAP-deficient thymocytes. Loss of CBAP led to reduced TCR-induced phosphorylation of proteins involved in both proximal and distal signaling events, including ZAP70, LAT, PLCγ1, and JNK1/2. Moreover, TCR-induced association of LAT signalosome components was reduced in CBAP-deficient thymocytes. Our data demonstrate that CBAP is a novel component in the TCR signaling pathway and modulates thymocyte apoptosis during negative selection.

NK cell development and homeostasis require IL-15 produced by both hematopoietic and parenchymal cells. Certain hematopoietic IL-15 sources, such as macrophages and dendritic cells, are known, whereas the source of parenchymal IL-15 remains elusive. Using two types of adipocyte-specific Il15(-/-) mice, we identified adipocytes as a parenchymal IL-15 source that supported NK cell development nonredundantly. Both adipocyte-specific Il15(-/-) mice showed reduced IL-15 production specifically in the adipose tissue but impaired NK cell development in the spleen and liver in addition to the adipose tissue. We also found that the adipose tissue harbored NK progenitors as other niches (e.g. spleen) for NK cell development, and that NK cells derived from transplanted adipose tissue populated the recipient's spleen and liver. These findings suggest that adipocyte IL-15 contributes to systemic NK cell development by supporting NK cell development in the adipose tissue, which serves as a source of NK cells for other organs., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The role of β-catenin in thymocyte development has been extensively studied, however, the function of β-catenin in thymic epithelial cells (TECs) remains largely unclear. Here, we demonstrate a requirement for β-catenin in keratin 5 (K5)-expressing TECs, which comprise the majority of medullary TECs (mTECs) and a progenitor subset for cortical TECs (cTECs) in the young adult thymus. We found that conditionally ablated β-catenin in K5(+)-TECs and their progeny cells resulted in thymic atrophy. The composition of TECs was also aberrantly affected. Percentages of K5(hi)K8(+)-TECs, K5(+)K8(-)-TECs and UEA1(+)-mTECs were significantly decreased and the percentage of K5(lo)K8(+)-TECs and Ly51(+)-cTECs were increased in β-catenin-deficient thymi compared with that in the control thymi. We also observed that β-catenin-deficient TEC lineage could give rise to K8(+)-cTECs more efficiently than wild-type TECs using lineage-tracing approach. Importantly, the expression levels of several transcription factors (p63, FoxN1 and Aire), which are essential for TEC differentiation, were altered in β-catenin-deficient thymi. Under the aberrant differentiation of TECs, development of all thymocytes in β-catenin-deficient thymi was impaired. Interleukin-7 (IL-7) and chemokines (Ccl19, Ccl25 and Cxcl12) levels were also downregulated in the thymic stromal cells in the mutants. Finally, introducing a BCL2 transgene in lymphoid lineages, which has been shown to rescue IL-7-deficient thymopoiesis, partially rescued the thymic atrophy and thymocyte development defects caused by induced ablation of β-catenin in K5(+)-TECs. Collectively, these findings suggest that β-catenin is required for the differentiation of TECs, thereby contributing to thymocyte development in the postnatal thymus.

IL-15 is an essential survival factor for CD8αα(+) intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL-15-induced survival signals in primary CD8αα(+) iIELs remains elusive. Although Bcl-2 level in CD8αα(+) iIELs positively correlates with IL-15Rα expression in the intestinal epithelial cells, overexpression of Bcl-2 only moderately restores CD8αα(+) γδ iIELs in Il15(-/-) mice. Here, we found that IL-15 promptly activated a Jak3-Jak1-PI3K-Akt pathway that led to the upregulation of Bcl-2 and Mcl-1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl-2 but also resulted in the phosphorylation of extra-long Bim at Ser(65) . The latter event facilitated the dissociation of Bim from Bcl-2 without affecting Bim abundance in IL-15-treated CD8αα(+) iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl-2 or removal of Bim from CD8αα(+) iIELs promoted their survival in Il15ra(-/-) mice. Taken together, IL-15 promotes CD8αα(+) iIEL survival by both increasing Bcl-2 levels and dissociating Bim from Bcl-2 through activation of a Jak3-Jak1-PI3K-Akt-ERK1/2 pathway, which differs from a previously reported IL-15-induced survival signal., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Osteoclasts and osteoblasts are not exist alone,while communicating with each other through direct contact, diffusible paracrine factors and cell-bone matrix interaction. Co-culture system of osteoblast with osteoclast,including direct co-culture and indirect co-culture. It should be according to the ratio of osteoclasts and osteoblasts under the pathology, choosing the same species. Compared with lonely culture of osteoblasts or osteoclasts,co-culture system is much closer to the microenvironment in vivo. It benefits to explain the interactions between osteoblasts and osteoclasts, exploring molecular communication in bone diseases. It was mainly used to investigate the pharmacological mechanism of herbal and western medicine in bone remodeling. Some osteoporosis drugs (such as epimedium,sanchi, fructus psoraleae, ranelate strontium) not only promoted osteoblastic bone formation, but also inhibited osteoclastic bone resorption in the system,so as to balance bone homeostasis. At the same time,it has been used to study medical physics and assess biomedical materials in recent years. Considerably,the co-cultrue system will be used to study the subchondral bone remodeling and its pharmacological mechanism of herbal and western medicine in osteoarthritis.

Hepatocytes have a direct necrotic role in acetaminophen (APAP)-induced liver injury (AILI), prolonged secondary inflammatory response through innate immune cells and cytokines also significantly contributes to APAP hepatotoxicity. Interleukin 15 (IL-15), a multifunction cytokine, regulates the adaptive immune system and influences development and function of innate immune cells. To better understand the role of IL-15 in liver injury, we treated wild-type (WT) and IL-15-knockout (Il15⁻/⁻) mice with a hepatotoxic dose of APAP to induce AILI and evaluated animal survival, liver damage, APAP metabolism in livers and the inflammatory response. Production of pro-inflammatory cytokines/chemokines was greater in Il15⁻/⁻ than WT mice. Subanalysis of hepatic infiltrated monocytes revealed greater neutrophil influx, along with greater hepatic induction of inducible nitric oxide synthase (iNOS), in Il15⁻/⁻ than WT mice. In addition, the level of hepatic hemeoxygenase 1 (HO-1) was partially suppressed in Il15⁻/⁻ mice, but not in WT mice. Interestingly, elimination of Kupffer cells and neutrophils did not alter the vulnerability to excess APAP in Il15⁻/⁻ mice. However, injection of galactosamine, a hepatic transcription inhibitor, significantly reduced the increased APAP sensitivity in Il15⁻/⁻ mice but had minor effect on WT mice. We demonstrated that deficiency of IL-15 increased mouse susceptibility to AILI. Moreover, Kupffer cell might affect APAP hepatotoxicity through IL-15.

NK cell development requires IL-15, which is "trans-presented" to IL-15Rβγ on NK cells by IL-15Rα on other cells. In this study, we report that different levels of IL-15 trans-presentation are required for different NK cell developmental events to reach full maturation status. Because the IL-15Rα intracellular domain has the capacity to recruit signaling molecules, we generated knockin and transgenic (Tg) mice that lack the intracellular domain to assess the role of the IL-15 trans-presentation level independent of the function of this domain. The level of IL-15Rα on various cells of these mice follows the order WT > Tg6 > knockin > Tg1 ≥ knockout. Bone marrow (BM)-derived dendritic cells prepared from these mice induced Stat5 phosphorylation in NK cells. The level of phospho-Stat5 correlated with the level of IL-15Rα on BMDCs, thus offering the opportunity to study quantitative effects of IL-15 trans-presentation on NK cell development in vivo. We found that NK cell homeostasis, mature NK cell differentiation, and acquisition of Ly49 receptor and effector functions require different levels of IL-15 trans-presentation input to achieve full status. All NK cell developmental events examined were quantitatively regulated by the IL-15Rα level of BM-derived and radiation-resistant accessory cells, but not by IL-15Rα of NK cells. We also found that IL-15Rα of radiation-resistant cells was more potent than IL-15Rα of BM-derived accessory cells in support of stage 2 to stage 3 splenic mNK differentiation. In summary, each examined developmental event required a particular level of IL-15 trans-presentation by accessory cells.

The development of invariant NKT (iNKT) cells depends on the thymus. After positive selection by CD4(+)CD8(+)CD1d(+) cortical thymocytes, iNKT cells proceed from CD44(low)NK1.1(-) (stage 1) to CD44(high)NK1.1(-) (stage 2), and then to CD44(high)NK1.1(+) (stage 3) cells. The programming of cytokine production occurs along the three differentiation stages, whereas the acquisition of NK receptors occurs at stage 3. Stage 3 thymic iNKT cells are specifically reduced in Il15ra(-/-) mice. The mechanism underlying this homeostatic deficiency and whether the IL-15 system affects other thymic iNKT cell developmental events remain elusive. In this study, we demonstrate that increased cell death contributed to the reduction of stage 3 cells in Il15ra(-/-) mice, as knockout of Bim restored this population. IL-15-dependent upregulation of Bcl-2 in stage 3 cells affected cell survival, as overexpression of hBcl-2 partially restored stage 3 cells in Il15ra(-/-) mice. Moreover, thymic iNKT cells in Il15ra(-/-) mice were impaired in functional maturation, including the acquisition of Ly49 and NKG2 receptors and the programming of cytokine production. Finally, IL-15Rα expressed by radiation-resistant cells is necessary and sufficient to support the survival as well as the examined maturation events of thymic iNKT cells.

Translationally controlled tumor protein (TCTP) is expressed throughout T cell development and prominently induced following T cell activation. However, its function(s) during these processes is unclear. Here, we demonstrated that conditional deletion of TCTP before the beta selection checkpoint resulted into a partial block of thymocyte development at the double-negative (DN) 3 stage. Deletion of TCTP in the double-positive (DP) stage did not cause any significant phenotype in the thymus except a slight increase of mature CD8 single-positive (SP) thymocytes. In contrast to the very modest phenotype observed in the thymus, a significant reduction of mature T cells was observed in the peripheral lymphoid organs of these two conditional null TCTP mutant mice. Detailed analysis revealed that the latter phenotype (peripheral T cell lymphopenia) was largely due to a decreased viability of mature TCTP-deficient (TCTP(-/-)) T cells. Transgenic expression of the anti-apoptotic protein Bcl-2 rescued the partial block of early thymocyte development, but not peripheral T cell lymphopenia of T-lineage-specific TCTP(-/-) mice, suggesting that the signaling networks of TCTP in these two processes are not identical. Last, we demonstrated that TCTP(-/-) T cells manifested a significant defect in T cell Ag receptor (TCR)-mediated cell proliferation. Further analysis revealed that such defect was due to a marked delay in the initial cell-cycle entry of TCTP(-/-) T cells following TCR stimulation. Together, these results indicate that TCTP plays a very modest role in thymocyte development, but is critical for peripheral T cell maintenance and TCR-mediated cell proliferation.

Objective: To observe the efficacy of polyglycoside of Tripterygium wilfordii(GTW) in preventing postoperative recurrence of Crohn disease(CD)., Methods: Thirty-nine post-operative CD patients in whom all of the diseased gut had been removed from January 2005 to December 2006 were enrolled in a randomized, placebo-controlled trial. The patients took GTW(21 cases) or SASP(18 cases) in two weeks after operation. Crohn disease activity index(CDAI), ESR and CRP were collected at week 0, 13, 26, 52 or at the onset of symptoms. Ileocolonoscopy was performed at the end of the trial or at the onset of symptoms., Results: One patient in GTW group and 2 patients in SASP group were lost and 2 patients in GTW were excluded from the trial for non-compliance. Clinical recurrence was ascertained in one patient (5.6%) received GTW and in four (25.0%) received SASP. Four of eighteen patients in GTW(22.2%) had endoscopic recurrence compared with nine of sixteen(56.2%) in SASP. There were significant differences between the two groups(P<0.05)., Conclusion: Tripterygium wilfordii showed good efficacy in preventing recurrence of postoperative CD which can maintain remission and prevent recurrence.

We previously demonstrated that IL-3 stimulates transcription of the antiapoptotic gene mcl-1 via two promoter elements designated as the SIE and CRE-2 sites. To further study the functional role of these two DNA elements, mutant mice with targeted mutations of both SIE and CRE-2 sites (SC mutants) were generated. Homozygous SC mutants manifested a markedly reduced level of Mcl-1 in thymus but not in other major organs such as spleen, liver, lung, or heart. Reduced expression of Mcl-1 in SC mutant thymus resulted in attenuated positive selection of double-positive thymocytes into both CD4 and CD8 lineages, a result likely due to reduced survival of SC mutant double-positive thymocytes that were supposed to be positively selected. In contrast, in the peripheral lymphoid organs, only CD8(+) but not CD4(+) T cells were significantly reduced in homozygous SC mutant mice, a result consistent with a more dramatic decrease both of Mcl-1 expression and cell viability in mutant CD8(+) compared with mutant CD4(+) T cells. Impaired T cell development and peripheral CD8(+) lymphopenia in homozygous SC mutant mice were both cell autonomous and could be rescued by enforced expression of human Mcl-1. Together, the promoter-knock-in mouse model generated in this study not only revealed a role of Mcl-1 in thymocyte-positive selection, but also uncovered that Mcl-1 expression is regulated in a tissue or cell lineage-specific manner.

Mice devoid of the IL-15 system lose over 90% of CD8alphaalpha(+) TCRalphabeta and TCRgammadelta intestinal intraepithelial lymphocytes (iIELs). Previous work revealed that IL-15Ralpha and IL-15 expressed by parenchymal cells, but not by bone marrow-derived cells, are required for normal CD8alphaalpha(+) iIEL homeostasis. However, it remains unclear when and how the IL-15 system affects CD8alphaalpha(+) iIELs through their development. This study found that IL-15Ralpha is dispensable for the thymic stage of CD8alphaalpha(+) TCRalphabeta and TCRgammadelta iIEL development but is required for the maintenance and/or differentiation of the putative lineage marker negative precursors in the intestinal epithelium, especially for the most mature CD8 single positive subset. Moreover, the IL-15 system directly supports the survival of mature CD8alphaalpha(+) iIEL in vivo. Taken together, this study suggests that regulation of CD8alphaalpha(+) iIEL homeostasis by the IL-15 system does not occur in the thymus but involves mature cells and putative precursors in the intestine.

Using a model of 2,4-dinitro-1-fluorobenzene-induced contact hypersensitivity (CHS) we found that, as compared with wild-type mice, IL-15 receptor alpha chain (IL-15Ralpha)-deficient mice showed significantly less ear swelling. This decreased response was associated with diminished expression of CCL5/RANTES and CXCL10/IP-10, chemokines critical for effector cell recruitment, in the inflamed tissue. We determined that both the number of CD8(+) T cells infiltrating the affected skin and the production of CCL5/RANTES by antigen-stimulated CD8(+) T cells were decreased in IL-15Ralpha(-/-) mice. The lower levels of CXCL10/IP-10 suggested that the IL-15Ralpha(-/-) mice had reduced production of IFN-gamma, the primary inducer of CXCL10/IP-10, which was in fact the case. However, by contrast with CCL5/RANTES, the diminished levels of IFN-gamma were likely due to the decreased number of skin-infiltrating CD8(+) T cells, since IFN-gamma production by antigen-stimulated CD8(+) T cells was comparable between wild-type and IL-15Ralpha(-/-) mice. Our data suggest a positive, pro-inflammatory feedback loop involving CCL5/RANTES, IFN-gamma and CXCL10/IP-10 that underlies the CHS reaction and that is disrupted, likely primarily by a defect in CCL5/RANTES production, in mice lacking IL-15Ralpha, resulting in impaired leukocyte recruitment and inflammation. Moreover, it is particularly noteworthy that the defect in CCL5/RANTES expression in CD8(+) T cells is intrinsic to the absence of IL-15Ralpha, indicating that IL-15Ralpha is critical for CCL5/RANTES expression in CD8(+) T cells.

Although IL-15 is known to be a T cell growth factor, the function in T cells of IL-15Ralpha, its high affinity receptor, remains unclear. We found that murine IL-15Ralpha(-/-) CD4(+) T cells hyperproliferated in response to TCR stimulation, in vitro and in vivo, and displayed a lower TCR activation threshold than wild-type CD4(+) T cells. TCR-induced activation of Zap70 and of the phospholipase C-gamma1-NFATp, Ras-ERK-c-Fos, and Rac-JNK-c-Jun pathways was all augmented in IL-15Ralpha(-/-) CD4(+) T cells. This in turn led to earlier IL-2Ralpha induction and higher IL-2 production, which most likely contribute to the hyperproliferation of IL-15Ralpha(-/-) CD4(+) T cells. Exogenous IL-15 reduced levels of TCR-activated signals, transcription factors, IL-2, and IL-2Ralpha, and division in wild-type CD4(+) T cells. These results reveal IL-15Ralpha to be a negative regulator for CD4(+) T cell activation and demonstrate a novel layer of regulation of TCR signaling by a cytokine system.

A previous study showed that a sodium hexafluorosilicate-calcium chloride-based two-solution fluoride (F) rinse containing 6 mmol/l of F was more effective than a 12 mmol/l F sodium fluoride rinse in depositing F on tooth surfaces and increasing oral F levels. The present study compared the remineralization effects of these two rinses in an intraoral de- and remineralization model. The results showed that the 6 mmol/l F two-solution rinse produced greater remineralization in increasing lesion mineral contents and reducing lesion depths. The results demonstrated that the effectiveness of an F regimen depends less on the F dose and more on the ability of the treatment to utilize F efficiently for remineralization., (Copyright 2002 S. Karger AG, Basel)

Mice that lack IL-15 or the IL-15R alpha-chain (IL-15Ralpha) are deficient in peripheral CD8(+), but not in CD4(+), T cells. This CD8(+) T cell-specific deficiency has now been investigated further by characterization of a new strain of IL-15Ralpha(-/-) mice. The adult mutant mice exhibited a specific reduction in the percentage of CD8-single positive TCR(high) thymocytes. The expression of Bcl-2 was reduced in both CD8(+) thymocytes and naive T cells of the mutant animals, and the susceptibility of these cells to death was increased. Memory CD8(+) cells were profoundly deficient in IL-15Ralpha(-/-)mice, and the residual memory-like CD8(+) cells contained a high percentage of dead cells and failed to up-regulate Bcl-2 expression compared with naive CD8(+) cells. Moreover, exogenous IL-15 both up-regulated the level of Bcl-2 in and reduced the death rate of wild-type and mutant CD8(+) T cells activated in vitro. These results indicate that IL-15 and IL-15Ralpha regulate the expression of Bcl-2 in CD8(+) T cells at all developmental stages. The reduced Bcl-2 content in CD8(+) cells might result in survival defect and contribute to the reduction of CD8(+) cells in IL-15Ralpha(-/-)mice.

Enterovirus 71 (EV71), the newest member of Enteroviridae, is notable for its etiological role in epidemics of severe neurological diseases in children. Developing effective vaccines is considered a top choice among all control measures. We compared the inactivated virus vaccine (10 microg protein/mouse) with subunit vaccines--VP1 DNA vaccine (100 microg/mouse) or recombinant VP1 protein (10 microg/mouse)--in its ability to elicit maternal antibody and to provide protection against lethal infection of EV71 in suckling mice. Prior to gestation, all three groups of vaccinated dams possessed similar levels of neutralizing antibody. With a challenge dose of 2300 LD(50) virus/mouse, suckling mice born to dams immunized with inactivated virus showed 80% survival. The subunit vaccines provided protection only at a lower challenge dosage of 230 LD(50) per mouse, with 40% survival for DNA vaccine and 80% survival for VP1 protein. The cytokine profile produced by splenocytes showed a high level of IL-4 in the inactivated virus group, high levels of IFN-gamma and IL-12 in the DNA vaccine group, and high levels of IL-10 and IFN-gamma in the VP1 protein group. Overall, the inactivated virus elicited a much greater magnitude of immune response than the subunit vaccines, including total IgG, all four IgG subtypes, and T-helper-cell responses; these antibodies were shown to be protective against lethal infection when passively transferred to susceptible newborn mice. Our data indicated that inactivated virus is the choice of vaccine preparation capable of fulfilling the demand for effective control, and that VP1 subunit vaccines remain promising vaccine strategies that require further refinement.

CD8 single-positive cells, including CD8alphaalpha+ and CD8alphabeta+ subsets, constitute the majority of TCRalphabeta+ intestinal intraepithelial lymphocytes (alphabeta iIEL) in mice. CD8+ alphabeta iIEL show significantly weaker responses to TCR stimulation in the presence of exogenous IL-2 than do CD8+ T cells of the central immune system. IL-15 is a T cell growth factor likely expressed in the intestine mucosa. To understand the role of IL-15 in CD8+ alphabeta iIEL biology, we compared the effects of exogenous IL-15 and IL-2 on the survival and primary responses of the two CD8+ alphabeta iIEL subsets in vitro. In contrast to the death of approximately 60% of both CD8alphaalpha+ and CD8alphabeta+ iIEL cultured in IL-2 with or without TCR stimulation, IL-15 promoted survival of the CD8alphaalpha+ subset in the presence of TCR stimulation and promoted survival of both subsets in the absence of TCR stimulation. The higher proliferation level of TCR stimulated CD8alphaalpha+ alphabeta iIEL cultured in IL-15 compared with those cultured in IL-2 is likely due to IL-15's prosurvival effects. In addition, unlike exogenous IL-2, exogenous IL-15 did not support the effector functions of either iIEL subsets, including IFN-gamma production, IL-4-induced Th2 cytokine production, and anti-TCR mAb-redirected cytotoxicity. These findings demonstrate that IL-15 and IL-2 are functionally distinct and suggest that IL-15 plays a unique role in the maintenance of the CD8+ alphabeta iIEL pool in the absence of Ag stimulation and in the survival and expansion of CD8alphaalpha+ alphabeta iIEL upon Ag stimulation.

TCR gamma delta+ cells are enriched in the intestine mucosa and constitute approximately half of the intestinal intraepithelial lymphocytes (iIEL) in mice. They are likely activated by self and foreign Ags in situ, but little is known about how the activated gamma delta iIEL are regulated. In the iIEL compartment, IL-2 is produced by activated TCR alpha beta+ iIEL, and IL-15 message is detected in iIEL and in the epithelial cells. We found surface expression of IL-2 as well as IL-15Rs on activated gamma delta iIEL, and examined the effects of IL-2 and IL-15 on the survival and death of gamma delta iIEL during secondary stimulation through TCR. We found that both cytokines supported growth of the restimulated gamma delta iIEL, but exerted different effects on their survival. A significant higher number of live cells were recovered from the gamma delta iIEL cultures restimulated in IL-15 than in IL-2. Quantitation of apoptotic cells showed more cell death in the IL-2 group than in the IL-15 group. The cell death was associated with restimulation through TCR and was not caused by insufficient growth factor, thus representing activation-induced cell death. Western blot analyses found no difference in the levels of Bcl-2 and Bax proteins between the two groups. However, the level of Bcl-xL protein diminished with time in the IL-2 group whereas the level was sustained in the IL-15 group, which may contribute to the pro-survival effect of IL-15. These results demonstrated that the survival of activated gamma delta iIEL is differentially regulated by IL-2 and IL-15.

CD8 single-positive (CD8+) T cells in murine intestinal intraepithelial lymphocytes (iIEL) consist of alpha alpha-CD8+ and alpha beta-CD8+ subpopulations. Cytotoxicity represents an important function of peripheral CD8+ T cells, so we examined perforin-granzymebased and Fas-based cytotoxicity of activated CD8+ TCR-alpha beta+ iIEL subsets. We found that allospecific CTL activity was induced from alpha beta-CD8+ iIEL but not from alpha alpha-CD8+ iIEL even when allospecific TCR were present on the iIEL, as demonstrated by using 2C TCR transgenic mice. On the other hand, both CD8+ iIEL subsets proliferated upon allostimulation with a lower responder frequency than CD8+ LN cells. The alpha alpha-CD8+ TCR-alpha beta+ iIEL appeared to lose their ability to perform perforin-based killing after activation through TCR because fresh cells lysed P815 cells coated with anti-TCR beta-chain (TCR-beta) mAb, whereas cells activated by plate-bound anti-TCR mAb did not. Of interest, both activated CD8+ TCR-alpha beta+ iIEL subsets, but not fresh cells, were able to mediate Fas-based killing when triggered with PMA and CA2+ ionophore.

The CD8+CD4- (CD8+) murine small intestinal intraepithelial lymphocytes (IELs) contain two subpopulations, one expressing alpha alpha-CD8 homodimers and another alpha beta-CD8 heterodimers. In this study, plate-bound anti-TCR beta-chain (TCR-beta) mAb alone or combined with anti-CD28 mAb is used as a model system to study activation requirement of these two CD8+ IEL subsets. In contrast to CD8+ lymph node (LN) cells that require both TCR and CD28 triggering for activation, alpha beta-CD8+ IELs proliferate and produce IL-2 and IFN-gamma when stimulated with anti-TCR-beta mAb alone, and soluble CTLA-4 Ig has no effect on their responses. On the other hand, alpha alpha-CD8+ IELs neither make IL-2 or IFN-gamma nor proliferate even when both stimuli are provided. However, alpha alpha-CD8+ IELs are capable of proliferation in both CD8+ IEL subsets is lower than in CD8+ LN cells, which contributes to the weaker and delayed response of CD8+ IELs.

Minor lymphocyte-stimulating (Mls) Ag are alloantigens that stimulate T cells expressing specific V beta regions. Recent studies have established that Mls Ag are examples of endogenous superantigens encoded by the products of endogenous mouse mammary tumor virus (MLV) genomes. In a mouse strain that expresses a given mammary tumor virus (Mls) Ag, reactive T cells expressing the corresponding V beta region are profoundly deficient, due at least in part to clonal deletion of the cells during their development. Expression of Mls and other endogenous superantigens, therefore, results in profound alterations in the ultimate repertoire of T cells in an animal. A role for endogenous superantigens in positive selection of T cells has not been previously established. Here we present evidence that expression of Mls-1a leads to a specific increase in the abundance of V beta 14+ T cells. Genetic studies indicate linkage of the effect to the Mls-1a gene. Neonatal tolerance studies argue against the possibility that the increase is due solely to the deletion of Mls-reactive V beta 14- T cells. The results are consistent with the Mls-1a product playing a role in the positive selection of V beta 14+ T cells.

The specificity of T cells bearing gamma delta T-cell receptors (gamma delta+ T cells) is poorly characterized. Earlier studies suggest that like alpha beta+CD8+ T cells, some gamma delta+ T cells may recognize antigens associated with class I major histocompatibility complex molecules. alpha beta+CD8+ T cells are nearly absent in class I-deficient mice (mutant for beta 2-microglobulin), reflecting a requirement for intrathymic "positive selection" of these cells by class I molecules. Here, we examine whether the development of gamma delta+ T cells is altered in the beta 2-microglobulin mutant mice. We show that the cellularity, marker expression, repertoire, and functional competence of gamma delta+ T cells are not detectably deficient in beta 2-microglobulin mutant mice. We conclude that class I expression is unnecessary for the development of most gamma delta+ T cells.

The role of major histocompatibility complex (MHC) class I expression in natural killer (NK) cell target recognition is controversial. Normal T cell blasts from MHC class I-deficient mutant mice were found to serve as target cells for NK cells in vitro, which suggests that MHC class I molecules are directly involved in NK cell recognition. Spleen cells from the mutant mice were deficient in their ability to lyse MHC class I-deficient target cells or NK-susceptible tumor targets, and mutant mice could not reject allogeneic bone marrow. Thus, class I molecules may participate in the positive selection or tolerance induction of NK cells.