mTOR signaling promotes rapid m6A mRNA methylation to regulate NK-cell activation and effector functions.

Natural killer (NK) cells can be rapidly activated in response to cytokines during host defense against malignant cells or viral infection. However, it remains unclear what mechanisms precisely and rapidly regulate the expression of the numerous genes involved in activating NK cells. In this study, we discovered that NK-cell N6-methyladenosine (m6A) methylation levels were rapidly upregulated upon short-term NK-cell activation and were repressed in the tumor microenvironment. Deficiency of methyltransferase-like 3 (METTL3) or METTL14 moderately influenced NK-cell homeostasis, while double knockout of METTL3/14 significantly impacted NK-cell homeostasis, maturation, and antitumor immunity. This suggests a cooperative role of METTL3 and METTL14 in regulating NK-cell development and effector functions. Using methylated RNA immunoprecipitation sequencing (MeRIP-seq), we demonstrated that genes involved in NK-cell effector functions, such as Prf1 and Gzmb, were directly modified by m6A methylation. Furthermore, inhibiting mTOR complex 1 (mTORC1) activation prevented m6A methylation levels from increasing when NK cells were activated, and this could be restored by S-adenosylmethionine (SAM) supplementation. Collectively, we have unraveled crucial roles for rapid m6A mRNA methylation downstream of the mTORC1-SAM signal axis in regulating NK-cell activation and effector functions.

The ω-3 Polyunsaturated Fatty Acid Docosahexaenoic Acid Enhances NK-Cell Antitumor Effector Functions.

ω-3 polyunsaturated fatty acids (PUFA) are known to directly repress tumor development and progression. In this study, we explored whether docosahexaenoic acid (DHA), a type of ω-3 PUFA, had an immunomodulatory role in inhibiting tumor growth in immunocompetent mice. The number of natural killer (NK) cells but not the number of T or B cells was decreased by DHA supplementation in various tissues under physiologic conditions. Although the frequency and number of NK cells were comparable, IFNγ production by NK cells in both the spleen and lung was increased in DHA-supplemented mice in the mouse B16F10 melanoma tumor model. Single-cell RNA sequencing revealed that DHA promoted effector function and oxidative phosphorylation in NK cells but had no obvious effects on other immune cells. Using Rag2-/- mice and NK-cell depletion by PK136 antibody injection, we demonstrated that the suppression of B16F10 melanoma tumor growth in the lung by DHA supplementation was dependent mainly on NK cells. In vitro experiments showed that DHA directly enhanced IFNγ production, CD107a expression, and mitochondrial oxidative phosphorylation (OXPHOS) activity and slightly increased proliferator-activated receptor gamma coactivator-1α (PGC-1α) protein expression in NK cells. The PGC-1α inhibitor SR-18292 in vitro and NK cell-specific knockout of PGC-1α in mice reversed the antitumor effects of DHA. In summary, our findings broaden the current knowledge on how DHA supplementation protects against cancer growth from the perspective of immunomodulation by upregulating PGC-1α signaling-mediated mitochondrial OXPHOS activity in NK cells.

Single-cell landscape of the cellular microenvironment in three different colonic polyp subtypes in children.

BACKGROUND: The understanding of the heterogeneous cellular microenvironment of colonic polyps in paediatric patients with solitary juvenile polyps (SJPs), polyposis syndrome (PJS) and Peutz-Jeghers syndrome (PJS) remains limited. METHODS: We conducted single-cell RNA sequencing and multiplexed immunohistochemistry (mIHC) analyses on both normal colonic tissue and different types of colonic polyps obtained from paediatric patients. RESULTS: We identified both shared and disease-specific cell subsets and expression patterns that played important roles in shaping the unique cellular microenvironments observed in each polyp subtype. As such, increased myeloid, endothelial and epithelial cells were the most prominent features of SJP, JPS and PJS polyps, respectively. Noticeably, memory B cells were increased, and a cluster of epithelial-mesenchymal transition (EMT)-like colonocytes existed across all polyp subtypes. Abundant neutrophil infiltration was observed in SJP polyps, while CX3CR1hi CD8+ T cells and regulatory T cells (Tregs) were predominant in SJP and JPS polyps, while GZMAhi natural killer T cells were predominant in PJS polyps. Compared with normal colonic tissues, myeloid cells exhibited specific induction of genes involved in chemotaxis and interferon-related pathways in SJP polyps, whereas fibroblasts in JPS polyps had upregulation of myofiber-associated genes and epithelial cells in PJS polyps exhibited induction of a series of nutrient absorption-related genes. In addition, the TNF-α response was uniformly upregulated in most cell subsets across all polyp subtypes, while endothelial cells and fibroblasts separately showed upregulated cell adhesion and EMT signalling in SJP and JPS polyps. Cell-cell interaction network analysis showed markedly enhanced intercellular communication, such as TNF, VEGF, CXCL and collagen signalling networks, among most cell subsets in polyps, especially SJP and JPS polyps. CONCLUSION: These findings strengthen our understanding of the heterogeneous cellular microenvironment of polyp subtypes and identify potential therapeutic approaches to reduce the recurrence of polyps in children.

mTORC2 acts as a gatekeeper for mTORC1 deficiency-mediated impairments in ILC3 development.

Group 3 innate lymphoid cells (ILC3s) are mediators of intestinal immunity and barrier function. Recent studies have investigated the role of the mammalian target of rapamycin complex (mTOR) in ILC3s, whereas the mTORC1-related mechanisms and crosstalk between mTORC1 and mTORC2 involved in regulating ILC3 homeostasis remain unknown. In this study, we found that mTORC1 but not mTORC2 was critical in ILC3 development, IL-22 production, and ILC3-mediated intestinal homeostasis. Single-cell RNA sequencing revealed that mTORC1 deficiency led to disruption of ILC3 heterogeneity, showing an increase in differentiation into ILC1-like phenotypes. Mechanistically, mTORC1 deficiency decreased the expression of NFIL3, which is a critical transcription factor responsible for ILC3 development. The activities of both mTORC1 and mTORC2 were increased in wild-type ILC3s after activation by IL-23, whereas inhibition of mTORC1 by Raptor deletion or rapamycin treatment resulted in increased mTORC2 activity. Previous studies have demonstrated that S6K, the main downstream target of mTORC1, can directly phosphorylate Rictor to dampen mTORC2 activity. Our data found that inhibition of mTORC1 activity by rapamycin reduced Rictor phosphorylation in ILC3s. Reversing the increased mTORC2 activity via heterozygous or homozygous knockout of Rictor in Raptor-deleted ILC3s resulted in severe ILC3 loss and complete susceptibility to intestinal infection in mice with mTORC1 deficiency (100% mortality). Thus, mTORC1 acts as a rheostat of ILC3 heterogeneity, and mTORC2 protects ILC3s from severe loss of cells and immune activity against intestinal infection when mTORC1 activity is diminished.

Effect of dietary supplemental vitamin C and betaine on the growth performance, humoral immunity, immune organ index, and antioxidant status of broilers under heat stress.

Heat stress (HS) has become one of the important factors affecting the development of animal husbandry. The purpose of this experiment was to investigate whether vitamin C (Vc) and betaine (Bet) improve immune organ index and humoral immunity by enhancing the antioxidant status of immune organs, thus protecting broilers from HS-induced injuries. A total of 200 28-day-old Ross 308 broilers were randomly assigned into 5 groups (n = 4 replicates/group, 10 broilers/replicate) which were reared at different ambient temperatures (24 ± 1°C or 33 ± 1°C). The control group fed basal diet, while high-temperature groups were either fed a basal diet (HS group) or a basal diet supplemented with 250-mg Vc/kg diet (HSVc group), 1000-mg Bet/kg diet (HSBet group), and 250-mg Vc plus 1000 mg Bet/kg diet (HSVcBet group), respectively. On day 42, growth performance, humoral immune function, immune organ index, and antioxidant capacity were measured. HS reduced the productive performance of broilers, antibody potency against the Newcastle disease virus (NDV) and sheep red blood cells (SRBC), indices of thymus and bursa, and antioxidant capacity of immune organs. Adding Vc alone or in combination with Bet improved performance, NDV and SRBC antibody potency, thymus and bursa indices, and antioxidant capacity of immune organs in heat-stressed broilers, with the most effective being combination. In summary, HS reduces the antioxidant capacity and immune organ development status of broiler immune organs. Vc and/or Bet can improve the development of immune organs and restore part of the production performance by regulating the antioxidant status of immune organs, among which the combined addition of Vc and Bet has the best effect.

Novel Loss of Function (G15D) Mutation on RAC2 in a Family with Combined Immunodeficiency and Increased Levels of Immunoglobulin G, A, and E.

Ras-related C3 botulinum toxin substrate 2 (RAC2) is a small guanine nucleotide binding molecule that is exclusively expressed in hematopoietic cell lineages as a switcher. Based on in vivo and/or in vitro model experiments, RAC2 plays important roles in different cells through proliferation, secretion, and phagocytosis. It also performs a suppressing function in immunoglobulin (Ig) switching in Rac2-/- animals or cells. Several RAC2 natural mutations have been described in patients with primary immunodeficiency. RAC2 mutations can be classified into loss-of-function inactivating (LoF-I) and gain-of-function activating mutations according to their functional effects. Only two LoF-I mutations on RAC2 have been reported, including a dominant D57N mutation in several cases that exhibit granulocyte function defects and a recessive D56X mutation in cases with common variable immunodeficiency. Regardless of the type of mutation, most of the reported RAC2 mutant cases have shown reduced IgG, IgA, and IgM levels. Herein, we report on a family with three members that suffer from persistent HPV infection, recurrent respiratory infections, bronchiectasis, and autoimmune disease. The immunologic profile suggests that the family was affected by combined immunodeficiency (CID) with increased serum levels of IgG, IgA, and IgE. Exome sequencing identified a de novo RAC2 mutation (c.44G > A/p.G15D) that was co-segregated with the disease in the family. Gene functional experiments identified that such mutation results in reduced guanosine triphosphate binding activity and RAC2 protein expression. In patients' lymphocytes, impaired aggregation and proliferation effects, decreased mitochondrial membrane potential, and increased levels of cell apoptosis were observed, although no functional abnormalities were detected in neutrophils. To our knowledge, this study was the first to identify a LoF-I mutation of RAC2 affecting lymphocyte function that consequently led to CID and increased levels of serum IgG, IgE, and IgA. This study presents a novel subtype of RAC2-related immune disorder.

Effects of betaine on viability, apoptosis, function protein expression and oxidative status in heat-stressed IEC-6 cells.

Intestinal epithelial dysfunction is one of the key factors in the pathogenesis of heat stress-induced disease. The purpose of this experiment was to investigate whether betaine protects IEC-6 cells from dysfunction induced by heat stress (HS) through antioxidative mechanism. The IEC-6 cells were divided into four groups: control group incubated at 37 °C, while those in heat treated groups (41 °C for 24 h) were pretreated with 0, 0.5 and 1 mmol/L betaine, respectively. Cell viability, apoptosis, barrier function protein and oxidative status were analyzed. Compared to control group, the rate of apoptosis and the Bax and caspase-3 expressions significantly increased in HS group (P < 0.05), however, cell activity, total antioxidative capacity (T-AOC), activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and the expression of Bcl-2, claudin-1 and occludin decreased significantly (P < 0.05). Betaine (0.5 mmol/L) can significantly enhance IEC-6 cell viability, while significantly reduce the apoptosis rate of cell during HS (P < 0.05). Meanwhile, the expression of Bcl-2, claudin-1 and occludin proteins were also significantly upregulated (P < 0.05) when compared to HS group. HS had a negative impact on IEC-6 cells, while betaine protected from damage caused by HS via increasing the antioxidative capacity. This suggested that betaine might be an effective dietary additive to protect animals from detrimental intestinal reactions caused by HS.

Sex differences exist in adult heart group 2 innate lymphoid cells.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are the most dominant ILCs in heart tissue, and sex-related differences exist in mouse lung ILC2 phenotypes and functions; however, it is still unclear whether there are sex differences in heart ILC2s. RESULTS: Compared with age-matched wild-type (WT) male mice, 8-week-old but not 3-week-old WT female mice harbored an obviously greater percentage and number of heart ILC2s in homeostasis. However, the percentage of killer-cell lectin-like receptor G1 (Klrg1)- ILC2s was higher, but the Klrg1+ ILC2s were lower in female mice than in male mice in both heart tissues of 3- and 8-week-old mice. Eight-week-old Rag2-/- mice also showed sex differences similar to those of age-matched WT mice. Regarding surface marker expression, compared to age-matched male mice, WT female mice showed higher expression of CD90.2 and Ki67 and lower expression of Klrg1 and Sca-1 in heart total ILC2s. There was no sex difference in IL-4 and IL-5 secretion by male and female mouse heart ILC2s. Increased IL-33 mRNA levels within the heart tissues were also found in female mice compared with male mice. By reanalyzing published single-cell RNA sequencing data, we found 2 differentially expressed genes between female and male mouse heart ILC2s. Gene set variation analysis revealed that the glycine, serine and threonine metabolism pathway was upregulated in female heart ILC2s. Subcluster analysis revealed that one cluster of heart ILC2s with relatively lower expression of Semaphorin 4a and thioredoxin interacting protein but higher expression of hypoxia-inducible lipid droplet-associated. CONCLUSIONS: These results revealed greater numbers of ILC2s, higher expression of CD90.2, reduced Klrg1 and Sca-1 expression in the hearts of female mice than in male mice and no sex difference in IL-4 and IL-5 production in male and female mouse heart ILC2s. These sex differences in heart ILC2s might be due to the heterogeneity of IL-33 within the heart tissue.

Docosahexaenoic acid supplementation represses the early immune response against murine cytomegalovirus but enhances NK cell effector function.

BACKGROUND: Docosahexaenoic acid (DHA) supplementation is beneficial for several chronic diseases; however, its effect on immune regulation is still debated. Given the prevalence of cytomegalovirus (CMV) infection and because natural killer (NK) cells are a component of innate immunity critical for controlling CMV infection, the current study explored the effect of a DHA-enriched diet on susceptibility to murine (M) CMV infection and the NK cell effector response to MCMV infection. RESULTS: Male C57BL/6 mice fed a control or DHA-enriched diet for 3 weeks were infected with MCMV and sacrificed at the indicated time points postinfection. Compared with control mice, DHA-fed mice had higher liver and spleen viral loads at day 7 postinfection, but final MCMV clearance was not affected. The total numbers of NK cells and their terminal mature cell subset (KLRG1+ and Ly49H+ NK cells) were reduced compared with those in control mice at day 7 postinfection but not day 21. DHA feeding resulted in higher IFN-γ and granzyme B expression in splenic NK cells at day 7 postinfection. A mechanistic analysis showed that the splenic NK cells of DHA-fed mice had enhanced glucose uptake, increased CD71 and CD98 expression, and higher mitochondrial mass than control mice. In addition, DHA-fed mice showed reductions in the total numbers and activation levels of CD4+ and CD8+ T cells. CONCLUSIONS: These results suggest that DHA supplementation represses the early response to CMV infection but preserves NK cell effector functions by improving mitochondrial activity, which may play critical roles in subsequent MCMV clearance.

Identification of nafamostat mesylate as a selective stimulator of NK cell IFN-γ production via metabolism-related compound library screening.

Natural killer (NK) cells play important roles in controlling virus-infected and malignant cells. The identification of new molecules that can activate NK cells may effectively improve the antiviral and antitumour activities of these cells. In this study, by using a commercially available metabolism-related compound library, we initially screened the capacity of compounds to activate NK cells by determining the ratio of interferon-gamma (IFN-γ)+ NK cells by flow cytometry after the incubation of peripheral blood mononuclear cells (PBMCs) with IL-12 or IL-15 for 18 h. Our data showed that eight compounds (nafamostat mesylate (NM), loganin, fluvastatin sodium, atorvastatin calcium, lovastatin, simvastatin, rosuvastatin calcium, and pitavastatin calcium) and three compounds (NM, elesclomol, and simvastatin) increased the proportions of NK cells and CD3+ T cells that expressed IFN-γ among PBMCs cultured with IL-12 and IL-15, respectively. When incubated with enriched NK cells (purity ≥ 80.0%), only NM enhanced NK cell IFN-γ production in the presence of IL-12 or IL-15. When incubated with purified NK cells (purity ≥ 99.0%), NM promoted NK cell IFN-γ secretion in the presence or absence of IL-18. However, NM showed no effect on NK cell cytotoxicity. Collectively, our study identifies NM as a selective stimulator of IFN-γ production by NK cells, providing a new strategy for the prevention and treatment of infection or cancer in select populations.

Prenatal inflammation exposure-programmed hypertension exhibits multi-generational inheritance via disrupting DNA methylome.

The multi-generation heredity trait of hypertension in human has been reported, but the molecular mechanisms underlying multi-generational inheritance of hypertension remain obscure. Recent evidence shows that prenatal inflammatory exposure (PIE) results in increased incidence of cardiovascular diseases, including hypertension. In this study we investigated whether and how PIE contributed to multi-generational inheritance of hypertension in rats. PIE was induced in pregnant rats by intraperitoneal injection of LPS or Poly (I:C) either once on gestational day 10.5 (transient stimulation, T) or three times on gestational day 8.5, 10.5, and 12.5 (persistent stimulation, P). Male offspring was chosen to study the paternal inheritance. We showed that PIE, irrespectively induced by LPS or Poly (I:C) stimulation during pregnancy, resulted in multi-generational inheritance of significantly increased blood pressure in rat descendants, and that prenatal LPS exposure led to vascular remodeling and vasoconstrictor dysfunction in both thoracic aorta and superior mesenteric artery of adult F2 offspring. Furthermore, we revealed that PIE resulted in global alteration of DNA methylome in thoracic aorta of F2 offspring. Specifically, PIE led to the DNA hypomethylation of G beta gamma (Gßγ) signaling genes in both the F1 sperm and the F2 thoracic aorta, and activation of PI3K/Akt signaling was implicated in the pathologic changes and dysregulated vascular tone of aortic tissue in F2 LPS-P offspring. Our data demonstrate that PIE reprogrammed DNA methylome of cells from the germline/mature gametes contributes to the development of hypertension in F2 PIE offspring. This study broadens the current knowledge regarding the multi-generation effect of the cumulative early life environmental factors on the development of hypertension.

Screening for Active Compounds Targeting Human Natural Killer Cell Activation Identifying Daphnetin as an Enhancer for IFN-γ Production and Direct Cytotoxicity.

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ+ NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments.

Conditional knockout of Tsc1 in RORγt-expressing cells induces brain damage and early death in mice.

BACKGROUND: Tuberous sclerosis complex 1 (Tsc1) is known to regulate the development and function of various cell types, and RORγt is a critical transcription factor in the immune system. However, whether Tsc1 participates in regulating RORγt-expressing cells remains unknown. METHODS: We generated a mouse model in which Tsc1 was conditionally deleted from RORγt-expressing cells (Tsc1RORγt) to study the role of RORγt-expressing cells with Tsc1 deficiency in brain homeostasis. RESULTS: Type 3 innate lymphoid cells (ILC3s) in Tsc1RORγt mice displayed normal development and function, and the mice showed normal Th17 cell differentiation. However, Tsc1RORγt mice exhibited spontaneous tonic-clonic seizures and died between 4 and 6 weeks after birth. At the age of 4 weeks, mice in which Tsc1 was specifically knocked out in RORγt-expressing cells had cortical neuron defects and hippocampal structural abnormalities. Notably, over-activation of neurons and astrogliosis were observed in the cortex and hippocampus of Tsc1RORγt mice. Moreover, expression of the γ-amino butyric acid (GABA) receptor in the brains of Tsc1RORγt mice was decreased, and GABA supplementation prolonged the lifespan of the mice to some extent. Further experiments revealed the presence of a group of rare RORγt-expressing cells with high metabolic activity in the mouse brain. CONCLUSIONS: Our study verifies the critical role of previously unnoticed RORγt-expressing cells in the brain and demonstrates that the Tsc1 signaling pathway in RORγt-expressing cells is important for maintaining brain homeostasis.

α-Galactosylceramide and its analog OCH differentially affect the pathogenesis of ISO-induced cardiac injury in mice.

Immunotherapies for cancers may cause severe and life-threatening cardiotoxicities. The underlying mechanisms are complex and largely elusive. Currently, there are several ongoing clinical trials based on the use of activated invariant natural killer T (iNKT) cells. The potential cardiotoxicity commonly associated with this particular immunotherapy has yet been carefully evaluated. The present study aims to determine the effect of activated iNKT cells on normal and ß-adrenergic agonist (isoproterenol, ISO)-stimulated hearts. Mice were treated with iNKT stimulants, α-galactosylceramide (αGC) or its analog OCH, respectively, to determine their effect on ISO-induced cardiac injury. We showed that administration of αGC (activating both T helper type 1 (Th1)- and T helper type 2 (Th2)-liked iNKT cells) significantly accelerated the progressive cardiac injury, leading to enhanced cardiac hypertrophy and cardiac fibrosis with prominent increases in collagen deposition and TGF-ß1, IL-6, and alpha smooth muscle actin expression. In contrast to αGC, OCH (mainly activating Th2-liked iNKT cells) significantly attenuated the progression of cardiac injury and cardiac inflammation induced by repeated infusion of ISO. Flow cytometry analysis revealed that αGC promoted inflammatory macrophage infiltration in the heart, while OCH was able to restrain the infiltration. In vitro coculture of αGC- or OCH-pretreated primary peritoneal macrophages with primary cardiac fibroblasts confirmed the profibrotic effect of αGC and the antifibrotic effect of OCH. Our results demonstrate that activating both Th1- and Th2-liked iNKT cells is cardiotoxic, while activating Th2-liked iNKT cells is likely cardiac protective, which has implied key differences among subpopulations of iNKT cells in their response to cardiac pathological stimulation.

Unique Phenotypes of Heart Resident Type 2 Innate Lymphoid Cells.

Innate lymphoid cells (ILCs), including ILC1s, ILC2s, and ILC3s, play critical roles in regulating immunity, inflammation, and tissue homeostasis. However, limited attention is focused on the unique phenotype of ILCs in the heart tissue. In this study, we analyzed the ILC subsets in the heart by flow cytometry and found that ILC2s were the dominant population of ILCs, while a lower proportion of type 1 ILCs (including ILC1 and NK cells) and merely no ILC3s in the heart tissue of mice. Our results show that ILC2 development kinetically peaked in heart ILC2s at the age of 4 weeks after birth and later than lung ILC2s. By conducting parabiosis experiment, we show that heart ILC2s are tissue resident cells and minimally replaced by circulating cells. Notably, heart ILC2s have unique phenotypes, such as lower expression of ICOS, CD25 (IL-2Rα), and Ki-67, higher expression of Sca-1 and GATA3, and stronger ability to produce IL-4 and IL-13. In doxorubicin-induced myocardial necroptosis model of mouse heart tissue, IL-33 mRNA expression level and ILC2s were remarkably increased. In addition, IL-4 production by heart ILC2s, but not lung ILC2s, was also dramatically increased after doxorubicin treatment. Our results demonstrate that heart-resident ILC2s showed tissue-specific phenotypes and rapidly responded to heart injury. Thus, further studies are warranted to explore the potential for IL-33-elicited ILC2s response as therapeutics for attenuating heart damage.

Crosstalks between mTORC1 and mTORC2 variagate cytokine signaling to control NK maturation and effector function.

The metabolic checkpoint kinase mechanistic/mammalian target of rapamycin (mTOR) regulates natural killer (NK) cell development and function, but the exact underlying mechanisms remain unclear. Here, we show, via conditional deletion of Raptor (mTORC1) or Rictor (mTORC2), that mTORC1 and mTORC2 promote NK cell maturation in a cooperative and non-redundant manner, mainly by controlling the expression of Tbx21 and Eomes. Intriguingly, mTORC1 and mTORC2 regulate cytolytic function in an opposing way, exhibiting promoting and inhibitory effects on the anti-tumor ability and metabolism, respectively. mTORC1 sustains mTORC2 activity by maintaining CD122-mediated IL-15 signaling, whereas mTORC2 represses mTORC1-modulated NK cell effector functions by restraining STAT5-mediated SLC7A5 expression. These positive and negative crosstalks between mTORC1 and mTORC2 signaling thus variegate the magnitudes and kinetics of NK cell activation, and help define a paradigm for the modulation of NK maturation and effector functions.

Downregulation of ATG5-dependent macroautophagy by chaperone-mediated autophagy promotes breast cancer cell metastasis.

Recent data have shown that the expression of lysosome-associated membrane protein type 2 A (LAMP2A), the key protein in the chaperone-mediated autophagy (CMA) pathway, is elevated in breast tumor tissues. However, the exact effects and mechanisms of CMA during breast cancer metastasis remain largely unknown. In this study, we found that the LAMP2A protein level was significantly elevated in human breast cancer tissues, particularly in metastatic carcinoma. The increased LAMP2A level was also positively correlated with the histologic grade of ductal breast cancer. High LAMP2A levels also predicted shorter overall survival of breast cancer patients. Downregulation of CMA activity by LAMP2A knockdown significantly inhibited the growth and metastasis of both MDA-MB-231 and MDA-MB-468 breast cancer cells in vivo and in vitro, while upregulation of CMA activity by LAMP2A overexpression had the opposite effect. Mechanistically, we found that elevated CMA activity mediated increased growth and metastasis of human breast cancer cells by downregulating the activity of autophagy-related gene 5 (ATG5)-dependent macroautophagy. Collectively, these results indicate that the anti-macroautophagic property is a key feature of CMA-mediated tumorigenesis and metastasis and may, in some contexts, serve as an attractive target for breast cancer therapies.

Sustained elevation of NF-κB activity sensitizes offspring of maternal inflammation to hypertension via impairing PGC-1α recovery.

Growing evidence has demonstrated that maternal detrimental factors, including inflammation, contribute to the development of hypertension in the offspring. The current study found that offspring subjected to prenatal exposure of inflammation by lipopolysaccharide (LPS) challenge during the second semester showed significantly increased systolic blood pressure. In addition, these offspring also displayed augmented vascular damage and reactive oxygen species (ROS) levels in thoracic aortas when challenged with deoxycorticosterone acetate and high-salt diet (DOCA-salt). Interestingly, the antioxidant N-acetyl-L-cysteine markedly reversed these changes. Mechanistically, prenatal LPS exposure led to pre-existing elevated peroxisome proliferators-activated receptor-γ co-activator (PGC)-1α, a critical master of ROS metabolism, which up-regulated the ROS defense capacity and maintained the balance of ROS generation and elimination under resting state. However, continued elevation of NF-κB activity significantly suppressed the rapid recovery of PGC-1α expression response to DOCA-salt challenge in offspring that underwent prenatal inflammatory stimulation. This was further confirmed by using a NF-κB inhibitor (N-p-Tosyl-L-phenylalanine chloromethyl ketone) that restored PGC-1α recovery and prevented blood pressure elevation induced by DOCA-salt. Our results suggest that maternal inflammation programmed proneness to NF-κB over-activation which impaired PGC-1α-mediated anti-oxidant capacity resulting in the increased sensitivity of offspring to hypertensive damage.

Maternal inflammation activated ROS-p38 MAPK predisposes offspring to heart damages caused by isoproterenol via augmenting ROS generation.

Maternal inflammation contributes to the increased incidence of adult cardiovascular disease. The current study investigated the susceptibility of cardiac damage responding to isoproterenol (ISO) in adult offspring that underwent maternal inflammation (modeled by pregnant Sprague-Dawley rats with lipopolysaccharides (LPS) challenge). We found that 2 weeks of ISO treatment in adult offspring of LPS-treated mothers led to augmented heart damage, characterized by left-ventricular systolic dysfunction, cardiac hypertrophy and myocardial fibrosis. Mechanistically, prenatal exposure to LPS led to up-regulated expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, antioxidant enzymes, and p38 MAPK activity in left ventricular of adult offspring at resting state. ISO treatment exaggerated ROS generation, p38 MAPK activation but down-regulated reactive oxygen species (ROS) elimination capacity in the left ventricular of offspring from LPS-treated mothers, while antioxidant N-acetyl-L-cysteine (NAC) reversed these changes together with improved cardiac functions. The p38 inhibitor SB202190 alleviated the heart damage only via inhibiting the expression of NADPH oxidases. Collectively, our data demonstrated that prenatal inflammation programs pre-existed ROS activation in the heart tissue, which switches on the early process of oxidative damages on heart rapidly through a ROS-p38 MAPK-NADPH oxidase-ROS positive feedback loop in response to a myocardial hypertrophic challenge in adulthood.