Splenic Natural Killer Cell Research Articles

Ephrin-B2-expressing natural killer cells induce angiogenesis

BackgroundTherapeutic angiogenesis aims to induce new blood vessel growth in ischemic tissues; however, previous clinical trials have had limited success. Studies of uterine angiogenesis revealed a specialized subset of natural killer (NK) cells, called uterine NK (uNK) cells, which have unique proangiogenic abilities. MethodsWe show that uNK cells in mice express ephrin-B2, a regulator of angiogenesis, to induce tubule formation in an ex vivo coculture tubule formation assay. We next induced the expression of ephrin-B2 by splenic NK (sNK) cells harvested from male mice. ResultsWe showed that induced NK (iNK) cells can also instruct endothelial cells to form tubules using ephrin-B2. ConclusionsWe concluded that Ephrin-B2 is a marker of proangiogenic uNK cells and that a proangiogenic phenotype characterized by ephrin-B2 can be induced in sNK cells to induce therapeutic angiogenesis.

Immunomodulatory and antitumor activities of the exopolysaccharide produced by potential probiotic Lactobacillus plantarum YW11 in a HT-29 tumor-burdened nude mouse model

In this study, the exopolysaccharides (EPSs)-producing L. plantarum YW11 was initially evaluated for its tolerance to simulated artificial gastric juice and bile salts, and cell surface hydrophobicity. Subsequent study using a HT-29 tumor-burdened nude mouse model showed that the EPS had potent immunomodulatory and antitumor activities. Administration of the EPS had no influence on body weight of the mice, and it could significantly prevent HT-29 tumor cells induced acute liver and kidney damages. The purified EPS at a dose of 5 mg/mL was able to enhance effectively the killing activities of splenic natural killer cells and cytotoxic T lymphocyte, and to stimulate the secretion of serum cytokines IL-2 and TNF-α. As evidenced by a cell culture assay, the EPS exhibited obvious inhibition on growth of colon cancer HT-29 cells treated with 600 μg/mL of the EPS for 72 h with an inhibition rate of 58.02%. Administration of the EPS obviously inhibited growth of HT-29 xenograft tumor to an extent near that of the positive control (5-Fu, 25 mg/kg bw). Therefore, the results of this study indicated the important roles of the EPS in the potential probiotic functions of the strain.

Tcf1 Sustains the Expression of Multiple Regulators in Promoting Early Natural Killer Cell Development.

T cell factor 1 (Tcf1) is known as a critical mediator for natural killer (NK) cell development and terminal maturation. However, its essential targets and precise mechanisms involved in early NK progenitors (NKP) are not well clarified. To investigate the role of Tcf1 in NK cells at distinct developmental phases, we employed three kinds of genetic mouse models, namely, Tcf7 fl/fl Vav Cre/+, Tcf7 fl/fl CD122 Cre/+ and Tcf7 fl/fl Ncr1 Cre/+ mice, respectively. Similar to Tcf1 germline knockout mice, we found notably diminished cell number and defective development in BM NK cells from all strains. In contrast, Tcf7 fl/fl Ncr1 Cre/+ mice exhibited modest defects in splenic NK cells compared with those in the other two strains. By analyzing the published ATAC-seq and ChIP-seq data, we found that Tcf1 directly targeted 110 NK cell-related genes which displayed differential accessibility in the absence of Tcf1. Along with this clue, we further confirmed that a series of essential regulators were expressed aberrantly in distinct BM NK subsets with conditional ablating Tcf1 at NKP stage. Eomes, Ets1, Gata3, Ikzf1, Ikzf2, Nfil3, Runx3, Sh2d1a, Slamf6, Tbx21, Tox, and Zeb2 were downregulated, whereas Spi1 and Gzmb were upregulated in distinct NK subsets due to Tcf1 deficiency. The dysregulation of these genes jointly caused severe defects in NK cells lacking Tcf1. Thus, our study identified essential targets of Tcf1 in NK cells, providing new insights into Tcf1-dependent regulatory programs in step-wise governing NK cell development.

Reevaluation of NOD/SCID Mice as NK Cell-Deficient Models.

Objective Natural killer (NK) cell-deficient mice are useful models in biomedical research. NOD/SCID mice have been used as a model of this type in research. However, the actual status of NK cells in NOD/SCID mice and CB17/SCID mice in comparison with that in BALB/c mice has not been sufficiently evaluated. Methods Splenocytes from naïve or poly(I:C)-treated mice were isolated for phenotyping and analysis of cytotoxicity-related molecules and inhibitory receptors; for cytotoxicity assay, purified NK cells were also used. Results The proportion of splenic NK cells did not differ significantly between NOD/SCID and CB17/SCID mice. The perforin levels in NK cells were similar between the poly(I:C)-treated CB17/SCID and NOD/SCID mice, while the granzyme B and NKG2A/C/E levels in NK cells from NOD/SCID mice were significantly lower than those from CB17/SCID mice. Moreover, the NKG2D and Ly49A levels in NK cells from NOD/SCID mice were higher than those from CB17/SCID. The splenocytes from CB17/SCID mice showed higher cytotoxicity than those from NOD/SCID mice, while the cytotoxicity of purified NK cells basically did not differ between the two strains. After in vitro stimulation with cytokines, the splenocytes from CB17/SCID mice showed higher IFN-γ production than those from NOD/SCID mice; however, NK cells did not. Conclusion There was no significant difference in the proportion of splenic NK cells between CB17/SCID and NOD/SCID mice, and the function of NK cells was only partially compromised in NOD/SCID mice. Caution should be taken when considering the use of NOD/SCID mice as an NK-deficient model.

Lactobacillus rhamnosus and Bifidobacterium longum alleviate colitis and cognitive impairment in mice by regulating IFN-γ to IL-10 and TNF-α to IL-10 expression ratios

Gut lactobacilli and bifidobacteria on the immune homeostasis. Therefore, to understand the mechanism in vivo, we selected human fecal Lactobacillus rhamnosus NK210 and Bifidobacterium longum NK219, which strongly suppressed the IFN-γ to IL-10 expression (IIE) ratio in lipopolysaccharide-stimulated macrophages. Thereafter, we examined their effects on the endotoxin, antibiotics, or antitumor drug-stimulated immune imbalance in mice. Intraperitoneal injection of lipopolysaccharide and oral gavage of ampicillin increased IFN-γ and TNF-α expression in the spleen, colon, and hippocampus, while IL-10 expression decreased. However, intraperitoneal injection of cyclophosphamide suppressed IFN-γ, TNF-α, and IL-10 expression. LPS exposure induced splenic natural killer cell cytotoxicity against YAC-1 cells (sNK-C) and peritoneal macrophage phagocytosis against Candida albicans (pMA-P) activities, while cyclophosphamide and ampicillin treatments suppressed sNK-C and pMA-P activities. However, LPS, ampicillin, cyclophosphamide all increased IIE and TNF-α to IL-10 expression (TIE) ratios. Oral administration of NK210 and/or NK219 significantly reduced LPS-induced sNK-C, pMA-P, and IFN-γ expression, while cyclophosphamide- or ampicillin-suppressed sNK-C and pMA-P activities, cyclophosphamide-suppressed IFN-γ, TNF-α, and IL-10 expression, and ampicillin-suppressed IL-10 expression increased. Nevertheless, they suppressed LPS-, ampicillin-, or cyclophosphamide-induced IIE and TIE ratios, cognitive impairment, and gut dysbiosis. In particular, NK219, but not NK210, increased the IIE expression ratio in vitro and in vivo, and enhanced sNK-C and pMA-P activities in normal control mice, while cognitive function and gut microbiota composition were not significantly affected. These findings suggest that NK210, Lactobacillus sp, and NK219, Bifidobacterium additively or synergistically alleviate gut dysbiosis, inflammation, and cognitive impairment with immune imbalance by controlling IIE and TIE ratios.

A detailed analysis of innate and adaptive immune responsiveness upon infection with Salmonella enterica serotype Enteritidis in young broiler chickens

Salmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate and adaptive immune cells as well as antibody titers were assessed. SE was observed in the ileum and spleen of SE-infected chickens at 7 days post-infection (dpi). At 1 dpi numbers of intraepithelial cytotoxic CD8+ T cells were significantly increased alongside numerically increased intraepithelial IL-2Rα+ and 20E5+ natural killer (NK) cells at 1 and 3 dpi. At both time points, activation of intraepithelial and splenic NK cells was significantly enhanced. At 7 dpi in the spleen, presence of macrophages and expression of activation markers on dendritic cells were significantly increased. At 21 dpi, SE-induced proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in sera of all SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells and we hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance to SE in young broiler chickens.

Radium-223 dichloride causes transient changes in natural killer cell population and cytotoxic function

Rationale Natural killer (NK) cells play an important role in both the innate and adaptive arms of the immune system. While previous studies have demonstrated the effects of ionizing radiation on cytotoxic function of NK cells, little is known about how a chronic exposure to high LET alpha particles emitted by radionuclides will affect both NK population size and function. This study was conducted to determine the effects of 223RaCl2 on splenic NK cell population size and function in Swiss Webster mice. Methods Swiss Webster mice were administered intravenously with 0, 50, or 600 kBq/kg 223RaCl2. Spleens were harvested at 5, 12, and 19 days post-administration. The numbers of splenocytes per spleen were enumerated and flow cytometry was used to assess changes in the distribution of splenocyte subpopulations of B, CD4 and CD8 T lymphocytes, and NK cells. NK functional activity was quantified using YAC-1 target cells and the 51Cr-release assay. Results The total number of splenocytes was unaffected by 223RaCl2. However, significant changes in the distribution of splenocyte subpopulations were observed for NK cells and CD8 T lymphocytes. NK functional activity was enhanced substantially relative to controls at 12 days post-administration, but decreased markedly by day 19. Conclusion NK functional activity is both diminished and enhanced by 223RaCl2 depending on both administered activity and time post-administration. These results suggest that there may be an optimum window of time to combine the 223RaCl2-induced antitumor NK cell response with other cancer therapies that elicit immune activation.

MiR-1224 contributes to ischemic stroke-mediated natural killer cell dysfunction by targeting Sp1 signaling

BackgroundBrain ischemia compromises natural killer (NK) cell-mediated immune defenses by acting on neurogenic and intracellular pathways. Less is known about the posttranscriptional mechanisms that regulate NK cell activation and cytotoxicity after ischemic stroke.MethodsUsing a NanoString nCounter® miRNA array panel, we explored the microRNA (miRNA) profile of splenic NK cells in mice subjected to middle cerebral artery occlusion. Differential gene expression and function/pathway analysis were applied to investigate the main functions of predicted miRNA target genes. miR-1224 inhibitor/mimics transfection and passive transfer of NK cells were performed to confirm the impact of miR-1224 in NK cells after brain ischemia.ResultsWe observed striking dysregulation of several miRNAs in response to ischemia. Among those miRNAs, miR-1224 markedly increased 3 days after ischemic stroke. Transfection of miR-1224 mimics into NK cells resulted in suppression of NK cell activity, while an miR-1224 inhibitor enhanced NK cell activity and cytotoxicity, especially in the periphery. Passive transfer of NK cells treated with an miR-1224 inhibitor prevented the accumulation of a bacterial burden in the lungs after ischemic stroke, suggesting an enhanced immune defense of NK cells. The transcription factor Sp1, which controls cytokine/chemokine release by NK cells at the transcriptional level, is a predicted target of miR-1224. The inhibitory effect of miR-1224 on NK cell activity was blocked in Sp1 knockout mice.ConclusionsThese findings indicate that miR-1224 may serve as a negative regulator of NK cell activation in an Sp1-dependent manner; this mechanism may be a novel target to prevent poststroke infection specifically in the periphery and preserve immune defense in the brain.

Synovial fibroblasts regulate the cytotoxicity and osteoclastogenic activity of synovial natural killer cells through the RANKL‐RANK axis in osteoarthritis

AbstractChronic inflammation plays an important role in the progression of osteoarthritis (OA). Cumulative evidence suggests that natural killer (NK) cells potentially participate in OA inflammation. To investigate the regulation of NK cell reactivity in OA synovium, we characterized the phenotypic and functional properties of infiltrating NK cells in the synovial tissue in a rat collagenase‐induced OA model. Our data indicated that in comparison with splenic NK cells, OA synovial NK cells were poorly responsive to the stimulation of IL‐12 plus IL‐15 and IL‐18, as evidenced by lower production of IFN‐γ, perforin and granzyme B. Consistently, synovial NK cells showed lower cytotoxicity to target cells than splenic NK cells. Further investigations revealed that synovial NK cells highly expressed receptor activator of nuclear factor‐κB (RANK), while OA synovial fibroblasts (SFs) upregulated the expression of RANK ligand (RANKL). Co‐culture of OA SFs with RANK‐expressing NK cells inhibited cytokine‐induced production of above cytotoxic mediators in NK cells and subsequently impaired NK cell cytotoxicity. Gene silencing of RANKL expression in SFs partially recovered the cytotoxicity of NK cells after co‐culture. Furthermore, through the RANKL‐RANK axis, OA SFs induced RANKL expression in NK cells and subsequently promoted the osteoclastogenic activity of NK cells in vitro. Taken together, our study showed that OA SF inhibited the cytotoxicity while augmenting the osteoclastogenic activity of NK cells through the RANKL‐RANK axis. This study reveals a novel mechanism by which SFs modulate the reactivity of synovial NK cells in OA.

Pathogenic ILC1 and NKp46+ ILC3 Are Defined by Core and Tissue-Specific Transcriptional Signatures in Autoimmune Neuroinflammation

Abstract During multiple sclerosis, myelin-specific CD4+T cells invade the central nervous system (CNS) where they initiate a chronic inflammatory disease, leading to demyelination and neurodegeneration. Therefore, it is crucial to identify immunological factors allowing disease-inducing T cells to enter the CNS. Our previous study revealed a novel function of T-bet-expressing NKp46+ innate lymphocytes (ILC) as CNS gatekeepers, which control functional stability and access of autoreactive Th17 cells to the CNS. However, the identity of NKp46+ ILC subset(s) that regulate neuroinflammation and their mechanism of action are still poorly characterized. T-bet-expressing NKp46+ ILC encompass three subsets – conventional natural killer (NK) cells, ILC1 and NKp46+ ILC3. Here, we demonstrate that the pathogenic role of T-bet-expressing NKp46+ ILC maps to ILC1 and NKp46+ ILC3 but not NK cells. Single cell RNA-Seq analysis revealed that meninges-infiltrating NK cells exhibit undifferentiated phenotype devoid of expression of effector molecules and reminiscent of blood NK cells, explaining the functional redundancy of NK cells in the disease process. By contrast, NKp46+ ILC1/3, which directly interact with myelin-specific CD4+ T cells, were defined by the core set of 13 genes, which was not shared by splenic or meningeal NK cells. A subset of the ILC-specific genes was highly expressed by meningeal, but not splenic, ILCs, suggesting their importance in disease pathology. Thus, our transcriptional analysis identified modules of gene expression that coordinate functional polarization of ILCs in meningeal microenvironment and provides a blueprint for the discovery of ILC-specific targeted therapy for the treatment of CNS autoimmune disorder.

Protective effect of myricetin, apigenin, and hesperidin pretreatments on cyclophosphamide-induced immunosuppression

Aim: Major side effects of cyclophosphamide administration are immunosuppression and myelosuppression. The immunomodulatory effects of plant bioactive compounds on chemotherapy drug-induced immunosuppression may have significant effects in cancer treatment. For this reason, we investigated the immunomodulatory effect of myricetin, apigenin, and hesperidin in cyclophosphamide-induced immunosuppression in rats. Methods: In our study, a total of 64 rats were used, and divided into eight equal groups. These groups were: control, cyclophosphamide, cyclophosphamide + myricetin (100 mg/kg), cyclophosphamide + myricetin (200 mg/kg), cyclophosphamide + apigenin (100 mg/kg), cyclophosphamide + apigenin (200 mg/kg), cyclophosphamide + hesperidin (100 mg/kg), and cyclophosphamide + hesperidin (200 mg/kg). Myricetin, apigenin, and hesperidin pretreatments were performed for 14 d, while cyclophosphamide application (200 mg/kg) was performed only on the 4th day of the study. Levels of humoral antibody production, quantitative hemolysis, macrophage phagocytosis, splenic lymphocyte proliferation, and natural killer cell cytotoxicity were determined. In addition, we measured pro-inflammatory cytokines, and followed lipid peroxidation and antioxidant markers and examined the histology of bone marrow, liver and spleen in all groups. Results: During cyclophosphamide treatment, all three phytochemicals increased the levels of humoral antibody production, quantitative hemolysis, macrophage phagocytosis, splenic lymphocyte proliferation, antioxidant markers, and natural killer cell cytotoxicity. Moreover, the agents decreased the levels of pro-inflammatory cytokines and mediators, reduced lipid peroxidation markers, and reduced tissue damage in liver, spleen, and bone marrow. Conclusion: Our study demonstrated that myricetin, apigenin, and hesperidin can reduce the immunosuppressive effect of cyclophosphamide by enhancing both innate and adaptive immune responses, and these compounds may be useful immunomodulatory agents during cancer chemotherapy.

Dual-Functional PLGA Nanoparticles Co-Loaded with Indocyanine Green and Resiquimod for Prostate Cancer Treatment.

PurposeWith the advance of screening techniques, there is a growing number of low-risk or intermediate-risk prostate cancer (PCa) cases, remaining a serious threat to men’s health. To obtain better efficacy, a growing interest has been attracted to develop such emerging treatments as immunotherapy and focal therapy. However, few studies offer guidance on whether and how to combine these modalities against PCa. This study was designed to develop dual-functional nanoparticles (NPs) which combined photothermal therapy (PTT) with immunotherapy and determine the anti-tumor efficacy for PCa treatment.MethodsBy a double emulsion technique, the drug nanocarrier, poly(lactic-co-glycolic acid) or PLGA, was applied for co-loading of a fluorescent dye, indocyanine green (ICG) and a toll-like receptor 7/8 (TLR7/8) agonist resiquimod (R848) to synthesize PLGA-ICG-R848 NPs. Next, we determined their characteristic features and evaluated whether they inhibited the cell viability in multiple PCa cell lines. After treatment with PLGA-ICG-R848, the maturation markers of bone marrow-derived dendritic cells (BMDCs) were detected by flow cytometry. By establishing a subcutaneous xenograft model of mouse PCa, we explored both the anti-tumor effect and immune response following the NPs-based laser ablation.ResultsWith a mean diameter of 157.7 nm, PLGA-ICG-R848 exhibited no cytotoxic effect in PCa cells, but they significantly decreased RM9 cell viability to (3.9±1.0)% after laser irradiation. Moreover, PLGA-ICG-R848 promoted BMDCs maturation with the significantly elevated proportions of CD11c+CD86+ and CD11c+CD80+ cells. Following PLGA-ICG-R848-based laser ablation in vivo, the decreased bioluminescent signals indicated a significant inhibition of PCa growth, while the ratio of splenic natural killer (NK) cells in PLGA-ICG-R848 was (3.96±1.88)% compared with (0.99±0.10)% in PBS group, revealing the enhanced immune response against PCa.ConclusionThe dual-functional PLGA-ICG-R848 NPs under laser irradiation exhibit the anti-tumor efficacy for PCa treatment by combining PTT with immunotherapy.

Effect of proximal and distal acupoint catgut-embedding on uterus prostaglandin, serum IL-2 and splenic NK cell activity in primary dysmenorrhea rats

To observe the effect of proximal and distal acupoint catgut-embedding on the contents of prostaglandin E2 (PGE2) and prostaglandin F2α(PGF2α) in the uterus, serum Interleukin-2 (IL-2) contents and splenic natural killer (NK) cell activity in primary dysmenorrhea (PD) rats，so as to explore the different effects of proximal and distal acupoint catgut-embedding on PD rats. Female Wistar rats were randomly divided into control, model, proximal catgut-embedding and distal catgut-embedding groups, with 8 rats in each group. The PD model was established by subcutaneous injection of estradiol ben-zoate (0.5 mg/rat on the first day and 10th day, and 0.2 mg/rat from the 2nd to the 9th day) and intraperitoneal injection of oxytocin (2 U/rat, on the 11th day). Catgut-embedding was applied at bilateral "Guanyuan"(CV4) and "Ciliao"(BL32) in the proximal catgut-embedding group, and bilateral "Sanyinjiao" (SP6) and "Diji"(SP8) in the distal catgut-embedding group. After the treatment, the body writhing times in 30 min and uterine mass were recorded, PGE2 and PGF2αin uterus and IL-2 in serum were assayed by using ELISA, the activity of NK cell in the spleen was detected using MTT colorimetry. Following modeling, the body writhing times of the model group was increased than that of the control group (P<0.01); After interventions, the body writhing times were decreased in the proximal and distal catgut-embedding groups than those of the model group (P<0.01). Compared with the control group, the uterine mass and uterus PGF2α content were increased (P<0.01), while uterus PGE2, serum IL-2 and splenic NK cell activity were decreased (P<0.01) in the model group. After interventions, the uterine mass and uterus PGF2α were down-regulated (P<0.01, P<0.05), while the contents of uterus PGE2, serum IL-2 and splenic NK cell activity up-regulated (P<0.01) in the proximal and distal catgut-embedding groups in contrast to the model group. The content of uterus PGF2α was down-regulated (P<0.05) and splenic NK cell activity was up-regulated (P<0.01) in the proximal catgut-embedding group than those of the distal catgut-embedding group. Both proximal and distal acupoint catgut-embeding can increase PGE2 and decrease PGF2α in the uterus, increase the level of serum IL-2 and the activity of NK cells in the spleen in PD model rats, thereby achieving analgesic effect. The effect of proximal catgut-embedding is better than that of distal catgut-embedding.

Dysregulation of murine immune functions on inhalational exposure to ammonia, dimethyl disulfide, 3-methylindole, or propionic acid.

Animal husbandry workers are exposed to various malodorous compounds in the workplace. Although these compounds cause severe nuisance, no systemic investigation of their effects on the immune system has been conducted. To address this issue, we evaluated the effects of inhalational exposure to ammonia, dimethyl disulfide, 3-methylindole (3-MI), and propionic acid (PA), representing four major groups of malodorous compounds, on humoral and cellular immunity in mice. Mice were exposed to the substances (low dose: 10 µL and high dose: 200 µL) for 10 min/day for 4 weeks in a modified standard mouse cage. Neutrophil% and splenic cytotoxic T cell% were significantly lower in the high-dose ammonia group than in the vehicle control. Exposure to ammonia and 3-MI increased immature thymic T lymphocyte% relative to control and concomitantly decreased both mature helper and cytotoxic T-cell populations in the thymus. In the ammonia exposure group, levels of serum immunoglobulin E and immunoglobulin A were elevated, and the IgG2a:IgG1 ratio in the serum was reduced in a dose-dependent manner. Splenic natural killer cell activity was significantly less in the PA exposure group than in the control. Overall, our findings suggest that inhalational exposure to these malodorous substances disturbs immune homeostasis in vivo.

Immunomodulation by systemic administration of human-induced pluripotent stem cell-derived mesenchymal stromal cells to enhance the therapeutic efficacy of cell-based therapy for treatment of myocardial infarction.

Rationale: Poor survival and engraftment are major hurdles of stem cell therapy in the treatment of myocardial infarction (MI). We sought to determine whether pre-transplantation systemic intravenous administration of human induced pluripotent stem cell (hiPSC)-derived mesenchymal stromal cells (hiPSC-MSCs) could improve the survival of hiPSC-MSCs or hiPSC-derived cardiomyocytes (hiPSC-CMs) following direct intramyocardial transplantation in a mouse model of MI.Methods: Mice were randomized to undergo intravenous administration of saline or 5×105 hiPSC-MSCs one week prior to MI, induced by ligation of the left anterior descending coronary artery. Mice were further assigned to undergo direct intramyocardial transplantation of hiPSC-MSCs (1×106) or hiPSC-CMs (1×106) 10 minutes following MI. Echocardiographic and invasive hemodynamic assessment were performed to determine cardiac function. In-vivo fluorescent imaging analysis, immunofluorescence staining and polymerase chain reaction were performed to detect cell engraftment. Flow cytometry of splenic regulatory T cells (Tregs) and natural killer (NK) cells was performed to assess the immunomodulatory effects.Results: Pre-transplantation systemic administration of hiPSC-MSCs increased systemic Tregs activation, decreased the number of splenic NK cells and inflammation, and enhanced survival of transplanted hiPSC-MSCs and hiPSC-CMs. These improvements were associated with increased neovascularization and decreased myocardial inflammation and apoptosis at the peri-infract zone with consequent improved left ventricular function four weeks later. Co-culture of splenic CD4 cells with hiPSC-MSCs also modulated their cytokine expression profile with a decreased level of interferon-γ, tumor necrosis factor-α, and interleukin (IL)-17A, but not IL-2, IL-6 and IL-10.Conclusion: Pre-transplantation systemic intravenous administration of hiPSC-MSCs induced immunomodulation and facilitated the survival of intramyocardially transplanted cells to improve cardiac function in MI.

Immunotoxicity studies of sulfolane following developmental exposure in Hsd:Sprague Dawley SD rats and adult exposure in B6C3F1/N mice

Sulfolane is a solvent used in the petrochemical industry and a groundwater contaminant in areas near refineries. The current studies were conducted to assess the impact of oral exposure to sulfolane on the immune system using two models: (1) a perinatal drinking water exposure to 0, 30, 100, 300, or 1000 mg/L from gestation day (GD) 6 until ∼13 weeks-of-age in Harlan Sprague Dawley rats; and, (2) a 90-day gavage exposure of adult female B6C3F1/N mice to 0, 1, 10, 30, 100, or 300 mg/kg/day. Immune parameters evaluated included measurement of antibody production against sheep red blood cells (SRBC) and keyhole limpet hemocyanin (KLH), ex vivo measurements of natural killer (NK) cell activity, cytotoxic T-cell (CTL) activity, and T-cell proliferation, as well as measures of splenic immune cell populations, hematological parameters, and histopathology of immune tissues. A decrease in ex vivo NK cell activity was observed in cells from female – but not male – F1 rats following developmental exposure. In adult female mice, splenic NK cell number was lower than the vehicle controls at doses ≥ 100 mg/kg; however, ex vivo NK cell activity was not affected by sulfolane treatment. In female mice, a decrease in the number of large unstained cells at doses ≥ 30 mg/kg was observed. In F1 rats, effects on white blood cells (WBC) were limited to a decreasing trend in leukocytes in females; no effects were observed in males. Under the conditions of this study, a no-observed-effect level (NOEL) of 3 mg/kg/day was identified based on reduced NK cell activity in female F1 rats. Overall, these findings suggest that oral exposure to sulfolane in rodents had minimal effects on the immune system.

Polysaccharide from Codium fragile Induces Anti-Cancer Immunity by Activating Natural Killer Cells.

Natural polysaccharides exhibit beneficial immune modulatory effects, including immune stimulatory and anti-cancer activities. In this study, we examined the effect of Codium fragile polysaccharide (CFP) on natural killer (NK) cell activation, and its effect on tumor-bearing mice. Intravenous CFP treatment of C57BL/6 mice resulted in the upregulation of CD69, which is a marker associated with NK cell activation. In addition, intracellular levels of interferon (IFN)-γ and the cytotoxic mediators perforin and granzyme B were markedly increased in response to the CFP treatment of splenic NK cells. IFN-γ production by NK cells was directly induced by CFP, whereas the upregulation of CD69 and cytotoxic mediators required IL-12. Finally, intraperitoneal treatment with CFP prevented CT-26 (murine carcinoma) tumor cell infiltration in the lungs, without significantly reducing the body weight. In addition, treatment with CFP prevented B16 melanoma cell infiltration in the lung of C57BL/6 mice. Moreover, the anti-tumor effect was diminished by the depletion of NK cells. Therefore, these data suggest that CFP may be used as an NK cell stimulator to produce a phenomenon that contributes to anti-cancer immunity.

Exogenous interleukin-33 promotes hepatocellular carcinoma growth by remodelling the tumour microenvironment

BackgroundInterleukin-33 (IL-33) is an effective inducer of pro-inflammatory cytokines regulating innate and adaptive immunity. Inflammation could be a double-edged sword, promoting or inhibiting tumour growth. To date, the roles and mechanisms of IL-33 in tumours remain controversial. Here, we examined the effect of exogenous IL-33 on the biological characteristics of hepatocellular carcinoma (HCC) and the possible mechanism of action.MethodsIn this study, IL-33 expression in the tissues of 69 HCC patients was detected and its relationship with prognosis was evaluated. After establishing a mouse HCC model and IL-33 treatment operation, the infiltration of splenic myeloid-derived suppressor (MDSCs), dendritic (DCs), regulatory T, and natural killer (NK) cells was detected by flow cytometry analysis, and the vascular density of the tumour tissues was detected by immunohistochemistry to reveal the mechanism of IL-33 in HCC proliferation. Finally, the Cancer Genome Atlas database was used to analyse Gene Ontology terms the and Kyoto Encyclopaedia of Genes and Genomes pathway. Moreover, the chi-square test, two-tailed unpaired Student’s t-test, and multiple t-tests were performed using SPSS version 23.0 and GraphPad Prism 8.0 software.ResultsThe IL-33 expression level was negatively correlated with the overall survival of HCC patients, suggesting its potential clinical significance in the prognosis of HCC. We found that systemic IL-33 administration significantly promoted the tumour size in vivo. Furthermore, the IL-33-treated mice presented decreased frequencies of tumouricidal NK and CD69+ CD8+ T cells. After IL-33 treatment, the incidence of monocytic MDSCs and conventional DCs increased, while that of granulocytic MDSCs decreased. Moreover, IL-33 promoted the formation of intracellular neovascularization. Therefore, IL-33 accelerated HCC progression by increasing the accumulation of immunosuppressive cells and neovascularization formation. Finally, we found that the transcription of IL-33 was closely related to the PI3K-Akt and MAPK pathways in Gene Set Enrichment Analysis plots, which were involved in the tumourigenesis and pathogenesis of HCC.ConclusionsTaken together, IL-33 may be a key tumour promoter of HCC proliferation and tumourigenicity, an important mediator, and a potential therapeutic target for regulating HCC progression.

High-Fat Diet and Feeding Regime Impairs Number, Phenotype, and Cytotoxicity of Natural Killer Cells in C57BL/6 Mice.

Overweight and obesity are major public health challenges worldwide. Obesity is associated with a higher risk for the development of several cancer types, but specific mechanisms underlying the link of obesity and cancer are still unclear. Natural killer (NK) cells are circulating lymphoid cells promoting the elimination of virus-infected and tumor cells. Previous investigations demonstrated conflicting results concerning the influence of obesity on functional NK cell parameters in small animal models. The aim of the present study was to clarify potential obesity-associated alterations of murine NK cells in vivo, implementing different feeding regimes. Therefore, C57BL/6 mice were fed a normal-fat diet (NFD) or high-fat diet (HFD) under restrictive and ad libitum feeding regimes. Results showed diet and feeding-regime dependent differences in body weight, visceral fat mass and plasma cytokine concentrations. Flow cytometry analyses demonstrated significant changes in total cell counts as well as frequencies of immune cell populations in peripheral blood comparing mice fed NFD or HFD in an ad libitum or restrictive manner. Mice fed the HFD showed significantly decreased frequencies of total NK cells and the mature CD11b+CD27+ NK cell subset compared to mice fed the NFD. Feeding HFD resulted in significant changes in the expression of the maturation markers KLRG1 and CD127 in NK cells. Furthermore, real-time PCR analyses of NK-cell related functional parameters in adipose tissue revealed significant diet and feeding-regime dependent differences. Most notable, real-time cytotoxicity assays demonstrated an impaired cytolytic activity of splenic NK cells toward murine colon cancer cells in HFD-fed mice compared to NFD-fed mice. In conclusion, our data demonstrate that feeding a high-fat diet influences the frequency, phenotype and function of NK cells in C57BL/6 mice. Interestingly, restricted feeding of HFD compared to ad libitum feeding resulted in a partial prevention of the obesity-associated alterations on immune cells and especially on NK cells, nicely fitting with the current concept of an advantage for interval fasting for improved health.

Antigens of Mycobacterium tuberculosis Stimulate CXCR6+ Natural Killer Cells.

Natural killer (NK) cells participate in immunity against several pathogens by exerting cytotoxic and cytokine-production activities. Some NK cell subsets also mediate recall responses that resemble memory of adaptive lymphocytes against antigenic and non-antigenic stimuli. The C-X-C motif chemokine receptor 6 (CXCR6) is crucial for the development and maintenance of memory-like responses in murine NK cells. In humans, several subsets of tissue-resident and circulating NK cells with different functional properties express CXCR6. However, the role of CXCR6+ NK cells in immunity against relevant human pathogens is unknown. Here, we addressed whether murine and human CXCR6+ NK cells respond to antigens of Mycobacterium tuberculosis (Mtb). For this purpose, we evaluated the immunophenotype of hepatic and splenic CXCR6+ NK cells in mice exposed to a cell-wall (CW) extract of Mtb strain H37Rv. Also, we characterized the expression of CXCR6 in peripheral NK cells from active pulmonary tuberculosis (ATB) patients, individuals with latent TB infection (LTBI), and healthy volunteer donors (HD). Furthermore, we evaluated the responses of CXCR6+ NK cells from HD, LTBI, and ATB subjects to the in vitro exposure to CW preparations of Mtb H37Rv and Mtb HN878. Our results showed that murine hepatic CXCR6+ NK cells expand in vivo after consecutive administrations of Mtb H37Rv CW to mice. Remarkably, pooled hepatic and splenic, but not isolated splenic NK cells from treated mice, enhance their cytokine production capacity after an in vitro re-challenge with H37Rv CW. In humans, CXCR6+ NK cells were barely detected in the peripheral blood, although slightly significative increments in the percentage of CXCR6+, CXCR6+CD49a−, CXCR6+CD49a+, and CXCR6+CD69+ NK cells were observed in ATB patients as compared to HD and LTBI individuals. In contrast, the expansion of CXCR6+CD49a− and CXCR6+CD69+ NK cells in response to the in vitro stimulation with Mtb H37Rv was higher in LTBI individuals than in ATB patients. Finally, we found that Mtb HN878 CW generates IFN-γ-producing CXCR6+CD49a+ NK cells. Our results demonstrate that antigens of both laboratory-adapted and clinical Mtb strains are stimulating factors for murine and human CXCR6+ NK cells. Future studies evaluating the role of CXCR6+ NK cells during TB are warranted.