Natural Killer Cells In Spleen Research Articles

Siwu decoction mitigates radiation-induced immune senescence by attenuating hematopoietic damage

BackgroundTo investigate the long term effects of ionizing radiation (IR) on hematopoietic stem/progenitor cells (HSPCs), immune tissues and cells, and the effects of Siwu decoction (SWD) on immune senescence mice.MethodsC57BL/6 J mice were exposed to 6.0 Gy 60Co γ irradiation. After 8-weeks of IR, SWD (5, 10, 20 g/kg/d) was administered for 30 days. The changes of HSPCs in bone marrow (BM) and T, B type lymphocyte and natural killer (NK) cells in spleen were detected by flow cytometry. The changes of peripheral blood cells were also examined. Hematoxylin–eosin staining were used to detect the pathological lesions of hippocampus, spleen and thymus tissues. Absolute mouse telomere length quantification qPCR assay kit was used to measure the telomere length of BM cells. The expression of factors associated with inflammation and aging such as p16, β-galactosidase in spleen, thymus and BM was determined.ResultsAdministration of SWD could increase the proportion of LSK (Lin−, Sca-1 + , c-Kit−), multipotent progenitor cells and multipotent progenitor cells and decrease the proportion of common myeloid progenitors and granulocyte–macrophage progenitors in BM. The proportion of B cells and NK cells in spleen and the content of white blood cells, red blood cells, hemoglobin, lymphocytes and eosinophils in peripheral blood were increased, at the same time, the proportion of neutrophils and monocytes was reduced by SWD. The pathological lesions of hippocampus, spleen and thymus were improved. The expression of p16 and β-galactosidase in spleen, thymus and BM was reduced and shortening of the telomere of BM cells was inhibited after administration. In addition, SWD could reduce the content of Janus activated kinase (JAK) 1, JAK2 and signal transducer and activator of transcription 3 (STAT3) in BM and spleen.ConclusionsSWD could slow down IR-induced immune senescence by improving hematopoietic and immunologic injury. SWD might reduce the inflammation level of BM hematopoietic microenvironment by acting on JAK/STAT signaling pathway, while the immune damage of mice was improved by affecting the differentiation of HSPCs. The remission of hematopoietic and immunologic senescence was further demonstrated at the overall level.Graphical

Sepsis induced dysfunction of liver type 1 innate lymphoid cells

BackgroundSepsis is a life-threatening condition triggered by uncontrolled immune responses to infection, leading to widespread inflammation, tissue damage, organ dysfunction, and potentially death. The liver plays a crucial role in the immune response during sepsis, serving as a major site for immune cell activation and cytokine production. Liver type 1 innate lymphoid cells (ILCs) consist of NK cells and ILC1s. They maintain the local immune microenvironment by directly eliminating target cells and secreting cytokines. However, the specific roles and pathological changes of liver-resident NK cells and ILC1s during sepsis remain poorly understood.ResultsThis study aims to investigate the pathological changes of NK cells and ILC1s, which might contribute the dysfunction of liver. Sepsis mouse model was established by cecal ligation and puncture (CLP). Mouse immune cells from liver were isolated, and the surface makers, gene expression profiles, cytokine response and secretion, and mitochondrial function of NK (Natural Killer) cells and ILC1s (Innate Lymphoid Cell 1) were analyzed. A significant decrease in the number of mature NK cells was observed in the liver after CLP. Furthermore, the secretion of interferon-gamma (IFN-γ) was found to be reduced in spleen and liver NK cells when stimulated by IL-18. Mitochondrial activities in both liver NK cells and ILC1 were found to be increased during sepsis, suggesting an enhanced metabolic response in these cells to combat the infection. However, despite this heightened activity, liver NK cells exhibited a decreased level of cytotoxicity, which might impact their ability to target infected cells effectively. RNA sequencing supported and provided the potential mechanisms for the proinflammatory effects and exhaustion like phenotypes of liver NK cells.ConclusionsSepsis induces dysfunction and exhaustion-like phenotypes in liver NK cells and ILC1, which might further impair other immune cells and represent a potential therapeutic target for sepsis.

Androgens Modulate the Immune Profile in a Mouse Model of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is associated with a low-grade inflammation, but it is unknown how hyperandrogenism, the hallmark of PCOS, affects the immune system. Using a PCOS-like mouse model, it is demonstrated that hyperandrogenism affects immune cell populations in reproductive, metabolic, and immunological tissues differently in a site-specific manner. Co-treatment with an androgen receptor antagonist prevents most of these alterations, demonstrating that these effects are mediated through androgen receptor activation. Dihydrotestosterone (DHT)-exposed mice displayed a drastically reduced eosinophil population in the uterus and visceral adipose tissue (VAT). A higher frequency of natural killer (NK) cells and elevated levels of IFN-γ and TNF-α are seen in uteri of androgen-exposed mice, while NK cells in VAT and spleen displayed a higher expression level of CD69, a marker of activation or tissue residency. Distinct alterations of macrophages in the uterus, ovaries, and VAT are also found in DHT-exposed mice and can potentially be linked to PCOS-like traits of the model. Indeed, androgen-exposed mice are insulin-resistant, albeit unaltered fat mass. Collectively, it is demonstrated that hyperandrogenism causes tissue-specific alterations of immune cells in reproductive organs and VAT, which can have considerable implications on tissue function and contribute to the reduced fertility and metabolic comorbidities associated with PCOS.

Immune-Enhancing Activity of Vitis coignetiae Extract via Increasing Cytokine and Natural Killer Cell Activity in Splenocytes and Cyclophosphamide-Induced Immunosuppressed Rats

Plant and fruit extracts exhibit fewer side effects than pharmaceuticals and can display therapeutic qualities. Consequently, they have attracted increased attention among health-conscious individuals, and many studies related to their health-promoting effects are being actively conducted. Vitis coignetiae is well-known for its antioxidant, anti-inflammatory, and anticancer properties. However, the immune-enhancing effects of Vitis coignetiae have not yet been studied. In this study, Vitis coignetiae extract (VCE) increased immune-related activity in Wistar rat splenocytes and cyclophosphamide (Cy)-induced immunosuppressed rats. In splenocytes, VCE was nontoxic up to 300 μg/mL and increased cell viability in the presence of Cy. VCE also recovered Cy-induced decreases in cytokine (tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-2 (IL-2), and IL-12) levels and natural killer (NK) cell activity in splenocytes. In Cy-induced immunosuppressed rats, VCE protected against Cy-induced spleen tissue damage and decreases in body and spleen weight, cytokine (TNF-α, IFN-γ, IL-2, and IL-12) levels, and NK cell activity. We also observed recovery of immunoglobulin G (IgG) and IgM levels following VCE treatment. In conclusion, the ability of VCE to restore immune activity from an immunosuppressive state to normal levels suggests its immune-enhancing efficacy. Therefore, VCE may have the potential for application in the development of immune-enhancing functional foods and medicines.

Systems pharmacology reveals the mechanism of Astragaloside IV in improving immune activity on cyclophosphamide-induced immunosuppressed mice

Ethnopharmacological relevanceMyelosuppression, also known as bone marrow suppression (BMS), is a pathological phenomenon of the decrease in the production of blood cells and further lead to immune homeostasis disorder. Astragalus mongholicus Bunge (AM, checked with The World Flora Online, http://www.worldfloraonline.org, updated on January 30, 2023) is a traditional Chinese medicine with efficacy of tonifying Qi and strengthening body immunity in thousands of years of clinical practice in China. Astragaloside IV (AS-IV) is a major active ingredient of AM, which plays an important role in regulating immune system through different ways. Aim of the studyThis study was aimed to investigate the protective effect and mechanism of AS-IV on macrophages in vitro and cyclophosphamide (CTX)-induced immunosuppressive mice in vivo, and to provide experimental basis for the prevention and treatment of AS-IV in myelosuppression. Materials and methodsBased on network pharmacology and molecular docking technology, the core targets and signaling pathways of saponins of AM against myelosuppression were screened. And then, the immunoregulatory effect of AS-IV on RAW264.7 cells was investigated by cellular immune activity and cellular secretion analysis in vitro. In this way, the effects of AS-IV on the main potential targets of HIF-1α/NF-κB signaling pathway were analyzed by qRT-PCR and Western blot methods. Furthermore, comprehensive analysis of the effects of AS-IV against CTX-induced mice were conducted on the basis of immune organs indices analysis, histopathological analysis, hematological analysis, natural killer cell activity analysis and spleen lymphocyte transformation activity analysis. In order to further verify the relationship between active ingredients and action targets, drug inhibitor experiments were finally conducted. ResultsAS-IV, as a potential anti-myelosuppressive compound, was screened by systematic pharmacological methods to act on target genes including HIF1A and RELA together with the HIF-1α/NF-κB signaling pathway. Further studies by molecular docking technology showed that AS-IV had good binding activity with HIF1A, RELA, TNF, IL6, IL1B and other core targets. Besides, cellular and animal experiments validation results showed that AS-IV could enhance the migration and phagocytosis of RAW264.7 cells, and protect the immune organs such as spleen and thymus together with bone tissues from damage. By this means, immune cell function including spleen natural killer cell and lymphocyte transformation activity were also enhanced. In addition, white blood cells, red blood cells, hemoglobin, platelets and bone marrow cells were also significantly improved in the suppressed bone marrow microenvironment (BMM). In kinetic experiments, the secretion of cytokines such as TNF-α, IL-6 and IL-1β were increased, and IL-10, TGF-β1 were decreased. The key regulatory proteins such as HIF-1α, NF-κB, PHD3 in HIF-1α/NF-κB signaling pathway were also regulated in the results of upregulated expression of HIF-1α, p–NF–κB p65 and PHD3 at the protein or mRNA level. Finally, the inhibition experiment results suggested that AS-IV could significantly improve protein response in immunity and inflammation such as HIF-1α, NF-κB and PHD3. ConclusionAS-IV could significantly relieve CTX-induced immunosuppressive and might improve the immune activity of macrophages by activating HIF-1α/NF-κB signaling pathway, and provide a reliable basis for the clinical application of AS-IV as a potentially valuable regulator of BMM.

Enhancing Natural Killer Cell-Mediated Cancer Immunotherapy by the Biological Macromolecule Nocardia rubra Cell-Wall Skeleton.

The biological macromolecule Nocardia rubra cell-wall skeleton (Nr-CWS) has well-established immune-stimulating and anti-tumor activities. However, the role of Nr-CWS on natural killer (NK) cells remains unclear. Here, we explore the function and related mechanisms of Nr-CWS on NK cells. Using a tumor-bearing model, we show that Nr-CWS has slightly effect on solid tumor. In addition, using a tumor metastasis model, we show that Nr-CWS suppresses the lung metastasis induced by B16F10 melanoma cells in mice, which indicates that Nr-CWS may up-regulate the function of NK cells. Further investigation demonstrated that Nr-CWS can increase the expression of TRAIL and FasL on spleen NK cells from Nr-CWS treated B16F10 tumor metastasis mice. The spleen index and serum levels of TNF-α, IFN-γ, and IL-2 in B16F10 tumor metastasis mice treated with Nr-CWS were significantly increased. In vitro, the studies using purified or sorted NK cells revealed that Nr-CWS increases the expression of CD69, TRAIL, and FasL, decreases the expression of CD27, and enhances NK cell cytotoxicity. The intracellular expression of IFN-γ, TNF-α, perforin (prf), granzyme-B (GrzB), and secreted TNF-α, IFN-γ, IL-6 of the cultured NK cells were significantly increased after treatment with Nr-CWS. Overall, the findings indicate that Nr-CWS could suppress the lung metastasis induced by B16F10 melanoma cells, which may be exerted through its effect on NK cells by promoting NK cell terminal differentiation (CD27lowCD11bhigh), and up-regulating the production of cytokines and cytotoxic molecules.

In situ injectable hydrogel-loaded drugs induce anti-tumor immune responses in melanoma immunochemotherapy.

Melanoma is a highly aggressive tumor located in the skin, with limited traditional therapies. In order to reduce the side effects caused by traditional administration method and amplify the killing effect of immune system against tumor cells, an in situ injectable hydrogel drug delivery system is developed for the first time which co-delivers doxorubicin (Dox) and imiquimod (R837) for the synergistic therapy of melanoma. The mechanical properties and stability of the hydrogel are characterized and the optimal doses of hydrogel and drugs are also identified. As a result, the co-delivery system effectively suppresses melanoma growth and metastatic progression both in vitro and in vivo. Further studies show that the co-delivery system causes immunogenic cell death, activation of antigen presenting cells, comprising dendritic cells and M1 macrophages, and secretion of related cytokines consisted of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), subsequently with the activation of T lymphocytes and natural killer cells in spleen and tumor area. The co-delivery system also decreases the suppressive immune responses, including infiltration of M2 macrophages and secretion of interleukin-10 (IL-10), in vivo. Besides, other death modes are induced by the co-delivery system, including apoptosis and non-apoptotic cell death. In a word, this co-delivery system induces melanoma cell death directly and activates immune system for further tumor killing simultaneously, which shows probability for precise targeted tumor therapy.

Tissue origin of cytotoxic natural killer cells dictates their differential roles in mouse digit tip regeneration and progenitor cell survival.

SummaryRegeneration of amputated digit tips relies on mesenchymal progenitor cells and their differentiation into replacement bone and tissue stroma. Natural killer (NK) cells have well-characterized roles in antigen-independent killing of virally infected, pre-tumorous, or stressed cells; however, the potential for cytotoxic activity against regenerative progenitor cells is unclear. We identified NK cell recruitment to the regenerating digit tip, and NK cytotoxicity was observed against osteoclast and osteoblast progenitors. Adoptive cell transplants of spleen NK (SpNK) or thymus NK (ThNK) donor cells into immunodeficient mice demonstrated ThNK cell-induced apoptosis with a reduction in osteoclasts, osteoblasts, and proliferative cells, resulting in inhibition of regeneration. Adoptive transfer of NK cells deficient in NK cell activation genes identified that promotion of regeneration by SpNK cells requires Ncr1, whereas inhibition by ThNK cells is mediated via Klrk1 and perforin. Successful future therapies aimed at enhancing regeneration will require a deeper understanding of progenitor cell protection from NK cell cytotoxicity.

Analysis of chicken intestinal natural killer cells, a major IEL subset during embryonic and early life

Restrictions on antimicrobials demand alternative strategies to improve broiler health, such as supplying feed additives which stimulate innate immune cells like natural killer (NK) cells. The main objective of this study was to characterize intestinal NK cells in broiler chickens during embryonic and early life and compare these to NK cells in spleen, blood and bone marrow. Also T-cell subsets were determined. The majority of intestinal NK cells expressed IL-2Rα rather than 20E5 and 5C7, and showed low level of activation. Within intestinal NK cells the activation marker CD107 was mostly expressed on IL-2Rα+ cells while in spleen and blood 20E5+ NK cells primarily expressed CD107. High percentages of intestinal CD8αα+, CD8αβ+ and from 2 weeks onward also gamma delta T cells were found.Taken together, we observed several intestinal NK subsets in broiler chickens. Differences in NK subsets were mostly observed between organs, rather than differences over time. Targeting these intestinal NK subsets may be a strategy to improve immune-mediated resistance in broiler chickens.

1666-P: Increased Levels of Autophagy in a Rodent Model of Type 1 Diabetes

Beta-cell apoptosis is a hallmark of type 1 diabetes (T1D) and type 2 diabetes (T2D). In T2D loss of autophagy has been associated to increased apoptosis and diminished beta-cell mass. In autoimmunity, dysfunctional autophagy alters negative selection of T cells in thymus driving autoreactive immune cells into periphery. Autophagy contributes to presentation of antigens in antigen presenting cells shaping the repertoire of peptides presented to T cells. The aim of this project is to investigate the role of autophagy in the etiology of T1D. To investigate the effect of spermidine (SPD, a known inducer of autophagy) in vivo, non-obese diabetic (NOD) mice received spermidine (10 mM; n=30) or pure drinking water (n=28) from the age of 4 weeks until the age of 35 weeks or diabetes onset. Mice were analyzed for diabetes rate, autophagy markers (LC3-II to LC3-I ratio and p62) and immune cell phenotype in spleen and blood. Spermidine led to a higher diabetes rate in SPD group (80 %) compared to control group (60,7 %) with p-value of 0,026 (log-rank test). Autophagy was induced by diabetes (plasma glucose > 200 mg/dL) and by age. Autophagy levels were higher at 21-35 weeks in old diabetic animals compared to nondiabetic animals at 35 weeks and compared to young diabetic animals at 14-20 weeks (p< 0,0001, and p< 0,0001, respectively). Spermidine treatment increased autophagy levels in pancreas, but diabetic mice at 21-35 weeks still had higher autophagy levels compared to nondiabetic animals at week 35. (LC3-II/LC3-I: p < 0,05; p62: p < 0,05). Spermidine elevated the frequency of natural killer cells in spleen (p < 0,01) and blood (p < 0,01), natural killer T cells (p < 0,01), naïve CD4+ T cells (p < 0,01), naïve CD8+ T cells (p < 0,01) and central memory CD4+ T cells (p < 0,01) in blood. Our experiments indicate that autophagy is induced upon T1D and spermidine-induced autophagy might increase diabetes incidence. Immune cell phenotyping revealed that spermidine promotes immune cells potentially provoking inflammation. Disclosure C. Karacay: None. P. Kotzbeck: None. B. Prietl: None. C. Harer: None. T. Pieber: Advisory Panel; Self; ADOCIA, Arecor, AstraZeneca, Eli Lilly and Company, Novo Nordisk A/S, Sanofi. Research Support; Self; AstraZeneca, Novo Nordisk A/S. Speaker’s Bureau; Self; Novo Nordisk A/S, Roche Diagnostics K.K.

Endogenous glucocorticoids control host resistance to viral infection through the tissue-specific regulation of PD-1 expression on NK cells

Controlling the balance between immunity and immunopathology is crucial for host resistance to pathogens. After infection, activation of the hypothalamic–pituitary–adrenal (HPA) axis leads to the production of glucocorticoids. However, the pleiotropic effects of these steroid hormones make it difficult to delineate their precise role(s) in vivo. Here we found that the regulation of natural killer (NK) cell function by the glucocorticoid receptor (GR) was required for host survival after infection with mouse cytomegalovirus (MCMV). Mechanistically, endogenous glucocorticoids produced shortly after infection induced selective and tissue-specific expression of the checkpoint receptor PD-1 on NK cells. This glucocorticoid–PD-1 pathway limited production of the cytokine IFN-γ by spleen NK cells, which prevented immunopathology. Notably, this regulation did not compromise viral clearance. Thus, the fine tuning of NK cell functions by the HPA axis preserved tissue integrity without impairing pathogen elimination, which reveals a novel aspect of neuroimmune regulation.

CXCR6+ NK Cells in Human Fetal Liver and Spleen Possess Unique Phenotypic and Functional Capabilities.

Tissue-resident Natural Killer (NK) cells vary in phenotype according to tissue origin, but are typically CD56bright, CXCR6+, and CD69+. NK cells appear very early in fetal development, but little is known about when markers of tissue residency appear during gestation and whether the expression of these markers, most notably the chemokine receptor CXCR6, are associated with differences in functional capability. Using multi-parametric flow cytometry, we interrogated fetal liver and spleen NK cells for the expression of a multitude of extracellular markers associated with NK cell maturation, differentiation, and migration. We analyzed total NK cells from fetal liver and spleen and compared them to their adult liver and spleen counterparts, and peripheral blood (PB) NK. We found that fetal NK cells resemble each other and their adult counterparts more than PB NK. Maturity markers including CD16, CD57, and KIR are lower in fetal NK cells than PB, and markers associated with an immature phenotype are higher in fetal liver and spleen NK cells (NKG2A, CD94, and CD27). However, T-bet/EOMES transcription factor profiles are similar amongst fetal and adult liver and spleen NK cells (T-bet−/EOMES+) but differ from PB NK cells (T-bet+EOMES−). Further, donor-matched fetal liver and spleen NK cells share similar patterns of expression for most markers as a function of gestational age. We also performed functional studies including degranulation, cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) assays. Fetal liver and spleen NK cells displayed limited cytotoxic effector function in chromium release assays but produced copious amounts of TNFα and IFNγ, and degranulated efficiently in response to stimulation with PMA/ionomycin. Further, CXCR6+ NK cells in fetal liver and spleen produce more cytokines and degranulate more robustly than their CXCR6− counterparts, even though CXCR6+ NK cells in fetal liver and spleen possess an immature phenotype. Major differences between CXCR6− and + NK cell subsets appear to occur later in development, as a distinct CXCR6+ NK cell phenotype is much more clearly defined in PB. In conclusion, fetal liver and spleen NK cells share similar phenotypes, resemble their adult counterparts, and already possess a distinct CXCR6+ NK cell population with discrete functional capabilities.

Assessment of PD-1 expression in human resting and activated natural killer cells and murine tumor models.

36 Background: Blockade of the PD-1/PD-L1/2 axis has revolutionized cancer therapy. Although reinvigorated PD-1+ T cells are the main effectors in the response to checkpoint blockade, the contribution of Natural Killer (NK) cells to PD-1/PD-L1 inhibition is under debate. While PD-1 has been identified on NK cells, this appears to be restricted to small populations under limited conditions. We sought to evaluate the extent of PD-1 expression in mouse and human resting and activated NK cells. Methods: Human NK cells were isolated from healthy donor PBMCs and cancer patients. Ex vivo activation and proliferation techniques included recombinant human cytokine and feeder line co-culture. Murine NK cells were isolated from splenocytes, and PBMCs from wild type and immunodeficient mice. We assessed NK cell surface markers and intracellular cytokine by flow cytometry, and gene expression by quantitative RT-PCR. Results: Over 21-days of ex vivo expansion, expression of PD-1 or PD-L1 on human NK cells was < 1% at all time points, while TIGIT+ expression increased to > 85%. Conversely, ConA stimulation of T cells increased PD-1 expression with no change in TIGIT expression. QRT-PCR demonstrated absent PD-1 expression in purified NK cells compared to a 5-fold increase in PD-1 gene expression in ConA stimulated PBMCs. PD-1/PD-L1 was also < 1% in the NK92 cell line and < 2.5% in peripheral CD56+CD3- NK cells from patients with soft tissue sarcoma (STS). NK cells from digested freshly resected STS show variable PD-1 ( < 10%) and minimal PD-L1 ( < 1%) expression with a small, but measurable population of intra-tumoral NK cells (1% of immune cells). In vivo mouse studies showed < 5% PD-1+ NK cells in spleen and tumor of CT26 tumor-bearing mice, while PD-L1+ NK cells increased in frequency from spleen (5-35%) to tumor (40-95%) in both wild type BALB/C and SCID mice. Conclusions: In contrast to prior studies, we did not observe a substantial PD-1+ population on human or murine NK cells after multiple activation strategies compared to T cells. Contrary to its application in T cells, our data suggest that PD-1 is not a useful marker for NK cell exhaustion/dysfunction. PD-L1 on NK cells may represent an important link between NK and T cell immunotherapy.

Laminarin enhances the activity of natural killer cells in immunosuppressed mice.

The aim of the study was to investigate the effects of laminarin on natural killer (NK) cell cytotoxicity of immunosuppressive mice and its mechanism. Cyclophosphamide (cy) was used to make an immunosuppressive model of mice. The mice of two groups were given interventions by gavage with laminarin 500 mg/kg and 1000 mg/kg every day for 10 days. MACS was adopted to isolate spleen NK cells, and cytotoxicity of NK cells and IL-12, IFN-γ level in serum were detected in vivo. Cytotoxicity of NK92-MI cells, activating receptors (NKp30, NKp44, NKp46 and NKG2D) and perforin and granzyme B expression were detected in vitro. Compared to the normal control group, the cytotoxicity of NK cells, IL-12 and IFN-γ level in serum in the cy model group were all reduced significantly (p < 0.01). Compared to the cy model group, laminarin increased the cytotoxicity of NK cells, IL-12 and IFN-γ levels in serum significantly (p < 0.05). In vitro, laminarin increased the cytotoxicity, NKp30 and NKG2D, perforin and granzyme B expressions of NK92-MI cells (p < 0.01). This research showed that laminarin can promote NK cell cytotoxicity in immunosuppressive mice by increasing the levels of IL-12 and IFN-γ in serum and expressions of NKp30 and NKG2D, perforin and granzyme B.

CD47 Expression in Natural Killer Cells Regulates Homeostasis and Modulates Immune Response to Lymphocytic Choriomeningitis Virus

CD47 is a ubiquitous cell surface receptor that directly regulates T cell immunity by interacting with its inhibitory ligand thrombospondin-1 and limits clearance of cells by phagocytes that express its counter-receptor signal-regulatory protein-α. Murine natural killer (NK) cells express higher levels of CD47 than other lymphocytes, but the role of CD47 in regulating NK cell homeostasis and immune function remains unclear. Cd47−/− mice exhibited depletion of NK precursors in bone marrow, consistent with the antiphagocytic function of CD47. In contrast, antisense CD47 knockdown or gene disruption resulted in a dose dependent accumulation of immature and mature NK cells in spleen. Mature Cd47−/− NK cells exhibited increased expression of NK effector and interferon gene signatures and an increased proliferative response to interleukin-15 in vitro. Cd47−/− mice showed no defect in their early response to acute Armstrong lymphocytic choriomeningitis virus (LCMV) infection but were moderately impaired in controlling chronic Clone-13 LCMV infection, which was associated with depletion of splenic NK cells and loss of effector cytokine and interferon response gene expression in Cd47−/− NK cells. Broad CD47-dependent differences in NK activation, survival, and exhaustion pathways were observed in NK cell transcriptional signatures in LCMV infected mice. These data identify CD47 as a cell-intrinsic and systemic regulator of NK cell homeostasis and NK cell function in responding to a viral infection.

Нарушение функциональной активности естественных киллеров при экспериментальном инсульте различной степени тяжести

Актуальность. Изучение факторов риска развития инфекционно-воспалительных осложнений и путей их снижения при инсульте является актуальной неврологической проблемой. Цель: оценка функциональной активности клеток естественных киллеров в остром периоде экспериментального инсульта различной степени тяжести. Методы. В проведенном экспериментальном исследовании на животных (крысы линии Вистар, массой 200-220 г, n = 50) изучали функциональная активность естественных киллеров селезенки, при моделировании монополушарного левостороннего экспериментального инсульта (ЭИ) различной степени тяжести. Результаты. Показано, что у животных на 7-й день инсульта отмечается статистически значимое снижение функциональной активности естественных киллеров селезенки: ЭИ легкой степени - 20 (19; 23)%, р = 0,001, ЭИ средней степени - 19 (17; 21)%, р &lt; 0,001, ЭИ тяжелой степени - 18 (16; 22)%, р &lt; 0,001. Нарушения функциональной активности естественных киллеров более выражены на модели ЭИ средней и тяжёлой степени тяжести. Заключение. Показано, что при ЭИ наблюдается снижение функциональной активности естественных киллеров селезёнки, которое нарастает при увеличении степени тяжести инсульта. Background. Studying risk factors for development of infectious and inflammatory complications and ways to reduce them in stroke is a modern challenge to neurology. Aim: Evaluating the functional activity of natural killer (NK) cells in the acute period of experimental stroke (ES) of different severity. Methods. In this experimental study, the functional activity of spleen NK cells was studied in Wistar rats weighing 200-220 g (n = 50) with ES of different severity. Results. On the 7th day of stroke. the functional activity of spleen NK was significantly decreased: in mild ES, 20 (19; 23)%, р = 0.001, in moderate ES, 19 (17; 21)%, р &lt;0.001, and in severe ES, (16; 22)%, р &lt; 0.001. Disorders of the NK functional activity were more pronounced in moderate and severe ES. Conclusion. ES was associated with impaired functional activity of NK cells, which progressed with increasing grade of ES severity.

Myc Regulates the Development and Anti-Tumor Immunity of Natural Killer Cells

Natural Killer (NK) cells play an essential role in the innate immunity. We recently reported that in aggressive NK cell leukemia, MYC is hyperactivated and responsible for the survival and proliferation of leukemic cells through the activation of JAK/STAT pathway (Huang et al., Cell Research 2017). However, the role of Myc in normal NK cell development or function has not been fully explored. Here we addressed the important question on whether Myc is required for NK cell development and its anti-tumor immunity.By crossing Mycf/f allele with Ncr1Cre allele, we generated a NK cell specific Myc conditional knock out (cKO) mouse model. Mycf/f mice were used as control. The percentages of NK1.1+DX5+ mature NK cells (mNK) decreased significantly in both the peripheral blood (PB) and spleen compared to the control mice. In addition, the percentages of terminally differentiated CD11b+CD27- cells within the mNK population decreased, while CD11b-CD27+ cells increased. The expression of terminal differentiation marker Klrg1 also significantly decreased. Further analyzing the NK cells in the bone marrow (BM) revealed the CD122+% and mNK% in the BM did not change. In contrast, the NK1.1-DX5-% in lin-CD122+ population (NK progenitors) increased by 1-fold. This suggested that the development of NK cells was impaired after Myc deletion.To investigate the effects of overexpressed Myc on normal NK cell development, we generated a MYC inducible mouse model (iMYC) by crossing Tg(tetO-MYC) allele with Ncr1Cre and Rosa26Loxp-Stop-Loxp-rtTA alleles. Mice without iMYC allele were used as controls. We transplanted the BM cells from the control or iMYC mice into lethally irradiated BoyJ recipients. We observed that mNK% in CD45.2+ cells increased significantly in liver and PB 3 weeks after the transplantation and dox induction. The ratio of CD27-:CD27+ cells in NK1.1+DX5+CD11b+ cells and Klrg1+mNK% also increased in all tissues analyzed (BM, liver, PB, spleen). In addition, Brdu incorporation rates were increased in spleen and BM NK cells from the iMYC mice. NK cells isolated from iMYC mice showed an enhanced proliferation ability in vitro by dox induction in a dose-dependent manner. These suggested MYC promoted the NK cell commitment, maturation and proliferation.To explore whether the NK cell cytotoxic functions were also affected by Myc, we measured the IFN-γ secretion and CD107a degranulation by PMA/ionomycin or YAC-1 stimulation. Both of the abilities were significantly impaired in Myc cKO mice. Furthermore, the direct lysis of YAC-1 cells by NK cells was also decreased. In contrast, the CD107a degranulation did not change in iMYC mice. The secretion of IFN-γ of splenic NK cells after PMA/Ionomycin stimulation was slightly decreased in iMYC mice, which may be due to the increased population of terminally differentiated NK cells with lower cytotoxicity. This suggested that the physiological level of Myc was important for the cytotoxic functions of NK cells.To investigate whether Myc deletion in NK cells affects the tumor surveillance, we used 2 syngeneic tumor models B16F10 (melanoma) and MC38 (colon adenocarcinoma). The lung metastatic dots were significantly increased in Myc cKO mice compared to control 2 weeks after the inoculation of B16F10. All of the mice in the Myc cKO group were dead on day 21 while the survival rates were 100% in the control group. The weights and volumes of tumors in mice subcutaneously inoculated with MC38 were also significantly higher in Myc cKO mice. This indicated a key role of Myc in NK cell mediated immunity.Finally, we performed transcriptome analysis of splenic NK cells sorted from Myc cKO mice and control mice. The top 2 enriched KEGG pathways in the down-regulated genes includes NK cell mediated cytotoxicity. Interestingly, the expression of Cish, which is an important NK cell anti-tumor immunity checkpoint, was significantly increased in the Myc cKO group, suggesting Myc may affect the NK cell functions through the downregulation of Cish.In summary, our data showed that Myc promoted NK cell development and proliferation. A physiological level of Myc was critical for NK cell cytotoxicity and anti-tumor immunity. Our data also indicated that targeting Myc could be a potential strategy in boosting NK cell-mediated immunotherapy or NK cell expansion ex vivo. Further studies are needed to uncover the underlying mechanism and explore the translational application. DisclosuresNo relevant conflicts of interest to declare.

Endogenous glucocorticoids control host resistance to viral infection through the tissue-specific regulation of PD-1 expression on NK cells.

Controlling the balance between immunity and immunopathology is crucial for host resistance to pathogens. After infection, activation of the hypothalamic–pituitary–adrenal (HPA) axis leads to the production of glucocorticoids. However, the pleiotropic effects of these steroid hormones make it difficult to delineate their precise role(s) in vivo. Here we found that the regulation of natural killer (NK) cell function by the glucocorticoid receptor (GR) was required for host survival to mouse cytomegalovirus infection. Mechanistically, endogenous glucocorticoids produced shortly after infection induced the selective and tissue-specific expression of the checkpoint molecule PD-1 on NK cells. This glucocorticoid–PD-1 pathway limited the production of the cytokine interferon-γ (IFN-γ) by splenic NK cells, which prevented lethal immunopathology. Importantly, this regulation did not compromise viral clearance. Thus, the fine-tuning of NK cell functions by the HPA axis preserved tissue integrity without impairing pathogen elimination, which reveals a novel aspect of neuroimmune regulation.

Immune enhancement effect of an herb complex extract through the activation of natural killer cells and the regulation of cytokine levels in a cyclophosphamide-induced immunosuppression rat model

Objective: To investigate the effects of a herb complex extract (HCE) prepared from Cornus officinalis Sieb. Et Zucc., Eriobotrya japonica Lindley, and olive leaves on immune response of mouse spleen NK cells in vitro and in vivo analysis. Methods: The activity of natural killer (NK) cells was measured in splenocytes and YAC-1 cells. Mice were immunosuppressed using cyclophosphamide (5 mg/kg body weight). Three different doses of HCE (200, 400, and 800 mg/kg body weight) and red ginseng extract (800 mg/kg body weight) which was used as standard immunomodulatory herb were administered orally for 4 weeks. The body weight, dietary, water intake, organs (liver, thymus, and spleen) weight, completed blood count, and cytokines (tumor necrosis factor alpha, interferon gamma, and interleukin-2) production was measured. Results: At the maximum concentration of HCE, the activity of NK cells was increased by 48.5%. HCE increased liver, spleen, and thymus weights without altering numbers of white blood cells, lymphocytes, and neutrophils in a cyclophosphamide-induced immunosuppression rat model. However, HCE recovered the inhibited cytokine expression; HCE (800 mg/kg) increased cytokines levels. The results indicate the immune enhancement potential of this HCE. Conclusion: The HCE enhances immunity by increasing NK cell activity, regulating cytokine levels, and maintaining spleen weight. Therefore, it may be used as a potential immunity enhancer.

Effect of intrathecal morphine and fentanyl on level of natural killer cells in hippocampus of rats with incisional pain

Objective To evaluate the effect of intrathecal morphine and fentanyl on the level of natural killer(NK)cells in the hippocampus of rats with incisional pain. Methods Ninety-six healthy male Sprague-Dawley rats, weighing 180-220 g, aged 6-8 weeks, in which intrathecal catheters were successfully implanted, were divided into 4 groups(n=24 each)using a random number table: control group(C group), incisional pain group(P group), intrathecal morphine-fentanyl group(MF group)and intrathecal morphine-fentanyl plus incisional pain group(MFP group). A 1-cm longitudinal incision was made through skin, fascia and muscle of the plantar aspect of the right hindpaw in sevoflurane-anesthetized rats.At 20 min before establishment of the incisional pain model, the mixture of morphine 5 μg/kg and fentanyl 0.25 μg/kg was intrathecally injected in MFP group, while the equal volume of normal saline was given instead in NS and P groups.Six rats were selected at 24 h before establishment of the model(T0, baseline)and at 1, 6, 24, 48 and 72 h after establishment of the model(T1-5), and the mechanical paw withdrawal threshold(MWT)and thermal paw withdrawal latency(TWL)were measured.Six rats from each group were selected at T0, T2, T3 and T5, venous blood samples were taken from the peripheral vein, the animals were then sacrificed immediately, and the hippocampi and spleen were quickly harvested.The levels of NK cells in peripheral blood, hippocampi and spleen were measured. Results Compared with group C, the MWT was significantly decreased, the TWL was shortened, the levels of NK cells in hippocampi were increased at T2-5, the levels of NK cells in the spleen were increased at T2 and decreased at T3, 5, and the levels of NK cells in peripheral blood were decreased at T2, 3 in group P(P 0.05), the levels of NK cells in the spleen were increased at T2 and decreased at T5, the levels of NK cells in peripheral blood were decreased at T2, 5(P 0.05). Compared with group P, the MWT was significantly increased, the TWL was prolonged, the levels of NK cells in hippocampi were decreased at T2-5, and the levels of NK cells in the spleen and peripheral blood were decreased at T2-5 and increased at T3 in group MFP(P<0.05). Conclusion Intrathecal morphine and fentanyl can maintain the level of NK cells stable in the hippocampus, which may be helpful for maintenance of immune function of rats with incisional pain. Key words: Morphine; Piperidines; Pain, postoperative; Injections, spinal; Killer cells, natural; Hippocampus