NK cells require type I IFN receptor for antiviral responses during genital HSV-2 infection

Type I interferon (IFN) signalling, NK cells and NK cell-derived IFN-γ are critical in the early control of genital HSV-2 infection. We have recently reported that NK cells are the source of early IFN-γ in the genital tract in response to HSV-2. However, the response of NK cells to genital HSV-2 infection is not well defined in the context of type I IFN signalling. Here we show that HSV-2 replication was significantly higher in mice deficient in the type I IFN receptor or NK cells compared to wild type controls. There was no detectable IFN-γ production in the genital washes from IFN-α/βR^−/− mice or NK cell depleted mice in response to HSV-2 infection compared to control mice. Absence of the type I IFN receptor does not alter homing of NK cells to the genital mucosa. Moreover, the absence of IL-12 had no significant effect on NK cell-derived IFN-γ. Surprisingly, IFN-α/βR^−/− mice had more IL-15 positive cells in the genital mucosa in response to HSV-2 infection compared to control mice. We then examined the expression of IL-15 receptors on NK cells. There was no significant differences in the levels of IL-15 receptor expression on NK cells from IFN-α/βR^−/− or control mice. Our data clearly suggest that type I IFN receptor signalling is essential for NK cell activation in response to genital HSV-2 infection, and propose that NK cell activation by IL-15 may involve type I IFNs.     

The interactions of multiple cytokines control NK cell maturation

Although NK cells are well known for their cytotoxic functions, they also produce an array of immunoregulatory cytokines and chemokines. During an immune response, NK cells are exposed to complex combinations of cytokines that influence their differentiation and function. In this study, we have examined the phenotypic and functional consequences of exposing mouse NK cells to IL-4, IL-12, IL-15, IL-18, and IL-21 and found that although all factors induced signs of maturation, characterized by decreased proliferation and IFN-γ secretion, distinct combinations induced unique cytokine secretion profiles. In contrast, the immunosuppressive factors IL-10 and TGF-β had little direct effect on NK cell effector functions. Sustained IL-18 signals resulted in IL-13 and GM-CSF production, whereas IL-12 and IL-21 induced IL-10 and TNF-α. Surprisingly, with the exception of IL-21, all cytokines suppressed cytotoxic function of NK cells at the expense of endogenous cytokine production suggesting that "helper-type" NK cells were generated. The cytokine signals also profoundly altered the cell surface phenotype of the NK cells-a striking example being the downregulation of the activating receptor NKG2D by IL-4 that resulted in decreased NKG2D-dependent killing. IL-4 exposure also modulated NKG2D expression in vivo suggesting it is functionally important during immune responses. This study highlights the plasticity of NK cell differentiation and suggests that the relative abundance of cytokines at sites of inflammation will lead to diverse outcomes in terms of NK cell phenotype and interaction with the immune system.     

Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children

Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD₆₅ or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-γ and TNF-α) and chemokines (IP-10, MCP-1, MIP-1α, MIP-1β and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-β mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-γ and MCP-1, and mRNA expression of FOXP3 and TGF-β, was detected after cryopreservation. Stimulation with GAD₆₅ induced higher levels of IL-6, IFN-γ, TNF-α and MIP-1α, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCP-1 and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.     

Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children

Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD₆₅ or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-γ and TNF-α) and chemokines (IP-10, MCP-1, MIP-1α, MIP-1β and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-β mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-γ and MCP-1, and mRNA expression of FOXP3 and TGF-β, was detected after cryopreservation. Stimulation with GAD₆₅ induced higher levels of IL-6, IFN-γ, TNF-α and MIP-1α, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCP-1 and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.     

Neutrophil microparticles modulate cytokine production by natural killer cells

Neutrophil microparticles (NMs) are scarce in the circulation but are highly enriched at sites of inflammation and exert profound effects on immune cells. In the present study, we addressed whether NMs modulate cytokine-producing capacity of natural killer (NK) cells. NMs inhibited the production of IFN-γ and TNF-α but enhanced the release of TGF-β1 by IL-2/IL-12-activated NK cells. The inhibitory effect of NMs was strongly attenuated by blockade of phosphatidylserine exposed on NMs. Thus, NMs skew the cytokine profile of NK cells from pro-inflammatory toward anti-inflammatory, potentially favoring the resolution of inflammation.     

Combinatory responses of proinflamamtory cytokines on nitric oxide-mediated function in mouse calvarial osteoblasts

Combinatory responses of proinflamamtory cytokines have been examined on the nitric oxide-mediated function in cultured mouse calvarial osteoblasts. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induced iNOS gene expression and NO production, although these actions were inhibited by L-NG-monomethylarginine (L-NMMA) and decreased alkaline phosphatase (ALPase) activity. Furthermore, NO donors, sodium nitroprusside (SNP) and NONOate dose-dependently elevated ALPase activity. In contrast, transforming-growth factor-β (TGF-β) decreased NO production stimulated by IL-1β, TNF-α and interferon-γ (IFN-γ). iNOS was expressed by mouse calvarial osteoblast cells after stimulation with IL-1β, TNF-α, and IFN-γ. Incubation of mouse calvarial osteoblast cells with the cytokines inhibited growth and ALPase activity. However, TGF-β-treatment abolished these effects of IL-1β, TNF-α and IFN-γ on growth inhibition and stimulation of ALPase in mouse calvarial osteoblast cells. In contrast, IL-1β, TNF-α, and IFN-γ exerted growth-inhibiting effects on mouse calvarial osteoblast cells which were partly NO-dependent. The results suggest that NO may act predominantly as a modulator of cytokine-induced effects on mouse calvarial osteoblast cells and TGF-β is a negative regulator of the NO production stimulated by IL-1β, TNF-α and IFN-γ.     

IFN-α production by plasmacytoid dendritic cells stimulated with RNA-containing immune complexes is promoted by NK cells via MIP-1β and LFA-1

Several systemic autoimmune diseases display a prominent IFN signature. This is caused by a continuous IFN-α production by plasmacytoid dendritic cells (pDCs), which are activated by immune complexes (ICs) containing nucleic acid. The IFN-α production by pDCs stimulated with RNA-containing IC (RNA-IC) consisting of anti-RNP autoantibodies and U1 small nuclear ribonucleoprotein particles was recently shown to be inhibited by monocytes, but enhanced by NK cells. The inhibitory effect of monocytes was mediated by TNF-α, PGE(2), and reactive oxygen species, but the mechanisms for the NK cell-mediated increase in IFN-α production remained unclear. In this study, we investigated the mechanisms whereby NK cells increase the RNA-IC-induced IFN-α production by pDCs. Furthermore, NK cells from patients with systemic lupus erythematosus (SLE) were evaluated for their capacity to promote IFN-α production. We found that CD56(dim) NK cells could increase IFN-α production >1000-fold after RNA-IC activation, whereas CD56(bright) NK cells required costimulation by IL-12 and IL-18 to promote IFN-α production. NK cells produced MIP-1α, MIP-1β, RANTES, IFN-γ, and TNF-α via RNA-IC-mediated FcγRIIIA activation. The IFN-α production in pDCs was promoted by NK cells via MIP-1β secretion and LFA-mediated cell-cell contact. Moreover, NK cells from SLE patients displayed a reduced capacity to promote the RNA-IC-induced IFN-α production, which could be restored by exogenous IL-12 and IL-18. Thus, different molecular mechanisms can mediate the NK cell-dependent increase in IFN-α production by RNA-IC-stimulated pDCs, and our study suggests that the possibility to therapeutically target the NK-pDC axis in IFN-α-driven autoimmune diseases such as SLE should be investigated.     

FTY720 and SEW2871 reverse the inhibitory effect of S1P on natural killer cell mediated lysis of K562 tumor cells and dendritic cells but not on cytokine release

The aims of this study are to examine the effect of sphingosine 1-phosphate (S1P) on IL-2-activated natural killer (NK) cell lysis of K562 tumor cells and immature dendritic cells (iDCs), and to investigate the mechanisms involved in S1P activity. Our results show that S1P protected K562 cells or iDCs from NK cell lysis, which was reversed by FTY720 and SEW2871, the antagonists of S1P₁. S1P did not modulate the expression of NKG2D, NKp30, NKp44 or CD158 on the surface of NK cells, and neither affected the expression of CD80, CD83, or CD86 on the surface of DCs. In contrast, it increased the expression of HLA-I and HLA-E on DCs, an activity that was inhibited by FTY720 or SEW2871. Similarly, the inhibitory effect of S1P for NK cell lysis of K562 cells was directed toward S1P₁ expressed on the tumor cells but not on NK cells. Further analysis indicates that NK cells secreted various cytokines and chemokines with various intensities: (1) low (IL-4, IL-6, IL-12, TNF-α and MCP-1); (2) intermediate (IL-1β, IL-10, TGF-β1, and IL-17A); (3) high (IFN-γ, and MIP-1α); and (4) very high (MIP-1β). S1P significantly reduced the release of IL-17A and IFN-γ from NK cells, but this inhibition was S1P₁-independent. These results indicate that S1P is an anti-inflammatory molecule, and that S1P₁ is important for the interaction among NK cells and tumor cells or DCs leading to up-regulation of HLA-I and HLA-E on the surface of DCs, but not in S1P inhibition of the release of inflammatory cytokines from NK cells. Further, the results suggest that FTY720 and SEW2871 may potentially be used as prophylactic and/or therapeutic drugs to treat cancer patients.     

The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis

In tegumentary leishmaniasis caused by Leishmania braziliensis, there is evidence that increased production of IFN-γ, TNF-α and absence of IL-10 is associated with strong inflammatory reaction and with tissue destruction and development of the lesions observed in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). We evaluate the role of regulatory cytokines and cytokine antagonists in the downregulation of immune response in L. braziliensis infection. Peripheral blood mononuclear cells from CL and ML were stimulated with soluble Leishmania antigen in the presence or absence of regulatory cytokines (IL-10, IL-27 and TGF-β) or antagonists of cytokines (α-TNF-α and α-IFN-γ). Cytokines production (IL-10, IL-17, TNF-α and IFN-γ) was measured by ELISA. IL-10 and TGF-β downmodulate TNF-α and IL-17 production, whereas IL-27 had no effect in the production of TNF-α, IFN-γ and IL-17 in these patients. Neutralization of TNF-α decreased IFN-γ level and the neutralization of IFN-γ decreased TNF-α level and increased IL-10 production. This study demonstrate that IL-10 and TGF-β are cytokines that appear to be more involved in modulation of immune response in CL and ML patients. IL-10 might have a protective role, since the neutralization of IFN-γ decreases the production of TNF-α in an IL-10-dependent manner.     

p53对TGEV感染PK-15细胞4种免疫调节因子mRNA转录水平的影响

为探究p53对IFN-α、MIP-1α、PGK-1、TGF-β1四种免疫调节因子在TGEV感染PK-15细胞中的影响,本研究首先采用CRISPR-Cas9慢病毒系统靶向于PK-15细胞的p53基因构建p53基因敲除(p53-/-)的细胞;再以感染复数为0.1 MOI的TGEV感染p53野生型(p53+/+)和p53-/-PK-15细胞,于不同的感染时间收集细胞并提取细胞总RNA,应用实时荧光定量PCR(qRT-PCR)技术检测四种细胞因子的转录水平。结果表明,构建的靶向于p53基因敲除的PK-15细胞中,p53基因的454碱基位点缺失一个碱基T,细胞的p53蛋白已检测不到;TGEV感染后IFN-αmRNA的相对表达量在两种细胞中均表现为先上升后下降的趋势,但在病毒感染的36 h之前,p53-/-PK-15细胞中的表达量显著低于p53+/+PK-15细胞(p<0.05);MIP-1αmRNA相对表达量随着病毒感染时间的推移而递增,且在p53+/+PK-15细胞中显著高于p53-/-PK-15细胞(p<0.05);TGF-β1 mRNA的相对表达量在p53+/+PK-15细胞中随时间推移总体呈递减趋势,并在病毒感染(post infection,p.i.)12 h之后显著低于p53-/-PK-15细胞(p<0.05);PGK-1 mRNA相对表达量在病毒感染的12 h p53+/+PK-15细胞中虽略有上升,但差异不显著,而在p53-/-PK-15细胞中呈现时间依赖性递增,并显著高于p53+/+PK-15细胞(p<0.05)。以上结果表明:p53对TGEV感染PK-15细胞后的细胞免疫因子起到了关键的调节作用,推测其可能在宿主抗TGEV感染中发挥着重要作用。     

Cytokine and cytokine receptor gene polymorphisms and their functionality

Cytokines, signaling proteins produced by a variety of cell types, are essential for the development and functioning of both innate and adaptive immune response. Cytokine gene expression is tightly regulated, and aberrant expression from environmental and genetic polymorphism has been implicated in a range of diseases, susceptibility to infections, and responses to treatment. This review concentrates on the functionality of cytokine and cytokine receptor gene polymorphisms; it is through these variants that genuine disease-associations are based. Several mechanisms for single nucleotide polymorphism (SNP) functionality are present within cytokine genes including: amino acid changes (IL-6R, IL-13, IL-1α), exon skipping (IL-7Rα), proximal promoter variants (IL-1β, IL-Ra, IL-2, IL-6, IL-10, IL-12, IL-13, IL-16, TNF, IFN-γ, TGF-β), distal promoter variants (IL-6, IL-18) and intronic enhancer variants (IL-8).     

Analysis of the expression of toll-like receptors 2 and 4 and cytokine production during experimental Leishmania chagasi infection

Toll-like receptors (TLRs) recognise pathogen-derived molecules and influence immunity to control parasite infections. This study aimed to evaluate the mRNA expression of TLRs 2 and 4, the expression and production of the cytokines interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-17, IL-10 and transforming growth factor (TGF)-β and the production of nitric oxide (NO) in the spleen of mice infected with Leishmania chagasi. It also aimed to evaluate any correlations between mRNA expression TLR2 and 4 and cytokines and NO production. Infection resulted in increased TLR2-4, IL-17, TNF-α and TGF-β mRNA expression during early infection, with decreased expression during late infection correlating with parasite load. IFN-γ and IL-12 mRNA expression decreased at the peak of parasitism. IL-10 mRNA expression increased throughout the entire time period analysed. Although TGF-β, TNF-α and IL-17 were highly produced during the initial phase of infection, IFN-γ and IL-12 exhibited high production during the final phase of infection. IL-10 and NO showed increased production throughout the evaluated time period. In the acute phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17, NO, IL-10 and TGF-β expression and parasite load. During the chronic phase of infection, there was a positive correlation between TLR2-4, TNF-α, IL-17 and TGF-β expression and parasite load. Our data suggest that infection by L. chagasi resulted in modulation of TLRs 2 and 4 and cytokines.     

Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection

NK cells are enriched in the liver, constituting around a third of intrahepatic lymphocytes. We have previously demonstrated that they upregulate the death ligand TRAIL in patients with chronic hepatitis B virus infection (CHB), allowing them to kill hepatocytes bearing TRAIL receptors. In this study we investigated whether, in addition to their pathogenic role, NK cells have antiviral potential in CHB. We characterised NK cell subsets and effector function in 64 patients with CHB compared to 31 healthy controls. We found that, in contrast to their upregulated TRAIL expression and maintenance of cytolytic function, NK cells had a markedly impaired capacity to produce IFN-γ in CHB. This functional dichotomy of NK cells could be recapitulated in vitro by exposure to the immunosuppressive cytokine IL-10, which was induced in patients with active CHB. IL-10 selectively suppressed NK cell IFN-γ production without altering cytotoxicity or death ligand expression. Potent antiviral therapy reduced TRAIL-expressing CD56(bright) NK cells, consistent with the reduction in liver inflammation it induced; however, it was not able to normalise IL-10 levels or the capacity of NK cells to produce the antiviral cytokine IFN-γ. Blockade of IL-10 +/- TGF-β restored the capacity of NK cells from both the periphery and liver of patients with CHB to produce IFN-γ, thereby enhancing their non-cytolytic antiviral capacity. In conclusion, NK cells may be driven to a state of partial functional tolerance by the immunosuppressive cytokine environment in CHB. Their defective capacity to produce the antiviral cytokine IFN-γ persists in patients on antiviral therapy but can be corrected in vitro by IL-10+/- TGF-β blockade.     

Synergy between TLR3 and IL-18 promotes IFN-γ dependent TRAIL expression in human liver NK cells

Natural killer (NK) cells are a component of innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. However, intra-hepatic NK cells’ ability to respond to virus is still mostly unknown. Our results show that the synthetic dsRNA polyinosinic–polycytidylic acid (poly I:C), a mimic of a common product of viral infections, activates NK cells directly in the context of cytokines found in the liver, i.e.: poly I:C plus inflammatory cytokines (IL-18, IL-12, and IL-2) induced NK cell IFN-γ production and TRAIL expression, and anti-inflammatory cytokines (TGF-β and IL-10) inhibit NK cell IFN-γ production. Neutralization of IFN-γ blocks poly I:C plus inflammatory cytokines-induced NK cell TRAIL expression, suggesting that IFN-γ is an autocrine differentiation factor for these cells. A better understanding of the intra-hepatic NK cell activation against viral infection may help in the design of therapies and vaccines for the control of viral hepatitis.     

Roles of leptin and ghrelin in the regulation of the phenotype and cytokine production by NK cells from peripheral blood

Both leptin and ghrelin used separately at the concentrations corresponding to trimesters II–III of pregnancy increase the number of CD56^bright NK cells in mononuclear cell suspension; their combination also enhances the L-selectin expression on the surface of these cells in the culture. These hormones do not affect the production of TGF-β1, IL-17А, or IFN-γ by NK cells, and they inhibit the production of IL-10. Leptin decreses the IL-4 production by NKp46⁺ cells, but the presence of ghrelin abrogates this effect.     

Phagocytosis, a potential mechanism for myeloid-derived suppressor cell regulation of CD8+ T cell function mediated through programmed cell death-1 and programmed cell death-1 ligand interaction

CD8(+) T cells become exhausted, inducing cell surface protein programmed cell death-1 (PD-1) as chronic virus diseases or tumors progress, but underlying mechanisms of this are unclear. We previously showed that M-CSF is important for developing tolerogenic dendritic cells (DCs) from human CD14(+) monocytes. In this article, we identify M-CSF-derived DCs (M-DCs) after stimulation with IL-10 as myeloid-derived suppressor cells with additional tolerogenic activities to CD8(+) T cells. IL-10 increased PD-1 ligand expression on M-DC, and IL-10-stimulated M-DCs (M-DC/IL-10) induced expression of PD-1 on, and apoptosis of, CD8(+) T cells and phagocytosed CD8(+) T cells. Enhanced phagocytic activity of M-DC/IL-10 required IFN-γ, which further increased PD-1 ligand and PD-2 ligand expression on M-DC/IL-10. IFN-γ-stimulated M-DC/IL-10 cells were phenotypically macrophage-like cells with little or no expression of CD86, a costimulatory molecule, but with high expression levels of CD14, CD200R, and CD80. No phagocytic activity was detected with GM-CSF-derived DCs. We propose that phagocytosis by IFN-γ-stimulated M-DC/IL-10 cells, which may be DCs or, alternatively, a unique subset of macrophages, may be a mechanism by which IFN-γ-producing CD8(+) T cells are tolerized after type 1 immune responses to chronic virus or tumor, and that IFN-γ links effector CD8(+) T cells to their phagocytic clearance.     

NK cell tolerance to TLR agonists mediated by regulatory T cells after polymicrobial sepsis

As sensors of infection, innate immune cells are able to recognize pathogen-associated molecular patterns by receptors such as TLRs. NK cells present in many tissues contribute to inflammatory processes, particularly through the production of IFN-γ. They may display a protective role during infection but also a detrimental role during sterile or infectious systemic inflammatory response syndrome. Nevertheless, the exact status of NK cells during bacterial sepsis and their capacity directly to respond to TLR agonists remain unclear. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors. The aim of this study was to gain insight into TLR2/TLR4/TLR9 expression on/in murine NK cells at the protein level and determine if their agonists were able to induce cytokine production. We show, by flow cytometry, a strong intracellular expression of TLR2 and a low of TLR4 in freshly isolated murine spleen NK cells, similar to that of TLR9. In vitro, purified NK cells respond to TLR2, TLR4, and TLR9 agonists, in synergy with activating cytokines (IL-2, IL-15, and/or IL-18), and produce proinflammatory cytokines (IFN-γ and GM-CSF). Finally, we explored the possible tolerance of NK cells to TLR agonists after a polymicrobial sepsis (experimental peritonitis). For the first time, to our knowledge, NK cells are shown to become tolerant in terms of proinflammatory cytokines production after sepsis. We show that this tolerance is associated with a reduction of the CD27(+)CD11b(-) subset in the spleen related to the presence of regulatory T cells and mainly mediated by TGF-β.