NK cells play a critical protective role in host defense against acute extracellular Staphylococcus aureus bacterial infection in the lung

Staphylococcus aureus remains a common cause of nosocomial bacterial infections and are often antibiotic resistant. The role of NK cells and IL-15 and their relationship in host defense against extracellular bacterial pathogens including S. aureus remain unclear. We have undertaken several approaches to address this issue using wild type (WT), IL-15 gene knock-out (KO), and NK cell-depleted mouse models. Upon pulmonary staphylococcal infection WT mice had markedly increased activated NK cells, but not NKT or gammadelta T cells, in the airway lumen that correlated with IL-15 production in the airway and with alveolar macrophages. In vitro exposure to staphylococcal products and/or coculture with lung macrophages directly activated NK cells. In contrast, lung macrophages better phagocytosed S. aureus in the presence of NK cells. In sharp contrast to WT controls, IL-15 KO mice deficient in NK cells were found to be highly susceptible to pulmonary staphylococcal infection despite markedly increased neutrophils and macrophages in the lung. In further support of these findings, WT mice depleted of NK cells were similarly susceptible to staphylococcal infection while they remained fully capable of IL-15 production in the lung at levels similar to those of NK-competent WT hosts. Our study thus identifies a critical role for NK cells in host defense against pulmonary extracellular bacterial infection and suggests that IL-15 is involved in this process via its indispensable effect on NK cells, but not other innate cells. These findings hold implication for the development of therapeutics in treating antibiotic-resistant S. aureus infection.     

Interleukin-10 suppresses natural killer cell but not natural killer T cell activation during bacterial infection

Interleukin (IL)-10 is an anti-inflammatory cytokine known to modulate the outcome of sepsis by decreasing pro-inflammatory cytokine production, including IL-12, a main activator of natural killer (NK) cells. We hypothesized that neutralization of IL-10 would increase NK and natural killer T (NKT) cell activation through increased IL-12 in a mouse model of bacterial peritonitis. NK and NKT cell activations were measured by CD69 expression on NK1.1+/CD3- and NK1.1+/CD3+ cells after cecal ligation and puncture (CLP). NK cells were significantly more activated in mice treated with anti-IL-10 antibodies, whereas no such effect was observed in NKT cells. Similarly, intracellular interferon gamma (IFN-gamma) levels were increased in NK cells of anti-IL-10-treated mice, but not in NKT cells. IL-12 and IL-18 levels were increased in both CLP groups, but in anti-IL-10-treated mice, early IL-12 and late IL-18 levels were significantly higher than in controls. Survival at 18 h after CLP was lower in anti-IL-10 mice, which was associated with increased liver neutrophil accumulation. In summary, these data show an activating effect of IL-10 on NK, but not on NKT cells after CLP, which corresponded with decreased survival, higher IFN-gamma production, and increased remote organ neutrophil accumulation. These effects were not mediated by IL-12 and IL-18 alone, and reinforce a role for NK cells in remote organ dysfunction following peritonitis.     

Interleukin-15 treatment induces weight loss independent of lymphocytes

Obesity is a chronic inflammatory condition characterized by activation and infiltration of proinflammatory immune cells and a dysregulated production of proinflammatory cytokines. While known as a key regulator of immune natural killer (NK) cell function and development, we have recently demonstrated that reduced expression of the cytokine Interleukin-15 (IL-15) is closely linked with increased body weight and adiposity in mice and humans. Previously, we and others have shown that obese individuals have lower circulating levels of IL-15 and NK cells. Lean IL-15 overexpressing (IL-15 tg) mice had an accumulation in adipose NK cells compared to wildtype and NK cell deficient obese IL-15(-/-) mice. Since IL-15 induces weight loss in IL-15(-/-) and diet induced obese mice and has effects on various lymphocytes, the aim of this paper was to determine if lymphocytes, particularly NK cells, play a role in IL-15 mediated weight loss. Acute IL-15 treatment resulted in an increased accumulation of NK, NKT, and CD3(+) T cells in adipose tissue of B6 mice. Mice depleted of NK and NKT cells had similar weight loss comparable to controls treated with IL-15. Finally, IL-15 treatment induces significant weight loss in lymphocyte deficient RAG2(-/-)γc(-/-) mice independent of food intake. Fat pad cross-sections show decreased pad size with cytokine treatment is due to adipocyte shrinkage. These results clearly suggest that IL-15 mediates weight loss independent of lymphocytes.     

Intradermal delivery of IL-12 naked DNA induces systemic NK cell activation and Th1 response in vivo that is independent of endogenous IL-12 production.

In this study four murine IL-12 naked DNA expression plasmids (pIL-12), containing both the p35 and p40 subunits, were shown to induce systemic biological effects in vivo after intradermal injection. Three of the four IL-12 expression vectors augmented NK activity and induced expression of the IFN-gamma and IFN-gamma-inducible Mig genes. Both IL-12 p70 heterodimer and IFN-gamma proteins were documented in the serum within 24 h after intradermal injection of the pIL-12o- plasmid, which also induced the highest level of NK activity in the spleen and liver among the IL-12 constructs. Interestingly, both p40 mRNA expression at the injection site and serum protein levels followed a biphasic pattern of expression, with peaks on days 1 and 5. Subsequent studies revealed that the ability of intradermally injected pIL-12o- to augment NK lytic activity was prevented by administration of a neutralizing anti-IL-12 mAb. Finally, injection of the pIL-12o- into BALB/c IL-12 p40-/- mice also resulted in a biphasic pattern of IL-12 p70 appearance in the serum, and induced IFN-gamma protein and activated NK lytic activity in liver and spleen. These results demonstrate that injection of delivered naked DNA encoding the IL-12 gene mediates the biphasic systemic production of IL-12-inducible genes and augments the cytotoxic function of NK cells in lymphoid and parenchymal organs as a direct result of transgene expression. The results also suggest that these naked DNA plasmids may be useful adjuvants for vaccines against infectious and neoplastic diseases.     

Reversible NK1.1 surface expression on invariant liver natural killer T cells during Listeria monocytogenes infection

The invariant (i) natural killer (NK)T cells consistently express the Valpha14 chain of the T cell receptor (TCR) and recognize alpha-galactosylceramide (alpha-GalCer) presented by the nonpolymorphic presentation molecule CD1d. Despite their name, the iNKT cells represent a heterogeneous population, which can be divided on the basis of NK1.1 surface expression. Here we show that NK1.1 surface expression on liver iNKT cells in mice fluctuates during Listeria monocytogenes infection. At early stages of listeriosis, iNKT cells expressing NK1.1 were numerically reduced and those lacking NK1.1 were increased. At later time points, the NK1.1(-) iNKT cell population contracted, whereas NK1.1(+) iNKT cells reemerged. Alterations in NK1.1 surface expression on iNKT cells were paralleled by numerical changes of interleukin (IL)-12 producers in the liver and were completely prevented by endogenous IL-12 neutralization, whereas NK1.1 surface alterations on iNKT cells following alpha-GalCer stimulation were not prevented. Adoptive cell transfer experiments revealed that the liver NK1.1(-) iNKT cells from NK1.1(+) cell-depleted L. monocytogenes-infected mice accumulated in the liver of recipient recombination-activating gene-1-deficient mice where they acquired NK1.1 surface expression. Thus, we present first evidence that NK1.1 surface expression on liver iNKT cells is reversible during L. monocytogenes infection, and that different mechanisms underlie stimulation by TCR and IL-12.     

Differential roles for IL-15R alpha-chain in NK cell development and Ly-49 induction

IL-15Ralpha-deficient (IL-15Ralpha(-/-)) mice lack NK cells. However, when bone marrow (BM) progenitors from IL-15Ralpha(-/-) mice were cultured with IL-7, stem cell factor and flt3 ligand, followed by IL-15, they were able to differentiate into functional NK cells, indicating that IL-15Ralpha is not critical for NK cell development. Whereas NK cells generated in vitro from IL-15Ralpha(-/-) BM progenitors expressed CD94/NKG2, they failed to express Ly-49 receptors. In keeping with this, when IL-15Ralpha(-/-) BM cells were transferred into wild type recipients, they gave rise to NK cells in vivo, but with greatly reduced expression of Ly-49 receptors. Furthermore, the small numbers of NK cells found in IL-15(-/-) as well as IL-15Ralpha(-/-) but not flt3 ligand(-/-) mice expressed much lower levels of Ly-49 receptors than those from wild type mice. These results indicate a novel role for IL-15Ralpha-chain in Ly-49 induction on developing NK cells.     

Interleukin-15 enhances natural killer cell cytotoxicity in patients with acute myeloid leukemia by upregulating the activating NK cell receptors

Interleukin-15 (IL-15) has a major role in NK-cell homeostasis. Modulation of the relative frequency and expression intensity of the NK-cell receptors by IL-15 may increase NK cell-mediated cytotoxicity in cancer patients. We investigated the receptor repertoire and measured NK-cell activity in newly diagnosed AML patients and evaluated the ex vivo effects of IL-15. The expression of the activating NK cell receptors was significantly decreased in the AML patients compared to that in NK cells of healthy donors. When NK cells obtained from AML patients were cultured with IL-15, expression of the activating receptors was significantly upregulated compared to pre-culture levels. Concomitantly, cytotoxic activity of NK cells against autologous leukemic blasts increased following IL-15 stimulation. This IL-15 induced increase in activity was blocked by neutralizing antibodies specific for the NK cell activating receptors. These pre-clinical data support the future use of IL-15 for NK cell- based therapies for AML patients.     

IL-2 induces expression of serine protease enzymes and genes in natural killer and nonspecific T killer cells

The expression of serine protease genes was examined in murine NK cells that were purified by panning spleen cells with PMA. Although unstimulated NK cells were cytolytic, they were found not to express the C11 (chymotrypsin-like) mRNA. Culturing these cells in IL-2 (500 to 800 U/ml) for 5 to 7 days induced both the lytic activities and the protease enzymes by 20- to 30-fold. Concomitant to these activation events, the total steady state mRNA of both C11 and HF (trypsin-like) genes were also elevated. The activation of lysis, serine protease enzymes, and C11 and HF mRNA all peaked around day 5 in culture and was dose dependent. In order to exclude the possibility that PMA synergizes with IL-2 in this system, spleen cells from SCID mice, which contained mainly NK cells, were cultured under the same conditions (800 U/ml IL-2, with or without PMA) and PMA did not appear to enhance the expression of these mRNA. Similarly, IL-2 also induced the lytic activities, enzyme levels, and mRNA in the non-Ag-specific T killer cells isolated from spleens of normal mice. Lytic activity of T killer cells was not as high as the NK cells, however, the addition of PHA into the lytic assay resulted in enhanced lysis comparable to that of NK cells. These results showed that lytic activity increased along with protease enzyme levels and mRNA expression in both NK and resting T cells. Therefore, elevated levels of the protease enzymes could be one mechanism involved in optimal lytic activity of IL-2-induced lymphokine activated killer cells.     

IFN-gamma production from liver mononuclear cells of mice in burn injury as well as in postburn bacterial infection models and the therapeutic effect of IL-18

Hosts after severe burn injury are known to have a defect in the Th1 immune response and are susceptible to bacterial infections. We herein show that liver NK cells are potent IFN-gamma producers early after burn injury. However, when mice were injected with LPS 24 h after burn injury, IFN-gamma production from liver mononuclear cells (MNC; which we previously showed to be NK cells) was suppressed, and the serum IFN-gamma concentration did not increase, while serum IL-10 conversely increased compared with control mice. Interestingly, a single injection of IL-18 simultaneously with LPS greatly restored the serum IFN-gamma concentration in mice with burn injury and also increased IFN-gamma production from liver MNC. Nevertheless, a single IL-18 injection into mice simultaneously with LPS was no longer effective in the restoration of serum IFN-gamma and IFN-gamma production from the liver MNC at 7 days after burn injury, when mice were considered to be the most immunocompromised. However, IL-18 injections into mice on alternate days beginning 1 day after burn injury strongly up-regulated LPS-induced serum IFN-gamma levels and IFN-gamma production from liver and spleen MNC of mice 7 days after burn injury and down-regulated serum IL-10. Furthermore, similar IL-18 therapy up-regulated serum IFN-gamma levels in mice with experimental bacterial peritonitis 7 days after burn injury and greatly decreased mouse mortality. Thus, IL-18 therapy restores the Th1 response and may decrease the susceptibility to bacterial infection in mice with burn injury.     

Enforced expression of Bcl-2 restores the number of NK cells,but does not rescue the impaired development of NKT cells or intraepithelial lymphocytes,in IL-2/IL-15 receptor beta-chain-deficient mice

IL-2/IL-15Rbeta-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15Rbeta-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15Rbeta-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15Rbeta-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.     

IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells

Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL-10 had no effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 or IL-15.     

Interleukin-15-mediated inflammation promotes non-alcoholic fatty liver disease

Interleukin-15 (IL-15) is essential for the homeostasis of lymphoid cells particularly memory CD8⁺ T cells and NK cells. These cells are abundant in the liver, and are implicated in obesity-associated pathogenic processes. Here we characterized obesity-associated metabolic and cellular changes in the liver of mice lacking IL-15 or IL-15Rα. High fat diet-induced accumulation of lipids was diminished in the livers of mice deficient for IL-15 or IL-15Rα. Expression of enzymes involved in the transport of lipids in the liver showed modest differences. More strikingly, the liver tissues of IL15-KO and IL15Rα-KO mice showed decreased expression of chemokines CCl2, CCL5 and CXCL10 and reduced infiltration of mononuclear cells. In vitro, IL-15 stimulation induced chemokine gene expression in wildtype hepatocytes, but not in IL15Rα-deficient hepatocytes. Our results show that IL-15 is implicated in the high fat diet-induced lipid accumulation and inflammation in the liver, leading to fatty liver disease.     

Sustained expression of Fc-fusion cytokine following in vivo electroporation and mouse strain differences in expression levels

We previously demonstrated that cytokine expression following intramuscular gene transfer of a naked plasmid is increased 2 logs by in vivo electroporation, but the relatively low expression levels of the encoded protein is still a limitation for successful gene therapy and gene function studies. We recently reported that the serum viral IL-10 levels achieved by electroporation-mediated intramuscular delivery of pCAGGS-vIL10, a viral IL-10-expressing plasmid, can be further enhanced by modifying the plasmid into an immunoglobulin fusion protein expression plasmid, pCAGGS-vIL10/Fc. Here we examined the applicability of this approach to the expression of an endogenous cytokine, IL-10, in two different inbred mouse strains. We obtained sustained high serum levels of IL-10 in C3H/HeJ mice (C3H), but the level and duration of the gene expression was mouse-strain dependent. Although the serum IL-10 level was also increased by using the IL-10/Fc gene plasmid in C57BL/6 mice (B6), IL-10/Fc and a luciferase reporter showed significantly lower levels in B6 than in C3H mice, and the persistence of pCAGGS-IL10/Fc expression ranged from several days in B6 mice to more than one month in C3H mice. These results suggest that the electroporation-mediated intramuscular delivery of the immunoglobulin fusion protein expression plasmid is simple and very efficient, but mouse strain differences in transgene expression should be taken into consideration in its use.     

Age-associated augmentation of the synthetic ligand- mediated function of mouse NK1.1 ag(+) T cells: their cytokine production and hepatotoxicity in vivo and in vitro

We recently reported that the direct antitumor effectors in the liver induced by alpha-galactosylceramide (alpha-GalCer) are NK cells that are activated by the IFN-gamma produced from NK1.1 Ag(+) T cells (NKT cells) specifically stimulated with alpha-GalCer, whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand pathway. In the present study, we investigated how mouse age affects the alpha-GalCer-induced effect using young (6-wk-old), middle-aged (30-wk-old), and old (75-wk-old) mice. The serum IFN-gamma and IL-4 concentrations as well as alanine aminotransferase levels after the alpha-GalCer injection increased in an age-dependent manner. An alpha-GalCer injection also induced an age-dependent increase in the Fas ligand expression on liver NKT cells. Under the stimulus of alpha-GalCer in vitro, the liver mononuclear cells from old and middle-aged mice showed vigorous proliferation, remarkable antitumor cytotoxicity, and enhanced production of both IFN-gamma and IL-4 in comparison to those of young mice, all of which were mediated mainly by NK1.1(+) cells. Furthermore, liver mononuclear cells from old mice stimulated with alpha-GalCer showed a more potent Fas-Fas ligand-mediated cytotoxicity against primary cultured hepatocytes than did those from young mice. Most alpha-GalCer-injected old mice, but no young mice, died, while anti-IFN-gamma Ab pretreatment completely inhibited mouse mortality. However, alpha-GalCer-induced hepatic injury did not improve at all by anti-IFN-gamma Ab treatment, and the Fas-ligand expression of liver NKT cells did not change. Taken together, the synthetic ligand-mediated function of NKT cells is age-dependently up-regulated, and the produced IFN-gamma is responsible for alpha-GalCer-induced antitumor immunity and the mouse mortality, while hepatic injury was unexpectedly found to be independent of IFN-gamma.     

Virulizin, a novel immunotherapy agent, activates NK cells through induction of IL-12 expression in macrophages

Virulizin, a novel biological response modifier, has demonstrated significant antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. The significant role of macrophages and NK (Natural killer) cells was implicated in the antitumor mechanism of Virulizin where expansion as well as increased activity of macrophages and NK cells were observed in mice treated with Virulizin. Depletion of macrophages compromised Virulizin-induced NK1.1⁺ cell infiltration into xenografted tumors and was accompanied by reduced antitumor efficacy. In the present study, involvement of macrophages in NK cell activation was investigated further. We found that depletion of NK cells in CD-1 nude mice by anti-ASGM1 antibody significantly compromised the antitumor activity of Virulizin. Cytotoxicity of NK cells isolated from Virulizin-treated mice was enhanced against NK-sensitive YAC-1 cells and C8161 human melanoma cells, but not against NK-insensitive P815 cells. An increased level of IL-12 was observed in the serum of mice treated with Virulizin. IL-12 mRNA and protein levels were also increased in peritoneal macrophages isolated from Virulizin-treated mice. Moreover, Virulizin-induced cytotoxic activity of NK cells isolated from the spleen was abolished when an IL-12 neutralizing antibody was co-administered. In addition, depletion of macrophages in mice significantly impaired Virulizin-induced NK cell cytotoxicty. Taken together, the results suggest that Virulizin induces macrophage IL-12 production, which in turn stimulates NK cell-mediated antitumor activity.     

Augmented IL-15Rα expression by CD40 activation is critical in synergistic CD8 T cell-mediated antitumor activity of anti-CD40 antibody with IL-15 in TRAMP-C2 tumors in mice

IL-15 has potential as an immunotherapeutic agent for cancer treatment because it is a critical factor for the proliferation and activation of NK and CD8(+) T cells. However, monotherapy of patients with malignancy with IL-15 that has been initiated may not be optimal, because of the limited expression of the private receptor, IL-15Rα. We demonstrated greater CD8 T cell-mediated therapeutic efficacy using a combination regimen of murine IL-15 administered with an agonistic anti-CD40 Ab (FGK4.5) that led to increased IL-15Rα expression on dendritic cells (DCs), as well as other cell types, in a syngeneic established TRAMP-C2 tumor model. Seventy to one hundred percent of TRAMP-C2 tumor-bearing wild-type C57BL/6 mice in the combination group manifested sustained remissions, whereas only 0-30% in the anti-CD40-alone group and none in the murine IL-15-alone group became tumor free (p < 0.001). However, the combination regimen showed less efficacy in TRAMP-C2 tumor-bearing IL-15Rα(-/-) mice than in wild-type mice. The combination regimen significantly increased the numbers of TRAMP-C2 tumor-specific SPAS-1/SNC9-H(8) tetramer(+)CD8(+) T cells, which were associated with the protection from tumor development on rechallenge with TRAMP-C2 tumor cells. Using an in vitro cytolytic assay that involved NK cells primed by wild-type or IL-15Rα(-/-) bone marrow-derived DCs, we demonstrated that the expression of IL-15Rα by DCs appeared to be required for optimal IL-15-induced NK priming and killing. These findings support the view that anti-CD40-mediated augmented IL-15Rα expression was critical in IL-15-associated sustained remissions observed in TRAMP-C2 tumor-bearing mice receiving combination therapy.