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Immunity. Author manuscript; available in PMC Mar 17, 2009.
Published in final edited form as:
PMCID: PMC2656759
EMSID: UKMS4208

Post-transcriptional regulation of IL-10 gene expression allows NK cells to express immunoregulatory function

Summary

NK cells play a well-recognised role in early pathogen containment and in shaping acquired cell-mediated immunity. However, indirect evidence in man and experimental models has suggested that NK cells also play negative regulatory roles during chronic disease. To formally test this hypothesis, we employed a well-defined experimental model of visceral leishmaniasis. Our data demonstrate that NKp46+CD49b+CD3- NK cells are recruited to the spleen and into hepatic granulomas where they inhibit host protective immunity in an IL-10-dependent manner. Although IL-10 mRNA could be detected in activated NK cells 24h after infection, the inhibitory function of NK cells was only acquired later during infection, coincident with increased IL-10 mRNA stability and an enhanced capacity to secrete IL-10 protein. Our data support a growing body of literature that implicate NK cells as negative regulators of cell-mediated immunity and suggest that NK cells, like CD4+ Th1 cells, may acquire immunoregulatory functions as a consequence of extensive activation.

Introduction

Natural killer (NK) cells are known to provide a critical host defence system during the early phases of infection with a variety of viruses, fungi, bacteria and parasites (Bancroft, 1993; Biron et al., 1999; Cerwenka and Lanier, 2001; Farag et al., 2002; Smyth et al., 2002; Trinchieri, 1989). NK cells do not possess conventional clonotypic antigen-specific receptors but are capable of spontaneously killing tumour and virus-infected cells that have down-regulated one or more MHC molecules and/or expressed certain stress antigens on their surface (Diefenbach and Raulet, 2003; Mehrotra et al., 1998).. NK cells have also been shown to play an important role during pregnancy (Moffett-King, 2002), autoimmunity and tissue inflammation (Homann et al., 2002; Shi et al., 2000).

Target recognition and cytokine stimulation are the two major triggering mechanisms for NK cells and both shape their effector responses (Alli and Khar, 2004; Chakir et al., 2001; Fehniger et al., 1999; Lauwerys et al., 2000; Mehrotra et al., 1998). Considerable advances have been made in understanding the receptors that activate and inhibit functionally mature NK cells and in the cytokines leading to IFNγ production and cytoloytic activity. However, less is known about the regulation of other aspects of NK cell differentiation. Recent in vitro studies have suggested that NK cells can also differentiate to produce IL-10 (Bodas et al., 2006; Grant et al., 2008; Moretta et al., 2002) and may have regulatory activity (Deniz et al., 2008). Although a negative regulatory role for NK cells mediated through IL-10 has been suggested during tumour development, in pregnancy and most recently during persistent HCV infection (Barber et al., 2007; De Maria et al., 2007; Vigano et al., 2001), direct experimental evidence for a negative regulatory role of NK cells during infectious disease is lacking.

We have been investigating the underlying cellular events that regulate the effectiveness of the host protective immunity following infection with the protozoan parasite Leishmania donovani. Resistance to this pathogen operates largely through the development of granulomatous inflammation, positively regulated by both Th1 and Th2 cytokines (Stager et al., 2003) and negatively regulated largely by IL-10 (Murray et al., 2002). Although natural T regs have received much attention in models of cutaneous leishmaniasis caused by L. major (Belkaid et al., 2002), in experimental visceral leishmaniasis (VL) caused by L. donovani (Stager et al., 2006), chronic cutaneous leishmaniasis caused by L. major Seidman (Anderson et al., 2007) and also during human kala azar (Nylen et al., 2007), the bulk of IL-10 producing CD4+ T cells are inducible FoxP3- and CD25- regulatory T cells. A large fraction of these IL-10+ CD4+ T cells also co-express IFNγ, suggesting that they may, as in chronic toxoplasmosis (Jankovic et al., 2007) represent a further stage of Th1 cell differentiation (O’Garra and Vieira, 2007). Here, we show that following L. donovani infection, NK cells accumulate in the spleen and in hepatic granulomas of infected mice and that these NK cells represent another source of IL-10 during infection. Using adoptive transfer, we now provide formal evidence that NK cells from infected mice suppress host resistance in an IL-10-dependent manner. Although up-regulation of IL-10 gene expression was also found to be a feature of early NK cell activation, only NK cells isolated from mice with established infection could suppress host resistance, a function associated with the acquisition of increased IL-10 mRNA stability and an enhancement of IL-10 protein secretion. Thus, our data suggests a mechanism whereby prolonged in vivo activation gives rise to a population of NK cells with altered post-transcriptional regulation of IL-10 gene expression, heightened capacity for IL-10 protein production and inhibitory function.

Results

NK cells accumulate IL-10 mRNA during the development of experimental VL

IL-10 is a major contributor to disease progression in many diseases (Murphy et al., 2001; Murray et al., 2002; Sharma et al., 1999), and IL-10 production in experimental leishmaniasis has been attributed to CD4+ T cells and macrophages (Anderson et al., 2007; Miles et al., 2005; Stager et al., 2006). As part of a more complete analysis of the IL-10 response to L. donovani infection, we examined IL-10 mRNA accumulation in a variety of highly enriched (>98% pure) cell populations in the absence of in vitro re-stimulation. In accord with our recent description of the late emergence of IL-10-producing CD25-Foxp3- CD4+T cells (Stager et al., 2006), the accumulation of IL-10 mRNA within the splenic CD4+ T cell population increased over time. However, we were surprised to note that CD49b+ (DX5+) NK cells, and not CD4+ or CD8+ T cells, B cells, CD11chi cells or macrophages, had accumulated most IL-10 mRNA by day 14 post infection (p.i.) and that their mRNA accumulation equalled that of CD4+ T cells at day 28 p.i. (Figure 1A). Thus, within the splenic populations examined here, NK cells represented a previously unrecognised potential source of IL-10 during this infection.

Figure 1
Cellular accumulation of IL-10 mRNA during L. donovani infection

NK cells expand in number and enter hepatic granulomas

During L. donovani infection, the spleen undergoes extensive architectural remodelling associated with splenomegaly, both characteristics also associated with human disease (Kaye et al., 2004). However neither the number of NK cells nor their distribution has previously been reported. The latter has been problematic due to the lack of appropriate means to unambiguously identify NK cells in tissue sections in BALB/c mice. NKp46 has recently been described as a marker for NK cells in the mouse (Walzer et al., 2007) and is suitable for in situ detection of NK cells using immunohistochemistry. First, we confirmed expression of NKp46 on CD49b+ splenic NK cells in naïve BALB/c mice. As shown in Figure 1B (upper panels), >95% of CD3-CD49b+ cells stained brightly for NKp46. Using NKp46, we therefore determined the distribution of NK cells in the spleen of naïve mice (Figure 2B). As suggested by previous studies (Andrews et al., 2001; Dokun et al., 2001; Walzer et al., 2007), NKp46+ cells were located predominantly within the marginal zone and red pulp. As with CD49b+ cells found in naïve mice, almost all CD49b+ NK cells in L. donovani-infected mice co-expressed NKp46 (Figure 2A, lower panels). In addition, of CD3-CD49b+CD122+ cells, 95.5±1.03% expressed NKp46 (compared to 97.4±1.38% in naïve mice; Supplementary S1). In infected mice, a subset of CD49b+ cells also expressed CD11c at low level, potentially allowing their mis-classification as immature cDC or plasmacytoid DC (Blasius et al., 2007; Caminschi et al., 2007; Vosshenrich et al., 2007)}. However, the CD11cloCD49b+ cells observed in infected mice were uniformly MHCII- by flow cytometry, lacked intracellular MHCII by immunocytochemistry, did not express the costimulatory markers CD80 and CD86, and did not express either Gr-1 or B220 (Supplementary Data: Figures S2-S4 and data not shown). Staining with NKp46 therefore allowed us to unambiguously define NK cells in the spleen of mice infected with L. donovani (Figure 1D). NK cells were located within the red pulp and marginal zone, in a similar distribution to NK cells in naïve mice.

Figure 2
Splenic and Hepatic IL-10+ NK cells expand in number during experimental visceral leishmaniasis

In the liver of infected mice, immunity to L. donovani is expressed in granulomas (Engwerda and Kaye, 2000). To determine whether NK cells contributed to the cellular composition of hepatic granulomas, we first confirmed NKp46 expression on hepatic CD49b+CD3- cells in both naïve and infected mice (Figure 1E). Next, we performed immunohistochemistry using NKp46. The response of BALB/c mice has been well characterised previously and at 14-28d p.i., granulomas can be seen in a variety of stages of maturation (Stager et al., 2003). NKp46+CD3- cells were readily identified within immature and mature granulomas and also within the parenchyma (Figure 1F-H). By thin section analysis, ~75% of all detectable hepatic NK cells were within granulomas and NK cells were observed in ~60% of the granulomas examined. Given the relatively low frequency of NK cells per granuloma, we cannot rule out that all granulomas might contain NK cells if examined across their entire volume.

To determine whether splenic and hepatic NK cells increased in number during infection, we quantified NK cells by flow cytometry. As shown in Figure 2A, in mice infected with L. donovani, the absolute number of splenic NK cells increased by 3-fold. A similar, though less pronounced increase in hepatic NK cells was also observed (Figure 2B). To confirm that kinetics of acquisition of IL-10mRNA by hepatic CD49b+NKp46+ NK cells in relation to CD4+ and CD8+ T cells, we again performed qRT-PCR on flow sorted cells at different times post infection. As shown in Figure 2C and D, both splenic and hepatic NK cells with high levels of IL-10 mRNA accumulation were readily detected by day 7 post infection. Hepatic CD4+ T cells with elevated levels of IL-10 mRNA appeared more rapidly than in spleen, whereas the kinetics of the IL-10 response in CD8+ T cells was similar in both organs. Together, these data demonstrate that during the course of L. donovani infection, NK cells are one of the first populations to increase their potential for IL-10 production, that their numbers increase in infected tissues, and that they can home specifically into both the infected spleen and into sites of granulomatous inflammation.

IL-10-producing NK cells suppress hepatic resistance to L. donovani

Next, we wished to determine whether NK cells had the capacity to regulate the outcome of infection, as might be suggested by their increased accumulation of IL-10 mRNA. We first depleted NK cells in vivo using anti-ASGM1 antibodies. Treatment of mice with anti-ASGM1 has been commonly used to deplete NK cells, though this antibody has known reactivity against other cells as well (Slifka et al., 2000). . Antibodies were administered over the first 6 days of infection (see Methods) and led to a rapid decrease in the frequency of CD49b+ cells (from 2.27% in control-treated mice to 0.04% in mice treated for 24h with anti-ASGM1) which was sustained over the first 7 days post infection. Treatment with anti-ASGMI significantly reduced parasite burden in the spleen (to 55±8.5% of control; p<<0.001,) and liver (to 48±9% of control; p< 0.001) of infected mice, as measured at day 28 post infection. Whereas ASGM1 stained <2% of CD4+ and <5% of CD8+ spleen cells in naïve BALB/c mice, ASGM1 expression was markedly increased on T cells in infected mice (~15% and ~32% of CD4+ and CD8+ T cells, respectively at 21d p.i; data not shown), making this approach unsuitable for analysing the role of NK cells at later stages of infection, To circumvent this problem, we used an adoptive transfer approach to test whether NK cells could influence the outcome of infection in otherwise unmanipulated hosts. Recipient BALB/c mice were infected with L. donovani and at 21d p.i. they were adoptively transferred with 106 NK cells derived from either naïve or d21-infected mice. Parasite burden was then evaluated 7 days later (28d p.i.). At this time, hepatic resistance was beginning to be expressed, whereas parasite numbers were increasing in the spleen (Engwerda and Kaye, 2000). Transfer of NK cells isolated from the spleen of naïve mice into animals that had been previously infected for 21 days had no effect on splenic (Figure 3A) or hepatic (Figure 3B) parasite burden. In contrast, adoptive transfer of splenic NK cells from 21d-infected mice into 21d-infected recipients significantly suppressed host resistance in both organs. In four independent experiments (n=13-18 mice), NK cells from d21 infected mice increased parasite burden by 2.47±0.17 and 1.86±0.16 fold in spleen and liver respectively (p<0.001), whereas NK cells from naïve mice increased parasite burden by 1.1±0.16 and 1.25±0.02 fold in spleen and liver respectively (p=ns). These data indicated that NK cells from infected but not naïve mice could inhibit host resistance to L. donovani. To confirm the fate of the adoptively transferred NK cells, we CFSE-labelled NK cells from naïve and infected mice prior to transfer, to allow visualisation by immunohistochemistry and flow cytometry. Transferred NK cells could be detected in the spleen (Figure 3C and D), with a localisation similar to that seen for endogenous NK cells (Figure 1D). By flow cytometry, the number of recovered NK cells was similar at 18h and 7 days post transfer (Figure 3E). These data suggest that the differences in suppressive function of NK cells from naïve and infected mice were not due to differential survival or homing. Using this approach, we also confirmed that NK cells directly home into hepatic granulomas (Figure 3F and G). As with endogenous NK cells, transferred NK cells were observed in most, but not all granulomas (Figure 3F).

Figure 3
Adoptive transfer of NK cells demonstrates their capacity to inhibit host resistance

As ~50% of NK cells in infected BALB/c mice expressed CD11c (Supplementary Data; Figure S2), we wished to determine if this inhibitory function might be attributable to this emergent CD11clo population of NK cells. However, as shown in Figure 3H and I, sorted CD11- and CD11clo subsets of CD49b+ NK cells equally inhibited host resistance (by 3.05±0.5 and 3.41±0.56 fold in the spleen and 2.59±0.25 and 3.22±0.24 fold in the liver for CD11c- and CD11lo NK cells respectively; n=5 and p<0.01 in all cases vs. no transfer or transfer of naïve NK cells). In spite of this inhibitory activity, both CD11- and CD11clo subsets also exhibited enhanced cytotoxic effector function compared to NK cells from naïve mice (Supplementary Data; Figure S5). By these assays, diverse functions of NK cells did not, therefore, segregate on the basis of CD11c expression.

Although providing the first functional evidence that NK cells can suppress host resistance, they did not directly address whether this is mediated via IL-10. To address this question, we developed a mixed chimera approach, which allowed both progression of L. donovani infection and also the activation of both IL-10-sufficient and IL-10-deficient NK cells within the same infected host. We first generated mixed chimeras on the BALB background, transferring bone marrow derived from Thy1.2+ BALB.IL-10-/- and Thy1.1+ BALB.IL.10+/+ into Thy1.1+ BALB.IL.10+/+ recipients. However, due to limited expression of Thy1 on NK cells (Dunn and North, 1991; Rahal et al., 1991), it was not possible to recover sufficient IL-10-/- and IL-10+/+ NK cells for subsequent adoptive transfer.

We therefore used CD45.1+ B6.IL-10+/+ and CD45.2+ B10.IL-10-/- mice as an alternate genetic system. As in BALB/c mice, the number of CD3-NKp46+ NK cells in the spleen of infected B6 mice also increased during infection (from 2.7±0.2 to 3.9±1.5) and these NK cells had increased accumulation of IL-10 mRNA compared to naïve mice (8.4±0.3 fold). However, the magnitude of the NK response in B6 mice, as measured by both these parameters, was clearly more muted than in BALB/c mice (Figures (Figures11 and and2).2). Nevertheless, we generated (CD45.1+ B6.IL-10+/+ + CD45.2+ B10.IL-10-/-)→CD45.1 B6.IL-10+/+ irradiation chimeras and after 8 weeks of reconstitution, these mice were infected with L. donovani (Figure 4A). At 28d p.i., IL-10-sufficient (CD49b+CD45.1+) and IL-10-deficient (CD49b+ CD45.2+) NK cells (Figure 4B) were present in similar ratios. Sorted (>98%purity) NK cells were then transferred into 21d-infected.CD45.1+ B6.IL-10+/+ recipient mice. Whereas IL-10-sufficent NK cells inhibited splenic (Figure 4C) and hepatic (Figure 4D) resistance to L. donovani, the transfer of IL-10-/- NK cells had no effect compared to mice receiving no cell transfer. In two independent experiments, IL-10+/+ NK cells increased parasite burden by 2.58±0.41 and 2.2±0.29 fold in spleen and liver respectively (n=10, p<0.01) whereas IL-10-/- NK cells increased parasite burden by only 1.3±0.22 and 1.12±0.01 fold in spleen and liver respectively (n=10, ns). Even given the caveat that the NK cell response in B6 mice was less dramatic than that seen in BALB/c mice, these data provide a formal demonstration that the ability to produce IL-10 is essential for NK cells to suppress host resistance in vivo.

Figure 4
Adoptive transfer of NK cells demonstrates their IL-10-dependent capacity to inhibit host resistance

Acquisition of an IL-10-secreting phenotype requires sustained NK cell activation

NK cells are activated within the first few days of Leishmania infection and to secrete IFNγ (Martin-Fontecha et al., 2004; Schleicher et al., 2007), but their ability to produce IL-10 early after infection has not been assessed. To further explore the kinetics of the IL-10 response, CD49b+ NK cells were sorted from naïve BALB/c mice and BALB/c mice infected for either 24h or for 21d with L. donovani. Unexpectedly, RT-PCR analysis indicated similar accumulation of IL-10 mRNA at both time points (Figure 5A). We then examined the capacity of NK cells isolated from 24h- and 21d-infected mice to inhibit host resistance. In contrast to NK cells isolated at 21d p.i, NK cells isolated at 24h p.i had no suppressive effect (Figure 5B). Thus, from 3 independent experiments (n=10-14), NK cells from d21 and 24h infected mice increased parasite burden by 2.47 ± 0.17 vs. 1.01 ± 0.13 fold in the spleen (P<0.001) and 2.16 ± 0.16 vs.1.05 ± 0.15 fold in the liver (P<0.001). These data suggested that IL-10 mRNA accumulation could not reliably indicate the inhibitory function of NK cells in this assay. We therefore determined whether both populations of NK cells were similarly capable of secreting IL-10, as well as IFNγ (Figure 5C-E). NK cells were cultured in vitro in the presence or absence of rIL-12, as this cytokine has also been shown to induce both IL-10 and IFNγ from NK cells (Mehrotra et al., 1998). NK cells from naïve mice were readily activated in a dose-dependent manner by rIL-12 to produce IFNγ (Figure 5C). In contrast, only low levels of IL-10 (60-120 pg/ml) were produced at the highest dose of rIL-12 tested (5ng/ml). NK cells from mice 21d-infected mice produced high levels of IL-10 (500-1200pg/ml), with even low doses of rIL-12 tested (Figure 5E). IL-10 secretion did not affect the production of IFNγ, which was produced in similar concentrations and with similar kinetics to that found in cultures of NK cells from naïve mice. In contrast, NK cells isolated from mice 24h p.i. had an enhanced IFNγ response, yet produced minimal quantities of IL-10, requiring high doses of rIL-12 to elicit a response (Figure 5D). These data suggest that NK cells acquired the capacity to secrete high levels of IL-10 as infection progressed.

Figure 5
Post-transcriptional regulation of IL-10 gene expression and increased IL-10 secretion by NK cells from L. donovani-infected mice

Post-transcriptional regulation of IL-10 gene expression has been shown to control IL-10 secretion in a variety of cell types, including macrophages and keratinocytes (Powell et al., 2000). To explore whether this mechanism might account for the disparity between IL-10 mRNA accumulation and IL-10 protein secretion noted above, we used actinomycin D to block transcription and examine IL-10 mRNA stability. In NK cells isolated at 24h p.i, IL-10 mRNA decayed by almost 95% within 60 minutes of addition of actinomycin D (Figure 5F), similar to a recent report using cultured NK cells (Grant et al., 2008). In contrast, NK cells isolated at 21d p.i. had retained ~60% of their initial IL-10 mRNA over a similar time period, representing an approximate doubling in relative mRNA half life (~75 min vs. ~30 min in NK cells from 21d- vs. 24h-infected mice, respectively). These data suggest that altered mRNA stability contributes to the increased capacity of NK cells from 21d-infected mice to secrete IL-10. Together, our data indicate that over time, NK cells acquire the capacity to suppress hepatic resistance to L. donovani by a mechanism that involves post-transcriptional regulation of IL-10 gene expression leading to elevated IL-10 protein production.

Discussion

NK cells play a vital role in early pathogen containment and by virtue of their capacity to produce IFNγ, they are widely regarded as beneficial for host protection against intracellular pathogens. In this report, we demonstrate that NK cells have the capacity to act in the opposing manner, inhibiting host resistance to an intracellular pathogen through their production of IL-10.

Reports implicating NK cells as negative regulators of immunity are infrequent. Studies in IFNγ -/- mice have suggested that pulmonary NK cells contribute to the immunosuppressive environment in the lung after mycoplasma infection, and depletion of NK cells in IFNγ-deficient animals prior to infection reduced IL-10 levels in BAL fluid (Woolard et al., 2005). NK cells can inhibit CD4+ T cell IFNγ production after murine cytomegalovirus infection (Su et al., 2001), and have been described to inhibit autoimmunity (Baxter and Smyth, 2002; Flodstrom et al., 2002; Horwitz et al., 1997), with a correlation between NK cell number and / or activity and periods of disease progression or remission being observed in multiple sclerosis and Systemic Lupus Erythematosus (reviewed in (French and Yokoyama, 2004). Additionally, prior depletion of NK cells aggravates peptide-induced experimental allergic encephalomyelitis (Zhang et al., 1997). Uterine NK cells at the foetal/maternal interface are also thought to be essential at mediating tolerance to the fetus (Moffett-King, 2002). Nevertheless, NK cells are almost universally regarded as promoting anti-microbial immunity. Our finding that NK cells can inhibit host protection by production of IL-10 now dispels the notion that NK cells always operate to facilitate pathogen clearance.

IL-10 is well known as a critical regulator of immunity to Leishmania infection. IL-10-/- mice are highly resistant to L. donovani infection (Murphy et al., 2001; Murray et al., 2002), and IL-10 is essential for L. major persistence (Belkaid et al., 2002). Three sources of IL-10 have been examined in great detail. Mosser and colleagues have highlighted the capacity of immune complexes to induce IL-10 production from macrophages (Miles et al., 2005) and subsequently a number of studies have indirectly shown a role for Ig in the establishment and maintenance of disease (Woelbing et al., 2006). Belkaid et. al.,established a critical role for IL-10 producing CD25+Foxp3+ natural Tregs in L. major infection (Treg; (Belkaid et al., 2002)). These cells act as a rheostat for the emerging CD4+ Th1 response (Belkaid et al., 2002), and are vital for long term memory to re-infection (Belkaid et al., 2002). Finally, CD25-Foxp3- IL-10-producing CD4+ T cells expand during both L. donovani (Stager et al., 2006) and L. major Seidman infection (Anderson et al., 2007). IL-10-producing CD4+ T cells appear in significant numbers after 21 d of L. donovani infection in BALB/c mice, regulated by IL-6 produced by DC (Stager et al., 2006). CD25-Foxp3- CD4+ T cells have also been identified as a source of IL-10 in humans infected with L. donovani (Nylen et al., 2007). Here, we demonstrate that NK cells isolated from L. donovani-infected mice also produce IL-10 and confirm their ability to regulate disease outcome in vivo. Our data indicate that the well-defined early role for NK cells in enhancing Th1 differentiation (Cerwenka and Lanier, 2001) is not the full extent of NK cell function, and other immunoregulatory functions are acquired as infection progresses..

To date, our analysis of mice after NK cell transfer has not revealed any significant alterations in the number of CD4+ or CD8+ T cells nor in the number or activation status of the major cDC.subsets (data not shown). The ability to observe transferred NK cells using 2-photon intravital microscopy (Beattie et.al., unpublished) and the availability of mice with cell-specific deficiency of IL-10R expression (Jack, R. and Muller, W., unpublished; Aidinis et al., 2008) should help elucidate their mechansism of action.

Although Nylen and colleagues did not identify NK cells as a major source of IL-10 mRNA in human disease, NK cells account for only ~3% of cells in splenic aspirate and IL-10 secretion was not directly examined. Furthermore, the role of NK cells may well become overshadowed by that of CD4+ T cells as disease progresses, as indeed is suggested by our data. Given that the presence of granulomatous inflammation has been associated with sub-clinical infection with L. donovani and that in the mouse model of VL, disease is less progressive than in man (Kaye et al., 2004), our data demonstrating the modulation of parasite burden by NK cells suggests that IL-10-producing NK cells might have a more dominant role at early stages of human VL, perhaps contributing to the immunoregulatory balance that governs the transition from sub-clinical infection to clinical disease. Nevertheless, NK cell numbers do increase during fatal experimental visceral leishmaniasis in the hamster, a model often regarded as better reflecting end stage human disease (Sartori et al., 1999).

Our data demonstrate that the capacity of NK cells to produce IL-10 protein is acquired over the course of infection, whilst the capacity to produce IFNγ remains largely unaltered. The precise molecular mechanism(s) involved remain to be defined, though our data points to enhanced IL-10 mRNA stability as a contributing factor. Although post-transcriptional regulation of cytokine genes in NK cells has not been described, a precedent for the control of IL-10 secretion by such a mechanism is found in macrophages, where IL-10 mRNA stability has been shown to be associated with regulatory elements in the 3′ UTR (Powell et al., 2000). Further studies will be required to determine whether analogous mechanisms exist in murine NK cells. Studies carried out by Nutt et. al. demonstrated that stimulating NK cells with IL-2 and IL-21 can induce IL-10 production (Brady et al., 2004)., we show here that IL-12 can induce NK cells from infected mice to secrete IL-10, and human NK cells respond to IL-2 and IL-12 with IL-10 secretion (Moretta et al., 2002). Furthermore, recent studies indicate that in cultured murine NK cells, these cytokines act synergistically to drive Stat4-dependent but T-bet independent induction of IL-10 mRNA (Grant et al., 2008). Thus, it is tempting to speculate that IL-2 and IL-12 may also play a role in the development and / or maintenance of IL-10-producing NK cells during experimental leishmaniasis. In support of such an association, we have previously shown that the frequency of IL-12p40+ cells in hepatic granulomas increases significantly between day 14 and day 28 p.i. (Engwerda et al., 1998), mirroring the kinetics of emergence of IL-10-producing NK cells described here. IL-2 has also been linked to the generation of IL-10 during L. donovani infection (Bodas et al., 2006). Whether these cytokines, or other environmental factors, are ultimately responsible for modulating NK cell function remains to be established, however.

Finally, our data suggest striking, if not mechanistic, parallels in the functional differentiation of NK cells and CD4+ Th1 cells. Recent studies (Jankovic et al., 2007) have identified conventional IFNγ-producing Th1 cells as a major source of IL-10 following Toxoplaasm gondii infection, proposing that expression of IL-10 helps to suppress APC function as a regulatory mechanism to limit immunopathology. Our data suggest that NK cells, under the influence of an evolving inflammatory response, likewise further differentiate from a population characterised by cytolytic effector activity and IFN production, into one which also the ability to secrete IL-10. Identifying the factors responsible for promoting this switch in cytokine profile may open new avenues for therapeutic regulation of NK cell activity in a variety of chronic infectious and non-infectious inflammatory states.

Materials and Methods

Mice and infection

BALB/c and C57BL6 mice were obtained from Charles River (UK), B10.IL-10-/- mice were originally developed by DNAX and were obtained from Dr A. Sher (NIAID, NIH), and BALB.Thy1.1 and BALB.IL-10-/- (Thy1.2) and hCD2-GFP (de Boer et al., 2003) mice were the kind gift of Drs J. Langhorne, A. O’Garra and D. Kioussis, respectively (NIMR, London). Mice were housed under specific pathogen free conditions and used at 6-8 weeks of age. The Ethiopian strain of Leishmania donovani (LV9) was maintained by serial passage in Syrian hamsters. Amastigotes were isolated from infected spleens, as previously described (Stager et al., 2000) and mice were infected with 2 × 107 L. donovani amastigotes intravenously via the tail vein in 200 μl of RPMI 1640 (Gibco, Paisley, UK). Mice were sacrificed by cervical dislocation, and parasite burden in the liver and the spleen were determined from Giemsa stained impression smears (Engwerda et al., 1998). Parasite burden was expressed as Leishman Donovan Units (LDU; i.e. the number of parasites per 1000 host cell nuclei x organ weight) (Smelt et al., 1997). All experiments were approved by the LSHTM and University of York Animal Procedures and Ethics Committee and performed under U.K. Home Office license.

For the generation of chimeras, donor mice were irradiated twice with 1100 rads using a split-dose regimen (550rad on d1 and d2) and then were reconstituted i.v. via the tail vein on d2 with 2×106 T cell-depleted bone marrow cells per mouse, comprising a 1:1 ratio of IL-10+/+ and IL-10 -/- cells (see text for details). At 6-8 weeks after reconstitution, mice were examined for chimerism by flow cytometry and infected as described above. For adoptive transfer, sorted splenic NK cell subsets from either naïve or infected mice were injected (1 × 106/mouse) i.v. into 21d-infected mice and 1 week later splenic and hepatic parasite loads were determined.

Splenic DC and NK Enrichment

Splenic NK cells from naïve and infected mice were enriched by digesting the spleens in RPMI supplemented with 0.1 mg/ml collagenase for 25 minutes at RT. B220+, CD19+, CD3ε+ and highly phagocytic cells were separated using magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and a MidiMACS separation column (Miltenyi Biotec). CD3-CD49b+ NK cells were then purified using either a FACS Vantage (BD Biosciences, Mountain View, CA) or a MoFlo (Dakocytomation) cell sorter. Sort gates are described in the Results. Sorted cells exceeded >98% purity.

Flow cytometry

For flow cytometry, cells were incubated with 10 μg/ml 2.4G2 anti-FcRγII/III mAb (ATCC, Rockville, MD) followed by staining with directly-conjugated monoclonal antibodies. Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-MHCII (2G9), anti-CD3 (145-2C11) antibodies; phycoerythrin (PE)-conjugated anti-CD45RB (16A), anti-CD44 (IM7), anti-Ly49b (DX5), anti-NK 1.1 (PK136) anti-Gr-1 (RB6-8C5) antibodies; biotinylated anti-CD11c (HL3), anti-MHC-II (2G9), CD4 (RM4-5), CD8 (53-6.7), CD80 (16.10A1), CD86 (GL1), CD19 (1D3), B220/CD45R (RA3-6B2) CD3e (145-2C11) antibodies and APC-conjugated CD11c (clone HL3) antibody (all from BD Pharmingen, San Diego, CA). Biotinylated antibodies were visualized with PerCP-streptavidin (BD Pharmingen). NKp46 expression was detected by using a polyclonal antibody against mouse NKp46 (R&D systems). Secondary staining was carried out with a donkey anti-goat IgG alexa 488 antibody (Invitrogen). Some samples were also stained with rabit anti-ASGM1 (Cedarlane, Ontario, Canada) followed by anti-rabbit alexa 647 (molecular probes). Minimal background staining was observed using appropriate control FITC-conjugated, PE-conjugated, biotinylated and APC antibodies. All staining was performed on ice in phosphate buffered saline (PBS) containing 2% FCS and 5 mM EDTA for 30min. Flow cytometric analysis was performed with a FACSCalibur (BD Biosciences, Mountain View, CA) or a Cyan (Dakocytomation). 50,000 cells were acquired and analysed using either CellQuest (BD Biosciences) or Summit (Dakocytomation) software. To obtain absolute numbers of cells, samples were spiked with a known concentration of microbeads (Polysciences) prior to acquisition.

In vivo depletion of ASGM1+ Cells

BALB/c mice were injected with 20μl of rabbit polyclonal anti-ASGM1 (Cedarlane) or control polyclonal normal rabbit IgG (Sigma) in a total volume of 200μl/mouse 24h prior to infection and days 2, 4 and 6 post infection. Liver LDU’s were determined 28 days after infection.

Cytotoxicity assay

YAC-1 cells (103/well) were used as targets for NK cell-mediated lysis. Briefly, NK cell subsets isolated from naïve and day 28 infected animals were seeded in 96-well round bottomed plates at different E/T ratios and incubated at 37°C for 4 hrs. Each test sample was plated in triplicate. Cytotoxcity was measured using the LIVE/DEAD Viability/Cytotoxity kit (Invitrogen) according to manufacturer’s instruction and published protocols (Papadopoulos et al., 1994).

Real-time RT-PCR

RNA was isolated from sorted cell subsets with an RNeasy kit, according to manufacturer’s instructions (Qiagen). RNA was reverse transcribed into cDNA using the first strand cDNA synthesis kit according to the manufacturer’s instructions (Invitrogen). Oligonuclotides (5′-3′) used for the specific amplification of IL-10 and HPRT were as described in (Ato et al., 2002). The real-time quantitative PCR was performed with the SYBR Green PCR kit in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) according to manufacturer’s instructions. Expression of IL-10 was normalised to HRPT and expressed as either absolute copy number (target molecules / 1000 HPRT molecules) or relative expression using the change in cycle threshold (ΔΔCT) analysis method (relative expression in infected vs. naïve mice).

Immunofluorescence

For immunofluorescence, cytospun cells were fixed in 4% paraformaldehyde for 30 minutes at room temperature (RT). After fixing, samples were quenched with 50 mM NH4Cl in PBS for 30 minutes at RT, and then blocked and permeablized by incubation with 1.5% v/v normal goat serum in 0.1% v/v saponin in PBS for 45 minutes at RT. Samples were incubated with biotin-conjugated rat anti-MHC-II (clone 2G9, BD Pharmingen) and hamster anti-CD11c (clone N418, Serotec) diluted in 0.1% v/v saponin in PBS and incubated for 45 minutes at RT. After washing specific staining was detected by Alexa 488 conjugated streptavidin and Alexa 546 conjugated goat-anti hamster IgG (Invitrogen). Slides were washed 3x with 0.1% v/v saponin in PBS, then coverslips were mounted in Prolong Gold (Invitrogen) and visualized with a 63x (NA 1.4) Plan-Apochromat oil immersion objective using a Zeiss Axioplan LSM 510 confocal microscope.

Confocal microscopy

Confocal was performed on 8μM thick serial frozen sections. Sections were fixed with ice-cold acetone before staining with anti-CD3 PE, anti-F4/80 biotin and goat anti-mouse NKp46 followed by secondary donkey anti-goat IgG alexa 488 (molecular probes) and streptavidin 546. DAPI was used as a nuclear counterstain. Slides were analysed by confocal microscopy (Zeiss LSM 510).

mRNA stability determination

Purified NK cells isolated from infected BALB/c mice at 24h and 21d p.i. were incubated in the presence of actinomycin D (5μg/ml). The cells were collected for RNA extraction at 60min intervals for 3h following the addition of actinomycin D and data are plotted as % IL-10 mRNA remaining relative to that determined at the addition of actinomycin D.

ELISA

Purified CD3-CD49b+ NK cells were cultured (1×105 cells / well) in various doses of IL-12. Supernatants were harvested at 24h intervals for 4 days. IL-10 and IFNγ levels in supernatants were assayed using a sandwich ELISA kit (BioSource) according to manufacturer’s instruction.

Statistics

Statistical analysis was performed using a paired Student t test or a Mann Whitney U test, as appropriate, with p<0.05 considered significant.

Supplementary Material

Figures S1-S5

Acknowledgements

The authors thank the staff of the LSHTM and University of York Biological Services Facilities for animal husbandry, staff of the Technology Facility for assistance with flow cytometry and imaging, Drs Langhorne, O’Garra and Kioussis for generously providing mice and Dr M. Coles and M. Kullberg for critical review of the manuscript. This work was supported by grants from The Wellcome Trust and the U.K. Medical Research Council.

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