Systemic inflammatory response syndrome is a whole-body reaction to a triggering insult that often results in life-threatening illness. Contributing to the development of this inflammatory cascade are numerous cellular partners, among which NK cells were shown to play a key role. Accumulating evidence points to organ-specific properties of systemic inflammation and NK cells. However, little is known about compartment-specific activation of NK cells during systemic inflammatory response syndrome or the relative contribution of NK cell–intrinsic properties and microenvironmental cues. In this study, we undertook a sequential characterization of NK responses in the spleen, lungs, bone marrow, peritoneum, and blood using a mouse model of endotoxemia. We report that, despite similar systemic dynamics of NK cell responses, expression of activation markers (CD69 and CD25) and effector molecules (IFN-γ, granzyme B, and IL-10) display organ-specific thresholds of maximum activation. Using adoptive transfers of spleen and lung NK cells, we found that these cells have the capacity to quickly adapt to a new environment and adjust their response levels to that of resident NK cells. This functional adaptation occurs without significant alterations in phenotype and independently of subpopulation-specific trafficking. Thus, using a dynamic in vivo–transfer system, to our knowledge our study is the first to report the compartmentalization of NK cells responses during systemic inflammation and to show that NK cell–intrinsic properties and microenvironmental cues are involved in this process, in a sequential manner.

Systemic inflammatory response syndrome, a whole-body reaction to an inflammatory stimulus, is associated with sepsis, severe trauma, or invasive surgery, among other conditions, and represents a life-threatening medical emergency. Although systemic inflammatory response syndrome is defined as a systemic event, it does not affect the entire organism uniformly, but rather in a compartmentalized manner, with organ-specific microenvironments shaping the inflammatory response (1). Experimental evidence suggests that inflammatory mediators, like cytokines and chemokines among others, are distinctively regulated in an organ-specific manner at the transcript and protein levels (24). The organs themselves and their damage responses are differentially affected by specific factors during systemic inflammation (5, 6). In addition, our group (7, 8) showed previously that endotoxin tolerance, a consequence of systemic inflammation, is also compartment specific.

Systemic inflammation involves a large panel of immune and nonimmune cells, as well as a vast network of cell-associated or soluble molecules (9, 10). Within this myriad array of factors, NKs have often been considered responsible for amplifying the inflammatory cascade, especially via secretion of IFN-γ (11, 12). However, NKs are far more than IFN-γ producers. Initially recognized for their tumor-killing role, NKs are now known as cells that are able to secrete a multitude of cytokines and chemokines, kill transformed and hyperactive or senescent immune cells, thus shaping the immune response, and even mediate recall or memory responses (13). NKs also were shown to have organ-specific features in mice and humans (1418). Functional differences among NKs in lungs, spleen, and blood have been pointed out since the 1980s (19, 20). However, recent studies demonstrated that NKs have different organ-specific developmental requirements, phenotypes, functional capacity, and ability to generate memory in compartments like the spleen, lungs, liver, uterus, bone marrow, and peritoneal cavity, among others (14, 2125). Although evidence on the compartment-specific characteristics of NKs is growing, the mechanisms behind these organ-specific functional and phenotypical differences remain elusive. Similarly, an important unanswered question is whether tissue-specific NK responses shape the compartmentalization of inflammation or are the result of it.

We undertook a study to define NK-activation dynamics during endotoxemia in different compartments to tease apart NK-intrinsic and microenvironmental influences. We observed that NK responses vary in a compartment-specific manner not in dynamics of activation but with regard to their maximal activation levels. We also show that, during endotoxemia, NKs do not just secrete IFN-γ, they upregulate granzyme B (GzmB) expression and express IL-10, thus presenting inflammatory and anti-inflammatory properties. Using a series of adoptive transfers, we went on to show that NK compartmentalized responses are dependent on NK origin and the local microenvironment, in a time-dependent manner, suggesting that rapidly mobilized NKs are able to respond in consequence to their potentially new environment, regardless of the organ from which they were mobilized.

All protocols for animal experiments were reviewed and approved by the Comité d’Ethique pour l’Expérimentation Animale - Ethics Committee for Animal Experimentation “Paris Centre et Sud” (approval number 2013-0004) and were performed in accordance with national laws and institutional guidelines for animal care and use.

Wild-type (CD45.2) and CD45.1 mice, male and females of 8–12 wk of age, on a C57BL/6 background were used for the experiments. IL-10–GFP (VertX) mice, as previously described (26), were generously provided by Prof. Claude Leclerc (Institut Pasteur). Wild-type mice were purchased from Janvier (France), and transgenic mice were obtained from the Institut Pasteur animal breeding facility.

Mice were injected i.p. with conventional LPS from Escherichia coli O111:B4 (Sigma-Aldrich) at a dose of 10 mg/kg in saline, in a 200 μl volume. Control mice received saline. Mice were sacrificed at several experimental time points, and organs or biological fluids were harvested immediately. Typically, one LPS-activation dynamic experiment included five to seven mice receiving LPS plus one or two control mice receiving saline for each experimental time point.

Sampling procedures involved the following steps. Blood was collected by retro-orbital bleeding on EDTA. Peritoneal cells were harvested by lavaging the peritoneal cavity with 3 ml of wash buffer (0.5% FCS, 2 mM EDTA in PBS). Spleen, lungs, and bone marrow were harvested subsequently. EDTA blood was spun down, and cell pellets were suspended in wash buffer, layered on top of lymphocyte separation media (Eurobio), and centrifuged at 580 × g for 20 min without brake to remove RBCs. Spleens were passed through a 70-μm cell strainer. Lung samples were processed with a commercial enzymatic digestion kit (Miltenyi Biotec), according to the manufacturer’s instructions, and passed through 100- and 70-μm strainers to remove debris. Bone marrow cells were flushed out of both femurs with 3 ml of wash buffer, disrupted by pipetting, and passed through a 70-μm cell strainer. All obtained cells were homogenized by pipetting, washed twice in wash buffer, suspended in appropriate volume for counting, and kept on ice until further processing.

The following Abs (clones) were used: NK1.1 (PK136), CD3 (145-2C11), CD11b (M1/70), CD27 (LG.3A10), CD69 (H1.2F3), CD25 (PC61), Ly6C (HK1.4), CD11c (HL3), B220 (RA3-6B2), CD45.1 (A20), CD45.2 (104), IFN-γ (XMG1.2), and GzmB (GB11) (purchased from BioLegend, eBioscience, or BD Biosciences).

Single-cell suspensions from all organs were counted and prepared for surface staining in 96-well plates. Unspecific binding was blocked by incubation with anti-mouse CD16/CD32 (BD Biosciences) for 10 min, before the addition of surface-labeling Abs for an additional 20 min, in 0.5% FCS at 4°C. Cells were washed in PBS in preparation for viability staining using fixable viability dye (eFluor 780; eBioscience) for 5 min at 4°C. Cell were washed in wash buffer and fixed using commercial fixation buffer (BioLegend). For intracellular cytokine and GzmB staining, cells were permeabilized and washed with buffers from commercial kits (Inside Stain Kit; Miltenyi Biotec and Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent; eBioscience, respectively). Following permeabilization and wash, cells were stained with respective Abs for 30–45 min at 4°C and, after a final wash, were suspended in wash buffer for analysis. Cell counting and flow cytometry sample acquisition were performed on a MACSQuant device (Miltenyi Biotec). Data analysis was performed using FlowJo software (TreeStar). Unless otherwise indicated, NK parameters were assessed on NK1.1+CD3 cells from a lymphocyte gate among live singlet events.

NKs were isolated from spleens and lungs of CD45.1- or CD45.2-congenic mice using a negative enrichment kit (eBioscience), according to the manufacturer’s instructions. Enriched NKs were checked regularly for viability and purity and were routinely >95 and 85%, respectively. Phenotypes of purified cell preparations reflected organ origin in terms of CD27-CD11b staining. NKs were transferred i.v. (retro-orbital injection) or i.p. to recipient congenic mice (0.75–1 × 106 or 0.05–0.1 × 106 cells/recipient, respectively). Immediately after cell transfer, mice were injected i.p. with LPS (10 mg/kg; Sigma-Aldrich). At 3, 6, 12, or 24 h after LPS injection, single-cell suspensions were obtained from spleen and lungs of recipient mice, and FACS analysis was performed. In the case of VertX mice, NKs were stained with CellVue Lavender dye (eBioscience), according to the manufacturer’s instructions, to allow tracking of transferred cells in recipient VertX mice.

Statistical significance was tested using Prism 5.0 Software (GraphPad). The Mann–Whitney U test was used for single comparisons. Two-way ANOVA with Bonferroni post hoc comparisons was used for multiple comparisons. Unless otherwise specified in figure legends, error bars in all figures represent SEM, with the midlines representing the mean value.

We investigated the behavior of NKs in the different organs during the course of LPS-driven systemic inflammation. As previously reported (14), we found that NKs are present in different proportions in the different organs at homeostasis, with the highest frequencies among lymphocytes observed in the lungs and peritoneal cavity, followed by blood, spleen, and bone marrow, which have varying, but lower, levels of NKs. However, the spleen contains the greatest absolute numbers of NKs (Fig. 1). Although NK percentages and numbers declined steadily in the spleen during the course of endotoxemia, pulmonary NKs showed a transient early increase in NK percentages between 1 and 3 h, followed by a return to basal levels; however, these variations were not mirrored by absolute numbers (Fig. 1B). Percentages of NKs in the bone marrow, peritoneum, and blood compartment had a tendency to decrease during endotoxemia, but with various dynamics in terms of time. However, absolute NK counts were severely reduced in all compartments (Fig. 1C).

FIGURE 1.

NK proportions are compartment specific but are severely depleted during endotoxemia. C57BL/6 mice were injected with LPS (10 mg/kg, i.p.) and sacrificed at 1.5, 3, 6, 12, 24, and 48 h. Single-cell suspensions were prepared from spleen, lungs, bone marrow, peritoneum, and blood and stained for flow cytometric analysis. (A) Gating strategy showing the lymphocyte gate (upper panels) and the NK (NK1.1+CD3) gate (lower panels) in the different organs. (B) Summary of NK percentages throughout the course of endotoxemia in the different organs. (C) Summary of NK absolute numbers throughout the course of endotoxemia in the different organs. Data are representative of one of at least three repeats (n ≥ 5 mice per time point) (A) and the mean of three experimental repeats (B and C).

FIGURE 1.

NK proportions are compartment specific but are severely depleted during endotoxemia. C57BL/6 mice were injected with LPS (10 mg/kg, i.p.) and sacrificed at 1.5, 3, 6, 12, 24, and 48 h. Single-cell suspensions were prepared from spleen, lungs, bone marrow, peritoneum, and blood and stained for flow cytometric analysis. (A) Gating strategy showing the lymphocyte gate (upper panels) and the NK (NK1.1+CD3) gate (lower panels) in the different organs. (B) Summary of NK percentages throughout the course of endotoxemia in the different organs. (C) Summary of NK absolute numbers throughout the course of endotoxemia in the different organs. Data are representative of one of at least three repeats (n ≥ 5 mice per time point) (A) and the mean of three experimental repeats (B and C).

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As described previously (14), we found that, in control mice, the blood and lung compartments are made up almost entirely of mature CD27CD11b+ NKs, whereas the spleen has a better represented immature CD27+ NK subpopulation (Fig. 2A, 2B). However, in the peritoneal cavity and bone marrow, CD27+ cells represent the majority of NKs (Fig. 2B). During the course of endotoxemia, NKs from different compartments maintain their organ-specific phenotypic profiles even though a tendency to upregulate expression of CD11b, particularly in the peritoneum, is seen throughout the observation period (Fig. 2B). The percentage of NKs expressing B220 and CD11c, two alternative phenotypic markers, also varies among compartments at homeostasis: blood NKs and lung NKs (LngNKs) express the lowest levels of CD11c and B220, whereas the spleen, bone marrow, and peritoneal cavity NKs express higher levels (Supplemental Fig. 1A, 1B). These two markers were generally upregulated following LPS injection, with organ-specific dynamics being apparent (Supplemental Fig. 1A, 1B). Ly6C, another phenotypic marker associated with NK maturation, had the highest expression on lung and blood NKs and the lowest expression on bone marrow NKs at homeostasis. Interestingly, the percentage of NKs expressing Ly6C in the different compartments remained fairly constant throughout endotoxemia (Supplemental Fig. 1C). Thus, our data show that NKs decrease in numbers in all studied compartments during LPS-induced systemic inflammation but mostly maintain their tissue-specific phenotypes.

FIGURE 2.

NK phenotypes are compartment specific and relatively stable during endotoxemia. C57BL/6 mice were injected with LPS (10 mg/kg, i.p.) and sacrificed at 1.5, 3, 6, 12, 24, and 48 h. Single-cell suspensions were prepared from spleen, lungs, bone marrow, peritoneum, and blood and stained for flow cytometric analysis. (A) Representative plot of CD27CD11b subpopulation gating strategy, with staining shown in black and isotype staining shown in light gray. (B) Representative overlay graphs for CD11b and CD27 expression on NK1.1+CD3 cells (left panels) and summary bar graphs depicting the proportions of CD27+CD11b, CD27+CD11b+, CD27CD11b+, and CD27CD11b subsets within the NK population in the different organs at the indicated time points (right panels). Black vertical lines in line graphs represent the values of isotype controls. Data are representative of one of at least three repeats (n ≥ 5 mice per time point) for the left panels and the mean of three experimental repeats for the right panels.

FIGURE 2.

NK phenotypes are compartment specific and relatively stable during endotoxemia. C57BL/6 mice were injected with LPS (10 mg/kg, i.p.) and sacrificed at 1.5, 3, 6, 12, 24, and 48 h. Single-cell suspensions were prepared from spleen, lungs, bone marrow, peritoneum, and blood and stained for flow cytometric analysis. (A) Representative plot of CD27CD11b subpopulation gating strategy, with staining shown in black and isotype staining shown in light gray. (B) Representative overlay graphs for CD11b and CD27 expression on NK1.1+CD3 cells (left panels) and summary bar graphs depicting the proportions of CD27+CD11b, CD27+CD11b+, CD27CD11b+, and CD27CD11b subsets within the NK population in the different organs at the indicated time points (right panels). Black vertical lines in line graphs represent the values of isotype controls. Data are representative of one of at least three repeats (n ≥ 5 mice per time point) for the left panels and the mean of three experimental repeats for the right panels.

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To investigate NK activation in the different compartments during endotoxemia, we analyzed surface expression of CD69 and CD25 and expression of intracellular cytokines IFN-γ and GzmB by fluorescent Ab labeling, as well as IL-10 by GFP expression, at different time points after LPS injection. As early as 1.5 h following LPS injection, CD69 is upregulated on NKs to significant levels only on peritoneal, splenic, and bone marrow cells, whereas blood and lung NKs only upregulate CD69 starting at 3 h (Fig. 3A). Although splenic NKs continue to upregulate CD69 expression, peaking at 6–12 h, peritoneal NKs maintained expression levels close to those from 3 h and sustained these elevated levels until 24 h. However, bone marrow NKs seem to reach peak CD69 expression by 3 h, which decreased slightly by 6 h and was maintained through 24 h (Fig. 3A). Lung and blood NKs also upregulated CD69; their expression levels were significantly lower compared with the spleen until 48 h, when NKs from all compartments still show elevated levels of CD69 compared with homeostasis (Fig. 3A, Supplemental Fig. 2A). CD25 expression by NKs started relatively later, becoming obvious at 6 h (Fig. 3B, Supplemental Fig. 2B). Compared with CD69 expression, CD25 upregulation was clearly confined to spleen, peritoneum, and bone marrow NKs, whereas lung and blood NKs showed only marginal CD25 positivity. In all compartments, CD25 peaked at 12–24 h and returned to homeostatic levels by 48 h (Fig. 3B). In terms of cytokine expression, similarly to CD69-upregulation dynamics, splenic NKs showed the highest percentages of IFN-γ positivity, reaching >80% at 6 h (Fig. 3C, Supplemental Fig. 2C). The other compartments followed similar dynamics but at lower frequencies, with the blood and bone marrow compartments having the least IFN-γ expression. GzmB expression at homeostasis differs among NKs from different compartments, with the highest levels observed in the lung (Fig. 3D, Supplemental Fig. 2D). However, during endotoxemia, expression of GzmB increased in NKs from all compartments. Interestingly, levels of GzmB expression remained high even at 48 h after LPS injection (Fig. 3D). As previously reported (27), NKs can also express IL-10 during systemic inflammation. We used IL-10–GFP–expressing VertX mice to monitor expression of this cytokine by NKs during endotoxemia. We found that NKs did not start expressing IL-10–GFP before 24 h. High levels were detected only in the blood and lungs, and low levels were detected in the spleen, bone marrow, and peritoneum (Fig. 3E, Supplemental Fig. 2E). However, the proportion of IL-10–expressing NKs continued to increase between 24 and 48 h in all compartments.

FIGURE 3.

NK activation is compartment specific during endotoxemia. C57BL/6 or VertX mice were injected with LPS (10 mg/kg, i.p.) and sacrificed at 1.5, 3, 6, 12, 24, and 48 h. Single-cell suspensions were prepared from spleen, lungs, bone marrow, peritoneum, and blood and stained for flow cytometric analysis. CD69 (A), CD25 (B), IFN-γ (C), GzmB (D), and IL-10-GFP (E) expression was assessed on NKs (NK1.1+CD3) and are presented as representative graphs at the indicated time points (left panels) or summary of data (right panels). Vertical lines on graphs represent values of isotype controls (A–D) and or of cells from control unstimulated mice (E). Data are representative of one of at least three repeats (n ≥ 5 mice per time point) for the left panels and the mean of three experimental repeats for the right panels. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, p values of interaction determined by two-way ANOVA between the respective groups.

FIGURE 3.

NK activation is compartment specific during endotoxemia. C57BL/6 or VertX mice were injected with LPS (10 mg/kg, i.p.) and sacrificed at 1.5, 3, 6, 12, 24, and 48 h. Single-cell suspensions were prepared from spleen, lungs, bone marrow, peritoneum, and blood and stained for flow cytometric analysis. CD69 (A), CD25 (B), IFN-γ (C), GzmB (D), and IL-10-GFP (E) expression was assessed on NKs (NK1.1+CD3) and are presented as representative graphs at the indicated time points (left panels) or summary of data (right panels). Vertical lines on graphs represent values of isotype controls (A–D) and or of cells from control unstimulated mice (E). Data are representative of one of at least three repeats (n ≥ 5 mice per time point) for the left panels and the mean of three experimental repeats for the right panels. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, p values of interaction determined by two-way ANOVA between the respective groups.

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We performed adoptive-transfer experiments to investigate whether the differences observed in NK activation between one compartment and another are NK intrinsic or are dependent on the local microenvironment. NKs from CD45.1 mice were enriched from either spleen or lung and transferred i.v. to CD45.2-congenic mice. LPS was administered immediately after cell transfer, and NK responses in transferred versus local host NKs in spleen and lungs were assessed by flow cytometry at several time points after LPS injection. LPS administration right after cell transfer was chosen with the aim of leaving no time for transferred cells to adapt to their new environment before the initiation of systemic inflammation. Reflecting the initial percentage of resident NKs, transferred spleen or lung NK were found at higher percentages among LngNKs compared with those found in the spleen (Fig. 4A, 4E), regardless of their tissue origin (data not shown). At 3 h, transferred LngNKs found in the spleen had significantly lower CD69 and IFN-γ expression compared with transferred or resident spleen NKs (SplNKs) (Fig. 4B, 4C), with activation levels equivalent to those of LngNKs (Fig. 4F, 4G). However, GzmB expression was higher among transferred LngNKs in the spleen compared with resident and transferred SplNKs, reflective again of the lung compartment (Fig. 4D, 4H). In the lung compartment, although transferred LngNKs behaved as resident NKs, the transferred SplNKs mirrored splenic levels of activation, surpassing resident cells in CD69 and IFN-γ expression but exhibiting lower levels of GzmB expression (Fig. 4F–H). The fact that transferred spleen and lung NKs behaved like host NKs in the homologous compartment in terms of CD69, IFN-γ, and GzmB expression confirmed that pretransfer manipulation did not affect their functional status. These data indicated that NKs from different compartments maintain their original responsiveness at early time points after an inflammatory stimulus. To explore the duration of this phenomenon, we repeated the same experiment at later time points. Interestingly, we found that, by 6 h, transferred spleen and lung NKs behaved similarly to resident NKs in the organ they had reached. We observed that transferred NKs are still represented at higher proportions in the lung compared with the spleen (Fig. 5A, 5E). At this time point, transferred LngNKs found in the spleen expressed similar levels of CD69, IFN-γ, and GzmB as resident and transferred SplNKs (Fig. 5B–D), which, as described earlier, were higher for CD69 and IFN-γ than in the lung. Moreover, transferred SplNKs found in the lung behaved like resident and transferred LngNKs (Fig. 5F, 5G). However, for GzmB, the levels observed in the lung did not surpass the levels observed in the spleen. This might be due to the fact that, in this set of independent experiments, the overall responses (i.e., levels of IFN-γ at 6 h) were lower compared with previous experiments. However, the same adaptation phenomenon for CD69 and IFN-γ was observed (data not shown). The same results were obtained at 12 and 24 h for CD69 and IFN-γ (Fig. 6A–D) and IL-10 (Fig. 6E) expression.

FIGURE 4.

Transferred NKs maintain their original organ-specific activation profiles at 3 h after endotoxemia induction. NKs enriched from spleen and lungs of CD45.1 mice were transferred i.v. to CD45.2 mice (106 cells/100 μl PBS). Recipient mice were injected with LPS (10 mg/kg, i.p.), and NK activity in spleen and lungs was assessed at 3 h after LPS injection. Gating strategy for cytometric identification of resident versus transferred cells among NKs in the spleen (A) and lungs (E). Representative line graphs (left panels) and percentages (right panels) of CD69+ NKs (B and F), IFN-γ+ NKs (C and G), and GzmB+ NKs (D and H) among the respective populations in the spleen and lungs, respectively. Each circle represents an individual mouse. Data are representative of one of three independent repeats (n = 5 mice per transfer group). *p < 0.05, **p < 0.01, Mann–Whitney test, resident spleen or lung NKs versus transferred spleen or lung NKs, with the latter also compared between themselves. ns, not significant.

FIGURE 4.

Transferred NKs maintain their original organ-specific activation profiles at 3 h after endotoxemia induction. NKs enriched from spleen and lungs of CD45.1 mice were transferred i.v. to CD45.2 mice (106 cells/100 μl PBS). Recipient mice were injected with LPS (10 mg/kg, i.p.), and NK activity in spleen and lungs was assessed at 3 h after LPS injection. Gating strategy for cytometric identification of resident versus transferred cells among NKs in the spleen (A) and lungs (E). Representative line graphs (left panels) and percentages (right panels) of CD69+ NKs (B and F), IFN-γ+ NKs (C and G), and GzmB+ NKs (D and H) among the respective populations in the spleen and lungs, respectively. Each circle represents an individual mouse. Data are representative of one of three independent repeats (n = 5 mice per transfer group). *p < 0.05, **p < 0.01, Mann–Whitney test, resident spleen or lung NKs versus transferred spleen or lung NKs, with the latter also compared between themselves. ns, not significant.

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FIGURE 5.

Transferred NKs behave like resident NKs at 6 h after endotoxemia induction. NKs enriched from spleen and lungs of CD45.1 mice, were transferred i.v. to CD45.2 mice (106 cells/100 μl PBS). Recipient mice were injected with LPS (10 mg/kg, i.p.), and NK activity in spleens and lungs was assessed at 6 h after LPS injection. (A and E) Gating strategy for cytometric identification of resident versus transferred cells among NKs in the spleen and lungs, respectively. Representative line graphs (left panels) and summary (right panels) of CD69+ NKs (B and F), IFN-γ+ NKs (C and G) and GzmB+ NKs (D and H) among respective populations in the spleen and lungs, respectively. Circles represent individual mice. Data are representative of one experiment of three independent repeats (n = 5 mice per transfer group). *p < 0.05, **p < 0.01, resident spleen or lung NKs versus transferred spleen or lung NKs, with the latter also compared between themselves by Mann–Whitney test. ns, not significant.

FIGURE 5.

Transferred NKs behave like resident NKs at 6 h after endotoxemia induction. NKs enriched from spleen and lungs of CD45.1 mice, were transferred i.v. to CD45.2 mice (106 cells/100 μl PBS). Recipient mice were injected with LPS (10 mg/kg, i.p.), and NK activity in spleens and lungs was assessed at 6 h after LPS injection. (A and E) Gating strategy for cytometric identification of resident versus transferred cells among NKs in the spleen and lungs, respectively. Representative line graphs (left panels) and summary (right panels) of CD69+ NKs (B and F), IFN-γ+ NKs (C and G) and GzmB+ NKs (D and H) among respective populations in the spleen and lungs, respectively. Circles represent individual mice. Data are representative of one experiment of three independent repeats (n = 5 mice per transfer group). *p < 0.05, **p < 0.01, resident spleen or lung NKs versus transferred spleen or lung NKs, with the latter also compared between themselves by Mann–Whitney test. ns, not significant.

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FIGURE 6.

Transferred NKs respond like resident cells up to 12 and 24 h. NKs isolated from the spleen and lungs of CD45.1 or VertX mice were transferred i.v. (106 cells/100 μl PBS) into CD45.2 or VertX mice, respectively, and recipients were challenged with LPS (10 mg/kg). At 12 and 24 h after challenge, the activation of transferred and resident NKs was assessed by FACS and compared. Summary of percentages of CD69+ (A) and IFN-γ–expressing (B) NKs among resident spleen and lung NKs, transferred SplNKs, and transferred LngNKs at 12 h. Summary of percentages of CD69 (C) or IFN-γ–expressing (D) NKs among resident spleen and lung NKs, transferred SplNKs, and transferred LngNKs at 24 h. For IL-10–GFP studies, NKs isolated from spleen and lungs of VertX mice were labeled with CellVue Lavender and transferred into VertX mice prior to LPS injection. (E) Gating strategy for transferred cells in the spleen (upper left panel) and in the lungs (upper middle panel; gated on NKp46+CD3 cells) and overlay line graph of IL-10–GFP expression in resident spleen and lung NKs. Overlay line graph of IL-10–GFP expression on transferred spleen and lung NKs in the respective compartments (lower left panel). Summary of percentages of IL-10–GFP–expressing NKs among resident spleen and lung NKs, transferred SplNKs, and transferred LngNKs at 24 h (lower right panels). Circles represent individual mice. Data are representative of three independent experiments (n ≥ 3 mice per group).

FIGURE 6.

Transferred NKs respond like resident cells up to 12 and 24 h. NKs isolated from the spleen and lungs of CD45.1 or VertX mice were transferred i.v. (106 cells/100 μl PBS) into CD45.2 or VertX mice, respectively, and recipients were challenged with LPS (10 mg/kg). At 12 and 24 h after challenge, the activation of transferred and resident NKs was assessed by FACS and compared. Summary of percentages of CD69+ (A) and IFN-γ–expressing (B) NKs among resident spleen and lung NKs, transferred SplNKs, and transferred LngNKs at 12 h. Summary of percentages of CD69 (C) or IFN-γ–expressing (D) NKs among resident spleen and lung NKs, transferred SplNKs, and transferred LngNKs at 24 h. For IL-10–GFP studies, NKs isolated from spleen and lungs of VertX mice were labeled with CellVue Lavender and transferred into VertX mice prior to LPS injection. (E) Gating strategy for transferred cells in the spleen (upper left panel) and in the lungs (upper middle panel; gated on NKp46+CD3 cells) and overlay line graph of IL-10–GFP expression in resident spleen and lung NKs. Overlay line graph of IL-10–GFP expression on transferred spleen and lung NKs in the respective compartments (lower left panel). Summary of percentages of IL-10–GFP–expressing NKs among resident spleen and lung NKs, transferred SplNKs, and transferred LngNKs at 24 h (lower right panels). Circles represent individual mice. Data are representative of three independent experiments (n ≥ 3 mice per group).

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We also assessed the phenotype of the transferred cells compared with resident NKs. We found that, at 6 h after LPS injection, SplNKs found in the lung only partially maintained their original phenotype (Fig. 7A, 7C). In contrast, transferred LngNKs found in the spleen maintained a lung phenotypic profile, with an underrepresentation of CD27+CD11b cells but a higher percentage of CD11b+ cells (Fig. 7A, 7B). These results indicate that tissue-specific responsiveness of NKs is eventually changed by the local inflammatory microenvironment, leading to a compartment-dictated level of activation without necessarily involving phenotypic changes.

FIGURE 7.

Transferred NKs maintain their original phenotype in other organs. NKs isolated from the spleen and lungs of CD45.1 mice were transferred i.v. (106 cells/100 μl PBS) into CD45.2 mice, and recipients were challenged with LPS (10 mg/kg). At 6 h after challenge, the phenotypes of transferred versus resident NKs were assessed by FACS and compared. (A) Representative contour plots showing CD27CD11b subpopulations among resident, transferred SplNKs, and transferred LngNKs gated on NK1.1+CD3 cells from recipients spleen and lungs. Summary of CD27CD11b NK subpopulations among resident, transferred SplNKs, and transferred LngNKs in the spleen (B) and lung (C). Circles represent individual mice. Data are representative of three independent experiments (n = 5 mice per group). ***p < 0.001, Mann–Whitney test, resident spleen and lung NKs versus transferred lung and spleen NKs, respectively.

FIGURE 7.

Transferred NKs maintain their original phenotype in other organs. NKs isolated from the spleen and lungs of CD45.1 mice were transferred i.v. (106 cells/100 μl PBS) into CD45.2 mice, and recipients were challenged with LPS (10 mg/kg). At 6 h after challenge, the phenotypes of transferred versus resident NKs were assessed by FACS and compared. (A) Representative contour plots showing CD27CD11b subpopulations among resident, transferred SplNKs, and transferred LngNKs gated on NK1.1+CD3 cells from recipients spleen and lungs. Summary of CD27CD11b NK subpopulations among resident, transferred SplNKs, and transferred LngNKs in the spleen (B) and lung (C). Circles represent individual mice. Data are representative of three independent experiments (n = 5 mice per group). ***p < 0.001, Mann–Whitney test, resident spleen and lung NKs versus transferred lung and spleen NKs, respectively.

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We also assessed the possibility that the microenvironment-induced responses of NKs are a consequence of differential trafficking of a subset of the transferred cells; for this, we turned to a system that allows no trafficking. As reported by Gonzaga et al. (23), NKs transferred into the peritoneal cavity are trapped therein. We confirmed these findings, even under systemic inflammatory conditions, because we observed that splenic or lung NKs transferred i.p. into CD45.2 mice were found in the peritoneal cavity and not the spleen or the lungs (Fig. 8A, 8B). When comparing transferred cells with resident peritoneal NKs, we found the same time-dependent acquisition of compartment-driven responsiveness, with spleen and lung NKs behaving differently at 3 h but similarly to their peritoneal counterparts by 6 h (Fig. 8C, 8D). These results underline that compartment-specific adaptation of transferred NK responses can occur independently of differential trafficking and, thus, represent a functional adaptation. Overall, our data outline that during systemic inflammation, compartment-specific microenvironmental factors can eventually override NK compartment-specific responsiveness, independently of the compartment in question.

FIGURE 8.

Transferred cells are trapped in the peritoneum but eventually behave like resident NKs. NKs isolated from CD45.1 mice were transferred i.p. (105 cells/100 μl PBS) into CD45.2 mice, and recipients were challenged with LPS (10 mg/kg). At 3 and 6 h after challenge, transferred and resident NKs were assessed by FACS and compared. (A) Representative contour plots showing NK gating (upper panels) and transferred cell identification (lower panels) in the peritoneum, spleen, and lungs of recipient mice. (B) Summary of transferred NK percentages among total NKs pooled from experiments performed at 3 and 6 h. Summary of percentages of IFN-γ–expressing NKs among resident peritoneal NKs (PerNKs), transferred SplNKs, and transferred LngNKs at 3 h (C) and 6 h (D). Circles represent individual mice. Data are representative of at least three independent experiments (n ≥ 5 mice per group). *p < 0.05, **p < 0.01, Mann–Whitney test, resident PerNKs versus transferred SplNKs and LngNKs.

FIGURE 8.

Transferred cells are trapped in the peritoneum but eventually behave like resident NKs. NKs isolated from CD45.1 mice were transferred i.p. (105 cells/100 μl PBS) into CD45.2 mice, and recipients were challenged with LPS (10 mg/kg). At 3 and 6 h after challenge, transferred and resident NKs were assessed by FACS and compared. (A) Representative contour plots showing NK gating (upper panels) and transferred cell identification (lower panels) in the peritoneum, spleen, and lungs of recipient mice. (B) Summary of transferred NK percentages among total NKs pooled from experiments performed at 3 and 6 h. Summary of percentages of IFN-γ–expressing NKs among resident peritoneal NKs (PerNKs), transferred SplNKs, and transferred LngNKs at 3 h (C) and 6 h (D). Circles represent individual mice. Data are representative of at least three independent experiments (n ≥ 5 mice per group). *p < 0.05, **p < 0.01, Mann–Whitney test, resident PerNKs versus transferred SplNKs and LngNKs.

Close modal

In the current study we show that NK activation during LPS-induced systemic inflammation happens in a compartmentalized manner. In a review on the topic, Shi et al. (14) asked a couple of outstanding questions about NKs in different organs. Among these questions, one in particular caught our attention: “Are specific features of NKs within individual organs the cause or the consequence of inflammatory responses within that organ?” Using a series of adoptive transfers, we provide evidence that supports a role for NK-intrinsic responsiveness and organ-specific microenvironmental factors in the compartmentalization of NK responses during systemic inflammation that act in a sequential manner.

We undertook a serial analysis of NKs in the spleen, lungs, bone marrow, peritoneum, and blood during the course of experimental endotoxemia. We observed a severe reduction in NK numbers in all of the compartments that we investigated. We consider that this is the result of a high rate of apoptosis, as we observed at 6 h (data not shown) and was reported for lymphocytes during systemic inflammation (28), including NKs (29). We cannot exclude that NKs relocalize to other organs that we did not investigate, such as the liver. In addition, we investigated compartment-specific phenotypes. Similar to many previous studies, we found homeostatic phenotypical differences between NKs from different tissues (21, 23, 3033) but observed that these are relatively stable throughout the course of systemic inflammation. NKs were described to evolve through a CD27CD11b→CD27+CD11b→CD27+CD11b+→CD27CD11b+ maturation process (21). In addition to the known CD27/CD11b profiles, with CD27 expression being mostly absent from the lungs and blood, we found that Ly6C levels on NKs from different organs are also different, and these are remarkably stable during inflammation. In contrast, expression of B220 and CD11c progressively changed over time, with the two markers behaving like activation indicators, as they were used previously (34). Activation of NKs during endotoxemia mostly followed the same dynamic systemically, while maintaining compartment-specific thresholds of maximum activation. Although CD69 acted as an early activation marker in all compartments, CD25 expression was limited to the spleen, bone marrow, and peritoneum and peaked only at 24 h. IFN-γ expression by NKs peaked at 6 h, as previously reported during LPS challenge, at least in the spleen (35), and gradually declined until 24–48 h. Interestingly, GzmB expression was upregulated continuously, remaining at very high levels even at 48 h in all compartments, with the exception of the bone marrow. This finding expands the described upregulation of GzmB expression by TLR agonists (36) to other compartments; however, its significance remains difficult to interpret, because, as opposed to granzymes M and A, GzmB does not contribute to lethality during endotoxemia (37). NKs were also shown to express IL-10 during systemic inflammation but not during local pulmonary inflammation (27). Like in the aforementioned study, we detected IL-10 expression only at 24 h after LPS injection, mostly expressed by circulating and lung NKs; however, there was significant upregulation of IL-10 expression in the other compartments by 48 h. Certain limitations of IL-10–GFP reporter mice must be acknowledged, such as the fact that in a dynamic study such transcriptional reporters might be biased due to differential half-times of IL-10 versus the GFP protein (38). Nevertheless, our results show that NKs from all studied compartments expressed IL-10 after endotoxemia, with the lung and blood compartments being the first to do so. These observations suggest that NKs play pro- and anti-inflammatory roles during systemic inflammation. Although a clear role for inflammatory IFN-γ–producing NKs is established in systemic inflammation (11, 12), defining a role for IL-10–producing NKs requires more experimental evidence. However, it is possible that NK–derived IL-10 plays a regulatory role in preventing further damage to the organism, as was shown in the case of myocardial infarction (39).

Thus, our study shows that, during LPS-induced systemic inflammation, NKs from all investigated compartments get activated, in a systemic dynamic but with compartment-specific thresholds of maximum effector function. NKs in the spleen showed the highest level of activation marker and IFN-γ expression, followed by the peritoneum and bone marrow cells, whereas NK activation in the blood and lungs is markedly lower. However, these less activated compartments were the ones to display higher levels of GzmB expression and the first to express IL-10.

We considered two possibilities for these compartment-specific differences in NK activation during endotoxemia: a distinct NK-intrinsic responsiveness between various organs or particular microenvironmental cues that affect NK activation at the compartment level. Functional differences between NKs from different compartments were described. For example, LngNKs were shown to be less responsive than SplNKs to ex vivo stimulation in terms of cytokine production, degranulation, and cytotoxicity (30). However, in a different study, spleen and lung NKs were shown to have similar cytotoxic capacity (40). Nevertheless, earlier studies showed that the impaired responsiveness of LngNKs was due to the microenvironment and that lung NK responses increased after overnight culture and were boosted by inflammatory cytokines (41). Considering these findings, we assessed the second possibility by making use of an adoptive-transfer model in which NKs from different compartments of congenic mice could be assessed for responsiveness in comparison with resident NKs in a different organ of the host congenic mouse. We found that, upon adoptive transfer and LPS stimulation, functional differences mimicking their organ of origin were observed in transferred NKs at early moments. As previously reported (20, 25, 30, 41), pulmonary NKs were hyporesponsive in comparison with splenic NKs in terms of IFN-γ expression, but they upregulated GzmB significantly, indicating an alternative program of activation rather than a general lack of response. However, at later times, responses of transferred cells were similar to those of resident NKs in all parameters analyzed, from CD69 expression to IFN-γ, GzmB, and even IL-10 expression. A certain degree of variability accompanied some of these experiments, in which, at 6 h after transfer, we observed a lower level of NK activation and no difference in GzmB levels between spleen and lungs. Nevertheless, adaptation of transferred NKs to resident cell levels of activation was always observed. Although priming of pulmonary NK responses by inflammatory cytokines was shown previously (41, 42), the finding that SplNKs can downregulate their responsiveness to lung or peritoneal levels was very interesting. The latter implies that organ-specific microenvironmental cues precisely regulate the degree of NK responses by activatory and inhibitory factors. The fact that NK responses were aligned to resident levels without losing original phenotypes, at least in the spleen, suggest that only a functional adaptation occurs, at least at the time points that we investigated. Of note, extended periods of time were shown to be necessary for phenotypical adaptation of cell after transfer under homeostatic conditions (21, 23). These results were recapitulated in the peritoneum, where transferred spleen and lung NKs responded differently at 3 h but similarly to resident peritoneal NKs by 6 h. Finally, our peritoneal transfer model allowed us to exclude a role for a preferential trafficking of a specific NK subpopulation to a certain organ, because as originally described, the peritoneum traps transferred NKs at this level (23). In addition to confirming this finding, our study expands on it by introducing the inflammatory setting in which we observe the same phenomenon. These conditions allowed us to confirm that the bulk population of spleen or lung NKs can adapt to the peritoneal environment and respond in consequence, differently at 3 h but adapting by 6 h.

A multitude of soluble factors were reported to affect NK activation. During endotoxemia, IL-2, IL-12, IL-15, IL-18, and type one IFNs were shown to play crucial and nonredundant roles (35). However, these effects were assessed only in the spleen, and it is quite possible that these factors affect the various compartments differentially, as previously suggested (1, 2, 4, 8). Differences between non-NK compositions of different organs most probably also have a role in shaping the compartmentalization of NK responses, especially because organ-specific features were reported for macrophages (43), T cells (44), NKT cells (45), and innate lymphoid cells (46). For example, specific cross-talk between alveolar macrophages or Kupffer cells and NKs was reported previously (47, 48). These particular cell types, in combination with NK-intrinsic properties, might contribute to the regulation of compartmentalized responses during systemic inflammation.

Despite mounting evidence on the tissue-specific properties of NKs, comparative in vivo analyses of NKs from different tissues are scarce. Most of the data come from ex vivo phenotypical characterizations and functionality assays. Although this is sufficient to identify homeostatic differences between different tissue-resident NKs, the inflammatory environment might alter these respective properties. Our results showed exactly this: although at early moments after LPS challenge NKs of different origins maintain their source-specific responsiveness, this is quickly lost, and environmental factors take over to control and tune the degree of NK responses. This is probably set in place as a control mechanism, because more responsive splenic NKs that could get recruited to the lung environment (e.g., as shown during inflammation or infection for lymph nodes or the liver) (49, 50) might be too inflammatory for the local requirements and cause unnecessary collateral damage. To our knowledge, this is the first study to address the compartmentalization of NK responses during systemic inflammation and to show a role for tissue-specific NK-intrinsic properties and microenvironmental cues using a dynamic in vivo transfer system. Further research on the topic is sure to determine which specific soluble or cellular factors contribute to shaping organ-specific inflammatory responses and how these interact with resident and recruited NKs. Our findings support the notion of compartmentalization during systemic inflammation and underline the fact that analysis of readily available samples, like peripheral blood cells in humans or spleen cells in mice, might hide more than they reveal, leading to a potentially false under- or overestimation of the inflammatory response.

We thank Dr. Françoise Guinet for constructive comments on the manuscript.

This work was supported by the Institut Pasteur. O.R. was supported by a stipend from the Pasteur - Paris University International Ph.D. Program. I.S.C. was supported by a European Federation of Immunological Societies – Immunology Letters Short-Term Fellowship.

The online version of this article contains supplemental material.

Abbreviations used in this article:

GzmB

granzyme B

LngNK

lung NK

SplNK

spleen NK.

1
Cavaillon
J. M.
,
Annane
D.
.
2006
.
Compartmentalization of the inflammatory response in sepsis and SIRS.
J. Endotoxin Res.
12
:
151
170
.
2
Chinnaiyan
A. M.
,
Huber-Lang
M.
,
Kumar-Sinha
C.
,
Barrette
T. R.
,
Shankar-Sinha
S.
,
Sarma
V. J.
,
Padgaonkar
V. A.
,
Ward
P. A.
.
2001
.
Molecular signatures of sepsis: multiorgan gene expression profiles of systemic inflammation.
Am. J. Pathol.
159
:
1199
1209
.
3
Thorgersen
E. B.
,
Pischke
S. E.
,
Barratt-Due
A.
,
Fure
H.
,
Lindstad
J. K.
,
Pharo
A.
,
Hellerud
B. C.
,
Mollnes
T. E.
.
2013
.
Systemic CD14 inhibition attenuates organ inflammation in porcine Escherichia coli sepsis.
Infect. Immun.
81
:
3173
3181
.
4
Grotz
W. M. R.
,
van Griensven
M.
,
Stalp
M.
,
Rohde
F.
,
Hildebrand
F.
,
Krettek
C.
,
Pape
H.-C.
.
2001
.
Organ-specific cytokine gene expression in sepsis – an experimental study in a two-hit septic model.
Eur. J. Trauma
27
:
191
198
.
5
von Drygalski
A.
,
Furlan-Freguia
C.
,
Ruf
W.
,
Griffin
J. H.
,
Mosnier
L. O.
.
2013
.
Organ-specific protection against lipopolysaccharide-induced vascular leak is dependent on the endothelial protein C receptor.
Arterioscler. Thromb. Vasc. Biol.
33
:
769
776
.
6
van Meurs
M.
,
Castro
P.
,
Shapiro
N. I.
,
Lu
S.
,
Yano
M.
,
Maeda
N.
,
Funahashi
T.
,
Shimomura
I.
,
Zijlstra
J. G.
,
Molema
G.
, et al
.
2012
.
Adiponectin diminishes organ-specific microvascular endothelial cell activation associated with sepsis.
Shock
37
:
392
398
.
7
Fitting
C.
,
Dhawan
S.
,
Cavaillon
J. M.
.
2004
.
Compartmentalization of tolerance to endotoxin.
J. Infect. Dis.
189
:
1295
1303
.
8
Philippart
F.
,
Fitting
C.
,
Cavaillon
J. M.
.
2012
.
Lung microenvironment contributes to the resistance of alveolar macrophages to develop tolerance to endotoxin*.
Crit. Care Med.
40
:
2987
2996
.
9
Wiersinga
W. J.
,
Leopold
S. J.
,
Cranendonk
D. R.
,
van der Poll
T.
.
2014
.
Host innate immune responses to sepsis.
Virulence
5
:
36
44
.
10
Ulloa
L.
,
Tracey
K. J.
.
2005
.
The “cytokine profile”: a code for sepsis.
Trends Mol. Med.
11
:
56
63
.
11
Souza-Fonseca-Guimaraes
F.
,
Cavaillon
J. M.
,
Adib-Conquy
M.
.
2013
.
Bench-to-bedside review: natural killer cells in sepsis - guilty or not guilty?
Crit. Care
17
:
235
.
12
Chiche
L.
,
Forel
J. M.
,
Thomas
G.
,
Farnarier
C.
,
Vely
F.
,
Bléry
M.
,
Papazian
L.
,
Vivier
E.
.
2011
.
The role of natural killer cells in sepsis.
J. Biomed. Biotechnol.
2011
:
986491
.
13
Vivier
E.
,
Tomasello
E.
,
Baratin
M.
,
Walzer
T.
,
Ugolini
S.
.
2008
.
Functions of natural killer cells.
Nat. Immunol.
9
:
503
510
.
14
Shi
F. D.
,
Ljunggren
H. G.
,
La Cava
A.
,
Van Kaer
L.
.
2011
.
Organ-specific features of natural killer cells.
Nat. Rev. Immunol.
11
:
658
671
.
15
Sun
H.
,
Sun
C.
,
Tian
Z.
,
Xiao
W.
.
2013
.
NK cells in immunotolerant organs.
Cell. Mol. Immunol.
10
:
202
212
.
16
Sharma
R.
,
Das
A.
.
2014
.
Organ-specific phenotypic and functional features of NK cells in humans.
Immunol. Res.
58
:
125
131
.
17
Lysakova-Devine
T.
,
O’Farrelly
C.
.
2014
.
Tissue-specific NK cell populations and their origin.
J. Leukoc. Biol.
96
:
981
990
.
18
Björkström
N. K.
,
Ljunggren
H. G.
,
Michaëlsson
J.
.
2016
.
Emerging insights into natural killer cells in human peripheral tissues.
Nat. Rev. Immunol.
16
:
310
320
.
19
Nolibe
D.
,
Berel
E.
,
Masse
R.
,
Lafuma
J.
.
1981
.
Characterization of a major natural killer activity in rat lungs.
Biomedicine
35
:
230
234
.
20
Lauzon
W.
,
Yang
H.
,
Lemaire
I.
.
1990–1991
.
Comparative analysis of natural killer function in lung, spleen, and peripheral blood lymphocytes: evidence of differential characteristics.
Reg. Immunol.
3
:
145
150
.
21
Chiossone
L.
,
Chaix
J.
,
Fuseri
N.
,
Roth
C.
,
Vivier
E.
,
Walzer
T.
.
2009
.
Maturation of mouse NK cells is a 4-stage developmental program.
Blood
113
:
5488
5496
.
22
Daussy
C.
,
Faure
F.
,
Mayol
K.
,
Viel
S.
,
Gasteiger
G.
,
Charrier
E.
,
Bienvenu
J.
,
Henry
T.
,
Debien
E.
,
Hasan
U. A.
, et al
.
2014
.
T-bet and Eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow.
J. Exp. Med.
211
:
563
577
.
23
Gonzaga
R.
,
Matzinger
P.
,
Perez-Diez
A.
.
2011
.
Resident peritoneal NK cells.
J. Immunol.
187
:
6235
6242
.
24
Montaldo
E.
,
Vacca
P.
,
Chiossone
L.
,
Croxatto
D.
,
Loiacono
F.
,
Martini
S.
,
Ferrero
S.
,
Walzer
T.
,
Moretta
L.
,
Mingari
M. C.
.
2016
.
Unique eomes(+) NK cell subsets are present in uterus and decidua during early pregnancy.
Front. Immunol.
6
:
646
.
25
Wang
J.
,
Li
F.
,
Zheng
M.
,
Sun
R.
,
Wei
H.
,
Tian
Z.
.
2012
.
Lung natural killer cells in mice: phenotype and response to respiratory infection.
Immunology
137
:
37
47
.
26
Madan
R.
,
Demircik
F.
,
Surianarayanan
S.
,
Allen
J. L.
,
Divanovic
S.
,
Trompette
A.
,
Yogev
N.
,
Gu
Y.
,
Khodoun
M.
,
Hildeman
D.
, et al
.
2009
.
Nonredundant roles for B cell-derived IL-10 in immune counter-regulation.
J. Immunol.
183
:
2312
2320
.
27
Perona-Wright
G.
,
Mohrs
K.
,
Szaba
F. M.
,
Kummer
L. W.
,
Madan
R.
,
Karp
C. L.
,
Johnson
L. L.
,
Smiley
S. T.
,
Mohrs
M.
.
2009
.
Systemic but not local infections elicit immunosuppressive IL-10 production by natural killer cells.
Cell Host Microbe
6
:
503
512
.
28
Hotchkiss
R. S.
,
Tinsley
K. W.
,
Swanson
P. E.
,
Chang
K. C.
,
Cobb
J. P.
,
Buchman
T. G.
,
Korsmeyer
S. J.
,
Karl
I. E.
.
1999
.
Prevention of lymphocyte cell death in sepsis improves survival in mice.
Proc. Natl. Acad. Sci. USA
96
:
14541
14546
.
29
Inoue
S.
,
Unsinger
J.
,
Davis
C. G.
,
Muenzer
J. T.
,
Ferguson
T. A.
,
Chang
K.
,
Osborne
D. F.
,
Clark
A. T.
,
Coopersmith
C. M.
,
McDunn
J. E.
,
Hotchkiss
R. S.
.
2010
.
IL-15 prevents apoptosis, reverses innate and adaptive immune dysfunction, and improves survival in sepsis.
J. Immunol.
184
:
1401
1409
.
30
Michel
T.
,
Poli
A.
,
Domingues
O.
,
Mauffray
M.
,
Thérésine
M.
,
Brons
N. H.
,
Hentges
F.
,
Zimmer
J.
.
2012
.
Mouse lung and spleen natural killer cells have phenotypic and functional differences, in part influenced by macrophages. [Published erratum appears in 2013 PLoS One 8(1).]
PLoS One
7
:
e51230
.
31
Okada
K.
,
Sato
S.
,
Sato
A.
,
Mandelboim
O.
,
Yamasoba
T.
,
Kiyono
H.
.
2015
.
Identification and analysis of natural killer cells in murine nasal passages.
PLoS One
10
:
e0142920
.
32
Victorino
F.
,
Sojka
D. K.
,
Brodsky
K. S.
,
McNamee
E. N.
,
Masterson
J. C.
,
Homann
D.
,
Yokoyama
W. M.
,
Eltzschig
H. K.
,
Clambey
E. T.
.
2015
.
Tissue-resident NK cells mediate ischemic kidney injury and are not depleted by anti-asialo-GM1 antibody.
J. Immunol.
195
:
4973
4985
.
33
Hayakawa
Y.
,
Smyth
M. J.
.
2006
.
CD27 dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity.
J. Immunol.
176
:
1517
1524
.
34
Elpek
K. G.
,
Rubinstein
M. P.
,
Bellemare-Pelletier
A.
,
Goldrath
A. W.
,
Turley
S. J.
.
2010
.
Mature natural killer cells with phenotypic and functional alterations accumulate upon sustained stimulation with IL-15/IL-15Ralpha complexes.
Proc. Natl. Acad. Sci. USA
107
:
21647
21652
.
35
Zanoni
I.
,
Spreafico
R.
,
Bodio
C.
,
Di Gioia
M.
,
Cigni
C.
,
Broggi
A.
,
Gorletta
T.
,
Caccia
M.
,
Chirico
G.
,
Sironi
L.
, et al
.
2013
.
IL-15 cis presentation is required for optimal NK cell activation in lipopolysaccharide-mediated inflammatory conditions.
Cell Rep.
4
:
1235
1249
.
36
Lucas
M.
,
Schachterle
W.
,
Oberle
K.
,
Aichele
P.
,
Diefenbach
A.
.
2007
.
Dendritic cells prime natural killer cells by trans-presenting interleukin 15.
Immunity
26
:
503
517
.
37
Anthony
D. A.
,
Andrews
D. M.
,
Chow
M.
,
Watt
S. V.
,
House
C.
,
Akira
S.
,
Bird
P. I.
,
Trapani
J. A.
,
Smyth
M. J.
.
2010
.
A role for granzyme M in TLR4-driven inflammation and endotoxicosis.
J. Immunol.
185
:
1794
1803
.
38
Bouabe
H.
2012
.
Cytokine reporter mice: the special case of IL-10.
Scand. J. Immunol.
75
:
553
567
.
39
Yan
X.
,
Hegab
A. E.
,
Endo
J.
,
Anzai
A.
,
Matsuhashi
T.
,
Katsumata
Y.
,
Ito
K.
,
Yamamoto
T.
,
Betsuyaku
T.
,
Shinmura
K.
, et al
.
2014
.
Lung natural killer cells play a major counter-regulatory role in pulmonary vascular hyperpermeability after myocardial infarction.
Circ. Res.
114
:
637
649
.
40
Ballas
Z. K.
,
Buchta
C. M.
,
Rosean
T. R.
,
Heusel
J. W.
,
Shey
M. R.
.
2013
.
Role of NK cell subsets in organ-specific murine melanoma metastasis.
PLoS One
8
:
e65599
.
41
Robinson
B. W.
,
Pinkston
P.
,
Crystal
R. G.
.
1984
.
Natural killer cells are present in the normal human lung but are functionally impotent.
J. Clin. Invest.
74
:
942
950
.
42
Lauzon
W.
,
Lemaire
I.
.
1991
.
Effects of biological response modifiers on lung natural killer activity.
Immunopharmacol. Immunotoxicol.
13
:
237
250
.
43
Lavin
Y.
,
Winter
D.
,
Blecher-Gonen
R.
,
David
E.
,
Keren-Shaul
H.
,
Merad
M.
,
Jung
S.
,
Amit
I.
.
2014
.
Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment.
Cell
159
:
1312
1326
.
44
Mueller
S. N.
,
Mackay
L. K.
.
2016
.
Tissue-resident memory T cells: local specialists in immune defence.
Nat. Rev. Immunol.
16
:
79
89
.
45
Lee
Y. J.
,
Wang
H.
,
Starrett
G. J.
,
Phuong
V.
,
Jameson
S. C.
,
Hogquist
K. A.
.
2015
.
Tissue-specific distribution of iNKT cells impacts their cytokine response.
Immunity
43
:
566
578
.
46
Björkström
N. K.
,
Kekäläinen
E.
,
Mjösberg
J.
.
2013
.
Tissue-specific effector functions of innate lymphoid cells.
Immunology
139
:
416
427
.
47
Lauzon
W.
,
Lemaire
I.
.
1994
.
Alveolar macrophage inhibition of lung-associated NK activity: involvement of prostaglandins and transforming growth factor-beta 1.
Exp. Lung Res.
20
:
331
349
.
48
Michel
T.
,
Hentges
F.
,
Zimmer
J.
.
2013
.
Consequences of the crosstalk between monocytes/macrophages and natural killer cells.
Front. Immunol.
3
:
403
.
49
Pak-Wittel
M. A.
,
Yang
L.
,
Sojka
D. K.
,
Rivenbark
J. G.
,
Yokoyama
W. M.
.
2013
.
Interferon-γ mediates chemokine-dependent recruitment of natural killer cells during viral infection.
Proc. Natl. Acad. Sci. USA
110
:
E50
E59
.
50
Wang
J.
,
Xu
J.
,
Zhang
W.
,
Wei
H.
,
Tian
Z.
.
2005
.
TLR3 ligand-induced accumulation of activated splenic natural killer cells into liver.
Cell. Mol. Immunol.
2
:
449
453
.

The authors have no financial conflicts of interest.

Supplementary data