IL-27 promotes NK cell effector functions via Maf-Nrf2 pathway during influenza infection

Influenza virus targets epithelial cells in the upper respiratory tract. Natural Killer (NK) cell-mediated early innate defense responses to influenza infection include the killing of infected epithelial cells and generation of anti-viral cytokines including interferon gamma (IFN-γ). To date, it is unclear how the underlying cytokine milieu during infection regulates NK cell effector functions. Our data show during influenza infection myeloid cell-derived IL-27 regulates the early-phase effector functions of NK cells in the bronchioalveolar and lung tissue. Lack of IL-27R (Il27ra−/−) or IL-27 (Ebi3−/−) resulted in impaired NK cell effector functions including the generation of anti-viral IFN-γ responses. We identify CD27+CD11b+ NK cells as the primary subset that expresses IL-27R, which predominantly produces IFN-γ within the upper respiratory tract of the infected mice. IL-27 alone was incapable of altering the effector functions of NK cells. However, IL-27 sensitizes NK cells to augment both in vitro and in vivo responses mediated via the NKG2D receptor. This ‘priming’ function of IL-27 is mediated partly via transcriptional pathways regulated by Mafs and Nrf2 transcriptionally regulating TFAM and CPT1. Our data for the first time establishes a novel role for IL-27 in regulating early-phase effector functions of NK cells during influenza infection.

Each year thousands of people are hospitalized due to complication related to influenza virus infections. Innate and adaptive immune cells mediate the host immune responses to influenza virus infections. NK cells provide the first line of innate defense against influenza virus by killing infected epithelial cells and by producing anti-viral cytokine interferon (IFN)-γ 1,2 . NK cells express the multiple activating and inhibitory receptors to execute anti-viral or anti-tumor effector functions 3 . Virally-infected cells express H60, Rae, and Mult1 or Hemagglutinin (HA) ligands for NK cells activating receptor NKG2D and NCR1, respectively 4 . Recognition of ligands by NKG2D or NCR1 results in lysis of infected/tumor cells and the generation of IFN-γ from NK cells 5,6 . NK cells constitutively express or upregulate the expression of activating receptors to mount anti-viral responses; however, virally-infected/tumor cells evade NK cell-mediated recognition through various mechanisms. Virus down regulates ligands for NK cell-activating receptor or enhances engaging inhibitory receptors 4,7,8 .
Effect of cytokines in modulating NK cell responses has been an area of intense research. The common gamma receptor (γcR)-interacting cytokines IL-2, IL-7, IL-15, and IL-21 have been used to expand NK cells for adoptive transfer experiments in the clinical setting 9 . Unique α-chains define the receptors for these cytokines. IL-2 and IL-15 share a β-chain and the γcR along with cytokine-specific IL-2Rα and IL-15Rα, respectively 10,11 . Historically, IL-2 has been extensively used to expand murine and human NK cells 12,13 . IL-15 activates PI(3)K-mediated mTORC1 pathway 14,15 . IL-12 is a heterodimeric cytokine consists of p35 and p40 subunits, and it binds to the IL-12 receptor (IL-12Rβ1 and IL-12Rβ2) 16,17 . IL-18 belongs to an IL-1 family that interacts with a heterodimeric receptor composed of IL-18Rα and IL-18Rβ 18,19 . IL-12 and IL-18 enhance NK cell effector functions including IFN-γ production 20,21 . However, IL-12 or IL-18 responses are acute and independent of NK cell activating and inhibitory receptors 22 . IL-23 is another heterodimeric cytokine composed of p19 and p40 subunits, and its receptor is made up of IL-23Rα and IL-12Rβ1 23 . IL-23 activates NK cells to produce IL-22 24,25 . IL-35 contains p35 and EBI3 subunits, and its recently defined receptor consists of IL-12Rβ2 and gp130 [26][27][28] . gp130 is the shared receptor subunit of an IL-6 family of cytokine receptors 29 . IL-27 is another heterodimeric cytokine that belongs to the IL-12 family and consists of p28 and Epstein-Barr virus-induced gene 3 (EBI3) 30 . Receptor for IL-27 is composed of gp130 and WSX1 31 .
IL-27 and has been shown to modulate NK cells anti-tumor cytotoxicity responses [32][33][34][35] . These studies demonstrate that IL-27 augments NK cells cytotoxic responses to a variety of tumor cell lines in perforin, granzyme, TRAIL, and Fc-γR-III-dependent mechanisms 32,33,[36][37][38][39] . The role of IL-27 in NK cell-mediated anti-tumor immunity has been defined 39 . However, the underlying molecular mechanism is not well-defined. Notably, the mechanism by which IL-27 regulate NK cells effector functions during viral infections is yet to be fully understood.
In this study, we determined the role of IL-27 signaling in regulating NK cells effector responses during influenza infection as well as dissecting molecular mechanism of its action. Our data show that NK cells upregulate IL-27R following influenza infection. IL-27 but not IL-12 or IL-35 is obligatory for promoting the early NK cell-mediated responses. Ebi3 −/− and Il27ra −/− mice exhibited significantly reduced NK cell effector functions (IFN-γ and cytotoxicity) during influenza infection. Our in vivo and in vitro findings strongly suggest that defect in effector responses were NK cells intrinsic and involve CD27 + CD11b + subset. Mechanistically, IL-27 regulates NK cells effector functions via small Maf-F and Nrf2. Expressions of γ-glutamylcysteine ligase catalytic (GCLC), mitochondrial transcription factor A (TFAM), and carnitine palmitoyltransferase 1 (CPT1) were significantly reduced in NK cells derived from Ebi3 −/− mice, demonstrating a unique and exclusive functional role for IL-27. These findings provide a novel insight into how IL-27 plays a central role in containing viral infections by sensitizing NK cells to recognize infected cells and provide protective immunity to the host.

Influenza infection leads to IL-27 generation and induction of IL-27R. Activation of innate and
adaptive immune responses is natural host defense against influenza infection. The innate immune response includes infiltration of myeloid cells and NK cells in the upper respiratory tract. To determine the functional relevance of NK cells during influenza infection, we intranasally infected C57BL/6 (wild-type, WT) mice with 500 PFU of mouse-adapted human A/PR/8/34 H1N1 (PR8) influenza virus. The lumen side of the trachea consists of a thick layer of columnar epithelial cells that are exposed to and are the targets of influenza virus. Our earlier study has shown that effector lymphocytes including NK cells infiltrate into the epithelial layer via the basement membrane 40 . We found only fewer NK cells were present on days post-infection (DPI) 0 and their number steadily increased on DPI 4 and 7. Our earlier work has shown that NK cells are abundantly present within the alveolar space and conducting airways of the lungs during influenza infection 5,[40][41][42] . To define the functional relevance, we analyzed the production of IFN-γ by NK cells on DPI 0, 2, 4, 7, and 10. Percentages of IFN-γ + NK cells considerably increased on DPI 2 and DPI 4 ( Fig. 1A. Expression of CD107a (LAMP1), which is a surrogate marker for cytotoxicity peaked on DPI 4 (Fig. 1B).
Inflammatory cytokine milieu within the infected trachea and alveolar space regulate the production of IFN-γ from NK cells during influenza infection. To identify the relative contribution of IL-12, IL-27, and IL-23, we infected WT mice with influenza virus. Single cell suspensions from bronchoalveolar lavage (BAL) and the lung tissues were used to quantify the transcript levels of Il12p35, Il12p40, Il23p19, and Il27p28. Cells from both BAL and lung tissues contained transcripts encoding these cytokines. We found Il27p28 transcripts consistently appeared earlier (DPI 4) than Il12p35 and Il12p40 or Il23p19 (DPI 7) in both BAL and lung tissues (Fig. 1C).
We next analyzed the expression of the IL-27 receptor (IL-27R) temporally during influenza infection. The IL-27R is composed of IL-27Rα (Wsx1) and the shared IL-6 family receptor subunit, gp130. To determine its expression, we used an antibody specific for IL-27Rα (Wsx1). We performed confocal analyses of NK cells from lung tissues ( Fig. 2A) and flow cytometry analyses (Fig. 2B) of NK cells from BAL, lung tissues, and spleen of infected mice from different DPIs. Our data reveal expression of IL-27Rα in NK cells peaked on DPI 4 coinciding with the production of IL-27. Next, we characterized the cell type that produces the IL-27. Cells from both BAL and lung tissues were stained define Ly6G + cells myeloid population as the predominant producer of IL-27 during this early period of influenza infection (Supplementary Fig. 1A and B). Consistent with published data, production of IL-27 by Ly6G-positive cells temporally aligned with the expression of IL-27R on NK cells during influenza infection 43 .

IL-27 augments NKG2D but not IL-12-mediated IFN-γ production. Proinflammatory cytokines
IL-12 and IL-18 can stimulate the production of IFN-γ. IL-23 is primarily responsible for the production of IL-22 from effector lymphocytes including NK cells 40,44,45 . Generation of IL-12 during the early phase of influenza infection indicates that these cytokines may play a role in regulating the IFN-γ gene transcription in NK cells during the early phase of infection. Influenza infection also leads to the expression of inducible stress proteins such as H60, Rae-1, and Mult1 in infected cells that are the cognate ligands for the activation receptor NKG2D 46 . Infected epithelial cells also express viral haemagglutinin, which is one of the defined ligands of the activating NK cell receptor, NCR1 (Nkp46). To distinguish the role of IL-27 on activation receptor (such as NKG2D or Ly49D) and cytokine receptors (such as IL-12 or IL-18), we stimulated IL-2-cultured NK cells with combinations of stimuli (Fig. 3). Recombinant IL-27 (rIL-27) augmented only NKG2D-but not IL-12 or IL-18-mediated activation (Fig. 3A). Although IL-12 is a potent stimulator of IFN-γ, the presence of exogenous IL-27 did not augment IFN-γ production. In contrast, the presence of IL-18 along with IL-12 resulted in the maximal production of intracellular IFN-γ (Fig. 3B). These observations were further validated for other cytokines and chemokines including GM-CSF, RANTES, and MIP1-α by testing the culture supernatants via multiplex assays (Fig. 3C).
To further corroborate these findings, we stimulated IL-2-cultured splenic NK cells from WT and Il27ra −/− mice with anti-NKG2D or anti-Ly49D mAbs in the presence or absence of recombinant IL-27. IL-27 alone was not able to induce the generation of IFN-γ (Fig. 3D). However, IL-27 significantly augmented anti-NKG2D mAb-mediated production of cytokines and chemokines in NK cells from WT mice. Role of exogenous IL-27 on anti-Ly49D mAb-mediated generation of GM-CSF and RANTES was significant; however, its effect on IFN-γ and MIP-1α was only moderate in NK cells from the WT. Importantly, the addition of rIL-27 did not have any effect on NK cells from Il27ra −/− mice confirming the specific role of IL-27 on activation receptor-mediated cytokine and chemokine production (Fig. 3D). IL-12 and IL-18 together promote optimal production of IFN-γ. rIL-27 along with IL-12 did not significantly augment the production of IFN-γ. Therefore, we next tested whether the combination of IL-12, IL-18, and IL-27 has an additive effect on NK cells. There were no significant differences between IL-12 and IL-18 or the combination of all three cytokines in the production of IFN-γ production (Fig. 3E). These observations confirm that IL-27 plays a central role in the co-stimulation activation receptor (such as NKG2D)-mediated NK cell effector functions.
IL27R and IL-35R belong to the IL-6R family, and they utilize a common receptor chain, gp130 (Ref). Also, to distinguish the unique role of IL-27 and its receptor IL-27R (IL-27Rα/gp130) from IL-6R in NK cells, we stimulated IL-2-cultured NK cells from WT mice with anti-NKG2D mAb in the presence of increasing concentrations of recombinant IL-6 protein (rIL-6). We found that in contrast to rIL-27, rIL-6 was unable to augment anti-NKG2D mAb-mediated activation and production of IFN-γ even at a higher concentration of 10 ng/ml (Fig. 3F). IL   www.nature.com/scientificreports www.nature.com/scientificreports/ To eliminate the possibility of an NK cell extrinsic defect in the observed effector functions in mice lacking IL-27R (Il27ra −/− ) mice, we performed adoptive transfer experiments. Splenocytes from WT (B6.SJL, CD45.1 + ) and Il27ra −/− (CD45.2 + ) mice were isolated, and a mixture (1:1) of these cells were adoptively transferred into lymphocytes-deficient Rag2 −/− γc −/− mice on (Fig. 4C). Host mice were infected with influenza. On DPI 2, we were able to detect NCR1 + WT and Il27ra −/− NK cells in the lung tissue of Rag2 −/− γc −/− mice (Fig. 4D). The percentages of NK cells transferred from Il27ra −/− mice that produced IFN-γ were significantly lower compared to that of B6.SJL (Fig. 4E,F). However, the overall percentages of LAMP1 + NK cells did not vary between the B6.SJL (CD45.1) and Il27ra −/− CD45.2) mice. Collectively these data suggest IL-27 plays a primary role in the production of IFN-γ from NK cells during the early phase of influenza infection in vivo.

IL-27 regulates CD27 + CD11b + effector NK cell population in vivo. Expression of CD27 on T cells
along with CD44 marks the onset of early effector memory phenotype 47 . The absence of CD27 on CD44 + CD62L − T cells define the late effector memory and effector T cell subsets. In the case of NK cells, expression of CD27 and CD11b define functional subsets of NK cells 48,49 . CD27 + CD11b + and CD27 + CD11b − NK cells are specialized to secrete cytokines, whereas CD27 − CD11b + NK cells are more cytotoxic 48,49 . To identify the primary subset of NK cells that produces IFN-γ during the early phase of influenza infection, we analyzed the lungs of the WT mice on DPI 2. Lung-derived NK cells were gated based on their CD27/CD11b positivity (Fig. 5A). We found that the majority of NK cells that produced IFN-γ were the CD27 + CD11b + compared to the other two subsets. Next, we gated the NK cells from the lung of the infected mice (DPI 2) based on the expression of IL-27R into NCR1 + IL-27R + or NCR1 + IL-27R − cells. These two subsets were further divided into CD27 − CD11b + , CD27 + CD11b + , and CD27 + CD11b − NK cells and the production of IFN-γ examined. Among these, IL-27R + CD27 + CD11b + NK cells www.nature.com/scientificreports www.nature.com/scientificreports/ were proportionately most positive for intracellular IFN-γ compared any other subsets (Fig. 5B). Also, among the IL-27R − NK cells, CD27 + CD11b + NK cells were predominant in producing IFN-γ (Fig. 5B). Our data suggest that the CD27 + CD11b + NK cell subset may have an inherent maturation or recruitment defect in Il27ra −/− mice.
Distinct stages of development and acquisition of unique functional receptors characterize NK cell maturation in the BM. CD27 + CD11b − subset represents the earliest stage of functional maturation of NK cell development in the BM 50,51 . These single positive subset transition into the CD27 + CD11b + intermediate stage and eventually into CD27 + CD11b − NK cells. CD27 − CD11b + NK cells were predominant in circulation as well as in the lung tissue 49 . Furthermore, CD27 + CD11b + , as well as CD27 + CD11b − NK cells, have the more proliferative capacity 49,52 . Irrespective of this knowledge, it is not clear if NK cells subset specification changes under inflammatory condition. To define this, we analyzed the BAL and the lung tissues of influenza-infected WT and Il27ra −/− mice on DPI 0, 4, and 7 (Fig. 5C,D). We found a significant reduction in the percentages of CD27 + CD11b + NK cell effector population in the alveolar space of Il27ra −/− but not WT in influenza-infected mice. Also, there was a proportionate and a concomitant increase in the percentages of CD27 − CD11b + NK cell subset at an earlier stage of influenza infection. The changes in the percentages of these subsets were not due to a change in the absolute number of NK cells in the BAL of the infected mice. Absolute number of NK cells per million lymphocytes in the BAL ranged between 17,400 to 35,550 in the WT on DPI4. In mice that lacked IL-27Rα, it ranged between 11,500 to 20,000 NK cells. On DPI7, it was between 62,400 to 123,250 in the WT mice while in Il27ra −/− mice between 128,250 to 179,550. CD27+ CD11b−, CD27+ CD11b+, CD27− CD11b+ NK cells were distributed as shown with the percentages. Thus, in fact the absolute number of NK cells in the BAL during influenza infection between the WT and the Il27ra −/− mice were either comparable or moderately increased in the Il27ra −/− mice. We next examined the bone marrow (BM) of naïve mice for these different NK cell subsets. Interestingly, Il27ra −/− mice have significantly less CD27 + CD11b + NK cells in the BM, suggesting IL-27 may have a role in NK cells development or maturation or proliferation ( Supplementary Fig. 4A and B). Our data further show a similar level of various inhibitory and activating NK cell receptors in naïve BM-derived WT and Il27ra −/− mice, suggesting a more specific role of IL-27 in regulating CD27 + NK cell subsets ( Supplementary Fig. 4C-E).

Lack of EBI3 reduces the ability of NK cells to produce IFN-γ during influenza infection. IL-27
cytokine consists of two subunits, EBI3, and IL-27p28. To further confirm the role of IL-27 in regulating NK cells www.nature.com/scientificreports www.nature.com/scientificreports/ effector functions in vivo, we infected WT and Ebi3 −/− (the critical subunit of both IL-27 and IL-35 cytokines) mice with influenza virus. NK cells upregulate IL-27R in WT mice in response to influenza infection ( Fig. 2A,B). Our data suggests Ebi3 −/− mice are more susceptible to influenza infection as revealed by weight loss (Fig. 6A), mortality curve (Fig. 6B) and increased collagen deposition in the lung tissue as detected by Masson's trichrome staining (Fig. 6C). Interestingly, lymphocytes (Fig. 6D) and specifically NK and T cells (Fig. 6E) recruitment to the bronchoalveolar space (BAL), but not lung tissue/parenchyma were significantly reduced in Ebi3 −/− mice early point of infection. In line with these observations, NK cells in the bronchoalveolar space were not capable of mounting early-phase anti-viral effector functions as revealed by reduced intracellular IFN-γ in Ebi3 −/− mice at the early time point of infection (Fig. 6F). Ebi3 −/− mice displayed reduced IFN-γ production at the transcriptional level (Fig. 6G), suggesting IL-27 regulates NK cells responses at an early phase of influenza infection.

IL-27 regulates NKG2D-dependent cytotoxicity.
Our data suggest IL-27 enhance NKG2D-mediated cytokines generation. Earlier work suggested that IL-27-augmented cytotoxicity of NK cells is dependent on the upregulation of NKG2D ligands on the epithelial tumor and via ADCC 53 . We next examined the requirement of IL-27 in regulating NKG2D-mediated NK cells cytotoxicity. We used two stable EL4 cell lines (induced-self), RMA-S (missing-self) and YAC1 (non-self) in cytotoxicity assays. IL-2-cultured NK cells from Ebi3 −/− or Il27ra −/− mice have shown similar cytotoxicity towards EL4-H60 low , EL4-H60 high , RMA-S, and YAC1 cell lines as compared to WT mice, suggesting there is no inherent defect in the cytotoxic potentials of NK cells ( Supplementary  Fig. 5A). We found that recombinant IL-27 enhances the cytotoxic capacity of NKG2D-mediated WT and Ebi3 −/− but not Il27ra −/− mice IL-2-cultured NK cells (Supplementary Fig. 5B).

IL-27-dependent activation of NF-κB, T-bet, MafF, and Nrf2 regulates NK cells effector function.
Our data strongly suggest a critical role of IL-27 in regulating NK cells effector functions in vivo and in vitro. We www.nature.com/scientificreports www.nature.com/scientificreports/ next investigated the molecular mechanisms by which IL-27 regulate the effector functions of NK cells. Towards this, we purified NK cells from WT, and Ebi3 −/− mice on DPI 4 following influenza infection, purified mRNA, and performed gene array analyses for a panel of 48 transcription factors using Biomark chips. We found transcript levels of Ahr (Aryl hydrocarbon receptor), Ccnd1 (Cyclin D1), Elk1 (ETS domain-containing protein), and cKit (CD117) were considerably increased (Fig. 7A). In contrast, Foxo1 (Forkhead box 1), Foxo4 (Forkhead box 4), Irf4 (Interferon regulatory factor 4), Myc (bHLH transcription factor), Nfatc1 (NF-AT), cRel (NF-κB subunit), Rela (NF-κB subunit), and Tbx21 (T-bet) were substantially reduced (Fig. 7A). Among these, transcription factors such as c-Rel, Rel-a, T-bet are known to play a direct role in the transcription of IFN-γ and thus providing a mechanistic explanation of how IL-27 regulates cytokine production in NK cells. Also, our data showed a reduction in the expression of v-Maf musculoaponeurotic fibrosarcoma oncogene (Maf) homolog F (MafF) but not c-Maf in NK cells from Ebi3 −/− mice (Fig. 7A).
Nuclear factor E2-related factor 2 (Nrf2) binds to the ARE sequence as a heterodimer with one of the small bZIP proteins, Mafs, and activates specific gene transcriptions. We confirmed gene array data using RT-qPCR analyses of c-Maf, MafF, and MafK expression in sorted NK cells from influenza-infected WT and Ebi3 −/− mice (Fig. 7B). The gene array data also show that the expressions Nrf1 and Nrf2 were significantly reduced in Ebi3 −/− mice sorted NK cells (Fig. 7B), suggesting IL-27 stimulate NK cells through MafF/MafG-Nrf2 pathways. Nrf2 plays an indispensable role in augmenting the expression of Phase-II detoxifying and the anti-oxidant enzymes. Therefore, we investigated the expressions of quinone oxidoreductase (NQO1) and γ-glutamylcysteine ligase catalytic (GCLC). Although the transcript levels of Nqo1 did not change, levels of Gclc was significantly reduced in NK cells from Ebi3 −/− mice (Fig. 7C). In addition, we found that transcripts encoding both mitochondrial transcription factor A (TFAM) and carnitine palmitoyltransferase 1 (CPT1) were also significantly reduced in NK cells from Ebi3 −/− mice compared to that of WT (Fig. 7C). Our study demonstrates that IL-27 has an augmenting www.nature.com/scientificreports www.nature.com/scientificreports/ effect on NKG2D-mediated IFN-γ production from NK cells (Fig. 3). To define whether Nrf2 gene was a direct target of this combined activation, we stimulated NK cells with anti-NKG2D mAb in the presence or absence of IL-27. Presence of IL-27 significantly augmented the expression of Nrf2 (Fig. 7D). Collectively, these findings provide a novel insight into the stimulatory functions of IL-27 on NK cells in the trachea and alveolar space of the lungs during influenza infections. Role of these changes should be further explored and could partially be responsible for the reduction in the production of IFN-γ in NK cells from Ebi3 −/− mice.
Consistent with Ebi3 −/− mice data, NK cells from Il27ra −/− mice ( Fig. 8A) but not Il12a −/− mice (Fig. 8B) also have reduced expression of MafF. Nrf2 can form a heterodimer with small Mafs, specifically MafF and MafG 54 . To further explore the relevance of reductions in Nrf2 and MafF, we stimulated NK cells using plate-bound anti-NKG2D mAb along with small molecule compounds AI-1 (activates Nrf2 by covalently modifying Keap1, a negative regulator of Nrf2 and Oltipraz that activate anti-oxidant response element, Nrf2 55 . Presence of either AI-1 or Oltipraz enhanced the IFN-γ generation following NKG2D stimulation (Fig. 8C) validating the ability of IL-27 activating a proinflammatory cascade via MafF/MafG-Nrf2 pathways.

Discussion
NK cells provide the early line of defense against viral infections [56][57][58] . NK cells execute effector functions through their non-clonotypic activating receptors such as NKG2D, NCR1, and Ly49D. Cytokines are required to regulate the initiation, amplification, and the maintenance of transcriptional memory of an immune response 59 . Cytokines also play a crucial role in establishing 'immunological priming' . This phenomenon has been well-documented for over two decades for B, and T cells that form the basis for the adaptive immunity 60,61 . However, the occurrence of similar functional adaptations for NK cells is not yet fully understood. Myeloid cell-derived cytokines such as IL-12, IL-15, IL-18, and IL-27 coordinate the receptor-mediated activation of NK cells [62][63][64] . While the intricate temporal relationship between these cytokines and activation receptor-mediated stimulation of NK cells, in vivo, remains a paradox.
Whether and how the IL-12 family of cytokines plays an essential role in transcriptionally-priming NK cells are of active investigations 39 . IL-12 transcriptionally-prime both T and NK cells 65,66 . In T cells, cytokines function as a 'third signal' along with the activation receptor (primary signal) and co-stimulation (second signal) 67  www.nature.com/scientificreports www.nature.com/scientificreports/ coordinating the activation via receptors such as NKG2D or NCR1 is not clear. In this study, we utilized an influenza infection model and examined the role of IL-12 cytokine family in coordinating the activation of NK cells, which trafficked into the alveolar space (BAL) and lung tissue within 2-4 days post-infection. Entry of NK cells into the lumen-side of the trachea coincided with the destruction of epithelial cell layer 40,45 . Importantly, a significant percentage of NK cells in the lung tissue produced IFN-γ compared to that of spleens between DPIs 2-4, within the same mice. However, expression of CD107a (Lamp1) a surrogate marker for the release of the enzyme, granzyme B, from the cytotoxic vesicles peaked between DPIs 4-7. Through our earlier work, we showed that this increase in cytotoxic granules coincided with significant damage to the epithelial cell layer between DPIs 4-7 40 . Damage to the ciliary structure of the epithelial cells and an injury to the epithelial layer can make patients susceptible to secondary bacterial infections leading to developing pneumonia and death. Therefore, an efficient immune response to clear the pathogens and rapid regeneration of the epithelial layer is essential to re-establish this protective barrier.
Analyses of transcripts encoding Il12p35, Il12p40, Il23p19, and Il27p28 indicated that IL-27 was produced during the early phase of influenza infection between DPIs 4-7 both in the alveolar space and lung tissues. IL-27 is one of the IL-12 family members and a proinflammatory cytokine [70][71][72] . Further analyses demonstrated that a Ly6G + myeloid population was the predominant cell type that produced IL-27 as described earlier 43 . We predict that these Ly6G + myeloid cells are neutrophils and the support for this notion is coming from the findings that human neutrophils along with monocytes/macrophages in patients with melioidosis, a severe form of septicemia caused by the gram-negative bacterium, Burkholderia pseudomallei 73 . Production of IL-27 concurred with the expression of IL-27Rα (Wsx1) in NK cells on DPI 4 both in the alveolar space (BAL) and within the lung tissues. Earlier studies have demonstrated that Il-27 reduced lung inflammation by dampening neutrophil recruitment and T H 1 and T H 17 functions during influenza infection 43 . However, compared our study that focuses on the early phase of infection, this study explored the role of IL-27-induced IL-10 on T cells at DPI 8 43 .
Myeloid populations including dendritic cells produce IL-12, IL-18, and IL-27 that can function as the 'third' signal to prime effector lymphocytes 74,75 . In CD8 + T cells, IL-12 can open Ifng gene open along with select few hundred genes by chromatin remodeling, relieving gene repression, and allowing continued transcription by promoting augmented histone acetylation 74 . IL-12 along with IL-18 can promote the production of IFN-γ in T and NK cells [76][77][78] . However, the role of these cytokines in transcriptionally-priming NK cells and to augment NKG2D-dependent effector functions has not been investigated. Our data strongly suggest the presence of IL-27 during the activation of NK cells via NKG2D or Ly49D significantly augmented the production of IFN-γ, GM-CSF, RANTES, and MIP-1α. However, a similar additive effect was not observed when NK cells were stimulated with IL-27 along with IL-12 or IL-18, demonstrating a unique transcriptional and functional role of IL-27. Similarly, IL-6, another proinflammatory cytokine that shares its receptor subunit with the IL-27 receptor, did not augment IFN-γ production either alone or along with anti-NKG2D mAb-mediated activation of NK cells. Thus, it www.nature.com/scientificreports www.nature.com/scientificreports/ is possible that during an acute viral infection, neutrophils and monocytes/macrophages may provide the 'third' signal to NK cells at the site of infection through the production of IL-27. This function may be distinct from the role of dendritic cells that produce both IL-12 and IL-18 within the secondary lymphoid organs (draining lymph nodes) that is to provide a full-fledged activation of NK cells to mediate effector functions. Detailed transcriptomic and genomic analyses are required to further define the temporal and independent roles of these cytokines and their producers.
Earlier reports have suggested that IL-27 can selectively regulate NK cell subsets in human 33 . Through this study, we have found that IL-27 regulates the development of CD27 + CD11b + NK cell subset. Based on CD27 and CD11b expression, different organs of the body has unique NK cells subset repertoire 49 . Lung NK cells are largely CD27 − CD11b + , whereas BM NK cells are mainly CD27 + CD11b − . These subsets define maturation stages of NK cells 49,79,80 . CD27 high CD11b low define the earliest stage of NK cells maturation. Our data show that after influenza infection CD27 + CD11b + effector NK cells appear in the alveolar space and lung tissue of WT mice but not in Il27ra −/− mice, suggesting a critical role for IL-27 in regulating this NK subset. We found a defect in CD27 + CD11b + population; however, no changes were seen in the expression of NK cells activating or inhibitory receptor in the BM of naïve Il27ra −/− mice. It is possible that myeloid cells in the BM constitutively produce a low level of IL-27 to regulate NK cells maturation, thereby modulates NK effector function. It could also be possible that CD27 + CD11b + subset may be recruited to lung tissue from BM after influenza infection. It has also been shown that CD27 expression on NK cells determines migratory capacity and thus IL-27 may play a selective role in promoting the trafficking of a subset of NK cells to the site of infection 49,81 .
Lack of IL-27Rα (Wsx1) or IL-27 subunit EBI3 resulted in impaired NK cell-mediated effector functions and ineffective clearance of influenza infection. In line with earlier work using influenza infection on Il27ra −/− mice 43 , we found similar weight loss and mortality trend in Ebi3 −/− mice. Mixed chimera experiments using splenocytes from WT (CD45.1) and Il27ra −/− (CD45.2) further provide strong support that the reduced production of IFN-γ in NK cells from Il27ra −/− mice are cell-intrinsic. Our study strongly suggests that IL-27 plays a crucial role during the early phase of influenza infection (DPI 1-4). After DPI 5, the requirement for IL-27 decreased. On DPI 7, the absolute numbers of NK cells and their effector responses were comparable in WT and Ebi3 −/− or Il27ra −/− mice, suggesting a possible role of other cytokines/factors in regulating NK cells functions. It is also important to note, the adaptive immune response by T cells against infected epithelial cells and the B cells producing neutralizing antibody start to occur after DAP 7. Indeed, IL-27 is known to regulate cytoprotective IL-10 generation from T cells 43,82 . NK cells from CD27 −/− mice develop phenotypically normal with a similar or enhanced expression of activating or inhibitory receptor 81 . This observation is in line with our Il27ra −/− mice data, where there is no defect in NK cells activating or inhibitory receptor expression. Thus, IL-27 plays an essential role in the early NK cell-mediated functions as well as regulates adaptive immune response determining the pathophysiological outcome of influenza infection.
Transcriptional priming of effector lymphocytes by cytokines lead to genomic imprinting, resulting in unique cell fate decisions, and distinct functional outcomes. IL-12 mediates such prototypical chromatin modifications on signature cytokine genes such as Ifng and Il4 in T cells primarily via signal transducer and activator of transcription 4 (STAT4)-dependent chromatin remodeling 83,84 . Earlier studies have shown that the interaction of IL-27 with IL-27Rα/gp130 heterodimer leads to the recruitment of Jak1, Jak2, Tyk2 triggering the phosphorylation of STAT1, STAT2, STAT3, and STAT5 in CD4 + T cells 85,86 . STAT1/STAT3 heterodimers translocate into the nucleus, binds to Tbx21 (T-bet) promoter, increases T-bet levels, which in turn initiates the transcription of Ifng gene. Ifng gene promoter is known to contain at least three T-bet, two NF-AT, two NF-κB, one AP-1, and one CREB/ATF-2 binding sites. Thus, a substantial reduction in NF-AT, NF-κB, and T-bet can account for the significant reduction in IFN-γ production in NK cells from Ebi3 −/− or Il27ra −/− mice.
Given these premier roles of STAT proteins in the transcription of signature cytokines are well-established, we next focused on the role of IL-27 on Maf proteins. We found that NK cells from Ebi3 −/− as well as Il27ra −/− mice have reduced MafF expression with the highest fold change during influenza infection. Maf-F belongs to small Maf family which are basic-region and basic-leucine zipper (bZIP)-type transcription factors. Nuclear factor E2related factor 2 (Nrf2) heterodimerizes with small bZIP protein, Maf-F, and activates specific gene transcriptions. This notion is confirmed by our findings that the expression Nrf2 is also reduced in Ebi3 −/− mice, suggesting IL-27 is also functioning through these transcription factors. These findings are further validated by the earlier observation that Nrf2 indeed interacts with Maf-F and Maf-G 54 . In summary, our findings demonstrate the crucial role played by IL-27 during the early phase of influenza infection. IL-27 plays a central role in augmenting the stimulation via NKG2D and NCR1. Future studies are warranted to define the temporal and functional relationship of IL-27 with IL-12, IL-18, and IL-15.

Methods
Mice and stable cell lines. C57BL/6, B6.SJL (H-2 b CD45.1 + ), Il12a −/− mice and Ebi3 tm1Rsb (Ebi3 −/− ) mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in pathogen-free conditions at the Biological Resource Center at the Medical College of Wisconsin. Rag2 −/− γc −/− mice were purchased from Jackson Laboratory (Bar Harbor, ME). Generation of Il27ra −/− gene knockout mice has been described earlier 87 . Female and male mice between the ages of 6 and 12 weeks were used. EL4 (ATCC, Manassas, VA), EL4 H60 (a derivative of EL4 and was generated by our laboratory), RMA/S (a kind gift from Dr Nilabh Shastri, UC Berkeley), and YAC1 (ATCC, Manassas, VA) cells were maintained in RPMI-1640 medium containing 10% heat-inactivated FBS (Life Technologies, Carlsbad, CA). Those cell lines were periodically tested to exclude the possibility of mycoplasma contamination. The generation of stable H60-expressing EL4 cell lines has been described 88 .
Ethics Statement. All animal experiments in this study were conducted in accordance with the guidelines of the US Government Animal Welfare Act (AWA) 7 U.S.C. § 2131. Institutional Animal Care approved all animal www.nature.com/scientificreports www.nature.com/scientificreports/ protocols and Use Committees of the IACUC at the Medical College of Wisconsin, Milwaukee, WI. Medical College of Wisconsin is formally accredited by AAALAC and all the animal care and use-protocols used in this study fully adhere to the specified guidelines of AAALAC. The unique animal protocols that are approved by the IACUC and used in this study are AUA1500 and AUA1512.
In vivo infection. WT mice 6-8 weeks of age were deeply anesthetized and were intranasally challenged with 500 plaque-forming unit of PR8 virus in sterile phosphate-buffered saline in a total volume of 30 μl through one nostril as described 40 . Mock infections were carried out using only sterile phosphate-buffered saline without the virus. After infections, mice were observed for weight loss and mortality for two weeks.
BAL fluid collection. Mice were euthanized, and thoracic cavity was cut open, and a 1 cm incision was made parallel to the trachea through the fur of the mouse to expose it. A midline incision was made on the ventral aspect of the trachea slightly above the thoracic inlet 0.3 ml of phosphate-buffered saline-1% bovine serum albumin was infused into the lung through the thoracic inlet using a sterile 1 ml syringe. Lavage fluid was aspirated, aliquoted, and frozen until use.  Adoptive transfer. Single-cell suspensions from the splenocytes of B6.SJL (H-2 b CD45.1 + ) and Il27ra −/− (H-2 b , CD45.2 + ) mice were mixed 1:1 and adoptively transferred into Rag2 −/− γc −/− (CD45.2 + ) intravenously as described earlier 6 . Donor CD45.1 + and CD45.2 + NK cells were detected, and the generation of IFN-γ was analyzed by flow cytometry following day two post adoptive transfer.