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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: J Immunol. 2012 Mar 2;188(7):3000–3008. doi: 10.4049/jimmunol.1101273

Neutrophilic granulocytes modulate invariant natural killer T cell function in mice and humans

Gerhard Wingender *,†, Marcus Hiss §, Isaac Engel *, Konrad Peukert §, Klaus Ley ‡, Hermann Haller §, Mitchell Kronenberg *, Sibylle von Vietinghoff ‡,§
PMCID: PMC3311698  NIHMSID: NIHMS353803  PMID: 22387552

Abstract

Invariant natural killer T (iNKT) cells are a conserved αβTCR+ T cell population that can swiftly produce large amounts of cytokines, thereby activating other leukocytes, including neutrophilic granulocytes (neutrophils). Here we investigated the reverse relationship, showing that high neutrophil concentrations suppress the iNKT cell response in mice and humans. Peripheral Vα14i NKT cells from spontaneously neutrophilic mice produced reduced cytokines in response to the model iNKT cell antigen αGalCer and expressed lower amounts of the T-bet and GATA3 transcription factors than did wild-type controls. This influence was extrinsic, as iNKT cell transcription factor expression in mixed chimeric mice depended on neutrophil count, not iNKT cell genotype. Transcription factor expression was also decreased in primary iNKT cells from the neutrophil rich bone marrow compared to spleen in wild-type mice. In vitro, the function of both mouse and human iNKT cells was inhibited by co-incubation with neutrophils. This required cell-cell contact with live neutrophils. Neutrophilic inflammation in experimental peritonitis in mice decreasediNKT cell T-bet and GATA3 expression and αGalCer induced cytokine production in vivo. This was reverted by blockade of neutrophil mobilization. Similarly, iNKT cells from the human peritoneal cavity expressed lower transcription factor levels during neutrophilic peritonitis. Our data reveal a novel regulatory axis whereby neutrophils reduce iNKT cell responses, which may be important in shaping the extent of inflammation.

Introduction

Invariant natural killer T (iNKT) cells are T lymphocytes characterized by the expression of an invariant Vα14-Jα18 TCR rearrangement (Vα14i NKT cells) in mice and a homologous Vα24-Jα18 TCR (Vα24i NKT cells) in humans (1–3). They recognize glycolipids, such as the foreign antigen alpha-galactosyl ceramide (αGalCer), components of bacterial cell walls, and also endogenous lipids (2) when presented by CD1d, a non-polymorphic MHC class I antigen presenting molecule homolog (2, 4). iNKT cell antigenic stimulation rapidly induces effector functions such as cytokine production and cytotoxicity (1–3). iNKT cell cytokines include IFNγ, generally controlled in T lymphocytes by the transcription factor T-bet (T-box transcription factor 21), and IL-4 controlled by GATA3 (5). iNKT cells play a role in host defense particularly against some gram-negative bacteria such as Borrelia, Sphingomonas and Salmonella (6), but also gram-positive pathogens such as S. pneumonia, as well as virus (7). They are also involved in a range of chronic and acute inflammatory processes including allergic asthma, ischemia-reperfusion injury and atherosclerosis (1, 3). Clinical trials are currently exploring the potential of in vitro expanded iNKT cells for the treatment of metastatic neoplasms (8).

Neutrophilic granulocytes (neutrophils) are the most abundant innate immune cells in blood (9). While neutrophil counts are remarkably stable under resting conditions (10, 11), many bacterial and fungal infections, and also hormones such as catecholamines and glucocorticoids, rapidly up-regulate circulating blood neutrophil counts, making them a dynamic indicator of the extent of inflammation (9). In a number of adhesion molecule deficient mice, such as β2-integrin deficient (CD18, Itgb2−/−) and E- and P-selectin (Sele−/−Selp−/−) deficient mice, circulating neutrophil counts are spontaneously elevated (12).

iNKT cells modulate inflammatory processes, including neutrophilic inflammation. For example, iNKT cells stimulated neutrophil infiltration into the lung (13), the ischemic kidney (14), and the liver during listeria infection (15), but inhibited neutrophil invasion in cholestatic liver damage (16). Furthermore, iNKT cells altered cytokine production by neutrophils, inhibiting IL-10 and increasing IL-12 secretion (17). However, data on the reverse relationship, i.e. neutrophils modulating iNKT cells, has not been reported. Here we investigated whether neutrophil concentration influences iNKT cell function, and demonstrate a marked suppressive effect for both mouse and human iNKT cells.

Materials and Methods

Animals, bone marrow transplantation and adoptive thymocyte transfer

Animal experiments were approved by the Animal Care Committee at the La Jolla Institute for Allergy & Immunology (LIAI). Wild-type (wt, CD45.2) C57BL/6 mice and congenic B6. SJL-PtprcaPepcb/BoyJ (CD45.1) were from Jackson Labs (Bar Harbor, ME). B6.129-Tcra-Jtm1Tgi (Jα18−/−), β2-integrin (CD18)- deficient (Itgb2−/−) (96% B6) (18), and E- and P-selectin-deficient (Sele−/−selp−/−) (19) (on a C57BL/6 background for at least six generations) were bred at LIAI in specific-pathogen-free conditions. Mice were genotyped by PCR and used in age- and sex-matched groups. Lethal irradiations were performed in a 137Cesium irradiator (600 rad twice, three hours apart) and mice were reconstituted with un-fractioned bone marrow from wild-type (CD45.1+) and/or Itgb2−/−(CD45.2+) mice as indicated. Mice were treated with trimethoprim-sulfomethoxazole in drinking water for two weeks after transplantation. Experiments were performed 3–4 months after bone marrow transplantation. Adoptive transfer of CD5 enriched (Miltenyi Biotec, Auburn, CA) thymocytes and splenocytes was done after a single irradiation (400 rad) and cells analyzed at the indicated time points. Blood for leukocyte counts was taken via tail bleeding into EDTA-coated capillary tubes, analyzed with an automatic analyzer (Hemavet 950FS, DREW Scientific, Oxford, CT).

αGalCer application and peritonitis model

αGalCer ((2S,3S,4R)-1-O-(α-D-galactopyranosyl)-N-hexacosanoyl-2-amino-1,3,4-octadecanetriol) (KRN7000, Kirin Pharma, Gunma, Japan) was given by intravenous injection 90 min before analysis (1 µg/mouse). For induction of peritonitis, 1 ml BBL fluid thioglycollate medium (Becton-Dickinson, Sparks, MD) was injected intraperitoneally and cells were recovered by washing twice with 5 ml PBS at the indicated time points as described (20). Anti-CXCR2 (R&D Systems, Minneapolis, MN) was injected i.v. (30 µg/mouse). Cell preparation from liver, spleen and thymus was essentially as described (21).

Human samples

Blood and peritoneal fluid were recovered after local ethics board approval (MHH 2010/807) and written informed consent according to the declaration of Helsinki. In stable peritoneal dialysis (PD) patients (n=10, 64% male, mean age 55 years (range 20–73), mean time on PD 34 months (5–124), 9 previous peritonitis episodes in 4 patients) and patients with acute peritonitis (n=4, 75% male, mean age 53 years (range 28–69), mean time on PD 33 months (6–84), 3 previous peritonitis episodes in 1 patient), cells were recovered from peritoneal outflow of overnight dwells or the first peritonitic outflow before initiation of therapy. Leukocyte counts were assessed in the clinical laboratory at Hannover Medical School.

Ex vivo stimulation, human iNKT cell expansion, stimulation and cytotoxicity assay

For ex vivo stimulation, 106 murine splenocytes or thymocytes were co-incubated in 200 µl full RPMI medium (with penicillin/streptomycin and 10% FCS) with 2 × 106 bone marrow neutrophils (unless otherwise stated) recovered by flushing the bones with pyrogen-free HBSS without calcium and magnesium and enriched by density gradient centrifugation as described (22). Alternatively, bone marrow neutrophils were purified using a “Neutrophil Enrichment Kit” (#19762) (StemCell Technologies, Vancouver, Canada) according to the manufacturer’s instructions. Cells were counted in a hemocytometer and viability was assessed by trypan blue exclusion. Human peripheral blood mononuclear cells (PBMC) and granulocytes were isolated by density gradient centrifugation as described (23). 3 × 106 PBMC and 2.5 × 107 PMN were co-incubated in 500 µl full media.

For human iNKT cell expansion, total peripheral blood mononuclear cells (PBMC) were cultured in full medium with 100 ng/ml αGalCer for 7 days as described (24, 25), washed, and re-suspended in full media. Transwells (0.4 µm pore size) were from Corning (Corning, NY). For the cytotoxicity assay, fresh PBMC were incubated with 100 ng/ml αGalCer in full RPMI for 1h, washed and labeled with CFSE (Invitrogen, Carlsbad, CA) at 1 µM (αGalCer loaded) and 0.1 µM (control) according to the manufacturer's instructions (26). Stimulation of iNKT cells with αGalCer with and without neutrophils was done for 4h unless otherwise indicated, cytotoxicity was allowed to proceed for 6h before cells were washed, stained and analyzed by flow cytometry. Human IFNγ ELISA was from BioLegend (SanDiego, CA).

Cell preparation and staining for flow cytometry

The following antibodies were used for flow cytometry: anti-mouse: CD1d (1B1), CD3hε(145.2C11, 17A2), CD19 (1D3, 6D5), CD45 (30-F11), CD45.1-PE (A20), CD45.2 (104), CD69 (H1.2F3), CD122 (TM-β1), CD154 (CD40L, MR1), 7/4, Ly6G (1A8), Ly6C(HK1.4), Gr1 (RB6-8C5), T-bet (4B10), GATA3 (L50-823), TNFα(MP6-XT22), IL-4 (11B11), and IFNγ(XMG1.2), anti-human: CD1d (51.1), CD3ε (HIT3a), CD19 (HIB19), iVα24Jα18 (6B11), IFNγ (45.B3), T-bet (4B10). Antibodies were purchased from Abcam(Cambridge, MA), BD Biosciences (San Diego, CA), BioLegend (SanDiego, CA), eBioscience (San Diego, CA), or Invitrogen (Carlsbad, CA). Near infra-red LIVE/DEAD® Fixable Dead Cell Stain Kit (Invitrogen, Carlsbad, CA) and BD-Fix-Perm for intracellular staining (BD PharMingen, San Jose, CA) were used according to the manufacturer's instructions. Purification of mouse CD1d and preparation of αGalCer-loaded CD1d tetramers was as described. (27) Mouse iNKT cells were defined as CD8α−CD19−tetramer+TCRβ+, human iNKT cells as CD19−CD3ε+Vα24i+cells. Flow cytometry analysis was performed on a Becton-Dickinson FACS Calibur, Canto or LSRII. Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

Statistical Analysis

Two-tailed student t-test or ANOVA with appropriate post-hoc test was used as indicated in figure legends, p-values <0.05 were considered significant. Data are expressed as mean ±SEM. P values are indicated with *p<0.05, **p<0.01 and ***p<0.001.

Results

Decreased function of Vα14i NKT cells from spontaneously neutrophilic mice

Mice deficient for the β subunit of β2 integrins (CD18, Itgb2−/−) are spontaneously neutrophilic (figure 1A, (12, 18)). We did not observe a significant difference in the number of splenic iNKT cells, but the number of iNKT cells in the liver, where in mice a major iNKT cell population is located (1–3), was reduced in agreement with previous reports (28)(supplemental figure 1). Wt and neutrophilic Itgb2−/− mice were injected with the potent iNKT cell antigen αGalCer (2) and cytokine production was determined via intracellular staining. The proportion of iNKT cells that produced IFNγ, TNFα or IL-4 was significantly smaller in neutrophilic Itgb2−/− than in wt mice (figure 1B and supplemental figure 2). Similar decreases in cytokine production were seen in cells from spleen and liver (figure 1B). Baseline expression of the activation marker CD122, which constitutes a part of the IL-15 receptor and is important for iNKT cell survival, (29) and the TNF family member CD154 (CD40L) were similar (figure 1C and data not shown). However, while induction of CD69 after activation was normal, CD154 up-regulation on Itgb2−/−iNKT cells upon αGalCer stimulation was significantly reduced (figure 1C). Decreased cytokine production in response to αGalCer was apparent in both NK1.1 and CD4 negative and positive subpopulations to a very similar degree (data not shown). NK cells are rapidly activated downstream of iNKT cell stimulation, a process referred to as trans-activation (30, 31). In line with the reduced iNKT cell cytokine production and CD154 expression, NK cell trans-activation, measured by IFNγ production by NK cells, was greatly reduced in neutrophilic Itgb2−/− mice (figure 1D). These data demonstrate impaired activation and cytokine production by iNKT cells from neutrophilic mice in vivo.

Figure 1. Decreased function of Vα14i NKT cells from spontaneously neutrophilic mice.

Figure 1

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(A) Peripheral blood neutrophil counts (PMN) were significantly elevated in β2-integrin deficient (Itgb2−/−) compared to wild-type (wt) mice (n=18 (wt) and 3 (Itgb2−/−)). (B) Mice were injected with αGalCer 90 min before sacrifice and iNKT cell cytokine production in spleen and liver was assessed (n=4 per group from 2 independent experiments). (C) iNKT cell surface expression of the activation markers CD69 and CD154 without and after 90 min in vivo activation with αGalCer (n=2). (D) NK cell trans-activation is a major amplifying loop after iNKT cell stimulation. Production of IFNγ by splenic NK cells 90 min after αGalCer in wt and Itgb2−/− mice (typical example from n=4 in 2 independent experiments).

Decreased T-bet and GATA3 transcription factor expression of Vα14i NKT cells from neutrophilic mice

To gain insight into the mechanism for the decreased cytokine responses by Vα14i NKT cells, we analyzed the expression of the T-bet and GATA3 transcription factors, critical for IFNγ and IL-4 expression, respectively, in conventional CD4+ T lymphocytes (5). Transcription factors were analyzed by flow-cytometry after intracellular staining in thymic and peripheral iNKT cells from Itgb2−/− and wild-type (wt) mice (figure 2A,B). Itgb2−/− splenic iNKT cells contained significantly less of either transcription factor than wild-type cells (figure 2C,D). In contrast, the T-bet and GATA3 expression levels in thymic iNKT cells were similar in wt and Itgb2−/−mice (figure 2), arguing against a developmental cause of this difference. Also, CD1d expression was not different in spleens or thymus of Itgb2−/− compared to wild-type mice (data not shown).

Figure 2. Decreased transcription factor expression in Vα14i NKT cells from neutrophilic mice.

Figure 2

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(A,B) T-bet and GATA3 expression in iNKT cells recovered from thymus and spleen was analyzed by flow–cytometry after intracellular staining. (C,D) Both were significantly reduced in peripheral iNKT cells recovered from spleen of Itgb2−/− compared to wt mice (expressed as mean fluorescence intensity relative to wt cells) (n=8–10 from 3–4 independent experiments).

To test whether or not decreased iNKT cell transcription factor expression was due to β2-integrin deficiency, we employed mice deficient in endothelial and platelet, but not leukocyte selectins (19). These mice were neutrophilic to a similar degree as Itgb2−/− mice (supplemental figure 3A,(12)). iNKT cell characterization of this mouse strain is shown in supplemental figure 1. Also in this strain, T-bet and GATA3 expression in splenic and hepatic iNKT cells were reduced (supplementary figure 3B,C), suggesting neutrophilia as a possible cause of the observed iNKT cell phenotype.

Modulation of Vα14i NKT cells by neutrophils is cell extrinisic and reversible

To test if the phenotype of peripheral iNKT cells from neutrophilic Itgb2−/− mice was cell intrinsic or environmental, we employed adoptive thymocyte and splenocyte transfers and bone marrow transplantations. We transferred thymocytes from wt or Itgb2−/− mice into normal and neutrophilic hosts. Thymocytes were used as the cell source as they expressed similar levels of T-bet and GATA3 in both donor strains (figure 2). Four to six weeks later, donor and recipient iNKT cell T-bet and GATA3 expression levels were analyzed by flow cytometry. Wt and Itgb2−/−thymocytes transferred into iNKT deficient host mice (Jα18−/−), which have normal neutrophil counts (data not shown), expressed similar levels of T-bet and GATA3 in iNKT cells (figure 3A). When thymocytes were transferred to mice with endogenous iNKT cell populations, host neutrophil counts had a similar influence. For example, Itgb2−/−thymocytes transferred into wt hosts retained relatively higher T-bet and GATA-3 expression, similar to their host counterparts (figure 3B). To create a cohort of neutrophilic recipients, we created bone marrow chimeric recipients with 100% Itgb2−/− bone marrow. Transfer of wt thymocytes into these neutrophilic host mice resulted in lower T-bet and GATA3 transcription factor expression, similar to the host Itgb2−/−iNKT cells (figure 3B). These data suggest that the decreased expression levels of T-bet and GATA3 observed in Itgb2−/−iNKT cells was not a cell intrinsic phenomenon, but rather a consequence of the environment.

Figure 3. T-bet and GATA3 expression in Vα14i NKT cells is modulated by neutrophil counts.

Figure 3

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(A) Wt and Itgb2−/− thymocytes were transferred to iNKT cell deficient Jα18−/− mice with normal neutrophil counts and splenic iNKT cells analyzed after 6 weeks (an example of n=4 from 2 experiments) for T-bet (left) and GATA3 (right). (B) To test for wild type iNKT cell transcription factor expression in neutrophilia, wt (CD45.1) thymocytes were transferred into normal wt (CD45.2) hosts (top panel) or neutrophilic hosts previously reconstituted with Itgb2−/− (CD45.2) bone marrow (bottom panel). Four weeks later the expression levels of T-bet and GATA3 were analyzed. They were lower in splenic iNKT cells from neutrophilic mice than normal controls, but identical for wt and Itgb2−/−iNKT cells in the same environment (examples from n=4 transplanted mice per group). (C) Wt (CD45.1) and Itgb2−/−iNKT cell T-bet and GATA3 from mixed 50%wt/50%Itgb2−/− bone marrow chimeras were indistinguishable (examples from n=9). (D) Wt (CD45.1) and Itgb2−/− (CD45.2) splenocytes were transferred into Jα18−/− mice to assess the effect of normal neutrophil counts on peripheral Itgb2−/−iNKT cells. T-bet and GATA3 transcription factor expression within iNKT cells on day 0 and day 3 after transfer is given (example of n=3 from 2 experiments).

To confirm these results in an experimental system where wt and Itgb2−/− iNKT cells develop in the same animal, we reconstituted lethally irradiated wt mice with bone marrow from wt and Itgb2−/−mice mixed at an equal ratio. As described (12), transfer of 50% wt/50% Itgb2−/− bone marrow resulted in normal peripheral blood neutrophil counts (in wt bone marrow transplanted mice 2.1±0.2 PMN/µl (mean±SEM), in 50% wt/50% Itgb2−/− 2.4±0.4, and inItgb2−/− bone marrow transplanted mice 16.9±6.5 PMN/µl (mean±SEM)). Consistent with the results from transfer of mature cells, in mixed bone-marrow chimeras, the expression of T-bet and GATA3 in wt and Itgb2−/− iNKT cells from the same mouse were similar, irrespective of their genotype (figure 3C).

To test if the decrease in T-bet and GATA3 expression in peripheral iNKT cells in neutrophilic Itgb2−/− mice was reversible, we adoptively transferred splenocytes from wt and Itgb2−/− mice at an equal ratio into iNKT cell deficient, normo-neutremichost mice. This completely normalized the transcription factor expression of the Itgb2−/− iNKT cells by day three after transfer (figure 3D), indicating that the down-regulation was a reversible phenotype. It is of note that both wt and Itgb2−/− bone marrow neutrophils were devoid of CD49d (data not shown), which has recently been proposed as a marker of myeloid derived suppressor cells (32), a cell type induced in a variety of pathophysiologic conditions, but not present in healthy mice and humans (33–35).

Together, these results indicate that lower iNKT cell T-bet and GATA3 expression in neutrophilic mice is not cell-intrinsic, but determined by the environment, and they suggest neutrophil counts as the likely responsible factor.

Neutrophils modulate Vα14i NKT cell T-bet and GATA3 from wild type mice

The murine bone marrow harbors large numbers of mature neutrophils (11). Therefore, if exposure to increased numbers of neutrophils decreased T-bet and GATA3 expression, wild type bone marrow Vα14i NKT cells might display lower transcription factor expression than cells from other organs. Flow cytometric analyses indeed showed decreased expression of T-bet and GATA3 in bone marrow iNKT cells compared to cells from spleen (figure 4A) and thymus (data not shown) of the same wt animal. To test whether or not such down-regulation could also be induced in vitro, primary mouse splenocytes and thymocytes were co-incubated with mouse bone marrow neutrophils in vitro. This decreased T-bet and GATA3 expression in both splenic and thymic Vα14i NKT cells (figure 4B), demonstrating that high local concentrations of resting neutrophils can induce iNKT cell down-regulation of these transcription factors also in unmanipulated wt mice and that it can be replicated in vitro in short term cultures.

Figure 4. Neutrophils modulate Vα14i NKT cell transcription factor expression in vivo and in vitro.

Figure 4

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(A) T-bet and GATA3 expression in wt spleen and bone marrow (BM) derived iNKT cells. (B) Mouse splenocytes and thymocytes were cultured in the presence or absence of neutrophils (107/ml) for 6 h and T-bet and GATA3 expression determined (examples from 2 independent experiments). (C) Wt splenocytes were cultured with increasing concentrations of wt bone marrow neutrophils purified by density gradient BM PMN) and negative antibody selection (BM-PMN neg.-sel.). iNKT cell T-bet and GATA3 expression is shown in relation to actual neutrophil concentration calculated from flow cytometry (Ly6C+ Ly6G+ 7/4+ cells) (example from n=3). (D) Co-culture with neutrophils (5 × 105/ml for 4 h) was performed with or without physical contact (tw = transwell with 0.4 µm pore size, one of two independent experiments shown).

Most causes of neutrophilia in vivo also involve activation of neutrophils by inflammatory mediators leading to activation, degranulation and distinct forms of cell death (36). Stimulation of neutrophils by TNFα, N-formylmethionyl-leucyl-phenylalanine or phorbol-12-myristate-13-acetate did not alter the neutrophil-mediated decrease in T-bet expression (data not shown). However, spontaneously apoptotic and heat killed neutrophils lost their ability to affect Vα14i NKT cell T-bet and GATA3 expression in our in vitro co-culture systems (data not shown), indicating that live neutrophils were required. Even after density gradient purification, bone marrow contains other cell types. We therefore employed negative selection to obtain highly purified neutrophils. Comparing the decrease in T-bet and GATA3 transcription factor expression relative to absolute neutrophil numbers, measured by flow cytometry, revealed highly similar dose responses (figure 4C), indicating that indeed the neutrophils in the mixture were responsible for the iNKT- cell inhibitory effect.

Co-culture with neutrophils did not alter PD-1, BTLA, GITR or CD152 on the iNKT cell surface (data not shown). Wt and CD1d−/− bone marrow neutrophils did not differ in their ability to induce down-regulation of T-bet and GATA3 in iNKT cells (data not shown). To further investigate whether iNKT cell inhibition was due to a soluble factor or cell-cell contact dependent, neutrophils were separated from Vα14i NKT cells using a transwell (figure 4D). This completely abolished the neutrophil inhibitory effect on Vα14i NKT cell transcription factor expression.

Neutrophils modulate Vα24iNKT cell function in vitro

To test if neutrophils would similarly impact primary human Vα24i NKT cells, peripheral blood mononuclear cells were co-cultured with elevated human neutrophil concentrations for 4 h. This significantly decreased iNKT cell T-bet and GATA3 expression (figure 5A). Similar to the murine system, neutrophil stimulation did not alter the neutrophil-mediated decrease in Vα24i NKT cell T-bet expression and IFNγ production (data not shown).

Figure 5. Neutrophils modulate Vα24i NKT cell function in vitro.

Figure 5

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(A) Human peripheral blood mononuclear cells isolated by density gradient centrifugation were cultured in the presence or absence of neutrophils (5 × 107/ml) for four hours. Neutrophils significantly decreased T-bet and GATA3 expression in Vα24i NKT cells (n=4). (B) T-bet expression of in vitro expanded Vα24i NKT cells was assessed after 4 h co-incubation with neutrophils (PMN, 107/ml). (C) In vitro expanded Vα24i NKT cells were exposed to αGalCer (100 ng/ml) in the presence and absence of neutrophils (PMN, 107/ml). IFNγ concentration in the supernatant after 4 h was determined by ELISA (n=3). (D) Individual iNKT cell (CD3+Vα24i+CD19−) IFNγ was determined by flow cytometry (n=5). (E,F) iNKT cell cytotoxicity against fresh PBMC loaded with 100ng/ml αGalCer. PBMC were differentially stained with CFSE (1 µM for αGalCer exposed, 0.1 µM for control cells), mixed and incubated in full RPMI for 6 h with and without iNKT cells and freshly isolated neutrophils (PMN, 106/ml). The proportion of αGalCer labeled (CFSEhi) and CFSElow (control PBMC) was determined by flow cytometry and is expressed as αGalCer labeled relative to control PBMC in E (n=3 independent experiments). (G) αGalCer stimulation of in vitro expanded human iNKT cells was conducted in the presence or absence of 107/ml neutrophils with or without physical contact (transwell with 0.4 µm pore size) for 10 h (n=4, Bonferroni after 1 way ANOVA).

Vα24i NKT cells are infrequent in human peripheral blood, but can be expanded in vitro (24, 25). In our hands, this expansion resulted mainly in IFNγ producing iNKT cells with IL-4 barely above detection limit. When in vitro expanded human iNKT cells were exposed to neutrophils, T-bet expression decreased significantly (figure 5B). Re-stimulation with αGalCer in the presence of neutrophils resulted in significantly less IFNγ secretion to the supernatant than control cells (figure 5C). Individual cell IFNγ production was assessed by flow cytometry after intracellular staining. Co-incubation with neutrophils resulted in a significantly smaller proportion of IFNγ+iNKT cells (figure 5D). Vα24i NKT cell cytotoxicity was assessed after six hours co-culture with αGalCer loaded, CFSE labeled PBMC. Addition of freshly isolated neutrophils significantly decreased αGalCer-mediated cytotoxicity (figure 5E,F). However, the use of neutrophil derived supernatants, or the separation of the neutrophils in culture using a transwell, abolished their effect on Vα24i NKT cells (figure 5G and data not shown). These data show that inhibition of iNKT cells by high neutrophil concentrations applies similarly to mouse and human cells and demonstrate that cell-cell contact is required for neutrophils to impair iNKT cell function.

Neutrophilic inflammation decreases Vα14i NKT cell cytokine production in vivo

To investigate the effect of inflammatory neutrophilia on iNKT cell function in vivo, peritonitis was induced in wt mice by injection of thioglycollate (figure 6). After three days, iNKT cells were stimulated in vivo by injection of αGalCer and analyzed after 90 min. αGalCer did not alter the inflammatory peritoneal cavity leukocyte count (figure 6A). However, iNKT cell cytokine production was significantly lower in cells recovered from a neutrophilic compared to a normal environment (figure 6B). iNKT cell T-bet and GATA3 expression levels were also decreased in mice with peritonitis, and correlated well with iNKT cell cytokine production (figure 6C). Accumulation of peritoneal leukocytes, mostly neutrophils, was significant at six hours after thioglycollate injection (figure 6D) (20). When we assessed the time course of T-bet and GATA3 expression levels in peritoneal iNKT cells, we found them to be already decreased at this time (figure 6E). Neutrophil recruitment in peritonitis is to large degree CXCL1 chemokine dependent and can be prevented by CXCR2 chemokine receptor blockade (figure 6D) (20). CXCR2 blockade also normalized the expression of the transcription factors in peritoneal iNKT cells (figure 6E), demonstrating that the reduction was not due to a direct effect of thioglycollate on iNKT cells. Altogether, these data indicate that acute, inflammatory neutrophilia induces down-regulation of T-bet and GATA3 and impaired cytokine production following iNKT cell antigen stimulation in vivo.

Figure 6. Neutrophilic inflammation decreases Vα14i NKT cell cytokine production in vivo.

Figure 6

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(A) Peritoneal cell counts were elevated three days after intraperitoneal thioglycollate injection, but un-affected by additional αGalCer injection 90 min before sacrifice (n=4–8). (B) αGalCer-stimulated splenic iNKT cell cytokine expression was lower after induction of peritoneal leukocyte accumulation (n=6–9 from 2 independent experiments). (C) T-bet and GATA3 expression (expressed as % of ctrl iNKT cells) was also reduced in mice with peritonitis compared to ctrl and correlated with reduction in cytokine production. (D) Peritoneal leukocyte recruitment 6 h after thioglycollate injection with and withoutblockade of neutrophil recruitment by an anti-CXCR2 antibody (Bonferroni post ANOVA). (E) T-bet and GATA3 expression in peritoneal iNKT cells 6 h after thioglycollate injection with and without anti-CXCR2 antibody treatment (typical of n=4 from 2 independent experiments).

Vα24i NKT cell T-bet expression is decreased in neutrophilic peritonitis

To test whether inflammatory neutrophilia in vivo also decreased human iNKT cell function, we investigated peripheral blood and peritoneal cavity Vα24i NKT cells at baseline and in peritonitis. Peritoneal iNKT cells were recovered from the outflow fluid of patients treated with chronic peritoneal dialysis for renal replacement therapy. Total peritoneal fluid leukocyte concentrations were very low under resting conditions (figure 7A,B). Most leukocytes from peritoneal cavity of stable peritoneal dialysis patients were lymphocytes (data not shown), most likely a resident population (37, 38). Vα24i NKT cells in the human peritoneal cavity have not been described, but were readily detected among CD3ε+ T cells (figure 7C). Conventional T cells in human peritoneum predominantly produce IFNγ(38). Indeed, expression of the TH2 transcription factor GATA3 was at the detection limit in peritoneal Vα24i NKT cells (data not shown). However, the TH1 transcription factor T-bet was expressed and was significantly higher in Vα24i NKT cells from the peritoneal cavity than from peripheral blood (figure 7D). Acute peritonitis results in a massive neutrophil influx into the peritoneum, accounting for >90% of the leukocytes (figure 7B and data not shown), resulting in a concentration (cells/µl) similar to peripheral blood. In peritonitis, T-bet expression in peritoneal Vα24i NKT cells was similar to or lower than in blood iNKT cells from the same patient (figure 7D). These data show that T-bet expression of primary Vα24i NKT cells from the neutrophil-poor peritoneal cavity is higher than in peripheral blood and decreases in response to neutrophilic inflammation in humans in vivo.

Figure 7. Vα24i NKT cell T-bet expression is decreased in neutrophilic peritonitis.

Figure 7

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(A,B) Leukocyte concentrations in peripheral blood were significantly higher than in peritoneal fluid (PF) of chronic peritoneal dialysis patients, peritonitis significantly increased leukocyte counts in the peritoneal cavity. (C,D) Vα24i NKT cells in peripheral blood and peritoneal fluid were analyzed by flow cytometry. T-bet expression was significantly higher in peritoneal than peripheral blood iNKT cells in stable patients (n=10) but not during peritonitis (n=4) (##p<0.01 blood versus PF in healthy patients, *p<0.05 control versus peritonitis PF).

Discussion

Our data show for the first time that neutrophilic granulocytes inhibit iNKT lymphocyte function in mice and humans, both under resting conditions and during inflammation in vivo. We observed down-regulation of iNKT cell baseline T-bet and GATA3 expression, and decreased responses to the iNKT cell antigen αGalCer, regarding both cytokine production and CD154 (CD40L) up-regulation. NK cell trans-activation to produce IFNγ, an important pathway for amplification of immune responses downstream of iNKT cell activation, was also impaired. These effects were reversible.

Mouse and human iNKT cells and neutrophils differ in numbers and tissue distribution. While neutrophils are more frequent in human than mouse blood, and constitute the most common leukocyte population there, the mouse bone marrow contains a large pool of mature neutrophils (11). However, similar effects of neutrophil concentration were observed in mice and humans. The expression by iNKT cells of T-bet and GATA3 was decreased in the neutrophil-rich bone marrow environment in mice below the amount in splenic iNKT cells under resting conditions. Similarly, human blood iNKT cells expressed lower T-bet than iNKT cells from the relatively neutrophil poor peritoneal cavity. The down-regulation of these transcription factors and iNKT cell functions could readily be reproduced in vitro by exposing human or mouse iNKT cells to neutrophils. Taken together, our data indicate similar iNKT cell responses to neutrophil concentrations in both species.

Depression of iNKT cell function in neutrophilic mice could have been a developmentally induced phenotype. However, the expression levels of T-bet and GATA3 were similar in the thymus of wt and neutrophilic mice. Furthermore, when we tested for the effect of neutrophilia on mature cells, iNKT cell depression was readily observed in mature splenic and thymic Vα14i NKT cells in vivo and in vitro, and in human peripheral blood Vα24i NKT cells. Also, Vα14i NKT cells from neutrophilic mice readily recovered T-bet and GATA3 expression after adoptive transfer to a wild type environment. These data suggest that neutrophils provide a short term and reversible modulatory effect on iNKT cell activation. Acute neutrophilia is an early and often short-lived response to infection, stress and trauma (9). In such conditions, neutrophils are often activated, degranulate and produce reactive oxygen species. Activated neutrophils retained their inhibitory function for iNKT cells, although it was not increased, indicating that iNKT cell inhibition by neutrophils is not restricted to resting conditions, and therefore could be of general importance during inflammations in vivo.

T-bet and GATA3 transcription factors are critical for the expression of IFNγ and IL-4/IL-13, respectively, in peptide-reactive or conventional CD4+ T lymphocytes (5). T-bet and GATA3 deficiency severely affect Vα14i NKT cell differentiation (39, 40) and therefore data on their role in mature iNKT cells is limited. However, retroviral-mediated expression of T-bet (41) or GATA3 (42, 43) in Vα14i NKT cells increased cytokine production in response to αGalCer in vitro, suggesting a functional role in cytokine production by activated iNKT cells. Our data (figure 6C) demonstrate a correlation of T-bet and GATA3 expression with iNKT cell cytokine content assessed by intracellular cytokine staining. However, given the broad suppressive effect of neutrophils on the production of other cytokines by iNKT cells, including TNFα, IL-13 and GM-CSF, it is likely that interaction with neutrophils leads to down-regulation of additional transcription factors.

It is of note that CD49d negative granulocytes isolated from normal mice and a large number of healthy donors suppressed iNKT cell function in our study. This suggests the suppressive cells are not exclusively MSDC, which although heterogeneous, are at least in part a CD49d+(32) population with both granulocytic and monocytic phenotypes. Furthermore, MSDC typically are found in disease, e.g. infection and different forms of malignancies, and they are not usually present in healthy organisms, which provided the sources of the neutrophils in our study (33–35). Regulation of conventional T lymphocytes by neutrophils has been suggested by enhanced T cell activation in neutropenic animals (44, 45). Data on a mechanism for this, however, are controversial. Some reports described roles for soluble molecules such as NO induced by IFNγ(46), IL-10 (47), or arginase liberated from dying cells (48). However, in other settings, direct interaction of neutrophils and antigen presenting cells appeared to be required (17, 45). In our experiments, iNKT cell inhibition by neutrophils depended on the presence of live cells and required cell-cell contact. Currently no cell-cell contact dependent mechanism for T cell suppression by neutrophils has been described and therefore the inhibition of iNKT cells by neutrophils we observed here likely represents a novel mechanism. Potential candidates for cell surface molecules that could be important in the iNKT cell - neutrophil interaction are inhibitory molecules that have been reported to be involved in T cell - APC interactions (49–53). While we did not observe changes in PD-1, BTLA, GITR or CD152 iNKT cell surface expression, whether they have a role in iNKT cell - neutrophil interaction remains to be established.

Inhibition of iNKT cells by neutrophilic granulocytes could play an important role in a number of pathophysiologic conditions that activate iNKT cells. Inhibition of iNKT cells by neutrophilia may be beneficial in settings of otherwise overwhelming iNKT cell activation, e.g. during sepsis and bronchial asthma (1, 3), and it may contribute to reduced iNKT cell function in chronic inflammatory conditions such as atherosclerosis (54). However, an increased concentration of neutrophils also may inhibit beneficial iNKT cell responses, such as cytotoxicity or cytokine secretion required for the elimination of malignancies (1, 3) or pathogenic bacteria (7). Interestingly, several, although not all (55), pathogens where host protection requires an iNKT cell response, such as Borrelia burgdorferi and Rickettsiae, and several viral infections, do not usually elicit a strong neutrophilic response (7).

In summary, our report describes inhibition of iNKT cell activation by neutrophils, both in vitro and in vivo, and in mice as well as humans in both steady state conditions and during inflammatory conditions. Deliberate modulation of this interaction may be potentially beneficial for induction of stronger iNKT cell responses to neoplasms and pathogens, and for limiting allergic or autoimmune activity.

Supplementary Material

1

Acknowledgements

We would like to thank blood donors, patients and staff at Hannover Medical School peritoneal dialysis unit for participating in this study, Archana Khurana for preparation of the CD1d tetramers and Hui Ouyang and Barbara Hertel for expert technical assistance.

Grant Support:

This work was supported by an Outgoing International Fellowship by the Marie Curie Actions (G.W.), NIH grants RO1AI45053, R37AI71922 (M.K.) and HL58108 (K.L), and grants from Deutsche Forschungsgemeinschaft and Hannover Medical School (HiLF 09.10) (S.v.V.).

Nonstandard Abbreviations

iNKT

invariant natural killer T cell

αGalCer

alpha-galactosyl ceramide

T-bet

T-box transcription factor 21

Itgb2

β2-integrin gene

PD

peritoneal dialysis

Footnotes

The authors have no competing financial interests regarding this work.

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