Neutrophilic granulocytes modulate invariant NKT cell function in mice and humans - PubMed (original) (raw)

Neutrophilic granulocytes modulate invariant NKT cell function in mice and humans

Gerhard Wingender et al. J Immunol. 2012.

Abstract

Invariant NKT (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). In this study, we investigated the reverse relationship, showing that high neutrophil concentrations suppress the iNKT cell response in mice and humans. Peripheral Vα14 iNKT cells from spontaneously neutrophilic mice produced reduced cytokines in response to the model iNKT cell Ag α-galactosyl ceramide and expressed lower amounts of the T-box transcription factor 21 and GATA3 transcription factor 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 with spleen in wild-type mice. In vitro, the function of both mouse and human iNKT cells was inhibited by coincubation with neutrophils. This required cell-cell contact with live neutrophils. Neutrophilic inflammation in experimental peritonitis in mice decreased iNKT cell T-box transcription factor 21 and GATA3 expression and α-galactosyl ceramide-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.

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Figures

Figure 1. Decreased function of Vα14_i_ NKT cells from spontaneously neutrophilic mice

(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 _i_NKT cell cytokine production in spleen and liver was assessed (n=4 per group from 2 independent experiments). (C) _i_NKT 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 _i_NKT 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).

Figure 2. Decreased transcription factor expression in Vα14_i_ NKT cells from neutrophilic mice

(A,B) T-bet and GATA3 expression in _i_NKT cells recovered from thymus and spleen was analyzed by flow–cytometry after intracellular staining. (C,D) Both were significantly reduced in peripheral _i_NKT 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).

Figure 3. T-bet and GATA3 expression in Vα14_i_ NKT cells is modulated by neutrophil counts

(A) Wt and Itgb2−/− thymocytes were transferred to _i_NKT cell deficient Jα18−/− mice with normal neutrophil counts and splenic _i_NKT 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 _i_NKT 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 _i_NKT cells from neutrophilic mice than normal controls, but identical for wt and _Itgb2−/−i_NKT cells in the same environment (examples from n=4 transplanted mice per group). (C) Wt (CD45.1) and _Itgb2−/−i_NKT 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−/−i_NKT cells. T-bet and GATA3 transcription factor expression within _i_NKT cells on day 0 and day 3 after transfer is given (example of n=3 from 2 experiments).

Figure 4. Neutrophils modulate Vα14_i_ NKT cell transcription factor expression in vivo and in vitro

(A) T-bet and GATA3 expression in wt spleen and bone marrow (BM) derived _i_NKT 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.). _i_NKT 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).

Figure 5. Neutrophils modulate Vα24_i_ NKT cell function in vitro

(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 Vα24_i_ NKT cells (n=4). (B) T-bet expression of in vitro expanded Vα24_i_ NKT cells was assessed after 4 h co-incubation with neutrophils (PMN, 107/ml). (C) In vitro expanded Vα24_i_ 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 i_NKT cell (CD3+Vα24_i+CD19−) IFNγ was determined by flow cytometry (n=5). (E,F) _i_NKT 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 _i_NKT 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 _i_NKT 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).

Figure 6. Neutrophilic inflammation decreases Vα14_i_ NKT cell cytokine production in vivo

(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 _i_NKT 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 _i_NKT 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 _i_NKT cells 6 h after thioglycollate injection with and without anti-CXCR2 antibody treatment (typical of n=4 from 2 independent experiments).

Figure 7. Vα24_i_ NKT cell T-bet expression is decreased in neutrophilic peritonitis

(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) Vα24_i_ NKT cells in peripheral blood and peritoneal fluid were analyzed by flow cytometry. T-bet expression was significantly higher in peritoneal than peripheral blood _i_NKT 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).

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Neutrophilic granulocytes modulate invariant NKT cell function in mice and humans - PubMed (original) (raw)