The Natural Cytotoxicity Receptor 1 Contribution to Early Clearance of Streptococcus pneumoniae and to Natural Killer-Macrophage Cross Talk

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligandhigh lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-liganddull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC

NK cells activation and cytokines mRNA levels in lungs following intranasal challenge with <i>S. pneumoniae</i>.

<p>Lungs were harvested from <i>Ncr1</i>^+/gfp (Het group) and <i>Ncr1</i>^gfp/gfp (KO group) C57BL/6 mice 3h after intranasal inoculation with 5×10⁷ CFU of <i>S. pneumoniae</i> strain WU2, and from non infected mice. (A) NK cells were gated as CD45⁺NK1.1⁺ and analyzed for the expression of membrane-associated CD107a by flow cytometry. The bar graphs show the fraction of CD107a positive NK cells (average of 3 mice per group, ±SD). ** p<0.01 compared to Het group in 3h-infected mice (ANOVA test). Total RNA was extracted from naïve lungs and from 3h-infected lungs of <i>Ncr1</i>^+/gfp (Het) and <i>Ncr1</i>^gfp/gfp (KO). Infection was performed with 5×10⁷ CFU of <i>S</i>. <i>pneumoniae.</i> Level of cytokines mRNA of IFNγ (n = 4), TNFα (n = 4) and IL-6 (n = 3) were analyzed by RT-PCR and calibrated to mRNA level of GAPDH. Results are from one representative experiment of two. *** p<0.001, ** p<0.01, compared with the Het group +SD (ANOVA test).</p

NCR1 involvement in NK cells activation following infection of BMMQ/ BMDC with <i>S. pneumoniae.</i>

<p>(A-D) Six-day culture of BMMQ and BMDC grown from <i>Ncr1</i>^+/+ BM cells (C57BL/6) were stained with mNCR1-Ig, mNKG2D-Ig and LIR1-Ig fusion proteins. Shown are representative overlays of F4/80⁺CD115⁺ BMMQ (A, C) and CD11c⁺ BMDC (B, D) demonstrating staining with mNCR1-Ig (solid line, A-D), mNKG2D-Ig (dotted line, C–D), LIR1-Ig (dashed line, A–B) and with goat anti-human IgG (grey-filled, A–D). Shown is one representative experiment out of four to five independent experiments. In some experiments, NKG2D-Ig staining was higher compared to NCR1-Ig staining. (E–F) Six-day culture- BMMQ (E) and -BMDC (F) grown from <i>Ncr1</i>^+/+ BM cells (C57BL/6) were infected with 1 MOI of <i>S. pneumoniae</i> for 1.5 h, then bacteria were removed and antibiotics were added. In parallel, NK cells were isolated from splenocytes of <i>Ncr1</i>^+/+ (‘NK WT’) and <i>Ncr1</i>^gfp/gfp (‘NK KO’) C57BL/6 mice. Isolated NK were incubated with uninfected BMMQ or BMDC, or with <i>S. pneumoniae</i>-infected BMMQ or BMDC (‘BMMQ+SP’, ‘BMDC+SP’) after removal of bacteria for additional 18 hours. NK-BMMQ ratio was 1∶4 and NK-BMDC ration was 1∶10. IFNγ concentration levels in the co-culture supernatants were determined by ELISA. Results are from one representative experiment out of two. ** p<0.01 compared with IFNγ levels of NK WT incubated with infected BMMQ/BMDC, ± SD (ANOVA test).</p

NCR1 ligand expression on <i>S</i>. <i>pneumoniae</i> and naïve macrophages and DC.

<p>(A) Fixed bacteria were stained with mNCR1-Ig (left panel) and mNKG2D-Ig (right panel) fusion proteins. Shown are representative overlays of demonstrating staining with the fusion protein (bold line) and with goat anti-human IgG (plain line). Both lines overlapped. Cells from lung lavage (B, E, F), BM (C, E, F, G) and spleen (D) of C57BL/6 <i>Ncr1</i>^+/+ (WT), <i>Ncr1</i>^+/gfp (Het) and <i>Ncr1</i>^gfp/gfp (KO) naive mice were stained with mNCR1-Ig, mNKG2D-Ig, LIR1-Ig (B) or CD99-Ig (C, D) and with macrophages/DC markers (F4/80, CD115, CD11c). Panels B-F were gated on macrophages (F4/80⁺CD115⁺) and panel G was gated on DC (CD11c⁺). Panels B-D show overlays of mNCR1-Ig staining as follows: bold line for <i>Ncr1</i>^+/+ (B1, C1, D1) or <i>Ncr1</i>^gfp/gfp (B2, C2, D2), hairline for <i>Ncr1</i>^+/gfp (C1, D1); LIR1-Ig staining (grey line for B1, B2); CD99-Ig staining (dashed line for C1, C2, D1, D2); and goat anti-human IgG (grey line for B3, C3, D3). mNKG2D-Ig staining for all mice types is shown is the right panel (B3, C3 and D3: <i>Ncr1</i>^+/+ in bold line, <i>Ncr1</i>^+/gfp in dashed line and <i>Ncr1</i>^gfp/gfp in dotted line). The bar graphs (E–G) show the fraction of NCR1-ligand^high BAL and BM-gated macrophages for <i>Ncr1</i>^+/+ vs. <i>Ncr1</i>^gfp/gfp (E), the fraction of NCR1-ligand^high BAL and BM-gated macrophages for <i>Ncr1</i>^+/gfp vs. <i>Ncr1</i>^gfp/gfp (F) and NCR1-ligand^high BM-gated DC (G). Results in E are normalized to <i>Ncr1</i>^+/+ (n = 12 and 16 mice per group). Results in F are normalized to <i>Ncr1</i>^+/gfp (n = 12 and 15 mice per group). *** p<0.001 and * p<0.05 compared to WT/Het group +SD (two-tailed student t-test). NS, non significant compared to WT/Het group. Results (B–D) are from one representative experiment out of six to seven.</p

Survival of mice after intranasal inoculation with <i>S. pneumoniae</i>.

<p>(A) <i>Ncr1</i>^+/+ (WT, n = 13), <i>Ncr1</i>^+/gfp (Het, n = 6) and <i>Ncr1</i>^gfp/gfp (KO, n = 12) C57BL/6 mice were challenged intranasally with high lethal dose of <i>S. pneumoniae</i> strain WU2 in 25 µl PBS. Survival was monitored daily for 8 days. Results are the summary of two independent experiments. Statistical analysis revealed significant differences in survival rates between <i>Ncr1</i>^+/+ and <i>Ncr1</i>^gfp/gfp mice, while <i>Ncr1</i>^+/+ mice had higher survivel rate (*p<0.05, two-tailed Mann-Withney U-test, line is for geometric mean).</p

Bacterial load in lungs following <i>in vivo</i> depletion of NK cells.

<p><i>Ncr1</i>^+/+ and <i>Ncr1</i>^gfp/gfp C57BL/6 mice treated intraperitoneally either with 50 µl of anti asialo GM1 (WT-anti NK and KO-anti NK) or with 50 µl of PBS (mock treatment, WT-mock and KO-mock). 24 h after anti asialo GM1 or mock treatment, all mice were challenged with 5×10⁷ CFU of <i>S. pneumoniae</i> strain WU2 and lungs were harvested 3 h after challenge. (A) CFU of <i>S. pneumoniae</i> in lungs, 3 h after bacterial challenge (n = 3 to 5 per group). Results are presented as box plot of CFU. ** p<0.01 compared with WT-mock; * p<0.05, compared with WT-anti NK, ± SD (ANOVA test). (B) Flow cytometry analysis of spleen cells from depleted and non-depleted mice for CD3 negative-gated cell population showing NK1.1 versus GFP expression. Analysis was made 24 h after depletion and 3 h following bacterial challenge (representative mouse per each panel). Numbers indicate percentage of gated cells (LR+UR) from CD3⁻ cells. Results are from one representative experiment of two.</p

NCR1 contribution to activation of naïve alveolar macrophages.

<p>BALF cells were harvested from <i>Ncr1</i>^+/+ and <i>Ncr1</i>^gfp/gfp naïve C57BL/6 lungs. Alveolar macrophages were gated by staining with mAbs to CD45 and F4/80 (A) and gated cells were analyzed for the expression of CD11b (B) by flow cytometry. Numbers indicate percentage of alveolar macrophages in BALF (A) and fraction of CD11b positive alveolar macrophages (B). Results are from one representative experiment of five. (C) BALF cells taken from <i>NCR1</i>^+/+ (WT) and <i>NCR1</i>^gfp/gfp (KO) mice were incubated with 1 MOI CFDA-labeled <i>S. pneumonia</i> for 2 h. Cells were then washed and visualized with a fluorescent microscope. The bar graph (C) shows the average of fluorescent BALF cells (bac positive) from 11 microscopic fields. Numbers were normalized to the average of fluorescent BALF cells from <i>NCR1</i>^gfp/gfp mice, which was considered as 1. *p<0.05 compared to <i>NCR1</i>^+/+ group, +SD (ANOVA test). Results are from one representative experiment out of two. (D–E) <i>Ncr1</i>^+/+ BALF were incubated with 1 MOI CFDA-labeled <i>S. pneumonia</i> for 1 hr. Following wash of bacteria, cells were fixed and stained with mNCR1-Ig and biotin-conjugated-rat anti mouse F4/80. Secondary staining was performed using APC-conjugated-F(ab')2 goat-anti-human-IgG-Fc and PE-Cy7-Strepavidin. Hoechst 33342 was employed for nuclear staining. Cells were washed and visualized with Confocal microscope. (D) Representative confocal image of NCR1-ligand^high (bac positive) and NCR1-ligand^none (bac negative) macrophages (F4/80 staining is not shown) (magnification X400). (E) We analyzed 52 random fields from 6 independent experiments and counted F4/80⁺ NCR1-ligand^high and NCR1-ligand^none cells, either positive or not for bacteria. The average number of NCR1-ligand^high and NCR1-ligand^none is shown normalized to NCR1-ligand^none, which its average was considered as 1. (left two columns of the graph, **p<0.01 compared to NCR1-ligand^high group, +SD (two-tailed student t-test)). We then calculated the ratio of bacteria-positive for each of the two populations and normalized the results according to the fraction of NCR1-ligand^none&bac+ of the total NCR1-ligand^none cells. Right two columns of graph shows the normalized fraction of bacteria positive cells for the two macrophage populations (NCR1-ligand^high and NCR1-ligand^none). *p<0.05 compared to NCR1-ligand^high group, +SD (two-tailed student t-test).</p

Bacterial load in lungs.

<p><i>Ncr1</i>^+/+ (WT), <i>Ncr1</i>^+/gfp (Het) and <i>Ncr1</i>^gfp/gfp (KO) C57BL/6 mice were challenged intranasally with 5×10⁷ concentration of <i>S. pneumoniae</i> strain WU2. Lungs were harvested for CFU assessment: (A) 3 h after inoculation (n = 6 to 8), (B) 6 h (n = 5 to 9), and (C) 24 h (n = 5 to 6). Results are the summary of 3 independent experiments and were normalized according to the CFU of the Het group that its CFU average was considered as 1 in each experiment. ** p<0.01, compared with WT or with Het groups ±SD (two-tailed student t-test). NS, not significant compared with WT or with Het groups. Results are presented as vertical scatter plot of normalized CFU.</p