Additional file 1: of Adaptively introgressed Neandertal haplotype at the OAS locus functionally impacts innate immune responses in humans

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Additional file 2: of Adaptively introgressed Neandertal haplotype at the OAS locus functionally impacts innate immune responses in humans

Supplementary tables. (XLSX 1030 kb

Specific Dysregulation of IFNγ Production by Natural Killer Cells Confers Susceptibility to Viral Infection

<div><p>Natural Killer (NK) cells contribute to the control of viral infection by directly killing target cells and mediating cytokine release. In C57BL/6 mice, the Ly49H activating NK cell receptor plays a key role in early resistance to mouse cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. Here we show that transgenic expression of Ly49H failed to provide protection against MCMV infection in the naturally susceptible A/J mouse strain. Characterization of Ly49H⁺ NK cells from <i>Ly49h</i>-A transgenic animals showed that they were able to mount a robust cytotoxic response and proliferate to high numbers during the course of infection. However, compared to NK cells from C57BL/6 mice, we observed an intrinsic defect in their ability to produce IFNγ when challenged by either m157-expressing target cells, exogenous cytokines or chemical stimulants. This effect was limited to NK cells as T cells from C57BL/6 and <i>Ly49h</i>-A mice produced comparable cytokine levels. Using a panel of recombinant congenic strains derived from A/J and C57BL/6 progenitors, we mapped the genetic basis of defective IFNγ production to a single 6.6 Mb genetic interval overlapping the <i>Ifng</i> gene on chromosome 10. Inspection of the genetic interval failed to reveal molecular differences between A/J and several mouse strains showing normal IFNγ production. The chromosome 10 locus is independent of MAPK signalling or decreased mRNA stability and linked to MCMV susceptibility. This study highlights the existence of a previously uncovered NK cell-specific <i>cis</i>-regulatory mechanism of <i>Ifnγ</i> transcript expression potentially relevant to NK cell function in health and disease.</p></div

Chromosome 10 controls both IFNγ production and MCMV resistance.

<p>(A, B) Freshly isolated splenocytes from B6, Css10 and BcA9 mice were stimulated with P/I for 4 h. The plots are gaited (A) on CD3⁻DX5⁺ NK cells (B) CD3⁺DX5⁻ T cells and the percentage of cells expressing intracellular IFNγ is shown. (C) Splenocytes from indicated strains were incubated for 4 h with RMAS-m157 and the percentage of Ly49H⁺ NK cells expressing intracellular IFNγ is shown. Data were analyzed using one way ANOVA with Bonferoni post-test and presented as mean ± SEM. **<i>P</i><0.01, ***<i>P</i><0.001. (D) Indicated mice were infected with MCMV and viral load was quantified from the spleen at day 3 p.i. Data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM. ***<i>P</i><0.001. Similar results were obtained in another independent experiment.</p

Mapping of IFNγ production by NK cells reveals a single locus on chromosome 10.

<p>(A) Genome-wide linkage analysis was done using mice from the 33 RCS strains outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004511#ppat-1004511-g004" target="_blank">Figure 4A</a>. IFNγ production by NK cells following P/I treatment was used as the mapping trait. The negative log genome-wide <i>p</i> values are shown. (B) Chr 10 negative log genome-wide <i>p</i> values of IFNγ production by NK cells upon P/I treatment. (C) Map of the 6.6 Mbp relevant interval in chr 10 harboring 45 genes in black rectangles (adapted from UCSC mouse genome browser, mm9).</p

Decreased IFNγ production by BcA9 NK cells is independent of impaired MAP kinase pathway signaling or RNA instability.

<p>(A) B6 and BcA9 splenocytes were stimulated with indicated Pathogen-associated molecular patterns for 16–18 h and IFNγ in NK cells was quantified intracellularly, 3 mice per group were used. (B), NK cells from indicated strains were stimulated with P/I and intracellular TNFα was analyse by FACS. (C, D and E) NK cells from indicated strains were generated by culturing splenocytes in IL-2 media for 6 days. (C) NK cells were stimulated for 3 h with P/I and intracellular IFNγ was analyzed by flow cytometry. (D) IFNγ production was quantified from the supernatants at the indicated times (E) Expression levels of IFNγ were determined by qPCR at the indicated times. Data represent means ± SEM of triplicates. Sample data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM and significant <i>P</i> values are indicated. *<i>P</i><0.05, ***<i>P</i><0.001. Similar results were obtained in three independent experiments. (F) Western blot analyses p-p38, p38, ERK, pERK, and actin were performed with equal protein loading of each sample. Quantification of p-P38 and pERK against βActin are shown. (G) B6 and BcA9 NK cells were cultured in rIL-2 and stimulated for 1 h with P/I. Cells were treated or not with actinomycin D (Act) for 4 h and IFNγ transcript was quantified by qPCR. Results of 3 mice per group are shown. (H) B6 and BcA9 NK cells were cultured 6 days, and treated or not with 5-aza-2-deoxycytidine (Aza). Cells were then stimulated or not with P/I for 1 h and IFNγ was analysed by FACS as described before. Results of 3 mice per group are shown. Similar results were obtained in another independent experiment. **<i>P</i><0.01. NK cells from indicated strains were generated by culturing splenocytes in IL-2 media for 6 days. (I) L-2 derived NK cells were left none stimulated or stimulated for 1 h with P/I subjected to ChIP against H3 and H3K4me1. Levels of the H3K4me1 active chromatin mark were measure relative to levels of H3 for known <i>Ifnγ</i> regulatory regions. (J) Fresh NK cells were stimulated for 1 h with P/I subjected to ChIP against H3 and H3K4me1 and level of H3K4me1 active chromatin mark was measured as before.</p

Decreased IFNγ production by NK cells in A-<i>Ly49h</i> mice occurs independently of the NKC and H2 and is linked to MCMV susceptibility.

<p>(A) Freshly isolated splenocytes from 33 RCS strains and parental strains were stimulated with P/I for 4 h. The percentage of CD3⁻DX5⁺ NK cells expressing intracellular IFNγ is shown and represents data from three pooled experiments. Data were analyzed using a two-way ANOVA. (B) Splenocytes from 18 BcA strains expressing Ly49H receptor were co-cultured with RMAs-m157 for 3 h. Intracellular IFNγ was analyzed on CD3⁻DX5⁺ Ly49H⁺ NK cells by flow cytometry. Data were analyzed using a two way ANOVA with a bonferroni post test. (C) 18 BcA strains expressing Ly49H receptor and the parental B6 strains were infected with low dose of MCMV. Spleen viral titer was quantified 3 days later by PA. Data were analyzed using a two way ANOVA with a bonferroni post test. (D) B6 and BcA9 mice were infected with high dose of MCMV. Spleen viral load was quantified at day 3 p.i. Data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM and <i>P</i> values of significant results between groups are indicated. (A, B,C and D) Statistical differences between B6 and BcA9 strains are shown *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Ly49H expression by NK cells does not rescue A/J mice from lethal MCMV infection.

<p>(A) Expression of Ly49H (top) and binding of M157-Ig (bottom) gaited on CD3⁻DX5⁺ NK cells. (B) Quantification of Ly49H staining or M157-Ig binding in indicated strains. Data are representative of one experiment out of 2 (B6: n = 3, FVB-<i>Ly49h</i>: n = 3, A-<i>Ly49h</i> n = 3). (C, D) Survival and weight loss following infection with 7000 PFU (high dose) of MCMV. Two independent experiments pooled together are shown (B6, n = 10; FVB-<i>Ly49h</i>, n = 8; A-WT, n = 6, A-<i>Ly49h,</i> n = 10, B6-<i>Ly49h^−/−,</i> n = 8). (E, F) Viral titer in the spleen and liver of mice infected with 2500PFU (Low dose) of MCMV for 3 days. Data are presented as mean ± SEM and significant <i>P</i> values are indicated. (G, H) Serum from Wt and <i>Ly49h</i> transgenic mice was prepared at 36 h after MCMV infection with high viral titer and proteins were quantified by ELISA. Results from 3–6 mice per group are shown. Results shown are representative of one experiment out 2. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Decreased IFNγ production by Ly49H⁺ NK cells in A/J mice.

<p>Splenocytes from B6 and A-<i>Ly49h</i> mice were harvested and stimulated with RMAs-m157, BAF-m157, IL12/IL18 or PMA and ionomycin (P/I) for 3–5 h. (A) Representative dot plots demonstrating IFNγ production following stimulation and gaited on CD3⁻DX5⁺ Ly49H⁺ NK cells. The numbers represent the percentage of Ly49H⁺ producing IFNγ. (B) Graphical representation of IFNγ production by CD3⁻DX5⁺ Ly49H⁺ NK cells following stimulation. (C) Representative dot plots showing IFNγ production following stimulation and gaited on CD3⁺ or CD3⁻ cells. Numbers represent the percentage of cells producing IFNγ. (D) Graphical representation of IFNγ production by CD3⁻ DX5⁺ (T cells) or CD3⁺ DX5⁻ (NK cells) after P/I stimulation. Data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM and <i>P</i> values of significant results between groups are indicated. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

NK cells from A/J mice can proliferate and produce Granzyme B and Perforin following MCMV inoculums.

<p>Splenocytes were harvested from naïve B6 and A-<i>Ly49h</i> mice (day 0) or mice infected with 2500 PFU MCMV (n = 3 mice/time point). Time points post-infection are indicated in the figure. (A) BrdU incorporation was analyzed on CD3⁻DX5⁺ Ly49H⁺ NK cells by flow cytometry. (B) Spleen viral titers were determined by PA. Intracellular (C) Granzyme (D) Perforin expression was analyzed by flow cytometry on CD3-DX5+ Ly49H+ NK cells. Representative plots from individual mice are shown. The percent of Ly49H+ NK cells positive for Gzmb, and Prf1 are summarized for one experiment. Data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM and significant <i>P</i> values are indicated. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. These data are representative of 2–3 independent experiments.</p