Bacterial Manipulation of NK Cell Regulatory Activity Increases Susceptibility to Listeria monocytogenes Infection

Abstract

Natural killer (NK) cells produce interferon (IFN)-γ and thus have been suggested to promote type I immunity during bacterial infections. Yet, Listeria monocytogenes (Lm) and some other pathogens encode proteins that cause increased NK cell activation. Here, we show that stimulation of NK cell activation increases susceptibility during Lm infection despite and independent from robust NK cell production of IFNγ. The increased susceptibility correlated with IL-10 production by responding NK cells. NK cells produced IL-10 as their IFNγ production waned and the Lm virulence protein p60 promoted induction of IL-10 production by mouse and human NK cells. NK cells consequently exerted regulatory effects to suppress accumulation and activation of inflammatory myeloid cells. Our results reveal new dimensions of the role played by NK cells during Lm infection and demonstrate the ability of this bacterial pathogen to exploit the induction of regulatory NK cell activity to increase host susceptibility.

Fig 1. NK cells increase susceptibility to systemic L. monocytogenes (Lm) infection despite an independent from early IFNγ production.

(A) Lm burdens from C57BL/6 (B6) mice treated with control (IgG2a) or NK1.1⁺ cell depleting (αNK1.1) antibodies (Abs) and infected 24 h later with 10⁴ Lm. Shown are total colony forming units (CFU) per tissue as determined by dilution plating of tissue lysates at 96 h post-infection (hpi). Symbols represent individual mice with mean (lines). Shown is one of five experiments using n = 3–5 mice/group. (B) Survival curve for B6 mice treated with IgG2a or αNK1.1 antibodies and infected 24 h later with 5 x 10⁴ Lm. Data are pooled from two experiments using n = 5–7 mice/group (C) Lm burdens from livers of B6 and NKT cell deficient (B6.CD1d ^-/-) mice at 96 hpi. B6 mice were treated with IgG2a or αNK1.1 24 h before infection. Liver CFU from one of three experiments using n = 3–4 mice/group. (D) Lm burdens from livers of B6 mice treated 24 h before infection with αNK1.1⁺, anti-asialoGM1 (αGM1), or a non-depleting anti-NKG2A/C/E (αNKG2) Ab. Liver CFU at 96 hpi are shown for two pooled experiments representing n = 6 mice/group. (E) Proportions and total cell numbers of splenic NK1.1⁺CD3^- gated NK cells staining positive for intracellular IFNγ at the indicated hpi. Shown are mean ± SEM for data pooled from 3–5 experiments representing n = 9–18 mice/time point. (F) Serum IFNγ measured for control IgG2a or αNK1.1 treated B6 mice at 24 or 96 hpi. (G) B6.ifnar1 ^-/- mice were treated with indicated Abs 24h before infection as above. Shown are liver bacterial burdens at 96 hpi. Bars depict mean ± SEM values from 3 pooled experiments using n = 3–5 mice/group. (H) B6.ifngr1 ^-/- mice were treated with indicated Abs 24h before infection with 4 x 10³ CFU Lm. Livers were harvested for CFU counts at 72 hpi. Bars depict mean ± SEM values from 3 poled experiments using n = 3–5 mice/group. (I) B6 mice were treated with antibodies and infected as in (A). Liver bacterial burdens at 24, 48, 72, and 96 h after Lm infection. Data are mean ± SEM from 3–5 pooled experiments representing n = 9–18 mice/time point. (J) B6 mice were left untreated or administered αNK1.1 at 24 h before or 24 h after Lm infection. Data are pooled from two experiments with n = 3–4 mice/group. *P<0.05 as measured by (A, F-H) t-test or (B, D, I, J) ANOVA.

Fig 2. NK cells responding to Lm infection acquire the ability to produce IL-10.

(A) Serum IL-10 concentrations at the indicated times after Lm infection of control IgG2a or αNK1.1 treated B6 mice. Data are pooled from 3 experiments for n = 9–12 mice/group. (B) Histogram depicting the staining for IL-10-gfp reporter in gated CD3^-NK1.1⁺ NK cells from livers of uninfected IL-10-gfp+ (B6.tiger) mice or B6.tiger and control B6 mice at 72 hpi with Lm. (C) Shown are mean ± SEM percentages of gated CD3^-NK1.1⁺ cells that stained positive for IL-10-gfp prior to infection or at 24 or 72 hpi with Lm. Gated NK cells from spleen, liver, and blood of B6.tiger mice were analyzed. Data are pooled from 3 experiments and represent n = 9–14 mice per time point. (D) Proportions of IFNγ⁺ and IL-10⁺ populations of NK1.1⁺CD3^- gated NK cells from the indicated tissues at 24 or 96 hpi. Data are pooled from three experiments with n = 3–5 mice/group. Pooled from three experiments representing n = 9–12 mice/group. *P< 0.05 as determined by (A, C) ANOVA.

Fig 3. The L1S region of the Lm p60 protein is sufficient to induce NK cell IL-10 production.

(A) Serum IL-10 at 96 hpi in B6 mice infected with wt or p60-deficient (Δp60) Lm strains. (B) Schematic of co-culture system consisting of BMDC infected with Lm for one hour, followed by washing and the addition of splenic purified NK cells at 2 h post-infection. (C) 24 h supernatant IFNγ after infection of BMDC with wt or p60-deficient Lm in co-culture with wt NK cells. (D) 24 h and 72 h supernatant IL-10 after infection of IL-10 deficient BMDC with wt or p60-deficient Lm in co-culture with wt NK cells. (E) Schematic of co-culture system consisting of BMDC treated with a TLR agonist ± L1S for one hour, followed by washing and the addition of splenic purified NK cells at 2 h post-stimulation. (F) 24 h supernatant IFNγ after treatment of BMDC with LPS ± L1S in co-culture with wt NK cells. (G) 24 h and 72 h supernatant IL-10 after treatment of IL-10 deficient BMDC with LPS ± L1S in co-culture with wt NK cells. (H) 24 h supernatant IFNγ after treatment of human DC with polyI:C ± L1S in co-culture with PBMC from unrelated donors. (I) 72 h supernatant IL-10 after treatment of human DC with polyI:C ± L1S in co-culture with PBMC from unrelated donors. (J) 72 h supernatant IL-10 after treatment of IL-10 deficient BMDC with LPS + L1S ± stimulation with 50 ng/mL IFNγ in co-culture with wt NK cells. (K) 72 h supernatant IL-10 after stimulation of IL-10 deficient BMDC with LPS + L1S in co-culture with NK cells from wt or IFNGR^-/- mice. (L) IL-10 was measured in supernatants collected at 24, 48, and 72 h from L1S-stimuated co-cultures of IL-10 deficient BMDC and NK cells from wt or IFNGR^-/- mice. Data for (C, D, F, G, H, I, J, K, L) pooled from three or more experiments. *P<0.05 as determined by t-test.

Fig 4. NK cell IL-10 production drives increased Lm burdens.

(A) Liver CFU from individual B6 and congenic IL-10 deficient B6 (B6.il10 ^-/-) mice at 72 hpi. IgG2a or αNK1.1 Abs were given 24 h before infection. Pooled from two experiments representing n = 7–9 mice/group. (B) Design of cell transfer experiments. NK cells were purified from naïve wt CD45.1⁺ (B6.ptprc ^a), IL-10-deficient CD45.2⁺ (B6.il-10 ^-/-), or IFNγ-deficient (GKO) CD45.2⁺ mice for transfer into infected B6.il-10 ^-/- recipients. (C) Detection of CD45.1⁺ donor NK cells in B6.il10 ^-/- recipients at 96 hpi. Live gated CD3^-NK1.1⁺ splenic NK cells are shown. (D) IL-10 from splenic lysates of infected B6.il-10 ^-/- recipients 72 h after NK cell transfers. Shown are mean ± SEM for one of three experiments using n = 3–4 mice/group. (E) Lm burdens from indicated tissues of B6.il-10 ^-/- recipients 96 hpi. *P< 0.05 as determined by (A) ANOVA or (D, E) t-test.

Fig 5. NK cells suppress innate responses and their production of IL-10 increases susceptibility to Lm.

(A) Splenocytes were harvested from uninfected or 72 hpi B6 mice and evaluated for the indicated myeloid cell markers. Top row depicts total live-gated population bottom row depicts the indicated gated population. Data are from individual mice and representative of more than n = 20 mice from over 5 experiments. (B) Splenocytes were harvested and analyzed as above. Shown are the mean ± SEM number of each indicated cell population per spleen as calculated from data gated as in (A). Data are from one of more than 5 experiments and represent n = 5 individual mice per group. (C) Leukocytes were isolated from livers of the indicated mice and analyzed as above. Shown are the mean ± SEM number of each indicated cell population per liver for one of 3 experiments and represent n = 5 individual mice per group. (D) Serum IL-12p70 for control IgG2a and αNK1.1-treated mice at 96 hpi. Shown are mean ± SEM from four pooled experiments representing n = 12–16 mice/group. (E) DCF fluorescence was measured at the indicated times after plating of labeled splenocytes harvested from uninfected (mock) or Lm-infected control IgG2a and αNK1.1 treated mice at 72 hpi. Shown are mean fluorescence values for triplicate wells from one of three experiments. *P<0.05 as measured by (B, C) ANOVA, (D) t-test, or (E) 2 way ANOVA.