IFN-γ extends the immune functions of Guanylate Binding Proteins to inflammasome-independent antibacterial activities during Francisella novicida infection

Abstract

Guanylate binding proteins (GBPs) are interferon-inducible proteins involved in the cell-intrinsic immunity against numerous intracellular pathogens. The molecular mechanisms underlying the potent antibacterial activity of GBPs are still unclear. GBPs have been functionally linked to the NLRP3, the AIM2 and the caspase-11 inflammasomes. Two opposing models are currently proposed to explain the GBPs-inflammasome link: i) GBPs would target intracellular bacteria or bacteria-containing vacuoles to increase cytosolic PAMPs release ii) GBPs would directly facilitate inflammasome complex assembly. Using Francisella novicida infection, we investigated the functional interactions between GBPs and the inflammasome. GBPs, induced in a type I IFN-dependent manner, are required for the F. novicida-mediated AIM2-inflammasome pathway. Here, we demonstrate that GBPs action is not restricted to the AIM2 inflammasome, but controls in a hierarchical manner the activation of different inflammasomes complexes and apoptotic caspases. IFN-γ induces a quantitative switch in GBPs levels and redirects pyroptotic and apoptotic pathways under the control of GBPs. Furthermore, upon IFN-γ priming, F. novicida-infected macrophages restrict cytosolic bacterial replication in a GBP-dependent and inflammasome-independent manner. Finally, in a mouse model of tularemia, we demonstrate that the inflammasome and the GBPs are two key immune pathways functioning largely independently to control F. novicida infection. Altogether, our results indicate that GBPs are the master effectors of IFN-γ-mediated responses against F. novicida to control antibacterial immune responses in inflammasome-dependent and independent manners.

Fig 1. Gbp^chr3 and the inflammasome control the in vivo host resistance to F. novicida in a divergent manner.

Mice of the indicated genotypes were subcutaneously (sc) injected with 5×10³ (A-B) or 4×10⁵ (C-E) F. novicida. (A) Survival was monitored twice a day for 10 days. (B) Bacterial burden in the spleen, (C) IFN-γ or (D, E) IL-18 levels in the serum were quantified at 16 h (D) and 48 h post-inoculation (C, E). IFN-γ concentration at 16 h post-inoculation was under the limit of detection. Each symbol represents the value of an individual mouse; the bars indicate the geometric mean. (A-E) one experiment representative of one (D), to at least three (A-C, E) independent experiments is shown. Mantel Cox log-rank test (A), Kruskal-Wallis analysis with Dunn's correction for multiple comparisons (B, C, E) and Mann-Whitney test (D) were performed.

Fig 2. IFN-γ-induced GBPs control F. novicida-mediated cell death in an inflammasome-dependent and -independent manner.

Cell death was measured in real-time by assessing propidium iodide fluorescence in (A, B) unprimed or (C, D) IFN-γ-primed (100 U/ml for 16 h) BMDMs from the indicated genotypes infected or not with F. novicida at a MOI of 10. (B, D) Kinetics were quantified by calculating the corresponding area under the curve. (E-F) The number of propidium iodide positive cells was quantified from time-lapse video-microscopy images (see S3 Fig and S1 and S2 Movies) at 12 h (E) and 18 h (F) post-infection. (G) ATP-based cell viability was monitored at 24 h post-infection. Cell death is shown. (A-G) Mean and s.d. are shown. One experiment representative of two (E, F) to at least three independent experiments is shown. (B, D, E-G) One-way ANOVA analysis was performed with Tukey's correction for multiple comparisons.

Fig 3. F. novicida infection triggers AIM2-, NLRP3-dependent canonical and caspase-11 non-canonical inflammasomes activation in a hierarchical manner.

(A-D) Cell death was measured in real-time by assessing propidium iodide fluorescence in BMDMs of the indicated genotype infected with F. novicida at an MOI of 10. (B, D) Kinetics were quantified by calculating the corresponding area under the curve. (C) Aim2^-/- BMDMs were treated with Non-Targeting (NT) or indicated gene-specific siRNA. (E-G) IL-1β levels in the supernatant of BMDMs of the indicated genotype treated (E, F) or not (G) with the indicated siRNA and infected with F. novicida at an MOI of 10 were quantified at 6 or 9 h post-infection as indicated. Graphs show mean and s.d. of triplicate assays. Data are representative of three independent experiments. (B, D, E-G) One-way ANOVA analysis was performed with Tukey's correction for multiple comparisons. (NI: Not infected).

Fig 4. GBPs control apoptosis in inflammasome-deficient BMDMs.

(A-C) Cell retraction in propidium iodide positive cells was assessed by quantifying wheat germ agglutinin staining intensity at (A) 90 minutes post Nigericin (Ng, a pyroptotis stimulus) or post-Gliotoxin (GlioTx, an apoptotic stimulus) or at (B, C) 21 h post-infection in (A, B) unprimed or (C) IFN-γ-primed (100 U/ml for 16 h) BMDMs from the indicated genotypes infected (B, C) or not with F. novicida at a MOI of 10. (D) Lysates from BMDMs from the indicated genotypes infected or not with F. novicida at a MOI of 10 were analyzed by Western blotting analysis. IFN-γ-priming (100 U/ml for 16 h) is indicated. The dotted vertical lines in Casp9 Western blot illustrate that the samples from a single original Western blot gel/ image were reorganized to fit the indicated order without any other image manipulation. The dotted horizontal lines in Casp8 and Casp3 Western blot indicate images from two different exposure times or from the use of two different primary antibodies (pro- and cleaved Casp3), respectively. (E, F) DEVDase activity was analyzed using a fluorogenic caspase3/7 substrate at 4 h post-GlioTx addition or at the indicated time post-infection in BMDMs from the indicated genotypes infected or not with F. novicida at a MOI of 10 and primed (F) or not (E) with IFN-γ (100 U/ml for 16 h). (A-C, E) Mean and s.d. are shown. (A-F) One experiment representative of two independent experiments is shown. Unpaired t-test (A) and one-way ANOVA analysis with Tukey's correction for multiple comparisons (B-C) were performed. (NI: non-infected).

Fig 5. IFN-γ-induced GBPs control intracellular bacterial replication independently of inflammasomes.

(A-F) BMDMs from the indicated genotypes were primed or not overnight with 100 U/ml of IFN-γ. BMDMs were infected with (A-E) F. novicida or (F) F. tularensis LVS at a multiplicity of infection (MOI) of 1 and 0.4, respectively. Intracellular bacterial burden was assessed by determination of viable counts at the indicated times post-infection. Results were normalized with viable counts detected at 2h post-infection. The corresponding raw data are presented in S7 Fig. (B) Flow cytometry-based quantification of infected cells (GFP⁺) among live (propidium iodide^-) BMDMs after 10h of infection with GFP-expressing F. novicida at a MOI of 10. (C) Quantification of bacterial loads in single cells after 10h of infection with GFP-expressing F. novicida at a MOI of 10, assessed by high-resolution microscopy in flow and presented as a comparison of both genotypes, with bacteria-per-cell values grouped by increments of 10. (D) Immunofluorescence of BMDMs infected for 10h with GFP-expressing F. novicida at a MOI of 1 (stained with DAPI (blue) and with an antibody to F. novicida (red); scale bars: 5 μm. (E) GFP intensity was quantified in single GFP-expressing F. novicida in BMDMs from the indicated genotypes primed or not overnight with 100 U/ml of IFN-γ and infected for 10 h. Cumulative data from two independent experiments are shown. The GFP intensity of single bacteria was normalized in each experiment to the average intensity of single bacteria in unprimed WT BMDMs. Dots represent the normalized GFP intensity of a single bacteria, the bar represents the mean. Fluorescence of ≈300 bacteria per sample and per experiment was analyzed. The dotted lines indicate the average intensity in unprimed WT BMDMs (100%) and the threshold set to quantify GFP^low bacteria (80%). Data are representative of two (C, D, F) or at least three (A, B) independent experiments. (A, B, F) Mean and s.d. of triplicate wells are shown. One-way ANOVA analysis with Tukey's correction for multiple comparisons (A-B, F) were performed. The distribution of GFP intensity in the different sample was analyzed using Komogorov-Smirnov test with Bonferroni correction.

Fig 6. rIFN-γ administration largely rescues the in vivo antimicrobial function of Asc^-/- mice but fails to complement Gbp^chr3 deficiency.

(A-D) Survival of mice treated by daily intraperitoneal injection of PBS or 10⁵ U of rIFN-γ during 5 days after sc inoculation with 5×10⁴ F. novicida (A) or 5×10³ F. novicida (B-D). (A-C) effect of IFN-γ treatment in each genotype, (D) comparison of IFN-γ -treated Asc^-/- and Gbp^chr3-KO mice. (E-F) Bacterial burden in the liver and spleen 2 days after sc inoculation with 5×10⁴ F. novicida (E) or or 5×10³ F. novicida (F). Each symbol represents the value of an individual mouse, geometric mean is shown. Data are representative of two independent experiments. Log-rank Cox-Mantel test (A-D), Mann-Whitney (E) and Kruskal-Wallis analysis with Dunn's correction (F) were performed.