IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila

Abstract

Interferon gamma (IFN-γ) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-γ confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-γ. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-γ-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-γ-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN-γ. Instead, we found that 2DG rescues bacterial replication by inhibiting the expression of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune-responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirmed that loss of both iNOS and IRG1, but not individual deficiency in either gene, partially reduced IFN-γ-mediated restriction of L. pneumophila Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we found that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3, and NOX2) resulted in a total loss of L. pneumophila restriction by IFN-γ in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-γ-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages.IMPORTANCELegionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN-γ) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has identified the exact molecular factors induced by IFN-γ that are required for its activity. We generated macrophages lacking different combinations of IFN-γ-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN-γ-mediated restriction of L. pneumophila We identified six genes that comprise the totality of the IFN-γ-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN-γ and highlight how redundancy downstream of IFN-γ is key to prevent exploitation of macrophages by pathogens.

Keywords: Legionella pneumophila; host-pathogen interactions; innate immunity; interferons; macrophages.

FIG 1

IFN-γ restricts L. pneumophila replication in many different immune gene-deficient macrophages. (A) Luminescence measured in relative light units (log₁₀ RLU, left) and recovery of CFU (log₁₀ CFU, right) of LP02 ΔflaA lux L. pneumophila from infected wild-type C57BL/6 BMMs either not stimulated (no stim) or stimulated with 6.0 ng/ml IFN-γ. (B) Log₁₀ RLU from LP02 ΔflaA lux L. pneumophila from infected Ifngr1^−/− and Nos2^−/− BMMs either not stimulated or stimulated with 6.0 ng/ml IFN-γ. (C) Log₁₀ RLU from LP02 ΔflaA lux L. pneumophila from infected Myd88^−/− and Myd88^−/− Nod1^−/− Nod2^−/− BMMs either not stimulated or stimulated with 6.0 ng/ml IFN-γ. (D) Log₁₀ RLU from LP02 ΔflaA lux L. pneumophila from infected LysMCre⁺ Atg5^fl/fl and LysMCre⁺ BMMs either not stimulated or stimulated with 6.0 ng/ml IFN-γ. (E) Log₁₀ RLU from LP02 ΔflaA lux L. pneumophila from infected Gbp^chr3−/− BMMs, wild-type C57BL/6 control BMMs derived in parallel with Gbp^chr3−/− BMMs, and Casp1/11^−/− BMMs either not stimulated or stimulated with 6.0 ng/ml IFN-γ. Data reflect individual experiments that represent at least two independent experiments. Error bars in all graphs represent standard deviation of the mean from at least three technical replicates. P < 0.001, comparing no stim versus IFN-γ curves in all genotypes of BMMs (except Ifngr1^−/−) by 2-way analysis of variance (ANOVA). No-stim and IFN-γ curves do not differ significantly in Ifngr1^−/− BMMs.

FIG 2

2DG rescues L. pneumophila replication in IFN-γ-stimulated macrophages. (A) Lactate secretion (left) and glucose consumption (right) measured in cell culture medium following infection of wild-type C57BL/6 BMMs with LP02 ΔflaA ΔuhpC lux L. pneumophila and stimulated with 6.0 ng/ml IFN-γ and 1.0 mM 2DG as indicated. **, P < 0.01; *, P < 0.05; ns, not significant, comparing indicated curves by 2-way ANOVA. (B) Log₁₀ RLU (left) and log₁₀ CFU (right) of LP02 ΔflaA ΔuhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs either not stimulated (no stim), stimulated with 6.0 ng/ml IFN-γ (IFN-γ), or stimulated with 6.0 ng/ml IFN-γ plus 2.0 mM 2DG (IFN-γ/2DG). P < 0.001, comparing all curves to each other in each graph by 2-way ANOVA. (C) Log₁₀ RLU from LP02 ΔflaA ΔuhpC lux L. pneumophila from infected LysMCre⁺ Hif1a^fl/fl and LysMCre⁺ BMMs not stimulated or stimulated with 6.0 ng/ml IFN-γ and 2.0 mM 2DG as indicated. P < 0.01, comparing all curves to each other in each graph by 2-way ANOVA. (D) Log₁₀ RLU from LP02 ΔflaA ΔuhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs stimulated with 6.0 ng/ml IFN-γ and 2.0 mM 2DG as indicated and cultured in infection medium containing 11.11 mM glucose (left) or 11.11 mM galactose in the absence of glucose (center) and in glucose-free medium with no additional source of sugar (right). P < 0.001, comparing all curves to each other in each graph by 2-way ANOVA. Data reflect results of individual experiments that represent at least three independent experiments. Error bars in all graphs represent standard deviation of the mean from at least three technical replicates.

FIG 3

Differential effect of UPR stress stimuli on rescue of L. pneumophila replication in IFN-γ-stimulated macrophages. (A) Log₁₀ RLU from LP02 ΔflaA ΔuhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs stimulated for 48 h postinfection with 6.0 ng/ml IFN-γ, 2.0 mM 2DG, 2.0 μM geldanamycin (geld.), 1.0 μg/ml brefeldin A (BfA), and 2.0 mM dithiothreitol (DTT) as indicated. (B) Log₁₀ RLU from LP02 ΔflaA ΔuhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs stimulated for 48 h postinfection with 6.0 ng/ml IFN-γ, 10.0 μM tunicamycin (tunic.), and 25.0 nM thapsigargin (thaps.) as indicated. (C) Log₁₀ RLU from LP02 ΔflaA ΔuhpC lux L. pneumophila from infected wild-type C57BL/6 BMMs stimulated for 48 h postinfection with 6.0 ng/ml IFN-γ, 2.0 mM 2DG, 2.0 μM geldanamycin, and 0.1 μM ISRIB as indicated. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant, comparing indicated means by unpaired t test. Data reflect results of individual experiments that represent at least three independent experiments. Error bars in all graphs represent standard deviation of the mean from at least two technical replicates. Concentrations of UPR stimuli displayed represent a single point in a titration at which we observed maximum effect on L. pneumophila replication in combination with IFN-γ stimulation relative to a minimum effect on L. pneumophila replication in the absence of IFN-γ.

FIG 4

BMMs lacking IRG1 retain IFN-γ-mediated restriction of L. pneumophila while BMMs lacking both INOS and IRG1 lose the ability to fully restrict L. pneumophila. (A) Log₁₀ RLU from LP02 ΔflaA lux L. pneumophila from infected iCas9 BMMs in which Acod1 was targeted with two guide RNAs (iCas9::Acod1) or Ifngr1 was targeted with one guide RNA (iCas9::Ifngr1) or that were not manipulated (iCas9) either not stimulated (no stim) or stimulated with 6.0 ng/ml IFN-γ. (B) Log₁₀ RLU from LP02 ΔflaA lux L. pneumophila from infected primary Acod1^−/− and wild-type C57BL/6N BMMs either not stimulated or stimulated with 6.0 ng/ml IFN-γ. Wild-type C57BL/6N BMMs were included as a control for BMMs derived from Acod1^−/− mice, which were generated on the C57BL/6N background. (C) Log₁₀ RLU from LP02 ΔflaA lux L. pneumophila from infected Acod1^−/− and wild-type C57BL/6N BMMs either not stimulated (no stim), stimulated with 6.0 ng/ml IFN-γ (IFN-γ), or stimulated with 6.0 ng/ml IFN-γ plus 100 μM 1400W (IFN-γ/1400W). (D) Log₁₀ RLU from LP02 ΔflaA lux L. pneumophila from infected iCas9 BMMs in which Nos2 was targeted with two guide RNAs (iCas9::Nos2), Acod1 was targeted with two guide RNAs (iCas9::Acod1), or both Nos2 and Acod1 were targeted with two guide RNAs each (iCas9::Nos2Acod1) or that were not manipulated (iCas9) either not stimulated or stimulated with 6.0 ng/ml IFN-γ. (E) Log₁₀ RLU from LP02 ΔflaA lux L. pneumophila from infected primary BMMs derived from Nos2^−/− Acod1^−/− and littermate Nos2^+/− Acod1^−/− mice either not stimulated or stimulated with 6.0 ng/ml IFN-γ. (A to E) ***, P < 0.001; **, P < 0.01; ns, not significant, comparing indicated curves by 2-way ANOVA. (F) Log₁₀ CFU of LP02 ΔflaA lux L. pneumophila recovered 48 h postinfection from BMMs derived from Nos2^−/− Acod1^−/− and littermate Nos2^+/− Acod1^−/− mice either not stimulated or stimulated with 6.0 ng/ml IFN-γ. ***, P < 0.001; **, P < 0.01; ns, not significant, comparing means by unpaired t test. Data reflect results of individual experiments that represent at least two independent experiments. Error bars in all graphs represent standard deviation of the mean from at least three technical replicates.

FIG 5

QKO BMMs that additionally lack CASP11, IRG1, or both factors display partial to complete lack of restriction of L. pneumophila replication when stimulated with IFN-γ. (A) Log₁₀ RLU from LP02 ΔflaA lux L. pneumophila from infected wild-type C57BL/6, QKO, QKO/C11, QKO/IRG1, and 6KO BMMs either not stimulated (no stim) or stimulated with 6.0 ng/ml IFN-γ (IFN-γ). ***, P < 0.001; ns, not significant, comparing indicated curves by 2-way ANOVA. (B) Log₁₀ CFU recovered from WT C57BL/6, QKO, QKO/C11, QKO/IRG1, and 6KO BMMs 48 h following infection with LP02 ΔflaA lux L. pneumophila. ***, P < 0.001; *, P < 0.05; ns, not significant, comparing means by unpaired t test. Data in panels A and B are pooled from multiple independent experiments using BMMs derived from two or more different mice per genotype. Error bars in panels A and B represent standard deviation of the mean as follows: C57BL/6, RLU, n = 5, and CFU, n = 5 independent experiments; QKO, RLU, n = 9, and CFU, n = 3 independent experiments; QKO/C11, RLU, n = 4, and CFU, n = 3 independent experiments; QKO/IRG1, RLU, n = 8, and CFU, n = 3 independent experiments; 6KO, RLU, n = 5, and CFU, n = 3 independent experiments. (C) Log₁₀ RLU from LP02 ΔflaA ΔuhpC lux L. pneumophila from infected QKO, Nos2^−/− Acod1^−/−, and QKO/IRG1 BMMs stimulated with 6.0 ng/ml IFN-γ and 2.0 mM 2DG as indicated. ***, P < 0.001; ns, not significant, comparing indicated curves by 2-way ANOVA. Data in panel C reflect results of individual experiments that represent at least two independent experiments.