Mice Lacking Fatty Acid-Binding Protein 5 Are Resistant to Listeria monocytogenes

Abstract

To investigate the role of fatty acid-binding protein 5 (FABP5) in infectious diseases, FABP5-deficient mice were challenged with Listeria monocytogenes, a facultative intracellular bacterial pathogen. Interestingly, FABP5-deficient animals were able to clear the infection within 3 days whereas control wild-type (WT) animals showed comparatively higher bacterial burdens in the liver and spleen. Sections of infected tissues showed an increase in inflammatory foci in WT mice compared to FABP5-deficient mice. FABP5-deficient mice had lower circulating inflammatory cytokines and increased inducible nitric oxide synthase production. FABP5-deficient mouse bone marrow-derived macrophages produced higher levels of nitrite anion than their WT counterparts in response to various stimuli. Additionally, in contrast to FABP5-/- mice, transgenic mice overexpressing FABP5 in myeloid cells (LysM-Cre driven) showed decreased survival rates and increased bacterial burden and inflammatory cytokines. Overall, these findings suggest that increased FABP5 levels correlate with a higher L. monocytogenes bacterial burden and elevated subsequent inflammation.

Keywords: FABP5; Listeria monocytogenes infection; Macrophages.

Fig. 1

Enhanced resistance of FABP5^−/− mice to lethal infection with L. monocytogenes. a Kaplan-Meier survival plot of mice i.v. infected with L. monocytogenes. Six WT (dashed line) and 10 FABP5^−/− (continuous line) mice were infected with 1.7 × 10⁵ CFUs. b Changes in body weight loss following infection from mice described in a. cL. monocytogenes burden in the liver and spleen of WT (open circles) and FABP5^−/− (black squares) mice 48 h postinfection (7 mice per group) infected with 2.4 × 10⁴ CFUs. d Serum was collected from the same WT (open circles) and FABP5^−/− (black squares) mice described in c and ALT was quantified using a modified Reitman-Frankel colorimetric end-point reaction. e Stained WT and FABP5^−/− livers 48 h after L. monocytogenes (Lm) infection. Cellular infiltrates are surrounded by black lines. HE. ×20. f Quantification of cellular infiltrates in WT (open circles) and FABP5^−/− (black squares) livers (8 mice per group). Data are representative of 4 independent experiments.

Fig. 2

Decreased levels of multiple cytokines in L. monocytogenes-infected FABP5^−/− mice in comparison to WT mice. a IFNγ. b IL-12p70. c IL-1β. d IL-6. e KC. f TNFα. g IL-10. Cytokines were measured in the serum of WT (open circles) or FABP5^−/− (black squares) mice infected with 4.5 × 10⁴ CFUs 48 h postinfection using the multiplex MSD plate (n = 7 mice/group). Data are representative of 4 independent experiments.

Fig. 3

Increased in vivo iNOS activation by Ly6C⁺ monocytes in FABP5^−/− mice and increased in vitro NO₂⁻ production by FABP5^−/− macrophages. a Representative density plots of iNOS expression in CD11b⁺/Ly6G⁻/Ly6C⁺ gated spleen monocytes (n = 5–6 mice/group) Lm, L. monocytogenes. b Absolute numbers of iNOS⁺ monocytes in WT (open circles) and FABP5^−/− (black squares) spleen 48 h postinfection (n = 5–6 mice/group). c Proportion of neutrophils (Ly6G⁺) in WT (open circles) and FABP5^−/− (black squares) spleen 48 h postinfection (n = 5–6 mice/group). d Absolute numbers of iNOS⁺ neutrophils in WT (open circles) and FABP5^−/− (black squares) spleen 48 h postinfection (n = 5–6 mice/group). e FABP5^−/− BMDMs secrete more NO₂⁻ than WT BMDMs in response to inflammatory stimuli. WT and FABP5^−/− BMDMs were left untreated or stimulated with 10 ng/mL TNFα, 100 ng/mL LPS, and 10 U/mL IFNγ in combination as indicated. Supernatants were obtained 24 h after stimulation and NO₂⁻ production was measured by Griess reaction. Bars represent mean nitrite (NO₂⁻, μM) ± SEM. Data are representative of 3 independent experiments.

Fig. 4

Bone marrow chimeras confirm the resistance to L. monocytogenes infection in FABP5^−/− mice. L. monocytoges burden in the liver (a) and spleen (b) of bone marrow chimeric mice 48 h postinfection (5 mice per group) infected with 3.8 × 10⁴L. monocytogenes. IFNγ (c) and KC (d) were measured in the serum of bone marrow chimeric mice infected with 3.8 × 10⁴ CFUs 48 h postinfection using the multiplex MSD plate. Data are representative of 2 independent experiments.

Fig. 5

Increased susceptibility of myeloid cell-specific FABP5 transgenic mice to lethal infection with L. monocytogenes. a Kap­lan-Meier survival plot of mice i.v. infected with L. monocytogenes. Five LysM-FABP5-Tg⁻ (continuous line) and 7 LysM-FABP5-Tg⁺ (dashed line) mice were infected with 5.55 × 10⁴ CFUs. b Changes in body weight loss following infection from mice described in a. cL. monocytogenes burden in liver and spleen of LysM-FABP5-Tg⁻ (open circles) and LysM-FABP5-Tg⁺ (black squares) mice 48 h postinfection (5–8 mice per group) infected with 4.2 × 10⁴L. monocytogenes. d Serum was collected from the same LysM-FABP5-Tg⁻ (open circles) and LysM-FABP5-Tg⁺ (black squares) mice as described in c and ALT was quantified using a modified Reitman-Frankel colorimetric end-point reaction. e IFNγ. f IL-12p70. g KC. h IL-1β. Cytokines were measured in the serum of LysM-FABP5-Tg⁻ (open circles) or LysM-FABP5-Tg⁺ (black squares) mice infected with 4.2 × 10⁴Lm 48 h postinfection using the multiplex MSD plate. Data are representative of 3 independent experiments.

Fig. 6

FABP5^−/− bone marrow-derived macrophages (BMDMs) have increased bactericidal activity and kill intracellular L. monocytogenes more efficiently than WT or LysM-FABP5-Tg⁺ cells. BMDMs from WT, FABP5^−/−, and LysM-FABP5-Tg⁺ mice were plated on glass coverslips (n = 3 per group), allowed to adhere, and treated with 100 ng/mL LPS ± 10 U/mL IFNγ for 18 h. Cells were infected for 4 h with L. monocytogenes at a MOI = 10 and the coverslips were lysed for enumeration of the bacterial load by plating serial dilutions of lysate. Bars represent mean intracellular L. monocytogenes CFUs ± SEM from 1 of 3 independent experiments.