A Phenome-Wide Association Study Uncovers a Pathological Role of Coagulation Factor X during Acinetobacter baumannii Infection

Abstract

Coagulation and inflammation are interconnected, suggesting that coagulation plays a key role in the inflammatory response to pathogens. A phenome-wide association study (PheWAS) was used to identify clinical phenotypes of patients with a polymorphism in coagulation factor X. Patients with this single nucleotide polymorphism (SNP) were more likely to be hospitalized with hemostatic and infection-related disorders, suggesting that factor X contributes to the immune response to infection. To investigate this, we modeled infections by human pathogens in a mouse model of factor X deficiency. Factor X-deficient mice were protected from systemic Acinetobacter baumannii infection, suggesting that factor X plays a role in the immune response to A. baumannii Factor X deficiency was associated with reduced cytokine and chemokine production and alterations in immune cell population during infection: factor X-deficient mice demonstrated increased abundance of neutrophils, macrophages, and effector T cells. Together, these results suggest that factor X activity is associated with an inefficient immune response and contributes to the pathology of A. baumannii infection.

Keywords: Acinetobacter; PheWAS; coagulation; innate immunity.

FIG 1

Factor X-deficient mice demonstrate enhanced bacterial burdens in the liver following systemic S. aureus infection. Approximately 2 × 10⁷ CFU of S. aureus Newman was injected via the retro-orbital venous sinus. Mice were euthanized and tissues removed at 96 hpi and bacterial burdens were quantified. Each dot represents a single mouse, and data are compiled from three experiments. Shown is the median with interquartile range; the y axis is set to the limit of detection. *, P < 0.05 by Mann-Whitney test.

FIG 2

Factor X-deficient mice demonstrate reduced bacterial burdens after systemic A. baumannii infection. Approximately 2 × 10⁸ to 3 × 10⁸ CFU of A. baumannii 17978 was injected via the retro-orbital venous sinus. Mice were euthanized and tissues removed at 4 (A) and 24 (B) hpi and bacterial burdens were quantified. (C) Bacterial burdens 24 hpi following infection with approximately 2 × 10⁸ CFU A. baumannii 17978 after treatment with PBS (vehicle) or 10 mg/kg of body weight of enoxaparin, intraperitoneally, at the time of infection. Each dot represents a single mouse, and data are compiled from two (A) or three (B and C) experiments. Shown is the median with interquartile range; the y axis is set to the limit of detection. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann-Whitney test).

FIG 3

Factor X deficiency does not affect infection induced coagulation. Mice were injected with approximately 3 × 10⁸ CFU of A. baumannii 17978 and euthanized at 24 hpi. Tissue were fixed and scored blindly (A) or hepatic thrombosis was quantified using anti-fibrin/fibrinogen immunohistochemistry (B). Each dot represents a single mouse, and data are compiled from a single experiment. For panel A, the whiskers of box-and-whisker plots indicate minimum to maximum. Horizontal lines in panel B indicate mean. No statistical difference (P < 0.05) was found by two-way analysis of variance (ANOVA) with Sidak’s correction for multiple comparisons.

FIG 4

F10^F/F mice demonstrate reduced serum cytokine and chemokine levels during systemic A. baumannii infection. Serum cytokines (A) and chemoattractants (B) at 4 and 24 h following injection in the retro-orbital venous sinus of approximately 3 × 10⁸ CFU of A. baumannii 17978 were quantified by Luminex analysis. Each dot represents data from a single mouse, from two experiments; the horizontal line indicates the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA with Sidak’s correction for multiple comparisons, comparing genotypes at each time point). For graphing and statistical tests, values that exceeded or were below the limit of detection were set to the limit for each analyte.

FIG 5

FX activity is associated with a pathogenic immune response and mortality following injection of heat-killed A. baumannii. Serum cytokines (A) and chemoattractants (B) following retro-orbital injection with PBS (mock) or approximately 1 × 10⁹ to 2 × 10⁹ CFU of heat-killed A. baumannii ATCC 17978 were quantified by Luminex analysis after mice were euthanized at 24 hpi. (C) Survival of mice injected with approximately 1 × 10⁹ to 2 × 10⁹ CFU of heat-killed A. baumannii ATCC 17978. For panels A and B, each dot represents data from a single mouse, from two experiments; the horizontal line indicates the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA with Sidak’s correction for multiple comparisons, comparing genotypes within each treatment group). For graphing and statistical tests, values that exceeded or were below the limit of detection were set to the limit for each analyte. For panel C, data are combined from two independent experiments (n = 13 for B6 mice and n = 17 for F10^F/F mice). *, P < 0.05 by log rank (Mantel-Cox) survival test.

FIG 6

F10^F/F mice have increased numbers of proinflammatory immune cells during A. baumannii infection. Immune cells from spleen, kidneys, and blood of mice injected with either PBS (mock) or approximately 2 × 10⁸ to 3 × 10⁸ CFU of A. baumannii ATCC 17978 were quantified at 24 hpi using flow cytometry. (A) Neutrophils (CD11b⁺ Ly6G⁺ CD11c⁻ F4/80⁻) and dendritic cells (CD11b⁺ CD11c⁺ Ly6G⁻ F4/80⁻). (B) Macrophages (CD11b⁺ F4/80⁺ Ly6G⁻ CD11c⁻) and monocytes (CD11b⁺ F4/80⁻ Ly6G⁻ CD11c⁻). (C) CD8⁺ T_eff (CD4⁻ CD8⁺ C45RA⁺ CD62L⁻) and T_n (CD4⁻ CD8⁺ C45RA⁺ CD62L⁺) cells. (D) CD4⁺ T_eff (CD4⁺ CD8⁻ C45RA⁺ CD62L⁻), T_n (CD4⁺ CD8⁻ C45RA⁺ CD62L⁺), and T_reg (CD4⁺ CD8⁻ FOXP3⁺) cells. Each dot represents data from a single mouse, from two experiments. The horizontal line indicates the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (two-way ANOVA with Tukey’s correction for multiple comparisons).