Restraint of Fumarate Accrual by HIF-1α Preserves miR-27a-Mediated Limitation of Interleukin 10 during Infection of Macrophages by Histoplasma capsulatum

Abstract

Hypoxia-inducible factor 1α (HIF-1α) regulates the immunometabolic phenotype of macrophages, including the orchestration of inflammatory and antimicrobial processes. Macrophages deficient in HIF-1α produce excessive quantities of the anti-inflammatory cytokine interleukin 10 (IL-10) during infection with the intracellular fungal pathogen Histoplasma capsulatum (R. A. Fecher, M. C. Horwath, D. Friedrich, J. Rupp, G. S. Deepe, J Immunol 197:565-579, 2016, https://doi.org/10.4049/jimmunol.1600342). Thus, the macrophage fails to become activated in response to proinflammatory cytokines and remains the intracellular niche of the pathogen. Here, we identify the tricarboxylic acid (TCA) cycle metabolite fumarate as the driver of IL-10 during macrophage infection with H. capsulatum in the absence of HIF-1α. Accumulation of fumarate reduced expression of a HIF-1α-dependent microRNA (miRNA), miR-27a, known to mediate decay of Il10 mRNA. Inhibition of fumarate accrual in vivo limited IL-10 and fungal growth. Our data demonstrate the critical role of HIF-1α in shaping appropriate TCA cycle activity in response to infection and highlight the consequences of a dysregulated immunometabolic response. IMPORTANCE Histoplasma capsulatum and related Histoplasma species are intracellular fungal pathogens endemic to broad regions of the globe, including the Americas, Africa, and Asia. While most infections resolve with mild or no symptoms, failure of the host to control fungal growth produces severe disease. Previously, we reported that loss of a key transcriptional regulator, hypoxia-inducible factor 1α (HIF-1α), in macrophages led to a lethal failure to control growth of Histoplasma (R. A. Fecher, M. C. Horwath, D. Friedrich, J. Rupp, G. S. Deepe, J Immunol 197:565-579, 2016, https://doi.org/10.4049/jimmunol.1600342). Inhibition of phagocyte activation due to excessive interleukin 10 by HIF-1α-deficient macrophages drove this outcome. In this study, we demonstrate that HIF-1α maintains contextually appropriate TCA cycle metabolism within Histoplasma-infected macrophages. The absence of HIF-1α results in excessive fumarate production that alters miRNA-27a regulation of interleukin-10. HIF-1α thus preserves the capacity of macrophages to transition from a permissive intracellular niche to the site of pathogen killing.

Keywords: Histoplasma; hypoxia inducible factor 1; innate immunity; lung; mitochondrial metabolism.

FIG 1

MiR-27a regulates the decay of Il10 during Hc infection. (A) Il10 mRNA decay curve, measured by reverse transcription-qPCR (RT-qPCR) at 0 to 180 min of cessation of transcription via actinomycin D treatment in Lyz2cre and Lyz2cre HIF1α^fl/fl BMDMs at 24 h postinfection. Data are normalized to the quantity of Il10 in samples isolated at 0 min. (B) Expression of HIF-1α-dependent miRNAs, quantified by RT-qPCR, in BMDMs at 24 h postinfection. Data are normalized to uninfected controls. (C) miR-27a expression measured by RT-qPCR in BMDMs infected with H. capsulatum (Hc). Data are normalized to uninfected controls. Il10 mRNA (D) was measured by RT-qPCR at 24 h postinfection, and IL-10 protein (E) was quantified by ELISA at 48 h postinfection in BMDMs previously transfected with a miR-27a mimic (agomir), miR-27a inhibitor (antagomir), or Il10 3′ UTR target site blocker. IL-10 protein was below the level of detection in uninfected BMDMs (data not shown). (F) Il10 mRNA was quantified by RT-qPCR at 0 to 180 min of cessation of transcription via actinomycin D treatment in BMDMs at 24 h postinfection. BMDMs were transfected with scrambled RNA control or miR-27a mimic 18 h prior to infection. Data are normalized to the quantity of Il10 in samples isolated at 0 min. All data are means and standard errors of the means (SEM). In panels A to D and F, data are from 3 biologically independent samples, representative of 3 experiments. In panel E, data are from 5 biologically independent samples, representative of 3 experiments. For panels B to E, two-way ANOVA with Student-Newman-Keuls post hoc testing was used to compare data between the groups. *, P < 0.05; ***, P < 0.001. For the Il10 mRNA decay curves, two-way ANOVA with Student-Newman-Keuls post hoc testing was used to compare data between the infected Lyz2cre and Lyz2cre HIF1α^fl/fl BMDMs (A) or to compare data between the scrambled RNA and mimic-treated Lyz2cre HIF1α^fl/fl BMDMs (F). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

HIF-1α regulates the metabolism of Hc-infected BMDMs. (A) Serial measurements of extracellular lactate were quantified with a commercial kit. Basal glycolysis (glycolytic proton efflux rate [glycoPER]) (B) and basal mitochondrial respiration (oxygen consumption rate [OCR]) (C) were measured using the Seahorse XFe96 analyzer (Agilent). (D) Intracellular pyruvate quantity was measured with a commercial kit. (E) Extracellular IL-10 protein production by BMDMs was measured by ELISA at 48 hpi. (F) miR-27a expression by BMDMs was measured by qPCR at 27 hpi. (A to F) Data are means and SEM and are representative of 2 separate experiments. Data in panels A and F are from 4 biologically independent samples; data in panels B to E are from 6 biologically independent samples. For all panels, two-way ANOVA with Student-Newman-Keuls post hoc testing was used to compare data between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

FIG 3

TCA cycle activity is elevated in macrophages during Hc infection in the absence of HIF-1α. (A) TCA cycle metabolites were quantified by LC-MS at 24 h postinfection. Data are pooled from 3 separate experiments and represent the mean log₂ fold change versus uninfected Lyz2cre BMDMs from 4 biologically independent samples per experiment. Serial measurements of intracellular fumarate (B) and succinate (C) were quantified by commercial kit and are presented as means and SEM from 4 biologically independent samples (representative of 2 experiments). (D) Expression of TCA cycle enzyme mRNAs was quantified by glycolysis RT² Profiler PCR array (Qiagen); data were pooled from 3 separate experiments with 1 biologically independent sample per experiment. (E and F) Intracellular activities of SDH and FH were quantified by microplate activity assay. Data are means and SEM from 4 biologically independent samples (representative of 2 experiments). (A and D) Two-tailed Student’s t tests were used to compare data between infected Lyz2cre and Lyz2cre Hif1α^fl/fl groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B, C, E, and F) Two-way ANOVA with Student-Newman-Keuls post hoc testing was used to compare data between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G) Model of the TCA cycle in infected HIF-1α-deficient macrophages.

FIG 4

Inhibition of the TCA cycle restrains IL-10 during H. capsulatum infection. (A to C) IL-10 cytokine production was quantified by ELISA at 48 h after concurrent Hc infection and treatment with (A) UK5099, (B) DMM, or (C) dimethyl fumarate. (D) BMDMs were transfected with 20 nM scrambled RNA control, 20 nM siRNA-Sdha, or 20 nM siRNA-Fh1 for 18 h prior to Hc infection. IL-10 was quantified by ELISA at 48 h postinfection. Intracellular fumarate was quantified with a commercial kit at 24 h postinfection in cells treated with DMM (E) or siRNAs (F). (A to D) Data are means and SEM for 4 biologically independent samples per group and are representative of 3 separate experiments. (E and F) Data are mean fold change versus uninfected wild-type control and SEM (n = 10), pooled from 2 separate experiments. For all panels, two-way ANOVA with Student-Newman-Keuls post hoc testing was used to compare data between groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Fumarate decreases miR-27a expression. (A) miR-27a expression was quantified by RT-qPCR at 24 h after concurrent Hc infection and treatment with 10 μM UK5099 or DMM. Data are mean fold difference relative to uninfected Lyz2cre control, with SEM, for 4 biologically independent samples, representative of 2 experiments. (B) BMDMs were transfected with 20 nM scrambled RNA control, 20 nM siRNA-Sdha, or 20 nM siRNA-Fh1 for 18 h prior to Hc infection. MiR-27a expression was measured by RT-qPCR at 24 h postinfection. Data are means and SEM for 3 biologically independent samples, representative of 2 experiments. (C) IL-10 protein was quantified by ELISA after 48 h of concurrent Hc infection and treatment with 1 mM exogenous α-ketoglutarate (α-kg) and 10 nM dimethyl fumarate. Data are means and SEM for 5 biologically independent samples, representative of 2 experiments. (D) KDM5 demethylase activity was quantified by fluorometric assay after 24 h of concurrent Hc infection and treatment with 10 μM dimethyl malonate (DMM). (E) H3K4me3 was quantified by ELISA using total protein samples extracted from BMDMs at 24 h postinfection. Data are means and SEM for 7 biologically independent samples, pooled from 2 separate experiments. For all panels, two-way ANOVA with Student-Newman-Keuls post hoc testing was used to compare data between groups; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

TCA cycle activity drives IL-10 and increased fungal burden in vivo. (A) F4/80⁺ cells were sorted from lung homogenate at day 3 postinfection in Lyz2cre and Lyz2cre Hif1α^fl/fl mice. Il10 mRNA and miR-27a were measured by RT-qPCR, with data normalized to expression in uninfected F4/80⁺ lung cells. Data are means and SEM for 6 biologically independent samples, pooled from 2 experiments. (B and C) Lungs were collected from Lyz2cre and Lyz2cre Hif1α^fl/fl mice at day 3 postinfection, after 4 days of treatment with DMSO (vehicle), 12 mg/kg UK5099, or 600 mg/kg DMM. (B) Lung IL-10, quantified by ELISA. (C) Lung fungal burden, quantified by CFU. Data are means and SEM for 6 biologically independent samples, pooled from 2 experiments. For all panels, two-way ANOVA with Student-Newman-Keuls post hoc testing was used to compare data between the groups. **, P < 0.01; ***, P < 0.001.