<i>Burkholderia pseudomallei</i> modulates host iron homeostasis to facilitate iron availability and intracellular survival

<div><p>Background</p><p>The control over iron homeostasis is critical in host-pathogen-interaction. Iron plays not only multiple roles for bacterial growth and pathogenicity, but also for modulation of innate immune responses. Hepcidin is a key regulator of host iron metabolism triggering degradation of the iron exporter ferroportin. Although iron overload in humans is known to increase susceptibility to <i>Burkholderia pseudomallei</i>, it is unclear how the pathogen competes with the host for the metal during infection. This study aimed to investigate whether <i>B</i>. <i>pseudomallei</i>, the causative agent of melioidosis, modulates iron balance and how regulation of host cell iron content affects intracellular bacterial proliferation.</p><p>Principal findings</p><p>Upon infection of primary macrophages with <i>B</i>. <i>pseudomallei</i>, expression of ferroportin was downregulated resulting in higher iron availability within macrophages. Exogenous modification of iron export function by hepcidin or iron supplementation by ferric ammonium citrate led to increased intracellular iron pool stimulating <i>B</i>. <i>pseudomallei</i> growth, whereas the iron chelator deferoxamine reduced bacterial survival. Iron-loaded macrophages exhibited a lower expression of NADPH oxidase, iNOS, lipocalin 2, cytokines and activation of caspase-1. Infection of mice with the pathogen caused a diminished hepatic ferroportin expression, higher iron retention in the liver and lower iron levels in the serum (hypoferremia). <i>In vivo</i> administration of ferric ammonium citrate tended to promote the bacterial growth and inflammatory response, whereas limitation of iron availability significantly ameliorated bacterial clearance, attenuated serum cytokine levels and improved survival of infected mice.</p><p>Conclusions</p><p>Our data indicate that modulation of the cellular iron balance is likely to be a strategy of <i>B</i>. <i>pseudomallei</i> to improve iron acquisition and to restrict antibacterial immune effector mechanisms and thereby to promote its intracellular growth. Moreover, we provide evidence that changes in host iron homeostasis can influence susceptibility to melioidosis, and suggest that iron chelating drugs might be an additional therapeutic option.</p></div

Iron loading promotes HO-1 and FTH1 expression, intracellular iron availability and <i>B</i>. <i>pseudomallei</i> growth.

<p>BMM were treated with ferric ammonium citrate (FAC, 100 μM) or corresponding vehicle (A. bidest) for 20 hours followed by infection with <i>B</i>. <i>pseudomallei</i> at MOI 50. <b>(A)</b> 24 hours after infection gene expression of TfR1 (n = 6), Dmt1 (n = 5), HO-1 (n = 6), FPN (n = 5), and FTH1 (n = 6) was analysed by qRT-PCR. Expression of HO-1 was detected by immunoblot in cell lysates. Data are presented as mean with SEM. <b>(B)</b> Intracellular free iron levels were determined after 24 hours using the iron-sensitive fluorescent probe Phen Green SK. Data are expressed as mean with SEM of triplicate determinations (n = 3). (A, B) Comparison of groups was done using one-way ANOVA and the Bonferroni post-hoc test (*p<0.05, **p<0.01, ***p<0.001). <b>(C)</b> BMM or murine hepatoma Hepa1-6 cells were treated with FAC (100 μM) or corresponding vehicle for 20 hours followed by infection with <i>B</i>. <i>pseudomallei</i> at MOI 50 (BMM) or MOI 200 (Hepa1-6). Invasion (0 h) and intracellular growth (6 h, 24 h) of <i>B</i>. <i>pseudomallei</i> were examined by kanamycin protection assay. Data are presented as mean with SEM of triplicate determinations (n = 3). <b>(D)</b> LB broth or M9 minimal medium with or without FAC (100 μM) or corresponding vehicle was inoculated with <i>B</i>. <i>pseudomallei</i>. The optical density (OD) at 650 nm and colony forming units (CFU)/ml were determined at indicated time points. Data are presented as mean and SEM of duplicates. (C, D) Statistical analyses were conducted using Student’s <i>t-</i>test (*p<0.05, **p<0.01).</p

Limitation of iron availability ameliorates bacterial clearance and survival of infected mice.

<p>DFO (100 mg/kg) or vehicle (D-PBS)-treated C57BL/6 mice were intranasally inoculated with <i>B</i>. <i>pseudomallei</i> at 500 CFU. <b>(A)</b> Cumulative survival rate between groups was compared using (Log-rank Kaplan-Meier test, *<i>p</i><0.005 compared to vehicle-treated mice (2 independent experiments, n = 10)). <b>(B)</b> 48 hours after infection, the bacterial load (CFU) in BALF (n = 8) and organs (n = 10) was determined. Data from two experiments are expressed as box and whisker plots indicating minimum, maximum, quartiles and median. <b>(C)</b> Cytokine production (IL-6, MCP-1, TNFα, IFNγ) in BALF (n = 8) and serum (n = 10), and <b>(D)</b> myeloperoxidase (MPO) levels in serum (n = 5) were measured 48 hours after infection. (C, D) Data from two experiments are presented as mean with SEM. (B-D) Statistical analyses were done using a Student’s <i>t-</i>test (*p<0.05, **p<0.01).</p

Exogenously added hepcidin leads to increased iron levels stimulating intramacrophage replication of <i>B</i>. <i>pseudomallei</i>.

<p>BMM or murine hepatoma Hepa1-6 cells were treated with hepcidin (1 μg/ml) or corresponding vehicle (A. bidest) for 20 hours followed by infection with <i>B</i>. <i>pseudomallei</i> at MOI 50 (BMM) or MOI 200 (Hepa1-6). <b>(A)</b> Intracellular free iron levels were determined after 24 hours in BMM using the iron-sensitive fluorescent probe Phen Green SK. Data are presented as mean with SEM of triplicate determinations (n = 3). Statistical analyses were performed using one-way ANOVA and the Bonferroni post-hoc test (*p<0.05, ***<0.001). <b>(B)</b> Invasion (0 h) and intracellular growth (6 h, 24 h) of <i>B</i>. <i>pseudomallei</i> were examined by kanamycin protection assay. Data are expressed as mean with SEM of triplicate determinations (BMM, n = 3; Hepa1-6, n = 4). Statistical analyses were done using Student’s <i>t-</i>test (*p<0.05). <b>(C)</b> LB broth or M9 minimal medium with or without hepcidin (1 μg/ml) or corresponding vehicle was inoculated with <i>B</i>. <i>pseudomallei</i>. The optical density (OD) at 650 nm and colony forming units (CFU)/ml were determined at indicated time points. Data are presented as mean with SEM of duplicates. Statistical analyses were performed using Student’s <i>t-</i>test (*p<0.05).</p

Iron chelation limits intracellular <i>B</i>. <i>pseudomallei</i> growth by direct bacteriostatic properties.

<p>BMM were treated with deferoxamine (DFO, 50 μM) or corresponding vehicle (A. bidest) for 20 hours followed by infection with <i>B</i>. <i>pseudomallei</i> at MOI 50. <b>(A)</b> 24 hours after infection gene expression of TfR1 (n = 8), Dmt1 (n = 8), HO-1 (n = 6), FPN (n = 6), and FTH1 (n = 5) was analysed by qRT-PCR. Expression of HO-1 was detected by immunoblot in cell lysates. Data are expressed as mean with SEM. <b>(B)</b> Intracellular free iron levels were determined after 24 hours using the iron-sensitive fluorescent probe Phen Green SK. Data are presented as mean with SEM of triplicate determinations (n = 3). (A, B) Comparison of groups was done using one-way ANOVA and the Bonferroni post-hoc test (*p<0.05, **p<0.01, ***p<0.001). <b>(C)</b> BMM or murine hepatoma Hepa1-6 cells were treated with DFO (50 μM) or corresponding vehicle for 20 hours followed by infection with <i>B</i>. <i>pseudomallei</i> at MOI 50 (BMM) or MOI 200 (Hepa1-6). Invasion (0 h) and intracellular growth (6 h, 24 h) of <i>B</i>. <i>pseudomallei</i> were examined by kanamycin protection assay. Data are shown as mean with SEM of triplicate determinations (BMM, n = 3; Hepa1-6, n = 4). Statistical analyses were done using Student’s <i>t-</i>test (*p<0.05, **p<0.01, ***p<0.001). <b>(D)</b> LB broth or M9 minimal medium with or without DFO (50 μM) or corresponding vehicle was inoculated with <i>B</i>. <i>pseudomallei</i>. The optical density (OD) at 650 nm and colony forming units (CFU)/ml were determined at indicated time points. Data are expressed as mean and SEM of duplicates. Statistical analyses were conducted using Student’s <i>t-</i>test (*p<0.05, **p<0.01). <b>(E)</b> BMM were exposed to DFO (50 μM, dashed bars) or vehicle (unfilled bars) as indicated and infected with <i>B</i>. <i>pseudomallei</i>. Treatment was carried out both 20 hours prior to and directly (0 h) after infection, directly (0 h), three hours (3 h), or six hours (6 h) after infection. Intracellular bacterial growth was examined by kanamycin protection assay at 0, 6, and 24 hours. Data are presented as mean with SEM of triplicate determinations (n = 2). Statistical analyses were done using Student’s <i>t-</i>test (**p<0.01, ***p<0.001).</p

Iron availability promotes bacterial growth at the primary site of infection and increases the inflammatory response.

<p>FAC (5 mg/kg) or vehicle (D-PBS)-treated C57BL/6 mice were intranasally inoculated with <i>B</i>. <i>pseudomallei</i> at 500 CFU. <b>(A)</b> Cumulative survival rate between groups was compared using Log-rank Kaplan-Meier test (2 independent experiments, n = 10). <b>(B)</b> 48 hours after infection, the bacterial load (CFU) in BALF (n = 8) and organs (n = 10) was determined. Data from two experiments are expressed as box and whisker plots indicating minimum, maximum, quartiles and median. <b>(C)</b> Cytokine production (IL-6, MCP-1, TNFα, IFNγ) in BALF (n = 8) and serum (n = 10), and <b>(D)</b> myeloperoxidase (MPO) levels in serum (n = 5) were measured 48 hours after infection. (C, D) Data from two experiments are presented as mean with SEM. (B-D) Statistical analyses were done using a Student’s <i>t-</i>test (*p<0.05, **p<0.01).</p

Iron supplementation impairs immune response pathways in <i>B</i>. <i>pseudomallei</i>-infected macrophages.

<p>BMM were treated with FAC (100 μM) or corresponding vehicle (A. bidest) for 20 hours followed by infection with <i>B</i>. <i>pseudomallei</i> at MOI 50. <b>(A)</b> 24 hours after infection gene expression of neutrophil cytosolic factor 1 (Ncf1, n = 6), inducible nitric oxide synthase (iNOS, n = 4), tumor necrosis factor alpha (TNFα, n = 5), interleukin-6 (IL-6, n = 5), interleukin-1β (IL-1β, n = 4), NLR family pyrin domain containing 3 (Nlrp3, n = 4), and lipocalin 2 (Lcn2, n = 6) was analysed by qRT-PCR. <b>(B)</b> Lcn2 secretion in supernatants was measured by ELISA (n = 3). (A, B) Data are expressed as mean with SEM. Comparison of groups was done using one-way ANOVA and the Bonferroni post-hoc test (*p<0.05, **p<0.01, ***p<0.001). <b>(C)</b> Cleavage of caspase-1 and -7 and expression of GAPDH were detected by immunoblot in cell lysates of FAC- or vehicle-treated BMM at 24 hours after infection with <i>B</i>. <i>pseudomallei</i>. <b>(D)</b> Cytotoxicity was measured as lactate dehydrogenase (LDH) release in cell supernatants of FAC- or vehicle-treated <i>B</i>. <i>pseudomallei</i>-infected BMM. Data are presented as mean with SEM of triplicate determinations (n = 3). Statistical analyses were conducted using Student’s <i>t-</i>test (***p<0.001).</p

Enhanced Susceptibility of ADAP-Deficient Mice to Infection Is Associated With an Altered Phagocyte Phenotype and Function.

The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date, only limited data exist regarding the role of ADAP in pathogen-specific immunity during in vivo infection, and its contribution in phagocyte-mediated antibacterial immunity remains elusive. Here, we show that mice lacking ADAP (ADAPko) are highly susceptible to the infection with the intracellular pathogen Listeria monocytogenes (Lm) by showing enhanced immunopathology in infected tissues together with increased morbidity, mortality, and excessive infiltration of neutrophils and monocytes. Despite high phagocyte numbers in the spleen and liver, ADAPko mice only inefficiently controlled pathogen growth, hinting at a functional impairment of infection-primed phagocytes in the ADAP-deficient host. Flow cytometric analysis of hallmark pro-inflammatory mediators and unbiased whole genome transcriptional profiling of neutrophils and inflammatory monocytes uncovered broad molecular alterations in the inflammatory program in both phagocyte subsets following their activation in the ADAP-deficient host. Strikingly, ex vivo phagocytosis assay revealed impaired phagocytic capacity of neutrophils derived from Lm-infected ADAPko mice. Together, our data suggest that an alternative priming of phagocytes in ADAP-deficient mice during Lm infection induces marked alterations in the inflammatory profile of neutrophils and inflammatory monocytes that contribute to enhanced immunopathology while limiting their capacity to eliminate the pathogen and to prevent the fatal outcome of the infection

Enhanced Susceptibility of ADAP-Deficient Mice to Listeria monocytogenes Infection Is Associated With an Altered Phagocyte Phenotype and Function

The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date, only limited data exist regarding the role of ADAP in pathogen-specific immunity during in vivo infection, and its contribution in phagocyte-mediated antibacterial immunity remains elusive. Here, we show that mice lacking ADAP (ADAPko) are highly susceptible to the infection with the intracellular pathogen Listeria monocytogenes (Lm) by showing enhanced immunopathology in infected tissues together with increased morbidity, mortality, and excessive infiltration of neutrophils and monocytes. Despite high phagocyte numbers in the spleen and liver, ADAPko mice only inefficiently controlled pathogen growth, hinting at a functional impairment of infection-primed phagocytes in the ADAP-deficient host. Flow cytometric analysis of hallmark pro-inflammatory mediators and unbiased whole genome transcriptional profiling of neutrophils and inflammatory monocytes uncovered broad molecular alterations in the inflammatory program in both phagocyte subsets following their activation in the ADAP-deficient host. Strikingly, ex vivo phagocytosis assay revealed impaired phagocytic capacity of neutrophils derived from Lm-infected ADAPko mice. Together, our data suggest that an alternative priming of phagocytes in ADAP-deficient mice during Lm infection induces marked alterations in the inflammatory profile of neutrophils and inflammatory monocytes that contribute to enhanced immunopathology while limiting their capacity to eliminate the pathogen and to prevent the fatal outcome of the infection

ADAP Promotes Degranulation and Migration of NK Cells Primed During vivo Listeria monocytogenes Infection in Mice.

The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date only limited and moreover conflicting data exist regarding the role of ADAP in NK cells. To extend existing knowledge we investigated ADAP-dependency of NK cells in the context of in vivo infection with the intracellular pathogen Listeria monocytogenes (Lm). Ex vivo analysis of infection-primed NK cells revealed impaired cytotoxic capacity in NK cells lacking ADAP as indicated by reduced CD107a surface expression and inefficient perforin production. However, ADAP-deficiency had no global effect on NK cell morphology or intracellular distribution of CD107a-containing vesicles. Proteomic definition of ADAPko and wild type NK cells did not uncover obvious differences in protein composition during the steady state and moreover, similar early response patterns were induced in NK cells upon infection independent of the genotype. In line with protein network analyses that suggested an altered migration phenotype in naïve ADAPko NK cells, in vitro migration assays uncovered significantly reduced migration of both naïve as well as infection-primed ADAPko NK cells compared to wild type NK cells. Notably, this migration defect was associated with a significantly reduced expression of the integrin CD11a on the surface of splenic ADAP-deficient NK cells 1 day post-Lm infection. We propose that ADAP-dependent alterations in integrin expression might account at least in part for the fact that during in vivo infection significantly lower numbers of ADAPko NK cells accumulate in the spleen i.e., the site of infection. In conclusion, we show here that during systemic Lm infection in mice ADAP is essential for efficient cytotoxic capacity and migration of NK cells