Defense Mechanisms of Hepatocytes Against Burkholderia pseudomallei

The Gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyze the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNγ can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNγ, IL-1β, and TNFα is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by inducible nitric oxide synthase-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNγ-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes

Restricting Microbial Exposure in Early Life Negates the Immune Benefits Associated with Gut Colonization in Environments of High Microbial Diversity

Background: Acquisition of the intestinal microbiota in early life corresponds with the development of the mucosal immune system. Recent work on caesarean-delivered infants revealed that early microbial composition is influenced by birthing method and environment. Furthermore, we have confirmed that early-life environment strongly influences both the adult gut microbiota and development of the gut immune system. Here, we address the impact of limiting microbial exposure after initial colonization on the development of adult gut immunity. Methodology/Principal Findings: Piglets were born in indoor or outdoor rearing units, allowing natural colonization in the immediate period after birth, prior to transfer to high-health status isolators. Strikingly, gut closure and morphological development were strongly affected by isolator-rearing, independent of indoor or outdoor origins of piglets. Isolator-reared animals showed extensive vacuolation and disorganization of the gut epithelium, inferring that normal gut closure requires maturation factors present in maternal milk. Although morphological maturation and gut closure were delayed in isolatorreared animals, these hard-wired events occurred later in development. Type I IFN, IL-22, IL-23 and Th17 pathways were increased in indoor-isolator compared to outdoor-isolator animals during early life, indicating greater immune activation in pigs originating from indoor environments reflecting differences in the early microbiota. This difference was less apparent later in development due to enhanced immune activation and convergence of the microbiota in all isolator-reared animals. This correlated with elevation of Type I IFN pathways in both groups, although T cell pathways were still more affected in indoor-reared animals. Conclusions/Significance: Environmental factors, in particular microbial exposure, influence expression of a large number of immune-related genes. However, the homeostatic effects of microbial colonization in outdoor environments require sustained microbial exposure throughout development. Gut development in high-hygiene environments negatively impacts on normal succession of the gut microbiota and promotes innate immune activation which may impair immune homeostasis

<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

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

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

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 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

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

Caspase-1-Dependent and -Independent Cell Death Pathways in <i>Burkholderia pseudomallei</i> Infection of Macrophages

<div><p>The cytosolic pathogen <i>Burkholderia pseudomallei</i> and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during <i>B. pseudomallei</i> infection have not been addressed so far in detail. Furthermore, the role of <i>B. pseudomallei</i> factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a <i>B. pseudomallei</i> flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of <i>B. pseudomallei</i> infection.</p></div