Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

Flagellin-independent effects of a Toll-like receptor 5 polymorphism in the inflammatory response to Burkholderia pseudomallei.

BACKGROUND: Toll-like receptors (TLRs) are sentinel receptors of the innate immune system. TLR4 detects bacterial lipopolysaccharide (LPS) and TLR5 detects bacterial flagellin. A common human nonsense polymorphism, TLR5:c.1174C>T, results in a non-functional TLR5 protein. Individuals carrying this variant have decreased mortality from melioidosis, infection caused by the flagellated Gram-negative bacterium Burkholderia pseudomallei. Although impaired flagellin-dependent signaling in carriers of TLR5:c.1174C>T is well established, this study tested the hypothesis that a functional effect of TLR5:c.1174C>T is flagellin-independent and involves LPS-TLR4 pathways. METHODOLOGY/PRINCIPAL FINDINGS: Whole blood from two independent cohorts of individuals genotyped at TLR5:c.1174C>T was stimulated with wild type or aflagellated B. pseudomallei or purified bacterial motifs followed by plasma cytokine measurements. Blood from individuals carrying the TLR5:c.1174C>T variant produced less IL-6 and IL-10 in response to an aflagellated B. pseudomallei mutant and less IL-8 in response to purified B. pseudomallei LPS than blood from individuals without the variant. TLR5 expression in THP1 cells was silenced using siRNA; these cells were stimulated with LPS before cytokine levels in cell supernatants were quantified by ELISA. In these cells following LPS stimulation, silencing of TLR5 with siRNA reduced both TNF-α and IL-8 levels. These effects were not explained by differences in TLR4 mRNA expression or NF-κB or IRF activation. CONCLUSIONS/SIGNIFICANCE: The effects of the common nonsense TLR5:c.1174C>T polymorphism on the host inflammatory response to B. pseudomallei may not be restricted to flagellin-driven pathways. Moreover, TLR5 may modulate TLR4-dependent cytokine production. While these results may have broader implications for the role of TLR5 in the innate immune response in melioidosis and other conditions, further studies of the mechanisms underlying these observations are required.

Survey of innate immune responses to Burkholderia pseudomallei in human blood identifies a central role for lipopolysaccharide.

B. pseudomallei is a gram-negative bacterium that causes the tropical infection melioidosis. In northeast Thailand, mortality from melioidosis approaches 40%. As exemplified by the lipopolysaccharide-Toll-like receptor 4 interaction, innate immune responses to invading bacteria are precipitated by activation of host pathogen recognition receptors by pathogen associated molecular patterns. Human melioidosis is characterized by up-regulation of pathogen recognition receptors and pro-inflammatory cytokine release. In contrast to many gram-negative pathogens, however, the lipopolysaccharide of B. pseudomallei is considered only weakly inflammatory. We conducted a study in 300 healthy Thai subjects to investigate the ex vivo human blood response to various bacterial pathogen associated molecular patterns, including lipopolysaccharide from several bacteria, and to two heat-killed B. pseudomallei isolates. We measured cytokine levels after stimulation of fresh whole blood with a panel of stimuli. We found that age, sex, and white blood cell count modulate the innate immune response to B. pseudomallei. We further observed that, in comparison to other stimuli, the innate immune response to B. pseudomallei is most highly correlated with the response to lipopolysaccharide. The magnitude of cytokine responses induced by B. pseudomallei lipopolysaccharide was significantly greater than those induced by lipopolysaccharide from Escherichia coli and comparable to many responses induced by lipopolysaccharide from Salmonella minnesota despite lower amounts of lipid A in the B. pseudomallei lipopolysaccharide preparation. In human monocytes stimulated with B. pseudomallei, addition of polymyxin B or a TLR4/MD-2 neutralizing antibody inhibited the majority of TNF-α production. Challenging existing views, our data indicate that the innate immune response to B. pseudomallei in human blood is largely driven by lipopolysaccharide, and that the response to B. pseudomallei lipopolysaccharide in blood is greater than the response to other lipopolysaccharide expressing isolates. Our findings suggest that B. pseudomallei lipopolysaccharide may play a central role in stimulating the host response in melioidosis.

microRNAs regulate human embryonic stem cell division.

microRNAs (miRNAs) regulate numerous physiological processes such as cell division and differentiation in many tissue types including stem cells. To probe the role that miRNAs play in regulating processes relevant to embryonic stem cell biology, we used RNA interference to silence DICER and DROSHA, the two main miRNA processing enzymes. Consistent with a role for miRNAs in maintaining normal stem cell division and renewal, we found that perturbation of miRNA pathway function in human embryonic stem cells (hESCs) attenuates cell proliferation. Normal cell growth can be partially restored by introduction of the mature miRNAs miR-195 and miR-372. These miRNAs regulate two tumor suppressor genes, respectively: WEE1, which encodes a negative G2/M kinase modulator of the CycB/CDK complex and CDKN1A, which encodes p21, a CycE/CDK cyclin dependent kinase inhibitor that regulates the G1/S transition. We show that in wild-type hESCs, WEE 1 levels control the rate of hESC division, whereas p21 levels must be maintained at a low level for hESC division to proceed. These data support a model for hESC cell cycle control in which miRNAs regulate negative cell cycle modulators at two phases of the cell cycle to ensure proper replenishment of the stem cell population.

Differential responses of FLIPLong and FLIPShort-overexpressing human myeloid leukemia cells to TNF-alpha and TRAIL-initiated apoptotic signals.

OBJECTIVE: Clonal marrow cells from patients with early myelodysplastic syndrome (MDS) undergo apoptosis in response to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Cells from advanced MDS are resistant to TRAIL. Two isoforms of the Flice inhibitory protein (FLIP) short (FLIPS) and FLIP long (FLIPL), which modulate TRAIL signals, showed disease-stage-dependent differential regulation. Therefore, we aimed at characterizing potential differential effects of FLIPL and FLIPS, on TRAIL and TNF-alpha-induced apoptosis in model leukemic cell lines. MATERIALS AND METHODS: Using lentiviral constructs, FLIPL and FLIPS, as well as a green fluorescent protein control were overexpressed in ML-1 cells, which constitutively express very low levels of FLIP and are highly sensitive to apoptosis induction. Cells were then exposed to TRAIL or TNF-alpha, and effects on the extrinsic and intrinsic pathways of apoptosis induction were assessed. RESULTS: Overexpression of FLIP reduced TRAIL and TNF-alpha-induced apoptosis in ML-1 cells. However, while FLIPL completely abrogated apoptosis, FLIPS allowed for BID cleavage and caspase-3 activation. Concurrently, there was a decline of Bcl-xL and X-linked inhibitor of apoptosis protein (XIAP) in FLIPS cells followed by apoptosis. Further, inhibition of nuclear factor-kappaB (NF-kappaB) activation in TNF-alpha-treated cells resulted in profound apoptosis in FLIPS, but not in FLIPL-overexpressing cells, consistent with the observations in patients with early stage MDS. Inhibition of NF-kappaB had only minimal effects on TRAIL signaling. CONCLUSION: Thus, FLIPL and FLIPS exerted differential effects in myeloid leukemic cell lines in response to TRAIL and TNF-alpha. It might be possible to therapeutically exploit those differences with effector molecules specific for the FLIP isoforms.

Differential effects of bexarotene on intrinsic and extrinsic pathways in TRAIL-induced apoptosis in two myeloid leukemia cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces programmed cell death (apoptosis) preferentially in tumor cells. However, not all cancer cells are sensitive to TRAIL. We determined whether ligation of the retinoid receptor, RXR, would sensitize cells to TRAIL-mediated apoptosis. The leukemic cell lines KG1a (apoptosis-resistant) and ML-1 (apoptosis-sensitive) were treated with the RXR-specific retinoid bexarotene, TRAIL, or both, and apoptosis was determined. In KG1a cells, bexarotene downregulated FLIP(Long) and activated caspase-8, thereby allowing for TRAIL-triggered apoptosis. Overexpression of FLIP(Long) in ML-1 cells abrogated apoptosis. In unmodified ML-1 cells bexarotene enhanced programmed cell death via truncation of Bid and release of cytochrome C. Blockade of caspase-8 prevented enhancement in both cell lines; blockade of caspase-9 had a significant effect only in ML-1 cells. Thus, the effect of bexarotene on TRAIL-mediated programmed cell death involved proximal events of the extrinsic pathway; however, downstream signals involved the intrinsic pathway in ML-1 but not in KG1a cells. These studies add further information to the regulation of programmed cell death in leukemic cells that have to be considered when designing therapeutic strategies.

NF-kappaB and FLIP in arsenic trioxide (ATO)-induced apoptosis in myelodysplastic syndromes (MDSs).

Tumor necrosis factor (TNF)-alpha, a potent stimulus of nuclear factor-kappaB (NF-kappaB), is up-regulated in myelodysplastic syndrome (MDS). Here, we show that bone marrow mononuclear cells (BMMCs) and purified CD34+ cells from patients with low-grade/early-stage MDS (refractory anemia/refractory anemia with ring sideroblasts [RA/RARS]) have low levels of NF-kappaB activity in nuclear extracts comparable with normal marrow, while patients with RA with excess blasts (RAEB) show significantly increased levels of activity (P = .008). Exogenous TNF-alpha enhanced NF-kappaB nuclear translocation in MDS BMMCs above baseline levels. Treatment with arsenic trioxide (ATO; 2-200 microM) inhibited NF-kappaB activity in normal marrow, primary MDS, and ML1 cells, even in the presence of exogenous TNF-alpha (20 ng/mL), and down-regulated NF-kappaB-dependent antiapoptotic proteins, B-cell leukemia XL (Bcl-XL), Bcl-2, X-linked inhibitor of apoptosis (XIAP), and Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein (FLIP), leading to apoptosis. However, overexpression of FLIP resulted in increased NF-kappaB activity and rendered ML1 cells resistant to ATO-induced apoptosis. These data are consistent with the observed up-regulation of FLIP and resistance to apoptosis with advanced MDS, where ATO as a single agent may show only limited efficacy. However, the data also suggest that combinations of ATO with agents that interfere with other pathways, such as FLIP autoamplification via NF-kappaB, may have considerable therapeutic activity.