Sage Journals: Discover world-class research

Abstract

Innate immunity coordinates LPS detection via TLR4 on polymorphonuclear leukocytes (PMNs) to elicit responses to many Gram-negative bacteria. In this study, we describe the effects of five subtypes of LPS [isolated from Escherichia coli B4, Pseudomonas aeruginosa PAO1, multidrug-resistant P. aeruginosa (MDRP), Acinetobacter baumannii and multidrug-resistant A. baumannii (MDRA)] on gene expression in PMNs. LPS isolated from B4, PAO1, and A. baumannii did not significantly alter TLR2 expression. However, LPS from MDRP and MDRA caused a 0.6-fold decrease and 2.7-fold increase, respectively, in TLR2 expression. Similarly, TLR4 expression was not significantly altered by LPS isolated from B4, PAO1 and A. baumannii but was down-regulated by LPS isolated from MDRP and MDRA by 0.1- and 0.6-fold, respectively. All LPS subtypes, excluding PAO1, down-regulated CD14 expression in PMNs. However, all five LPS subtypes up-regulated TNFA, IL1B, IL6, IL10 and TREM1 expression in a concentration-dependent manner, with the most substantial responses observed following exposure to LPS from MDRP and MDRA. These different effects on the gene expression in PMNs may depend on variation in LPS structural modifications related to acquired drug resistance, such as acylation and/or glycosylation.

Keywords

LPS polymorphonuclear leukocyte MDRA MDRP RT-PCR

Introduction

The human innate immune system identifies bacterial components via PRRs.¹ LPS present in the outer membrane of Gram-negative bacteria is often a diphosphorylated, polar macromolecule that contains hydrophobic elements within its lipid A core structure, as well as hydrophilic elements in its repeating polysaccharide surface. This molecule is recognized by several extracellular and cell surface proteins including the LPS-binding protein, CD14, MD-2 and TLR4, which are expressed by polymorphonuclear leukocytes (PMNs) and macrophages. The abundance of proteins involved in LPS detection reflects the requirement for specific protein–LPS and protein–protein interactions.¹ TLR4 induces both innate and adaptive immune responses to LPS.² This system triggers abundant inflammatory responses that enable clearance of bacterial infections; however, improper regulation can also result in immune system malfunction.

Studies on LPS structure during the course of infection have shown that LPS can be modified in response to environmental stimuli.³ Lipid A acylation patterns have been shown to be altered in LPS isolated from Pseudomonas aeruginosa strains infecting the lung of patients with cystic fibrosis, and O-antigen composition has similarly been shown to vary with environmental conditions.^4,5 However, gene expression analysis in PMNs stimulated by LPS from different Gram-negative bacteria strains has not been reported. In this study, we selected clinically important opportunistic organisms—P. aeruginosa, Acinetobacter baumannii (A.b) and their respective, multidrug-resistant strains—to elucidate their effects on PMN gene expressions.

Materials and methods

Reagents

Endotoxin-free Hanks’ balanced salt solution (HBSS), PBS and deionized water were purchased from Life Technology Japan (Tokyo, Japan). All reagents were checked and confirmed to be endotoxin free, DNase free, RNase free and pyrogen free by Life Technology Japan. Furthermore, all reagents and solutions included in the commercial kits in this study were confirmed to be free from contamination by endotoxin testing, which was performed in compliance with the following guidelines: ANSI/AAMI ST72: 2011; Bacterial endotoxins-Test methods, routine monitoring, and alternative to batch testing; USP <85> Bacterial Endotoxin Test.⁶

LPSs

LPSs isolated from Escherichia coli O111:B4 (B4), P. aeruginosa (PAO1), multidrug-resistant P. aeruginosa (MDRP), A.b and multidrug-resistant A.b (MDRA) were purchased from Wako Pure Chemical Industries (Tokyo, Japan). LPS was prepared by phenol extraction.⁷ The phenol extract contained < 1% protein. LPSs contained endotoxin levels of not less than 500,000 EU (endotoxin units)/mg.

PMN preparation

Human PMNs were isolated from healthy volunteers’ peripheral blood.⁸ Briefly, 20 ml whole blood was mixed with 4.5% dextran solution and allowed to stand for 40 min at room temperature. The leukocyte-rich plasma was centrifuged at 400 g on an endotoxin-free Ficoll-Paque Plus gradient (Amersham Bioscience, Milwaukee, WI, USA) for 20 min. Hypotonic (0.2%) saline was used to lyse erythrocytes and osmolality was restored by hypertonic (1.6%) saline. PMNs were only used in experiments if the viability was 99% or more (as assessed by trypan blue exclusion) and the purity was at least 95% (as assessed by morphological analysis). PMNs were adjusted to a final concentration of 1 × 10⁷ cells/ml in endotoxin-free HBSS. We obtained blood from nine volunteer donors [six men and three women, aged 29–58 yr (mean age 39 yr)]. Each donor donated blood more than once and the mRNA expression levels of 11 genes (one was the reference gene, ACTB) were averaged. Healthy individuals were confirmed, at the start of the study, through consent forms and interviews to be physically healthy, non-smokers who were not taking any medication and were informed about the study purpose; consent was obtained from all participants. The protocol was approved by the ethical review committee at the Teikyo University School of Medicine (No. 07-104).

PMN stimulation with LPS

LPS, resuspended in 100% DMSO (endotoxin-free), was inoculated into PMN suspension (5 × 10⁶ cells/ml) at 10, 100, or 500 ng/ml. The control group in this study was untreated PMNs (5 × 10⁶ cells/ml) without any added LPS. The mixtures were incubated with controls for 10 and 30 min in an environment containing 5% CO₂. Following stimulation, PMNs were collected and washed with cold endotoxin-free PBS.

RNA preparation

Human PMNs (5 × 10⁶ cells/ml) were washed with 1 ml of cold endotoxin-free PBS. Total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. The quantity and quality of total RNA samples were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany).

Complimentary DNA synthesis

Total RNA was reverse-transcribed to cDNA by using SuperScript VILO cDNA synthesis kit (Invitrogen Life Technologies, Carlsbad, CA, USA). Briefly, 1 µg of total RNA was incubated with 2.5 μM oligo (dT)₂₀, 50 ng of random hexamers and 200 U SuperScript III RT enzyme in a 20-μl reaction volume at 42℃ for 60 min, followed by 50℃ for 20 min. The reactions were terminated by heating the solution at 85℃ for 5 min.

Quantitative real-time PCR (qPCR) analysis

The mRNA expression levels of all genes were quantified in PMNs after treatment with LPS for 10 and 30 min.⁹ In gene expression analysis, the control group was untreated PMNs, i.e. intact PMNs resuspended in endotoxin-free HBSS. The gene expression levels of TREM1 (GenBank accession no. MN_018643.2) in PMNs were quantified using the StepOne real-time PCR system (Applied Biosystems, Foster City, CA, USA). Complementary DNA was amplified with SYBR Green by using the Power SYBR Green PCR master mix (Invitrogen). Briefly, qPCR was performed for TREM1 and the housekeeping gene ACTB (GenBank accession no. MN_001101.1). The PCR primer sets are described in Table 1. The following cDNA amplification program was used: 95℃ for 10 min; 40 cycles of 95℃ for 15 s; 60℃ for 1 min. All PCR reactions were performed in 20-μl reaction volumes comprising the following components: 1 μl of cDNA solution, 0.9 U Ampli Taq Gold DNA polymerase, 1 × reaction buffer [20 mM Tris-HCl pH 8.4, 3 mM MgCl₂, 200 μM dVTPs (mixture of dATP, dCTP, and dGTP), 400 μM dUTP, 500 nM ROX reference dye and 0.6 U uralic glycosylase] and 200 nM primers. TREM1 mRNA expression levels in PMNs were normalized to the gene expression levels of ACTB. Fold changes of PMN TREM1 mRNA levels between LPS-stimulated samples and untreated controls were determined using the Sequence Detection System software (Applied Biosystems).¹⁰

Table 1.

Primer sets for quantitative polymerase chain reaction.

Genes	Sequences	Amplicon sizes
TLR2	F: 5′-TCTGCTATGATGCATTTGTTT-3′	150 bp
	R: 5′-TATTGTCAATGATCCACTTGC-3′
TLR4	F: 5′-ATTTCAGCTCTGCCTTCACTA-3′	212 bp
	R: 5′-CTTCTGCAGGACAATGAAGAT-3′
CD14	F: 5′-CGCTCGAGGACCTAAAGATA-3′	243 bp
	R: 5′-CAGACAGGTCTAGGCTGGTAA-3′
TNFA	F: 5′-AGACCAAGGTCAACCTCCT-3′	194 bp
	R: 5′-AAAGTAGACCTGCCCAGAC-3′
IL1B	F: 5′-AACAAGTGGTGTTCTCCATGT-3′	185 bp
	R: 5′-AAATCGCTTTTCCATCTTCTT-3′
IL6	F: 5′-AGCTATGAACTCCTTCTCCAC-3′	170 bp
	R: 5′-GTTTGTCAATTCGTTCTGAAG-3′
IL8Rs	F: 5′-GGTCATCTTTGCTGTCGTCC-3′	191 bp
	R: 5′-CGTAGATGATGGGGTTGAG-3′
ITGAM	F: 5′-AAGGTGTCCACACTCCAGAAC-3′	204 bp
	R: 5′-GAGGAGCAGTTTGTTTCCAAG-3′
IL10	F: 5′-AAGCCTTGTCTGAGATGATCC-3′	161 bp
	R: 5′-GTTTTCACAGGGAAGAAATCG-3′
TREM1	F: 5′-GTCTCCACTCCTGACTCTGAA-3′	158 bp
	R: 5′-TAGGGTACAAATGACCTCAGC-3′
ACTB	F: 5′-TTAAGGAGAAGCTGTGCTACG-3′	205 bp
	R: 5′-TTGAAGGTAGTTTCGTGGATG-3′

F: forward primer; R: reverse primer; bp: base pairs.

Briefly, in this study, we employed the absolute quantification method for qPCR analysis. The copy numbers of each target gene were divided by the copy number of the same gene in untreated PMNs. All copy numbers of the target gene were adjusted relative to the number of copies of the housekeeping gene ACTB.

Statistical analysis

All P-values were determined by the nonparametric unpaired or paired t-tests (two-tailed) using Excel 2011 (Microsoft, Tokyo, Japan). We considered P < 0.05 to be significant and the degree of significance is indicated as **P < 0.01.

Results

We did not find any significant differences in gene expression levels of the 11 genes tested in PMNs between healthy men and women stimulated with LPS under the same conditions (data not shown).

PRRs in LPS-stimulated PMNs

TLR2 gene expression levels in B4, PAO1 and A.b LPS-stimulated PMNs were not significantly altered. MDRP LPS treatment, however, down-regulated TLR2 expression in PMNs 0.6-fold, while MDRA LPS stimulation up-regulated it 2.7-fold (Figure 1A).

Figure 1.

Gene expression analysis of PRRs in LPS-stimulated PMNs. mRNA expression levels of (A) TLR2, (B) TLR4,and (C) CD14 can be seen. LPS was administered at 0, 10, 100 and 500 ng/ml. All reaction tubes were incubated at 37℃ for 10 min. mRNA expression levels observed for 0 ng/ml LPS were considered as baseline and designated as 1.0. The fold-change is indicated as a ratio. *P < 0.05; **P < 0.01 compared with healthy subjects. Error bars represent SEM. Data are representative of at least three separate experiments.

Similarly, B4, PAO1 and A.b LPS did not significantly alter TLR4 expression in PMNs. In comparison, MDRA and MDRP LPS-stimulated PMNs expressed 0.1- and 0.6-fold lesser levels of TLR4 transcript, respectively (Figure 1B).

PMNs stimulated by all LPS subtypes, excluding PAO1, down-regulated CD14 expression. MDRA and MDRP LPS elicited the most significant repression, which caused an average 0.3-fold reduction in CD14 transcript levels (Figure 1C).

Inflammatory cytokines in LPS-stimulated PMNs

All LPS subtypes up-regulated TNFA, IL1B and IL6 expression in PMNs in a dose-dependent manner. Moreover, the transcript levels of these inflammatory cytokines from MDRA and MDRP LPS-stimulated PMNs were higher than those in PMNs stimulated by A.b and PAO1 LPS (Figures 2A–C) and induced an average fold change of 3.2—7.5.

Figure 2.

Gene expression analysis of inflammatory cytokines in LPS-stimulated PMNs. mRNA expression levels of (A) TNFA, (B) IL1B and (C) IL6 can be seen. Sample treatments and data analyses were as outlined in Figure 1.

Chemokine receptors in LPS-stimulated PMNs

IL8Rs expression levels in LPS-treated PMNs were not significantly altered (Figure 3A, B). However, MDRA and MDRP LPS stimulated higher levels of ITGAM expression in PMNs compared with A.b and PAO1 LPS (Figure 3B). MDRA and MDRP LPS-stimulated PMNs up-regulated ITGAM expression by 1.5-and 1.7-fold, respectively.

Figure 3.

Gene expression analysis of chemokine receptors in LPS-stimulated PMNs. mRNA expression levels of (A) IL8Rs and (B) ITGAM can be seen. Sample treatments and data analyses were as outlined in Figure 1.

Anti-inflammatory cytokine in LPS-stimulated PMNs

All LPS subtypes up-regulated IL10 expression in a dose-dependent manner. In particular, IL10 expression in MDRA LPS-stimulated PMNs was higher than that in A.b-stimulated PMNs (Figure 4). On average, IL10 expression was up-regulated 3.8-fold in response to LPS.

Figure 4.

Gene expression analysis of the anti-inflammatory cytokine IL10 in LPS-stimulated PMNs. mRNA expression levels of IL10 can be seen. Sample treatments and data analyses were as outlined in Figure 1.

Pro-inflammatory myeloid cell receptor in LPS-stimulated PMNs

All LPS subtypes, excluding A.b, induced up-regulation of TREM1 in a dose-dependent manner. TREM1 levels in MDRA and MDRP LPS-stimulated PMNs were higher than those in A.b and PAO1 LPS-stimulated PMNs (Figure 5). LPS from these multidrug-resistant subtypes up-regulated TREM1 expression 2.7- and 2.2-fold, respectively.

Figure 5.

Gene expression analysis of the pro-inflammatory myeloid cell receptor TREM1 in LPS-stimulated PMNs. mRNA expression levels of TREM1 can be seen. Sample treatments and data analyses were as outlined in Figure 1.

Discussion

Even at a minute dose, LPS can induce endotoxemia, which causes increased levels of circulating pro-inflammatory cytokines, exacerbated hypothermia and an often-fatal outcome. It can also activate non-transcriptional cellular responses, including autophagy, endocytosis, phagocytosis, and oxidative burst.^11–13 In human cells, three receptors for LPS have been identified: (1) soluble or membrane-bound CD14–MD2–TLR4 complex, (2) ITGAM (β2 integrins) and (3) scavenger receptors for lipid molecules. Soluble and membrane-bound CD14 greatly potentiates the host response to small quantities of LPS and other microbial mediators.¹⁴

MD2–TLR4 best detects bisphosphorylated diglucosamine to which six saturated fatty acyl chains with lengths of 12, 14 or 16 carbons are attached; this structure is called ‘hexa-acyl LPS’.¹⁵ Escherichia coli (E. coli) and A. b. produce LPS with predominantly hexa-acyl lipid A structures. However, P. aeruginosa produces LPS with predominantly penta-acyl lipid A structures.¹⁵ Lipid A is recognized as the most toxic and/or inflammatory region of LPS, although the polysaccharide portion of the molecule also has potent immunomodulatory and immunostimulatory properties.¹⁶ In genetic analysis, the activation of a two-component system (TCS), PmrA/B, is triggered by environmental stimuli and specific mutations within the TCS that result in their constitutive activation and subsequent overexpression of LPS-modifying genes.^17,18 Activation of the PmrA/B TCS leads to the up-regulation of the pmrCAB and arnBCADTEF-pmrE operons, which mediate the synthesis and transfer of pEtN and 4-amino-4-deoxy-L-arabinopyranose (Arap4N), respectively, to lipid A.^19,20

Modification of lipid A with pEtN occurs as a result of mutations in the pmrA/B TCS in A. b. Mutations in pmrA and/or pmrB have been found to induce the constitutive expression of pmrA (the cognate response regulator) and the subsequent autoregulation of the promoter region of the pmrCAB operon, which leads to the modification of the 4′- or 1-phosphate group of lipid A with pEtN. Mutations have been reported in different domains of both PmrA and PmrB.^21,22 Additionally, pmrB appears to be the most commonly mutated gene in A. b.²³ Moreover, glycosylation of lipid A with galactosamine (hexosamine) at the 1-phosphate group has been recently reported in resistant strains.²⁴

Unlike A. b, P. aeruginosa has both the pmrA/B and phoP/Q TCSs, each of which can separately regulate the arnBCADTEF operon.²⁵ Several studies have shown that P. aeruginosa can develop resistance to polymyxins via the constitutive modification of its LPS with Arap4N, which is stimulated by the pmrA/B and phoP/Q TCSs.^25,26 The synthesis and transfer of Arap4N to the lipid A are accomplished by the arnBCADTEF operon in P. aeruginosa. These genetic alterations are known to lead to the modification of lipid A with Arap4N. For P. aeruginosa, only one study has reported a mutation in pmrA that may be responsible for resistance;²⁷ the other mutations that have been described are mainly localized to PmrB or generally distributed to cognate regulators of TCSs. In terms of lipid A modifications in resistant bacteria, there appears to be an absolute relationship between colistin resistance and Arap4N addition and a significant relationship between colistin resistance and the loss of secondary lauroyl chain hydroxylation. Moreover, there is wide structural variation in lipid A in polymyxin-resistant P. aeruginosa in terms of the presence of either penta- or hexa-acylated lipid A.²⁸

Once TLR4 binds to LPS, two possible pathways of cellular activation can occur through either the myeloid differentiation factor MyD88 or the TLR domain adaptor-inducing IFN-β (TRIF) pathway.^29,30 The cascade of inflammatory cytokines and other inflammatory mediators following LPS exposure contributes to generalized inflammation, procoagulant activity, tissue injury and septic shock.^31,32 Similarly, TLR4 activation in response to LPS signaling can also lead to LPS toxicity and plays a role in septic shock.³³

Core genes in innate immunity, such as PRRs, inflammatory cytokines, chemokine receptors, anti-inflammatory cytokines and the pro-inflammatory myeloid cell receptor were selected,³⁴ and quantitative real-time RT-PCR was performed to validate their expression. Specifically, we evaluated TLR2, which recognizes bacterial lipoproteins; TLR4 and CD14, which recognize LPS from Gram-negative rods; TNFA, IL6 and IL1B, which were induced by activation of PRRs (for IL1B, this occurred via the inflammasome); IL8 (a strong chemoattractant of PMNs) and ITGAM, which are recognized by IL8Rs and mediate PMN migration; IL10, an anti-inflammatory and resolving cytokine; and TREM1, an amplifier of inflammation and useful biomarker of bacterial infections.

The mRNA expression levels of TLR2, TLR4, CD14, IL8Rs and ITGAM after 30 min of LPS stimulation were not significant or demonstrated a similar pattern as that observed after 10 min of LPS stimulation (data not shown). However, the mRNA levels of TNFA, IL1B, IL6, IL10 and TREM1 after 10 and 30 min LPS stimulation were significantly increased in a dose-dependent manner. The results of TLR2, TLR4 and CD14 expression did not indicate a dose-dependent effect (Figure 1). We speculate that this was mainly attributed to an overdose of LPS. PRRs recognize specific PAMPs and are sensitive because they guard the entrance to the signal-transduction cascade.¹⁴ The minimum concentration of LPS for recognition may therefore be < 10 ng/ml (data not shown). In our study, gene expression levels of PRRs (TLR2, TLR4 and CD14), inflammatory cytokines (TNFA, IL1B and IL6), chemokine receptors (IL8Rs and ITGAM), an anti-inflammatory receptor (IL10), and a pro-inflammatory myeloid cell receptor (TREM1) in LPS-stimulated PMNs were altered between multidrug-resistant organisms (MDROs) and non-MDROs. In MDROs, various modifications (i.e. acylation and glycosylation) of lipid A can occur. MD2–TLR4 best detects hexa-acyl LPS. E. coli and A. b produce LPS with predominantly hexa-acyl lipid A structures. However, P. aeruginosa produces LPS with predominantly penta-acyl lipid A structures.¹⁵

In MDROs, such as MDRA and MDRP, the presence of mutations in pmrA and/or pmrB can cause modification in the lipid A structure. In MDRA, hexa-acyl lipid A may be predicted to be modified by glycosylation, whereas penta-acyl lipid A in MDRP may be characterized as either penta- or hexa-acylated lipid A.²⁸

In this study, LPS generally up-regulated pro-inflammatory genes (Table 2). This dramatic increase in the expression of these inflammatory cytokines evokes systemic inflammation and cytokine storm. Furthermore, TREM1 overexpression can promote severe systemic inflammation because of its role as an amplifier of inflammation.³⁵ MDROs can cause persistent infection in patients, resulting in longer durations of inflammation than that caused by non-MDROs, even after antibiotic treatment.³⁶ In this study, LPS derived from MDROs up-regulated the expression levels of genes encoding inflammatory cytokines in PMNs to a greater extent than LPS derived from non-MDRO. Taken together, our findings suggested that patients with MDRP or MDRA pneumonia may be expected to experience a more severe response than patients with non-MDRO pneumonia.³⁶

Table 2.

Average fold change of gene expression levels in LPS-stimulated PMNs related to untreated controls. Positive numbers indicate up-regulation and negative values represent down-regulation.

	LPS
Immunomodulatory Genes	B4	PAO1	MDRP	A.b	MDRA
Pattern recognition receptors
TLR2	1.7	1.3	−0.6	1.6	2.7
TLR4	1.1	1.1	−0.1	1.0	−0.6
CD14	−0.2	1.0	−0.3	−0.1	−0.3
Inflammatory cytokines
TNF-α	7.2	4.5	6.7	4.1	7.5
IL-1β	3.9	2.5	4.7	3.1	3.2
IL-6	6.6	2.6	3.9	2.8	5.0
Chemokine receptors
IL-8Rs	1.3	−0.3	1.1	1.2	1.1
ITGAM	1.3	−0.2	1.7	−0.1	1.5
Anti-inflammatory cytokine
IL-10	1.6	3.0	3.2	2.4	3.8
Pro-inflammatory myeloid cell receptor
TREM1	2.3	2.4	2.7	1.2	2.2

B4: E. coli O111:B4 strain; PAO1: P. aeruginosa.

These results suggest that two common single-nucleotide polymorphisms in TLR2 and TLR4—Arg753Gln and Asp299Gly, respectively—are associated with shorter time to onset of severe sepsis or septic shock in patients admitted to the intensive care unit.³⁷ Additionally, it is possible that gene-specific epigenetic reprogramming occurs in endotoxin-tolerant murine macrophages. For example, TLR4-dependent processes remodel chromatin at distinct classes of genes such that pro-inflammatory genes are silenced, while anti-inflammatory and antimicrobial genes are activated.³⁸

It is also possible that these LPS subtypes contain different biochemical modifications, and are thus structurally distinct. Indeed, gene expression levels in PMNs in response to LPS from drug-sensitive strains (PAO1 or A.b) different from that to LPS from their respective drug-resistant strains (MDRP or MDRA). Several mechanisms of resistance to polymyxins such as colistin and other antimicrobial peptides have been reported; most of these pathways alter LPS synthesis to decrease the affinity between the antimicrobial agent and its target.³⁹ For example, two mechanisms of colistin resistance have been reported in A.b. The first involves inactivation of one of three genes required for lipid A biosynthesis—lpxA, lpxD, or lpxC—which results in total loss of LPS.^40,41 In a second mechanism, lipid A modification mediated by the addition of phosphoethanolamine results in loss of colistin affinity and subsequent resistance; these modifications are regulated by the PmrA/B TCS.^22,42

LPS and lipid A from A. b are implicated in diverse biological activities such as toxicity, pyrogenicity, mitogenicity and inflammation.^43,44 Particularly, Fernandez et al. reported that biofilm formation and swarming motility of MDRP led to multiresistant states.⁴⁵ Pelletier et al. predicted that Acinetobacter harbors lptA, which encodes pEtN transferase at the 4′ position of lipid A.²⁴ Mutation and pEtN addition to lipid A resulted in an increased inflammatory response via TLR4. These findings may also affect the virulence and cytotoxicity of MDRP and MDRA.

Furthermore, A.b is naturally competent for DNA uptake and demonstrates high rates of natural transformation.⁴⁶ Genomic studies have reported that some A.b strains have ≥ 28 genomic islands encoding antibiotic resistance determinants.⁴⁷

Among PAO1 variants, psrA mutants exhibit supersusceptibility to polymyxin B and indocilin because of outer membrane permeabilization. Multiple pathways are implicated in this increased sensitivity, including virulence factors such as type III secretion systems, biofilm formation, and adhesion or motility.⁴⁸

In addition, pure LPS is likely to exhibit more hydrophobic qualities, resulting in rapid binding to available host membranes³. Furthermore, the physical size of LPS complexes directly influences CD14-dependent internalization of these complexes.⁴⁹ Future studies about the genetic and cellular mechanisms that govern LPS-stimulated effects on gene expression are necessary to appreciate fully this fascinating recognition of PAMPs by PRRs.

Footnotes

Acknowledgements

We thank Ms. C Miyazaki for providing technical assistance.

Funding

Financial support for this study was provided by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24591490).

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

References

Gioannini

Weiss

. Regulation of interactions of Gram-negative bacterial endotoxins with mammalian cells. Immunol Res 2007; 39: 249–260.

Medzhitov

. Approaching the asymptote: 20 years later. Immunity 2009; 30: 766–775.

Ellis

Kuehn

. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiol Mol Biol R 2010; 74: 81–94.

Ernest

Guo

. Pseudomonas aeruginosa. Science 1999; 286: 1561–1565.

Sabra

Lundsdorf

Zeng

. Alterations in the formation of lipopolysaccharide and membrane vesicles on the surface of Pseudomonas aeruginosa PAO1 under oxygen stress conditions. Microbiology 2003; 149: 2789–2795.

http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm (last accessed 1 July 2015).

Leive

Morrison

. Isolation of lipopolysaccharides from bacteria. Methods Enzymol 1972; 28: 254–262.

Ubagai

Tansho

Ito

. Influences of aflatoxin B1 in reactive oxygen species generation and chemotaxis of human polymorphonuclear leukocytes. Toxicol in Vitro 2008; 22: 1115–1120.

Ubagai

Koshibu

Koshio

. Downregulation of immunomdulator gene expression in LPS-stimulated human polymorphonuclear leukocytes by the proton pump inhibitor lansoprazole. J Infect Chemother 2009; 15: 374–379.

10.

Ubagai

Nakano

Kikuchi

. Gene expression analysis of TREM1 and GRK2 in polymorphonuclear leukocytes as the surrogate biomarkers of acute bacterial infections. Int J Med Sci 2014; 11: 215–221.

11.

Blander

Medzhitov

. Regulation of phagosome maturation by signals from Toll-like receptors. Science 2004; 304: 1014–1018.

12.

Sanjuan

Dillon

Tait

. Toll-like receptor signaling in macrophages links the autophagy pathway to phagocytosis. Nature 2007; 450: 1253–1257.

13.

West

Brodssky

Rahner

. TLR signaling augments macrophage bactericidal activity through mitochondrial ROS. Nature 2011; 472: 476–480.

14.

Hoffman

Kafatos

Janeway

. Phylogenetic perspectives in innate immunity. Science 1999; 284: 1313–1317.

15.

Munford

. Sensing Gram-negative bacterial lipopolysaccharides: a human disease determinant? Infect Immun 2008; 76: 454–465.

16.

Raetz

Whitfield

. Lipopolysaccharide endotoxins. Annu Rev Biochem 2002; 71: 635–700.

17.

Abraham

Kwon

. A single amino acid substituteon in PmrB is associated with polymyxin B resistance in clinical isolate of Pseudomonas aeruginosa. FEMS Microbiol Lett 2009; 298: 249–254.

18.

Barrow

Kwon

. Alterations in two-component regulatory systems of phoPQ and pmrAB are associated with polymyxin B resistance in clinical isolates of Pseudomonas aeruginosa. Agents Chemother 2009; 53: 5150–5154.

19.

Gunn

. Bacterial modifyion of LPS and resistance to antimicrobial peptides. J Endotoxin Res 2001; 7: 57–62.

20.

Raetz

Reynolds

Trent

Bishop

. Lipid A modifyion systems in Gram-negative bacteria. Annu Rev Biochem 2007; 76: 295–329.

21.

Arroyo

Fernandez

Hankins

. The pmrCAB operon mediates polymyxin resistance in Acinetobacter baumannii ATCC 17987 and clinical isolates through phosphoethanolamine modification of lipid A. Antimicrob Agents Chemother 2011; 55: 3743–3751.

22.

Beceiro

Llobet

Aranda

. Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system. Antimicrob Agents Chemother 2011; 55: 3370–3379.

23.

Rolain

Diene

Kempf

. Real-time sequencing to decipher the molecular mechanism of resistance of a clinical pan-drug-resistant Acinetobacter baumannii isolate from Marseille, France. Antimicrob Agents Chemother 2013; 57: 592–596.

24.

Pelletier

Casella

Jones

. Unique structural modifications are present in the lipopolysaccharide from colistin-resistant strains of Acinetobacter baumannii. Antimicrob Agents Chemother 2013; 57: 4831–4840.

25.

McPhee

Lewenza

Hancock

. Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol Microbiol 2003; 50: 205–217.

26.

Macfarlane

Kwasnicka

Ochs

. PhoP-PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance. Mol Microbiol 1999; 34: 305–316.

27.

Lee

. Mutations and expression of PmrAB and PhoPQ related with colistin resistance in Pseudomonas aeruginosa clinical isolates. Diagn Microbiol Infect Dis 2014; 78: 271–276.

28.

Moskowitz

Brannon

Dasgupta

. PmrB mutations promote polymyxin resistance of Pseudomonas aeruginosa isolated from colistin-treated cystic fibrosis patients. Antimicrob Agents Chemother 2012; 56: 1019–1030.

29.

Akira

Uematsu

Takeuchi

. Pathogen recognition and innate immunity. Cell 2006; 124: 783–801.

30.

Beutler

Rietschel

. Innate immune sensing and its roots: the story of endotoxin. Nature Rev Immunol 2003; 3: 169–176.

31.

Calvano

Wenzhong

Rihards

. A network-based analysis of systemic inflammation in humans. Nature 2005; 473: 1032–1037.

32.

Suntharalingam

Perry

Ward

. Cytokine storm in a phase I trial of the anti-CD 28 monoclonal antibody TGN1412. New Engl J Med 2006; 355: 1018–1028.

33.

Opal

. The host response to endotoxin, antilipopolysaccharide strategies, and the management of severe sepsis. Int J Med Microbiol 2007; 297: 365–377.

34.

Kobayashi

Braughton

Whitney

. Bacterial pathogens modulate an apoptosis differentiation proGram in human neutrophils. Proc Natl Acad Sci 2003; 100: 10948–10953.

35.

Molloy

. Triggering receptor expressed on myeloid cells (TREM) family and the application of its antagonists. Rec Pat Antiinfect Drug Discov 2009; 4: 51–56.

36.

Martin-Loeches

Diaz

Valles

. Risks for multidrug-resistant pathogens in the ICU. Curr Opin Crit Care 2014; 20: 516–524.

37.

Nachtigall

Tamarkin

Tafelski

. Polymorphisms of the toll-like receptor 2 and 4 genes are associated with faster progression and a more severe course of sepsis in critically ill patients. J Int Med Res 2013; 42: 93–110.

38.

Foster

Hargreaves

Medzhitov

. Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature 2007; 477: 972–978.

39.

Beceiro

Tomas

Bou

. Antimicrobial resistance and virulence: a successful or deleterious association in the bacterial world? Clin Microbiol Rev 2013; 26: 185–230.

40.

Moffatt

Harper

Harrison

. Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production. Antimicrob Agents Chemother 2010; 54: 4971–4977.

41.

Henry

Vithanage

Harrison

. Colistin-resistant, lipopolysaccharide-deficient Acinetobacter baumannii responds to lipopolysaccharide loss through increased expression of genes involved in the synthesis and transport of lipoproteins, phospholipids, and poly-b-1:6-N-acetylglucosamine. Antimicrob Agents Chemother 2012; 56: 59–69.

42.

Adams

Nickel

Bajaksouzian

. Resistance to colistin in Acinetobacter baumannii associated with mutations in the PmrAB two-component system. Antimicrob Agents Chemother 2009; 53: 3628–3634.

43.

Lopez-Rojas

Jimenez-Mejias

Lepe

. Acinetobacter baumannii resistant to colistin alters its antibiotic resistance profile: a case report from Spain. J Infect Dis 2011; 204: 1147–1148.

44.

Rolain

Roch

Castanier

. Acinetobacter baumannii resistant to colistin with impaired virulence: a case report from France. J Infect Dis 2011; 204: 1146–1147.

45.

Fernandez

Alvarez-Ortega

Wiegand

. Characterization of the polymyxin B resistome of Pseudomonas aeruginosa. Antimicrob Agents Chemother 2013; 57: 110–119.

46.

Barbe

Vallenet

Fonknechten

. Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturaly transformation competent bacterium. Nucleic Acids Res 2004; 32: 5766–5779.

47.

Gomez

Neyfakh

. Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi. Antimicrob Agents Chemother 2006; 50: 3562–3567.

48.

Gooderham

Bains

McPhee

. Induction by cationic antimicrobial peptides and involvement in intrinsic polymyxin and antimicrobial peptide resistance, biofilm formation, and swarming motility of PsrA in Pseudomonas aeruginosa. J Bacteriol 2008; 190: 5624–5634.

49.

Kitchens

Munford

. CD14-dependent internalization of bacterial lipopolysaccharide (LPS) is strongly influenced by LPS aggregation but not by cellular responses to LPS. J Immunol 1998; 160: 1920–1928.

Gene expression analysis in human polymorphonuclear leukocytes stimulated by LPSs from nosocomial opportunistic pathogens

Abstract

Keywords

Introduction

Materials and methods

Reagents

LPSs

PMN preparation

PMN stimulation with LPS

RNA preparation

Complimentary DNA synthesis

Quantitative real-time PCR (qPCR) analysis

Statistical analysis

Results

PRRs in LPS-stimulated PMNs

Inflammatory cytokines in LPS-stimulated PMNs

Chemokine receptors in LPS-stimulated PMNs

Anti-inflammatory cytokine in LPS-stimulated PMNs

Pro-inflammatory myeloid cell receptor in LPS-stimulated PMNs

Discussion

Footnotes

Acknowledgements

Funding

Conflict of interest

References