human polymorphonuclear neutrophil responses to ... · 27/10/2008 · diabetic thai subjects. the...

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Human Polymorphonuclear Neutrophil Responses to Burkholderia

pseudomallei in Healthy and Diabetic Subjects

Sujin Chanchamroen1, Chidchamai Kewcharoenwong

1, Wattanachai Susaengrat

2, 5

Manabu Ato3, and Ganjana Lertmemongkolchai

1*

1The Center for Research and Development of Medical Diagnostic Laboratories,

Faculty of Associated Medical Sciences, Khon Kaen University, 2Department of

Medicine, Khon Kaen Hospital, Ministry of Public Health, Thailand. 3Department of 10

Immunology, National Institute of Infectious Diseases, Tokyo, Japan.

Running head: Human PMN responses to B. pseudomallei

*Corresponding author. Mailing address: Center for Research and Development in 15

Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences,

Khon Kaen University, Khon Kaen, Thailand. Phone: +66 4320 3825. Fax: +66 4320

3826. E-mail: [email protected]

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00503-08 IAI Accepts, published online ahead of print on 27 October 2008

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Abstract

The major predisposing factor to melioidosis is diabetes mellitus but no

immunological mechanisms have been investigated to explain this. In this study,

polymorphonuclear neutrophil (PMN) responses to Burkholderia pseudomallei, the

causative agent of melioidosis, were determined by flow cytometry in healthy and 5

diabetic Thai subjects. The results showed that B. pseudomallei displayed reduced

PMN uptake as compared to Salmonella enterica serovar Typhimurium and

Escherichia coli. Additionally, intracellular survival of B. pseudomallei was detected

throughout the 24 h period, indicating the intrinsic resistance of B. pseudomallei to

killing by PMNs. Moreover, PMNs from diabetic subjects displayed impaired 10

phagocytosis of B. pseudomallei, reduced migration in response to IL-8 and inability

to delay apoptosis. These data show that B. pseudomallei is intrinsically resistant to

phagocytosis and killing by PMNs. These observations, together with the impaired

migration and apoptosis in diabetes mellitus, may explain host susceptibility in

melioidosis. 15

Key words: B. pseudomallei; Melioidosis; Diabetes mellitus; PMN functions

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Introduction

Melioidosis is a serious infectious disease caused by the Gram-negative

bacillus, Burkholderia pseudomallei, and is endemic in northern Australia and

Southeast Asia, particularly northeast Thailand (4, 44). Infection occurs by

subcutaneous inoculation of contaminated soil, surface water or inhalation. The 5

clinical features vary from acute fulminant septicemia to chronic debilitating localized

infection. Therapeutic treatment is difficult and, even with recent improvements in

diagnosis and antibiotic regimens, the mortality rate associated with severe

melioidosis remains high at up to 50% (17). Moreover, recurrence of infection is

common despite adequate antimicrobial therapy (18). The risk factors for developing 10

melioidosis have been defined in several studies. Diabetes mellitus (DM), renal

diseases (renal calculi or renal failure), thalassemia and occupational exposure to

surface water are associated with an increased risk of melioidosis (4, 35).

Patients with diabetes mellitus, in particular, have a high incidence of

melioidosis, with up to 60% of patients having preexisting or newly diagnosed type 2 15

diabetes. The review of case records of 1,817 Thai patients with melioidosis revealed

that fewer than 10% of 382 patients with diabetes mellitus were insulin dependent or

had type I diabetes (31). However, no studies have been performed on the immune

functions of Thai diabetics with respect to B. pseudomallei.

In general, polymorphonuclear neutrophils (PMNs) play an important role in 20

the host inflammatory response against infection. Clinical investigations in subjects

who have diabetes mellitus and experimental studies in diabetic rats and mice clearly

demonstrate consistent defects of PMN chemotactic (12), phagocytic (19) and

antimicrobicidal activities (21). Up to now, the contribution of human PMNs to

resistance to B. pseudomallei infection has not been directly addressed, but indirect 25

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evidence suggests that they may play an important role in melioidosis. For example, a

previous study in Darwin, Australia that compared melioidosis patients who received

granulocyte colony-stimulating factor (G-CSF) with control subjects showed the

mortality rate decreased from 95% to 10% after the introduction of G-CSF (5). More

recently, a randomized controlled trial of G-CSF for the treatment of severe sepsis due 5

to melioidosis in Thailand resulted in a longer duration of host survival (6). These

results suggested the host benefit associated with G-CSF treatment that could involve

PMNs. Unfortunately, an in vitro whole blood assay was unable to explore the

mechanism of G-CSF action in the treatment of B. pseudomallei infection (5).

Additionally, it has been demonstrated in a murine model that the resistance against B. 10

pseudomallei infection is critically dependent on PMNs (9).

In this study, human PMN responses to B. pseudomallei, particularly in

diabetic Thai subjects who lived in an endemic area of melioidosis, were determined

in terms of bacterial killing, phagocytosis, migration and apoptosis.

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Materials and Methods

Human subjects

Forty-six diabetic and 26 healthy Thai subjects were enrolled into this study.

Diabetic subjects were defined as individuals who had preexisting diabetes mellitus 5

and were treated at the diabetes mellitus clinic, Outpatient Department, Khon Kaen

Hospital. Healthy subjects who lived in Khon Kaen, northeastern Thailand and had

normal blood counts, normal fasting blood glucose, and normal glycosylated

hemoglobin A1c (HbA1c) constituted the healthy control group. HbA1c is

glycosylated hemoglobin, which reflects the average blood glucose levels over the 10

previous 2–3 months and is generally used to monitor the degree of glycemic control

at DM clinics. In this study, DM subjects were defined as having good, poor or very

poor glycemic controls by the levels of HbA1c (5.5–7.5, 7.6–8.5 and greater than

8.5%, respectively). Characteristics of subjects are shown in Table 1. All were rice

farmers who were at risk of melioidosis, with no signs of acute infectious diseases 15

during the previous 3 months and at the time of the study. Any subjects with impaired

renal function defined by serum creatinine ≥ 2.0 mg/dl were excluded. The study was

reviewed and approved by the Khon Kaen University Ethics Committee for Human

Research and Khon Kaen Hospital Ethics committee. Written informed consent was

obtained from all study subjects. 20

Growth of bacteria

B. pseudomallei strain K96243 is the prototype strain for which the genome

sequence was derived. Salmonella enterica serovar Typhimurium and Escherichia

coli are clinical isolates that have been used extensively by our laboratory. The

bacteria were grown in Luria-Bertani (LB) broth for 18 h at 37°C, washed twice with 25

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PBS pH7.4, aliquoted and stored at −80°C. The number of viable bacteria was

determined by colony-forming counts and defined as colony-forming units (CFU)

prior to use. Live B. pseudomallei was handled under the Center for Disease Control

(CDC) regulations for biosafety containment level 3.

Labeling of bacteria with FITC 5

B. pseudomallei, S. Typhimurium and E. coli at 1×108 CFU/ml were incubated

with 1 µg/ml fluorescein isothiocyanate (FITC; Sigma, USA) in the dark at room

temperature for 60 min and analyzed for FITC intensity prior to use. FITC-labeled

bacteria were used in the experiment once only and discarded.

PMN isolation 10

Human PMNs were isolated from heparinized venous blood by 3.0% Dextran

T-500 sedimentation and Ficoll-PaquePLUS centrifugation (Amersham Biosciences,

UK). In all experiments, PMN purity was > 95%, as determined by Giemsa stain and

microscopy, while cell viability was > 98% as determined by trypan blue exclusion

(11). 15

Intracellular survival and replication of B. pseudomallei in human PMNs

Purified PMNs in RPMI 1640 were infected with B. pseudomallei at

Multiplicity of Infection (MOI) of 0.3:1 at 37°C for 30 min. Intracellular survival of

B. pseudomallei in PMNs was determined after the extracellular bacteria were killed

with 250 µg/ml kanamycin at 37°C for 30 minutes and culture supernatants were 20

checked for sterility by plating on LB agar plates.

Phagocytosis and oxidative burst assayed by flow cytometry

Diluted whole blood samples were stimulated in vitro with FITC-labeled

bacteria at MOI of 10:1 for 60 min or 800 ng/ml phorbol 12-myristate 13-acetate

(PMA; Sigma, USA) for 15 min at 37ºC, and 25 µl of 2800 ng/ml hydroethidine (HE; 25

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Sigma, USA) was added for a further 5 min at 37ºC. Erythrocytes were then lysed by

lysing buffer (BD Biosciences, USA), washed twice and fixed with 10%

paraformaldehyde for decontamination prior to analysis by flow cytometry

(FACSCalibur; BD Biosciences, USA) (24, 26, 43).

Determination of PMN migration 5

Purified PMNs at 5 × 106 cells/ml were incubated in the upper chamber of 3

µm pore Transwell plates (Corning Life sciences, Australia), and 20–100 ng/ml of

recombinant IL-8 (PeproTec, UK) were placed in the lower 0.5 ml chamber at 37°C

for 1 h. Transmigrated PMNs in the lower chamber were counted by flow cytometry

(FACSCalibur; BD Biosciences, USA), the migration index was calculated as: 10

number of transmigrated PMNs in response to IL-8/ number of transmigrated PMNs

in response to medium control (10). In some experiments, purified PMNs were

stimulated with intact heat killed B. pseudomallei at MOI of 1:1 or 1:10, at 37°C for 1

h prior to testing for migration.

PMN apoptosis assayed by flow cytometry 15

Apoptosis of PMNs was determined by flow cytometry using an annexin V

binding assay. Purified PMNs were cultured with medium alone or B. pseudomallei at

MOI of 1:1, 37ºC for 24 h. Intracellular survival of B. pseudomallei was quantified by

colony plating as described in the intracellular survival and replication assay. At the

indicated time points, cells were collected, washed with annexin V staining buffer (pH 20

7.4) and labeled with allophycocyanin (APC)-conjugated annexin V (BD Biosciences,

USA) for 15 min at room temperature. After washing, cells were fixed with 10%

paraformaldehyde and analyzed by flow cytometry (FACSCalibur; BD Biosciences,

USA) (8). In other experiments with heat-killed B. pseudomallei, propidium iodide

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(BD Biosciences, USA) was included with APC-conjugated annexin V and there was

no fixation step prior to analysis by flow cytometry.

Statistical analysis

Statistical analysis (Mann-Whitney test and paired t test) was performed by

Graphpad PRISM statistical software (GraphPad, San Diego, USA). A P value of < 5

0.05 was considered to be statistically significant.

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Results

Resistance of B. pseudomallei to phagocytosis and killing by human PMNs

We first monitored the intracellular survival of B. pseudomallei in PMNs. As

shown in Figure 1, after the initial 1 h of incubation (T0), the percentages of initial

inoculum of B. pseudomallei in diabetic PMNs (average 0.7%) was lower than that of 5

healthy PMNs (average 11.9%), even though it was not statistically significant (Mann

Whitney test, P> 0.05). After an additional 1 h of incubation, the numbers of

intracellular bacteria decreased 10–100-fold in PMN cells of both groups, but viable

B. pseudomallei could still be detected at 24 h. These results suggested that PMNs

from both healthy and DM groups can kill the majority of bacteria, but some B. 10

pseudomallei survive within human PMNs.

We then measured the uptake of B. pseudomallei by human PMNs in

comparison with other Gram negative bacteria, S. Typhimurium and E. coli by flow

cytometry. The results showed that B. pseudomallei was less efficiently phagocytosed

by PMNs than S. Typhimurium and E. coli (paired t test, P < 0.005) in both healthy 15

and diabetic groups (Figure 2). In addition, heat-killed B. pseudomallei, S.

Typhimurium and E. coli were assayed in the same condition and the results

confirmed the important finding that B. pseudomallei was phagocytosed by PMNs at a

lower rate than the other species (data not shown).

Poor glycemic control impaired B. pseudomallei phagocytosis 20

We next investigated whether the degree of glycemic control, as monitored by

percentage of glycosylated hemoglobin A1c (HbA1c) in DM, influenced the

antimicrobial activities of PMNs following encounter with B. pseudomallei. DM

subjects were classified into good, poor and very poor glycemic control by the levels

of HbA1c (5.5–7.5, 7.6–8.5, and above 8.5%, respectively). Firstly, phagocytosis of 25

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B. pseudomallei and oxidative burst by PMNs from diabetic and healthy subjects were

assessed as described above. Phagocytosis of B. pseudomallei was significantly

impaired in very poor glycemic control diabetic subjects (HbA1c>8.5%) when

compared with healthy subjects (Mann-Whitney test, P < 0.05) (Figure 3A and B).

Moreover, these results were in agreement with those obtained from our intracellular 5

survival assays suggesting that phagocytosis of B. pseudomallei from diabetic PMNs

were impaired in parallel with poor glycemic control. In addition, oxidative burst

induced by B. pseudomallei also tended to be impaired although this was not

statistically significant at 95% confidence interval (Mann-Whitney test, P = 0.0545)

while PMA induction was comparable between healthy and DM subjects (Figure 3A, 10

C and D). These results suggest that the extent of glycemic control influences the

impairment of PMN phagocytosis of B. pseudomallei, and might also affect the PMN

killing function via the oxidative burst.

Diabetic PMNs reduced migration in responses to interleukin-8 and this was

inhibited by intact B. pseudomallei 15

PMN migration in response to IL-8, a major chemokine responsible for this

function, was assessed in both subject groups. The results showed that PMNs from

diabetic subjects tended to reduce migration in responses to IL-8 in all doses that we

used when compared with healthy subjects (Figure 4A).

In other septic or bacteremic models of infection, pathogens are already in the 20

host system and several lines of evidence suggest that pathogens can interfere with

host immune responses. To investigate whether this PMN migration in response to IL-

8 could be altered upon encountering with B. pseudomallei, purified PMNs from five

healthy and five diabetic subjects were incubated with intact heat killed B.

pseudomallei at MOI of 1:1, 10:1 and 100:1 at 37ºC for 1 h. Then, stimulated PMNs 25

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were collected and tested for migration activity. Interestingly, the migration index of

B. pseudomallei stimulated PMNs was decreased when the numbers of B.

pseudomallei were increased (Figure 4B). These observations were significantly

demonstrated in healthy and DM subjects (paired t test, P < 0.005 and < 0.05,

respectively) suggesting that B. pseudomallei was capable of interfering with PMN 5

migration in both healthy and diabetic subjects.

B. pseudomallei decreased PMN apoptosis/necrosis

After phagocytosis, PMNs normally undergo apoptosis resulting in being

engulfed by macrophages but it has been reported in other pathogens such as

Anaplasma phagocytophilum (8), Leishmania major (1) and Chlamydia pneumoniae 10

(40) that these organisms could delay the spontaneous apoptosis of human PMNs. To

assess PMN apoptosis/necrosis after exposure to B. pseudomallei, purified PMNs

were incubated with live B. pseudomallei at MOI of 1:1 and analyzed for the kinetics

of annexin V positive PMNs at 0, 1, 3, 16 and 24 h post infection (Figure 5A). The

results showed that annexin V positive PMNs were clearly detected at 16 h and still 15

raised at 24 h, therefore this 24 h time point was selected for further studies in healthy

vs. DM subjects.

After the initial 1 h of incubation (T0), diabetic PMNs attained similar levels

of apoptosis/necrosis compared with healthy PMNs in the absence or presence of live

B. pseudomallei (Figure 5B). At 24 h of incubation (T24), the percentages of 20

spontaneous apoptotic/necrotic PMNs from healthy subjects were statistically reduced

in the presence of B. pseudomallei (paired t test, P < 0.05). However, this

phenomenon was not observed in diabetic subjects (paired t test, P > 0.05) (Figure

5C). These results indicated that live B. pseudomallei interfered with the spontaneous

apoptosis/necrosis of PMNs in healthy but not diabetic subjects. 25

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In addition, heat-killed bacteria were used to replace live B. pseudomallei

under the same conditions and PMNs were stained for annexin V and propidium

iodide (PI) (Figure 5D). The results showed that apoptotic PMNs, defined by annexin

V positive and PI negative cells were significantly decreased when compared with

medium alone (paired t test, P < 0.05 and < 0.005 in healthy and DM subjects, 5

respectively) (Figure 5E). These results indicated that apoptosis is a major event

during this 24 h with small numbers of necrotic cells (annexin V positive and PI

positive) and increasing MOI from 1:1 to 10:1 did not significantly change the

percentage of annexin V positive cells (data not shown). However, the mechanisms of

B. pseudomallei interference on PMN functions requires further investigation. 10

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Discussion

PMNs are the first line of host resistance against bacterial infection. The main

mechanisms that allow microbial killing are migration of PMNs to the site of

infection, phagocytosis and killing by both oxygen dependent and independent

mechanisms (20, 38). In addition, activated PMNs produce chemokines and cytokines 5

which recruit and activate other immune cells (38). Finally, activated PMNs undergo

apoptosis (16) resulting in phagocytosis by macrophages (41).

In this study, we have provided evidence that B. pseudomallei was

phagocytosed by PMNs at a lower rate than other Gram negative bacteria such as S.

Typhimurium and E. coli suggesting that B. pseudomallei might have antiphagocytic 10

activity; further studies are required to corroborate this conclusion. To avoid the

possible effects of varying bacterial doubling time, heat-killed bacteria of the three

pathogens were also studied and the results were consistent with the previous

observation with live bacteria indicating the distinctive character of the PMN–B.

pseudomallei interaction. However, the resistance to phagocytosis by other pathogens 15

involves several steps in the process of phagocytosis such as evasion of binding and

ingestion by interference with complement function. In B. pseudomallei infection, it

has been shown that capsular polysaccharide of this organism contributes to the

resistance to in vitro phagocytosis by reducing C3b deposition on the bacterial surface

(29). 20

Other mechanisms have been reported in Yersinia enterocolitica resistance to

phagocytosis: two adhesins, Inv and YadA, and the type III secretion systems

including effector proteins (7, 13, 42). These Yersinia effectors, which are referred to

as Yops (Yersinia outer proteins) are involved in inhibition of phagocytosis (13) and

YopT, which is an essential part of the antiphagocytic strategy, has been shown to 25

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disturb actin cytoskeleton (14, 30). Several reports have identified similar type III

secretion systems in B. pseudomallei contributing to bacterial virulence including

bipD, bipB and bopE (23, 32–34). However, type III secretion systems are unlikely to

be solely responsible for an antiphagocytic effect on the basis that it is an active

process that can be expected to be absent in heat inactivated bacteria. 5

Previous studies have documented the intracellular persistence of B.

pseudomallei; for example, B. pseudomallei triggered a poor killing mechanism (11,

27), PMNs were not capable of killing B. pseudomallei in the presence of 10% normal

serum (28) and the growth of B. pseudomallei was detected in PMNs after extended

incubation (15, 27). In our study, intracellular survival of B. pseudomallei in purified 10

PMNs was observed after extended incubation, consistent with the latter finding.

However, MOI ratio may affect the outcome of PMN functional assays, as in the other

studies, MOI varied from 4:1 to 100:1. We have used 0.3:1 and the result showed that

B. pseudomallei was still resistant to killing by human PMNs. In addition, it has been

demonstrated that B. pseudomallei is susceptible to the bactericidal effects of both 15

reactive nitrogen intermediate (RNI) and reactive oxygen intermediate (ROI) in a cell-

free system in vitro (22). The resistance of B. pseudomallei to the antimicrobial

activity of defensins may also facilitate intracellular survival in PMNs (15).

The major risk factor associated with severe melioidosis is diabetes mellitus

(35). One simple explanation is that innate immunity of diabetic patients, particularly 20

PMN functions is altered (2, 3, 36). Our study has demonstrated that diabetes mellitus

subjects have reduced PMN migration in response to IL-8 when compared with

healthy subjects. This may result in the delayed accumulation of PMNs at the site of

infection. Moreover, B. pseudomallei is a poor activator of IL-8 production from

human lung epithelial cell line A549 when compared with other Gram negative 25

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bacteria such as Salmonella enterica serovar Typhi (39). These findings suggest that

the signals initiated by the interaction of B. pseudomallei with epithelium cells at the

site of infection might not be sufficient for diabetic PMN recruitment.

In addition, diabetic PMNs exhibit reduced phagocytosis of B. pseudomallei in

poor glycemic control diabetic subjects. This is consistent with the result obtained 5

from intracellular survival assays that internalization of B. pseudomallei by diabetic

PMNs tended to be lower than that by PMNs from healthy subjects. A similar finding

has been reported in patients with poor glycemic control who showed impaired PMN

phagocytosis of virulent K1/K2 K. pneumoniae compared with patients with good

glycemic control and healthy volunteers (19). Therefore, persistently poor glycemic 10

control could have a progressively deleterious effect on phagocytic function. Further

investigation to reveal the mechanisms utilized by B. pseudomallei in antiphagocytic

activity and reduced PMN migration will be important. In addition, there was a trend

for the worst glycemic control DM subjects to display lower oxidative bursts in

response to B. pseudomallei and further studies are needed to address this observation 15

in more detail.

In the PMN apoptosis assay, B. pseudomallei infected PMNs from healthy

subjects delayed spontaneous apoptosis/necrosis up to 24 h while this phenomenon

was not significantly observed in diabetic subjects. It is not clear why the difference

has occurred. However, the delay of PMN apoptosis was significantly demonstrated 20

with heat-killed bacteria in both healthy and DM subjects, such a delay may favor

bacterial survival. A recent report demonstrated that PMNs produced their own

survival factors including cytokines and decreased Bax-α/Bcl-xL ratio during the early

steps of other infections when the number of bacteria was still low (25). PMN survival

may be extended in order to accomplish their functional activity in innate immunity. 25

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The reduced ability of diabetic PMNs to delay apoptosis following B. pseudomallei

exposure could result in the decrease of functional longevity of PMNs and increased

PMN clearance from the infectious sites. This would be consistent with previous data

which showing that diabetic PMNs underwent normal spontaneous apoptosis and did

not demonstrate LPS-induced inhibition of apoptosis (37). 5

Taken together, our results suggest that PMNs of diabetic subjects could be

defective within the early phase (24 h) of the inflammatory response against B.

pseudomallei. The alterations included not only migration, phagocytosis and apoptosis

but possibly also the killing mechanism via oxidative burst. Our experiments are the

first to directly address the immunological basis of diabetes as a major risk factor for 10

melioidosis. We believe that the impaired neutrophil functions of Thai diabetics with

poor glycemic control could contribute to their increased susceptibility to this

important disease.

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Acknowledgements

We thank Drs Gregory J. Bancroft and Mark P. Stevens for their critical

comments and reviewing the manuscript, Dr Debbie Smith, Ms Heidi Alderton and Dr

Jon Cuccui from the London School of Hygiene and Tropical Medicine, UK for

providing the training on biohazard containment level 3 at CMDL, Khon Kaen 5

University, Thailand and Ms Vicki Harley for her editorial help in preparing this

manuscript. This work was supported in part by Public Health Service Grant U01

AI061363 from the National Institute of Allergy and Infectious Diseases, USA.

10

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Figure legends

Figure 1 Intracellular survival of B. pseudomallei in purified PMNs from

healthy and diabetic subjects. Purified PMNs from 6 healthy (A) and 4

diabetic (B) subjects were co-cultured with live B. pseudomallei at an 5

MOI 0.3:1 for 30 min and extracellular organisms were killed by 250

µg/ml kanamycin for another 30 min prior to lysing the cells for

bacterial count (T0). Intracellular bacteria were quantified by colony

plating at the indicated time points and presented as percentages (%) of

initial inoculums for individuals calculated from the number of 10

recovered bacteria/total added number of B. pseudomallei.

Figure 2 Phagocytosis of B. pseudomallei, S. Typhimurium or E. coli by human

PMNs. Whole blood leukocytes were incubated with medium alone or

FITC-conjugated live bacteria at an MOI 10:1 for 60 min and analyzed 15

by flow cytometry. PMNs were analyzed for phagocytosis by mean

fluorescent intensity (MFI) of FITC (A), phagocytosis by PMNs from

7 healthy (B) and 14 diabetic (C) subjects. P-value was calculated by

paired t test, * P < 0.05, ** P < 0.005, *** P < 0.0005. Bps-B.

pseudomallei, Sal-S. Typhimurium, DM-diabetes mellitus. The vertical 20

lines represent mean ± SE of the group.

Figure 3 Phagocytosis and oxidative burst of PMNs from healthy, well

controlled, and poorly controlled diabetic subjects. Whole blood

leukocytes of healthy vs. diabetic subjects were incubated with 25

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medium alone, 800 ng/ml phorbol 12-myristate 13-acetate (PMA) or

FITC-conjugated live B. pseudomallei at an MOI 10:1 and analyzed by

flow cytometry. PMNs were analyzed for phagocytosis by MFI of

FITC-conjugated live B. pseudomallei and oxidative burst by MFI of

ethidium bromide or EB (A), phagocytosis of B. pseudomallei (B), 5

oxidative burst induced by PMA (C) or B. pseudomallei (D). P -value

was calculated by Mann Whitney test, * P < 0.05, ns-non significant,

(n)-number of subjects, DM-diabetes mellitus, HbA1c-glycosylated

hemoglobin A1c (good, poor and very poor glycemic control = 5.5–

7.5, 7.6–8.5 and above 8.5%, respectively). 10

Figure 4 Effect of B. pseudomallei on PMN migration in response to IL-8 of

healthy and diabetic subjects. Purified PMNs at 5 × 106 cells/ml of

healthy and diabetic subjects were examined for migration in a

transwell system in response to 20–100 ng/ml IL-8 (A) and the effect 15

of heat-killed B. pseudomallei on PMN migration responding to 100

ng/ml IL-8 by healthy (n = 5) (B) and diabetic (n = 5) subjects (C).

Transmigrated PMNs were counted by flow cytometry and migration

index was calculated as: number of transmigrated PMNs to IL-8/

number of transmigrated PMNs to medium control. DM-diabetes 20

mellitus, Hk-Bps-heat killed B. pseudomallei. P-value was calculated

by paired t test, * P < 0.05, ** P < 0.005.

Figure 5 PMN apoptosis in response to live B. pseudomallei of healthy and

diabetic subjects. PMNs were stained with annexin V-APC and 25

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analyzed for time kinetics by flow cytometry at 0–24 h (A). Purified

PMNs were co-incubated in vitro with medium alone or live B.

pseudomallei at an MOI 1:1, at 37ºC for 1 h = T0 (B) and 24 h = T24

(C). Annexin V and propidium iodide (PI) staining on PMNs incubated

with heat-killed B. pseudomallei for 24h (D) and calculated for 5

apoptosis in response to medium alone or B. pseudomallei by %

annexin V+ PI- PMNs (E). P-value was calculated by paired t test, * P

< 0.05, ** P < 0.005, ns-non significant, Bps-B. pseudomallei.

10

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Table 1

General Characteristics of Healthy and Diabetic Subjects

General characteristic Healthy Diabetes mellitus

Subjects (n) 36 56

Male (n) 19 26

Female (n) 17 30

Age (years)* 45±8 53±8

Fasting blood glucose (mg/dl)* 80±12 151±69

Hemoglobin A1c (%)* 5.1±0.5 8.0±1.9

Serum creatinine (mg/dl)* 1.0±0.2 1.2±0.4

Note: n= number, * = mean ± SD.

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human polymorphonuclear neutrophil responses to ... · 27/10/2008 · diabetic thai subjects. the...

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