Human Polymorphonuclear Neutrophil Responses to Burkholderia ...

INFECTION AND IMMUNITY, Jan. 2009, p. 456–463 0019-9567/09/$08.00⫹0 doi:10.1128/IAI.00503-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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Human Polymorphonuclear Neutrophil Responses to Burkholderia pseudomallei in Healthy and Diabetic Subjects䌤 Sujin Chanchamroen,1 Chidchamai Kewcharoenwong,1 Wattanachai Susaengrat,2 Manabu Ato,3 and Ganjana Lertmemongkolchai1* The Center for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University1, and Department of Medicine, Khon Kaen Hospital, Ministry of Public Health,2 Khon Kaen, Thailand, and Department of Immunology, National Institute of Infectious Diseases, Tokyo, Japan3 Received 23 April 2008/Returned for modification 29 May 2008/Accepted 26 September 2008

The major predisposing factor for 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, in healthy and diabetic Thai subjects were determined by flow cytometry. The results showed that B. pseudomallei displayed reduced uptake by PMNs compared to Salmonella enterica serovar Typhimurium and Escherichia coli. Additionally, intracellular survival of B. pseudomallei was detected throughout a 24-h period, indicating the intrinsic resistance of B. pseudomallei to killing by PMNs. Moreover, PMNs from diabetic subjects displayed impaired phagocytosis of B. pseudomallei, reduced migration in response to interleukin-8, and an 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. fection. Clinical investigations of subjects who have DM and experimental studies of diabetic rats and mice have clearly demonstrated consistent defects in PMN chemotactic (12), phagocytic (19), and antimicrobial activities (21). So far, the contribution of human PMNs to resistance to B. pseudomallei infection has not been directly addressed, but indirect evidence suggests that PMNs may play an important role in melioidosis. For example, a previous study done in Darwin, Australia, that compared melioidosis patients who received granulocyte colony-stimulating factor (G-CSF) with control subjects showed that 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 to melioidosis in Thailand resulted in survival of hosts for a longer time (6). These results suggested that there is a benefit to the host 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 treatment of B. pseudomallei infection (5). Additionally, it has been demonstrated using a murine model that the resistance against B. 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 area where melioidosis is endemic, were determined by examining bacterial killing, phagocytosis, migration, and apoptosis.

Melioidosis is a serious infectious disease caused by the gram-negative bacillus Burkholderia pseudomallei, and it is endemic in northern Australia and Southeast Asia, particularly northeast Thailand (4, 43). Infection occurs by subcutaneous inoculation of contaminated soil or surface water or by inhalation. The 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 (up to 50%) (17). Moreover, recurrence of infection is common despite adequate antimicrobial therapy (18). The risk factors for developing 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). In particular, patients with DM have a high incidence of melioidosis; up to 60% of patients have preexisting or newly diagnosed type 2 diabetes. A review of case records of 1,817 Thai patients with melioidosis revealed that fewer than 10% of 382 patients with DM were insulin dependent or had type I diabetes (31). However, no studies on the immune functions of Thai diabetics with respect to B. pseudomallei have been performed. In general, polymorphonuclear neutrophils (PMNs) play an important role in the host inflammatory response against in-

MATERIALS AND METHODS Human subjects. Fifty-six diabetic and 36 healthy Thai subjects were enrolled in this study. Diabetic subjects were defined as individuals who had preexisting DM and were treated at the DM clinic of the Outpatient Department, Khon Kaen Hospital. Healthy subjects who lived in Khon Kaen in northeastern Thailand and had normal blood counts, normal fasting blood glucose levels, and normal glycosylated hemoglobin A1c (HbA1c) levels constituted the healthy control group. HbA1c is a glycosylated hemoglobin which reflects the average

* Corresponding author. Mailing address: Center for Research and Development in 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: ganja_le @kku.ac.th. 䌤 Published ahead of print on 27 October 2008. 456

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TABLE 1. General characteristics of healthy and diabetic subjects Subjects

No.

No. of males

No. of females

Age (yr)

Fasting blood glucose level (mg/dl)a

HbAlc level (%)a

Serum creatine level (mg/dl)a

Healthy DM

36 56

19 26

17 30

45 ⫾ 8 53 ⫾ 8

80 ⫾ 12 151 ⫾ 69

5.1 ⫾ 0.5 8.0 ⫾ 1.9

1.0 ⫾ 0.2 1.2 ⫾ 0.4

a

The values are means ⫾ SD.

blood glucose levels over the previous 2 to 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 control on the basis of the levels of HbA1c (5.5 to 7.5, 7.6 to 8.5, and ⬎8.5%, respectively). Characteristics of subjects are shown in Table 1. All of the subjects were rice farmers who were at risk of melioidosis and had no signs of acute infectious diseases during the previous 3 months and at the time of the study. Any subjects with impaired renal function, defined by a serum creatinine level of ⱖ2.0 mg/dl, were excluded. The study was reviewed and approved by the Khon Kaen University Ethics Committee for Human Research and the Khon Kaen Hospital Ethics Committee. Written informed consent was obtained from all study subjects. Growth of bacteria. B. pseudomallei strain K96243 is the prototype strain whose genome sequence was determined. The Salmonella enterica serovar Typhimurium and Escherichia coli isolates used are clinical isolates that have been used extensively in our laboratory. The bacteria were grown in Luria-Bertanibroth for 18 h at 37°C, washed twice with phosphate-buffered saline (pH 7.4), aliquoted, and stored at ⫺80°C. The number of viable bacteria was determined by determining the number of CFU prior to use. Live B. pseudomallei was handled under the Centers for Disease Control regulations for biosafety containment level 3. Labeling of bacteria with FITC. B. pseudomallei, S. enterica serovar Typhimurium, and E. coli at a concentration of 1 ⫻ 108 CFU/ml were incubated with 1 ␮g/ml fluorescein isothiocyanate (FITC) (Sigma, United States) in the dark at room temperature for 60 min, and the FITC intensity was analyzed prior to use. FITC-labeled bacteria were used in an experiment once and discarded. PMN isolation. Human PMNs were isolated from heparinized venous blood by 3.0% dextran T-500 sedimentation and Ficoll-PaquePLUS centrifugation (Amersham Biosciences, United Kingdom). In all experiments, the PMN purity was ⬎95%, as determined by Giemsa staining and microscopy, while the cell viability was ⬎98%, as determined by trypan blue exclusion (11). Intracellular survival and replication of B. pseudomallei in human PMNs. Purified PMNs in RPMI 1640 were infected with B. pseudomallei at a multiplicity of infection (MOI) of 0.3:1 at 37°C for 30 min. The intracellular survival of B. pseudomallei in PMNs was determined after the extracellular bacteria were killed with 250 ␮g/ml kanamycin at 37°C for 30 min and culture supernatants were checked for sterility by plating on Luria-Bertani agar plates. Phagocytosis and oxidative burst assayed by flow cytometry. Diluted wholeblood samples were stimulated in vitro with FITC-labeled bacteria at an MOI of 10:1 for 60 min or with 800 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma, United States) for 15 min at 37°C; then 25 ␮l of a 2,800-ng/ml hydroethidine (Sigma, United States) solution was added and the preparation was incubated for 5 min at 37°C. Erythrocytes were then lysed with lysing buffer (BD Biosciences, United States), washed twice, and fixed with 10% paraformaldehyde for decontamination prior to analysis by flow cytometry (FACSCalibur; BD Biosciences, United States) (25, 26, 44). Determination of PMN migration. Purified PMNs at a concentration of 5 ⫻ 106 cells/ml were incubated in the upper chamber of 3-␮m-pore-size Transwell plates (Corning Life Sciences, Australia), 20 to 100 ng/ml of recombinant interleukin-8 (IL-8) (PeproTec, United Kingdom) was placed in the lower 0.5 ml chamber, and the plates were incubated at 37°C for 1 h. Transmigrated PMNs in the lower chamber were counted by flow cytometry (FACSCalibur; BD Biosciences, United States), and the migration index was calculated as follows: number of transmigrated PMNs in response to IL-8/number of transmigrated PMNs in response to the medium control (10). In some experiments, purified PMNs were stimulated with intact heat-killed B. pseudomallei at an MOI of 1:1 or 1:10 at 37°C for 1 h prior to the test for migration. PMN apoptosis assayed by flow cytometry. Apoptosis of PMNs was determined by flow cytometry using an annexin V binding assay. Purified PMNs were cultured with medium alone or with B. pseudomallei at an MOI of 1:1 at 37°C for 24 h. The intracellular survival of B. pseudomallei was quantified by colony plating as described above for the intracellular survival and replication assay. At the indicated time points, cells were collected, washed with annexin V staining

buffer (pH 7.4), and labeled with allophycocyanin (APC)-conjugated annexin V (BD Biosciences, United States) for 15 min at room temperature. After washing, cells were fixed with 10% paraformaldehyde and analyzed by flow cytometry (FACSCalibur; BD Biosciences, United States) (8). In other experiments with heat-killed B. pseudomallei, propidium iodide (PI) (BD Biosciences, United States) 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 using Graphpad PRISM statistical software (GraphPad, San Diego, CA). A P value of ⬍0.05 was considered statistically significant.

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 Fig. 1, after the initial 1 h of incubation (time zero), the percentages of the initial

FIG. 1. Intracellular survival of B. pseudomallei in purified PMNs from healthy and diabetic subjects. Purified PMNs from six healthy (A) and four diabetic (B) subjects were cocultured with live B. pseudomallei at an MOI of 0.3:1 for 30 min, and extracellular organisms were killed by incubation with 250 ␮g/ml kanamycin for another 30 min before the cells were lysed for bacterial counting (time zero). Intracellular bacteria were quantified by colony plating at the indicated time points, and the results are expressed as percentages of the initial inocula for individuals, which were calculated by dividing the number of recovered bacteria by the total number of B. pseudomallei cells added.

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FIG. 2. Phagocytosis of B. pseudomallei, S. enterica serovar Typhimurium, or E. coli by human PMNs. Whole-blood leukocytes were incubated with medium alone or FITC-conjugated live bacteria at an MOI of 10:1 for 60 min and analyzed by flow cytometry. (A) Phagocytosis by PMNs analyzed by using the mean fluorescence intensity (MFI) of FITC. (B) Phagocytosis by PMNs from seven healthy subjects. (C) Phagocytosis by PMNs from 14 diabetic subjects. P values were calculated by using the paired t test (*, P ⬍ 0.05; **, P ⬍ 0.005; ***, P ⬍ 0.0005.) Bps, B. pseudomallei; Sal, S. enterica serovar Typhimurium. The horizontal lines indicate means ⫾ standard errors for the groups.

inoculum of B. pseudomallei in diabetic PMNs (average, 0.7%) were lower than those in healthy PMNs (average, 11.9%), even though the difference was not statistically significant (P ⬎ 0.05, Mann-Whitney test). After an additional 1 h of incubation, the numbers of intracellular bacteria decreased 10- to 100-fold in PMNs of both groups, but viable B. pseudomallei could still be detected at 24 h. These results suggested that PMNs from both the healthy and DM groups could kill the majority of bacteria but some B. pseudomallei survived within human PMNs. We then measured the uptake of B. pseudomallei by human PMNs and compared it with the uptake of other gram-negative bacteria (S. enterica serovar Typhimurium and E. coli) by flow cytometry. The results showed that B. pseudomallei was less efficiently phagocytosed by PMNs than S. enterica serovar Typhimurium and E. coli (P ⬍ 0.005, paired t test) in both the healthy and diabetic groups (Fig. 2). In addition, heatkilled B. pseudomallei, S. enterica serovar Typhimurium, and E. coli were assayed under the same conditions, 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. We next investigated whether the degree of glycemic control, as determined by the percentage of glycosylated HbA1c in DM subjects, influenced the antimicrobial activities of PMNs following an encounter with B. pseudomallei. DM subjects were classified into good, poor, and very poor glycemic control

groups on the basis of the levels of HbA1c (5.5 to 7.5, 7.6 to 8.5, and ⬎8.5%, respectively). First, phagocytosis of 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 the very poor glycemic control diabetic subjects (HbA1c level, ⬎8.5%) compared with the healthy subjects (P ⬍ 0.05, Mann-Whitney test) (Fig. 3A and B). Moreover, these results were in agreement with those obtained in our intracellular survival assays, suggesting that phagocytosis of B. pseudomallei from diabetic PMNs was impaired parallel with poor glycemic control. In addition, the oxidative burst induced by B. pseudomallei also tended to be impaired, although the difference was not statistically significant at a 95% confidence level (P ⫽ 0.0545, Mann-Whitney test), while PMA induction was comparable for healthy and DM subjects (Fig. 3A, 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 exhibited reduced migration in response to IL-8, and this was inhibited by intact B. pseudomallei. PMN migration in response to IL-8, a major chemokine responsible for this function, was assessed for both groups of subjects. The results showed that PMNs from diabetic subjects tended to exhibit reduced migration in response to IL-8 at all the doses that we used compared with healthy subjects (Fig. 4A). In other septic or bacteremic models of infection, pathogens

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FIG. 3. Phagocytosis and oxidative burst of PMNs from healthy, well-controlled, and poorly controlled diabetic subjects. Whole-blood leukocytes of healthy and diabetic subjects were incubated with medium alone, 800 ng/ml PMA, or FITC-conjugated live B. pseudomallei at an MOI of 10:1 and analyzed by flow cytometry. (A) Phagocytosis by PMNs analyzed by using the mean fluorescence intensity (MFI) of FITC-conjugated live B. pseudomallei and oxidative burst analyzed by using the mean fluorescence intensity of ethidium bromide (EB). (B) Phagocytosis of B. pseudomallei. (C) Oxidative burst induced by PMA. (D) Oxidative burst induced by B. pseudomallei. P values were calculated by using the Mann-Whitney test (*, P ⬍ 0.05; ns, not significant). HbA1c with good, poor, and very poor glycemic control (5.5 to 7.5, 7.6 to 8.5 and ⬎8.5%, respectively) was used. Bps, B. pseudomallei.

are already in the host system, and several lines of evidence suggest that pathogens can interfere with host immune responses. To investigate whether the PMN migration in response to IL-8 could be altered by an encounter with B. pseudomallei, purified PMNs from five healthy and five diabetic subjects were incubated with intact heat-killed B. pseudomallei at MOIs of 1:1, 10:1, and 100:1 at 37°C for 1 h. Then stimulated PMNs were collected, and migration activity was tested. Interestingly, the migration index of B. pseudomallei-stimulated PMNs decreased when the number of B. pseudomallei cells was increased (Fig. 4B). The observations were significant for healthy and DM subjects (P ⬍ 0.005 and P ⬍ 0.05, respectively, paired t test), suggesting that B. pseudomallei was capable of interfering with PMN migration in both healthy and diabetic subjects. B. pseudomallei decreased PMN apoptosis/necrosis. After phagocytosis, PMNs normally undergo apoptosis and are engulfed by macrophages, but it has been reported that other pathogens, such as Anaplasma phagocytophilum (8), Leishmania major (1), and Chlamydia pneumoniae (40), can 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 an MOI of 1:1, and the kinetics of annexin V-positive PMNs at 0, 1, 3, 16, and 24 h postinfection were analyzed (Fig. 5A). The results

showed that annexin V-positive PMNs were clearly detected at 16 h and were still present at 24 h; therefore, the 24-h time point was selected for further studies of healthy and DM subjects. After the initial 1 h of incubation (time zero), diabetic PMNs exhibited levels of apoptosis/necrosis similar to those of healthy PMNs in the absence or presence of live B. pseudomallei (Fig. 5B). After 24 h of incubation, the percentage of spontaneous apoptotic/necrotic PMNs from healthy subjects was statistically reduced in the presence of B. pseudomallei (P ⬍ 0.05, paired t test). However, this phenomenon was not observed with diabetic subjects (P ⬎ 0.05, paired t test) (Fig. 5C). These results indicated that live B. pseudomallei interfered with the spontaneous apoptosis/necrosis of PMNs in healthy subjects but not in diabetic subjects. In addition, heat-killed bacteria were used to replace live B. pseudomallei under the same conditions, and PMNs were stained with annexin V and PI (Fig. 5D). The results showed that the number of apoptotic PMNs, defined as annexin Vpositive and PI-negative cells, was significantly decreased compared with the number of cells with medium alone (P ⬍ 0.05 and P ⬍ 0.005 for healthy and DM subjects, respectively, paired t test) (Fig. 5E). These results indicated that apoptosis was a major event during the 24 h with small numbers of necrotic cells (annexin V positive and PI positive), and increas-

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FIG. 4. Effect of B. pseudomallei on PMN migration in response to IL-8 of healthy and diabetic subjects. (A) Migration of purified PMNs (5 ⫻ 106 cells/ml) of healthy and diabetic subjects in a Transwell system in response to 20 to 100 ng/ml IL-8. (B and C) Effect of heat-killed B. pseudomallei on migration of PMNs responding to 100 ng/ml IL-8 in healthy (n ⫽ 5) (B) and diabetic (n ⫽ 5) (C) subjects. Transmigrated PMNs were counted by flow cytometry, and a migration index was calculated by dividing the number of transmigrated PMNs with IL-8 by the number of transmigrated PMNs with the medium control. Hk-Bps, heat-killed B. pseudomallei. P values were calculated by using the paired t test (*, P ⬍ 0.05; **, P ⬍ 0.005).

ing the 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 with PMN functions require further investigation. 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 oxygen-independent mechanisms (20, 38). In addition, activated PMNs produce chemokines and cytokines which recruit and activate other immune cells (38). Finally, activated PMNs undergo apoptosis (16), resulting in phagocytosis by macrophages (41). In this study, we obtained evidence that B. pseudomallei was phagocytosed by PMNs at a lower rate than other gram-negative bacteria, such as S. enterica serovar Typhimurium and E. coli, suggesting that B. pseudomallei might have antiphagocytic activity; further studies are required to corroborate this conclusion. To avoid the possible effects of different bacterial doubling times, heat-killed cells of the three pathogens were also studied, and the results were consistent with the observations obtained previously with live bacteria, indicating the distinctive character of the PMN-B. pseudomallei interaction.

However, the resistance to phagocytosis by other pathogens 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 the capsular polysaccharide of this organism contributes to the resistance to in vitro phagocytosis by reducing C3b deposition on the bacterial surface (29). Other mechanisms have been reported for Yersinia enterocolitica resistance to phagocytosis, including two adhesins (Inv and YadA) and the type III secretion systems including effector proteins (7, 13, 42). The 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 disturb the actin cytoskeleton (14, 30). Several reports have identified similar type III secretion systems in B. pseudomallei that contribute 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 since they are active processes that can be expected to be absent in heat-inactivated bacteria. 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

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FIG. 5. PMN apoptosis in response to live B. pseudomallei of healthy and diabetic subjects. PMNs were stained with annexin V-APC, and the time kinetics were analyzed by flow cytometry at 0 to 24 h (A). Purified PMNs were coincubated in vitro with medium alone or with live B. pseudomallei at an MOI of 1:1 at 37°C for 1 h (T0) (B) and 24 h (T24) (C). (D and E) Annexin V and PI staining of PMNs incubated with heat-killed B. pseudomallei for 24 h (D) and calculated for apoptosis in response to medium alone or B. pseudomallei by determining the percentage of annexin V-positive PI-negative PMNs (E). P values were calculated by using the paired t test (*, P ⬍ 0.05; **, P ⬍ 0.005; ns, not significant). Bps, B. pseudomallei; Hk-Bps, heat-killed B. pseudomallei.

serum (28), and growth of B. pseudomallei in PMNs was detected after extended incubation (15, 27). In our study, intracellular survival of B. pseudomallei in purified PMNs was observed after extended incubation, consistent with the latter finding. However, the MOI may affect the outcome of PMN functional assays, as in the other studies the MOI varied from 4:1 to 100:1. We used an MOI of 0.3:1, and the results 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 reactive nitrogen intermediates and reactive oxygen intermediates 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 DM (35). One simple explanation for this is that the innate immunity of diabetic patients, particularly PMN functions, is altered (2, 3, 36). Our study demonstrated that DM subjects have reduced PMN migration in response to IL-8 compared with healthy subjects. This may result in 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 compared with other gram-negative bacteria, such as S. 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 diabetic subjects with poor glycemic control. This is consistent with the results obtained in the intracellular survival assays, which showed that the internalization of B. pseudomallei by PMNs from diabetic patients tended to be lower than that by PMNs from healthy subjects. A similar finding has been reported for patients with poor glycemic control who showed impaired PMN phagocytosis of virulent K1/K2 Klebsiella pneumoniae compared with patients with good glycemic control and healthy volunteers (19). Therefore, persistently poor glycemic control could have a progressively deleterious effect on phagocytic function. Further investigation to reveal the mechanisms utilized by B. pseudomallei for antiphagocytic activity and reduced PMN migration will be important. In addition, there was a trend for the DM subjects

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with the worst glycemic control to display lower oxidative bursts in response to B. pseudomallei, and further studies are needed to address this observation 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 significant in diabetic subjects. It is not clear why the difference occurred. However, the delay of PMN apoptosis was significant with heatkilled bacteria in both healthy and DM subjects, and such a delay may favor bacterial survival. A recent report demonstrated that PMNs produced their own survival factors, including cytokines, and had decreased Bax-␣/Bcl-xL ratios during the early steps of other infections when the number of bacteria was still low (24). The survival of PMNs may be extended in order to accomplish their functional role in innate immunity. The reduced ability of diabetic PMNs to delay apoptosis following B. pseudomallei exposure could result in the decrease in functional longevity of PMNs and increased PMN clearance from the infectious sites. This would be consistent with previous data which showed that diabetic PMNs underwent normal spontaneous apoptosis and did not demonstrate lipopolysaccharide-induced inhibition of apoptosis (37). Taken together, our results suggest that PMNs of diabetic subjects could be defective in the early phase (24 h) of the inflammatory response against B. pseudomallei. The alterations included not only alterations in migration, phagocytosis, and apoptosis but possibly also alterations in the killing mechanism via oxidative burst. Our experiments were the first experiments to directly address the immunological basis of diabetes as a major risk factor for 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. ACKNOWLEDGMENTS We thank Gregory J. Bancroft and Mark P. Stevens for their critical comments on and review of the manuscript; Debbie Smith, Heidi Alderton, and Jon Cuccui of the London School of Hygiene and Tropical Medicine, United Kingdom, for providing the training for biohazard containment level 3 at CMDL, Khon Kaen University, Khon Kaen, Thailand; and Vicki Harley for her editorial help in preparing the manuscript. This work was supported in part by Public Health Service grant U01 AI061363 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Aga, E., D. M. Katschinski, G. van Zandbergen, H. Laufs, B. Hansen, K. Muller, W. Solbach, and T. Laskay. 2002. Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major. J. Immunol. 169:898–905. 2. Alba-Loureiro, T. C., S. M. Hirabara, J. R. Mendonca, R. Curi, and T. C. Pithon-Curi. 2006. Diabetes causes marked changes in function and metabolism of rat neutrophils. J. Endocrinol. 188:295–303. 3. Alba-Loureiro, T. C., C. D. Munhoz, J. O. Martins, G. A. Cerchiaro, C. Scavone, R. Curi, and P. Sannomiya. 2007. Neutrophil function and metabolism in individuals with diabetes mellitus. Braz. J. Med. Biol. Res. 40:1037–1044. 4. Cheng, A. C., and B. J. Currie. 2005. Melioidosis: epidemiology, pathophysiology, and management. Clin. Microbiol. Rev. 18:383–416. 5. Cheng, A. C., P. Dasari, and B. J. Currie. 2004. Granulocyte colony-stimulating factor and an in vitro whole blood model of melioidosis. Eur. J. Clin. Microbiol. Infect. Dis. 23:205–207. 6. Cheng, A. C., D. Limmathurotsakul, W. Chierakul, N. Getchalarat, V. Wuthiekanun, D. P. Stephens, N. P. Day, N. J. White, W. Chaowagul, B. J. Currie, and S. J. Peacock. 2007. A randomized controlled trial of granulocyte colony-stimulating factor for the treatment of severe sepsis due to melioidosis in Thailand. Clin. Infect. Dis. 45:308–314.

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