IMPAIRED PHAGOCYTOSIS AND OXIDATIVE BURST ACTIVITY...

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11 IMPAIRED PHAGOCYTOSIS AND OXIDATIVE BURST ACTIVITY OF MONOCYTES ON ESCHERICHIA COLI INFECTION IN THALASSEMIA Chayada Thiengtavor, 1 Sirikwan Siriworadetkun, 2 Kittiphong Paiboonsukwong, 2 Suthat Fucharoen, 2 Kovit Pattanapanyasat, 3 Saovaros Svasti, 2 Pornthip Chaichompoo, 1, * 1 Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand; 2 Thalassemia Research Center, Institute of Molecular biosciences, Mahidol University, Nakhon Pathom, Thailand 3 Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. *e-mail: [email protected] ___________________________________________________________________________ Abstract: Thalassemia is a common genetic disorder worldwide, with at least 60,000 severely affected individuals born every year. Infection is the first cause of death (46%) in developing country and the second cause of death (12.6%) in developed country, which Escherichia coli as major cause of severe infection (26%) and Klebsiella pneumoniae as minor (23%). Monocytes/macrophages are the main initial effectors of the innate immunity that can clear the invading pathogen and activate adaptive immunity through phagocytosis. Herein, thalassemic monocyte function on clearance ex vivo E. coli infection was investigated. A method for duo-measurement of phagocytosis and oxidative burst in whole blood using three- color flow cytometric method was developed. Lipopolysaccharide-primed whole blood samples showed increased phagocytosis and oxidative burst activity of phagocytes as measurement of percentages and mean fluorescent intensity of propidium iodide labeled E. coli and 2′,7′-dichlorofluorescin diacetate when compared to unprimed as dose- and time- dependent manner. Thalassemic monocytes had significant decreased efficiency on phagocytosis (13.9±5.2%) and oxidative burst (1.2±0.6%) when compared to normal subjects (44.3±10.4 and 12.8±6.8%, respectively, P < 0.05). Decreased phagocytosis could affect to pro-inflammatory and immunogenic peptide presentation to activate humoral and cell- mediated immune responses. These findings suggest that impaired phagocyte function of monocytes in thalassemia supports the clinical observation as severe anemia with splenectomized patients has an increased risk of infection. This study may provide a basic for monocyte/macrophage-centered therapeutic strategies in thalassemia. __________________________________________________________________________________________ Keywords : innate immunity, phagocytosis, oxidative burst, bacterial infection, flow cytometry Introduction : Thalassemia is a common genetic disorder worldwide, with at least 60,000 severely affected individuals born every year. In Thailand, approximately 3,000 children are born each year with the disease and 100,000 cases are reported in the population (Fucharoen and Winichagoon, 1987). Patients are suffering from severe anemia and other complications such as growth retardation, severe bone changes, hepatosplenomegaly, heavy iron overload, osteoporosis, endocrinopathies, heart failure, pulmonary hypertension, thromboembolic events, immune abnormalities and infection. Infection is a major complication and the leading cause of death in thalassemia, which Escherichia coli (26%) and Klebsiella pneumoniae (23%) as major pathogens. General management for prevention of infection in thalassemia is treatment with the immunization with pneumococcal and hepatitis vaccines,

Transcript of IMPAIRED PHAGOCYTOSIS AND OXIDATIVE BURST ACTIVITY...

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IMPAIRED PHAGOCYTOSIS AND OXIDATIVE BURST ACTIVITY OF

MONOCYTES ON ESCHERICHIA COLI INFECTION IN THALASSEMIA

Chayada Thiengtavor,1 Sirikwan Siriworadetkun,

2 Kittiphong Paiboonsukwong,

2

Suthat Fucharoen,2 Kovit Pattanapanyasat,

3 Saovaros Svasti,

2 Pornthip Chaichompoo,

1,*

1Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand;

2Thalassemia Research Center, Institute of Molecular biosciences, Mahidol University,

Nakhon Pathom, Thailand 3Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol

University, Bangkok, Thailand.

*e-mail: [email protected]

___________________________________________________________________________

Abstract:

Thalassemia is a common genetic disorder worldwide, with at least 60,000 severely affected

individuals born every year. Infection is the first cause of death (46%) in developing country

and the second cause of death (12.6%) in developed country, which Escherichia coli as major

cause of severe infection (26%) and Klebsiella pneumoniae as minor (23%).

Monocytes/macrophages are the main initial effectors of the innate immunity that can clear

the invading pathogen and activate adaptive immunity through phagocytosis. Herein,

thalassemic monocyte function on clearance ex vivo E. coli infection was investigated. A

method for duo-measurement of phagocytosis and oxidative burst in whole blood using three-

color flow cytometric method was developed. Lipopolysaccharide-primed whole blood

samples showed increased phagocytosis and oxidative burst activity of phagocytes as

measurement of percentages and mean fluorescent intensity of propidium iodide labeled E.

coli and 2′,7′-dichlorofluorescin diacetate when compared to unprimed as dose- and time-

dependent manner. Thalassemic monocytes had significant decreased efficiency on

phagocytosis (13.9±5.2%) and oxidative burst (1.2±0.6%) when compared to normal subjects

(44.3±10.4 and 12.8±6.8%, respectively, P < 0.05). Decreased phagocytosis could affect to

pro-inflammatory and immunogenic peptide presentation to activate humoral and cell-

mediated immune responses. These findings suggest that impaired phagocyte function of

monocytes in thalassemia supports the clinical observation as severe anemia with

splenectomized patients has an increased risk of infection. This study may provide a basic for

monocyte/macrophage-centered therapeutic strategies in thalassemia. __________________________________________________________________________________________

Keywords : innate immunity, phagocytosis, oxidative burst, bacterial infection, flow

cytometry

Introduction :

Thalassemia is a common genetic disorder worldwide, with at least 60,000 severely affected

individuals born every year. In Thailand, approximately 3,000 children are born each year

with the disease and 100,000 cases are reported in the population (Fucharoen and

Winichagoon, 1987). Patients are suffering from severe anemia and other complications such

as growth retardation, severe bone changes, hepatosplenomegaly, heavy iron overload,

osteoporosis, endocrinopathies, heart failure, pulmonary hypertension, thromboembolic

events, immune abnormalities and infection. Infection is a major complication and the

leading cause of death in thalassemia, which Escherichia coli (26%) and Klebsiella

pneumoniae (23%) as major pathogens. General management for prevention of infection in

thalassemia is treatment with the immunization with pneumococcal and hepatitis vaccines,

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oral penicillins especially in patients with splenectomy, removal of predisposing factors such

as gallstones, iron overload and appropriate antibiotics. Albeit largely improvement of

prognosis and therapeutics to protect infectious diseases, infection are still claim the lives of

too many thalassemic patients. Several factors are associated with immune abnormalities of

both innate and adaptive immunity on β-thalassemia patients such as severe anemia, multiple

blood transfusions, iron overload, iron chelator treatment and splenectomy. Desferrioxamine

(DFO) as iron chelator therapy might have increased risk for Yersinia enterocolitica infection

which may be localized to mesenteric nodes and tonsils or occur as a generalized form such

as septicemia (Wanachiwanawin, 2000). Defect in absolute number and function of immune

cells such as increased amounts and activity of CD8 suppressor T cells, decreased CD4/CD8

ratio, reduced T cell proliferation, increased amounts and activity of B cells, defective

chemotaxis and phagocytosis of neutrophils and macrophage and defective natural killer cell

function in β-thalassemia patients with susceptibility to infection have been reported

(Pattanapanyasat et al., 2000; Vento et al., 2006). Unfortunately, the mechanism of immune

abnormalities is poorly understood. It is important to notice that abnormal red blood cell

(RBC) membrane in thalassemia is continuously cleared by monocytes and macrophages that

eventually lead to hyperphagocytosis and hypersplenism. The standard approach for treating

hypersplenism is splenectomy. Unfortunately, it is increased risk to infection in thalassemia.

Additionally, commonly observed in long-term receipt of blood transfusions leads to

continuous alloantigenic stimulation that is associated with autoimmune hemolysis, T cell

and B cell changes and modification of monocyte and macrophage function (Vento et al.,

2006). Finally, defective functions of neutrophils, natural killer cells and complement system

have also been reported (Wanachiwanawin, 2000).

Monocytes/macrophages are cells in the innate immunity and play an important role of

antigen-presenting cells and release many inflammatory cytokines and chemokines that

contribute to infiltrate immune cells into site of infection and initiate the adaptive immunity.

Lipopolysaccharide (LPS), a major component of the outer membrane of gram-negative

bacteria, is a common antigen to activate immunity. During infection, patients with sepsis

due to gram-negative bacteria infection had 0.5-5 ng/mL plasma LPS (Bohmer et al., 1992).

Human monocytic cell line, THP-1, treated with LPS at low dosages (< 1 ng/mL) of leads to

increased synthesized the pro-inflammatory mediator interleukin (IL)-6, while higher dosages

(> 10 ng/mL) induced the expression of both IL-6 and IL-33 (Morris et al., 2014).

Monocytes/macrophages recognize and bind bacteria via microbe-associated molecular

patterns (MAMPs) and initiate phagocytosis. The phagocytosis is initiated by opsonizing

microbes with antibodies that bind with high-affinity to Fc receptor (CD16) on monocytes/

macrophages. Complement components are another predominant factor in blood circulation

that enables efficient opsonization. The C3b complement binds to surface of microbe act as

opsonin and recognized by macrophage-1 antigen (Mac-1). Mac-1 is a heterodimeric integrin

of two subunits; alpha-M (CD11b) and beta-2 (CD18). When the interaction between

microbe and receptor on monocytes/macrophages occur, the plasma membrane of monocytes/

macrophages begins to redistribute and form the cup shaped projection around microbe and

pinch off the interior of the cup to from an inside-out intracellular vesicle. Upon phagosome

closure, the maturing phagosome traverses an early and late phagosomal and a

phagolysosomal stage paralleling endosomal maturation. In the lumen of phagosome is

acidification by the proton pumping vesicular ATPase (Weiss and Schaible, 2015). The

phagocytosed microbes are destroyed to generate the peptides and presented to T cell via

major histocompatibility complex (MHC) to initiate adaptive immune response.

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In this study, the duo-measurement of phagocytosis and oxidative burst of monocytes

followed E. coli treatment in peripheral blood samples using three-color flow cytometric

method was developed. This procedure is based on an assay described earlier (Bohmer et al.,

1992; Keawvichit et al., 2012), which exploits the use propidium iodide (PI) for staining

bacteria, a fluorescent dye, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), as reactive oxygen

species (ROS) marker and monoclonal antibody specific to glycoprorin A (GPA) for

excluded immature RBCs, intact RBCs or ghost cells.

Methodology :

Subjects

Forty β-thalassemia/HbE patients (12 splenectomy and 16 non-splenectomy) and 12 normal

subjects with the age ranged from 24 to 48 years were recruited. This study approved by the

Mahidol University Central Institutional Review Board (MU-CIRB), approval number

2015/052.0104. Written informed consent was obtained from all individual participants

included in the study. All subjects had no hydroxyurea, prednisolone, blood transfusion and

iron chelator before blood sample collection at least 4 weeks. All blood samples were

collected at room temperature (RT) and processed within 2-3 h. Hematological parameters

and differential white blood cell (WBC) count were described in Table 1.

Table 1 Hematological parameters

Description Normal subjects -Thalassemia/HbE patients

Non-splenectomy Splenectomy

Number 12 16 12

RBC count (×106/L) 4.5 ± 0.2 4 ± 1.0* 3.3 ± 0.6*

Hb (g/dL) 13.3 ± 1.0 7.3 ± 1.5* 6.5 ± 1.2*

Hct (%) 39.7 ± 2.6 23.7 ± 4.5* 23.1 ± 3.2*

MCV (fL) 86.1 ± 4.5 59.7 ± 7.0* 70.3 ± 6.7*

MCH (pg) 28.8 ± 1.8 18.3 ± 2.5* 19.9 ± 2.3*

MCHC (g/dL) 33.5 ± 1.0 29.5 ± 5.4* 28.3 ± 1.6*

% RDW 12.5 ± 0.6 25 ± 3.5* 24.9 ± 2.1*

WBC count (×103/L) 6.5 ± 1.9 5.4 ± 2.1 10.4 ± 6.3

WBC differential count

%Blast cells 0 ± 0.4 0 ± 0.6 1 ± 1.3

%Neutrophils 62 ± 5.7 59 ± 12.3 48 ± 12.7*

%Eosinophils 2 ± 0 2 ± 2.2 2 ± 1.5

%Basophils 0 ± 0 0 ± 0 0 ± 0.3

%Lymphocytes 27 ± 7.3 29 ± 15.4 40 ± 14.9*

%Monocytes 8 ± 5.8 9 ± 6.8 10 ± 7.1

NRBCs (/100WBC) 0 ± 0 43 ± 66.3 456 ± 204.7*

Platelet count (×103/L) 270 ± 49.1 182 ± 96.7* 598 ± 142.9*

%Reticulocyte count 0.9 ± 0.2 6.7 ± 3.8* 13.9 ± 5.5*

Reticulocytest (×109/L) 39.8 ± 9.1 254.3 ± 135.4* 441 ± 136.6*

%HbA2/HbE 2.8 ± 0.3 54.6 ± 12.6* 53.5 ± 10.1*

%HbF 1.1 ± 0.8 32.9 ± 15* 34.7 ± 11*

%HbA 86.7 ± 0.8 21.5 ± 10.5* 19.6 ± 1.3*

RBC = red blood cells, Hb = hemoglobin, Hct = hematocrits, MCV = mean corpuscular volume, MCH = mean

corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, WBC = white blood cells,

NRBCs = nucleated red blood cells. *Significant different at P < 0.05 when compared to normal subjects

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Propidium iodide staining bacteria

Escherichia coli stain DH5α was fixed with cold-70% ethanol solution for 2 h at 4°C. After

incubation and wash, fixed E. coli was kept as master stock at 2×109 bacteria/mL in

phosphate buffer saline (PBS, Hyclone). One milliliter of fixed bacteria was stained with

100 μg/mL PI (Sigma) for 30 min at 4°C and used within 1 h.

Treatment of monocytes with Escherichia coli

Four hundreds forty microliter of whole blood samples in CPDA-1 anticoagulant were

primed with 1 and 10 ng/mL LPS (Sigma) for 60 min at 37°C. Then samples were pre-

incubated with 50 μM DCFH-DA (Sigma) for 10 min at 37°C. Fifty microliter of samples

were collected for analysis of dye loading and endogenous ROS levels (DCFT10), and then,

remained samples were co-incubated with PI stained E. coli (at 0.1-10×107 bacteria in 500 μl

final volume) for 0-180 min at 37°C (T0 to T180).

Phagocytosis and oxidative burst analysis

Each sample was placed in 1 mL 1×FACS lysing solution (BD Biosciences) for 10 min at

RT, after centrifugation, pellet samples were stained with PE conjugated anti-GPA (BD

Biosciences) by followed manufacture’s recommended. Data from at least 50,000 events of

leukocytes were acquired by BD FACScan flow cytometer (BD Biosciences) and analyzed

by BD CellQuest software (BD Biosciences) within 1 h after staining. The first analyzed

using the log amplification of FL-2 channel for gating negative population of GPA

(leukocytes) (Figure 1A). Then, FSC-H/SSC-H analysis of size and granularity was separated

leukocytes into granulocytes, monocytes (R2 region) and lymphocytes (Figure 1B). The

percentages and mean fluorescent intensity (MFI) of phagocytosis and oxidative burst were

analysis by quadrant analysis of FL-3 and FL-1 channel (Figure 1C-I).

CD16 and CD11b expression analysis

Fifty microliter of whole blood samples were placed in 1 mL 1×FACS lysing solution (BD

Biosciences) for 10 min at RT, after centrifugation, pellet samples were stained with PE

conjugated anti-GPA (BD Biosciences), BV421 conjugated anti-CD16 (BioLegend), BV510

conjugated anti-CD11b (BioLegend) and BV605 conjugated anti-CD45 (BioLegend) by

followed manufacture’s recommended. Data from at least 50,000 events of leukocytes were

acquired by BD FACS LSRII flow cytometer (BD Biosciences) and analyzed by BD Diva

software (BD Biosciences). The first analyzed using the log amplification of FL-2 channel

for gating negative population of GPA (leukocytes) (Figure 2A). Then, CD45-H/SSC-H

analysis of CD45 and granularity was separated leukocytes into granulocytes, monocytes (R2

region) and lymphocytes (Figure 2B). The percentages and mean fluorescent intensity (MFI)

of CD16 and CD11b were analysis by histogram analysis (Figure 2C-D).

Statistic analysis

All descriptive analysis (mean, SD, coefficient of variation and ranges) was performed using

GraphPad PRISM 6.0 (GraphPad Software, Inc.). Comparisons between parameters were

analyzed with non-parametric Mann-Whitney U test. Simple linear regression and

Spearman’s correlation coefficient (rs) were calculated. The threshold for statistical

significance for all comparisons was chose as P < 0.05.

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Figure 1 Flow cytometric analysis of phagocytosis and oxidative burst of thalassemic

monocytes. Thalassemic whole blood sample was incubated with 50 μM DCFH-DA for 10

min at 37°C water bath, and then, co-incubated with 5×107 PI-fixed Escherichia coli stain

DH5α. Flow cytometric analysis shown (A) Leukocyte population was gated from GPA- cells

in R1 region. (B) FSC-H/SSC-H analysis of monocytes in R2 region. (C-I) Phagocytosis (PI,

x-axis) and oxidative burst (DCF, y-axis) of monocytes at time 0-180 min.

Figure 2 Flow cytometric analysis of CD16 and CD11b expression on thalassemic

monocytes. Thalassemic whole blood sample was lyzed RBCs and stained with

fluorochromes specific to surface markers. (A) Leukocyte population was gated from GPA-

cells in R1 region. (B) CD45-H/SSC-H analysis of monocytes in R2 region. Histogram

analysis of (C) CD16 and (D) CD11b expression on thalassemic monocytes was determined.

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Results :

Analysis of Escherichia coli phagocytosis by monocytes

The PI labeled E. coli was prepared. Fixed E. coli at 2×109 bacteria/mL was stained with PI

at 1, 10 and 100 μg/mL. The yield of PI-fixed E. coli was 100% positive in FL-3 channel

compared to unstained fixed E. coli (Figure 3). The mean fluorescent intensity (MFI) of PI

labeled E. coli increased with the increasing concentration of PI. The E. coli labeled with 100

μg/mL PI had higher MFI (1,635±81.2). This concentration was further used for phagocytosis

and oxidative burst activity study.

The amount of E. coli used for monocyte phagocytosis analysis was determined. Normal

whole blood samples (n = 3) were treated with different doses of PI-fixed E. coli (0.1-10×107

bacteria) at 90 min 37°C. The percentages and MFI of monocyte that phagocytose E. coli

were increased as dose-dependent manner (Figure 4). At 1-10×107 bacteria incubated with

monocytes had 22-3820% phagocytosis with increased MFI levels from 40±9 to 377±111.

The PI-fixed E. coli at 1×107

and 5×107

was used for phagocytosis and oxidative burst

activity study.

Figure 3 Flow cytometric analysis of propidium iodide labeled 70%ethanal fixed Escherichia

coli. (A) Histogram and (B) mean fluorescent intensity (MFI) of fluorescent in FL-3 channel

of fixed E. coli labeled with different doses of propidium iodide (PI) (1-100 μg/mL).

(Experiment repeated 5 times) *Significant different when compared to unstained fixed E.

coli (P < 0.05).

Figure 4 Dose effect of PI-fixed E. coli treated normal monocytes. (A) Percentages and (B)

mean fluorescent intensity (MFI) of phagocytosis on monocytes treated with 0.1-10×107

bacteria at 37°C water bath for 90 min.

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Impaired phagocytosis and oxidative burst of thalassemic monocytes associated to CD16

expression

LPS-primed monocytes can induce phagocytosis and oxidative burst of peripheral blood

monocytes to destroyed microbial infection. The phenomena of LPS tolerance and priming of

phagocytosis and oxidative burst activity of monocytes were found. Normal blood samples

were pre-treated with 1 and 10 ng/mL LPS before co-incubated with either 1×107 or 5×10

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PI-fixed E. coli (Figure 5A and 5B). Percentages of double positive of phagocytosis and

oxidative burst were increased in time dependent manner. Moreover, increased percentages

of double positive of phagocytosis and oxidative burst were found to be LPS-dose dependent

manner in 1×107 PI-fixed E. coli co-incubation (Figure 5-A1). The percentages of double

positive of phagocytosis and oxidative of 10 ng/mL LPS primed monocytes at 60, 90 and 120

min (19±11%, 30±9% and 36±7, respectively) were significant higher than unprimed

monocytes (3±1%, 23±10% and 23±9, respectively). However, the there was no effect of

LPS in monocytes treated with 5×107 PI-fixed E. coli (Figure 5-B1). Interestingly, the

percentages of oxidative burst in monocytes treated with 1×107 PI-fixed E. coli was increased

at 15 min in unprimed, 1 ng/mL and 10 ng/mL LPS-primed (38±4%, 58±6% and 68±21%,

respectively) and then drop down within 15 min later (1±1%, 1±1% and 1±2%, respectively)

(Figure 5-A3). At the same time of oxidative burst reduction, the percentages of phagocytosis

were increased up to 19±5% in all 3 conditions at 30 min and reached to plateau phase at 60

min (34±9%, 30±11% and 27±9%, respectively) (Figure 5-A2). The MFI of both

phagocytosis and oxidative burst in all conditions had similar trend to the percentages of

them (Figure 5-A4 and 5-A5).

Phagocytosis and oxidative burst functions of thalassemic monocytes were compared to

normal monocytes. At 5×107 PI-fixed E. coli co-incubation with both normal and thalassemic

monocytes, phagocytic and oxidative burst activities were tolerance to LPS stimulation

(Figure 5B and 5C). There was no significant different double positive of phagocytosis and

oxidative burst between unprimed and LPS-primed both normal and Thalassemic monocytes.

Unprimed normal monocytes had 3-fold higher phagocytosis than thalassemic monocytes at

30 min after PI-fixed E. coli co-incubation (35±11% and 11±5% phagocytosis, respectively)

(Figure 5-B2 and 5-C2). The maximum of phagocytosis in thalassemia was observed at

90 min after PI-fixed E. coli co-incubation (35±11% phagocytosis and 88±14 MFI) while

normal monocytes achieved the same phagocytosis level as patients within 30 min after

incubation (35±11% phagocytosis and 152±49 MFI). These results showed that thalassemic

monocytes were impaired phagocytosis and oxidative burst activity.

To address how function of thalassemic monocytes defect, serological parameters including

the levels of immunoglobulin (Ig) A, IgG and IgM were examined. Thalassemia patients in

this cohort study had normal levels of Igs (data not shown). Next, the expression of CD16

and CD11b were further examined by using flow cytometry (Figure 6). The percentages of

both CD16+ and CD11b

+ monocytes in thalassemia patients were no significant different

when compared to normal subjects. However, the level of CD16 expression on surface of

thalassemic monocytes both non-splenectomy and splenectomy was decreased significantly

compared to normal subjects (MFI 1,907±870, 1,873±572 and 2,976±982, respectively)

(P < 0.05). The Spearman’s correlation coefficient (rs) of the CD16 MFI and the percentages

of double positive phagocytosis and oxidative burst of monocytes were 0.750 at P < 0.05.

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Figure 5 Effect of LPS and incubation time on phagocytosis and oxidative burst of

monocytes in thalassemia. Whole blood samples from (A-B) normal subjects (n = 6) and

(C) non-splenectomized β-thalassemia/HbE (n = 5) were treated with (A1-A5) 1×107 and

(B1-B5 and C1-C5) 5×107 PI-fixed E. coli at 37°C water bath for 0-180 min.

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Figure 6 The CD16 and CD11b expression on Thalassemic monocytes. Peripheral blood

samples obtained from normal subjects (normal) (n = 12), non-splenectomized β-thalassemia/

HbE (β-Thal/E-NS) (n = 16) and splenectomized β-thalassemia/HbE (β-Thal/E-S) (n = 12)

were stained with florochromes conjugated specific to leukocyte markers and analyzed the

percentages and the mean fluorescent intensity (MFI) of (A) CD16 and (B) CD11b on

monocytes by using flow cytometer. *Significant different when compared to normal

subjects (P < 0.05).

Discussion and Conclusion :

Our three-color flow cytometric analysis of whole blood was effective and simple technique

for assessing phagocytosis and oxidative burst of phagocytes including monocytes and

granulocytes to bacterial infection in single tube. This technique is required only 440 μL of

whole blood and useful for detection in samples with the intact RBC or the immature RBC

contamination, for example, thalassemia blood samples. PI-label bacteria was simple

preparation and useful for the kinetic measurement of intracellular killing analysis.

Similarly, the impairment of monocytic phagocytosis in thalassemia has been documented in

previous studies (Wiener, 2003). The mononuclear phagocyte system is heavily involved in

the pathology of thalassemia by Fc-receptor-mediated clearance of defective RBCs that might

be affect to monocytic function. However, there was no strongly evidenced to document the

mechanism of monocytic dysfunction in thalassemia yet. In this study finds that loss of CD16

expression on monocytes was associated to impaired phagocytosis and oxidative burst

activity of thalassemic monocytes against to gram-negative bacteria. It might be contribute to

the increased susceptibility to infections

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Acknowledgements :

This study was supported by Thailand Research Fund (TRF) (MRG5980043, DPG5980001,

IRG5780009 and IRG5780011), Office of the Higher Education Commission and Mahidol

University under the National Research University Initiative, Research Chair Grant, National

Science and Technology Development Agency, Thailand and Faculty of Science and Faculty

of Medicine Ramathibodi Hospital, Mahidol University.

References

1. Bohmer, R.H., Trinkle, L.S., and Staneck, J.L. (1992). Dose effects of LPS on

neutrophils in a whole blood flow cytometric assay of phagocytosis and oxidative

burst. Cytometry 13, 525-531.

2. Fucharoen, S., and Winichagoon, P. (1987). Hemoglobinopathies in Southeast Asia.

Hemoglobin 11, 65-88.

3. Keawvichit, R., Khowawisetsut, L., Chaichompoo, P., Polsrila, K., Sukklad, S.,

Sukapirom, K., Khuhapinant, A., Fucharoen, S., and Pattanapanyasat, K. (2012).

Platelet activation and platelet-leukocyte interaction in beta-thalassemia/hemoglobin

E patients with marked nucleated erythrocytosis. Annals of hematology 91, 1685-

1694.

4. Morris, M.C., Gilliam, E.A., Button, J., and Li, L. (2014). Dynamic modulation of

innate immune response by varying dosages of lipopolysaccharide (LPS) in human

monocytic cells. The Journal of biological chemistry 289, 21584-21590.

5. Pattanapanyasat, K., Thepthai, C., Lamchiagdhase, P., Lerdwana, S., Tachavanich,

K., Thanomsuk, P., Wanachiwanawin, W., Fucharoen, S., and Darden, J.M. (2000).

Lymphocyte subsets and specific T-cell immune response in thalassemia. Cytometry

42, 11-17.

6. Vento, S., Cainelli, F., and Cesario, F. (2006). Infections and thalassaemia. The

Lancet Infectious diseases 6, 226-233.

7. Wanachiwanawin, W. (2000). Infections in E-beta thalassemia. Journal of pediatric

hematology/oncology 22, 581-587.

8. Weiss, G., and Schaible, U.E. (2015). Macrophage defense mechanisms against

intracellular bacteria. Immunological reviews 264, 182-203.

9. Wiener, E. (2003). Impaired phagocyte antibacterial effector functions in beta-

thalassemia: a likely factor in the increased susceptibility to bacterial infections.

Hematology (Amsterdam, Netherlands) 8, 35-40.