The innate interferon gamma response of BALB/c and C57BL/6 mice to in vitro Burkholderia pseudomallei infection
© Koo and Gan; licensee BioMed Central Ltd. 2006
Received: 19 May 2006
Accepted: 18 August 2006
Published: 18 August 2006
Burkholderia pseudomallei is the causative agent for melioidosis. For many bacterial infections, cytokine dysregulation is one of the contributing factors to the severe clinical outcomes in the susceptible hosts. The C57BL/6 and BALB/c mice have been established as a differential model of susceptibility in murine melioidosis. In this study, we compared the innate IFN-γ response to B. pseudomallei between the C57BL/6 and BALB/c splenocytes and characterized the hyperproduction of IFN-γ in the relatively susceptible BALB/c mice in vitro.
Naïve BALB/c splenocytes were found to produce more IFN-γ in response to live bacterial infection compared to C57BL/6 splenocytes. Natural killer cells were found to be the major producers of IFN-γ, while T cells and Gr-1intermediate cells also contributed to the IFN-γ response. Although anti-Gr-1 depletion substantially reduced the IFN-γ response, this was not due to the contribution of Gr-1high, Ly-6G expressing neutrophils. We found no differences in the cell types making IFN-γ between BALB/c and C57BL/6 splenocytes. Although IL-12 is essential for the IFN-γ response, BALB/c and C57BL/6 splenocytes made similar amounts of IL-12 after infection. However, BALB/c splenocytes produced higher proinflammatory cytokines such as IL-1β, TNF-α, IL-6, IL-18 than C57BL/6 splenocytes after infection with B. pseudomallei.
Higher percentages of Gr-1 expressing NK and T cells, poorer ability in controlling bacteria growth, and higher IL-18 could be the factors contributing to IFN-γ hyperproduction in BALB/c mice.
Burkholderia pseudomallei is the causative agent for melioidosis, an infectious disease endemic in South-east Asia and northern Australia [1, 2]. It has also been increasingly reported in other tropical and subtropical regions . The bacillus is a facultative intracellular microbe and can invade and replicate in many different organs. Infection can result in a wide spectrum of clinical outcomes, ranging from an asymptomatic state, benign pulmonitis, acute or chronic pneumonia, and to fulminant septicemias . Furthermore, even after the apparent resolution of acute symptoms, the infection can persist for decades as a chronic and latent condition where relapse is possible . Despite appropriate antibiotic treatment, severe melioidosis with acute septicemia is associated with a high mortality rate .
In severe melioidosis, patients exhibit elevated serum levels of proinflammatory cytokines such as TNF-α , IFN-γ  and IFN-γ induced chemokines IP-10 and MIG . Murine models of acute melioidosis mimic human pathology. mRNA for proinflammatory cytokines such as TNF-α, IFN-γ and IL-6 were detected earlier and in more abundance in the organs of BALB/c mice with acute disease than the more resistant C57BL/6 mice when they were infected intravenously . We had previously established an intranasal murine model where BALB/c mice were susceptible while C57BL/6 mice were relatively more resistant to disease. We found high transient levels of IFN-γ both locally and systemically in susceptible mice, which exhibit acute disease followed by death within a week after infection . The high levels of IFN-γ correlated with high bacterial loads in the organs . In another study, administering CpG DNA prior to bacterial challenge could attenuate hyperproduction of IFN-γ in serum of BALB/c mice while lowering the bacterial load in the blood at the same time . So although IFN-γ was shown to be critical in host survival in the first 24 h after infection as neutralizing antibodies against IFN-γ lowered the LD50 by approximately 100, 000 fold , hyperproduction could contribute to immune pathology and severe disease. We are interested in comparing the innate IFN-γ response to B. pseudomallei between C57BL/6 and BALB/c mice, and in characterizing the hyperproduction of IFN-γ in BALB/c through the in vitro stimulation of naïve splenocytes with heat-killed or live bacteria. We found that naïve BALB/c splenocytes consistently produce more IFN-γ in response to live bacterial infection compared to C57BL/6 splenocytes. Through various comparisons between BALB/c and C57BL/6 splenocytes, factors which could contribute to the hyperproduction of IFN-γ in BALB/c splenocytes are discussed.
BALB/c and C57BL/6 splenocytes produce IFN-γ when stimulated with B. pseudomallei
Comparison of cytokine profile between BALB/c and C57BL/6 splenocytes
IL-12 and IL-18 are both required for optimal IFN-γ response but IL-10 has a suppressive effect
Similar cell-types produce IFN-γ in response to bacteria in BALB/c and C57BL/6
Gr-1 expressing populations in BALB/c and C57BL/6 splenocytes
Neutrophils make little IFN-γ
IFN-γ plays a critical role in pathological reactions which could lead to shock and death caused by Gram-negative bacteria or in response to LPS [23, 24]. In the murine model of B. pseudomallei infection, early hyperproduction of IFN-γ by two days after infection in BALB/c mice is correlated with susceptibility to disease eventually resulting in death [10, 11]. To understand the factors contributing to the acute hyperproduction of IFN-γ in response to infection in susceptible BALB/c mice as compared to the relatively resistant C57BL/6 mice, we used naïve splenocytes from both mouse strains to examine their response to B. pseudomallei. Our results show that BALB/c splenocytes made more IFN-γ compared to C57BL/6 splenocytes when stimulated with live bacteria, mimicking the IFN-γ hyperproduction by infected BALB/c mice in vivo. Although the in vitro model is an oversimplification as it excludes cell migration dynamics, it provides a convenient way of investigating inherent factors that contribute to the propensity of BALB/c cells to make IFN-γ in response to live bacteria. However, when stimulated with heat-killed bacteria, this trend is reversed. C57BL/6 and BALB/c mice have generally been classified as high and low IFN-γ responders respectively to account for their innate resistance and susceptibility to pathogens [25, 26]. In response to heat-killed bacteria, C57BL/6 splenocytes indeed made more IFN-γ than BALB/c, conforming to this model likely through the significantly higher IL-12 production.
We showed that BALB/c splenocytes were poorer in controlling bacterial infection. This would agree with in vivo data suggesting that high bacterial loads lead to hyperproduction of IFN-γ [11, 12]. It had been reported that peritoneal cells isolated from C57BL/6 mice were more potent at microbicidal activity against B. pseudomallei compared to those from BALB/c mice . However, higher IFN-γ observed in B. pseudomallei infected BALB/c than C57BL/6 splenocytes was not due to difference in the cell viability from the two strains of mice, although there could be more subtle damages to cells which the XTT assay failed to detect.
Although IL-12 is absolutely essential for IFN-γ production, we found that the higher IFN-γ production did not result directly from higher IL-12 concentrations in BALB/c when stimulated with live bacteria. In fact, BALB/c cells made more IL-10, abrogation of which increased IFN-γ production. This means that IL-12 concentration is not limiting and differences in IL-10 concentrations between the mouse strains are not big enough to tip the balance in IFN-γ production. IL-10 and IFN-γ are antagonistic to each another [28–30]. IL-10 can inhibit IFN-γ production by NK cells in the presence of antigen presenting cells (APCs) through the inhibition of IL-12 [31, 32]. This could explain why we did not see higher IL-12 level in BALB/c mice, and it also implies that co-stimulatory factors other than IL-12 (such as IL-15 and IL-18) were involved in IFN-γ response to B. pseudomallei infection [14, 16]. Since only live but not dead bacteria induced IL-10, this suggests that B. pseudomallei possess active mechanisms to trigger IL-10 production. It is possible that the high IL-10 seen in BALB/c in response to live bacteria could be a consequence of the immune system trying to counteract and suppress the increasing inflammation.
We did not find any obvious differences in the cell types making IFN-γ between BALB/c and C57BL/6 splenocytes. However, BALB/c splenocytes contained more Gr-1high and Gr-1intermediate cells, as well as NK cells. As NK cells are major IFN-γ producers in innate immunity, this translates to more IFN-γ. Although depletion of CD8 T cells did not affect IFN-γ production, CD8 T cells could produce IFN-γ in response to B. pseudomallei as examined by the IFN-γ capture assay (data not shown) and as shown by others [14, 16]. It is possible that the CD8 depletion from the bulk population also relieves a suppressive effect on the other cells making IFN-γ. The other explanation is they contribute only little in terms of quantity to the total amount so when they were depleted, the remaining population was enriched for the other cells making IFN-γ. In addition, we found that Gr-1intermediate cells contributed to the IFN-γ response. BALB/c splenocytes had higher numbers of Gr-1intermediate expressing CD8 T cells and NK cells compared to C57BL/6 and could explain why BALB/c cells made more IFN-γ. This apparently requires a live bacterial infection because although the same cells made IFN-γ in response to dead bacteria, they did not contribute to a higher response in BALB/c. A live infection allows for various pathogenic mechanisms of the bacteria, including the Type III secretion systems (TTSS), to operate. This may be necessary to allow for continual activation of cells or other factors such as IL-15 which could act as a co-stimulus for NK cells to make IFN-γ , and IL-27 which could act as a co-stimulus for T cells  to induce a high IFN-γ response. Live bacteria could also induce caspase-1 activation in macrophages, leading to secretion of IL-1β and IL-18 . As BALB/c splenocytes constitutively produced more IL-18 than C57BL/6, and IL-18 is at least partially required for IFN-γ, this could be an additional factor that contributes to the hyperproduction of IFN-γ in BALB/c splenocytes.
Several published studies employ Gr-1 antibodies to deplete neutrophils in vivo [36–38]. In our cell depletion studies, anti-Gr-1 depletion significantly reduced IFN-γ production. However, since the anti-Gr-1 antibody recognizes both Ly-6G and Ly-6C markers and are not exclusive for neutrophils, we excluded the involvement of neutrophils through depletion of Ly-6G+ cells. Ly-6G is found almost exclusively on neutrophils and represents the Gr-1high cells. This means that the Gr-1 antibody is depleting other cell-types expressing Ly-6C or the Gr-1intermediate cells. Haque et al. had recently shown F4/80 macrophages to be producing IFN-γ during B. pseudomallei infection in vivo . We did not further characterize the Gr-1intermediate population making IFN-γ, but we did find the expression of Gr-1intermediate on T cells and NK cells, which likely express Ly-6C as Ly-6G is not found on these cells. Others had shown that Ly-6C expressing NK cells  and CD8+ CD44hi Gr-1+ T cells  make IFN-γ. As BALB/c splenocytes contained more NK cells and Gr-1 expressing NK and T cells, this could be a factor contributing to higher IFN-γ production upon live bacterial encounter. Furthermore, we found that highly purified neutrophils make very little IFN-γ even in the presence of IL-12 and IL-18 and minute numbers of contaminating lymphocytes in neutrophil preparations could contribute to the IFN-γ effect. Thus, it is imperative for studies examining the production of IFN-γ by neutrophils to use highly purified neutrophils and to exclude the contribution of contaminating cell-types. In fact, Schleicher et al recently found that minute numbers of contaminating CD8+ T cells or CD11b+CD11c+NK cells are the source of IFN-γ in IL-12/IL-18-stimulated mouse macrophage populations . This highlights the need for caution in interpreting data due to the possible presence of minute numbers of contaminating but high IFN-γ producing cells.
It has been reported that IL-18 could induce neutrophilia in mice by acting on macrophages to produce IL-6, which in turn causes neutrophilia . Thus, the constitutively high IL-18 in BALB/c mice likely contributes to more neutrophils present in BALB/c splenocytes. Furthermore, addition of IL-18 to splenocytes in culture induced the production of GM-CSF, IFN-γ and IL-6 . As susceptible BALB/c mice show hyper-inflammation upon infection and succumb to acute disease, this seems to parallel patients with acute melioidosis who exhibit hyper-inflammation [7–9, 43]. As the most prominent risk factor for melioidosis is diabetes [1, 44], our studies in the susceptible mouse strain may have implication on the immune condition of diabetic individuals such as constitutive inflammation [45, 46] which predisposes them to acute disease.
An active infection is required to induce a hyperproduction of IFN-γ in BALB/c compared to C57BL/6 splenocytes. The higher production of TNF-α, IL-1β, IL-6 and IL-18, and the presence of more neutrophils contribute to a more intense inflammation seen in BALB/c splenocytes. The higher intracellular bacterial loads in BALB/c splenocytes likely contribute to more inflammation. Furthermore, the higher numbers of NK cells and Gr-1intermediate expressing cells in BALB/c splenocytes also contribute to more IFN-γ. Future investigations into how each cell population interacts with live bacteria could shed more light on the control of inflammation in the different mouse strains.
Virulent Burkholderia pseudomallei strain KHW used in this study is a local clinical isolate from a fatal case of melioidosis in Singapore. The isolate was identified as B. pseudomallei based on colonial morphology, API20 NE tests (BioMerieux, Marcy I'Etoile, France), and 16S RNA sequence.
Infection with B. pseudomallei
Bacteria were cultured on Trypticase soy agar (TSA) (Difco Laboratories, Detroit, MI) for 24 h at 37°C. Colonies were cultured overnight in LB medium, and a 1:20 dilution prepared and cultured for another 4 h. Bacterial number was estimated spectrophotometrically (1 OD600 = 2 × 106 CFU/μl). Heat-killed or heat-inactivated bacteria were prepared by treatment of overnight bacterial culture at 80°C for 1 h. Dead bacteria were centrifuged at 10,000 g, washed twice with sterile PBS and stored at -20°C.
Preparation and stimulation of splenocytes in vitro
Naïve mice were killed by CO2 asphyxiation. Spleens were removed aseptically and single cell suspensions prepared by passing through sterile mesh and erythrocytes were lysed. Cells were placed in antibiotic-free complete antibiotic-free RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% Fetal Bovine Serum (FBS) (Hyclone Laboratories, Logan, UT), 200 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin at a concentration of 2 × 106 cells in 0.5 ml of medium. Cells were stimulated with heat-killed B. pseudomallei at a ratio of bacteria to cell of 5:1 or infected with live B. pseudomallei at a multiplicity of infection (MOI) of 5:1. Treatment of cells with phorbol myristate acetate (PMA) (50 ng/ml) and ionomycin (1 μg/ml) served as positive control. The following antibodies were used for neutralization studies: anti-IL-12 (polyclonal goat IgG, R&D Systems, Minneapolis, MN), goat IgG isotype control, anti-IL-18 (clone 93–10C, MBL, Nagoya, Japan), anti-IL-10 (clone JES5-2A5, Biolegend, San Diego, CA), and rat IgG1 isotype control (R&D Systems). At 90 minutes after inoculation of bacteria, 250 μg/ml of kanamycin was added into each well to inhibit the growth of the extracellular bacteria. Supernatants were harvested at the indicated time points after treatment and stored at -20°C.
Cell viability determination
Viability of splenocytes was determined by the XTT assay kit (Roche Diagnostics, Mannheim, Germany) according to manufacturer's instructions. Briefly, 2 × 106 splenocytes were cultured in 96-well plate at a final volume of 100 μl antibiotic-free culture medium. Infection was performed as described above. Ninety minutes after inoculation of bacteria, 250 μg/ml of kanamycin was added into each well to kill extracellular bacteria. XTT labeling mixture was prepared by adding electron coupling reagent with XTT labeling reagent. 50 μl of labeling reagent were added into each well containing the splenocytes at 4 h before the measurements were recorded. The absorbance of the samples was measured spectrophotometrically using a microplate reader at a wavelength of 490 nm.
Bacterial load determination
To estimate the total bacteria count in vitro, splenocytes were cultured in antibiotic-free medium, and cell were harvested and lysed with 0.1% Triton-X100 at 4 h post-infection. For the intracellular bacteria load, cells were infected for 2 h before kanamycin was added. Cells were harvested and lysed with 0.1% Triton-X100 2 h later. Serial dilutions were made from the suspension and plated onto selective agar plates to obtain the bacterial counts.
Magnetic cell separation for cell-type purification
Single cell suspensions from spleens of mice were washed with MACS buffer (PBS supplemented with 2 mM EDTA and 1% FBS) and CD4, CD8, NK cells were purified using anti-CD4, anti-CD8, and anti-DX5 microbeads while the Gr-1+ cells were stained with anti-Gr-1 antibodies (RB6-8C5, Biolegend) followed by incubation with streptavidin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), neutrophils were stained with Ly-6G-PE followed by anti-PE microbeads before passing through the magnetic columns. All separation procedures were performed according to the manufacturer's guidelines. After incubation for 30 min at 4°C, the positive cells were depleted using MS column or LD column (Miltenyi Biotec), washed and resuspended in antibiotic-free medium.
Cytokine determination by ELISA
Supernatants were harvested at the indicated time points after treatment and stored at -20°C for ELISA. Mouse IFN-γ, IL-12, IL-10, TNF-α, and human IFN-γ ELISA assay kits were purchased from Bender MedSystem (Vienna, Austria), IL-1β and IL-18 ELISA kits from BD Biosciences, whereas IL-6 ELISA kit was purchased from Biolegend. All ELISAs were performed according to manufacturer's instructions. Cell culture supernatants were diluted 2 fold and assayed in duplicates. The detection limits for these assays were 15.6 pg/ml for IFN-γ, TNF-α, IL-6 and IL-1β, 31.2 pg/ml for IL-12 and IL-18, 39.0 pg/ml for IL-10, and 1.5 pg/ml for human IFN-γ.
Flow cytometric analysis
Antibodies used for cell surface marker staining were purchased from BD Pharmingen (San Diego, CA) unless otherwise stated: anti-CD8-PE (53-6.7, rat IgG2a), anti-CD4-biotin (GK1.5, rat IgG2b), anti-CD8-biotin (53-6.7, rat IgG2a), anti-CD3-FITC (145-2C11, Hamster IgG1) anti-Ly-6G-PE (1A8, rat IgG2a), anti-Gr-1-PerCP-Cy5.5 (RB6-8C5, rat IgG2b), anti-pan NK-FITC (DX5, rat IgM, Biolegend). Splenocytes were adjusted to 1 × 106 cells and stained with cell surface specific antibodies or isotype control antibodies for 30 min on ice. Where the biotin conjugated antibodies were used, staining with SAV-PE/SAV-FITC or secondary antibody was performed for 15 min at 4°C. After washing, the cells were analyzed by FACScan (Becton Dickinson, San Jose, CA) flow cytometer.
Interferon γ capture assay
Secretion of IFN-γ was analysed with an IFN-secretion assay kit (Miltenyi Biotec) according to the manufacturer's instructions. Briefly, infected splenocytes were cultured for 16 h at 37°C, 5% CO2 in medium. At the conclusion of the assay, cells were incubated with optimal concentration of antibodies for surface marker staining together with PE-conjugated anti-IFN-γ (Miltenyi Biotec) for 30 min at 4°C. Cells were than washed and analyzed by flow cytometry. Controls included isotype-matched control antibodies conjugated to identical fluorochromes.
Isolation of human neutrophils from peripheral blood
Human neutrophils were isolated from healthy adult blood donors (National University Hospital Blood Donation Centre). Briefly the buffy coats were diluted 2-fold in PBS and centrifuged through a Ficoll-Paque Plus (Amersham Pharmacia Biotech, Uppsala, Sweden) gradient to separate the erythrocytes/neutrophils from peripheral blood mononuclear cells (PMBC). The plasma, PBMC, and the Ficoll layers were removed by Pasteur pipette, and the erythrocytes/PMNC pellets were transferred into new tubes. Erythrocytes were lysed by osmotic shock using 0.2% NaCl solution and an equal volume of 1.6% NaCl were then added to reconstitute to a balance salt solution containing 0.9% NaCl. To remove minute contaminating lymphocytes, monocytes and NK cells, neutrophils were purified by positive selection with anti-CD15 microbeads (Miltenyi Biotec) according to manufacturer's instructions. Crude PMNC and CD15 positively selected cells were seeded at 2 × 106 cells per well in 0.5 ml of medium and stimulated with live or heat-killed B. pseudomallei in the presence or absence of human IL-12, IL-18 or both. Untreated cells or uninfected cells treated with cytokines serve as controls. Supernatant were harvested at 24 h for ELISA.
Statistical significance was determined by Student's t-test. A p value of < 0.05 is regarded as statistically significant.
This study is funded by grants from the Academic Research Fund (R-183-000-080-112), the Office of Life Sciences (R-183-000-602-712), National University of Singapore and the Biomedical Research Council (04/1/21/19/329) awarded to YHG.
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