Early Activation of iNKT Cells Increased Survival Time of BALB/c Mice in a Murine Model of Melioidosis

ABSTRACT

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. High interferon gamma (IFN-γ) levels in naive mice were reported to mediate protection against B. pseudomallei infection. Invariant natural killer T (iNKT) cells can produce and secrete several cytokines, including IFN-γ. When iNKT cell-knockout (KO) BALB/c mice were infected with B. pseudomallei, their survival time was significantly shorter than wild-type mice. Naive BALB/c mice pretreated intraperitoneally with α-galactosylceramide (α-GalCer), an iNKT cell activator, 24 h before infection demonstrated 62.5% survival at the early stage, with prolonged survival time compared to nonpretreated infected control mice (14 ± 1 days versus 6 ± 1 days, respectively). At 4 h after injection with α-GalCer, treated mice showed significantly higher levels of serum IFN-γ, interleukin-4 (IL-4), IL-10, and IL-12 than control mice. Interestingly, the IFN-γ levels in the α-GalCer-pretreated group were decreased at 4, 24, and 48 h after infection, while they were highly increased in the control group. At 24 h postinfection in the α-GalCer group, bacterial loads were significantly lower in blood (no growth and 1,780.00 ± 51.21, P < 0.0001), spleens (no growth and 34,300 ± 1,106.04, P < 0.0001), and livers (1,550 ± 68.72 and 13,400 ± 1,066.67, P < 0.0001) than in the control group, but not in the lungs (15,300 ± 761.10 and 1,320 ± 41.63, P < 0.0001), and almost all were negative at 48 h postinfection. This study for the first time shows that early activation of iNKT cells by α-GalCer helps clearance of B. pseudomallei and prolongs mouse survival.

INTRODUCTION

Burkholderia pseudomallei is a Gram-negative bacterium that is the causative agent of melioidosis. Melioidosis is an infectious disease that impacts humans and animals and is endemic in Northern Australia and Southeast Asia, including Thailand (1, 2). B. pseudomallei is known as a facultative intracellular pathogen that can survive inside host cells and spread from cell to cell (3). Both innate and adaptive immune responses play a central role against this infection (4). In a murine study, interferon gamma (IFN-γ) played a key role in C57BL/6 mice that are relatively resistant to B. pseudomallei compared to BALB/c mice (5). The immune cells and mechanisms reported to play a role in protection against B. pseudomallei infection in mice were found to be macrophages, neutrophils, T cells, natural killer (NK) cells (6, 7), and several cytokines, such as IFN-γ and tumor necrosis factor alpha (TNF-α) (8). During infection, B. pseudomallei activates IFN-γ production from NK cells and T cells, which then stimulate other immune cells, such as macrophages, to kill this pathogen (9). In addition, IFN-γ-knockout (KO) mice exhibit high bacterial burdens in the lungs, rendering early mortality after B. pseudomallei infection and highlighting the importance of IFN-γ in melioidosis (7). In humans, the IFN-γ responses during melioidosis are related to protection and survival against B. pseudomallei infection (10). A previous study showed that patients who recovered from melioidosis had a high number of IFN-γ-producing cells that recognized whole B. pseudomallei organisms and its proteins (11). These data demonstrated that IFN-γ is an essential mediator in protective immunity against B. pseudomallei infection in mice and humans (4, 12).

Invariant natural killer T (iNKT) cells are an unconventional T lymphocyte population that recognize glycolipid or lipid antigens (13). iNKT cells express a restricted repertoire of T cell receptors (TCRs), including the Vα14-Jα18 TCRα chain that combines with Vβ2, Vβ7, or Vβ8.2 in mice and the Vα24-Jα18 chain with Vβ11 in humans (14). These TCRs recognize lipid and glycolipid antigens such as α-galactosylceramide (α-GalCer) via CD1d on antigen-presenting cells (APCs) (15). Upon TCR engagement, iNKT cells secrete wide ranges of chemokines and cytokines, such as interleukin-4 (IL-4), IFN-γ, IL-10, and IL-17, to activate multiple immune cells, such as dendritic cells (DCs), NK cells, macrophages, T cells, and B cells (16). This feature allows iNKT cells to form a bridge between innate and adaptive immunity and to contribute to the immune response to tumors and during autoimmune reactions and microbial infections (17). The role of iNKT cells in melioidosis is still unknown. In a previous article, we demonstrated that human iNKT cells could be activated by this bacterium and are involved in melioidosis and that the levels of circulating iNKT cells in melioidosis patients were significantly lower than those in healthy controls (18). This result in humans led to the hypothesis that iNKT cells are reduced in number during B. pseudomallei infections. The research question of whether iNKT cells are required for immunity against B. pseudomallei infection became relevant and was further investigated here using iNKT cell-knockout (iNKT-KO) mice. Furthermore, it also led to the investigation of the role of α-GalCer-activated iNKT cells in the early protection against B. pseudomallei infection.

RESULTS

B. pseudomallei-infected mice have a significantly lower percentage of organ iNKT cells.

The percentages of iNKT cells in spleens, lungs, and livers of mice infected with 1,000 CFU of B. pseudomallei were analyzed by flow cytometry using gating strategies as shown in Fig. 1. The results showed that numbers of iNKT cells in every organ for 2 to 6 days after B. pseudomallei infection were significantly lower than those observed in the uninfected group (Fig. 2). This result indicates that B. pseudomallei may downregulate the number of iNKT cells in these organs.

FIG 1.

Gating strategy used to identify iNKT cells. Mouse organs and their mononuclear cells (MNCs) were harvested as described in the Materials and Methods. MNCs were stained with monoclonal antibodies (MAbs) to mouse T and iNKT cells and analyzed by flow cytometry. After selection for lymphocytes by forward scatter-side scatter (FSC-SSC), T cells were defined (CD3⁺/CD45⁺) by using anti-mouse CD45 and anti-mouse CD3. Finally, iNKT cells were then further identified (CD3⁺/α-GalCer/CD1d tetramer⁺) by anti-mouse CD3 and α-GalCer/CD1d tetramer (compared with unloaded/CD1d tetramer as a control).

FIG 2.

Number of iNKT cells in each organ during B. pseudomallei infection. (A to C) The percentages of iNKT cells in spleens (A), lungs (B), and livers (C) were compared between mice infected with 1,000 CFU (50LD₅₀) of B. pseudomallei (gray bars) and control-infected mice (black bars; 9 mice/group). The numbers of iNKT cells were determined by flow cytometry (Fig. 1). Data are shown as means ± SD. The results are representative of three independent experiments (total of 27 mice/group). Statistically significant differences were evaluated using the unpaired Student’s t test. The asterisks (*) indicate statistical significance (P < 0.05).

iNKT-KO mice are more susceptible to B. pseudomallei infection.

The role of iNKT cells in B. pseudomallei infection was investigated by using iNKT-KO mice. The B. pseudomallei-infected iNKT-KO mice died starting from day 8, and all died within 10 days after infection; all wild-type (WT) mice died within 14 days postinfection (p.i.). The survival time of iNKT-KO mice (9 ± 1 days) was significantly less than in WT mice (12 ± 2 days; P = 0.0246) (Fig. 3A). As IFN-γ was previously found to be significantly elevated after B. pseudomallei infection (19, 20), and iNKT cells are another innate immune cell that can produce IFN-γ (21, 22), IFN-γ levels in iNKT-KO mice and WT mice were monitored. There were no significant differences in IFN-γ concentrations between WT mice (2.22 ± 3.85 pg/mL) and iNKT-KO mice (2.74 ± 2.91 pg/mL; P = 0.8610) before B. pseudomallei infection. WT mice had significantly higher IFN-γ levels (579.06 ± 146.75 pg/mL) at 24 h after B. pseudomallei infection than iNKT-KO mice (5.59 ± 3.10; P = 0.0025), but not at 48 h (Fig. 3B). Taken together, these results suggest that iNKT cells could play a protective role in B. pseudomallei infection.

FIG 3.

Survival curves and IFN-γ concentrations of mice infected with B. pseudomallei. WT BALB/c mice (n = 3) and iNKT-KO mice (n = 3) were infected intraperitoneally with 100 CFU (5LD₅₀) of B. pseudomallei. (A) Survival of mice was observed for 14 days p.i. (x axis), and the percent survival (mean ± SD; y axis) of WT mice (white circles) was compared with that of iNKT-KO mice (black squares). Statistically significant differences were evaluated by Kaplan-Meier analysis and a log-rank test. (B) The amount of serum IFN-γ (x axis) of WT mice (white bars) and iNKT-KO mice (black bars) before and after 4, 24, and 48 h of infection with B. pseudomallei. The results are representative of two independent experiments (total of 6 mice/group). Statistically significant differences were evaluated using the unpaired t test. The asterisks (*) indicate statistical significance (P < 0.05), whereas “ns” indicates not significant (P > 0.05).

iNKT cell activation with α-GalCer prolongs the survival of BALB/c mice against B. pseudomallei infection.

As iNKT-KO mice showed more susceptibility to B. pseudomallei infection, we sought to determine if stimulating iNKT cells with α-GalCer, a specific iNKT cell antigen, could rescue mice from B. pseudomallei infection. WT BALB/c mice were pretreated with α-GalCer (or phosphate-buffered saline [PBS] control) 24 h before infection with B. pseudomallei. Interestingly, 62.5% (10/16) of the α-GalCer-treated mice survived until 14 days p.i., while all mice in the PBS control group died by day 8 (Fig. 4). The overall survival times for α-GalCer-treated mice were significantly longer than those for the control group (14 ± 1 and 6 ± 1 days, respectively; P < 0.0001).

FIG 4.

Survival of WT BALB/c mice pretreated with α-GalCer or PBS before infection with B. pseudomallei. The survival of BALB/c mice (8 mice/group) in percent (y axis) that received 2 μg of α-GalCer (black squares) or PBS (clear circles) 24 h before infection with 1,000 CFU (50LD₅₀) of B. pseudomallei was observed for 14 days. The results are representative of two independent experiments (total of 16 mice/group). Statistically significant differences were evaluated by using Kaplan-Meier analysis and a log-rank test, and asterisks (****) indicate statistical significance (P < 0.0001).

α-GalCer-treated mice had high serum IFN-γ at an early time point and controlled B. pseudomallei bacterial loads.

The serum levels of IFN-γ, IL-4, IL-10, and IL-12 were measured in the α-GalCer-treated and untreated infected mice. At 4 h after α-GalCer injection, the levels of IFN-γ were significantly higher (mean ± standard deviation [SD] = 1,012.26 ± 338.16 pg/mL) than in PBS controls (3.90 ± 2.27 pg/mL; P = 0.0357) (Fig. 5A). At the same time point, the animals also had significantly higher levels of IL-4 (1,436.00 ± 261.10 pg/mL; P = 0.0357), IL-10 (20.35 ± 5.79 pg/mL; P = 0.0357), and IL-12 (96.03 ± 82.70 pg/mL; P = 0.0357) than controls (Fig. 5B to D). Although IL-12 levels were low, IL-12 showed a similar pattern as observed for IFN-γ. IFN-γ levels in the α-GalCer-pretreated group, however, were decreased when measured at 4, 24, and 48 h following B. pseudomallei infection. In contrast, IFN-γ levels from the control group were increased at 24 h (1,017.84 ± 784.54 pg/mL; P = 0.0357) and reached their peaks at 48 h (1,511.87 ± 142.52 pg/mL; P = 0.0357) after infection (Fig. 5A). For IL-4, there was a significant difference between treatment and control groups after infection; however, the level of IL-4 was very low (Fig. 5B). The IL-10 level was significantly higher in the α-GalCer-pretreated group (Fig. 5C).

FIG 5.

Cytokine levels and bacterial loads in mice pretreated with α-GalCer before and after B. pseudomallei infection. (A to D) Mice (6 mice/group) were pretreated with α-GalCer (black bars) or with PBS (white bars). The levels of IFN-γ (A), IL-4 (B), IL-10 (C), and IL-12 (D) in sera were measured at different time points. The arrows indicate the time of α-GalCer and B. pseudomallei injections. (E and F) Bacterial counts in blood, spleens, lungs, and livers of α-GalCer-pretreated (black bars) and control (white bars) groups were assessed at 24 h (E) and 48 h (F) after infection. All data are presented as mean ± SE of mice per group per time point and are representative of two independent experiments (total of 12 mice/group). Statistically significant differences were evaluated by using the Mann-Whitney test, and asterisks (**) indicate statistical significance (P < 0.01).

The B. pseudomallei bacterial loads of the α-GalCer-pretreated group were significantly lower in blood (no growth and 1,780.00 ± 51.21 CFU/ml; P < 0.0001), spleens (no growth and 34,300 ± 1,106.04 CFU/ml; P < 0.0001), and livers (1,550 ± 68.72 and 13,400 ± 1,066.67 CFU/ml; P < 0.0001) than in the control group at 24 h, but not in the lungs (15,300 ± 761.10 and 1,320 ± 41.63 CFU/ml; P < 0.0001) (Fig. 5E and F). Almost all α-GalCer-pretreated infected mice showed negative bacterial loads after 48 h of infection (Fig. 5F). These results suggest that activation of iNKT cells by α-GalCer that lead to higher levels of IFN-γ may extend mouse survival time and provide early protection to mice against colonization after B. pseudomallei infection.

DISCUSSION

iNKT cells are a sublineage of T lymphocytes that recognize lipid or glycolipid antigens, and α-GalCer is a well-known ligand that stimulates iNKT cells (23). The protective functions of iNKT cells during infection are often the result of their ability to rapidly produce IFN-γ for the recruitment and activation of other cell types. Currently, iNKT cells are well known to play a crucial role in bacterial, viral, fungal, and parasitic infections (21, 24–26). In human melioidosis, iNKT cells are activated by B. pseudomallei, and their numbers in blood are significantly lower than in healthy controls (18). Moreover, data from our experiment in a BALB/c mouse model showed that iNKT cell numbers are reduced in spleens, lungs, and livers after infection with B. pseudomallei. The percentages of T cells and tetramer-negative cells in different infected organs were lower but not significantly different than in controls. It was then hypothesized that iNKT cells might decrease TCR expression after B. pseudomallei infection, rendering them undetectable by α-GalCer-loaded CD1d tetramer staining, similar to that found in other reports (27, 28). Another possibility is that B. pseudomallei infection could induce iNKT cell apoptosis similar to Mycobacterium tuberculosis infection (29). Therefore, iNKT cells might play a role in the immune response against melioidosis.

In this study, iNKT-KO (Traj18-deficient) mice were used as a model. Previously generated and widely used Traj18-deficient mice have been shown to have a biased TCR repertoire (30). To circumvent this confounding factor, the new strain of iNKT cell-deficient mice was generated by deleting the Traj18 locus using CRISPR technology, and these animals contain an unbiased TCR repertoire, including mucosal-associated invariant T (MAIT) cells (17). The current study used this newly generated Traj18-deficient mouse line in this study so that there are no limitations of the mouse model in the context of this study.

The hypothesis was explored in this study by using iNKT-KO mice infected with B. pseudomallei that lack iNKT cells (17), and the results showed that iNKT-KO mice died sooner than WT mice. Similar to these findings, mice lacking iNKT cells demonstrate reduced survival compared with WT mice when infected with Streptococcus pneumoniae (31). In addition, the serum IFN-γ levels of iNKT-KO mice were lower than those observed in WT controls at 24 h after B. pseudomallei infection, while the concentrations of IFN-γ at 48 h after infection did not differ between iNKT-KO mice and WT mice. The high levels of IFN-γ in both iNKT-KO- and WT-infected mice at 48 h were probably related to bacterial burden (32) that induced IFN-γ production not only from iNKT cells but also from other immune cells, such as NK cells, CD4⁺ T cells, and CD8⁺ T cells (33). The significantly lower IFN-γ levels in iNKT-KO mice together with the decrease in survival may indicate roles of iNKT cells that may help protect WT mice at the early stage of B. pseudomallei infection. To obtain better proof that iNKT cells are the source of IFN-γ, intracellular cytokine staining of IFN-γ-producing iNKT cells should be done. Unfortunately, in this current study, it could not be detected in IFN-γ-producing iNKT cells due to the low number of iNKT cells in the infections. The low number might be because after α-GalCer administration, mouse iNKT cells downregulate their T cell receptors, making iNKT cells undetectable by flow cytometry with α-GalCer/CD1d tetramer staining (27, 34). The next intervention experiment where early activation of iNKT cells using α-GalCer before B. pseudomallei infection was performed.

The 62.5% survival rate at 14 days was observed in α-GalCer-treated mice, whereas all mice in the control group died. Moreover, in this experiment, a higher dose (50 50% lethal dose [50LD₅₀]) of B. pseudomallei challenge to confirm such protection was used compared to 5LD₅₀ in a previous experiment with iNKT-KO mice. When α-GalCer-treated mice were observed for a further 30 days, 25% of mice still survived, and all these mice remained alive for 3 months until the experiment was terminated. This confirmed the protective role of iNKT cells. In the present study, 100 μg/kg (2 μg/20 g of body weight) of α-GalCer was used, as it is the maximum dose without causing side effects, such as liver damage (35). This suitable dose used to induce IFN-γ in mice has been investigated in several experiments (36–38). When α-GalCer was used to activate iNKT cells, a significantly higher level of IFN-γ production that predominated early before infection led to the activation of an innate immune response that remarkably cleared the bacteria in the spleen and liver, minimizing the robust IFN-γ response activated by the bacterium at 24 to 48 h after infection. The bacterial numbers in the lung, however, did not decrease at 24 h after infection This phenomenon might have been due to the different iNKT cell subtypes found in the lung, which mainly produce T helper 2 (Th2; IL-4 and IL-13), Th9 (IL-9 and IL-10), and Th17 (IL-17A and IL-22) cytokines after activation (39). A majority of iNKT cells in the lung are iNKT2 cells that produce Th2 cytokines, such as IL-13, IL-4, and IL-9, after activation by α-GalCer (39–41). These cytokines suppress IFN-γ production in other immune cells in the initial phase of activation. Moreover, Chen and colleagues reported that intraperitoneal injection of α-GalCer could stimulate lung iNKT cells and promote IL-10 production (42). IL-10, an anti-inflammatory cytokine that can downregulate IFN-γ production, leads to increased bacterial burdens (43). Systemic activation of iNKT cells by α-GalCer increased CD8⁺ T and NK cells. These immune cells later migrate to the lung and suppress Th2 immunity, thus initiating bacterial clearance by IFN-γ as observed at 48 h in the lungs.

It has been shown that excessive and unregulated production of proinflammatory cytokines in bacterial infections may be detrimental to the host and can result in septic shock and death depending on the bacterial loads and time of cytokine production relative to the time of bacterial exposure (44–46). The use of α-GalCer pretreatment therefore needs to be optimized and used at the appropriate time. Moreover, α-GalCer was not applied at the time of infection or at 24 to 48 h after infection, as administration will not occur in time to stimulate the innate immune response that protects mice from B. pseudomallei, similar to our previous report when CpG oligodeoxynucleotide (ODN) was used to activate IFN-γ (47). This protective role of using α-GalCer pretreatment in mice was also reported with Chlamydia muridarum, Pseudomonas aeruginosa, and Mycobacterium tuberculosis infection (48, 49). This approach was also successfully applied to parasitic and viral infections, such as Plasmodium yoelii, Plasmodium berghei, and herpes simplex virus 2 (50, 51).

The mechanism on how iNKT cells play a protective role is not yet clear, but our data suggest that early production of IFN-γ might lead to efficient control of B. pseudomallei in important organs. As B. pseudomallei is known to be an intracellular pathogen (4), there are two possible mechanisms by which iNKT cells could eliminate B. pseudomallei. First is the direct pathway, in which iNKT cells act directly on bacteria-infected macrophages to inhibit bacterial growth without the support of other immune cells, as demonstrated in M. tuberculosis, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes (52, 53). Second is the indirect pathway, in which iNKT cells could eliminate intracellular bacteria through modulating other immune cells, such as DCs and NK cells. For example, IFN-γ produced from iNKT cells could induce NK cell-secreted cytokines, which enhance cytotoxic activity and destroy bacteria inside macrophages (13). The indirect pathway has been reported to be broadly used in host defense against infections, such as influenza A virus, lymphocytic choriomeningitis virus, Leishmania major, and Chlamydia pneumoniae (54–57). In our mouse model, α-GalCer-treated mice with high levels of IFN-γ before infection might preactivate innate immunity effector cells, thus enhancing their antimicrobial power at the time of bacterial challenge with the uptake and intracellular killing of B. pseudomallei (47), leading to lower activation of IFN-γ levels by the bacteria. In contrast, in the control group, the robust production of IFN-γ noted on days 1 to 2 in infected animals was most probably related to the presence of very large bacterial loads, and it was then too late to stimulate IFN-γ to control the ongoing infection. The protective role of IFN-γ in a murine melioidosis model had been shown previously (58, 59). IFN-γ could increase expression of inducible nitric oxide synthase (iNOS) by mouse macrophages and enhance the uptake of B. pseudomallei (20, 60). In the current study, we showed that the higher levels of IFN-γ and faster bacterial clearance in α-GalCer-treated mice could delay death of mice after B. pseudomallei infection.

The α-GalCer ligand is specific for iNKT cells (61, 62) and can stimulate iNKT cells secreting Th1, Th2, and Th17 cytokines to orchestrate the other immune cells, including NK cells, macrophages, DCs, and T and B cells (61). In addition to IFN-γ, it was also found that α-GalCer could activate IL-4, IL-10, and IL-12 production. Although the IL-4 levels are different in the two groups, its small amount may not play a role in this early protection. Similar patterns of IL-12 and IFN-γ production were found. IL-12 is an important mediator of resistance to B. pseudomallei infection in mice, as depletion of IL-12 results in susceptibility of B. pseudomallei infection (58). IL-12 stimulates the production of IFN-γ and is involved in the differentiation into Th1 cells for killing of intracellular bacteria (63, 64). Although IL-10 increased 4 h after α-GalCer injection, its levels were much lower than those of IFN-γ. IL-10 is a potent anti-inflammatory cytokine (43). The increases in both IL-10 and IFN-γ observed in this study are consistent with other reports of responses to intracellular bacteria (65, 66), suggesting a potential beneficial role of IL-10 for increased host resistance during intracellular infection, regardless of the known anti-inflammatory action of this cytokine (67).

There have been several reports that α-GalCer can be used as a therapeutic agent for infectious diseases (68, 69). For example, Sada-Ovalle and colleagues reported that administration of α-GalCer decreased M. tuberculosis replication by improving iNKT cell production of IFN-γ and prolonged mouse survival (70). The present study has shown for the first time that injection of α-GalCer 1 day before infection with B. pseudomallei could activate iNKT cells and significantly reduce bacteremia and bacterial loads in organs, prolong survival times, and confer an early protection against sepsis melioidosis in a murine model. Because α-GalCer needed to be given before infection, therapeutic application would not be applicable. It can then be proposed that this immunostimulant can be used as an adjuvant for a B. pseudomallei vaccine. Previous studies have demonstrated the effectiveness of α-GalCer as an adjuvant for protection against microbial infections (71). For example, mice immunized with α-GalCer together with M. tuberculosis antigens showed stronger antigen-specific IFN-γ-producing T-cell responses than mice immunized with the antigen of M. tuberculosis alone, resulting in the reduction of bacterial burden in infected organs (72). Several B. pseudomallei antigens have been identified for subunit vaccines (73). Therefore, further investigation of whether α-GalCer together with these antigens enhances the protective immune response to B. pseudomallei might be helpful for effective vaccine development.

In summary, iNKT cells were shown to play an early protective role in B. pseudomallei infection. Pretreatment with α-GalCer could promote IFN-γ production in iNKT cells, which could be beneficial for protection against infection and prolong survival. These findings provide information for using iNKT cells as activators to enhance immune responses against B. pseudomallei infection.

MATERIALS AND METHODS

Mice.

Wild-type (WT), 6- to 8-week-old, male BALB/c mice were obtained from National Laboratory Animal Center, Nakorn Pathom, Thailand, and Nomura Siam International Co., Ltd.

Traj18-deficient BALB/c mice that selectively lack iNKT cells (17) were obtained from the Division of Stem Cell Cellomics, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan. As the signs of dying from infection are not clear enough for a humane endpoint, the mice that died in their cages were used for endpoint survival. The experiments operated with BALB/c mice were approved by the Animal Research Ethics Committee of Khon Kaen University, Thailand (reference number ACUC-KKU-65/2559).

Bacteria.

A clinical isolate of B. pseudomallei strain 1909a from the sputum of a melioidosis patient with diabetes mellitus in Ubonratchathani province, Thailand, was kindly provided by Vanaporn Wuthiekanun (Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand). The 50% lethal dose (LD₅₀) was 20 cells (8). B. pseudomallei from a −80°C glycerol stock was streaked on Ashdown’s agar and incubated at 37°C for 48 h. To prepare mid-log-phase bacteria, a single colony was inoculated into 3 mL of Luria-Bertani (LB) broth (Titan Biotech, Ltd., Rajasthan, India) and cultured overnight at 37°C with shaking at 200 rpm (J.P. Selecta, Barcelona, Spain). Subsequently, 1% of the culture was transferred into 50 mL of LB broth, and incubation was continued. When the cell culture reached mid-log phase (optical density at 550 nm [OD₅₅₀] = 0.3 to 0.5), the cells were harvested by centrifugation at 10,000 × g for 10 min, washed twice in 0.05 M phosphate-buffered saline (PBS; pH 7.4), and suspended in 1 mL of PBS. The numbers of bacteria were measured and adjusted to approximately 100 CFU (5LD₅₀) in 100 μL of PBS for the iNKT cell-knockout mice study and 1,000 CFU (50LD₅₀) in 100 μL of PBS for infection in the protective experiment.

Fluorescent antibodies.

Anti-mouse antibodies (BioLegend, San Diego, CA) used for iNKT cell detection by flow cytometry were CD3ε-Pacific Blue (clone 145-2C11) and CD45-allophycocyanin (APC)-eFluor780 (clone 30-F11). The phycoerythrin (PE)-conjugated CD1d tetramer-loaded α-GalCer and CD1d tetramer-unloaded α-GalCer were from the US NIH tetramer facility.

Flow cytometry analysis.

Mouse mononuclear cells (MNCs) were isolated from lungs, livers, and spleens. Briefly, each organ was preserved in R10⁺ medium comprised of RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 U mL⁻¹ penicillin-streptomycin, 10 mM HEPES buffer solution, and 5.5 μM 2-mercaptoethanol (2-ME) and homogenized and passed through a 100-μm cell strainer (Falcon). The filtered cell suspensions were centrifuged at 800 × g for 5 min at 4°C, the cell pellets were suspended in 1 mL of 10× red blood cell (RBC) lysing solution (Sigma, Zwijndrecht, Netherlands), and cells were incubated at room temperature for 2 min. The cells were diluted in R10⁺ medium and collected by centrifugation at 800 × g for 5 min at 4°C and then suspended in 100 μL of staining buffer (2% FBS and 0.1% sodium azide in PBS). The cells were stained with Pacific Blue-conjugated anti-CD3ε (1 μg/test), APC-eFluor780-conjugated anti-CD45 (0.125 μg/test), PE-conjugated CD1d tetramers loaded with α-GalCer, or PE-conjugated unloaded CD1d tetramers (control) according to the manufacturer’s protocols. The cells were mixed and incubated for 30 min at 4°C in the dark. After incubation, stained cells were washed twice with 1 mL of staining buffer, and cells were collected by centrifugation at 400 × g for 5 min at 4°C. Finally, the cells were suspended in 250 μL of staining buffer for analysis by flow cytometry (BD FACSCanto II, Becton, Dickinson USA). The complete details of iNKT cell gating, counting, and testing are shown in Fig. 1.

Number of iNKT cells during B. pseudomallei infection.

Spleens, lungs, and livers were collected from BALB/c mice on days 2, 3, and 6 after infection with 1,000 CFU (50LD₅₀) of B. pseudomallei in 100 μL of PBS or PBS alone for uninfected controls. The experiment was done in three independent studies with 9 mice/group (total = 27/group). Mononuclear cells were isolated from these organs and prepared as cell suspensions. The cells were stained with monoclonal antibodies (MAbs) to investigate the percentage of iNKT cells by flow cytometry (Fig. 1). The profiles of iNKT cells in each organ were compared between infected and control groups.

iNKT cell-knockout mice infection with B. pseudomallei.

Three iNKT-KO and three WT BALB/c mice (all male, sex and age matched) were infected with 5LD₅₀ (100 CFU) of B. pseudomallei strain 1909a in 100 μL of PBS by intraperitoneal injection (8). Subsequently, the survival times of infected mice were observed for 14 days. Two independent experiments were performed (total = 6/group). In addition, 100 μL of blood samples from each mouse was collected from the tail vein before infection and at 4, 24, and 48 h after B. pseudomallei infection for IFN-γ concentration determination using a mouse cytokine/chemokine magnetic bead panel (Merck, Germany), as described by the manufacturer.

iNKT cell activation by α-GalCer and bacterial infections.

The glycolipid α-galactosylceramide (α-GalCer) can effectively stimulate iNKT cells (23). For the protective study, eight male BALB/c mice received 2 μg of α-GalCer (Kirin Brewery Co., Ltd., Tokyo, Japan) in 100 μL of PBS via intraperitoneal injection, and another eight mice received PBS as a vehicle control. All mice were then infected intraperitoneally with 100 μL of 50LD₅₀ (1,000 CFU) log-phase cell culture of B. pseudomallei strain 1909a 24 h after α-GalCer administration and were then monitored for survival for 14 days. The experiments were done in two independent studies (total = 16/group). In other experiments, cytokine levels and bacterial load assessments were performed separately (6 mice/group and 2 independent studies [total = 12/group]). Experiments were designed similar to those of the protective experiment. One hundred microliters of blood was collected from tail veins at 0 and 4 h after α-GalCer administration for cytokine quantification. Blood samples from the tail vein of each mouse were collected at 4, 24, and 48 h after B. pseudomallei infection. Sera from the clotted blood samples were collected for cytokine determination.

Determination of bacterial loads in blood and tissues of the infected mice.

After infection, 200 μL of blood samples was collected from each mouse (4 mice/group) via cardiac puncture using a tuberculin syringe, and 100 μL was spread on LB agar for bacterial counts. In addition, spleens, livers, and lungs were homogenized in 1 mL of sterile normal saline, and 100 μL (undiluted or the appropriate dilutions) was spread on LB agar plates for bacterial counts (47). The bacterial colony was confirmed to be B. pseudomallei by using a latex agglutination test kit (74).

Cytokine assays.

IFN-γ, IL-4, IL-10, and IL-12 concentrations in mouse sera were measured using a mouse cytokine/chemokine magnetic bead panel (Merck, Germany) according to the manufacturer’s protocol. Briefly, 25 μL of undiluted mouse serum was incubated with antibody-immobilized beads in a 96-well plate in the dark at 4°C overnight. After washing, 25 μL of detection antibody was added into each well and incubated in the dark for 1 h at room temperature. Then, 25 μL of streptavidin-phycoerythrin was added and incubated in the dark for 30 min at room temperature. Finally, the plates were washed and resuspended with 150 μL of sheath fluid before obtaining the data on a Luminex 200 with xPONENT software.

Statistical analysis.

Statistical analysis was performed using SPSS version 23.0 and GraphPad Prism software. The distribution of data was tested by the Shapiro-Wilk test. Data that did not pass the normality test were analyzed by the Mann-Whitney test, while normally distributed data were analyzed using the unpaired Student’s t test. The results are shown as mean ± standard error (SE) or mean ± standard deviation (SD). The level of significance for all statistical analyses was set as a P value of <0.05.