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Open Access

Peer-reviewed

Research Article

Persistent infection due to a small-colony variant of Burkholderia pseudomallei leads to PD-1 upregulation on circulating immune cells and mononuclear infiltration in viscera of experimental BALB/c mice

Abstract

Background

Melioidosis is a neglected tropical disease endemic across South East Asia and Northern Australia. The etiological agent, Burkholderia pseudomallei (B.pseudomallei), is a Gram-negative, rod-shaped, motile bacterium residing in the soil and muddy water across endemic regions of the tropical world. The bacterium is known to cause persistent infections by remaining latent within host cells for prolonged duration. Reactivation of the recrudescent disease often occurs in elders whose immunity wanes. Moreover, recurrence rates in melioidosis patients can be up to ~13% despite appropriate antibiotic therapy, suggestive of bacterial persistence and inefficacy of antibiotic regimens. The mechanisms behind bacterial persistence in the host remain unclear, and hence understanding host immunity during persistent B. pseudomallei infections may help designing potential immunotherapy.

Methodology/Principal findings

A persistent infection was generated using a small-colony variant (SCV) and a wild-type (WT) B. pseudomallei in BALB/c mice via intranasal administration. Infected mice that survived for >60 days were sacrificed. Lungs, livers, spleens, and peripheral blood mononuclear cells were harvested for experimental investigations. Histopathological changes of organs were observed in the infected mice, suggestive of successful establishment of persistent infections. Moreover, natural killer (NK) cell frequency was increased in SCV- and WT-infected mice. We observed programmed death-1 (PD-1) upregulation on B cells of SCV- and WT-infected mice. Interestingly, PD-1 upregulation was only observed on NK cells and monocytes of SCV-infected mice. In contrast, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) downregulation was seen on NK cells of WT-infected mice, and on monocytes of SCV- and WT-infected mice.

Conclusions/Significance

The SCV and the WT of B. pseudomallei distinctly upregulated PD-1 expression on B cells, NK cells, and monocytes to dampen host immunity, which likely facilitates bacterial persistence. PD-1/PD-L1 pathway appears to play an important role in the persistence of B. pseudomallei in the host.

Author summary

B. pseudomallei is a bacterium that causes melioidosis, a disease endemic in Southeastern Asia and Northern Australia. It is estimated that melioidosis leads to 89,000 deaths worldwide each year. Nevertheless, melioidosis continues to remain a neglected tropical disease that is not even on the list of neglected tropical diseases of the World Health Organization. Furthermore, the disease has a high mortality and recurrence rate, which can be up to 40% and 13%, respectively. It has also been well documented that B. pseudomallei causes latent/persistent infections for a prolonged period without showing apparent symptoms in the infected individual. The mechanisms that are responsible for bacterial persistence in the host remain unclear. Our results demonstrated that B. pseudomallei were able to upregulate PD-1 expression on B cells, NK cells, and/or monocytes during persistent diseases, which likely diminish optimal host immunity. The weakened host immunity in turns facilitates persistence of the bacterium. Interestingly, the SCV had a higher PD-1 expression on distinct immune cells compared to the WT, which might explain its frequent association with persistent infections. Immunotherapies by targeting PD-1/PD-L1 pathway could serve as a better treatment than the conventional antibiotic regimens, which cause a high rate of recurrence in melioidosis patients.

Citation: See J-X, Chandramathi S, Abdulla MA, Vadivelu J, Shankar EM (2017) Persistent infection due to a small-colony variant of Burkholderia pseudomallei leads to PD-1 upregulation on circulating immune cells and mononuclear infiltration in viscera of experimental BALB/c mice. PLoS Negl Trop Dis 11(8): e0005702. https://doi.org/10.1371/journal.pntd.0005702

Editor: Mathieu Picardeau, Institut Pasteur, FRANCE

Received: January 12, 2017; Accepted: June 11, 2017; Published: August 18, 2017

Copyright: © 2017 See et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data are within the manuscript.

Funding: This work was supported by the University of Malaya Research Grant (UMRG: RG505-13HTM), Post-graduate Research Grant (PG314-2016A) and the University of Malaya-Ministry of Education (UM-MoE) High Impact Research (HIR) Grant (UM.C/625/1/HIR/MoE/CHAN/02: H-50001-A000013). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Burkholderia pseudomallei (B. pseudomallei) is the causative agent of melioidosis, an infectious disease, endemic across parts of South East Asia and Northern Australia [1]. Despite causing an estimated 89,000 deaths worldwide annually, melioidosis still remains a neglected tropical disease [2]. Being a major cause of community-acquired sepsis, melioidosis has a high mortality rate up to 40% [3]. Common routes of infection include percutaneous inoculation, inhalation, and/or ingestion of contaminated particles or aerosols [4]. Although melioidosis can manifest diverse symptoms such as pneumonia and abscesses in various organs including the brain, bacteremic melioidosis with pneumonia commonly leads to early mortality [5,6]. Apart from acute infection, B. pseudomallei can cause persistent disease with little or no clinical symptoms over a prolonged period of latency in the host, and only reactivate after years [7–9]. This suggests the likelihood of B. pseudomallei to reactivate only when the host immunity wanes. Indeed, B. pseudomallei can be considered also as an opportunistic pathogen, as melioidosis patients are commonly individuals with at least one or more underlying diseases (~80%) and the elderly [3]. Moreover, recurrence rates in patients can be up to ~13% despite appropriate antibiotic treatments[10], suggestive of bacterial persistence and inefficacy of antibiotic regimens. The mechanisms behind bacterial persistence in the host remain unclear.

Small-colony variants (SCVs) representing a sub-population of bacteria have been frequently associated with persistent infections [11–15]. As the name implies, SCVs are slow-growing and form pin-point colonies after 24–72 hours of incubation on agar medium [16]. Although the SCVs of Staphylococcus aureus (S. aureus) remain the most extensively studied variant, the morphotypes have also been described in many other bacteria including B. pseudomallei. SCVs are known to be relatively more resistant to antibiotics compared with their wild-type (WT) counterparts [17]. In B. pseudomallei, SCVs were reported to display a greater degree of drug resistance [18]. To the best of our knowledge, we are the only group till date that attempted to study WT and SCVs of B. pseudomallei. Our proteomic studies revealed that SCVs and WT pre- and post-infection of A549 lung epithelial cells showed distinct expressions of proteins involved in adhesion, invasion, and virulence (Al-Maleki et al., 2014; Al-Maleki et al., 2015). More importantly, our previous study also demonstrated that SCVs and WT triggered distinct host immune responses during persistent B. pseudomallei infections. Another study also demonstrated that B. pseudomallei can switch to different morphotypes during stress, and have distinct abilities to persist in vitro and in vivo [19]. Hence, these pieces of evidence together suggest that SCVs and WT could play different roles in persistent clinical melioidosis.

Programmed death-1 (PD-1) negatively regulates T cell functions, as its engagement with its ligand PD-L1 and PD-L2 arrest T cell proliferation, cytokine secretion, and cytolytic functions [20]. PD-1 is by far the best characterized co-inhibitory molecule associated with T-cell exhaustion in chronic viral infections [21,22]. Apart from chronically-infecting viruses [23–25], many bacteria that cause persistent infections, such as Mycobacterium tuberculosis and Helicobacter pylori (H. pylori), are known to upregulate PD-1 and PD-L1 [26–30]. Persistent B. pseudomallei infections in BALB/c mice also led to PD-1 upregulation on CD4+ and CD8+ T cells, suggestive of T cell exhaustion. This is in line with a previous study that reported on PD-L1 upregulation in polymorphonuclear neutrophils infected with B. pseudomallei, which consequently inhibited CD4+ T-cell functions as well [31]. These results suggest an important role of PD-1/PD-L1 pathway that might potentially be exploited by B. pseudomallei to facilitate persistence in the host. While the role of PD-1 in functional exhaustion is clearly established in T cells, accumulating lines of evidence indicate that PD-1 negatively regulates the functions of B cells, natural killer (NK) cells, and monocytes [32–37].

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) represents another co-inhibitory molecule that is inducibly expressed on T cells. CTLA-4 is homologous to CD28 (the co-stimulatory molecule that provides second signal for T cell activation), and inhibits T cell activation [38]. Both CTLA-4 and CD28 engage with two cognate ligands, B7-1 (CD80) and B7-2 (CD86), although CTLA-4 binds with a greater affinity [39]. Similar to PD-1, the role of CTLA-4 has been extensively studied in T cells. CTLA-4 upregulation on T cells has been well documented in hepatitis B (HBV) and human immunodeficiency virus (HIV) infections [38,40]. In bacterial infections, CTLA-4 has been reported to cause T cell anergy especially in H. pylori infections in mice, and pathogen clearance was improved following the blockade of CTLA-4 [41]. Notwithstanding, the role of CTLA-4 is well-studied in T cells, its role in other immune cells rather remains ambiguous. To date, very few studies have demonstrated that CTLA-4 inhibits the functions of B cells, NK cells, and monocytes [42–45]. Therefore, it is conceivable to hypothesize that PD-1 and CTLA-4 could dampen host immune responses leading to establishment of persistent infections. We demonstrated previously that persistent B. pseudomallei infections can lead to an increased expression of PD-1 on CD4+ and CD8+ T cells [46]. Here, we aimed to investigate into B cell and innate cell responses, including PD-1 and CTLA-4 expressions, during experimental persistent B. pseudomallei infections. We proposed that persistent B. pseudomallei infections can lead to upregulation of PD-1 on B cells, NK cells, and monocytes, resulting in suboptimal host immune responses. B. pseudomallei employs PD-1/PD-L1 pathway as an immune exhaustion strategy to persist in the host.

Materials and methods

Ethics statement

All mouse experiments were conducted according to the guidelines of the University of Malaya Animal Care and Use Policy (UM ACUP), and the protocols were reviewed and approved by the Animal Experimental Unit of University of Malaya, Kuala Lumpur, Malaysia (Ref. No.: 2014-08-05/MMB/R/JSV). The Animal Experimental Unit of University of Malaya is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), and conforms to all government laws and regulations. It provides for approved research and teaching activities, and safeguards the health and welfare of staff and students involved in scholarly activities using animals or animal parts derived from animals. Animals were maintained with controlled temperature, 12h light/dark cycles and given water and feed ad libitum. All efforts were made in order to minimize animal suffering. In addition, all bacterial isolates used in this study were analyzed anonymously.

Bacterial identification

A clinical isolate of B. pseudomallei from a melioidosis case in University Malaya Medical Center (UMMC) isolated as previously described was used in the study [47]. The isolate, when cultured on agar medium at 37°C, was found to differentiate into two colony morphotypes, OB (WT, INSDC: APLK00000000.1) and OS (SCV, INSDC: APLL00000000.1). OB and OS were characterized using a commercial analytical profile index API 20NE (BioMėrieux) test and an in house PCR assay specific for B. pseudomallei [48]. The two morphotypes were cultured on nutrient agar and a single colony of each morphotype was inoculated into Luria-Bertani (LB) broth (Becton Dickinson, Franklin Lakes, New Jersey, USA) at 37°C overnight in a shaker incubator at 200 revolutions per minute (rpm). Following culture, glycerol (Acros Organics, Geel, Belgium) with a final concentration of 30% (v/v) was added to the LB cultures and stored at -80°C as a stock culture for the entire duration of the study.

Bacterial inoculum

Bacterial inoculum of OB and OS was prepared as previously described [46]. Briefly, a single colony of OB and OS from nutrient agar was cultured in LB broth and incubated at 37°C overnight at 200rpm. Later, overnight cultures were adjusted to an OD600 of 0.05 with LB broth and incubated under similar conditions. Cultures that reached the mid-logarithmic phase (OD600 0.5–0.7) were harvested, washed, and re-suspended in phosphate-buffered saline (PBS). Subsequently, the bacterial suspensions were ten-fold serially diluted with PBS until the desired inoculum was obtained. The inoculum was plated on nutrient agar to enumerate colony-forming units (CFUs).

Persistent infection

Seven to eight-week-old female BALB/c mice obtained from University Putra Malaysia were used in the experiments. All mice were acclimatized for two weeks prior to infection. Mice were under ad libitum feeding conditions. Mice were anaesthetized with isoflurane (Piramal Healthcare Ltd, India), and 10μL of bacterial inoculum was administered via the intranasal route. A persistent B. pseudomallei infection was generated as described previously with minor modifications [49]. Sub-lethal bacterial dose (~2–8% of LD50) was determined as suggested by Goodyear et al. that used ~5% of LD50 to generate persistent infections in BALB/c mice. Recently, we confirmed that persistent infections with the sub-lethal bacterial dose led to bacterial colonization in the lungs, livers or spleens for ≥60 days by CFU enumeration of these organs, and development of macroscopic hepatic and splenic abscesses in infected mice [46]. Groups of six mice were infected with OB and OS morphotypes, respectively. Only mice that survived for ≥60 days were sacrificed for use in the downstream experiments. One experiment was performed to collect organ samples (n = 4 per group) from infected (OB or OS) and uninfected mice, respectively for histopathological analysis. Two independent experiments were performed in order to collect adequate sample size (n = 6 per group) from OB-infected, OS-infected, and uninfected mice for analysis of immune cells. Mice inoculated with PBS were used as controls, and will be referred to as uninfected mice for simplicity.

Peripheral blood mononuclear cells (PBMCs)

Mice with persistent B. pseudomallei infections were anaesthetized with isoflurane, and blood was drawn via terminal cardiac puncture. Heparinized blood samples were diluted with PBS at a 1:1 ratio. PBMCs were isolated as described [50,51]. Briefly, PBMCs were prepared by density-gradient centrifugation over Ficoll-Paque (Sigma Aldrich). PMBC layer was obtained and washed twice with PBS. Cell viability was determined by 0.4% trypan blue (Life Technologies) staining.

Haemotoxylin and eosin (H & E) staining

Lungs, livers, and spleens from mice were harvested after withdrawal of blood, and fixed immediately in 10% neutral buffered formalin for 24 hours. Organs were processed as parafilm blocks, followed by the H & E staining, and examined using a microscope. Representative images for each visceral organ were captured.

Polychromatic flow cytometry

PBMCs (1x106 cells in each tube) were stained with Alexa Fluor 488 hamster anti-mouse CD3e (BD Biosciences, clone 145-2C11), Pe-Cy7 rat anti-mouse CD4 (BD Biosciences, clone GK1.5), APC-H7 rat anti-mouse CD8a (BD Biosciences, clone 53–6.7), APC hamster anti-mouse PD-1 (BD Biosciences, clone J43), and PE hamster anti-mouse CTLA (BD Biosciences, clone UC10-4F10-11), Fixable Viability Stain 510 (BD Biosciences, cloneR35-95). Corresponding isotype control for each antibody was prepared for appropriate setting of gates during multicolor flow cytometry analysis. All antibodies were pre-titrated for optimal working concentration. Data were acquired on an 8-color FACSCanto II immunocytometry system (BD Biosciences) with BD FACSDiva software (BD Bioscience). Data were exported from BD FACSDiva and analyzed using Flowjo software version 10 (Tree Star, Oregon, USA).

B cell, NK cell, and monocyte immunophenotyping

We used a combination of positive and negative selection strategies to identify B cells, NK cells, and monocytes. NK cells express CD8, and monocytes express low levels of CD4 [52,53]. Therefore, we defined B cells as the lymphocyte population (FSC-A vs SSC-A) that was CD3-, CD4-, and CD8-, NK cells as the lymphocyte population that is CD3- and CD8+, and monocytes as the monocyte population (FSC-A vs SSC-A) that was CD3- and CD4dim.

Statistical analysis

Two-tailed Mann-Whitney U test was used to determine statistical significance among different groups, due to the assumption that samples might not follow Gaussian distribution. Results were illustrated using Box-Whisker Plots. All statistical analyses were done using GraphPad Prism 6 software (La Jolla, California, USA). The level of significance was first set at *P<0.05, ** P<0.01, ***P<0.001, and adjusted with appropriate Bonferroni correction.

Results

WT B. pseudomallei exhibited rapid growth on Ashdown’s Agar compared to SCV morphotype

Morphological differences between OB and OS morphotypes on Ashdown’s agar, which is a selective agar for B. pseudomallei [54] were compared following 24 and 48 hours incubation at 37°C under aerobic conditions (Fig 1A & 1B). OB is the WT, whereas OS is the SCV of B. pseudomallei isolated from the same melioidosis patient. OB developed clear and visible colonies on Ashdown’s agar, while OS could hardly be observed after 24 hours of incubation. Over 48 hours, OB continued to grow larger, whereas OS appeared as small pin-point colonies. This suggests that OS can be differentiated from OB by its slow-growth rate and morphology on Ashdown agar under aerobic conditions. In addition, OB and OS showed distinct morphologies after 72 hours of incubation (Fig 1C). OB appeared as pale purple, rough, wrinkled, and irregular colonies, whereas OS appeared as dark purple, smooth, round and ≥ 2mm diameter colony.

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Fig 1. Morphological differences between OB and OS on Ashdown’s agar.

(A & B) OB colonies were visible after 24 hours of incubation, and continued to grow larger up to 48 hours of incubation. OS morphotype only appeared as small colonies after 48 hours of incubation. (C) OB showed rough, wrinkled, and irregular morphology, while OS showed smooth and round morphology after 72 hours of incubation.

https://doi.org/10.1371/journal.pntd.0005702.g001

Persistent B. pseudomallei infections of BALB/c mice resulted in differential histopathological changes in visceral organs

Lungs, livers, and spleens harvested from OS-infected or OB-infected mice (n = 4 per group) were processed and stained with H & E to investigate the histopathological changes in a persistent B. pseudomallei infection. Non-necrotic solid lung lesions characterized by a discrete focus consisting primarily of mononuclear cells (Fig 2F) were observed in infected mice.

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Fig 2. Histopathological changes of lungs, livers, and spleens of mice with persistent B. pseudomallei infections.

(A) Lung (B) liver and (C-E) spleen of uninfected mice show normal histopathology. (F) Non-necrotic solid lung lesions characterized by a discrete focus consisting of primarily mononuclear cells (arrow). (G) Hepatic lesion with predominantly mononuclear cells (arrow). Cytoplasmic vacuolation of hepatocytes were observed in the area surrounding the lesion, which is characterized by swelling hepatocytes and clearing cytoplasm (arrow heads) Normal hepatocyte-H. (H) Splenomegaly with large encapsulated abscess cavity (arrow). (I) The encapsulated abscess-A was surrounded by foamy macrophages-FM, epithelioid macrophages-EM, and lymphocytes-L. (J) Magnification of the splenic encapsulated abscess, which contains neutrophils, mononuclear cells, bacteria, and necrotic cellular debris. Sections were stained with H & E. Scale bars: 10 μm (A, E, F, J), 50 μm (B & G), 100 μm (D & I) and 500 μm (C & H). Data are representative of one experiment (n = 4 per group).

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Livers of infected mice showed lesions with predominantly mononuclear cells (Fig 2G). Cytoplasmic vacuolation was also observed in the hepatocytes of surrounding lesions, characterized by swelling of hepatocytes and clearing of cytoplasm (Fig 2G). Cytoplasmic vacuolation in hepatocytes suggests the likelihood of mild-acute and sub-acute liver injury due to persistent B. pseudomallei infections.

Several mice showed splenomegaly with large encapsulated abscess cavities containing neutrophils, mononuclear cells, bacteria, and necrotic cellular debris surrounded by a layer of foamy macrophages, followed by epithelioid macrophages and lymphocytes (Fig 2H–2J). On the other hand, some mice (n = 2) after 60 days of persistent B. pseudomallei infections appeared to have normal red and white pulps with no lesion. Together, these results indicate that intranasal infection of sub-lethal dose B. pseudomallei causes persistent infections that can lead to histopathological changes and systemic spread of the bacteria from the lungs into the livers and spleens.

Persistent B. pseudomallei infections led to increased frequency of NK cells in BALB/c mice

Next, we sought to compare the frequencies of B cells, NK cells, and monocytes in the PBMCs of OB-infected, OS-infected, and uninfected mice. Gating strategy for selection of cell population was illustrated (Fig 3). Our results revealed that both OS-infected and OB-infected mice had a higher NK cell frequency relative to uninfected mice (Fig 4B). Interestingly, OS-infected mice had a higher NK cell frequency relative to the uninfected mice. No significant differences were found in monocyte and B cell frequencies among OS-infected, OB-infected, and uninfected mice (Fig 4A & 4C). Together, our results suggest a potential role of NK cell in persistent B. pseudomallei infections.

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Fig 3. Illustrations of the gating strategy employed in the immunophenotyping of B cells, NK cells, and monocytes.

Lymphocytes and monocytes were gated based on forward and side scatter characteristics. B cells were CD3-CD4-CD8-, NK cells were CD3-CD8+, and monocytes were defined as CD3-CD4dim. All gates were set using respective isotype controls.

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Fig 4. Immune cell frequencies in PBMCs of BALB/c mice infected with B. pseudomallei.

(A) B cell (B) NK cell and (C) monocyte frequencies in PBMCs isolated from uninfected, OB- and OS-infected mice after 60 days of infection. Data representative of two independent experiments (B-D; n = 6 per group). Scatter dot plots show the median value (line), the interquartile range (whiskers). P values were calculated using Mann-Whitney U test. *P<0.025, **P<0.005, ***P<0.0005 after Bonferroni correction for 2 comparisons.

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Persistent B. pseudomallei infections led to upregulation of PD-1, but not CTLA-4, on B cells in BALB/c mice

PD-1 and CTLA-4 belong to the B7-CD28 superfamily, and their expressions inhibit B cell functions [32,35,39,42,43]. Adaptive immune responses play a paramount role against persistent infections. However, the expression levels of PD-1 and CTLA-4 on B cells have seldom been investigated in B. pseudomallei infections. We found that the percentage of B cells that expressed PD-1 was increased in OS-infected and OB-infected compared with uninfected mice (Fig 5A & 5B). Nevertheless, no significant changes were found on B cells that expressed CTLA-4 among the three groups studied (Fig 5A & 5C). Taken together, our results indicate that expression of PD-1, but not CTLA-4, could attenuate optimal B cell functions during persistent B. pseudomallei infections.

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Fig 5. PD-1 and CTLA-4 expressions on Singlet/FVS 510-/Lymph/CD3-/CD4-/CD8- B cells.

Representative contour plot of (A) PD-1 and CTLA-4 expressions on B cells of uninfected, OB-infected and OS-infected mice after 60 days of infection. (B) PD-1+ and (C) CTLA-4+ percentage of uninfected, OB-infected and OS-infected mice. Data are pooled from two independent experiments (A-C; n = 6 per group). Scatter dot plots show the median value (line), the interquartile range (whiskers). P values calculated using Mann-Whitney U test. *P<0.025, **P<0.005, ***P<0.0005 after Bonferroni correction for 2 comparisons.

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SCV B. pseudomallei infections of BALB/c mice resulted in the upregulation of PD-1 on NK cells of BALB/c mice

Next, we looked into the innate immunity. PD-1 and CTLA-4 expressions arrest IFN-γ secretion capability of NK cells [34,45]. Thus, we examined the profile of PD-1 and CTLA-4 expressions on NK cells. Interestingly, OS-infected mice had a remarkable increase of NK cells expressing PD-1 relative to WT-infected and uninfected mice (Fig 6A & 6B). However, no changes in NK cells expressing PD-1 were observed between the OB-infected and uninfected mice. Interestingly, OB-infected mice had a lower NK cell frequency that expressed CTLA-4 as compared with OS-infected and uninfected mice (Fig 6A & 6C). Our findings demonstrate that SCV B. pseudomallei upregulates PD-1 expression on NK cells in mice during persistent infections, suggestive of NK cell exhaustion.

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Fig 6. PD-1 and CTLA-4 expressions on Singlet/FVS 510-/Lymph/CD3-/CD8+ NK cells.

Representative contour plot of (A) PD-1 and CTLA-4 expressions on NK cells of uninfected, OB-infected and OS-infected mice after 60 days of infection. (B) PD-1+ and C) CTLA-4+ percentage of uninfected, OB-infected and OS-infected mice. Data are pooled from two independent experiments (A-C; n = 6 per group). Scatter dot plots show the median value (line), the interquartile range (whiskers). P values calculated using Mann-Whitney U test. *P<0.025, **P<0.005, ***P<0.0005 after Bonferroni correction for 2 comparisons.

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SCV B. pseudomallei infection of BALB/c mice culminated in PD-1 upregulation and CTLA-4 downregulation on monocytes

Finally, we investigated the expressions of PD-1 and CTLA-4 on monocytes, as these two co-inhibitory molecules both negatively regulate monocyte functions [36,37,44]. We observed that OS-infected mice had a higher frequency of monocytes expressing PD-1 compared with OB-infected and uninfected mice (Fig 7A & 7B). No significant differences were observed between OB-infected and uninfected mice. Interestingly, we noticed a remarkable decrease in monocytes expressing CTLA-4 in OS-infected and OB-infected relative to uninfected mice (Fig 7A & 7C). OS-infected mice had a lower frequency of monocytes expressing CTLA-4 compared with OB-infected mice. Together, our results suggest that PD-1 and CTLA-4 are implicated in monocyte functions during persistent B. pseudomallei infections.

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Fig 7. PD-1 and CTLA-4 expressions on Singlet/FVS 510-/Monocyte/CD3-/CD4dim monocytes.

Representative contour plot of (A) PD-1 and CTLA-4 expressions on monocytes of uninfected, OB-infected and OS-infected mice after 60 days of infection. (B) PD-1+ and (C) CTLA-4+ percentage of uninfected, OB-infected and OS-infected mice. Data are pooled from two independent experiments (A-C; n = 6 per group). Scatter dot plots show the median value (line), the interquartile range (whiskers). P values calculated using Mann-Whitney U test. *P<0.025, **P<0.005, ***P<0.0005 after Bonferroni correction for 2 comparisons.

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Discussion

Our OS morphotype was stable and reproducible throughout the experiment, as it did not revert back to WT morphology when cultured from glycerol stock, during growth kinetic study and preparation of inoculum for infection. Our previous study showed that OS grew slower on nutrient agar compared to OB [46]. Moreover, our growth kinetic study demonstrated that OS had a defect in growth in vitro, as it grew much slower in Lunia-Bertani (LB) broth and reached a much lower OD600 density compared to OB. In this study, OS also grew slower on Ashdown’s agar and had different morphology when compared to OB. We categorized OB as type I (pale purple, irregular and rough colonies) and OS as type III (dark purple and smooth colonies) morphotype according to Chantratita et al. [19].

Despite previous studies on SCV and WT B. pseudomallei using both in vitro and in vivo model, literature on the pathogenesis of persistent infections due to SCVs and WT still remains scarce [46,55,56]. Tuchsherr et al. [57] revealed that intracellular infection of endothelial cells with SCVs of S. aureus did not cause any dramatic change in the genes that regulate innate immune responses compared to WT morphotypes. Nevertheless, the study by Tuchsherr et al. could only explain an acute-like intracellular infection in vitro as the infection assay was only conducted for a few hours. Hence, our experimental mouse model serves the purpose of comparing the pathogenesis between SCVs and WT during persistent B. pseudomallei infections. We previously showed that a higher bacterial burden was observed in spleens, but not in the lungs and livers of mice infected with the SCV compared with the WT morphotype [46]. Besides, mice that survived an infection with the SCV of B. pseudomallei for two months were more likely to develop macroscopic liver or splenic abscesses compared with the WT morphotype. Significant changes in lungs, despite using the intranasal route for infection were not observed. This observation can be supported by a previous study on the “persistence model” that demonstrated higher bacterial recovery percentage from livers and spleens compared with lungs after intranasal challenge [49].

To date, only Conejero et al. [58] attempted to characterize the histopathological changes in lungs, livers, and spleens in a chronic B. pseudomallei infection using C57BL/6 mice. In their study, four different types of lung lesions were observed, with two types of them forming granulomas. Additionally, the study also observed pyogranuloma with a necrotic center containing neutrophils surrounded by macrophages, plasma cells, and lymphocytes in the liver. Small pyogranulomas containing neutrophils and macrophages were also common in the liver. Moreover, multifocal to coalescent pyogranulomatous splenitis containing a necrotic center and nonnecrotic microgranulomas consisting epithelioid macrophages were also observed.

In our study, contrary to Conejero et al.’s findings, only one type of lung lesion was observed, which was characterized by a discrete focus of lymphocyte infiltration, with no granuloma (Fig 2F). However, similar findings were observed in many of the lungs of infected mice that had few to no significant lesions. In contrast to Conejero et al., minor hepatic lesions characterized by infiltration of predominantly mononuclear cells in most of the infected mice were observed. Notably, cytoplasmic vacuolation in hepatocytes of several infected mice was observed, suggesting mild-acute and subacute liver injury due to persistent B. pseudomallei infections (Fig 2G). In contrast to Conejero et al., normal spleen histology for several infected mice was observed in the present study. Several mice with macroscopic abscesses showed splenomegaly and necrotic pyogranulomas containing neutrophils, which was surrounded by a layer of foamy macrophages, followed by epithelioid macrophages and lymphocytes (Fig 2H and 2I).

Different observations compared with Conejero et al. were possibly caused by different strains of B. pseudomallei and mice used in the study. More importantly, Conejero et al. sacrificed mice with chronic B. pseudomallei infections after 20 to 60 days of infections, while our study sacrificed mice only after 60 days of persistent infections. The inconsistent duration of sacrificing mice for histopathological investigation in their study might have contributed significantly to different observations. Mice that were sacrificed on the 20th day could have not survived for a longer period due to a more serious B. pseudomallei infection. This would have led to a more severe histopathological changes and a biased observation. The present study sacrificed mice only after 60 days, which is the period considered as chronic melioidosis [59]. This duration leads to a more accurate and consistent histopathological investigation for persistent B. pseudomallei infections.

We speculate that the SCV is more likely to cause severe persistent disease, which was reflected by the higher bacterial load in spleens and more abscess formation in livers and spleens compared with the WT [46]. It is illogical that SCVs result in a more severe pathology than WT, as this process will not benefit the bacteria to persist for a longer duration due to massive host immune responses. However, a recent study by Dietrich et al. [60] demonstrated that non-replicating M. tuberculosis caused a higher CFU and an increased number of granulomas in mouse lungs compared with WT after six weeks. Dietrich et al. suggest that non-replicating M. tuberculosis might undermine host immunity leading to higher bacterial replication and severe pathology. This might be part of the resuscitation process for non-replicating or dormant bacteria to eventually facilitate its transmission. SCVs are similar to dormant bacteria in certain ways, including resistance to antibiotics and slow/zero growth rate. Our results demonstrate that SCVs might employ the same strategy as dormant M. tuberculosis by causing a greater degree of pathology in order to facilitate its transmission in persistent B. pseudomallei infections. This explanation is reasonable as one would anticipate SCVs to relapse in some time in future to transmit the disease.

There are only limited studies on the role of B cells, NK cells, and monocytes in melioidosis. Antibodies against B. pseudomallei appears to play a less significant role against melioidosis despite that many individuals still show high seropositivity across endemic regions [61]. B cells were found to play a lesser role in protecting against B. pseudomallei as evident from experiments conducted on B cell-deficient (μMT) mice. Nevertheless, it is now clear that μMT mice still produce B cells that could produce other isotypes, raising doubt of using this model to investigate the role of B cells [62–66]. Moreover, immunized mice which produced a high IgG level after lethal challenge had a survival rate of >80% after 40 days [67], suggestive of a protective role of B cells in experimental B. pseudomallei infections. Here, we showed an increase in B cells expressing PD-1 in the SCV-infected and WT-infected, compared with uninfected mice. This suggests that B. pseudomallei could upregulate PD-1 on B cells to limit optimal B cell functions, which likely affect antibody production, and their interaction with follicular Th cells (Tfh) however, may require more investigations.

NK cells are a unique population, as many studies have demonstrated that NK cells capture hallmarks of adaptive immunity including antigen specificity and memory responses [68,69]. In addition, NK cells can be functionally exhausted similar to T cells during chronic diseases [70,71]. NK cells have been identified as the major producer of interferon-γ (IFN-γ) in experimental and human melioidosis [66,72]. In experimental melioidosis, NK cell-derived IFN-γ showed functional redundancy with IFN-γ released by other immune cells in the first two days of infection [66]. Nevertheless, NK cells could still be playing an essential protective role over prolonged periods of B. pseudomallei infections [66]. Accordingly, our results demonstrated that persistent B. pseudomallei infections can lead to an increase in NK cell frequency regardless of bacterial morphotype differences. In this study, SCV-infected mice had a higher NK cell frequency as compared with WT-infected mice. Interestingly, only SCV-infected mice showed a higher percentage of PD-1+ NK cells, suggesting NK cell exhaustion. WT-infected mice had a lower frequency of CTLA-4+ NK cells relative to SCV-infected and uninfected mice. However, the role of differential expressions of PD-1 and CTLA-4 on the regulation of NK cell activities warrants further investigation.

Several findings have indicated that monocytes could possibly play a role in B. pseudomallei infections [73–75]. A recent study on human primary monocytes demonstrated that B. pseudomallei infections stimulated IL-23 production in these cells [75]. Interestingly, in our earlier study, it was demonstrated that persistent infections with SCV B. pseudomallei led to an increase in plasma IL-17A. Briefly, IL-23 is essential for inducing the production of IL-17,as well as expanding and stabilizing Th17 cells [76]. Thus, activated monocytes could be the major source of IL-23 in maintaining Th17 cells during persistent B. pseudomallei infections. Moreover, high mRNA expression of inflammatory genes in monocytes positively correlated with mortality in patients with sepsis due to B. pseudomallei [74]. Our findings affirmed that SCV-infected mice had a higher PD-1 expression on monocytes compared with WT-infected and uninfected mice. Strikingly, SCV-infected and WT-infected mice had a lower frequency of monocytes expressing CTLA-4 relative to uninfected mice, with the SCV resulting in a lower CTLA-4 expression on monocytes compared with the WT. The observed changes in PD-1 and CTLA-4 expression on monocyte functions would be an interesting future consideration.

In this study, we were not able to use definitive approaches including CD19, CD56, CD14/CD16 to identify B cells, NK cells, and monocytes. However, the markers used in the current study were based on previous studies conducted on HIV infection [52,53]. In addition, we were only able to characterize the expression of PD-1 and CTLA-4 levels on B cells, NK cells, and monocytes without dissecting the functional role of these molecules. Nevertheless, we demonstrated for the first time that persistent B. pseudomallei infections with SCVs can concurrently lead to PD-1 expression on B cells, NK cells, and monocytes in mice, clearly suggesting host immune exhaustion. Remarkably, SCVs caused a higher PD-1 upregulation on NK cells and monocytes compared with WT. Together with our previous work, we could conclude that SCVs caused PD-1 upregulation on adaptive (T and B cells) and innate immune cells (NK cells or monocytes), while WT caused PD-1 upregulation only on adaptive immune cells. These observations might be due to the more efficient ability of SCVs in causing host immune exhaustion, or in causing a greater pathology as compared with WT. We speculate that SCVs initiate a higher expression of PD-1 to suppress the host immune responses and facilitate their persistence, causing increased bacterial burden. Interestingly, SCVs and WT were shown to cause CTLA-4 downregulation on NK cells and monocytes. It is unclear whether PD-1 upregulation and/or CTLA-4 downregulation are playing the dominant role over the functions of these immune cells. Future studies should be aimed to investigate the functional role of PD-1 and CTLA-4 on various immune cells in B. pseudomallei infections using murine knockout models and checkpoint inhibitors using in vivo experiments.

References

  1. 1. Cheng AC, Currie BJ (2005) Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev 18: 383–416. pmid:15831829
  2. 2. Limmathurotsakul D, Golding N, Dance DAB, Messina JP, Pigott DM, et al. (2016) Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nature Microbiology 1: 15008.
  3. 3. Wiersinga WJ, Currie BJ, Peacock SJ (2012) Melioidosis. N Engl J Med 367: 1035–1044. pmid:22970946
  4. 4. Limmathurotsakul D, Peacock SJ (2011) Melioidosis: a clinical overview. Br Med Bull 99: 125–139. pmid:21558159
  5. 5. Padiglione A, Ferris N, Fuller A, Spelman D (1998) Brain abscesses caused by Burkholderia pseudomallei. J Infect 36: 335–337. pmid:9661950
  6. 6. Mukhopadhyay A, Lee KH, Tambyah PA (2004) Bacteraemic melioidosis pneumonia: impact on outcome, clinical and radiological features. Journal of Infection 48: 334–338. pmid:15066335
  7. 7. Koponen MA, Zlock D, Palmer DL, Merlin TL (1991) Melioidosis. Forgotten, but not gone! Arch Intern Med 151: 605–608. pmid:2001144
  8. 8. Mays EE, Ricketts EA (1975) Melioidosis: recrudescence associated with bronchogenic carcinoma twenty-six years following initial geographic exposure. Chest 68: 261–263. pmid:1149556
  9. 9. Ngauy V, Lemeshev Y, Sadkowski L, Crawford G (2005) Cutaneous melioidosis in a man who was taken as a prisoner of war by the Japanese during World War II. J Clin Microbiol 43: 970–972. pmid:15695721
  10. 10. Currie BJ, Fisher DA, Anstey NM, Jacups SP (2000) Melioidosis: acute and chronic disease, relapse and re-activation. Trans R Soc Trop Med Hyg 94: 301–304. pmid:10975006
  11. 11. Kahl B, Herrmann M, Everding AS, Koch HG, Becker K, et al. (1998) Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis. J Infect Dis 177: 1023–1029. pmid:9534977
  12. 12. Proctor RA, van Langevelde P, Kristjansson M, Maslow JN, Arbeit RD (1995) Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus. Clin Infect Dis 20: 95–102. pmid:7727677
  13. 13. von Eiff C, Bettin D, Proctor RA, Rolauffs B, Lindner N, et al. (1997) Recovery of small colony variants of Staphylococcus aureus following gentamicin bead placement for osteomyelitis. Clin Infect Dis 25: 1250–1251. pmid:9402396
  14. 14. Spanu T, Romano L, D'Inzeo T, Masucci L, Albanese A, et al. (2005) Recurrent ventriculoperitoneal shunt infection caused by small-colony variants of Staphylococcus aureus. Clin Infect Dis 41: e48–52. pmid:16080075
  15. 15. Abele-Horn M, Schupfner B, Emmerling P, Waldner H, Goring H (2000) Persistent wound infection after herniotomy associated with small-colony variants of Staphylococcus aureus. Infection 28: 53–54. pmid:10697795
  16. 16. Proctor RA, von Eiff C, Kahl BC, Becker K, McNamara P, et al. (2006) Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 4: 295–305. pmid:16541137
  17. 17. Proctor RA, von Humboldt A (1998) Bacterial energetics and antimicrobial resistance. Drug Resist Updat 1: 227–235. pmid:16904405
  18. 18. Haussler S, Rohde M, Steinmetz I (1999) Highly resistant Burkholderia pseudomallei small colony variants isolated in vitro and in experimental melioidosis. Med Microbiol Immunol 188: 91–97. pmid:10753061
  19. 19. Chantratita N, Wuthiekanun V, Boonbumrung K, Tiyawisutsri R, Vesaratchavest M, et al. (2007) Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei. J Bacteriol 189: 807–817. pmid:17114252
  20. 20. Riley JL (2009) PD-1 signaling in primary T cells. Immunol Rev 229: 114–125. pmid:19426218
  21. 21. Keir ME, Butte MJ, Freeman GJ, Sharpe AH (2008) PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 26: 677–704. pmid:18173375
  22. 22. Yi JS, Cox MA, Zajac AJ (2010) T-cell exhaustion: characteristics, causes and conversion. Immunology 129: 474–481. pmid:20201977
  23. 23. Wherry EJ, Ha SJ, Kaech SM, Haining WN, Sarkar S, et al. (2007) Molecular signature of CD8+ T cell exhaustion during chronic viral infection. Immunity 27: 670–684. pmid:17950003
  24. 24. Barathan M, Gopal K, Mohamed R, Ellegard R, Saeidi A, et al. (2015) Chronic hepatitis C virus infection triggers spontaneous differential expression of biosignatures associated with T cell exhaustion and apoptosis signaling in peripheral blood mononucleocytes. Apoptosis 20: 466–480. pmid:25577277
  25. 25. Velu V, Shetty RD, Larsson M, Shankar EM (2015) Role of PD-1 co-inhibitory pathway in HIV infection and potential therapeutic options. Retrovirology 12: 14. pmid:25756928
  26. 26. Jurado JO, Alvarez IB, Pasquinelli V, Martinez GJ, Quiroga MF, et al. (2008) Programmed death (PD)-1:PD-ligand 1/PD-ligand 2 pathway inhibits T cell effector functions during human tuberculosis. J Immunol 181: 116–125. pmid:18566376
  27. 27. Singh A, Mohan A, Dey AB, Mitra DK (2013) Inhibiting the programmed death 1 pathway rescues Mycobacterium tuberculosis-specific interferon gamma-producing T cells from apoptosis in patients with pulmonary tuberculosis. J Infect Dis 208: 603–615. pmid:23661793
  28. 28. Wu YY, Lin CW, Cheng KS, Lin C, Wang YM, et al. (2010) Increased programmed death-ligand-1 expression in human gastric epithelial cells in Helicobacter pylori infection. Clin Exp Immunol 161: 551–559. pmid:20646001
  29. 29. Beswick EJ, Pinchuk IV, Das S, Powell DW, Reyes VE (2007) Expression of the programmed death ligand 1, B7-H1, on gastric epithelial cells after Helicobacter pylori exposure promotes development of CD4+ CD25+ FoxP3+ regulatory T cells. Infect Immun 75: 4334–4341. pmid:17562772
  30. 30. Das S, Suarez G, Beswick EJ, Sierra JC, Graham DY, et al. (2006) Expression of B7-H1 on gastric epithelial cells: Its potential role in regulating T cells during Helicobacter pylori infection. Journal of Immunology 176: 3000–3009.
  31. 31. Buddhisa S, Rinchai D, Ato M, Bancroft GJ, Lertmemongkolchai G (2015) Programmed death ligand 1 on Burkholderia pseudomallei-infected human polymorphonuclear neutrophils impairs T cell functions. J Immunol 194: 4413–4421. pmid:25801435
  32. 32. Thibult ML, Mamessier E, Gertner-Dardenne J, Pastor S, Just-Landi S, et al. (2013) PD-1 is a novel regulator of human B-cell activation. International Immunology 25: 129-+. pmid:23087177
  33. 33. Benson DM, Bakan CE, Mishra A, Hofmeister CC, Efebera Y, et al. (2010) The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-011, a novel monoclonal anti-PD-1 antibody. Blood 116: 2286–2294. pmid:20460501
  34. 34. Alvarez IB, Pasquinelli V, Jurado JO, Abbate E, Musella RM, et al. (2010) Role Played by the Programmed Death-1-Programmed Death Ligand Pathway during Innate Immunity against Mycobacterium tuberculosis. Journal of Infectious Diseases 202: 524–532. pmid:20617899
  35. 35. Okazaki T, Maeda A, Nishimura H, Kurosaki T, Honjo T (2001) PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine. Proc Natl Acad Sci U S A 98: 13866–13871. pmid:11698646
  36. 36. Qorraj M, Bruns H, Bottcher M, Weigand L, Saul D, et al. (2016) The PD-1/PD-L1 axis contributes to immune metabolic dysfunctions of monocytes in chronic lymphocytic leukemia. Leukemia.
  37. 37. Huang X, Venet F, Wang YL, Lepape A, Yuan Z, et al. (2009) PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis. Proc Natl Acad Sci U S A 106: 6303–6308. pmid:19332785
  38. 38. Larsson M, Shankar EM, Che KF, Saeidi A, Ellegard R, et al. (2013) Molecular signatures of T-cell inhibition in HIV-1 infection. Retrovirology 10: 31. pmid:23514593
  39. 39. Sharpe AH, Freeman GJ (2002) The B7-CD28 superfamily. Nat Rev Immunol 2: 116–126. pmid:11910893
  40. 40. Ye B, Liu X, Li X, Kong H, Tian L, et al. (2015) T-cell exhaustion in chronic hepatitis B infection: current knowledge and clinical significance. Cell Death Dis 6: e1694. pmid:25789969
  41. 41. Anderson KM, Czinn SJ, Redline RW, Blanchard TG (2006) Induction of CTLA-4-mediated anergy contributes to persistent colonization in the murine model of gastric Helicobacter pylori infection. Journal of Immunology 176: 5306–5313.
  42. 42. Quandt D, Hoff H, Rudolph M, Fillatreau S, Brunner-Weinzierl MC (2007) A new role of CTLA-4 on B cells in thymus-dependent immune responses in vivo. Journal of Immunology 179: 7316–7324.
  43. 43. Pioli C, Gatta L, Ubaldi V, Doria G (2000) Inhibition of IgG1 and IgE production by stimulation of the B cell CTLA-4 receptor. J Immunol 165: 5530–5536. pmid:11067906
  44. 44. Laurent S, Carrega P, Saverino D, Piccioli P, Camoriano M, et al. (2010) CTLA-4 is expressed by human monocyte-derived dendritic cells and regulates their functions. Human Immunology 71: 934–941. pmid:20650297
  45. 45. Stojanovic A, Fiegler N, Brunner-Weinzierl M, Cerwenka A (2014) CTLA-4 Is Expressed by Activated Mouse NK Cells and Inhibits NK Cell IFN-gamma Production in Response to Mature Dendritic Cells. Journal of Immunology 192: 4184–4191.
  46. 46. See JX, Samudi C, Saeidi A, Menon N, Choh LC, et al. (2016) Experimental Persistent Infection of BALB/c Mice with Small-Colony Variants of Burkholderia pseudomallei Leads to Concurrent Upregulation of PD-1 on T Cells and Skewed Th1 and Th17 Responses. PLoS Negl Trop Dis 10: e0004503. pmid:26974441
  47. 47. Ramli NS, Eng Guan C, Nathan S, Vadivelu J (2012) The effect of environmental conditions on biofilm formation of Burkholderia pseudomallei clinical isolates. PLoS One 7: e44104. pmid:22970167
  48. 48. Suppiah J, Thimma JS, Cheah SH, Vadivelu J (2010) Development and evaluation of polymerase chain reaction assay to detect Burkholderia genus and to differentiate the species in clinical specimens. FEMS Microbiol Lett 306: 9–14. pmid:20345378
  49. 49. Goodyear A, Bielefeldt-Ohmann H, Schweizer H, Dow S (2012) Persistent gastric colonization with Burkholderia pseudomallei and dissemination from the gastrointestinal tract following mucosal inoculation of mice. PLoS One 7: e37324. pmid:22624016
  50. 50. Shankar EM, Che KF, Messmer D, Lifson JD, Larsson M (2011) Expression of a broad array of negative costimulatory molecules and Blimp-1 in T cells following priming by HIV-1 pulsed dendritic cells. Mol Med 17: 229–240. pmid:21103670
  51. 51. Che KF, Shankar EM, Muthu S, Zandi S, Sigvardsson M, et al. (2012) p38 Mitogen-activated protein kinase/signal transducer and activator of transcription-3 pathway signaling regulates expression of inhibitory molecules in T cells activated by HIV-1-exposed dendritic cells. Mol Med 18: 1169–1182. pmid:22777388
  52. 52. Ahmad F, Hong HS, Jackel M, Jablonka A, Lu IN, et al. (2014) High frequencies of polyfunctional CD8+ NK cells in chronic HIV-1 infection are associated with slower disease progression. J Virol 88: 12397–12408. pmid:25122796
  53. 53. Filion LG, Izaguirre CA, Garber GE, Huebsh L, Aye MT (1990) Detection of surface and cytoplasmic CD4 on blood monocytes from normal and HIV-1 infected individuals. J Immunol Methods 135: 59–69. pmid:1703191
  54. 54. Ashdown LR (1979) An improved screening technique for isolation of Pseudomonas pseudomallei from clinical specimens. Pathology 11: 293–297. pmid:460953
  55. 55. Al-Maleki AR, Mariappan V, Vellasamy KM, Tay ST, Vadivelu J (2015) Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells. PLoS One 10: e0127398. pmid:25996927
  56. 56. Al-Maleki AR, Mariappan V, Vellasamy KM, Shankar EM, Tay ST, et al. (2014) Enhanced intracellular survival and epithelial cell adherence abilities of Burkholderia pseudomallei morphotypes are dependent on differential expression of virulence-associated proteins during mid-logarithmic growth phase. J Proteomics 106: 205–220. pmid:24742602
  57. 57. Tuchscherr L, Heitmann V, Hussain M, Viemann D, Roth J, et al. (2010) Staphylococcus aureus small-colony variants are adapted phenotypes for intracellular persistence. J Infect Dis 202: 1031–1040. pmid:20715929
  58. 58. Conejero L, Patel N, de Reynal M, Oberdorf S, Prior J, et al. (2011) Low-dose exposure of C57BL/6 mice to burkholderia pseudomallei mimics chronic human melioidosis. Am J Pathol 179: 270–280. pmid:21703409
  59. 59. Currie BJ, Ward L, Cheng AC (2010) The epidemiology and clinical spectrum of melioidosis: 540 cases from the 20 year Darwin prospective study. PLoS Negl Trop Dis 4: e900. pmid:21152057
  60. 60. Dietrich J, Roy S, Rosenkrands I, Lindenstrom T, Filskov J, et al. (2015) Differential influence of nutrient-starved Mycobacterium tuberculosis on adaptive immunity results in progressive tuberculosis disease and pathology. Infect Immun 83: 4731–4739. pmid:26416911
  61. 61. Cheng AC, Wuthiekanun V, Limmathurotsakul D, Chierakul W, Peacock SJ (2008) Intensity of exposure and incidence of melioidosis in Thai children. Trans R Soc Trop Med Hyg 102 Suppl 1: S37–39.
  62. 62. Macpherson AJ, Lamarre A, McCoy K, Harriman GR, Odermatt B, et al. (2001) IgA production without mu or delta chain expression in developing B cells. Nat Immunol 2: 625–631. pmid:11429547
  63. 63. Ghosh S, Hoselton SA, Schuh JM (2012) mu-chain-deficient mice possess B-1 cells and produce IgG and IgE, but not IgA, following systemic sensitization and inhalational challenge in a fungal asthma model. J Immunol 189: 1322–1329. pmid:22732592
  64. 64. Perona-Wright G, Mohrs K, Taylor J, Zaph C, Artis D, et al. (2008) Cutting edge: Helminth infection induces IgE in the absence of mu- or delta-chain expression. J Immunol 181: 6697–6701. pmid:18981085
  65. 65. Melamed D, Miri E, Leider N, Nemazee D (2000) Unexpected autoantibody production in membrane Ig-mu-deficient/lpr mice. J Immunol 165: 4353–4358. pmid:11035071
  66. 66. Haque A, Easton A, Smith D, O'Garra A, Van Rooijen N, et al. (2006) Role of T cells in innate and adaptive immunity against murine Burkholderia pseudomallei infection. J Infect Dis 193: 370–379. pmid:16388484
  67. 67. Barnes JL, Ketheesan N (2007) Development of protective immunity in a murine model of melioidosis is influenced by the source of Burkholderia pseudomallei antigens. Immunol Cell Biol 85: 551–557. pmid:17563759
  68. 68. Sun JC, Lopez-Verges S, Kim CC, DeRisi JL, Lanier LL (2011) NK cells and immune "memory". J Immunol 186: 1891–1897. pmid:21289313
  69. 69. Cerwenka A, Lanier LL (2016) Natural killer cell memory in infection, inflammation and cancer. Nat Rev Immunol 16: 112–123. pmid:26806484
  70. 70. da Silva IP, Gallois A, Jimenez-Baranda S, Khan S, Anderson AC, et al. (2014) Reversal of NK-cell exhaustion in advanced melanoma by Tim-3 blockade. Cancer Immunol Res 2: 410–422. pmid:24795354
  71. 71. Gill S, Vasey AE, De Souza A, Baker J, Smith AT, et al. (2012) Rapid development of exhaustion and down-regulation of eomesodermin limit the antitumor activity of adoptively transferred murine natural killer cells. Blood 119: 5758–5768. pmid:22544698
  72. 72. Jenjaroen K, Chumseng S, Sumonwiriya M, Ariyaprasert P, Chantratita N, et al. (2015) T-Cell Responses Are Associated with Survival in Acute Melioidosis Patients. PLoS Negl Trop Dis 9: e0004152. pmid:26495852
  73. 73. Morris J, Williams N, Rush C, Govan B, Sangla K, et al. (2012) Burkholderia pseudomallei triggers altered inflammatory profiles in a whole-blood model of type 2 diabetes-melioidosis comorbidity. Infect Immun 80: 2089–2099. pmid:22473609
  74. 74. Wiersinga WJ, Dessing MC, Kager PA, Cheng AC, Limmathurotsakul D, et al. (2007) High-throughput mRNA profiling characterizes the expression of inflammatory molecules in sepsis caused by Burkholderia pseudomallei. Infect Immun 75: 3074–3079. pmid:17371859
  75. 75. Kulsantiwong P, Pudla M, Boondit J, Wikraiphat C, Dunachie SJ, et al. (2016) Burkholderia pseudomallei induces IL-23 production in primary human monocytes. Med Microbiol Immunol 205: 255–260. pmid:26563410
  76. 76. Korn T, Bettelli E, Oukka M, Kuchroo VK (2009) IL-17 and Th17 Cells. Annu Rev Immunol 27: 485–517. pmid:19132915