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Immune profiling of the progression of a BALB/c mouse aerosol infection by Burkholderia pseudomallei and the therapeutic implications of targeting HMGB1

Open AccessPublished:September 07, 2015DOI:https://doi.org/10.1016/j.ijid.2015.09.003

      Highlights

      • Burkholderia pseudomallei infection by an airborne route was modelled in mice, and groups were culled for further analysis as the infection progressed.
      • We found differences in the cytokines released by the different infected organs.
      • We found that the damage-associated molecular pattern HMGB1 was released during infection.
      • HMGB1 was removed from mice using an antibody during a B. pseudomallei infection.
      • HMGB1 knockdown reduced the expression of some pro-inflammatory cytokines and reduced bacterial load, but did not increase survival measurably.

      Summary

      Introduction

      The role of damage-associated molecular pattern HMGB1 signalling in a murine BALB/c model of severe respiratory melioidosis (Burkholderia pseudomallei infection) was explored in this study.

      Methods

      Time course experiments were performed.

      Results

      It was established that HMGB1 was released in concert with increasing weight of organs and increasing concentration of liver enzymes in the blood a short time after cytokine release. Differences in the cytokine response between organs were observed, where the lungs contained higher concentrations of chemokines and interleukin 17, while the spleen produced more interferon-gamma, which is essential in the host defence against B. pseudomallei. This is evidence as to why the disease is seemingly more severe in the respiratory form. The effect of depleting HMGB1 using an antibody was also evaluated. It was found that this treatment significantly reduced bacterial load in the liver, spleen, and, to a greater degree, in the lungs. Cytokine quantification indicated that this reduction in bacterial load is likely due to the treatment reducing the release of a variety of pro-inflammatory cytokines.

      Conclusion

      It is concluded that anti-HMGB1 treatment would be effective alongside other therapeutics, where it would reduce the characteristic over-inflammation associated with late stage infection.

      Keywords

      1. Introduction

      The Gram-negative bacterial species Burkholderia pseudomallei is the causative agent of melioidosis. Melioidosis is one of the leading causes of septicaemia in certain Southeast Asian countries.
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      From the environment, B. pseudomallei can enter the host via different routes, and the most common routes are believed to be through the drinking of contaminated water and through cuts and abrasions of the skin.
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      It is also recognized that B. pseudomallei can cause particularly unpleasant primary and secondary pneumonia,
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      Melioidosis can be modelled in the laboratory in mice.
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      • et al.
      Low-dose exposure of C57BL/6 mice to Burkholderia pseudomallei mimics chronic human melioidosis.
      One complication of melioidosis is that B. pseudomallei has intrinsic tolerance to a variety of antibiotic drugs, making treatment complex and difficult,
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      Treatment and prophylaxis of melioidosis.
      and this indicates the requirement for novel therapeutics to treat the disease.
      When considering the immune response to B. pseudomallei, much murine work has been performed to understand the infectious process, and some of this has been supported by clinical studies. The inability of the immune response to control infection in two specific ways results in sepsis and death. Firstly, it is known that neutrophils are important in limiting the morbidity of the disease. Murine neutrophils can be observed taking up the bacteria in in vivo models of respiratory disease,
      • Laws T.R.
      • Smither S.J.
      • Lukaszewski R.A.
      • Atkins H.S.
      Neutrophils are the predominant cell-type to associate with Burkholderia pseudomallei in a BALB/c mouse model of respiratory melioidosis.
      mice that have had neutrophils ablated are hyper-sensitive to infection by B. pseudomallei,
      • Easton A.
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      • Chu K.
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      • Bancroft G.J.
      A critical role for neutrophils in resistance to experimental infection with Burkholderia pseudomallei.
      and human melioidosis is often linked with disorders that impair neutrophil function.
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      • Lertmemongkolchai G.
      Effect of host factors on neutrophil functions in response to Burkholderia pseudomallei in healthy Thai subjects.
      However, neutrophils are not a ubiquitously beneficial cell and it has been demonstrated that the activity of neutrophils can contribute to the severe damage associated with disease.
      • Sahoo M.
      • Del Barrio L.
      • Miller M.A.
      • Re F.
      Neutrophil elastase causes tissue damage that decreases host tolerance to lung infection with Burkholderia species.
      Interferon-gamma (IFN-γ) signalling is also known to be essential in the control of B. pseudomallei. A plethora of work has been done to understand the cytokine response,
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      • Tan G.
      • et al.
      Caspase-1 mediates resistance in murine melioidosis.
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      • et al.
      Endogenous interleukin-18 improves the early antimicrobial host response in severe melioidosis.
      • Propst K.L.
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      • Schweizer H.P.
      • Dow S.W.
      Immunotherapy markedly increases the effectiveness of antimicrobial therapy for treatment of Burkholderia pseudomallei infection.
      • Koo G.C.
      • Gan Y.H.
      The innate interferon gamma response of BALB/c and C57BL/6 mice to in vitro Burkholderia pseudomallei infection.
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      • Lertmemongkolchai G.
      • et al.
      Role of T cells in innate and adaptive immunity against murine Burkholderia pseudomallei infection.
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      • Cai G.
      • Hunter C.A.
      • Bancroft G.J.
      Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens.
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      • Dance D.A.
      • Drasar B.S.
      • Bancroft G.J.
      Obligatory role of gamma interferon for host survival in a murine model of infection with Burkholderia pseudomallei.
      which has resulted in an understanding that B. pseudomallei seems to prevent pyroptosis, therefore reducing IFN-γ, interleukin (IL)-12, and IL-18 secretion; this results in increased IL-1 signalling, which is likely to contribute to the sepsis.
      • Ceballos-Olvera I.
      • Sahoo M.
      • Miller M.A.
      • Del Barrio L.
      • Re F.
      Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1beta is deleterious.
      The role of damage-associated molecular pattern (DAMP) signalling is diverse and has started to be elucidated.
      • Piccinini A.M.
      • Midwood K.S.
      DAMPening inflammation by modulating TLR signalling.
      Specifically, there is a growing body of work aimed at understanding the role of high-mobility group B protein 1 (HMGB1) signalling in a variety of inflammatory conditions.
      • Kang R.
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      • Zhang Q.
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      • Wu S.
      • Cao L.
      • et al.
      HMGB1 in health and disease.
      Until recently, little was known about whether DAMP signalling may contribute to the disease process of melioidosis. Charoensup et al. first established that increased levels of HMGB1 in human melioidosis patients correlated with a poor prognosis and secondly explored the therapeutic potential of targeting HMGB1 in a murine model of melioidosis.
      • Charoensup J.
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      • Paeyao A.
      • Promakhejohn S.
      • Punasee S.
      • Chularari C.
      • et al.
      High HMGB1 level is associated with poor outcome of septicemic melioidosis.
      They found that knockdown of HMGB1 had no effect on survival independent of other treatments, but had a modest effect when administered in combination with ceftazidime.
      Our study group also has an interest in HMGB1 as a target to treat infectious disease; in a previous study, it was found that HMGB1 had therapeutic benefit when used against infection by the bacterium Francisella tularensis.
      • D’Elia R.V.
      • Laws T.R.
      • Carter A.
      • Lukaszewski R.
      • Clark G.C.
      Targeting the “Rising DAMP” during a Francisella tularensis infection.
      In the present study, the therapeutic potential of anti-HMGB1 was investigated in a mouse model of melioidosis in which BALB/c mice were infected by aerosol route.

      2. Materials and methods

      2.1 Mice

      Six to eight-week-old female BALB/c mice (Charles River, UK) were transferred to a high-containment class III rigid isolator, where they were assigned randomly to groups and given unlimited access to food and water. Mice were challenged with B. pseudomallei strain K96243 by aerosol, as described previously, using a Henderson-type apparatus
      • Druett H.A.
      A mobile form of the Henderson apparatus.
      and a Collison nebulizer.
      • May K.R.
      • Harper G.J.
      The efficiency of various liquid impinger samplers in bacterial aerosols.
      Bacteria were grown in Luria broth at 37 °C on a rotary platform. Mice were checked twice daily and scored for clinical signs. Mice were culled at predetermined humane endpoints. Survival times were recorded for some mice, and others were culled for analysis of tissues at different time points. All procedures and housing were in accordance with the United Kingdom Animal (Scientific Procedures) Act (1986). Shinotest anti-HMGB1 and isotype control IgY were obtained from Oxford Biosystems Cadama (UK). A 600-μg dose of either antibody was administered by intraperitoneal route at 24 h and 48 h post-infection to the appropriate mice.

      2.2 Post-mortem analysis

      Whole blood was micro-centrifuged at 7000 rpm for 20 min, and the serum was removed and stored at −80 °C. Serum HMGB1 was measured with an ELISA kit (Shinotest, supplied through Oxford Biosystems Cadama, UK) in accordance with the manufacturer's instructions. Serum was also tested using a ‘dry-slide’ technology biochemistry analyser (VetTest; IDEXX Laboratories) in accordance with the manufacturer's instructions.
      The lung, liver, spleen, and blood were all processed at less than 1 h post-mortem. All organs were weighed and collected in 2 ml phosphate buffered saline (PBS) and then disrupted through a 40-μm cell sieve, and the resulting homogenate was collected. Subsequently, 100-μl aliquots of the cell suspension were used for enumeration of bacteria on agar following serial dilution in PBS and plating out on Luria agar. For cytokine analysis, 200-μl aliquots of cell suspension were centrifuged for 5 min at 2000 rpm. Supernatants were removed for cytokine analysis and stored at −80 °C. The levels of cytokine were measured via 23-plex murine Luminex array (Bio-Rad), used in accordence with the manufacturer's instructions. In addition, a magnetic plate washer (Bio-Rad) was used for wash steps and samples were ultimately fixed using 4% paraformaldehyde in PBS for at least 24 h at 4 °C.

      2.3 Statistical analysis

      Graphs were constructed using the program GraphPad PRISM v. 6.0. All statistical analyses were performed using IBM SPSS v. 21.0 software. For analysis, all data (with the exception of the organ weight data) were transformed by the logarithm of 10 to better fit the normal distribution. Data were analysed by general linear model (GLM), and validity of the test was established by Levene's tests for unequal variance. Data points were taken from multiple experiments and for this reason the experiment with which each data point was taken is included in the GLM as an additional factor. Individual comparisons between groups were performed by Bonferroni post-tests.

      3. Results

      3.1 HMGB1 release occurs with organ damage in a murine model of melioidosis

      To study the pathogenesis of the infection in mice, two time course experiments were performed, in which groups of five mice were culled prior to infection (as a baseline), and at 24, 48, and 60 h post infection with estimated doses of 75 and 217 colony-forming units (CFU) delivered by aerosol route.
      As the disease progressed, bacterial numbers increased in all organs (p < 0.001, using a multivariate GLM; Figure 1A) . At the same time as the increase in bacterial load, significant increases in organ mass of the lung and spleen were observed (both p < 0.001, using a multivariate GLM) and a significant reduction in overall mouse weight (p < 0.001, using a GLM) (Figure 1B). Moreover organ weights increased significantly when compared to naïve mice from the 48 h time point in the lung and 60 h time point in the spleen (p < 0.001, using Bonferroni post-tests). Mouse total body weight was significantly lower at 60 h post infection when compared to the control mice (p < 0.001, using Bonferroni post-tests). Serum was analysed using a blood chemistry analysis system. During infection, blood glucose decreased significantly (p < 0.001, using GLM, Figure 1C) to levels below the level of naïve mice at 48 h and 60 h (p < 0.001, in both cases using Bonferroni post-tests). Significant increases in the concentration of the liver enzymes alanine aminotransferase and aspartate aminotransferase were also observed as the infection progressed (both p < 0.001, using a multivariate GLM; Figure 1D). The level of these enzymes had risen significantly over background at 48 h and 60 h (p < 0.05, in all four cases, using Bonferroni post-tests). Finally, HMGB1 levels were also found to have increased significantly post infection (p < 0.001 using GLM; Figure 1E), rising by several fold after infection to levels significantly higher than background at 48 h and 60 h (p < 0.05, for both cases, using Bonferroni post-tests).
      Figure thumbnail gr1
      Figure 1Organ bacterial load, organ weight, blood glucose, blood liver enzyme, and HMGB1 in BALB/c mice at time points post infection with Burkholderia pseudomallei strain K96243 by the aerosol route. Data from two experiments are shown. Groups of five mice were culled before, or 24 h, 48 h or 60 h post infection. Estimated challenge doses for the two experiments were 75 and 217 CFU. Values for each mouse are shown with symbols and the median values are included as lines. Panel A shows organ weights (lung, liver, spleen, and whole mouse). Panel B shows the bacterial load in CFU/organ (lung, liver, and spleen). Panel C shows blood serum glucose. Panel D shows liver enzyme concentrations in the blood serum (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)). Panel E shows blood serum HMGB1 concentrations. Significance markers are indicative of Bonferroni post-test comparisons to naïve mice (*p < 0.05, **p < 0.01, ***p < 0.001).
      Luminex technology was used explore the cytokine response within the mouse organs at each time point post infection (representative results are shown in Figure 2A) . Furthermore, these data were analysed by multivariate GLM to show that the level of different cytokines changed as the infection developed in different organs. It was observed that some cytokine concentrations were significantly altered in one organ, but not in another. For example, significant changes in IFN-γ expression were observed in the liver and spleen (p < 0.001, using GLM), but not in the lung (p > 0.10, using GLM). In order to better understand the differences in cytokine dynamics between organs, the induction of cytokine (relative to baseline, naïve mice) was plotted against the bacterial load at all time points (Figure 2B). Here the bacterial load is regarded as a crude measure of antigen present to show how the different organs respond relative to each other. For IL-1α, IL-6, IL-12p40, IL-12p70, IL-17, granulocyte/monocyte colony stimulating factor (GM-CSF) chemokine, C–C motif ligand (CCL)4, and tumour necrosis factor alpha (TNF-α), induction of these cytokines relative to bacterial load was very similar between organs. This was not true of IL-1β, IL-4, IL-10, IFN-γ, chemokine, C–X–C motif ligand (CXCL)1, CCL2, CCL3, and CCL5. Seemingly, and relative to the infectious load, release of these cytokines is reduced in the lung.
      Figure thumbnail gr2a
      Figure 2Cytokine release at time points post infection with Burkholderia pseudomallei strain K96243 by the aerosol route. Data from two experiments are shown. Groups of five mice were culled before, or 24 h, 48 h or 60 h post infection. Estimated challenge doses for the two experiments were 75 and 217 CFU. Panel A shows the mean cytokine/bacterial concentration across all 10 mice per time point. The error bars are indicative of the 95% confidence intervals. The error bars are indicative of the 95% confidence intervals after the variation associated with experimental run had been removed using general linear model (GLM). Significance markers are indicative of multivariate GLM analysis to indicate whether cytokine levels are changing between groups (*p < 0.05, **p < 0.01, ***p < 0.001). Panel B shows the fold induction of cytokine (compared to naïve mice) relative to bacterial load, at each time point post infection (24, 48, and 60 h). Each data point is the mean of the 10 replicates. The circles are the lung, the squares the liver, and the crosses the spleen.
      Figure thumbnail gr2b
      Figure 2Cytokine release at time points post infection with Burkholderia pseudomallei strain K96243 by the aerosol route. Data from two experiments are shown. Groups of five mice were culled before, or 24 h, 48 h or 60 h post infection. Estimated challenge doses for the two experiments were 75 and 217 CFU. Panel A shows the mean cytokine/bacterial concentration across all 10 mice per time point. The error bars are indicative of the 95% confidence intervals. The error bars are indicative of the 95% confidence intervals after the variation associated with experimental run had been removed using general linear model (GLM). Significance markers are indicative of multivariate GLM analysis to indicate whether cytokine levels are changing between groups (*p < 0.05, **p < 0.01, ***p < 0.001). Panel B shows the fold induction of cytokine (compared to naïve mice) relative to bacterial load, at each time point post infection (24, 48, and 60 h). Each data point is the mean of the 10 replicates. The circles are the lung, the squares the liver, and the crosses the spleen.

      3.2 Anti-HMGB1 therapy reduces bacterial burden and alters the cytokine profile during a B. pseudomallei infection in mice

      The increase in the DAMP signal HMGB1 observed in mice post infection with B. pseudomallei opened up the possibility that reduction of this inflammatory signal may have a beneficial effect. Two experiments were performed in which mice were infected with B. pseudomallei strain K96423 by the aerosol route (receiving 188 CFU/mouse in one experiment and 246 CFU in the other). Mice were treated with either anti-HMGB1 (n = 20, 10 from each experiment) or with an isotype control (n = 20, 10 from each experiment). Survival was monitored and no significant difference in time to death was observed (p = 0.119, using a log-rank test stratified by experiment; Figure 3A) . Additional mice (n = 15, five receiving 188 CFU/mouse, five receiving 246 CFU/mouse, and five receiving 104 CFU/mouse) were culled at 48 h post infection in order to assess the progression of disease.
      Figure thumbnail gr3
      Figure 3Survival and 48-h post infection organ bacterial load of BALB/c mice treated with either anti-HMGB1 or an isotype and infected with Burkholderia pseudomallei strain K96243 by the aerosol route. Chicken anti-HMGB1 (600 mg) or IgY isotype (600 mg) was delivered at 24 h and at 48 h post infection by the intraperitoneal route. Survival is shown with a Kaplan–Meier plot in panel A, and includes survival data for 20 mice in each group combining two independent experiments where mice received an estimated dose of either 188 CFU (n = 10) or 246 CFU (n = 10). Bacterial load is shown in panel B, and three independent experiments (n = 5/group in each experiment) were performed; the median bacterial concentration across all mice (line) and the count for each mouse (symbols) is shown. The estimated doses were 188, 246, and 104 CFU per mouse for each experiment.
      It was found that anti-HMGB1 treatment reduced bacterial load across all three organs investigated (p < 0.001, using a repeated measures GLM; Figure 3B); this was most evident in the lung (p < 0.001, using GLM), but was also seen in the liver and spleen (p < 0.05, using GLM). No significant difference in organ weights was observed (data not shown). To further investigate how the anti-HMGB1 treatment may have brought about the change in bacterial load, Luminex analysis was performed to measure changes in cytokine release in different organs (Figure 4). Through analysis by multivariate GLM, significant reductions in several cytokines in the HMGB1 treated group were noted when compared to the isotype control treated group. These reductions included the following: in the lung IL-4 (p < 0.001), IL-6 (p < 0.05), IL-12p70 (p < 0.01), CXCL1 (p < 0.01), CCL3 (p < 0.05), and TNF-α (p < 0.05); in the liver IL-1β (p < 0.05) and IL-12p40 (p < 0.05); in the spleen TNF-α (p < 0.01).
      Figure thumbnail gr4
      Figure 4Cytokine release 48 h post infection in BALB/c mice organs treated with either anti-HMGB1 or isotype and infected with Burkholderia pseudomallei strain K96243 by the aerosol route. Anti-HMGB1 (600 mg) or IgY isotype (600 mg) was delivered at 24 h and at 48 h post infection by the intraperitoneal route. Three independent experiments (n = 5/group in each experiment) were performed; the mean cytokine/bacterial concentration across all mice is shown. The estimated doses were 188, 246, and 104 CFU per mouse for each experiment. The error bars are indicative of the 95% confidence intervals. When the variation associated with experimental run had been removed using general linear model (GLM). The significance markers are indicative of GLM main effect estimations, where the Bonferroni correction has been applied. Filled symbols are the anti-HMGB1 treated groups and open symbols are the isotype control treated group.

      4. Discussion

      Time course experiments were performed in the BALB/c mouse model to identify whether HMGB1 is released during respiratory disease caused by B. pseudomallei. In order to monitor and quantify the growth of bacteria and their effects, three organs were selected (lung, spleen, and liver) and bacterial growth, pathology (organ weight), and cytokine release were investigated. The bacterial growth that was observed occurred more rapidly than in some previously published infection studies;
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      however, these data are very similar to those of a recent study that used a similar and more substantial inoculum of bacteria
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      and to previous work within our institute.
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      Neutrophils are the predominant cell-type to associate with Burkholderia pseudomallei in a BALB/c mouse model of respiratory melioidosis.
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      The total weight of the mice dropped during infection and this has been observed elsewhere;
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      however this is the first time that organ weight in a murine B. pseudomallei infection model has been reported. Both the lung (from 48 h) and spleen (from 60 h) showed significantly increased weights, in the face of total weight loss. As an infection effect, this might be a consequence of immune infiltrate.
      • Laws T.R.
      • Smither S.J.
      • Lukaszewski R.A.
      • Atkins H.S.
      Neutrophils are the predominant cell-type to associate with Burkholderia pseudomallei in a BALB/c mouse model of respiratory melioidosis.
      • Lever M.S.
      • Nelson M.
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      • Beedham R.J.
      • Simpson A.J.
      Experimental acute respiratory Burkholderia pseudomallei infection in BALB/c mice.
      The reason why the spleen showed a delayed weight increase is likely because it takes between 3 and 10 h for bacteria to colonize the spleen from the lung.
      • Laws T.R.
      • Smither S.J.
      • Lukaszewski R.A.
      • Atkins H.S.
      Neutrophils are the predominant cell-type to associate with Burkholderia pseudomallei in a BALB/c mouse model of respiratory melioidosis.
      No significant increase in liver mass was observed during the course of infection. This is likely due to a number of factors, but is probably due to the liver having a substantially lower density of infection (similar numbers of bacteria per gram were observed between lung, liver, and spleen; however the liver is approximately 10-fold larger). The observed increase in liver enzymes in the blood and reduction in blood glucose might be indicative of liver damage and this warrants further investigation.
      It was observed that the concentrations of many cytokines in the lung, liver, and spleen increased with the progression of disease. Many of the cytokines titrated in this study had been measured in previous studies and the present data are broadly consistent with those of previous studies.
      • Laws T.R.
      • Smither S.J.
      • Lukaszewski R.A.
      • Atkins H.S.
      Neutrophils are the predominant cell-type to associate with Burkholderia pseudomallei in a BALB/c mouse model of respiratory melioidosis.
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      • et al.
      Burkholderia pseudomallei aerosol infection results in differential inflammatory responses in BALB/c and C57Bl/6 mice.
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      The present data go beyond these datasets and demonstrate that some cytokines are more prevalent within different organs. Specifically, it appears that, relative to bacterial load, the lung releases less IL-1β, IL-4, IL-10, IFN-γ, CXCL1, CCL2, CCL3, and CCL5. A previous study by Tan et al. showed that cytokine release is different in the lung and spleen at a single time point post infection.
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      • et al.
      Burkholderia pseudomallei aerosol infection results in differential inflammatory responses in BALB/c and C57Bl/6 mice.
      In this study by Tan et al., similar differences in cytokine release were observed: more TNF-α and CCL2 were released in the lung, while, adjusted for the lower infectious load, the spleen produced more IFN-γ. It is well-known that the lung is an organ rich in anti-inflammatory signals and this is assumed to prevent unnecessary inflammation against non-self-antigens, associated with the constant air exchange;
      • Hussell T.
      • Bell T.J.
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      this might be the cause of the cytokine deficit within the lung. It is unclear whether interventions that address this cytokine deficit would be of benefit. It is also feasible that we are observing the maximal output of IL-1β, IL-4, IL-10, IFN-γ, CXCL1, CCL2, CCL3, and CCL5 per gram of tissue and that the seeming reduction of cytokine release in the lung is simply a function of the greater infectious load being unable to further increase cytokine concentrations.
      To our knowledge this is the first report to publish the dynamics of IL-17 release in a murine model of melioidosis. The IL-17 concentration has previously been measured in a time course study in blood, and no time dynamics were observed.
      • Massey S.
      • Yeager L.A.
      • Blumentritt C.A.
      • Vijayakumar S.
      • Sbrana E.
      • Peterson J.W.
      • et al.
      Comparative Burkholderia pseudomallei natural history virulence studies using an aerosol murine model of infection.
      In humans, it was reported that patients with diabetes (a major risk factor in melioidosis) had reduced IL-17 production in response to Burkholderia antigens.
      • Pongcharoen S.
      • Ritvirool P.N.
      • Sanguansermsri D.
      • Chanchan P.
      • Jienmongkol P.
      • Butkhamchot P.
      • et al.
      Reduced interleukin-17 expression of Burkholderia pseudomallei-infected peripheral blood mononuclear cells of diabetic patients.
      IL-17 release in this mouse model of infection was low.
      In agreement with Charoensup et al., it was shown that concentrations of the DAMP HMGB1 increased after infection in mice in the present study.
      • Charoensup J.
      • Sermswan R.W.
      • Paeyao A.
      • Promakhejohn S.
      • Punasee S.
      • Chularari C.
      • et al.
      High HMGB1 level is associated with poor outcome of septicemic melioidosis.
      This is likely due to the infection and the immunopathological organ damage associated with the infection, causing the release of DAMPs. This has led us to the same conclusion as Charoensup and colleagues: that experimental interference with HMGB1 signalling may influence the course of disease. To investigate this, an HMGB1 specific antibody was used to reduce HMGB1 signalling in vivo and infection studies were performed comparing these mice to mice treated with an isotype control. This antibody intervention has been used previously in our laboratory in a different infection model.
      • D’Elia R.V.
      • Laws T.R.
      • Carter A.
      • Lukaszewski R.
      • Clark G.C.
      Targeting the “Rising DAMP” during a Francisella tularensis infection.
      The analysis of mouse survival without antibiotic treatment resulted in the same conclusions being drawn as in the similar experiment performed previously:
      • Charoensup J.
      • Sermswan R.W.
      • Paeyao A.
      • Promakhejohn S.
      • Punasee S.
      • Chularari C.
      • et al.
      High HMGB1 level is associated with poor outcome of septicemic melioidosis.
      that HMGB1 depletion as a monotherapy does not significantly impact on the progression of melioidosis. Despite the lack of effect, the possibility exists that anti-HMGB1 treatment may have had a limited effect, masked by the acute nature of B. pseudomallei infection, in this model. For this reason, controlled culls were performed at 48 h post infection and a variety of parameters were measured. In these experiments, inhibition of HMGB1 signalling reduced bacterial burden within all three organs and most significantly within the lung. This apparent disparity in results, where anti-HMGB1 treatment reduced bacterial load but did not enhance survival, is likely a consequence of statistical power. On a continuous scale, bacterial load can be analysed with powerful parametric techniques, whereas differences in survival data are limited to the quanta of the time intervals over which animals are observed (twice daily in this study). These quanta can be increased through tempering the infection with antibiotics, allowing more time points for differences in survival to manifest, and this was done with success in the previous study.
      • Charoensup J.
      • Sermswan R.W.
      • Paeyao A.
      • Promakhejohn S.
      • Punasee S.
      • Chularari C.
      • et al.
      High HMGB1 level is associated with poor outcome of septicemic melioidosis.
      In addition it was found that anti-HMGB1 treatment influenced cytokine concentration within the organs. Specifically the greatest effect of cytokine concentration was within the lung, where the anti-HMGB1 treatment significantly reduced levels of IL-4, IL-6, IL-10, IL12-p70, GM-CSF, CXCL1, CCL3, and TNF-α; however reductions in IL-1β and IL-12p40 were observed in the spleen and TNF-α in the liver. The present analysis did not exclude the possibility that any other cytokines are reduced by anti-HMGB1 infection; the experimental design was not sufficiently powerful to determine that cytokine levels were unaffected by treatment. Critically, these data do show that some pro-inflammatory cytokines are lowered by the treatment, and with the reduction in bacterial load in mind, this is consistent with our current understanding of the role of the immune response in melioidosis. Pro-inflammatory signals may not always be beneficial
      • Ceballos-Olvera I.
      • Sahoo M.
      • Miller M.A.
      • Del Barrio L.
      • Re F.
      Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1beta is deleterious.
      and even have the potential to be detrimental.
      • Asakrah S.
      • Nieves W.
      • Mahdi Z.
      • Agard M.
      • Zea A.H.
      • Roy C.J.
      • et al.
      Post-exposure therapeutic efficacy of COX-2 inhibition against Burkholderia pseudomallei.
      In short, it is believed that the anti-HMGB1 treatment tempered the immune response and thus reduced immune dysregulation, therefore allowing a better, more ‘managed’ immune response to attempt to control the infection. By this reasoning it might be considered that HMGB1 induced inflammation is additive and detrimental during acute infections. This theory is supported by the TNF-α levels, which were reduced in both the liver and lung suggesting that the overall immune response has been tempered. Previously, TNF-α was found to contribute to host protection and when it was ablated, mice were found to be hypersensitive to B. pseudomallei infection.
      • Santanirand P.
      • Harley V.S.
      • Dance D.A.
      • Drasar B.S.
      • Bancroft G.J.
      Obligatory role of gamma interferon for host survival in a murine model of infection with Burkholderia pseudomallei.
      The present data are different in that, rather than the ablation of TNF-α in its entirety, the presence of this cytokine was reduced and it is this that we hypothesize contributes to a better anti-microbial response.
      In our previous work with the bacterial pathogen F. tularensis, HMGB1 reduction increased IFN-γ release. It is unfortunate that during B. pseudomallei infection HMGB1 reduction did not increase IFN-γ release because this might have led to a greater beneficial effect. Charoensup et al. found that this approach with ceftazidime delivered a modicum of success, and the present in-depth immunological analysis and measurements of the bacterial burden compliments, supports, and explains why anti-HMGB1 treatment was beneficial.
      An important consideration is the type of antibiotic used. Charoensup et al. used ceftazidime in conjunction with anti-HMGB1 and demonstrated a positive effect associated with combining these drugs.
      • Charoensup J.
      • Sermswan R.W.
      • Paeyao A.
      • Promakhejohn S.
      • Punasee S.
      • Chularari C.
      • et al.
      High HMGB1 level is associated with poor outcome of septicemic melioidosis.
      The data are not reported here, but we also performed experiments combining anti-HMGB1 therapy with the antibiotic doxycycline at sub-optimal concentrations (data not shown). No survival benefit for the combination was observed; however this may indicate that serious consideration should be given to the antibiotic that should be taken, as many antibiotics have direct effects on the immune system.
      Anti-HMGB1 therapy is just one of several promising therapies reported in the literature. CpGs are DNA motifs that can instigate immune activity via toll-like receptors and these have been shown to be useful in generating a helpful immune response against B. pseudomallei.
      • Chen Y.S.
      • Hsiao Y.S.
      • Lin H.H.
      • Liu Y.
      • Chen Y.L.
      CpG-modified plasmid DNA encoding flagellin improves immunogenicity and provides protection against Burkholderia pseudomallei infection in BALB/c mice.
      • Judy B.M.
      • Taylor K.
      • Deeraksa A.
      • Johnston R.K.
      • Endsley J.J.
      • Vijayakumar S.
      • et al.
      Prophylactic application of CpG oligonucleotides augments the early host response and confers protection in acute melioidosis.
      • Puangpetch A.
      • Anderson R.
      • Huang Y.Y.
      • Saengsot R.
      • Sermswan R.W.
      • Wongratanacheewin S.
      Comparison of the protective effects of killed Burkholderia pseudomallei and CpG oligodeoxynucleotide against live challenge.
      • Easton A.
      • Haque A.
      • Chu K.
      • Patel N.
      • Lukaszewski R.A.
      • Krieg A.M.
      • et al.
      Combining vaccination and postexposure CpG therapy provides optimal protection against lethal sepsis in a biodefense model of human melioidosis.
      • Rozak D.A.
      • Gelhaus H.C.
      • Smith M.
      • Zadeh M.
      • Huzella L.
      • Waag D.
      • et al.
      CpG oligodeoxyribonucleotides protect mice from Burkholderia pseudomallei but not Francisella tularensis Schu S4 aerosols.
      • Puangpetch A.
      • Anderson R.
      • Huang Y.Y.
      • Sermswan R.W.
      • Chaicumpa W.
      • Sirisinha S.
      • et al.
      Cationic liposomes extend the immunostimulatory effect of CpG oligodeoxynucleotide against Burkholderia pseudomallei infection in BALB/c mice.
      • Goodyear A.
      • Kellihan L.
      • Bielefeldt-Ohmann H.
      • Troyer R.
      • Propst K.
      • Dow S.
      Protection from pneumonic infection with Burkholderia species by inhalational immunotherapy.
      Additionally, outer membrane vesicles are also potent instigators of inflammation and these have been shown to be effective against B. pseudomallei infection.
      • Nieves W.
      • Asakrah S.
      • Qazi O.
      • Brown K.A.
      • Kurtz J.
      • Aucoin D.P.
      • et al.
      A naturally derived outer-membrane vesicle vaccine protects against lethal pulmonary Burkholderia pseudomallei infection.
      In a similar way to anti-HMGB1 treatment, the immune response can also be directed to the benefit of the host during infection by B. pseudomallei, by the introduction of signals and inhibitors such as IL-1 inhibitors,
      • Ceballos-Olvera I.
      • Sahoo M.
      • Miller M.A.
      • Del Barrio L.
      • Re F.
      Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1beta is deleterious.
      the addition of endogenous IL-18,
      • Wiersinga W.J.
      • Wieland C.W.
      • van der Windt G.J.
      • de Boer A.
      • Florquin S.
      • Dondorp A.
      • et al.
      Endogenous interleukin-18 improves the early antimicrobial host response in severe melioidosis.
      the inhibition of COX-2 signalling,
      • Asakrah S.
      • Nieves W.
      • Mahdi Z.
      • Agard M.
      • Zea A.H.
      • Roy C.J.
      • et al.
      Post-exposure therapeutic efficacy of COX-2 inhibition against Burkholderia pseudomallei.
      or the direct application of IFN-γ.
      • Propst K.L.
      • Troyer R.M.
      • Kellihan L.M.
      • Schweizer H.P.
      • Dow S.W.
      Immunotherapy markedly increases the effectiveness of antimicrobial therapy for treatment of Burkholderia pseudomallei infection.
      • Mosovsky K.
      • Silva E.
      • Troyer R.
      • Propst-Graham K.
      • Dow S.
      Interaction of Interferon gamma-induced reactive oxygen species with ceftazidime leads to synergistic killing of intracellular Burkholderia pseudomallei.
      The present work, and the work described above that targets the immune system, may provide future treatment options, although substantial issues exist. It is very difficult to prioritize treatment options based on the scale of therapeutic effect, as much of this work has been performed using different infection models. Future work should be attempted to compare treatments using the same model/s. Additionally, therapeutic options should be ranked on the availability of clinical drugs and the likely side effects. Also, the possibility exists that these different therapeutics might be used in combination. For example, CpGs might be used where exposure seems certain; antibiotic would be used to reduce bacterial load, while anti-IL1 helps generate a more appropriate immune response. This immune response, which is balanced more towards IFN-γ signalling, might be further bolstered by the addition of further IFN-γ, and some of the unnecessary damage-associated inflammation might be tempered with anti-HMGB1. In combination, these therapies would increase the likelihood of a positive outcome.
      In conclusion, the release of the DAMP HMGB1 was observed and it is hypothesized that this may present a therapeutic target. Following further evaluation, it is concluded that an experimental reduction in HMGB1 signalling is beneficial during melioidosis and is likely driven by a reduction of unnecessary inflammation.

      Acknowledgements

      This work was funded by the UK Ministry of Defence. We are grateful for the invaluable assistance of our colleagues working as animal technicians or helping with the processing of samples. We are also heavily indebted to the DSTL aerosol challenge team. We thank our colleague Dr Dianne Williamson for invaluable discussion and help in writing this manuscript.
      Conflict of interest: The authors have no competing interests to declare.

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