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The effect of hyperglycaemia on in vitro cytokine production and macrophage infection with Mycobacterium tuberculosis.

Lachmandas E, Vrieling F, Wilson LG, Joosten SA, Netea MG, Ottenhoff TH, van Crevel R - PLoS ONE (2015)

Bottom Line: We examined the effects of hyperglycaemia, a hallmark of diabetes, on the cytokine response to and macrophage infection with Mycobacterium tuberculosis.Increasing in vitro glucose concentrations from 5 to 25 mmol/L had marginal effects on cytokine production following stimulation of peripheral blood mononuclear cells (PBMCs) with M. tuberculosis lysate, LPS or Candida albicans, while 40 mmol/L glucose increased production of TNF-α, IL-1β, IL-6 and IL-10, but not of IFN-γ, IL-17A and IL-22.The fact that only very high glucose concentrations were able to significantly influence cytokine production by macrophages suggests that hyperglycaemia alone cannot fully explain the increased susceptibility of diabetes mellitus patients to tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands; Radboud Centre for Infectious Diseases, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT
Type 2 diabetes mellitus is an established risk factor for tuberculosis but the underlying mechanisms are largely unknown. We examined the effects of hyperglycaemia, a hallmark of diabetes, on the cytokine response to and macrophage infection with Mycobacterium tuberculosis. Increasing in vitro glucose concentrations from 5 to 25 mmol/L had marginal effects on cytokine production following stimulation of peripheral blood mononuclear cells (PBMCs) with M. tuberculosis lysate, LPS or Candida albicans, while 40 mmol/L glucose increased production of TNF-α, IL-1β, IL-6 and IL-10, but not of IFN-γ, IL-17A and IL-22. Macrophage differentiation under hyperglycaemic conditions of 25 mmol/L glucose was also associated with increased cytokine production upon stimulation with M. tuberculosis lysate and LPS but in infection experiments no differences in M. tuberculosis killing or outgrowth was observed. The phagocytic capacity of these hyperglycaemic macrophages also remained unaltered. The fact that only very high glucose concentrations were able to significantly influence cytokine production by macrophages suggests that hyperglycaemia alone cannot fully explain the increased susceptibility of diabetes mellitus patients to tuberculosis.

No MeSH data available.


Related in: MedlinePlus

Effects of high glucose on phagocytic capacity of macrophages and infection with H37Rv.M2 macrophages differentiated in the presence of 5 or 25 mmol/L glucose were either incubated with P-beads and subjected to flow cytometry measurements to determine the percentage of phagocytic cells (A) or were infected for 1 hour with H37Rv at an MOI of 10:1 (B). After infection macrophages were washed three times and fresh RPMI containing the different glucose media was added. The first wash (Day 0 Sups) of each infection was collected and plated in serial dilutions to determine whether different amounts of bacilli were taken up by euglycaemic or hyperglycaemic macrophages. Simultaneously macrophages (D0) were lysed and plated for CFU counts. Infected macrophages were also lysed on Day 1 (D1), Day 3 (D3), Day 5 (D5) and Day 7 (D7) after infection. CFUs were counted at once after 2–3 weeks of growth at 37°C. Data are shown as mean ± SEM (n = 4).
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pone.0117941.g004: Effects of high glucose on phagocytic capacity of macrophages and infection with H37Rv.M2 macrophages differentiated in the presence of 5 or 25 mmol/L glucose were either incubated with P-beads and subjected to flow cytometry measurements to determine the percentage of phagocytic cells (A) or were infected for 1 hour with H37Rv at an MOI of 10:1 (B). After infection macrophages were washed three times and fresh RPMI containing the different glucose media was added. The first wash (Day 0 Sups) of each infection was collected and plated in serial dilutions to determine whether different amounts of bacilli were taken up by euglycaemic or hyperglycaemic macrophages. Simultaneously macrophages (D0) were lysed and plated for CFU counts. Infected macrophages were also lysed on Day 1 (D1), Day 3 (D3), Day 5 (D5) and Day 7 (D7) after infection. CFUs were counted at once after 2–3 weeks of growth at 37°C. Data are shown as mean ± SEM (n = 4).

Mentions: After investigating the cytokine profiles of macrophages stimulated with either LPS or H37Rv lysate in the presence of varying glucose concentrations the effect of hyperglycaemia on the in vitro infection of M2 macrophages with the H37Rv strain of MTB was determined. M2 macrophages were used for the infection experiments as we have previously shown that this macrophage subtype is more adept in supporting mycobacterial survival and could therefore serve as the primary bacterial reservoir in the lungs during MTB infection [25]. After 24 h of infection no differences in CFU counts (Fig. 3B) or cytokine production (Fig. 3C) were observed between euglycaemic (5 mmol/L) and hyperglycaemic (25 mmol/L) macrophages. We also examined the effect of hyperglycaemia on the phagocytic capacity of macrophages and outgrowth of H37Rv after prolonged infection. M2 macrophages were able to control MTB growth as was demonstrated by a 1 log reduction in CFU over time (Fig. 4B). However, differentiation and stimulation of M2 macrophages in the presence of 5 or 25 mmol/L of glucose were not associated with differences in the phagocytosis of fluorescent P-beads (Fig. 4A), mycobacterial uptake (D0 Sups) and H37Rv survival throughout the course of infection (Fig. 4B). We assessed macrophage viability during infection by measuring LDH release and staining the cells with Trypan Blue and found no differences in viability between glucose conditions (S3 Fig.). Together these data demonstrate that hyperglycaemia influences neither in vitro H37Rv infection and survival nor the infection-induced cytokine response in human M2 macrophages.


The effect of hyperglycaemia on in vitro cytokine production and macrophage infection with Mycobacterium tuberculosis.

Lachmandas E, Vrieling F, Wilson LG, Joosten SA, Netea MG, Ottenhoff TH, van Crevel R - PLoS ONE (2015)

Effects of high glucose on phagocytic capacity of macrophages and infection with H37Rv.M2 macrophages differentiated in the presence of 5 or 25 mmol/L glucose were either incubated with P-beads and subjected to flow cytometry measurements to determine the percentage of phagocytic cells (A) or were infected for 1 hour with H37Rv at an MOI of 10:1 (B). After infection macrophages were washed three times and fresh RPMI containing the different glucose media was added. The first wash (Day 0 Sups) of each infection was collected and plated in serial dilutions to determine whether different amounts of bacilli were taken up by euglycaemic or hyperglycaemic macrophages. Simultaneously macrophages (D0) were lysed and plated for CFU counts. Infected macrophages were also lysed on Day 1 (D1), Day 3 (D3), Day 5 (D5) and Day 7 (D7) after infection. CFUs were counted at once after 2–3 weeks of growth at 37°C. Data are shown as mean ± SEM (n = 4).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4322041&req=5

pone.0117941.g004: Effects of high glucose on phagocytic capacity of macrophages and infection with H37Rv.M2 macrophages differentiated in the presence of 5 or 25 mmol/L glucose were either incubated with P-beads and subjected to flow cytometry measurements to determine the percentage of phagocytic cells (A) or were infected for 1 hour with H37Rv at an MOI of 10:1 (B). After infection macrophages were washed three times and fresh RPMI containing the different glucose media was added. The first wash (Day 0 Sups) of each infection was collected and plated in serial dilutions to determine whether different amounts of bacilli were taken up by euglycaemic or hyperglycaemic macrophages. Simultaneously macrophages (D0) were lysed and plated for CFU counts. Infected macrophages were also lysed on Day 1 (D1), Day 3 (D3), Day 5 (D5) and Day 7 (D7) after infection. CFUs were counted at once after 2–3 weeks of growth at 37°C. Data are shown as mean ± SEM (n = 4).
Mentions: After investigating the cytokine profiles of macrophages stimulated with either LPS or H37Rv lysate in the presence of varying glucose concentrations the effect of hyperglycaemia on the in vitro infection of M2 macrophages with the H37Rv strain of MTB was determined. M2 macrophages were used for the infection experiments as we have previously shown that this macrophage subtype is more adept in supporting mycobacterial survival and could therefore serve as the primary bacterial reservoir in the lungs during MTB infection [25]. After 24 h of infection no differences in CFU counts (Fig. 3B) or cytokine production (Fig. 3C) were observed between euglycaemic (5 mmol/L) and hyperglycaemic (25 mmol/L) macrophages. We also examined the effect of hyperglycaemia on the phagocytic capacity of macrophages and outgrowth of H37Rv after prolonged infection. M2 macrophages were able to control MTB growth as was demonstrated by a 1 log reduction in CFU over time (Fig. 4B). However, differentiation and stimulation of M2 macrophages in the presence of 5 or 25 mmol/L of glucose were not associated with differences in the phagocytosis of fluorescent P-beads (Fig. 4A), mycobacterial uptake (D0 Sups) and H37Rv survival throughout the course of infection (Fig. 4B). We assessed macrophage viability during infection by measuring LDH release and staining the cells with Trypan Blue and found no differences in viability between glucose conditions (S3 Fig.). Together these data demonstrate that hyperglycaemia influences neither in vitro H37Rv infection and survival nor the infection-induced cytokine response in human M2 macrophages.

Bottom Line: We examined the effects of hyperglycaemia, a hallmark of diabetes, on the cytokine response to and macrophage infection with Mycobacterium tuberculosis.Increasing in vitro glucose concentrations from 5 to 25 mmol/L had marginal effects on cytokine production following stimulation of peripheral blood mononuclear cells (PBMCs) with M. tuberculosis lysate, LPS or Candida albicans, while 40 mmol/L glucose increased production of TNF-α, IL-1β, IL-6 and IL-10, but not of IFN-γ, IL-17A and IL-22.The fact that only very high glucose concentrations were able to significantly influence cytokine production by macrophages suggests that hyperglycaemia alone cannot fully explain the increased susceptibility of diabetes mellitus patients to tuberculosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands; Radboud Centre for Infectious Diseases, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT
Type 2 diabetes mellitus is an established risk factor for tuberculosis but the underlying mechanisms are largely unknown. We examined the effects of hyperglycaemia, a hallmark of diabetes, on the cytokine response to and macrophage infection with Mycobacterium tuberculosis. Increasing in vitro glucose concentrations from 5 to 25 mmol/L had marginal effects on cytokine production following stimulation of peripheral blood mononuclear cells (PBMCs) with M. tuberculosis lysate, LPS or Candida albicans, while 40 mmol/L glucose increased production of TNF-α, IL-1β, IL-6 and IL-10, but not of IFN-γ, IL-17A and IL-22. Macrophage differentiation under hyperglycaemic conditions of 25 mmol/L glucose was also associated with increased cytokine production upon stimulation with M. tuberculosis lysate and LPS but in infection experiments no differences in M. tuberculosis killing or outgrowth was observed. The phagocytic capacity of these hyperglycaemic macrophages also remained unaltered. The fact that only very high glucose concentrations were able to significantly influence cytokine production by macrophages suggests that hyperglycaemia alone cannot fully explain the increased susceptibility of diabetes mellitus patients to tuberculosis.

No MeSH data available.


Related in: MedlinePlus