Methylglyoxal modulates immune responses: relevance to diabetes

Abstract

Increased methylglyoxal (MG) concentrations and formation of advanced glycation end-products (AGEs) are major pathways of glycaemic damage in diabetes, leading to vascular and neuronal complications. Diabetes patients also suffer increased susceptibility to many common infections, the underlying causes of which remain elusive. We hypothesized that immune glycation damage may account for this increased susceptibility. We previously showed that the reaction mixture (RM) for MG glycation of peptide blocks up regulation of CD83 in myeloid cells and inhibits primary stimulation of T cells. Here, we continue to investigate immune glycation damage, assessing surface and intracellular cytokine protein expression by flow cytometry, T-cell proliferation using a carboxyfluorescein succinimidyl ester assay, and mRNA levels by RT-PCR. We show that the immunomodulatory component of this RM was MG itself, with MG alone causing equivalent block of CD83 and loss of primary stimulation. Block of CD83 expression could be reversed by MG scavenger N-acetyl cysteine. Further, MG within RM inhibited stimulated production of interleukin (IL)-10 protein from myeloid cells plus interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha from T cells. Loss of IL-10 and IFN-gamma was confirmed by RT-PCR analysis of mRNA, while TNF-alpha message was raised. Loss of TNF-alpha protein was also shown by ELISA of culture supernatants. In addition, MG reduced major histocompatibility complex (MHC) class I expression on the surface of myeloid cells and increased their propensity to apoptose. We conclude that MG is a potent suppressor of myeloid and T-cell immune function and may be a major player in diabetes-associated susceptibility to infection.

Figures

Fig 1

Block of CD83 expression driven by MG. (A) Mean percent expression of CD83 on CD14− (black bars), CD14+ (white bars) and CD14++ (grey bars) myeloid cells from PBMC cultured for 2.5 hrs with sodium phosphate buffer (BUFFER), 500 μM methylglyoxal (MG) or reaction mixture (RM). Extension bars represent standard deviations of means from three experiments, and P-values are given for paired t-test comparisons between treatments. (B) Paired data sets (from separate experiments to those in (A)) for percent expression of CD83 on CD14−, CD14+ and CD14++ myeloid cells from PBMC cultured for 2.5 hrs with sodium phosphate buffer (BUF), 500 μM methylglyoxal (MG) or reaction mixture (RM), with (+) and without (–) 600 μM N-acetyl cysteine (NAC). P-values from paired t-tests are shown for the effect of NAC.

Fig 2

MG inhibits proliferation in allogeneic culture. Figure shows example analysis histograms for division and activation of CD3+CD8+ (second column) and CD3+CD8− (third column) responders (CD19/CD14/HLA-DR-depleted PBMC) after a 5-day culture with LDC stimulators (non-adherent myeloid cells enriched for DC). Previous to culture, responders and LDC were treated or not treated for 2.5 hrs with sodium phosphate buffer (BUF) or methylglyoxal (MG) and untreated responders were mixed with treated LDC and vice versa. First column shows forward and side scatter properties of 5-day cell cultures and columns 2 and 3 show the CFSE and CD69 expression of CD8+ and CD8− responders, respectively. Final column shows CFSE fluorescence of the total gated population of cells (triangular gate on light scatter plot).

Fig 3

MG within RM drives altered cytokine protein expression. (A) Mean relative numbers of IL-10high CD14− (black bars), CD14+ (white bars) and CD14++ (grey bars) myeloid cells from PBMC cultured for 4 hrs with sodium phosphate buffer (BUFFER) or reaction mixture (RM) plus monensin with (stim) and without (unstim) 1 μg/ml of LPS. (B) Mean relative numbers of IFN-γ+ and TNF-α+ CD8+ (black bars) and CD4+ (grey bars) T cells from PBMC cultured for 2.5 hrs with buffer (BUF) or RM plus a mixture of monensin, PMA and ionomycin. Extension bars represent standard deviations of means from three experiments, and P-values are given from paired t-test comparisons between treatments.

Fig 4

Numbers of T cells and concentration of TNF-α in supernatants after stimulated culture. (A) Mean relative numbers of CD8+ (black bars) and CD4+ (grey bars) T cells from PBMC after 2.5 hrs of culture with either sodium phosphate buffer (BUFFER) or reaction mixture (RM) plus a mixture of monensin, phorbol ester and ionomycin in three similar experiments. Extension bars represent standard deviations of means. n/s = not significant. (B) Mean concentration of TNF-α from the supernatant of PBMC cultured for 2.5 hrs with (stim) or without (unstim) phorbol ester and ionomycin plus either sodium phosphate buffer (BUFFER) or reaction mixture (RM). Extension bars represent standard deviations of means from triplicate wells in three similar experiments, and P-value is from a paired t-test comparison between treatments.

Fig 5

MG within RM drives altered cytokine mRNA expression. (A) Amounts of mRNA for TNF-α, IFN-γ, IL-10 and housekeeping gene UbcH5B from CD3+ (positively selected by magnetic bead separation) PBMC cultured for 2.5 hrs with PMA and ionomycin plus RM (grey bars) relative to buffer (black bars, given as ±1 in each case). Extension bars represent standard deviations of means from three experiments. (B) Mean relative amounts of mRNA for IL-10, IFN-γ and TNF-α as well as the housekeeping gene UbcH5B in CD3+ cells from PBMC cultured for 24 hrs with either sodium phosphate buffer (black bars) or reaction mixture (grey bars) plus a mixture of monensin, phorbol ester and ionomycin (last 4 hrs). Amount of mRNA in reaction mixture culture is shown relative to buffer culture (in each instance given as ±1). Extension bars represent standard deviations of means from three similar experiments.

Fig 6

Loss of IFN-γ production can be attributed to MG. Figure shows example test histograms for analysis of IFN-γ protein within CD8+ and CD4+ T cells from PBMC or PBMC depleted of HLA-DR+, CD19+ and CD14+ cells cultured for 2.5 hrs with buffer, methylglyoxal (MG) or RM.

Fig 7

MG reduces intensity of MHC I staining. (A) Mean percent expression of MHC class I on CD14−, CD14+ and CD14++ myeloid cells from PBMC cultured for 4 hrs with buffer (black bars) or 500 μM MG (grey bars). Extension bars represent standard deviations of means from three experiments. (B) Paired data sets for mean fluorescence of MHC I staining on CD14−, CD14+ and CD14++ myeloid cells from the same three experiments. P-values from paired t-test comparisons between treatments are given.

Fig 8

MG induction of apoptosis. Figure shows mean percentage of total PBMC that stained as necrotic (propidium iodide+), early apoptotic (annexin V+) and late apoptotic (propidium iodide+ and annexin V+) after 2.5, 4 or 24 hrs of culture with sodium phosphate buffer or 500 μM MG. Extension bars represent standard deviations of means from three experiments, and P-value from paired t-test comparisons between treatments is given.