Detection of Increased Plasma Interleukin-6 Levels and Prevalence of Prevotella copri and Bacteroides vulgatus in the Feces of Type 2 Diabetes Patients

Aline Zazeri Leite, Nathália de Campos Rodrigues, Marina Ignácio Gonzaga, João Carlos Cicogna Paiolo, Carolina Arantes de Souza, Nadine Aparecida Vicentini Stefanutto, Wellington Pine Omori, Daniel Guariz Pinheiro, João Luiz Brisotti, Euclides Matheucci Junior, Vânia Sammartino Mariano, Gislane Lelis Vilela de Oliveira, Aline Zazeri Leite, Nathália de Campos Rodrigues, Marina Ignácio Gonzaga, João Carlos Cicogna Paiolo, Carolina Arantes de Souza, Nadine Aparecida Vicentini Stefanutto, Wellington Pine Omori, Daniel Guariz Pinheiro, João Luiz Brisotti, Euclides Matheucci Junior, Vânia Sammartino Mariano, Gislane Lelis Vilela de Oliveira

Abstract

Intestinal dysbiosis and metabolic endotoxemia have been associated with metabolic disorders, such as obesity, insulin resistance, and type 2 diabetes (T2D). The main goal of the present study was to evaluate the intestinal dysbiosis in Brazilian T2D patients and correlate these data with inflammatory cytokines and lipopolysaccharides (LPS) plasma concentrations. This study was approved by the Ethics Committees from Barretos Cancer Hospital and all individuals signed the informed consent form. Stool samples were required for DNA extraction, and the V3/V4 regions of bacterial 16S were sequenced using an Illumina platform. Peripheral blood was used to quantify inflammatory cytokines and plasma LPS concentrations, by CBA flex and ELISA, respectively. Statistical analyses were performed using Mann-Whitney and Spearman's tests. Analysis of variance, diversity indexes, and analysis of alpha- and beta-diversity were conducted using an annotated Operational Taxonomic Unit table. This study included 20 patients and 22 controls. We observed significant differences (P < 0.01) in the microbiota composition (beta-diversity) between patients and controls, suggesting intestinal dysbiosis in Brazilian T2D patients. The prevalent species found in patients' feces were the Gram-negatives Prevotella copri, Bacteroides vulgatus, Bacteroides rodentium, and Bacteroides xylanisolvens. The proinflammatory interleukin-6 (IL-6) was significantly increased (P < 0.05) in patients' plasma and LPS levels were decreased. We find correlations between the proinflammatory interferon-gamma with Gram-negatives Bacteroides and Prevotella species, and a positive correlation between the LPS levels and P. copri reads. The P. copri and B. vulgatus species were associated with insulin resistance in previous studies. In this study, we suggested that the prevalence of Gram-negative species in the gut and the increased plasma IL-6 in patients could be linked to low-grade inflammation and insulin resistance. In conclusion, the P. copri and B. vulgatus species could represent an intestinal microbiota signature, associated with T2D development. Furthermore, the identification of these Gram-negative bacteria, and the detection of inflammatory markers, such as increased IL-6, could be used as diabetes predictive markers in overweight, obese and in genetically predisposed individuals to develop T2D.

Keywords: dietary habits; inflammatory cytokines; interleukin-6; intestinal microbiota; metabolic endotoxemia; type 2 diabetes.

Figures

Figure 1
Figure 1
Alpha and beta-diversity in gut microbiota from type 2 diabetes (T2D) patients. (A,B) Rarefaction curves comparing the species richness, Chao1, and observed Operational Taxonomic Unit (OTU) numbers. (C,D) PcoA plots with weighted and unweighted UniFrac metric with Bonferroni’s correction.
Figure 2
Figure 2
Relative abundances of bacterial taxa in the feces of T2D patients. Prevalent phyla (A), classes (B), orders (C), families (D), genera (E), and species (F). Bars represent the reads percentages found in metagenomic analyses.
Figure 3
Figure 3
Negative correlation between the Ruminococcaceae reads with the HbA1C percentages. Statistical analyses were performed using the Spearman’s test. Significance was set at P < 0.05.
Figure 4
Figure 4
Cytokine profile in type 2 diabetes patients and control subjects. Plasma concentrations of IL-4 (A), interleukin-6 (IL-6) (B), IL-10 (C), IL-17A (D), interferon-gamma (IFN-γ) (E), and tumor necrosis factor (TNF) (F). Statistical analyses were performed by Mann–Whitney test. Significance was set at P < 0.05.
Figure 5
Figure 5
Correlations between the proinflammatory cytokines with relative abundances of bacterial taxa. Correlations found among interferon-gamma (IFN-γ) and Firmicutes phylum (A), Clostridia class (B), Bacteroides genus (C), and Prevotella genus (D). Positive correlation found between IL-17A with Enterobacteriaceae family (E). Positive correlation found between interleukin-6 (IL-6) with Negativicutes class (F). Statistical analyses were performed by Spearman’s test. Significance was set at P < 0.05.
Figure 6
Figure 6
Lipopolysaccharide (LPS) concentrations and the correlations with relative abundances of bacterial taxa. Plasma levels of the LPS in type 2 diabetes patients and controls (A). Negative correlation found between the LPS with Proteobacteria phylum (B). Positive correlation found between the LPS with Prevotella genus (C). Statistical analyses were performed using the Mann–Whitney and the Spearman’s test. Significance was set at P < 0.05.

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