Gut microbiota, innate immune pathways, and inflammatory control mechanisms in patients with major depressive disorder

Abstract

Although alterations in the gut microbiota have been linked to the pathophysiology of major depressive disorder (MDD), including through effects on the immune response, our understanding is deficient about the straight connection patterns among microbiota and MDD in patients. Male and female MDD patients were recruited: 46 patients with a current active MDD (a-MDD) and 22 in remission or with only mild symptoms (r-MDD). Forty-five healthy controls (HC) were also recruited. Psychopathological states were assessed, and fecal and blood samples were collected. Results indicated that the inducible nitric oxide synthase expression was higher in MDD patients compared with HC and the oxidative stress levels were greater in the a-MDD group. Furthermore, the lipopolysaccharide (an indirect marker of bacterial translocation) was higher in a-MDD patients compared with the other groups. Fecal samples did not cluster according to the presence or the absence of MDD. There were bacterial genera whose relative abundance was altered in MDD: Bilophila (2-fold) and Alistipes (1.5-fold) were higher, while Anaerostipes (1.5-fold) and Dialister (15-fold) were lower in MDD patients compared with HC. Patients with a-MDD presented higher relative abundance of Alistipes and Anaerostipes (1.5-fold) and a complete depletion of Dialister compared with HC. Patients with r-MDD presented higher abundance of Bilophila (2.5-fold) compared with HC. Thus, the abundance of bacterial genera and some immune pathways, both with potential implications in the pathophysiology of depression, appear to be altered in MDD, with the most noticeable changes occurring in patients with the worse clinical condition, the a-MDD group.

Fig. 1. Metataxonomic analysis and abundance of bacteria genera in fecal samples.

A Comparison of the relative abundance of the 20 most abundant genera (TSS; total-sum normalization) in the three groups of subjects. The dendrogram was based on genera similarity between the samples. Hclust with complete linkage method from the Calypso online software was used to compute the hierarchical clustering. PCoA plots of bacterial profiles based on Bray–Curtis similarity analysis (relative abundance) (B) and on the Jaccard’s coefficient for binary data (presence/absence) (C) from the three groups of subjects (blue triangle, control group; red circles, a-MDD group; orange squares, r-MDD group). The value given on each axis label represents the percentage of the total variance explained by that axis. Relative abundance of the genera Bilophila (D), Alistipes (E), Anaerostipes (F), and Dialister (G) in MDD patients compared with HC. Patients with r-MDD presented an increased presence of sequences belonging to the genus Bilophila (H). Patients with a-MDD showed increased abundance of sequences belonging to the genus Alistipes (I), while those corresponding to the genera Anaerostipes and Dialister were decreased (J, K). Differences in sample group genera were compared by using either the Wilcoxon rank test for variables with two groups or the Kruskal–Wallis test for variables with more than two groups. To correct for multiple comparisons, Bonferroni-adjusted significance levels were set for each analysis. *p < 0.05, **p < 0.01 vs. healthy controls (HC).

Fig. 2. Effects of the MDD on the innate immune pathway.

TLR-4 protein expression in PBMCs (A) and LPS plasma levels (B) were not affected by the MDD. Plasma levels of HMGB1 decreased in the MDD group (C). TLR-4 expression was not altered in the a-MDD group and was increased in the r-MDD group compared with HC (D). LPS plasma levels augmented in the a-MDD and were not affected in the r-MDD group compared with HC (E). HMGB1 plasma levels decreased in the r-MDD group compared with HC (F). Data are expressed as mean ± standard error of the mean (SEM). In the western blots the densitometric data of the respective bands of interest are normalized by β-actin (lower band). Samples were from parallel experiments and that gels/blots were processed in parallel. An unpaired two-tailed t-test was performed when comparing two groups. For more than two groups comparisons, a one-way ANOVA with a Tukey post-hoc test was employed, and in those cases in which the data did not follow a Gaussian distribution, a nonparametric ANOVA with a Kruskal–Wallis test followed by a Dunn’s post-hoc test was performed. *p < 0.05, **p < 0.01 vs. healthy controls (HC).

Fig. 3. Effects of MDD on intra- and intercellular inflammatory parameters.

The ratio between the activated ERK (pERK) and the ERK total form decreased in PBMCs from patients with MDD compared with HC (A). Patients with MDD did not show differences in the protein levels of NF-κB nor in the plasma levels of CRP and IL-6 compared with HC (B–D). a-MDD patients did not show differences when compared with HC (E–H). pERK/ERK ratio in PBMCs decreased (E) and plasma IL-6 increased (H) in r-MDD patients compared with HC. Plasma CRP levels in the r-MDD group decreased compared with the a-MDD group (G). Data are expressed as mean ± standard error of the mean (SEM). In the western blots the densitometric data of the respective bands of interest are normalized by β-actin or by GAPDH (lower band). Samples were from parallel experiments and that gels/blots were processed in parallel. An unpaired two-tailed t-test was performed when comparing two groups. For more than two groups comparisons, a one-way ANOVA with a Tukey post-hoc test was employed, and in those cases in which the data did not follow a Gaussian distribution, a nonparametric ANOVA with a Kruskal–Wallis test followed by a Dunn’s post-hoc test was performed. *p < 0.05, **p < 0.01, ***p < 0.001 vs. healthy controls (HC); ^#p < 0.05 vs. a-MDD.

Fig. 4. Effects of the MDD on the cyclooxygenase-2 pathway, on the oxidative/nitrosative response, and on counterbalancing mechanisms.

Patients with MDD presented lower levels of COX-2 expression in PBMCs (A) compared with HC, without changes in PGE₂ (B) and in 15d-PGJ₂ (C) plasma levels. The MDD did not affect the PPARγ expression (D). The a-MDD and r-MDD groups did not show any difference between them nor with the HC group (E–H). The MDD increased iNOS protein levels in PBMCs (I) but no affected the activity of TBARS, SOD and GPx (J–L). a-MDD and r-MDD groups presented increased iNOS expression in PBMCs (M). The a-MDD group shown increased TBARS levels compared with HC (N). There were no differences in the activity of SOD and GPx among groups (O, P). Data are expressed as mean ± standard error of the mean (SEM). In the western blots the densitometric data of the respective bands of interest are normalized by β-actin or by GAPDH (lower band). Samples were from parallel experiments and that gels/blots were processed in parallel. An unpaired two-tailed t-test was performed when comparing two groups. For more than two groups comparisons, a one-way ANOVA with a Tukey post-hoc test was employed, and in those cases in which the data did not follow a Gaussian distribution, a nonparametric ANOVA with a Kruskal–Wallis test followed by a Dunn’s post-hoc test was performed. *p < 0.05, ***p < 0.001 vs. healthy controls (HC).