The intestinal archaea Methanosphaera stadtmanae and Methanobrevibacter smithii activate human dendritic cells

Abstract

The methanoarchaea Methanosphaera stadtmanae and Methanobrevibacter smithii are known to be part of the indigenous human gut microbiota. Although the immunomodulatory effects of bacterial gut commensals have been studied extensively in the last decade, the impact of methanoarchaea in human's health and disease was rarely examined. Consequently, we studied and report here on the effects of M. stadtmanae and M. smithii on human immune cells. Whereas exposure to M. stadtmanae leads to substantial release of proinflammatory cytokines in monocyte-derived dendritic cells (moDCs), only weak activation was detected after incubation with M. smithii. Phagocytosis of M. stadtmanae by moDCs was demonstrated by confocal microscopy as well as transmission electronic microscopy (TEM) and shown to be crucial for cellular activation by using specific inhibitors. Both strains, albeit to different extents, initiate a maturation program in moDCs as revealed by up-regulation of the cell-surface receptors CD86 and CD197 suggesting additional activation of adaptive immune responses. Furthermore, M. stadtmanae and M. smithii were capable to alter the gene expression of antimicrobial peptides in moDCs to different extents. Taken together, our findings strongly argue that the archaeal gut inhabitants M. stadtmanae and M. smithii are specifically recognized by the human innate immune system. Moreover, both strains are capable of inducing an inflammatory cytokine response to different extents arguing that they might have diverse immunomodulatory functions. In conclusion, we propose that the impact of intestinal methanoarchaea on pathological conditions involving the gut microbiota has been underestimated until now.

Figure 1. M. stadtmanae induces significant higher release of cytokines in moDCs than M. smithii.

Cytokine release after stimulation of 2×10⁵ moDCs with 1×10⁶ and 1×10⁷ M. stadtmanae or M. smithii cells for 20 h was quantified using commercial ELISA-Kits (A–C). MoDCs were preincubated with 2 µM Cytochalasin D in DMSO (B) or 10 nM Bafilomycin A1 in DMSO (C) for 30 min prior stimulation. LPS (100 ng/ml) and medium were used as controls. Stated data are means of at least 3 independent biological replicates with their respective standard errors of the mean (SEM).

Figure 2. Phagocytosis of M. stadtmanae is crucial for immune cell activation.

A) After preincubation with 1 µM Cytochalasin D (in DMSO) and with DMSO alone (control) for 30 min 1×10⁵ moDCs were stimulated with 1×10⁷ methanoarchaeal cells in glass cover slips for a period of 4 h. Lysosomes in moDCs were stained with LysoTracker Red DND-99 during time of incubation. After incubation, moDCs were washed, fixed with 3% paraformaldehyde and labeled with Hoechst (DAPI-staining). Images were captured using LSM 510 confocal microscopy (Zeiss) with Leica confocal software and are representative of the respective samples. B) 1×10⁶ moDCs were stimulated with 1×10⁸ methanoarchaeal cells for a period of 4 h. After washing in PBS, moDCs were fixed for electron microscopy. Images are representative for the respective sample.

Figure 3. Increased expressions of cell-surface receptors on moDCs after stimulation with M. stadtmanae and M. smithii.

1×10⁶ moDCs were stimulated with 1×10⁷ M. stadtmanae or M. smithii cells or medium for 24 h and 48 h at 37°C. Subsequently, 2×10⁵ moDCs were incubated with antibodies (PE- or FITC-labeled) and analyzed with a FACS flow cytometer. MoDCs were selected by forward and side scattered signals before measuring the intensity of PE or FITC fluorescence signals of 10000 cells. Depicted graphics are original plots of FlowJo Software, Version 7.5.5.

Figure 4. Altered gene expression of AMPs in moDCs after stimulation with M. stadtmanae and M. smithii.

2–4×10⁶ moDCs were stimulated with 1×10⁷ M. stadtmanae or M. smithii cells and medium (for control) over a period of 24 h. Subsequently, RNA was isolated according to manufacturer's protocol and reverse-transcribed. Relative quantification of expression of genes encoding HBD1 and LL37 were calculated with the LightCycler 480 Software in relation to house-keeping gene hprt.