The Role of the Intestinal Microbiome in Chronic Psychosocial Stress-Induced Pathologies in Male Mice

Chronic psychosocial stress is a risk factor for the development of physical and mental disorders accompanied or driven by an activated immune system. Given that chronic stress-induced systemic immune activation is lacking in germ-free and antibiotics-treated mice, a causal role of the gut microbiome in the development of stress-related disorders is likely. To address this hypothesis in the current study we employed the chronic subordinate colony housing (CSC, 19 days) paradigm, a pre-clinically validated mouse model for chronic psychosocial stress, known to alter the gut microbial signature and to induce systemic low-grade inflammation, as well as physical and mental abnormalities. In detail, we investigated if (i) CSC-induced alterations can be prevented by repeated transplantation of feces (FT) from non-stressed single-housed control (SHC) mice during CSC exposure, and (ii) if the transplantation of a “stressed” CSC microbiome is able to induce CSC effects in SHC mice. Therefore, we repeatedly infused SHC and CSC recipient mice rectally with SHC donor feces at days 4 and 11 of the CSC paradigm and assessed anxiety-related behavior on day 19 as well as physiological, immunological, and bone parameters on day 20. Furthermore, SHC and CSC recipient mice were infused with CSC donor feces at respective days. To exclude effects of rectal infusions per se, another set of SHC and CSC mice was infused with saline, respectively. Our results showed that transplantation of SHC feces had mild stress-protective effects, indicated by an amelioration of CSC-induced thymus atrophy, anxiety, systemic low-grade inflammation, and alterations in bone homeostasis. Moreover, transplantation of CSC feces slightly aggravated CSC-induced systemic low-grade inflammation and alterations in bone homeostasis in SHC and/or CSC animals. In conclusion, our data provide evidence for a role of the host’s microbiome in many, but not all, adverse consequences of chronic psychosocial stress. Moreover, our data are consistent with the hypothesis that transplantation of healthy feces might be a useful tool to prevent/treat different adverse outcomes of chronic stress. Finally, our data suggests that stress effects can be transferred to a certain extend via FT, proposing therapeutic approaches using FT to carefully screen fecal donors for their stress/trauma history

Osteoblast-specific overexpression of complement receptor C5aR1 impairs fracture healing.

The anaphylatoxin receptor C5aR1 plays an important role not only in innate immune responses, but also in bone metabolism and fracture healing, being highly expressed on immune and bone cells, including osteoblasts and osteoclasts. C5aR1 induces osteoblast migration, cytokine generation and osteoclastogenesis, however, the exact role of C5aR1-mediated signaling in osteoblasts is not entirely known. Therefore, we hypothesized that osteoblasts are essential target cells for C5a and that fracture healing should be disturbed in mice with an osteoblast-specific C5aR1 overexpression (Col1a1-C5aR1). Osteoblast activity in vitro, bone phenotype and fracture healing after isolated osteotomy and after combined osteotomy with additional thoracic trauma were analyzed. The systemic and local inflammatory reactions were analyzed by determining C5a and IL-6 concentrations in blood, bronchoalveolar lavage fluid and fracture callus and the recruitment of immune cells. In vitro, osteoblast proliferation and differentiation were similar to wildtype cells, and phosphorylation of p38 and expression of IL-6 and RANKL were increased in osteoblasts derived from Col1a1-C5aR1 mice. Bone phenotype and the inflammatory reaction were unaffected in Col1a1-C5aR1 mice. Fracture healing was significantly impaired as demonstrated by significantly reduced bone content, bone mineral density and flexural rigidity, possibly due to significantly increased osteoclast numbers. C5aR1 signaling in osteoblasts might possibly affect RANKL/OPG balance, leading to increased bone resorption. Additional trauma significantly impaired fracture healing, particularly in Col1a1-C5aR1 mice. In conclusion, the data indicate that C5aR1 signaling in osteoblasts plays a detrimental role in bone regeneration after fracture

C5a and IL-6 concentrations in blood, BAL fluid and fracture hematoma 3 h after surgery.

<p>C5a and IL-6 concentrations in blood, BAL fluid and fracture hematoma 3 h after surgery.</p

Fracture healing in WT and Col1a1-C5aR1 mice 25 d after surgery.

<p>(A) Callus area, (B) amount of osseous tissue, (C) amount of cartilage and (D) relative flexural rigidity of wildtype (WT) and Col1a1-C5aR1 mice. Fx: mice with isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma (TXT). ^*p < 0.05, n = 6 per group.</p

Fracture healing in WT and Col1a1-C5aR1 mice 14 d after surgery.

<p>(A) Representative histological images of wildtype (WT) and Col1a1-C5aR1 mice with isolated fracture and fracture with additional thoracic trauma (TXT). (B) Callus area, (C) amount of osseous tissue and (D) amount of cartilage. (E) Number of osteoblasts per bone perimeter (N.Ob/B.Pm) and (F) number of osteoclasts per bone perimeter (N.Oc/B.Pm) of WT and Col1a1-C5aR1 mice. (G) Representative tartrate-resistant acid phosphatase (TRAP) staining of fractured calli of WT and Col1a1-C5aR1 mice with isolated fracture. Fx: mice with isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma. C: cortex, scale bar 100 μm, ^*p < 0.05, n = 6 per group.</p

Bone phenotype of WT and Col1a1-C5aR1 mice aged 12 and 55 weeks.

<p>Bone phenotype of WT and Col1a1-C5aR1 mice aged 12 and 55 weeks.</p

Fracture healing in WT and Col1a1-C5aR1 mice 21 d after surgery.

<p>(A) Representative histological images of wildtype (WT) and Col1a1-C5aR1 mice with isolated fracture and fracture with additional thoracic trauma (TXT). (B) Callus area, (C) amount of osseous tissue and (D) amount of cartilage. (E) Bone mineral density (BMD). (F) Relative flexural rigidity. (G) Number of osteoblasts per bone perimeter (N.Ob/B.Pm) and (H) number of osteoclasts per bone perimeter (N.Oc/B.Pm) of WT and Col1a1-C5aR mice. (I) Representative tartrate-resistant acid phosphatase (TRAP) staining of fractured calli of WT and Col1a1-C5aR1 mice with isolated fracture. Fx: mice with isolated fracture, Fx+TXT: mice with combined fracture and thoracic trauma. C: cortex, scale bar 100 μm, ^*p < 0.05; ^**p < 0.005; ^***p < 0.001, n = 6–7 per group.</p

Primer sequences.

<p>Primer sequences.</p

<i>In vitro</i> analysis of WT and Col1a1-C5aR1 osteoblasts.

<p>(A) Western blot showing C5aR1 expression in primary osteoblasts of wildtype (WT) and Col1a1-C5aR1 mice after cultivating for 14 d in normal proliferation medium (NM) and osteogenic differentiation medium (ODM). (B) Cell proliferation of WT and Col1a1-C5aR1 osteoblasts determined by BrdU incorporation. (C) Osteogenic differentiation capacity of osteoblasts assessed by alkaline-phosphatase staining after cultivating for 14 d in ODM with or without C5a and (D) by the expression of the osteogenic marker genes <i>Alpl</i> (alkaline phosphatase), <i>Ibsp</i> (bone sialoprotein) and <i>Bglap</i> (bone gamma carboxyglutamate protein/osteocalcin). (E) Western blot showing increased expression of phospho (p)-ERK 1/2 and phospho (p)-p38 after osteoblast stimulation with C5a for 6 h. F) Relative gene expression of interleukin (IL)-6, receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). ^*p < 0.05; ^***p < 0.001, n = 5–6 per group and treatment.</p