Microbial symbionts accelerate wound healing via the neuropeptide hormone oxytocin

Abstract

Wound healing capability is inextricably linked with diverse aspects of physical fitness ranging from recovery after minor injuries and surgery to diabetes and some types of cancer. Impact of the microbiome upon the mammalian wound healing process is poorly understood. We discover that supplementing the gut microbiome with lactic acid microbes in drinking water accelerates the wound-healing process to occur in half the time required for matched control animals. Further, we find that Lactobacillus reuteri enhances wound-healing properties through up-regulation of the neuropeptide hormone oxytocin, a factor integral in social bonding and reproduction, by a vagus nerve-mediated pathway. Bacteria-triggered oxytocin serves to activate host CD4+Foxp3+CD25+ immune T regulatory cells conveying transplantable wound healing capacity to naive Rag2-deficient animals. This study determined oxytocin to be a novel component of a multi-directional gut microbe-brain-immune axis, with wound-healing capability as a previously unrecognized output of this axis. We also provide experimental evidence to support long-standing medical traditions associating diet, social practices, and the immune system with efficient recovery after injury, sustained good health, and longevity.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Dietary supplementation with L. reuteri accelerates wound healing.

(a) Microscopy of formalin-fixed, paraffinized wounded skin of aged C57BL/6 mice (at 3, 6, and 12 days post- wounding). Wound margins are delineated with yellow outlines. The healing time course is faster in mice consuming L. reuteri evidenced by reduced wound sizes. (b) Histopathology of wound healing timecourse shows wound epidermal gaps [indicated by black brackets]. Accelerated epidermal closure in mice consuming the purified lactic acid bacteria led to complete re-epithelialization of wounds in 8/12 female mice by the 6th day post-wounding. By contrast, zero of 12 control animals had complete epidermal wound closure at the same time-point. 12 days after biopsy, the newly formed epidermis in L. reuteri-treated mice was normal and lacked regenerative hyperplasia, indicating a rapid rate of epithelial remodeling. (c) Wound area at each of three time points decreases significantly in both male and female mice fed L. reuteri. Female mice fed L. reuteri exhibit more significant wound closure compared to controls versus male mice at 6 and 12 days. (d) Wound size diminishes more rapidly in mice fed L. reuteri with the increased rate of wound closure, accompanying a smaller epidermal gap in both male and female mice. Hematoxylin and Eosin. Scale bars: a =  250 µm. (3 day: Male: Control (n = 6), Control + LR (n = 7), Female: CD (n = 6), CD+LR (n = 8); 6 day: Male: Control (n = 12), Control + LR (n = 12); Female: Control (n = 12), Control + LR (n = 12); 12 day: Male: Control (n = 7), Control + LR (n = 7), Female: Control (n = 9), Control+LR (n = 8)).

Figure 2. Immune cell profile of wounds in L. reuteri-treated mice differ from untreated counterparts.

(a) Compression of the wound healing cascade in L. reuteri-treated mice results in neutrophil departure by day 6 and the beginning of collagen deposition showing advanced healing. Control animals wounds' remain populated with neutrophils and other innate immune cell infiltrates, indicative of a comparatively early stage of wound healing. (b) Histopathology of the granulation tissue in wounds of male mice. Early granulation tissue in control mice characterized by activated plump fibroblasts, (c) minimal amount of collagen, and (d) abundant neutrophils with (e) small numbers of Treg cells. The granulation tissue of mice fed L. reuteri is more mature with (b) elongated fibroblasts and a chronic inflammatory component (lymphocytes), (c) increased collagen deposition, and (d) small numbers of neutrophils and (e) abundant Treg cells. (6 day: Male: Control (n = 12), Control + LR (n = 12); Female: Control (n = 12), Control + LR (n = 12)). (b) Hematoxylin and Eosin. (b) Masson's Trichrome. (d) and (e) Immunohistochemistry: Diaminobenzidine chromogen, Hematoxylin counterstain. Scale bars = 50 µm.

Figure 3. Adoptive cell transfer of Foxp3+ Tregs.

Spleen and mesenteric lymph nodes from C57BL/6 donor mice are sorted for CD4+GFP+ (Foxp3+) regulatory T cells. Collected cells are transferred to lymphocyte-naive Rag2–/– C57BL/6 recipients. Transferred Foxp3+ cells migrate to the skin, where they act during wound healing.

Figure 4. L. reuteri-primed Foxp3+ Tregs condense wound healing time course in Rag2–/– recipients.

(a) Direct microscopy of formalin-fixed, paraffinized wounded skin of aged Rag2–/– C57BL/6 mice (6 days post-wounding). Wound margins are delineated with yellow outlines. The healing time course is faster in mice receiving L. reuteri-primed Foxp3+ Tregs as evidenced by significantly reduced wound sizes in both male and female recipient mice. (b) Significantly advanced re-epithelialization of wounds of Rag2–/– mice after adoptive cell transfer of Foxp3+ cells from L. reuteri-treated donors. (c) Early granulation tissue in mice receiving Foxp3+ Tregs from untreated controls is characterized by minimal amount of collagen, and (d) abundant neutrophils with (e) small numbers of Foxp3+ lymphocytes. The granulation tissue of mice receiving L. reuteri-primed Tregs is more mature with (c) increased collagen deposition, and (d) occasional neutrophils and (e) increased accumulation of the transferred Foxp3+ cells lymphocytes. (LR Foxp3+GFP+ cells (n = 6), Untreated Foxp3+GFP+ cells (n = 5) for each gender.) b) Hematoxylin and Eosin. (b) Masson's Trichrome. (d) and (e) Immunohistochemistry: Diaminobenzidine chromogen, Hematoxylin counterstain. Scale bars (b) = 250 µm; (c), (d) and (e) = 50 µm.

Figure 5. Depletion of CD25+ cells abolishes the L. reuteri effect in wound healing while depletion of IL-17 restores the wound healing benefit.

(a) Male and female C57BL/6 mice (n = 8 per group) depleted of CD25+ cells by anti-CD25 antibody have larger wounds when compared with sham isotype IgG-treated control mice, despite uniform L. reuteri consumption in both groups. (b) The wounds of CD25 cell-depleted mice do not show the histopathological evidence of the typical L. reuteri-induced accelerated wound repair process, namely sham IgG exhibit complete epidermal closure and mature granulation tissue filling of the wound gap at 6 days after biopsy. (c) (d) Depletion of IL-17A benefits wound healing closure. Hematoxylin and Eosin (b and d). Scale bars =  250 µm.

Figure 6. L. reuteri accelerates wound repair via an oxytocin-associated mechanism in both male and female mice.

(a) Oxytocin-deficient male mice have significantly larger wounds in day 6 after wounding compared to wild-type control mice despite consumption of L. reuteri. Wound margins are delineated with yellow outlines. (b) Impaired wound healing in oxytocin-deficient mice is characterized by delayed re-epithelialization and granulation tissue formation. The mature granulation tissue of control mice is characterized by fibrosis and vessels running perpendicularly to the layers of elongated fibroblasts. In oxytocin-deficient mice there is still edematous (early) granulation tissue with an acute inflammatory component. (c) Oxytocin-deficient mouse wounds show minimal collagen deposition and significantly more (d) neutrophils. (b) Hematoxylin and Eosin. (c) Masson's Trichrome (d). Immunohistochemistry (Diaminobenzidine chromogen, Hematoxylin counterstain). Scale bars (b) = 25 µm; (c) (d) and (e)  = 50 µm.

Figure 7. The oxytocin-dependent effect of L. reuteri is mediated by CD4+CD45RBloCD25+ Tregs.

(a) Transferring CD4+CD45RBloCD25+ regulatory T (Treg) cells from oxytocin-potent L. reuteri-fed donor mice is sufficient to recapitulate the beneficial effects of probiotic consumption in the closure of cutaneous biopsy defects in Rag2–/– recipient mice. By contrast, Rag2 mice that got these same cells from L. reuteri-fed oxytocin-deficient donors failed to benefit, and instead presented large wounds at 6 days post- wounding. Whereas wounds of recipient mice of CD4+CD45RBloCD25+ cells of wild-type mice had accelerated wound healing, the recipients of oxytocin-deficient Tregs showed histopathological features of delayed wound healing, including significantly (b) delayed re-epithelialization, (c) decreased collagen deposition, (d) increased numbers of neutrophils, (e) and decreased regulatory T-cell in their wound counts. (b) Hematoxylin and Eosin. (c) Masson's Trichrome. (d and e) Immunohistochemistry; Diaminobenzidine chromogen, Hematoxylin counterstain. Scale bars: (b) =  250 µm; (c), (d) and (e) =  50 µm.

Figure 8. L. reuteri primes T regulatory cells via an oxytocin-dependent mechanism for enhanced wound healing.

Wild type mice fed L. reuteri in their drinking water exhibit enhanced wound healing over untreated controls. This effect is entirely transferable with Foxp3+ Tregs primed in a mouse host feeding on L. reuteri. In the absence of oxytocin, feeding mice with L. reuteri provides no wound healing benefit, and the CD4+CD25+CD45Rblo Tregs from these animals are unable to convey wound-healing advantages.