Accelerated wound healing in mice by on-site production and delivery of CXCL12 by transformed lactic acid bacteria

Abstract

Impaired wound closure is a growing medical problem associated with metabolic diseases and aging. Immune cells play important roles in wound healing by following instructions from the microenvironment. Here, we developed a technology to bioengineer the wound microenvironment and enhance healing abilities of the immune cells. This resulted in strongly accelerated wound healing and was achieved by transforming Lactobacilli with a plasmid encoding CXCL12. CXCL12-delivering bacteria administrated topically to wounds in mice efficiently enhanced wound closure by increasing proliferation of dermal cells and macrophages, and led to increased TGF-β expression in macrophages. Bacteria-produced lactic acid reduced the local pH, which inhibited the peptidase CD26 and consequently enhanced the availability of bioactive CXCL12. Importantly, treatment with CXCL12-delivering Lactobacilli also improved wound closure in mice with hyperglycemia or peripheral ischemia, conditions associated with chronic wounds, and in a human skin wound model. Further, initial safety studies demonstrated that the topically applied transformed bacteria exerted effects restricted to the wound, as neither bacteria nor the chemokine produced could be detected in systemic circulation. Development of drugs accelerating wound healing is limited by the proteolytic nature of wounds. Our technology overcomes this by on-site chemokine production and reduced degradation, which together ensure prolonged chemokine bioavailability that instructed local immune cells and enhanced wound healing.

Keywords: Lactobacillus reuteri; blood flow; chemokine; diabetes; macrophage.

Conflict of interest statement

Conflict of interest statement: The technology of transformed Lactobacillus reuteri-producing chemokines is filed for patent protection (PCT/EP2015/081146, WO2016/102660), and drug candidates using this technology are being developed by a company of which E.V., S.R., and M.P. are shareholders.

Copyright © 2018 the Author(s). Published by PNAS.

Figures

Fig. 1.

Local delivery of CXCL12 to wounds by L. reuteri. (A) Luminescent L. reuteri added to hind-limb wounds in anesthetized mice. (B) Quantification of plasmid expression (n = 5). (C) Overview image of a skin wound (red square shows area of analysis in the dermis). (Scale bar, 100 µm.) (D) Skin next to the wound stained for CXCL12 (green) in mice receiving no treatment (control) or different doses of CXCL12-producing L. reuteri (2 × 107, 1 × 109, and 4 × 109 cfu LB_CXCL12). Levels of CXCL12 were quantified in the dermis (E), epidermis (F), and hair follicles (G) after treatment with different doses of LB_CXCL12 (*P < 0.05).

Fig. 2.

Accelerated wound healing by treatment with CXCL12-producing L. reuteri. (A) Wounds receiving no treatment (control), treatment with control L. reuteri (LB), and CXCL12-expressing L. reuteri (LB_CXCL12) at the time of wound induction (0 h) and at 24 h. Wound sizes (B) and accumulated wound surface area at 2 d (C). (D) Fractions of wounds healed by different treatments at days 6, 8, and 10 (control, n = 4; LB, n = 3; LB_CXCL12, n = 8; *P < 0.05).

Fig. 3.

Accelerated wound healing by prolonged administration of rCXCL12. Wound size (A) and accumulated wound surface area (B) were quantified for 2 d in mice that were untreated (n = 15), treated with supernatants from induced LB (control supernatant, n = 4) or LB_CXCL12 (CXCL12 from LB, n = 5), or treated with 1.0 µg (n = 4), 0.6 µg (n = 5) or 0.2 µg (n = 10) rCXCL12 at one time point per day or with 0.2 µg rCXCL12 at six time points during 1 h each day (n = 5). (C) Cleavage of CXCL12 by CD26 at different pH over 30 min in vitro where the curves at pH 6.3 and pH 5.3 overlap. Wound size (D) and accumulated wound surface area (E) after 0–2 d in mice treated with rCXCL12 in buffers with different pH at one time point daily (control/saline, n = 12; rCXCL12 7.35, n = 10; rCXCL12 6.35, n = 4; rCXCL12 5.35, n = 4; *P < 0.05).

Fig. 4.

Early and local effects in the wounds treated with CXCL12-producing L. reuteri. (A and B) Densities of proliferating cells in dermis and epidermis close to the wound edge (0–250 µm) and (C) quantifications of dermal TGF-β levels. The density of F4/80+ macrophages is shown (D), along with fractions of macrophages expressing TGF-β (E) and MMR (F) in this area at 24 h after wound induction (A–F; n = 3 for all groups, n = 7–16). The density and fraction of macrophages expressing CXCR4 (G and H) or CXCL12 (I and J) in the wound area at 24 h after different treatments are shown: (K–N) efficacy of macrophage depletion using clodronate liposomes and the corresponding effect on wound healing in mice in which wounds were untreated (K and L) or treated with CXCL12-producing L. reuteri (M and N, control, n = 7; control and clodronate liposomes, both n = 5, n = 7–13; LB_CXCL12, n = 8; clodronate liposomes LB_CXCL12, n = 5, n = 7–9; *P < 0.05).

Fig. 5.

Reepithelialization of wounds inflicted in cultured disks of human skin after treatment with L. reuteri (LB) or LB expressing human CXCL12 (LB_hCXCL12). (A) Epidermis and dermis at the wound edge in cultured skin disks after 14 d (arrows indicate newly formed epidermal sleeves). Length of epidermal sleeves were measured from the wound edge (B), and the number of basal Ki67+ keratinocytes within 250 µm of the wound edge was measured (C) and quantified (D, n = 6, n = 6). (E) pH of medium after 24 h of culture. (F) Epidermis and dermis at the wound edge in skin disks after 14 d in culture. MHC class II+ cells appear in brown and are quantified in G (n = 6, n = 6); *P < 0.05 as indicated by braces or vs. 10% group.

Fig. 6.

Wound healing in mice with reduced skin perfusion or hyperglycemia. Wound size over time in mice with skin ischemia (A) and accumulated wound surface area quantified for 2 d (B). (C) Fractions of the wounds healed by the different treatments at days 6, 8, and 10 (n = 4 for all groups). (D) Wound size over time in severely hyperglycemic mice (blood glucose >16.4 mmol/L) and accumulated wound surface area at 2 d (E). (F) Fractions of wounds healed by different treatments at days 6, 8, and 10 (hyperglycemic control, n = 19; hyperglycemic LB, n = 8; hyperglycemic LB_CXCL12, n = 11). (G) Wound size over time in moderately hyperglycemic mice (blood glucose >11.1 mmol/L) and accumulated wound surface area at 2 d (H). (I) Fractions of the wounds healed by the different treatments at days 6, 8, and 10 (n = 4 for all groups; *P < 0.05).

Fig. 7.

Safety and pharmacokinetics of LB_CXCL12 for development of clinical wound treatment. (A) Plasma levels of CXCL12 in unaffected mice and in mice in which wounds were induced and blood collected after 3 h of treatment (n = 3 for all groups). Red line indicates detection limit. (B) Difference in perfusion in the skin surrounding the wound (0–300 µm) and an unaffected area of the skin used as reference, that is, delta perfusion (control and LB, n = 4; LB_CXCL12, n = 5). In vitro expression (C, n = 4–8) and in vivo expression (D, n = 5) of LB_Luc immediately resuspended from a freeze-dried formulation applied to the wound surface of 1-d-old cutaneous wounds. Wound size (E) and accumulated wound surface (F) over time in healthy mice in which wounds were treated with immediately revived control LB or LB_CXCL12 (n = 5 for all groups; *P < 0.05).