Time Heals All Wounds … But Wounds Heal Faster with Lactobacillus

Chronic nonhealing wounds represent a significant clinical problem and cost the healthcare system $19 billion annually. Recently, Vågesjö et al., 2018Vågesjö E. Öhnstedt E. Mortier A. Lofton H. Huss F. Proost P. Roos S. Phillipson M. Accelerated wound healing in mice by on-site production and delivery of CXCL12 by transformed lactic acid bacteria.Proc. Natl. Acad. Sci. USA. 2018; 115: 1895-1900Crossref PubMed Scopus (73) Google Scholar demonstrated a promising therapeutic approach for nonhealing wounds with topical application of CXCL12-producing Lactobacilli that enhanced healing through alterations in the wound microenvironment. Chronic nonhealing wounds represent a significant clinical problem and cost the healthcare system $19 billion annually. Recently, Vågesjö et al., 2018Vågesjö E. Öhnstedt E. Mortier A. Lofton H. Huss F. Proost P. Roos S. Phillipson M. Accelerated wound healing in mice by on-site production and delivery of CXCL12 by transformed lactic acid bacteria.Proc. Natl. Acad. Sci. USA. 2018; 115: 1895-1900Crossref PubMed Scopus (73) Google Scholar demonstrated a promising therapeutic approach for nonhealing wounds with topical application of CXCL12-producing Lactobacilli that enhanced healing through alterations in the wound microenvironment. Impaired wound healing in type 2 diabetes is a major clinical problem, as it is presently the leading cause of lower-extremity amputation in the United States. Importantly, amputation is associated with a 50% mortality rate at 5 years, a survival rate worse than most cancers. Wound healing consists of integrated and overlapping phases of hemostasis and coagulation, inflammation, proliferation, and resolution and remodeling (Gurtner et al., 2008Gurtner G.C. Werner S. Barrandon Y. Longaker M.T. Wound repair and regeneration.Nature. 2008; 453: 314-321Crossref PubMed Scopus (3865) Google Scholar). These phases must occur at prescribed times and continue for a specific duration, or pathological wound healing ensues. Chronic wounds, like those seen in type 2 diabetes, fail to progress through these discrete stages because of dysregulated inflammation that occurs during the inflammatory phase of wound healing. In normal wound healing, the early innate inflammatory response is critical for establishing the healing cascade. During the inflammatory phase, tissue damage recruits progenitor cells and immune cells, mainly macrophages, through the release of chemokines, such as CXCL12 (Stromal cell-Derived Factor 1α), to the site of tissue injury. As the course of wound healing progresses, macrophages transition to an anti-inflammatory phenotype and promote tissue repair through angiogenesis and growth factor release, including interleukin (IL)-10, transforming growth factor (TGF)-β, and vascular endothelial growth factor. This shift in macrophage phenotype can be driven by a variety of epigenetic and environmental factors, including changes in the wound microenvironment (Boniakowski et al., 2017Boniakowski A.E. Kimball A.S. Jacobs B.N. Kunkel S.L. Gallagher K.A. Macrophage-mediated inflammation in normal and diabetic wound healing.J. Immunol. 2017; 199: 17-24Crossref PubMed Scopus (225) Google Scholar). Impaired wound healing in diabetes is multifactorial; however, persistent, uncoordinated inflammation with a failure of macrophages to progress from a proinflammatory to an anti-inflammatory phenotype is a hallmark of chronic non-healing wounds in diabetes (Davis et al., 2018Davis F.M. Kimball A. Boniakowski A. Gallagher K. Dysfunctional wound healing in diabetic foot ulcers: new crossroads.Curr. Diab. Rep. 2018; 18: 2Crossref PubMed Scopus (125) Google Scholar). The ability to fully understand and control the initiation and resolution of inflammation in wound macrophages is critical to advancing wound repair. At present, what specifically drives changes in the wound microenvironment and macrophage phenotype throughout the course of healing is unknown. Over the past decade, substantial work has been devoted to identifying therapies that may augment the wound-healing process. The current standard of care for chronic wounds involves repeated dressing changes, wound debridement, antibiotics, and avoiding increased pressure to the injured site. Unfortunately, this treatment is relatively ineffective, with a substantial percentage of wounds remaining unhealed and at risk for infection. As such, a number of experimental and clinical trials have sought to investigate novel therapies for chronic wounds using biomaterials and local application of growth factors or stem cells (Everett and Mathioudakis, 2018Everett E. Mathioudakis N. Update on management of diabetic foot ulcers.Ann. N Y Acad. Sci. 2018; 1411: 153-165Crossref PubMed Scopus (262) Google Scholar). However, the ability to translate these preclinical studies into clinical therapies has often been hindered by a combination of clinical feasibility, regulation, or limited drug bioavailability in the wound microenvironment. The recent publication by Vågesjö et al. in Proceedings of the National Academy of Sciences provides a different avenue for the treatment of chronic wounds with the use of Lactobacilli to deliver CXCL12 locally, which results in alterations in the wound microenvironment to reinforce the immune cells that drive the healing process (Vågesjö et al., 2018Vågesjö E. Öhnstedt E. Mortier A. Lofton H. Huss F. Proost P. Roos S. Phillipson M. Accelerated wound healing in mice by on-site production and delivery of CXCL12 by transformed lactic acid bacteria.Proc. Natl. Acad. Sci. USA. 2018; 115: 1895-1900Crossref PubMed Scopus (73) Google Scholar). For their study, Vågesjö et al. used Lactobacillus reuteri (L. reuteri; a member of the human microbiome) transformed with a pSIP_CXCL12 plasmid that when applied topically resulted in vivo in elevated CXCL12 levels for the first hour and sustained CXCL12 levels in adjacent epidermis and hair follicles for 2 days after wound induction. To evaluate the effects on wound healing, the authors used the well-established murine hindlimb wound model and administered daily CXCL12-producing L. reuteri (2 × 107 cfu) to the wounds. This resulted in a significant improvement in the rate of wound healing, which was most prominent during the first 24 hr. The authors demonstrated that the improvement of wound healing was dependent not only upon the administration of CXCL12 but also on the combined presence of lactic acid producing L. reuteri. As such, only continuous delivery of recombinant CXCL12 (rCXCL12; 0.2 g given every tenth minute), but not daily topical application of supernatants from CXCL12-producing L. reuteri or rCXCL12, resulted in improved wound healing. The authors hypothesized that this may be secondary to degradation of CXLC12 by membrane-bound CD26. The CD26 enzyme is a pH-dependent enzyme which degrades CXCL12 and has an optimal activity at pH 8.3. To investigate their hypothesis, rCXCL12 was topically administered in buffers with a range of pH values (ranging from 5.35 to 7.35), as it is known that lactic acid production by L. reuteri causes a decrease in pH of 0.2–0.5 in vitro. Interestingly only at pH 6.35 did the rCXCL12 lead to improved wound closure. It was thereby inferred that production of lactic acid by L. reuteri may alter the local wound environment such that the pH is decreased, inhibiting CD26 and allowing for increased bioavailability of CXCL12. The local effects of sustained CXCL12 administration on wound healing were gleaned from analysis of growth factors and immune cells in the wound bed. Following application of CXCL12-producing L. reuteri, there were increased levels of proliferating cells, F4/80 resident macrophages, and the growth factor TGF-β within the dermis, suggesting bacteria-delivered CXCL12 may amplify the repair process (Figure 1). Unfortunately, the levels of infiltrating macrophages (CCR2, CX3CR1, Ly6C), and specifically the levels of proinflammatory (CX3CR1Lo, Ly6CHi) versus anti-inflammatory (CX3CR1Hi, Ly6CLo) macrophages, were not analyzed. Without such data, we are unable to determine if CXCL12 administration resulted in a phenotypic shift in the infiltrating wound monocyte and macrophages. Chronic wounds are often associated with underlying pathologic processes such as peripheral arterial disease or diabetes. Using murine models of limb ischemia and hyperglycemia, CXCL12-producing L. reuteri significantly improved the rate of wound healing over the first 4 days in ischemic animals but only improved the rate of wound healing for the first 24 hr in hyperglycemic mice. The absence of a substantial improvement in the rate of wound healing in diabetic mice may be secondary to systemic changes (i.e., epigenetic modification) in diabetic macrophages that result in the persistence of a proinflammatory phenotype (Kimball et al., 2017Kimball A.S. Joshi A. Carson 4th, W.F. Boniakowski A.E. Schaller M. Allen R. Bermick J. Davis F.M. Henke P.K. Burant C.F. et al.The histone methyltransferase MLL1 directs macrophage-mediated inflammation in wound healing and is altered in a murine model of obesity and type 2 diabetes.Diabetes. 2017; 66: 2459-2471Crossref PubMed Scopus (46) Google Scholar) that is not reversed by excess CXCL12. In order to translate their findings, L. reuteri delivering CXCL12 was also tested on healthy human skin samples in vitro and improved the rate of re-epithelialization. Current therapeutic options for the treatment of chronic nonhealing wounds rely heavily on the removal of necrotic tissue, offloading of pressure, and antibiotic therapies. Despite these “macroenvironment” treatment options, it is the wound “microenvironment,” consisting of macrophages, neutrophils, cytokines, fibroblasts, and proteases, that works in concert to drive the course of the healing. Recently, a number of preclinical studies have sought to intervene within the wound microenvironment and have shown beneficial results with local administration of growth factors or chemokines, alone or coupled with biogels. Indeed, several preclinical studies have demonstrated that application of CXCL12 resulted in improvement in wound healing (Gallagher et al., 2007Gallagher K.A. Liu Z.-J. Xiao M. Chen H. Goldstein L.J. Buerk D.G. Nedeau A. Thom S.R. Velazquez O.C. Diabetic impairments in NO-mediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha.J. Clin. Invest. 2007; 117: 1249-1259Crossref PubMed Scopus (553) Google Scholar, Henderson et al., 2011Henderson P.W. Singh S.P. Krijgh D.D. Yamamoto M. Rafii D.C. Sung J.J. Rafii S. Rabbany S.Y. Spector J.A. Stromal-derived factor-1 delivered via hydrogel drug-delivery vehicle accelerates wound healing in vivo.Wound Repair Regen. 2011; 19: 420-425Crossref PubMed Scopus (48) Google Scholar, Olekson et al., 2015Olekson M.A.P. Faulknor R. Bandekar A. Sempkowski M. Hsia H.C. Berthiaume F. SDF-1 liposomes promote sustained cell proliferation in mouse diabetic wounds.Wound Repair Regen. 2015; 23: 711-723Crossref PubMed Scopus (35) Google Scholar). In addition, there are multiple ongoing clinical trials evaluating the efficacy of local growth factor administration on wound healing (Everett and Mathioudakis, 2018Everett E. Mathioudakis N. Update on management of diabetic foot ulcers.Ann. N Y Acad. Sci. 2018; 1411: 153-165Crossref PubMed Scopus (262) Google Scholar). However, attempts to translate therapies from preclinical studies to clinical treatments are often limited by a combination of regulatory oversight, clinical feasibility, effectiveness, and side-effect profile. In addition, potentially effective treatment regimens that involve repeated dosing are costly and resource intensive. A drug-delivery vehicle that deposits agents in a time-release fashion and therefore harnesses the advantages of continuous dosing while still being delivered in a manner which is simple and cost effective would be highly beneficial to patients and healthcare providers alike, and would result in significant cost savings. The use of Lactobacilli as a vector to deliver CXCL12 to nonhealing wounds has the potential to provide many of these treatment advantages. In addition, Vågesjö et al. should be applauded for their attempt to address questions regarding clinical delivery, as they have pioneered freeze-dried formulations of CXCL12-producing bacteria which maintained stability and efficacy on wound healing. The ability to store and reconstitute a formulation of CXCL12-producing bacteria results in increased clinical applicability for healthcare providers. Of equal importance, the authors also demonstrated that topically applied CXCL12-producing L. reuteri did not result in systemic expression of CXCL12 or bacteremia. This is of great importance, as diabetic wounds are prone to bacterial infection resulting in severe illness, and increased systemic levels of CXCL12 have been associated with tumorigenesis and metastasis (Orimo et al., 2005Orimo A. Gupta P.B. Sgroi D.C. Arenzana-Seisdedos F. Delaunay T. Naeem R. Carey V.J. Richardson A.L. Weinberg R.A. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.Cell. 2005; 121: 335-348Abstract Full Text Full Text PDF PubMed Scopus (2908) Google Scholar). Ultimately, the overall clinical and translational appeal of this study is that Lactobacilli can likely be used as a vector to effectively deliver a wide array of chemokines and other proteins, not simply CXCL12, to the local wound tissue. In doing so, this represents a diverse strategy that can be harnessed for safe and efficient delivery of therapeutics that drive the wound microenvironment toward one of rapid healing. The Lactobacilli vector delivery system represents a promising technology for the unsolved problem of delayed cutaneous wound healing. We would like to acknowledge Robin Kunkel for her assistance with the graphical illustration. Research in the laboratory is supported by NIH RO1- HL137919 (K.G.), NIH F32 DK117545-01 (F.D.), NIH DK-102357 (K.G.), the Doris Duke Charitable Foundation (K.G.), and the Vascular and Endovascular Surgery Society (F.D.).