Macrophages are an essential component of innate immunity and have a broad role in the maintenance of tissue homeostasis. Two major polarization states have been described for macrophages, the pro-inflammatory type 1 (M1) and the anti-inflammatory type 2 (M2). The ratio between M1 and M2 phenotype determines the progression and/or resolution of chronic diseases, including wounds-healing. Wounds contain macrophagic phenotypes associated with both classical and alternative activation. During the early stages of inflammation, around 85% of macrophages have an M1 phenotype. This ratio switches 5-7 days post injury where only 15-20% of macrophages have an M1 phenotype and the wound is primarily populated by M2 phenotype. Taken together, these data indicate that a better understanding of the macrophagic role in wounds-healing is still required in order to design more efficient therapeutic strategies.
In a recent in vivo study, we have demonstrated that bovine lactoferrin (bLf), a milk-derivative glycoprotein, is able to facilitate the post-surgical wound-healing in subjects suffering from bisphosphonate-related osteonecrosis of the jaws that showing the progressive destruction of bone. The results consist in a significant shorter time of wound closure (1-2 weeks), after surgical removal of the necrotic bone, compared to that observed with classical treatment (2-3 months).
Furthermore, it has been reported that, depending on M1 or M2 polarization, macrophages express at different extent a set of genes related to iron homeostasis leading to an "iron-retention" or an "iron-release" phenotype, characteristic of M1 and M2 phenotypes, respectively. Recently, we have demonstrated that bLf modulates the entire iron homeostasis machinery switching macrophages from the "iron-retention" to the "iron-release" phenotype.
Consequently, the aim of this project is a better characterization of the bLf-induced macrophagic phenotype in order to investigate its role in wound-healing.
Wound-healing and tissue repair are critical biological processes that are fundamental to the survival of all living organisms(1). When tissues are injured during mechanical injury, an inflammatory response is induced. This inflammatory status is characterized by the recruitment, proliferation, and activation of a variety of hematopoietic and non-hematopoetic cells which together make up the cellular response that orchestrates wounds healing and tissue repair (2).
When the wound-healing response is well organized and controlled, the inflammatory response resolves quickly, and normal tissue architecture is restored. However, if the wound-healing response is chronic or becomes dysregulated, it can lead to the impairing normal tissue function and ultimately leading to organ failure and death (3). Therefore, wound-healing responses must be tightly regulated. Although many cell types are involved in tissue repair (4), macrophages have been shown to exhibit critical regulatory activity at all stages of wounds healing (5). As they represent potentially important therapeutic targets, there has been a great deal of interest over the past few years in deciphering the contributions of the different macrophage populations that control the initiation, maintenance, and resolution of wound-healing responses in different organ systems.
Wounds contain macrophages phenotypes associated with both classical and alternative activation, the ratio of which alters as the wound matures (6). During the early stages of inflammation, around 85% of macrophages have an M1 phenotype and avidly secrete pro-inflammatory mediators, while 15% have an M2 phenotype that secrete anti-inflammatory cytokines and growth factors (6). This ratio switches as the wound matures so that 5-7 days post injury only 15-20% of macrophages have an M1 phenotype and the wound is primarily populated by M2 phenotype (6,7).
In this respect, a recent in vivo study (8) emphasizes the role of bovine lactoferrin (bLf) in accelerating the wounds-healing, after surgical removal of the necrotic bone, in patients suffering from bisphosphonate-related osteonecrosis of the jaws.
Human lactoferrin (hLf), a multifunctional iron binding glycoprotein secreted by exocrine glands and neutrophils, exerts a relevant anti-inflammatory activity (9). BLf possesses a high homology of sequence with hLf and is used in vitro models and in human clinical trials. BLf is able (i) to chelate two ferric ions/molecule thus inhibiting microbial growth, (ii) to interact with cellular and molecular components due to its high pI>9, (iii) to enter inside the nucleus where, by binding to specific DNA sequences, inhibits pro-inflammatory cytokine synthesis and (iv) to bind at high affinity to hLf receptor (hLfR) and at low affinity to low density lipoprotein receptor related protein (LRP) (9,10). It has been proposed that hLfR and LRP could influence bLf internalization, localization into the nucleus, and consequently, anti-inflammatory activity (11-13) and cell damage repair (14,15).
Notably, recent studies have proposed bLf as an immune-modulator, able to counteract the inflammation-induced changes of the iron homeostasis system in macrophages (16,17). In this respect, it has been reported that, depending on M1 or M2 polarization, macrophages express at different extent a set of genes related to iron homeostasis leading to an "iron-retention" or an "iron-release" phenotype, characteristic of M1 and M2 phenotypes, respectively (18,19).
In this study, in M1 human macrophages, bLf modulates the entire iron homeostasis machinery switching macrophages from the "iron-retention" to the "iron-release" phenotype. In addition, bLf decreases IL-6 and IL-1ß while increases IL-10 levels (16).
Some Papers indicate a pivotal role of bLf in promoting the formation of granulation tissue and re-epithelialization, but the molecular mechanisms involves in healing-wounds are yet unclear (8,14,15). For this purpose, bLf could lead to switch of the macrophagic phenotype from M1 to M2. Consequently, the innovation of this project concerns a better characterization of the bLf-induced macrophagic phenotype in order to use this protein in accelerating wound-healing process and, then, to avoid the wounds chronicization that could lead to tissue damage.
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