This is the first report of a role for NKT cells in cutaneous wound repair. Using the excisional punch wound model, we demonstrated that early wound closure was accelerated in the absence of NKT cells. Importantly, we also made the novel observation that NKT cells themselves are a constituent of the early wound inflammatory infiltrate, and their kinetics of accumulation coincide with neutrophil influx. We propose that under normal, nonpathologic wound repair processes that occur within the wound environment, NKT cells exert a regulatory role, since their absence led to increased local production of a select subset of neutrophil and monocyte/macrophage chemokines. Specifically, the dermal production of MIP-2, KC, MIP-1α, and MIP-1β was transiently enhanced when NKT cells were absent. This transient enhancement occurred during the first 3 d after wounding and correlated with the kinetics of NKT cell infiltration.
Despite the elevation of select neutrophil and monocyte/macrophage chemoattractants, wounds from NKT-deficient animals did not contain greater numbers of neutrophils or macrophages. Additionally, while inflammatory infiltrates were identical, wounds from NKT cell deficient animals had 5-fold greater levels of the fibrogenic cytokine TGF-β1 at 1 d post-wounding, which correlated with markedly enhanced procollagen gene expression in wounds from NKT cell deficient animals at the same time point (d 1). Day three wounds from NKT cell deficient animals contained more collagen than wild type wounds. Therefore, wounds from NKT cell deficient animals display augmented matrix deposition, consistent with an accelerated rate of closure.
Our current findings alter, and in some cases challenge, conventional dogma regarding the functionality of both the chemokines within the wound environment and NKT cells in general. Within the wound, chemokines are typically associated with inflammation, chemotaxis of inflammatory cells, and local immunity [30
]. At first glance, these functions might seem inconsistent with accelerated wound closure as we observed among NKT cell deficient mice. NKT cells are traditionally associated with regulation of peripheral immunity and host defense. There are numerous reports that pair single NKT cell derived cytokines (either IL-4 or IFN-γ
) with target cell(s) activation and cytokine production. In the studies reported here, we demonstrated alterations in the local production of a select chemokine subset when NKT cells were absent, suggesting that their regulatory functions can be more global than single cell, single mediator pairings. We hypothesize that the NKT cell regulates neutrophil and macrophage functions. The exact nature of the NKT cell–inflammatory cell interaction also requires further study, but the NKT cell literature provides examples of such an interaction [19
While the traditionally cited function of chemokines is leukocyte chemoattraction to sites of inflammation, it is known that chemotactic responses reach a threshold for recruitment despite increasing concentrations of the chemokine [30
]. Thus, it is reasonable to suggest that a nonpathologic healing wound may already be at or near such a threshold and the increased levels of chemokines seen in the wounds of NKT cell deficient mice do not further elicit cell recruitment, but instead activate additional cells within the wound (i.e., keratinocytes and fibroblasts) to accelerate the epithelial closure and fibroproliferative aspects of wound healing. It is also important to emphasize the transient nature of the chemokine elevations, which may not have offered time for additional cell recruitment. The seemingly paradoxical finding of accelerated wound closure despite enhanced pro-inflammatory signals becomes explicable when one considers the myriad of functions the chemokines exhibit within the wound environment. Beyond leukocyte recruitment, chemokines have known roles in angiogenesis, fibrosis, and re-epithelialization [32
]. These alternative functions of chemokines might explain how augmented pro-inflammatory signals are not inconsistent with accelerated wound closure, especially given the transient nature of their elevation and the lack of additional inflammatory cell recruitment.
NKT cells are known to act locally in varied circumstances as others have described NKT cells infiltrating a variety of organs during states of inflammation [19
] including the skin, where NKT cells appear to instigate psoriatic plaques in susceptible models or individuals [36
]. It is not surprising, therefore, that NKT cells would infiltrate the skin during early wound healing, another instance of cutaneous inflammation, albeit more transient. Here, we provide the first in vivo
demonstration of NKT cells infiltrating the skin and, in contrast to these studies in psoriasis, NKT cells appear to negatively regulate the inflammatory process instead of propagating or perpetuating it. This difference is presumably due to differential cytokine secretion profiles resulting from APC-derived signals upon glycolipid antigen presentation. Our laboratory is currently investigating the wound-associated NKT cell phenotype further.
Other innate lymphocytes have been implicated in regulating cutaneous inflammation such as psoriasis. The γδ
TCR-bearing dendritic epidermal T cell (DETC) regulates epidermal homeostasis in normal skin via
its secretion of insulin-like growth factor 1 (IGF-1) [39
], and epidermal renewal via
its secretion of FGF-7 and 10 during wound repair [12
]. Although there is evidence of an NKT–DETC interaction in mouse models of airway hyper-responsiveness [40
], there is no known interaction between these two innate lymphocytes in the context of psoriasis or cutaneous injury.
In summary, this is the first description of NKT cells’ involvement in cutaneous wound repair. These studies describe the kinetics of NKT cell infiltration into cutaneous wounds during the first 3 d of wound repair. During this same period, the NKT cell also regulates the local production of selected chemokines, TGF-β1, and collagen deposition. NKT cells’ effect on the overall rate of wound closure also occurs during the first 3 d. During this early phase, their influence on wound repair is focused, transient, but dramatic. This specificity positions the NKT cell as an attractive therapeutic target for preventing nonhealing chronic wounds where the inflammatory phase becomes exaggerated or delayed.