Murine IL-17⁺ Vγ4 T lymphocytes accumulate in the lungs and play a protective role during severe sepsis

Sepsis triggers a complex immune response that involves both innate and adaptive systems. γδ T lymphocytes represent a link between these two branches of the immune system, by coordinating the activation of different cell populations via cytokine production [27]. γδ T lymphocytes have been described as a major source of IL-17 in peritoneum and lymphoid organs during experimental sepsis, a phenomenon shown to present either beneficial or deleterious effects, depending on the experimental model [33‐35, 38]. The data presented here identifies the Vγ4 subset as a dominant producer of IL-17 in the lungs of septic mice and as a central T cell population involved in host defense against sepsis.

The experimental model of severe sepsis used in the present work resulted in the accumulation of T lymphocytes in lung tissue, which were likely originated from lymphoid organs. Increased numbers of both γδ and αβ T lymphocyte subsets were detected in the lungs; however it is noteworthy that, differently from αβ T lymphocytes, γδ T cell numbers continually increased up to day 10 after CLP, mainly due to the accumulation of Vγ4 subset. The progressive accumulation of γδ T cells in the lungs of CLP-operated mice has been previously demonstrated by Hirsh and coworkers [7]; however, the presence of γδ T cell subtypes has not been described. The decrease in αβ T cell numbers observed at day 3 after CLP is in accordance with several reports in mice and humans that demonstrate a reduction in circulating CD3⁺ T lymphocytes during sepsis [39]. This reduction is explained by a massive apoptotic event of T lymphocytes, which is correlated with severity and mortality in experimental animals and patients [2, 39‐41]. The fact that the percentage of CD25⁺ T lymphocytes increased among γδ T lymphocytes in the lungs at early time points after CLP (day 1) suggests that γδ (but not αβ) T lymphocytes are constantly activated in lymphoid tissues during the course of sepsis and continuously migrate towards inflamed lungs. In accordance with our data, Matsushima and co-workers [40] demonstrated the early activation of γδ T lymphocytes from peripheral blood of patients with sepsis and systemic inflammatory response syndrome. These patients presented increased percentages of peripheral CD69⁺ γδ T cells at acute time points after injuries, whereas CD69 expression by αβ T cells did not increase during a 2-week period [40]. It is noteworthy that, in our study, such early activation was also evident for Vγ4 T cell population, as observed in mouse spleen and lungs 1 day after CLP.

The selective migration of γδ T lymphocyte subsets into the tissue during inflammation is dictated by elevated levels of chemoattractant mediators in the tissue and by the expression pattern of chemokine receptors on cell surface [9, 10, 15, 16, 42, 43]. Our results suggest that γδ and Vγ4 T cell migration into the lungs of CLP mice is likely accounted by the combined in situ accumulation of multiple chemokines. CLP-induced lung inflammation increased tissue levels of CCL2, CCL3 and CCL5, chemokines that are known to mediate γδ T lymphocyte migration in vivo and in vitro [44‐47]. Consistently, here we show that adoptively transferred γδ T cells preferentially accumulated in the lungs (rather than in blood or spleen) of recipient CLP mice. Even though CCL2, CCL3 and CCL5 are also chemoattractant for αβ T lymphocytes [48], the neutralization of these chemokines in CLP lung homogenates did not impair the chemotaxis of γδ^- T lymphocytes (data not shown), suggesting that these chemokines selectively dictate the migration of γδ T cells into the lungs in our model. The involvement of CCL25 in γδ T cell migration towards inflamed lungs during sepsis was also investigated by us, since CCL25 has been shown to attract IL-17⁺ γδ T cells into inflamed airways [15]. However, CCL25 was not enhanced in CLP mouse lungs (data not shown).

Our data demonstrate that the Vγ4 T lymphocyte subset predominated among the IL-17⁺ cell populations in CLP mouse lungs. In line with this, it has been demonstrated that mice lacking γδ T cells (but not αβ T cells) subjected to CLP failed to present elevated IL-17 levels in the plasma and peritoneal lavage, showing that γδ T cells are the major producers of IL-17 during experimental sepsis [33‐35, 38]. It has been established that, among murine γδ T lymphocytes, IL-17 production is restricted to Vγ4 and Vγ6 subtypes [27, 49]. Consistently with our data, Vγ4 T lymphocytes comprise the major subset that migrates into the lungs and have been shown to produce IL-17 in different experimental models [10, 13, 24‐30]. It is noteworthy that our supplemental data (Additional file 1: Figure S1A) demonstrate that IL-17 production by γδ T cells from CLP-mouse lungs predominated over the expression of other cytokines, including IFN-γ. These data are reinforced by the increase in the percentage of CD27^- γδ⁺ (and Vγ4⁺) population in CLP-mouse spleen and by the fact that, upon α-CD3 mAb in vitro stimulation, these cells were enriched for IL-17 but not for IFN-γ (Additional file 1: Figure S1B–C).

Increased numbers of γδ T lymphocytes in the blood, peritoneum and lungs have been correlated with sepsis positive outcome in patients and experimental animals [8, 14, 32, 35, 50]. Indeed, mice lacking γδ T lymphocytes and subjected to CLP presented increased mortality rate and decreased survival periods [14, 32]. The protective role of γδ T lymphocytes during sepsis results from the ability of these cells to produce inflammatory mediators capable to modulate other leukocyte populations, among which IL-17 is of particular importance [35, 51, 52]. Here we show that IL-17 production in the lungs of CLP mice depends on infiltrated Vγ4 γδ T cell subset, which likely contributes to host protective immune response. Since adverse roles have been proposed for IL-17 during experimental sepsis, the effect of IL-17 in the lungs of CLP mice needs further investigation. It has been described that IL-17 derived from γδ T cells promotes epithelial repair in different tissues [53‐55], suggesting that IL-17 produced by Vγ4 T cells might act on lung epithelium, promoting tissue repair and ameliorating mouse illness after CLP [33‐35, 56]. IL-17 has also been associated with neutrophil influx into inflamed tissue, which can lead to either protective or harmful outcomes [35, 37, 57, 58]. Concerning lung immune response, effective bacteria clearance by neutrophils reduces the risk of lung failure [36]; however, it is well known that excessive neutrophil activation and production of myeloperoxidase (MPO) can cause tissue damage [57]. In our study, we observed increased neutrophil numbers in the lungs of CLP mice, which was significantly reduced after anti-Vγ4 mAb treatment (data not shown), suggesting neutrophil involvement in the resolutive response. The involvement of tissue-recruited neutrophils coordinated by IL-17⁺ γδ T cells in tissue repair has been demonstrated in different experimental models. γδ T cell knockout (KO) mice submitted to inflammatory insults are shown to present reduced neutrophil and MPO accumulation in the lungs, liver and cornea, which correlated with increased lesions and delayed epithelial regeneration [53‐55]. Moreover, in a model of corneal epithelial abrasion, it was demonstrated that γδ T cells induced, via IL-17, the production of vascular endothelial growth factor (VEGF) by neutrophils, promoting corneal nerve regeneration [59]. Our study evidenced that IL-17⁺ Vγ4 T lymphocytes migrate into injured lungs of CLP mice, presenting a beneficial role during the course of sepsis.