Association between regulatory T cell activity and sepsis and outcome of severely burned patients: a prospective, observational study

There is accumulating evidence indicating that regulatory T cells (Tregs) play important roles in the maintenance of immunologic self-tolerance and in down-regulation of various immune responses [1]. Tregs have been shown to be important in regulating the immune responses in transplant rejection, tumor immunity, infectious diseases and allergy. Thus, there has recently been an increasing interest in investigating the biology of Tregs as well as its potential application in the treatment of immunity relevant illnesses.

Many types of Treg subsets have been reported in a variety of morbid conditions. It is now clear that immune regulatory cells consist of many distinct T cell subsets [2]. Among them, CD4⁺ Tregs have been demonstrated in a wide range of animal models and in humans [3, 4], and the forkhead/winged helix transcription factor p3 (FOXP3) has been suggested to represent a reliable intracellular marker for naturally occurring Tregs [5]. Most studies on CD4⁺ Tregs use a combination of CD25, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), FOXP3, IL-10 and/or transforming growth factor-β1 (TGF-β1) to define Treg populations [6].

The stress response to burn injury is similar to that of severe trauma or critical illness but differs in its severity and duration. The inflammatory response is triggered immediately after thermal injury and persists for almost five weeks postburn [7]. Superimposed severe infections can result in the suppression of one or more functions of the host immune system after major burns. Multiple mechanisms have been proposed to explain infection-induced immunosuppression, including an imbalance in the cellular T helper cell (Th1/Th2) or cytokine profile, induction of anergy, depletion of effector cells and most recently the activation of CD4⁺CD25⁺ Tregs [8]. The role of both naturally occurring CD4⁺CD25⁺ Tregs and IL-10-secreting Tregs in infection has been the subject of several recent excellent reviews [9, 10]. However, it seems that its response to trauma, burns, hemorrhagic shock, and microbial infection is associated with only a transient proinflammatory period followed by a more prolonged period of immune suppression [11]. Thus, it is speculated that there are some other factors involved in this process.

Numerous studies show that an increased burn size leads to higher mortality of burned patients [12, 13]. It was also implicated that the extent of burn size might be associated with the development of sepsis. It is now believed that along with the body's hyperinflammatory response designated to eliminate the invading pathogen, mechanisms primarily aimed at controlling this initial response are initiated, but may turn out to be deleterious with immune dysfunctions and even death. A similar state of immune suppression has been described after numerous forms of severe trauma [14‐16].

Although more and more evidence for immune dysfunction after sepsis has been accumulated the mechanisms underlying its development and how it acts to worsen the morbid state of the critically ill patient are yet to be elucidated. In this context, although the majority of clinical and basic researches conducted so far have focused on the roles of myeloid cell populations [17], the contribution of T lymphocytes [18, 19] and, in particular, of Tregs has been somewhat ignored. Whether CD4⁺CD25⁺ Tregs participate directly in sepsis-induced immunoparalysis resulting in poor outcomes remains to be investigated. The purpose of the present study was to investigate the significance of changes in activity of Tregs in severely burned patients, and its relation with pathogenesis of sepsis as well as the outcome of the patients following major burns.

Severe burn injury induces detrimental changes in immune function, often leaving the host highly susceptible to developing life-threatening opportunistic infections. Advances in our understanding of how burn influences host immune response suggest that thermal injury causes a phenotypic imbalance in the regulation of Th1- and Th2-type immune responses [24]. The immune response to infection represents a complex balance between the successful induction of proinflammatory antipathogen response and anti-inflammatory response required to limit damage to host tissues. The vast majority of clinical and basic science research on the immune consequences of burn injury and sepsis conducted during the past three decades has focused mainly on the roles of macrophages, neutrophils, and, to a lesser extent, conventional T lymphocytes [25, 26]. During recent years, however, it has become increasingly clear that minor subsets of innate immune cells, innate regulatory lymphocytes in particular, are central to processes involved in both protective immunity and immunopathology [27]. Tregs undoubtedly play an important role in controlling this balance during infection, and the results can range from highly detrimental to the host to highly beneficial to both the host and pathogen [28].

In our previous observations, significant proliferation of splenic T cells and IL-2 as well as IL-2Rα expression on T cells were simultaneously suppressed to a certain extent on PBD 1 to 7 in rats [29]. Nuclear factor of activated T cell activity of splenic T cells was markedly down-regulated on PBD 1 to 3. It was revealed that T cells were polarized to Th2 cells after burn injury. These data indicate that there is a marked suppression of T cell function following major burns. To collaborate with other findings, it has been reported that Tregs in mice can inhibit the proliferation of T cell and release of cytokine for polarization to antigen-specific Th1 cells after acute insults [30]. Similarly, we recently reported increased Treg activity after thermal injury in rats [31]. Because severe burn injury triggers both excessive inflammation and suppressed adaptive immunity, we would expect that there might be an activation of immune activity of Tregs isolated from burn-injury patients. Therefore, a major objective of this study was to define how thermal injury influenced the maturation of Tregs in peripheral blood in severely burned patients. We considered that this is an important question to be addressed as regulation of Th1- and Th2-type responses against infectious pathogens by Tregs can markedly affect host survival [30].

In the current study, increased expressions of CTLA-4 and FOXP3 on the surface of Tregs from burned patients were observed on PBD 1 to 21 compared with normal controls. This finding was consistent with a previous report that was published prior to the identification of Tregs [32]. In that study, the authors demonstrated that cell-surface CD25 expression on CD4⁺ T cells increased by five days after injury and remained elevated for at least two weeks. Meantime, it was presumed the increase in CD25 expression was due to injury-induced CD4⁺ T cell activation. However, we demonstrate here that the increased percentage of circulating Tregs in our burned patients was attributable to both T cell activation and a significant increase in the percentage of CD4⁺CD25^high cells. This increase in circulating CD4⁺CD25^high T cells may represent Tregs expansion or possibly migration of these cells from immunologically active sites instigated by the injury. These are important mechanistic issues that can be studied in more detail using animal models of injury.

Numerous studies have shown that an increased burn size leads to increased mortality in burn patients [12, 33]. In a large prospective clinical trial, Jeschke has indicated that different burn sizes are associated with differences in intensity of inflammation, in body composition, in protein synthesis, and in organ function. In the present study in vivo, we found there were obvious differences in the expressions of CTLA-4 and FOXP3 on the surface of Tregs among patients with various burn sizes. Taken together, these data suggest that acute insults can induce or amplify CD4⁺CD25⁺ Tregs function and that CD4⁺CD25⁺ T cells contribute to the development of postinjury immunosuppression.

Using a mouse burn injury model, Ni Choileain noticed that injury per se significantly enhances Tregs function [33]. Such increase in Tregs activity was apparent on day 7 after injury and was restricted to CD4⁺CD25⁺ T cells in lymph nodes draining the injury site. Moreover, our recent report implicated that the injury-induced increase in Tregs activity was cell-contact dependent and was mediated in part by increased cell surface TGF-β1 expression [31]. Depending on the different settings, cytokines (including TGF-β1 and IL-10) as well as direct cell killing of conventional T cells and antigen presenting cells (APCs) by the Tregs have been proposed as the one of the mechanism of immunosuppression [34‐36]. Similarly, our results in this study showed that gene/protein expression of IL-10 and TGF-β1 in Tregs from burn patients was augmented on PBD 1-21 in comparison to normal controls, and there were obvious differences among patients with different extent of burn injury. It appears that serious burn injury induces activitiy of Tregs resulting in high expressions of certain phenotypes, and this enhanced Tregs activity might play a key role in modulating cell-mediated immunity of T lymphocytes.

Sepsis and subsequent multiple organ dysfunction syndrome are frequent complications of major trauma or burns, and remain to be the most common cause of morbidity as well as mortality in critical illnesses. It is well known that marked immune depression is critically involved in the development of sepsis. Immunoparalysis has recently be thought to be a possible cause of explaining the failure of numerous clinical therapeutic trials in septic shock [37]. Severe burn injury induced a marked reduction in HLA-DR expression at both protein and messenger RNA levels [38]. Its persistent decrease was associated with mortality and the development of septic complications [39]. However, whether sepsis and mortality after burns are due to inflammation, immune suppression or other pathophysiologic contributing factors is not entirely elucidated. Furthermore, it is also not very clear whether Tregs induced by severe burns can contribute to the development of sepsis and outcome of the patients.

The studies presented here described that the expressions of activation markers of Tregs and cytokines produced by Tregs were significantly higher in patients with serious burns at all time points, and they were much higher in septic patients than those without sepsis on PBD 3 to 21. Among septic patients, the expressions of these parameters in the survival group were markedly lower than those with fatal outcome on PBD 3 to 21. These findings support the concept that CD4⁺CD25⁺ Tregs contribute to the control of immune response after being affected by thermal injury and sepsis. The persistence of a pronounced immunoparalysis induced by Tregs after severe sepsis is associated with a poor outcome after burns. Recently, similar findings have been reported by others [40‐42]. Some authors suggest that although CD4⁺CD25⁺ Tregs induced by IL-10 seem to contribute to sepsis-induced suppression of lymphoid dependent immunity, the removal of CD25⁺ cells does not provide a survival advantage or disadvantage.

We therefore speculated that Tregs might also play an essential role in initiating effective immunosuppression response to sepsis. This may be related to their ability to interact with components of the innate and adaptive immune response, and to their potentiality to be activated nonspecifically by bacterial products and/or cytokines, and to regulate through direct cell-cell and/or soluble mediators. It is our hope that a better understanding of the mechanism through which those rare lymphocyte subsets, which is found to exert such a profound effect on the immune response, may help in improving our clinical ability not only in diagnosis but also in treatment for the critically septic individual.

• Severe burn played an important role in activation and expansion of Treg cells. This feature might allow Treg to function for a prolonged period of time to regulate immune responses and induce suppression of T lymphocyte immune function.

• The elevated levels of cytokines producted by Treg and activation markers on Treg surface may also be involved in increased burn sizes, sepsis and mortality of burned patients.