Trauma-hemorrhage and dendritic cell functions : a critical review of splenic dendritic cell dysfunction following trauma-hemorrhage and therapeutic approach

Abstract Introduction Many studies demonstrated that trauma-hemorrhage induces marked alterations in various immune functions. The trauma-hemorrhage-induced immunosuppression is associated with an increased susceptibility to subsequent sepsis, organ failure and mortality. Previous studies demonstrated that trauma-hemorrhage induces immunosuppression in both innate immune systems and adaptive immune systems. Dendritic cell is the most potent antigen-presenting cell that initiates innate and adaptive immune response. The aim of this review was to discuss trauma-hemorrhage and dendritic cell functions. Conclusion Trauma-hemorrhage impairs splenic dendritic cell maturation. Suppressed TLR4 expression and MAPK activation contribute to the hyporesponsiveness of splenic dendritic cells following trauma-hemorrhage. 17β-oestradiol produces immunoprotective effects on splenic dendritic cells following trauma-hemorrhage. The immunomodulatory properties of 17β-oestradiol might be a potent therapeutic strategy for the treatment of depressed splenic dendritic cell functions following trauma-hemorrhage.

Trauma-hemorrhage and dendritic cell functions: a critical review of splenic dendritic cell dysfunction following trauma-hemorrhage and therapeutic approach T Kawasaki 1* , T Sata 1 The trauma-hemorrhage-induced immunosuppression is also associated with an increased susceptibility to subsequent sepsis, organ failure and mortality.Previous studies demonstrated that trauma-hemorrhage induces immunosuppression in both innate immune systems and adaptive immune systems.The in vitro capacity of peritoneal, alveolar and splenic macrophages to release interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-α in response to lipopolysaccharide (LPS) stimulation is diminished after traumahemorrhage.In contrast, Kupffer cells have been shown to have an enhanced capacity to produce pro-inflammatory cytokines following trauma-hemorrhage 5 .Despite the differential cytokine release capacities of macrophages from different microenvironments, the antigen-presenting capacity was depressed by macrophages in all tissue beds.Lymphocyte proliferative capacity and cytokines production capacity are also suppressed following trauma-hemorrhage 2,6,7 .These altered host defence mechanisms after trauma-hemorrhage are considered to play an important role in the development of infectious complications and sepsis.Dendritic cell (DC) is originally described by Steinman and Cohn in 1973 8 and is of increased interest recently.DC is the most potent antigen-presenting cell that initiates innate and adaptive immune response.Most DCs are in a non-activated 'immature' state.Once activated by microbial stimuli, immature DCs become activated and undergo maturation.Immature DCs are strategically located in pathogen entry routes such as skin and mucosa.After activated by pathogens, DCs undergo maturation and move to lymph nodes and the T cell area to present antigens to lymphocytes (Figure 1).However, it remains unknown whether trauma-hemorrhage influences DC functions.In this critical review, we describe the DC functions following trauma-hemorrhage.

Discussion
The authors have referenced some of their own studies in this review.These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.All human subjects, in these referenced studies, gave informed consent to participate in these studies.Animal care was also in accordance with the institution guidelines.

Does trauma-hemorrhage have any effects on splenic DC functions?
In the first part, we discuss the effect of trauma-hemorrhage on splenic DC functions.In a mice trauma-hemorrhage model, we demonstrated that the percentage of splenic DCs significantly decreased following traumahemorrhage when compared with sham mice 9 .The percentage of both Annexin V positive and PI negative cells (early phase of apoptosis) and Annexin V positive and PI positive cells (late phase of apoptosis) were significantly increased following trauma-hemorrhage.Our results demonstrate that apoptosis is a possible cause of splenic DC loss following trauma-hemorrhage.Previous reports 10,11 also suggested that the loss of splenic DC occurs both in patients with sepsis and in mouse sepsis Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY) Critical Review production capacity following traumahemorrhage.LPS-induced production of TNF-α and IL-6 was significantly suppressed in splenic DCs following trauma-hemorrhage.IL-12 and IFN-γ are important cytokines in DC maturation 16,17 .Activated DCs produce IL-12, which, in turn, upregulates the production of IFN-γ from Th1 and natural killer cells.IL-12 also activates DC autocrine production of IFN-γ.These processes in DCs could play a key role against early infections after trauma-hemorrhage by enhancing immunity against pathogens.LPS-or IL-12-induced IFN-γ production by splenic DCs decreased following trauma-hemorrhage.The release of IL-12p40 and IL-12p70 by DCs after LPS stimulation was also decreased following traumahemorrhage.These results suggest that suppressed IFN-γ and IL-12 production may be the cause of inhibition of DC maturation following trauma-hemorrhage.In addition, mature DC expresses high levels of  Immature DC and mature DC have different properties (Figure 2).Immature DCs express low CD80, CD86, CD40, and major histocompatibility complex class (MHC) antigen on their surface whereas mature DCs express high antigens 13 .Mature DCs specifically express CD83 14 .Immature DC has endocytic and phagocytic capacity.Mature DC enhances proinflammatory cytokine production and antigen-presenting capacity 15 .We investigated the effect of traumahemorrhage on DC maturation 9 .T-H influences the surface phenotype of splenic DC.Following trauma-hemorrhage, MHC-II and CD83 expression were significantly decreased.However, there were no significant differences between sham and trauma-hemorrhage mice in CD80 and CD86 expression.These results suggested that trauma-hemorrhage impairs DC maturation.To confirm the impairment of sDC maturation following trauma-hemorrhage, we further studied the changes of cytokine antigen-presenting capacity 18 .DCs isolated from trauma-hemorrhage mice showed a lower capacity to stimulate T-cell proliferation than those from sham controls.Traumahemorrhage depresses antigenpresenting function of DC.We found that: 1. Trauma-hemorrhage-induced apoptosis of splenic DC.

The DC maturation marker, CD83
and MHC-II expression on splenic DC were suppressed following trauma-hemorrhage.3. Trauma-hemorrhage suppressed the cytokine producing capacity of splenic DC. 4. Trauma-hemorrhage depressed the antigen-presenting function of splenic DC. 5. Trauma-hemorrhage impaired splenic DC maturation.

Does trauma-hemorrhage have any effect on toll-like receptor (TLR4) expression and mitogenactivated protein kinase (MAPK) activation of splenic DCs?
In the next part, we discuss how trauma-hemorrhage suppresses splenic DC functions.LPS is a potent activator of DCs and it induces the production of pro-inflammatory cytokines.TLR 4 has been shown to be essential for cellular responsiveness to LPS 19 .After LPS stimulation, activated signals go down from the TLR4 to the MAPK/NF-kB signalling pathways.
We investigated the expression of TLR4 and the activation of three different kinds of MAPKs, p38, extracellular signal-regulated protein kinase (ERK) and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) 23 .LPS stimulation significantly increased the expression of the active phosphorylated form of p38 MAPK in cells from both sham and trauma-hemorrhage mice.The activated p38 MAPK-positive cells percentage after LPS stimulation was significantly decreased in the traumahemorrhage group.The level of phosphorylated p38 MAPK was significantly greater than that from trauma-hemorrhage DCs.Total p38 MAPK expression was not significantly different between DCs from sham and trauma-hemorrhage mice at any time points after LPS stimulation.As well as p38, LPS-stimulated phospho ERK activation was significantly suppressed following traumahemorrhage.The percentage of phospho ERK positive cells was also decreased following trauma-hemorrhage.In DCs from sham mice, the level of phosphorylated ERK 1/2 was significantly greater than that from trauma-hemorrhage DCs.Total ERK 1/2 expression was not significantly different between DCs from sham or trauma-hemorrhage mice.There was no significant difference in SAPK/JNK activation between sham mice and trauma-hemorrhage mice.Previous studies have shown that p38 MAPK and ERK play a crucial role in the maturation of DCs 21,24 .Taken together, these pathways may contribute to the impaired maturation of DCs induced by trauma-hemorrhage.
The immunomodulatory effect of DCs relies mainly on the ligation of pattern recognition receptors, such as TLRs.Adaptive immunity to pathogens often initialises with the maturation of DCs after ligation of TLRs 25,26 .Next, we examined the effect of trauma-hemorrhage on TLR4 expression on splenic DC.The expression of TLR4-MD-2 on splenic DC suppressed significantly following trauma-hemorrhage.The percentage of TLR4-MD-2 positive cells also decreased significantly following trauma-hemorrhage.To determine whether traumahemorrhage influenced TLR4 gene expression, we used quantitative RT-PCR to assess changes in expression of TLR4 mRNA in purified splenic DCs from sham or trauma-hemorrhage mice.A 30% decrease in TLR4 mRNA levels in splenic DCs from traumahemorrhage mice was observed compared with shams.To determine whether trauma-hemorrhage induces splenic DCs hyporesponsiveness to other TLR agonists, we examined the effect of trauma-hemorrhage on TLR2 ligand zymosan-induced cytokines production of splenic DCs, as well as TLR4 ligand LPS, TLR2 ligand zymosan-induced cytokines production of splenic DC.Trauma-hemorrhage also induced hyporesponsiveness of zymosan.
The hyporesponsiveness of splenic DCs is a likely factor in the immunosuppression seen following traumahemorrhage.
Trauma-hemorrhage alters the LPS-induced activation of the MAPK cascade that controls different aspects of the LPS hyporesponsiveness of splenic DCs following trauma-hemorrhage.Trauma-hemorrhage appears to induce decreased cross-talk between the MAPKs pathways, as well as uncoupling of some LPS response from the MAPK cascade.Hyporesponsiveness of splenic DCs was also found after stimulation with TLR2 agonist zymosan following trauma-hemorrhage.Thus, altered MAPK activation and signal transduction contributes to the development of the hyporesponsiveness of splenic DCs which is central in the development of immune complications following trauma-hemorrhage.However, it remains unclear whether the hyporesponsiveness of splenic DCs following trauma-hemorrhage would occur after stimulation with other TLR agonists such as TLR5 agonist flagellin and TLR9 agonist CpG.We found that: 1. Trauma-hemorrhage downregulated MAPK activation of splenic DC.

Surface expression of TLR4-MD-2
was also suppressed following trauma-hemorrhage.

These changes in MAPK activation
and TLR4-MD-2 expression were associated with a suppressed ability of splenic DC to produce cytokines in response to LPS, a potent TLR4 ligand.4. Suppressed TLR4 expression and MAPK activation were involved in splenic DC dysfunction following trauma-hemorrhage.

Do female sex steroids adversely or beneficially affect the depressed function of DC in males with trauma-hemorrhage?
In the final part of this review, we would like to discuss the therapeutic strategy for trauma-hemorrhage-induced immunosuppression.Previous studies demonstrated that IL-15, Fms-like tyrosine kinase-3 ligand (Flt3L), IL-28 and IL-29 enhance DC function [27][28][29] .We focus on the effect of oestrogen, one of female sex hormones, on depressed splenic DC functions in this review [30][31][32] .
Bone reported a preponderance of morbidity and mortality from sepsis in males compared with females 33 .
McGowan et al. also reported a significantly higher incidence of bacteraemic infections in traumatised males than in females 34 .A retrospective study incorporating 30,286 trauma victims with an injury severity score (ISS) >15 demonstrated a significantly higher incidence of pneumonia in males 35 .Schroder et al. have shown a significantly higher survival rate in women (74%) compared with men (31%) following the onset of sepsis 36 .Similar genderdimorphic findings have been demonstrated in experimental studies following severe blood loss and the induction of sepsis 37,38 .Genderspecific immune response may be due to different effects and roles of sex hormones.Several studies were conducted in order to elucidate the effect of sex steroids on cell-mediated immune responses following traumahemorrhage.Male sex hormones play an important role in mediating immunosuppressive effects.Female sex hormones are immunoprotective.
Oestrogen is well known as the key regulator of cell growth, differentiation and function 39 .To evaluate whether female sex steroids adversely or beneficially affect the depressed function of DC in males with traumahemor rhage, we treated male C3H/ HeN trauma-hemorrhage mice with 17β-oestradiol (E2), oestrogen receptor (ER)-α agonist propyl pyrazoletriol (PPT) or ER-β agonist diarylpropionitrile (DPN) in the middle of resuscitation 30 .Plasma IL-6, IL-10, TNF-α and MCP-1 concentrations were significantly increased 2 h following trauma-hemorrhage.Administration of E2 or PPT following trauma-hemorrhage attenuated the increase in cytokine concentration under those conditions.Administration of DPN also reduced the elevation of these cytokine levels, but the levels of plasma cytokines still remained significantly higher than that in trauma-hemorrhage mice treated with E2 or PPT.Plasma IL-12p70 and IFN-γ concentrations were not detectable in sham and trauma-hemorrhage groups.Administration of E2 normalised the percentage of both early phase and late phase of apoptotic cells under those conditions.Although PPT administration following traumahemorrhage also normalised the percentage of apoptotic cells, DPN administration did not affect the apoptotic rate under those conditions.These results are in accordance with a previous study that reported E2 increases the viability of splenic DC in vitro 40 .Administration of E2 attenuated the suppression of CD40, CD83 and MHC-II expression under those conditions.PPT administration also normalised CD40, CD83 and MHC-II expression following traumahemorrhage; however, DPN treatment did not affect these expressions.Administration of E2 or PPT attenuated the suppressed antigenpresenting capacity of DCs under those conditions; however, ER-β agonist DPN did not improve DCs antigen-presenting function.Immature DCs have the ability to capture antigens by endocytosis or macropinocytosis; however, mature DCs lose this ability.A previous study demonstrated that E2 decreases the endocytosis of splenic DCs 40 .Therefore, these results strongly suggest that E2 administration following traumahemorrhage restores the maturation of splenic DCs.
The predominant biological effects of E2 are mediated through ER-α and ER-β 41 .These two receptors are differentially expressed in different tissues 42 .To determine whether trauma-hemorrhage influenced ER-α and ER-β gene expression in splenic DC, we used quantitative RT-PCR to assess changes in expression of ER-α and ER-β mRNA in purified splenic DCs from sham or trauma-hemorrhage mice.Although trauma-hemorrhage did not significantly influence the expression of ER-α and ER-β mRNA in the splenic DC, the splenic DC expresses ER-α mRNA predominantly.Taken together, these results suggest that the salutary effects of E2 on splenic DC functions are mediated predominantly via ER-α.We found that: 1. Administration of E2 was effective in normalising the cytokine production and antigen-presenting capacity of splenic DC. 2. E2 produced immunoprotective effects on splenic DC following trauma-hemorrhage.3. ER-α agonist PPT also produced immunoprotective effects, whereas ER-β agonist DPN did not attenuate splenic DC functions.4. The salutary effects of E2 on splenic DC functions were mediated predominantly via ER-α. 5. Splenic DC dysfunction following trauma-hemorrhage might be controlled by female sex steroid.

Conclusion
A major consequence of traumahemorrhage is the suppression of organ and immune cell functions.
The findings reviewed in this article suggest that trauma-hemorrhage impairs splenic DC maturation and oestrogen plays a decisive role in the depression or maintenance of DC functions following injury.The protective effects of oestrogen in restoring splenic DC function are through intracellular receptor, ER-α.Experimental studies clearly demonstrate that oestrogen and ER agonists are useful therapeutic adjuncts in protecting organ functions and improving outcome following traumahemorrhage.However, in the clinical setting, there are conflicting reports whether gender dimorphic responses are evident following injury.The reasons for the lack of uniform results appear to be due to the fact that most studies do not take into consideration the hormonal status of the host at the time of injury.Thus, studies reporting protective effects of the female gender following injury could be due to the patients having high oestrogen levels at the time of injury whereas those reporting a lack of protective effects in females could be due to low oestrogen levels.In view of this, it is important to carry out additional studies in which the hormonal status of the patient is measured as quickly as possible after injury and correlate sex steroid levels with the lack or prevalence of complications, circulating cytokine levels, incidence of organ dysfunction and failure and length of hospital stay.

Figure 1 :
Figure 1: Distribution of DC.Immature DCs are strategically located in pathogen entry routes such as skin and mucosa.After activated by pathogens, DCs undergo maturation and move to lymph nodes and T cell area to present antigen to lymphocyte.HSC, hematopoietic stem cells.

Figure 2 :
Figure 2: Maturation of DC.Immature DC and mature DC have different properties.Immature DCs express low CD80, CD86, CD40 and MHC antigens on their surface, whereas mature DCs express high levels of same antigens.Mature DCs specifically express CD83.Immature DCs possess endocytic and phagocytic capacity.Mature DC enhances pro-inflammatory cytokine production and antigen-presenting capacity.

Figure 3 :
Figure 3: Overview of TLR-triggered MAPK/NF-kB signalling pathways.After LPS stimulation, activated signals go down from the TLR4 to the MAPK/NF-kB signalling pathways.