Immune dysfunction following trauma-haemorrhage: influence of gender and age

Recent studies indicate that young female proestrus mice show an enhanced immune response following trauma-haemorrhage, as opposed to the immunodepression observed in males of comparable age. Testosterone is suggested as the cause of immunodepression in males, whereas oestradiol seems to be responsible for the enhanced immune response in females, however, sex hormone levels decrease with age. To determine if the sexual dimorphism in immune responses observed in young mice following trauma-haemorrhage changes with age, young (2-3 months) and aged (18-19 months) male and female CBA/J NIA mice were subjected to soft-tissue trauma (laparatomy) and haemorrhage (35+5 mmHg for 90 min and fluid resuscitation) or sham operation. Mice were killed 24 h later, and whole blood, as well as splenic and peritoneal macrophages (Mstraight phi) obtained. Plasma 17beta-oestradiol and free testosterone decreased in aged females and males, respectively. Mstraight phi from young females had enhanced IL-1beta and suppressed IL-10 production following trauma-haemorrhage, while aged females had unchanged production IL-1beta and IL-6 production and enhanced IL-10 release. In contrast, IL-1beta and IL-6 production by Mbeta from young males was suppressed and IL-10 production enhanced following trauma-haemorrhage, whereas Mstraight phi from aged males produced elevated levels of IL-1beta and IL-6 and suppressed levels of IL-10 following trauma-haemorrhage. Thus, the gender-related changes in the immune response to trauma-haemorrhage were reversed in aged mice.     

Immunoprotection in proestrus females following trauma-hemorrhage: the pivotal role of estrogen receptors

Immune responses in proestrus females are not altered after trauma-hemorrhage, whereas they are markedly depressed in males. Elevated levels of female sex steroids appear to be responsible for maintaining immune responses but it remains unknown, whether estrogen per se is responsible. To study this, proestrus female C3H/HeN mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35+/-5 mmHg for 90 min, then resuscitated) or sham operation and received the estrogen receptor antagonist EM-800 or vehicle during resuscitation. Two hours following trauma-hemorrhage, splenocyte proliferation, IL-2, IL-3, IFN-gamma release, and splenic macrophage IL-6 release was maintained in vehicle-treated females. In EM-800-treated females, however, these immune parameters were significantly depressed. Following trauma-hemorrhage, Kupffer cell TNF-alpha release and circulating TNF-alpha were increased only in EM-800-treated females. These findings indicate that the ability of proestrus females to maintain immune function following trauma-hemorrhage is estrogen-dependent and mediated via estrogen receptors.     

Impact of sex and age on bone marrow immune responses in a murine model of trauma-hemorrhage.

Although studies have demonstrated that trauma markedly alters the bone marrow immune responses, sex and age are crucial determinants under such conditions and have not been extensively examined. To study this, 21- to 27-day-old (premature), 6- to 8-wk-old (mature), and 20- to 24-mo-old (aged) male and female (proestrus) C3H/HeN mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (30 +/- 5 mmHg for 90 min) followed by fluid resuscitation. Twenty-four hours after resuscitation, bone marrow cells were harvested. Trauma-hemorrhage induced an increased number of the early pluripotent stem cell-associated bone marrow cell subsets (Sca1(+)CD34(-)CD117(+/-)lin(+/-)) in young mice. The CD117(+) proportion of these cell subsets increased in mature proestrus females, but not in males. Aged males displayed significant lower numbers of Sca1(+)CD34(-)CD117(+/-)lin(+/-) cells compared with young male mice. Trauma-hemorrhage also increased development of granulocyte/macrophage progenitor cells (CD11b(+)Gr-1(+)). Proliferative responses to granulocyte macrophage colony-stimulating factor were maintained in mature and aged proestrus females, but decreased in young mice and mature males. Augmented differentiation into monocyte/macrophage lineage in mature and aged proestrus females was observed and associated with the maintained release of TNF-alpha and IL-6. Conversely, increased IL-10 and PGE(2) production was observed in the male trauma-hemorrhage groups. Thus, sex- and age-specific effects in bone marrow differentiation and immune responses after trauma-hemorrhage occur, which are likely to contribute to the sex- and age-related differences in the systemic immune responses under such conditions.     

MyD88 and Src are differentially regulated in Kupffer cells of males and proestrus females following hypoxia

Hypoxia produces sex dimorphic immune responses in males and proestrus females. Because Kupffer cells are the major source of proinflammatory cytokines, studies were conducted to discern IL-6 production in mouse Kupffer cells following hypoxia. Hypoxia enhances TLR4 expression in Kupffer cells irrespective of sex. However, MyD88 and Src expression in Kupffer cells decreased significantly after hypoxia in proestrus females, whereas Src protein expression and phosphorylation increased in males in concurrence with differences in IL-6 production. 17beta-estradiol administration elevated MyD88 and Src expression in males to levels in normoxic proestrus females. Administration of Src inhibitor in hypoxic males prevented increased IL-6 production. Thus, differential regulation of MyD88 and Src in males and females plays an important role in sex-specific immune response following hypoxia.     

Immunomodulatory effects of dehydroepiandrosterone in proestrus female mice after trauma-hemorrhage.

Studies indicate that administration of the adrenal steroid dehydroepiandrosterone (DHEA) after trauma-hemorrhage in male mice improved cellular immune functions and reduced mortality rates from subsequent sepsis. There is evidence, however, that DHEA is converted to estrogens in males and that estrogens are immunoprotective after trauma-hemorrhage (TH). In contrast, DHEA in females can be converted to testosterone that has deleterious effects on immune functions. The aim of our study, therefore, was to determine whether administration of DHEA in proestrus females after TH would deteriorate immune responses. Proestrus female C3H/HeN mice (age 7-8 wk) were subjected to laparotomy (i.e., soft tissue trauma induced) and hemorrhagic shock (35 +/- 5 mmHg for 90 min) or sham operation. The mice then received DHEA (100 micro/25 g body wt) or vehicle subcutaneously followed by fluid resuscitation (4x the shed blood volume). Plasma IL-6, splenocyte proliferation, splenocyte IL-2, IL-3, IFN-gamma, IL-10 release, and splenic Mphi IL-1beta, IL-6, IL-10, and IL-12 release were determined 24 h after TH. Plasma IL-6 levels were significantly increased in vehicle-treated females, and DHEA administration markedly attenuated this response. In vehicle-treated females, splenocyte proliferation, IL-2, IL-3, and IFN-gamma release, and splenic Mphi IL-1 beta, IL-6, and IL-12 release were maintained or slightly enhanced after TH. In DHEA-treated females, however, these immune functional parameters were either unaltered compared with vehicle-treated animals or even further enhanced, but surprisingly were not depressed. Moreover, DHEA reduced splenocyte and splenic M phi anti-inflammatory cytokine (i.e., IL-10) production after TH compared with vehicle-treated females. Because DHEA further enhances the immune responsiveness in proestrus females after TH, this hormone might be a useful adjunct even in females for further enhancing immune responses and decreasing the mortality rate after trauma and severe blood loss.     

Is gut the major source of proinflammatory cytokine release during polymicrobial sepsis?

Although studies have shown that the gut is capable of being a cytokine-producing organ and that the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 are upregulated following the onset of sepsis, it remains unknown whether the gut is indeed the major source of the increased cytokine production under such conditions. To determine this, male rats were subjected to cecal ligation and puncture (CLP, a model of polymicrobial sepsis) or sham operation followed by the administration of normal saline solution subcutaneously (i.e., fluid resuscitation). Systemic and portal blood samples were taken simultaneously at 2, 5, 10, or 20 h after CLP or sham operation. Plasma levels of TNF-alpha, IL-1beta, and IL-6 were determined using an enzyme-linked immunosorbent assay. In additional animals, the small intestine was harvested at 10 h after CLP or sham operation and examined for TNF-alpha, IL-1beta, and IL-6 gene expression by RT-PCR. The results indicate that the levels of TNF-alpha, IL-1beta, and IL-6 in both systemic and portal blood samples were significantly elevated during sepsis with the exception that the increase in IL-1beta was not significant at 2 h after CLP. However, there were no significant differences in the levels of those proinflammatory cytokines between systemic and portal blood at any points after the onset of sepsis. Moreover, there were no significant alterations in the proinflammatory cytokine gene expression in the small intestine at 10 h after CLP. Since the levels of TNF-alpha, IL-1beta, and IL-6 were not significantly increased in portal blood as compared to systemic blood and since there was no upregulation of gene expression for these cytokines, it appears that organs other than the gut are responsible for the upregulated proinflammatory cytokines during polymicrobial sepsis.     

Gender-related differences in myocardial inflammatory and contractile responses to major burn trauma

Gender-related differences in immune responses to hemorrhage and sepsis have been described. However, most trauma studies continue to limit experimental models to males to avoid the variable responses associated with hormonal fluctuation in proestrus/estrus females. In the present study, male and female (either diestrus or proestrus/estrus) Sprague-Dawley rats (250-325 g) were given a third-degree scald burn over 40% total body surface area and fluid resuscitated (4 ml/kg per %burn of lactated Ringer solution); sham burn males and diestrus as well as sham burn proestrus/estrus female rats were included to provide controls. Twenty-four hours postburn, hearts were either perfused to examine mechanical function (Langendorff, n = 8 to 9 hearts/group) or to prepare cardiomyocytes (collagenase digestion, n = 4 to 5 hearts/group). Left ventricular developed pressure and the positive and negative first derivative of left ventricular pressure responses to increases in preload were significantly lower in burned males compared with responses measured in either burned proestrus/estrus or burned diestrus females; burn trauma increased cardiomyocyte secretion of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide to a lesser extent in proestrus/estrus females than levels secreted by either diestrus females or males. Similarly, myocytes from proestrus/estrus females accumulated significantly less sodium/calcium compared with values measured in males (P < 0.05). Our data confirm gender-related differences in myocardial function and myocardial inflammatory responses to burn injury.     

Diversity of interferon gamma and granulocyte-macrophage colony-stimulating factor in restoring immune dysfunction of dendritic cells and macrophages during polymicrobial sepsis

The development of immunosuppression during polymicrobial sepsis is associated with the failure of dendritic cells (DC) to promote the polarization of T helper (Th) cells toward a protective Th1 type. The aim of the study was to test potential immunomodulatory approaches to restore the capacity of splenic DC to secrete interleukin (IL) 12 that represents the key cytokine in Th1 cell polarization. Murine polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Splenic DC were isolated at different time points after CLP or sham operation, and stimulated with bacterial components in the presence or absence of neutralizing anti-IL-10 antibodies, murine interferon (IFN) gamma, and/or granulocyte macrophage colony-stimulating factor (GM-CSF). DC from septic mice showed an impaired capacity to release the pro-inflammatory and Th1-promoting cytokines tumor necrosis factor alpha, IFN-gamma, and IL-12 in response to bacterial stimuli, but secreted IL-10. Endogenous IL-10 was not responsible for the impaired IL-12 secretion. Up to 6 h after CLP, the combined treatment of DC from septic mice with IFN-gamma and GM-CSF increased the secretion of IL-12. Later, DC from septic mice responded to IFN-gamma and GM-CSF with increased expression of the co-stimulatory molecule CD86, while IL-12 secretion was no more enhanced. In contrast, splenic macrophages from septic mice during late sepsis responded to GM-CSF with increased cytokine release. Thus, therapy of sepsis with IFN-gamma/GM-CSF might be sufficient to restore the activity of macrophages, but fails to restore DC function adequate for the development of a protective Th1-like immune response.     

Cecal ligation and puncture-induced impairment of innate immune function does not occur in the absence of caspase-1

Mice that have been subjected to cecal ligation and puncture (CLP) have an impaired ability to clear a subsequent Pseudomonas aeruginosa challenge compared with that of sham CLP controls. We hypothesized that this outcome is dependent upon a caspase-1 mechanism and tested this hypothesis by measuring caspase-1 after CLP and by measuring clearance of a bacterial challenge in caspase-1-deficient mice after CLP. Wild-type mice subjected to CLP had increased caspase-1 activity as well as increased IL-1β and increased IL-18 production in splenocytes stimulated with heat-killed Pseudomonas and had increased plasma concentrations of IL-1β and IL-18 and impaired clearance of a P. aeruginosa challenge compared with sham controls. Healthy, uninjured caspase-1(-\-) mice did not differ from wild-type mice in their ability to clear a Pseudomonas challenge. However, unlike wild-type mice, caspase-1(-/-) mice subjected to CLP had no impairment of bacterial clearance of the Pseudomonas challenge, suggesting that caspase-1 induction after CLP played a role in impairment of bacterial clearance. This was further substantiated by the use of a specific caspase-1 inhibitor, Ac-YVAD-CMK. Wild-type mice treated with Ac-YVAD-CMK (10 mg/kg s.c. twice daily, initiated at time of CLP) did not have impaired clearance of a Pseudomonas challenge compared with that of sham mice and had significantly improved bacterial clearance compared with that of untreated CLP mice. Increased caspase-1 expression and activity after CLP injury appears to contribute to diminished innate immune function.     

Severe hypoxemia in the absence of blood loss causes a gender dimorphic immune response

A gender dimorphic immune response has beenobserved after trauma and severe hemorrhage, a condition believed to beassociated with tissue hypoxia. Although studies have shown thathypoxemia per se in males causes a systemic inflammatory response, itis unclear if the inflammatory response to hypoxemia exhibits gender dimorphic characteristics. To study this, male and female C3H/HeN micein the proestrus state of the estrous cycle were subjected to hypoxemia(95% N₂-5% O₂) or sham hypoxemia (room air)for 60 min. Later (2 h), plasma interleukin (IL)-6 and tumor necrosis factor (TNF)- levels were determined along with splenic immune responses. Plasma IL-6 and TNF- concentrations after hypoxemia weresignificantly increased in males but not in females. Splenocyte proliferation was depressed in males after hypoxemia but not in females. A shift toward an immunosuppressive Th-2 cytokine profile wasobserved in males after hypoxemia [decreased interferon- (Th-1) andincreased IL-10 (Th-2)], whereas no such shift was observed infemales. Splenic macrophage IL-6, IL-10, and IL-12 production weresuppressed in males after hypoxemia; however, such suppression was notobserved in females. These findings therefore indicate that a genderdimorphic immune response also exists after hypoxemia in the absence ofblood loss and tissue trauma, similar to trauma-hemorrhage.Furthermore, because no systemic inflammatory response or alterationsin T lymphocyte or macrophage functions are observed in proestrusfemales but such parameters are markedly altered after severe hypoxemiain males, these studies indicate that proestrus females can toleratehypoxemia better than males.

     

Hydrogen sulfide upregulates cyclooxygenase-2 and prostaglandin E metabolite in sepsis-evoked acute lung injury via transient receptor potential vanilloid type 1 channel activation

Hydrogen sulfide (H(2)S) has been shown to promote transient receptor potential vanilloid type 1 (TRPV1)-mediated neurogenic inflammation in sepsis and its associated multiple organ failure, including acute lung injury (ALI). Accumulating evidence suggests that the cyclooxygenase-2 (COX-2)/PGE(2) pathway plays an important role in augmenting inflammatory immune response in sepsis and respiratory diseases. However, the interactions among H(2)S, COX-2, and PGE(2) in inciting sepsis-evoked ALI remain unknown. Therefore, the aim of this study was to investigate whether H(2)S would upregulate COX-2 and work in conjunction with it to instigate ALI in a murine model of polymicrobial sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in male Swiss mice. dl-propargylglycine, an inhibitor of H(2)S formation, was administrated 1 h before or 1 h after CLP, whereas sodium hydrosulfide, an H(2)S donor, was given during CLP. Mice were treated with TRPV1 antagonist capsazepine 30 min before CLP, followed by assessment of lung COX-2 and PGE(2) metabolite (PGEM) levels. Additionally, septic mice were administrated with parecoxib, a selective COX-2 inhibitor, 20 min post-CLP and subjected to ALI and survival analysis. H(2)S augmented COX-2 and PGEM production in sepsis-evoked ALI by a TRPV1 channel-dependent mechanism. COX-2 inhibition with parecoxib attenuated H(2)S-augmented lung PGEM production, neutrophil infiltration, edema, proinflammatory cytokines, chemokines, and adhesion molecules levels, restored lung histoarchitecture, and protected against CLP-induced lethality. The strong anti-inflammatory and antiseptic actions of selective COX-2 inhibitor may provide a potential therapeutic approach for the management of sepsis and sepsis-associated ALI.     

Reversal of sexual dimorphism in splenic T lymphocyte responses after trauma-hemorrhage with aging

Previous studies have demonstrated that hemorrhagic shockproduces immunodepression in young male mice, whereas theimmunoresponsivness in young proestrus female mice is enhanced undersuch conditions. This sexually dimorphic immune response to hemorrhageappears to be related to high estrogen and testosterone levels infemales and males, respectively. Nonetheless, it is unknown what impact the age-related decline in the sex steroid levels has on the immune response after hemorrhage. To study this, young (2-3 mo) and aged (18-19 mo) male and female CBA/J NIA mice were subjected tolaparotomy (i.e., soft tissue trauma) and hemorrhage (35 ± 5 mmHg for90 min and fluid resuscitation) or sham operation. Twenty-four hours later, splenocyte responses were assessed in vitro. Splenic T lymphocyte responses [i.e., proliferation, interleukin-2 (IL-2) and interferon- (IFN-) release] were depressed in youngmales and enhanced in young females after trauma-hemorrhage. Incontrast, in the aged male and female groups these parameters ofsplenocyte function were reversed after trauma-hemorrhage (i.e.,increased proliferation and IL-2 release in aged males compared withsuppressed proliferation and IFN- release in aged females).Furthermore, the release of the immunosuppressive cytokine IL-10inversely correlated with the age- and gender-related changes insplenocyte responses after trauma-hemorrhage. Thus the sexuallydimorphic immune response in young males and females totrauma-hemorrhage appears to reverse as sex hormone levels decline with age.

     

Influence of gender and age on T-cell responses in a murine model of trauma-hemorrhage: differences between circulating and tissue-fixed cells.

Clinical studies indicate that peripheral blood lymphocyte functions are depressed following trauma; however, it is unclear whether tissue-fixed lymphocyte functions are also altered under those conditions. Moreover, the impact of gender and age on peripheral T-cell responses following trauma-hemorrhage (TH) are unknown. To study this, immature (approximately 3 wk of age), mature (approximately 7 wk of age), and aged (approximately 23 mo of age) male and proestrus female C3H/HeN mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (30+/-5 mmHg for 90 min). Twenty-four hours after resuscitation, blood and splenocytes were harvested and T-cell functions assessed. In immature animals, TH induced an enhanced immune response in the splenic compartment and a suppressed response in the peripheral blood mononuclear cells (PBMC) that was independent of gender. Differential responses were observed in cells from mature mice. Splenic responses were enhanced following TH, independent of gender, whereas PBMC displayed gender dimorphism with suppressed proliferation and T-cell helper 1 responses in males but not in females. A similar pattern was observed in cells from aged mice. Splenic T cells from male mice displayed a suppressed CD4-to-CD8 ratio after TH, whereas no such change was observed in cells from proestrus females. In contrast, only PBMC from mature males displayed a suppressed CD4-to-CD8 ratio after TH. Thus gender differences exist in PBMC responses after TH that do not necessarily correlate with changes in the tissue-fixed compartment. Age is also an important factor in the immune responses after TH. In view of this, both gender and age should be taken into consideration in evaluating the immune status and in treatment of TH shock.     

The immunomodulatory actions of prostaglandin E2 on allergic airway responses in the rat

PGE(2) has been reported to inhibit allergen-induced airway responses in sensitized human subjects. The aim of this study was to investigate the mechanism of anti-inflammatory actions of PGE(2) in an animal model of allergic asthma. BN rats were sensitized to OVA using Bordetella pertussis as an adjuvant. One week later, an aerosol of OVA was administered. After a further week, animals were anesthetized with urethan, intubated, and subjected to measurements of pulmonary resistance (R(L)) for a period of 8 h after OVA challenge. PGE(2) (1 and 3 micro g in 100 micro l of saline) was administered by insufflation intratracheally 30 min before OVA challenge. The early response was inhibited by PGE(2) (3 micro g). The late response was inhibited by both PGE(2) (1 and 3 micro g). Bronchoalveolar lavage fluid from OVA-challenged rats showed eosinophilia and an increase in the number of cells expressing IL-4 and IL-5 mRNA. These responses were inhibited by PGE(2). Bronchoalveolar lavage fluid levels of cysteinyl-leukotrienes were elevated after OVA challenge and were reduced after PGE(2) to levels comparable with those of sham challenged animals. We conclude that PGE(2) is a potent anti-inflammatory agent that may act by reducing allergen-induced Th2 cell activation and cysteinyl-leukotriene synthesis in the rat.     

Deficiency of gammadelta T lymphocytes contributes to mortality and immunosuppression in sepsis

Studies have indicated that gammadelta T lymphocytes play an important role in the regulation of immune function and the clearance of intracellular pathogens. We have recently reported that intraepithelial lymphocytes (IEL), which are rich in gammadelta T cells, within the small intestine illustrated a significant increase in apoptosis and immune dysfunction in mice subjected to sepsis. However, the contribution of gammadelta T cells to the host response to polymicrobial sepsis remains unclear. In this study, we initially observed that after sepsis induced by cecal ligation and puncture (CLP), there was an increase in small intestinal IEL CD8+gammadelta+ T cells in control gammadelta+/+ mice. Importantly, we subsequently found an increased early mortality in mice lacking gammadelta T cells (gammadelta-/- mice) after sepsis. This was associated with decreases in plasma TNF-alpha, IL-6, and IL-12 levels in gammadelta-/- mice compared with gammadelta+/+ mice after sepsis. In addition, even though in vitro LPS-stimulated peritoneal macrophages showed a reduction in IL-6 and IL-12 release after CLP, these cytokines were less suppressed in macrophages isolated from gammadelta-/- mice. Alternatively, IL-10 release was not different between septic gammadelta+/+ and gammadelta-/- mice. Whereas T helper (Th)1 cytokine release by anti-CD3-stimulated splenocytes was significantly depressed in septic gammadelta+/+ mice, there was no such depression in gammadelta-/- mice. However, gammadelta T cell deficiency had no effect on Th2 cytokine release. These findings suggest that gammadelta T cells may play a critical role in regulating the host immune response and survival to sepsis, in part by alteration of the level of IEL CD8+gammadelta+ T cells and through the development of the Th1 response.     

Mechanical ventilation of isolated septic rat lungs: effects on surfactant and inflammatory cytokines.

The effects of mechanical ventilation (MV) on the surfactant system and cytokine secretion were studied in isolated septic rat lungs. At 23 h after sham surgery or induction of sepsis by cecal ligation and perforation (CLP), lungs were excised and randomized to one of three groups: 1) a nonventilated group, 2) a group subjected to 1 h of noninjurious MV (tidal volume = 10 ml/kg, positive end-expiratory pressure = 3 cmH(2)O), or 3) a group subjected to 1 h of injurious MV (tidal volume = 20 ml/kg, positive end-expiratory pressure = 0 cmH(2)O). Nonventilated sham and CLP lungs had similar compliance, normal lung morphology, surfactant, and cytokine concentrations. Injurious ventilation decreased compliance, altered surfactant, increased cytokines, and induced morphological changes compared with nonventilation in sham and CLP lungs. In these lungs, the surfactant system was similar in sham and CLP lungs; however, tumor necrosis factor-alpha and interleukin-6 levels were significantly higher in CLP lungs. We conclude that injurious ventilation altered surfactant independent of sepsis and that the CLP lungs were predisposed to the secretion of larger amounts of cytokines because of ventilation.     

IL-10 mediation of activation-induced TH1 cell apoptosis and lymphoid dysfunction in polymicrobial sepsis

Recent studies suggest that increased activation-induced lymphocyte apoptosis (AICD) is detected in mouse splenocytes during polymicrobial sepsis which may contribute to lymphocyte immune dysfunction [i.e., decreased interleukin (IL-)2 and interferon-gamma (IFN-gamma) production] leading to the associated morbidity seen in those animals. Thus, we wanted to examine the hypothesis that immune suppressive agents, such as IL-4, IL-10 or prostaglandin E2(PGE2), known to be elevated in septic animals, also contribute to this increase in AICD. Here we demonstrate that the inclusion of monoclonal antibody (mAb) to IL-10, but not anti-IL-4 or ibuprofen (IBU), blunted this sepsis induced increase in splenocyte AICD. Additionally, septic mice deficient in the IL-10 gene product (-/-) showed neither an increase in AICD nor a loss of IL-2/IFN-gamma release capacity. Interestingly, mAb to IL-10 did not altered the extent of AICD in a Th2-cell line, but exogenous IL-10 did potentiate Th1-like cell line AICD. This was consistent with the finding that the increased AICD seen in septic mouse splenocytes was restricted largely to the CD4+ cells producing IL-2 (Th1-cells) and that mAb to IL-10 treatment suppressed this change. Furthermore, IL-10 appears to mediate its AICD effect by upregulation of the Fas receptor and Fas receptor signaling protein components, but not by altered expression of Bcl/Bax/Bad family members, in septic mouse splenocytes. To the extent that these processes contribute in a pathological fashion to the animal's capacity to survive sepsis we have previously observed that in vivo post-treatment of mice with mAb IL-10 markedly attenuated septic mortality. Collectively, these data indicate that in the septic mouse the Th2 cytokine IL-10 not only serves to actively induce Th1 lymphocyte immune dysfunction but also plays a role in their apoptotic depletion. These processes in turn appear to contribute to the animal's inability to ward off lethal septic challenge.     

Neutralisation of Peritoneal IL-17A Markedly Improves the Prognosis of Severe Septic Mice by Decreasing Neutrophil Infiltration and Proinflammatory Cytokines

Purpose

The current study aimed to elucidate the role of peritoneal fluid IL-17A in septic mice, and the effects of intraperitoneal or intravenous blockade of the IL-17A pathway by anti-IL17A antibody on survival, plasma, and peritoneal cavity cytokine profile in a murine caecal ligation and puncture (CLP) sepsis model. The main source of peritoneal fluid IL-17A in septic mice was identified.

Methods

Male C57BL/6 mice that underwent severe CLP or sham surgery were intraperitoneally or intravenously administered anti-IL17A antibodies or isotype antibodies. The survival rates were observed. IL-17A, TNF-α, and IL-6 cytokine levels were measured by ELISA. Surface and intracellular IL-17A immunofluorescence stains were detected by flow cytometry to identify the IL-17A–producing cells.

Results

The IL-17A level was elevated much higher and earlier in peritoneal fluid than in the blood of the CLP mice. The intraperitoneal IL-17A blockade more significantly protects against CLP-induced mortality than intravenous blockade because of decreased TNF-α and IL-6 levels both in peritoneal fluid and blood, neutrophil infiltration in the peritoneal cavity, and lung injury. γδ T lymphocytes were identified to be the main source of IL-17A in the peritoneal fluid of septic mice.

Conclusions

The earlier and higher elevated IL-17A derived from γδ T cells in peritoneal fluid plays a critical role during polymicrobial severe sepsis and effect of intraperitoneal IL-17A antibody administration superior to intravenous administration on survival of severe CLP-induced septic mice. The intraperitoneal blockade of IL-17A decreases proinflammatory cytokine production, neutrophil infiltration, and lung injury, thereby improving septic mice survival, which provides a new potential therapy target for sepsis.