Disparate Recruitment and Retention of Plasmacytoid Dendritic Cells to The Small Intestinal Mucosa between Young and Aged Mice.

Plasmacytoid dendritic cells (pDC), a highly specialized class of innate immune cells that serve as rapid sensors of danger signals in circulation or in lymphoid tissue are well studied. However, there remains knowledge gaps about age-dependent changes of pDC function in the intestinal mucosa. Here, we report that under homeostatic conditions, the proportion of pDC expressing C-C chemokine receptor 9 (CCR9) in the intestinal intraepithelial cell (iIEC) population is comparable between young (2-4 months) and aged (18-24 months) mice, but the absolute numbers of iIEC and pDC are significantly lower in aged mice. Employing the classic model of acute endotoxemia induced by lipopolysaccharide (LPS), we found a decrease in the proportion and absolute number of intraepithelial pDC in both young and aged mice despite the LPS-induced increased expression of the chemokine C-C ligand 25 (CCL25), the ligand of CCR9, in the intestinal mucosa of young mice. In adoptive transfer experiments, a significantly lower number of pDC was retained into the intestinal layer of aged host mice after LPS administration. This was associated with recoverable pDC numbers in the intestinal lumen. Furthermore, co-adoptive transfer of young and aged pDC into young hosts also showed significantly lower retention of aged pDC in the epithelial layer compared to the co-transferred young pDC. Collectively, these data show age-associated changes in mucosal CCL25 gene expression and in pDC number. These may underlie the reported inadequate responses to gastrointestinal pathogens during chronologic aging.

TLR9 signaling in fibroblastic reticular cells regulates peritoneal immunity.

Fibroblastic reticular cells (FRCs), a subpopulation of stromal cells in lymphoid organs and fat-associated lymphoid clusters (FALCs) in adipose tissue, play immune-regulatory roles in the host response to infection and may be useful as a form of cell therapy in sepsis. Here, we found an unexpected major role of TLR9 in controlling peritoneal immune cell recruitment and FALC formation at baseline and after sepsis induced by cecal ligation and puncture (CLP). TLR9 regulated peritoneal immunity via suppression of chemokine production by FRCs. Adoptive transfer of TLR9-deficient FRCs more effectively decreased mortality, bacterial load, and systemic inflammation after CLP than WT FRCs. Importantly, we found that activation of TLR9 signaling suppressed chemokine production by human adipose tissue-derived FRCs. Together, our results indicate that TLR9 plays critical roles in regulating peritoneal immunity via suppression of chemokine production by FRCs. These data form a knowledge basis upon which to design new therapeutic strategies to improve the therapeutic efficacy of FRC-based treatments for sepsis and immune dysregulation diseases.

Innate-Like Lymphocytes Are Immediate Participants in the Hyper-Acute Immune Response to Trauma and Hemorrhagic Shock.

Adverse outcomes following severe traumatic injury are frequently attributed to a state of immunological dysfunction acquired during treatment and recovery. Recent genomic evidence however, suggests that the trajectory toward development of multiple organ dysfunction syndrome (MODS) is already in play at admission (<2 h following injury). Improved understanding of the molecular events during the hyper-acute immunological response to trauma, <2 h following injury, may reveal opportunities to ameliorate organ injury and expedite recovery. Lymphocytes have not previously been considered key participants in this early response; however, two observations in human trauma patients namely, raised populations of circulating NKT and NK cells during the hyper-acute phase and persistent lymphopenia beyond 48 h show association with the development of MODS during recovery. These highlight the need for greater understanding of lymphocyte function in the hyper-acute phase of inflammation. An exploratory study was therefore conducted in a well-established murine model of trauma and hemorrhagic shock (T&HS) to investigate (1) the development of lymphopenia in the murine model and (2) the phenotypic and functional changes of three innate-like lymphocyte subsets, NK1.1+ CD3-, NK1.1+ CD3+, γδTCR+ CD3+ cells, focusing on the first 6 h following injury. Rapid changes in phenotype and function were demonstrated in these cells within blood and spleen, but responses in lung tissue lagged behind. This study describes the immediacy of the innate-like lymphocyte response to trauma in different body compartments and considers new lines for further investigation to develop our understanding of MODS pathogenesis.

Interleukin-33 contributes to ILC2 activation and early inflammation-associated lung injury during abdominal sepsis.

Sepsis is defined as infection with organ dysfunction due to a dysregulated immune response. The lung is one of the most vulnerable organs at the onset of sepsis. Interleukin (IL)-33 can be released by injured epithelial and endothelial cells in the lung and regulate immune activation and infiltration. Therefore, we postulated that IL-33 would contribute to the immune response in the lung during sepsis. Using the cecal ligation and puncture (CLP) sepsis model, we show here that IL-33 contributes significantly to both sepsis-induced inflammation in the lung and systemic inflammatory response in the early phase of sepsis. Despite the higher intra-peritoneal bacterial burden, the absence of IL-33 resulted in less infiltration of neutrophils and monocytes into the lungs in association with lower circulating, lung and liver cytokine levels as well as reduced lung injury at 6 h after sepsis. IL-33 was required for the upregulation of IL-5 in type 2 Innate Lymphoid Cells (ILC2), while IL-5 neutralization suppressed neutrophil and monocyte infiltration in the lungs during CLP sepsis. This reduction in leukocyte infiltration in IL-33-deficient mice was reversed by administration of recombinant IL-5. These results indicate that IL-33 plays a major role in the local inflammatory changes in the lung, in part, by regulating IL-5 and this axis contributes to lung injury early after the onset of sepsis.

Platelet HMGB1 is required for efficient bacterial clearance in intra-abdominal bacterial sepsis in mice.

Thrombocytopenia impairs host defense and hemostasis in sepsis. However, the mechanisms of how platelets regulate host defense are not fully understood. High-mobility group box 1 (HMGB1), a danger-associated molecular pattern protein, is released during infection and contributes to the pathogenesis of sepsis. Platelets express HMGB1, which is released on activation and has been shown to play a critical role in thrombosis, monocyte recruitment, and neutrophil extracellular trap (NET) production. However, the contribution of platelet HMGB1 to host defense is unknown. To determine the role of platelet HMGB1 in polymicrobial sepsis, platelet-specific HMGB1 knockout (HMGB1 platelet factor 4 [PF4]) mice were generated and were subjected to cecal ligation and puncture (CLP), a clinically relevant intra-abdominal sepsis model. Compared with HMGB1 Flox mice and wild-type (WT) mice, HMGB1 PF4 mice showed significantly higher bacterial loads in the peritoneum and blood, an exaggerated systemic inflammation response, and significantly greater mortality after CLP. Deletion of HMGB1 in platelets was associated with lower platelet-derived chemokines (PF4 and RANTES) in the peritoneal cavity, and a decrease of platelet-neutrophil interaction in the lung after CLP. In vitro, neutrophils cocultured with activated HMGB1 knockout platelets showed fewer platelet-neutrophil aggregates, reduced reactive oxygen species (ROS) burst as compared with control. Taken together, these data reveal an unrecognized role of platelet HMGB1 in the regulation of neutrophil recruitment and activation via modulation of platelet activation during sepsis.

Deep vein thrombosis in mice is regulated by platelet HMGB1 through release of neutrophil-extracellular traps and DNA.

Venous thromboembolic (VTE) disease, consisting of deep venous thrombosis (DVT) and pulmonary embolism (PE) is a leading cause of morbidity and mortality. Current prophylactic measures are insufficient to prevent all occurrence in part due to an incomplete understanding of the underlying pathophysiology. Mounting evidence describes interplay between activation of the innate immune system and thrombus development. Recent work has demonstrated that platelet release of HMGB1 leads to increased microvascular complications following injury. Additionally, platelet HMGB1 was found to enhance DVT and increase the formation of neutrophil extracellular traps (NETs), although the role of HMGB1 induced NET release in thrombosis remains unexplored. Utilizing a transgenic mouse lacking HMGB1 specifically from platelets and megakaryocytes we now demonstrate the specific role of platelet-derived HMGB1 in acute and subacute/chronic venous thrombosis. Platelets account for the majority of circulating HMGB1 and HMGB1 deposition within the developing clot. The pro-thrombotic effect of platelet-derived HMGB1 is mediated through enhanced neutrophil recruitment, NET formation and specifically release of extracellular DNA during NET formation. Taken together, these data suggest that platelet HMGB1 mediated NET release is a primary regulator of DVT formation in mice.

IL33-mediated ILC2 activation and neutrophil IL5 production in the lung response after severe trauma: A reverse translation study from a human cohort to a mouse trauma model.

BACKGROUND: The immunosuppression and immune dysregulation that follows severe injury includes type 2 immune responses manifested by elevations in interleukin (IL) 4, IL5, and IL13 early after injury. We hypothesized that IL33, an alarmin released early after tissue injury and a known regulator of type 2 immunity, contributes to the early type 2 immune responses after systemic injury. METHODS AND FINDINGS: Blunt trauma patients admitted to the trauma intensive care unit of a level I trauma center were enrolled in an observational study that included frequent blood sampling. Dynamic changes in IL33 and soluble suppression of tumorigenicity 2 (sST2) levels were measured in the plasma and correlated with levels of the type 2 cytokines and nosocomial infection. Based on the observations in humans, mechanistic experiments were designed in a mouse model of resuscitated hemorrhagic shock and tissue trauma (HS/T). These experiments utilized wild-type C57BL/6 mice, IL33-/- mice, B6.C3(Cg)-Rorasg/sg mice deficient in group 2 innate lymphoid cells (ILC2), and C57BL/6 wild-type mice treated with anti-IL5 antibody. Severely injured human blunt trauma patients (n = 472, average injury severity score [ISS] = 20.2) exhibited elevations in plasma IL33 levels upon admission and over time that correlated positively with increases in IL4, IL5, and IL13 (P < 0.0001). sST2 levels also increased after injury but in a delayed manner compared with IL33. The increases in IL33 and sST2 were significantly greater in patients that developed nosocomial infection and organ dysfunction than similarly injured patients that did not (P < 0.05). Mechanistic studies were carried out in a mouse model of HS/T that recapitulated the early increase in IL33 and delayed increase in sST2 in the plasma (P < 0.005). These studies identified a pathway where IL33 induces ILC2 activation in the lung within hours of HS/T. ILC2 IL5 up-regulation induces further IL5 expression by CXCR2+ lung neutrophils, culminating in early lung injury. The major limitations of this study are the descriptive nature of the human study component and the impact of the potential differences between human and mouse immune responses to polytrauma. Also, the studies performed did not permit us to make conclusions about the impact of IL33 on pulmonary function. CONCLUSIONS: These results suggest that IL33 may initiate early detrimental type 2 immune responses after trauma through ILC2 regulation of neutrophil IL5 production. This IL33-ILC2-IL5-neutrophil axis defines a novel regulatory role for ILC2 in acute lung injury that could be targeted in trauma patients prone to early lung dysfunction.

NK1.1+ cells promote sustained tissue injury and inflammation after trauma with hemorrhagic shock.

Various cell populations expressing NK1.1 contribute to innate host defense and systemic inflammatory responses, but their role in hemorrhagic shock and trauma remains uncertain. NK1.1+ cells were depleted by i.p. administration of anti-NK1.1 (or isotype control) on two consecutive days, followed by hemorrhagic shock with resuscitation and peripheral tissue trauma (HS/T). The plasma levels of IL-6, MCP-1, alanine transaminase (ALT), and aspartate aminotransferase (AST) were measured at 6 and 24 h. Histology in liver and gut were examined at 6 and 24 h. The number of NK cells, NKT cells, neutrophils, and macrophages in liver, as well as intracellular staining for TNF-α, IFN-γ, and MCP-1 in liver cell populations were determined by flow cytometry. Control mice subjected to HS/T exhibited end organ damage manifested by marked increases in circulating ALT, AST, and MCP-1 levels, as well as histologic evidence of hepatic necrosis and gut injury. Although NK1.1+ cell-depleted mice exhibited a similar degree of organ damage as nondepleted animals at 6 h, NK1.1+ cell depletion resulted in marked suppression of both liver and gut injury by 24 h after HS/T. These findings indicate that NK1.1+ cells contribute to the persistence of inflammation leading to end organ damage in the liver and gut.

Role of the IL-33-ST2 axis in sepsis.

Sepsis remains a major clinical problem with high morbidity and mortality. As new inflammatory mediators are characterized, it is important to understand their roles in sepsis. Interleukin 33 (IL-33) is a recently described member of the IL-1 family that is widely expressed in cells of barrier tissues. Upon tissue damage, IL-33 is released as an alarmin and activates various types of cells of both the innate and adaptive immune system through binding to the ST2/IL-1 receptor accessory protein complex. IL-33 has apparent pleiotropic functions in many disease models, with its actions strongly shaped by the local microenvironment. Recent studies have established a role for the IL-33-ST2 axis in the initiation and perpetuation of inflammation during endotoxemia, but its roles in sepsis appear to be organism and model dependent. In this review, we focus on the recent advances in understanding the role of the IL-33/ST2 axis in sepsis.

Graft-infiltrating host dendritic cells play a key role in organ transplant rejection.

Successful engraftment of organ transplants has traditionally relied on preventing the activation of recipient (host) T cells. Once T-cell activation has occurred, however, stalling the rejection process becomes increasingly difficult, leading to graft failure. Here we demonstrate that graft-infiltrating, recipient (host) dendritic cells (DCs) play a key role in driving the rejection of transplanted organs by activated (effector) T cells. We show that donor DCs that accompany heart or kidney grafts are rapidly replaced by recipient DCs. The DCs originate from non-classical monocytes and form stable, cognate interactions with effector T cells in the graft. Eliminating recipient DCs reduces the proliferation and survival of graft-infiltrating T cells and abrogates ongoing rejection or rejection mediated by transferred effector T cells. Therefore, host DCs that infiltrate transplanted organs sustain the alloimmune response after T-cell activation has already occurred. Targeting these cells provides a means for preventing or treating rejection.

Choosing and remaining in mental health nursing: perceptions of Western Australian nurses.

Mental health nursing has an ageing workforce with a critical shortage of nurses in Western Australia. Additionally, mental health is not the preferred career for many graduate nurses. Current challenges with recruitment and retention suggest that strategies are needed to address this issue. This research project adopted a novel approach that focused on exploring the positive aspects of why mental health nurses remain, rather than why they leave. A cross-sectional design was employed comprising a brief interview survey, and nurses working within one public mental health service in Western Australia were invited to participate. A total of 192 nurses participated across 5 months, from adult, older adult, forensic, and education/research programmes. Thematic analysis was conducted from five key questions, and responses from questions one and two are discussed in this paper: 'Why did you choose mental health nursing?' and 'Why do you remain in mental health nursing?'. The main themes extracted in response to choosing mental health nursing were wanting to make a difference, mental health captured my interest, encouraged by others, and opportunities. Subsequent themes extracted from responses to remaining in mental health nursing were facing reality, passion for mental health nursing, patient-centred caring, and workplace conditions. Findings will be utilized to inform strategies for recruitment and retention of graduate nurses; further development of support systems, such as preceptorship training and improving student clinical experiences; as well as improving professional development opportunities for existing mental health nurses.

Toll-like receptor 4 regulates platelet function and contributes to coagulation abnormality and organ injury in hemorrhagic shock and resuscitation.

BACKGROUND: Growing evidence indicates that the presence of toll-like receptor 4 (TLR4) on platelets is a key regulator of platelet number and function. Platelets exposed to TLR4 agonists may serve to activate other cells such as neutrophils and endothelial cells in sepsis and other inflammatory conditions. The functional significance of platelet TLR4 in hemorrhagic shock (HS), however, remains unexplored. METHODS AND RESULTS: Using thromboelastography and platelet aggregometry, we demonstrate that platelet function is impaired in a mouse model of HS with resuscitation. Further analysis using cellular-specific TLR4 deletion in mice revealed that platelet TLR4 is essential for platelet activation and function in HS with resuscitation and that platelet TLR4 regulates the development of coagulopathy after hemorrhage and resuscitation. Transfusion of TLR4-negative platelets into mice resulted in protection from coagulopathy and restored platelet function. Additionally, platelet-specific TLR4 knockout mice were protected from lung and liver injury and exhibited a marked reduction in systemic inflammation as measured by circulating interleukin-6 after HS with resuscitation. CONCLUSIONS: We demonstrate for the first time that platelet TLR4 is an essential mediator of the inflammatory response as well as platelet activation and function in HS and resuscitation.

B cells mediate chronic allograft rejection independently of antibody production.

Chronic rejection is the primary cause of long-term failure of transplanted organs and is often viewed as an antibody-dependent process. Chronic rejection, however, is also observed in mice and humans with no detectable circulating alloantibodies, suggesting that antibody-independent pathways may also contribute to pathogenesis of transplant rejection. Here, we have provided direct evidence that chronic rejection of vascularized heart allografts occurs in the complete absence of antibodies, but requires the presence of B cells. Mice that were deficient for antibodies but not B cells experienced the same chronic allograft vasculopathy (CAV), which is a pathognomonic feature of chronic rejection, as WT mice; however, mice that were deficient for both B cells and antibodies were protected from CAV. B cells contributed to CAV by supporting splenic lymphoid architecture, T cell cytokine production, and infiltration of T cells into graft vessels. In chimeric mice, in which B cells were present but could not present antigen, both T cell responses and CAV were markedly reduced. These findings establish that chronic rejection can occur in the complete absence of antibodies and that B cells contribute to this process by supporting T cell responses through antigen presentation and maintenance of lymphoid architecture.

Combined in silico, in vivo, and in vitro studies shed insights into the acute inflammatory response in middle-aged mice.

We combined in silico, in vivo, and in vitro studies to gain insights into age-dependent changes in acute inflammation in response to bacterial endotoxin (LPS). Time-course cytokine, chemokine, and NO2 (-/)NO3 (-) data from "middle-aged" (6-8 months old) C57BL/6 mice were used to re-parameterize a mechanistic mathematical model of acute inflammation originally calibrated for "young" (2-3 months old) mice. These studies suggested that macrophages from middle-aged mice are more susceptible to cell death, as well as producing higher levels of pro-inflammatory cytokines, vs. macrophages from young mice. In support of the in silico-derived hypotheses, resident peritoneal cells from endotoxemic middle-aged mice exhibited reduced viability and produced elevated levels of TNF-α, IL-6, IL-10, and KC/CXCL1 as compared to cells from young mice. Our studies demonstrate the utility of a combined in silico, in vivo, and in vitro approach to the study of acute inflammation in shock states, and suggest hypotheses with regard to the changes in the cytokine milieu that accompany aging.

Cognate antigen directs CD8+ T cell migration to vascularized transplants.

The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration - integrin-mediated firm adhesion followed by transendothelial migration - are dependent on the activation of Gαi-coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8+ effector T cells specific to graft antigens and that both steps occurred independent of Gαi signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gαi, but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.

Selective roles for toll-like receptors 2, 4, and 9 in systemic inflammation and immune dysfunction following peripheral tissue injury.

BACKGROUND: Toll-like receptors (TLRs) detect endogenous ligands released after trauma and contribute to the proinflammatory response to injury. Posttraumatic mortality correlates with the extent of the immunoinflammatory response to injury that is composed of a complex regulation of innate and adaptive immune responses. Although TLRs are known to modulate innate immune responses, their role in the suppression of lymphocyte responses following traumatic tissue injury is unclear. METHODS: This study used a murine model of severe peripheral tissue injury, involving muscle crush injury and injection of fracture components, to evaluate the roles of TLR2, TLR4, and TLR9 in the early and delayed immunoinflammatory phenotype. Posttraumatic immune dysfunction was measured in our trauma model using the following parameters: ex vivo splenocyte proliferation, TH1 cytokine release, and iNOS (inducible nitric oxide synthase) induction within splenic myeloid-derived suppressor cells. Systemic inflammation and liver damage were determined by circulating interleukin 6 levels and hepatocellular injury. RESULTS: Suppression of splenocyte responses after injury was dependent on TLR4 and TLR9 signaling as was posttraumatic iNOS upregulation in splenic myeloid-derived suppressor cells. TLR2 was found to have only a partial role through contribution to inhibition of splenocyte proliferation. This study also reveals the involvement of TLR2 and TLR4 in the initial systemic inflammatory response to traumatic tissue injury; however, this response was found to be TLR9 independent. CONCLUSION: These findings demonstrate the previously unidentified role of TLR2, TLR4, and TLR9 in the T cell-associated immune dysfunction following traumatic tissue injury. Importantly, this study also illustrates that TLRs play differing and selective roles in both the initial proinflammatory response and adaptive immune response after trauma. Furthermore, results in TLR9-deficient mice establish that the upregulation of early proinflammatory markers do not always correlate with the extent of sustained immune dysfunction. This suggests potential for targeted therapies that could limit immune dysfunction through selective inhibition of receptor function following injury.

Inducible nitric oxide synthase contributes to immune dysfunction following trauma.

Trauma results in a persistent depression in adaptive immunity, which contributes to patient morbidity and mortality. This state of immune paralysis following trauma is characterized by a change in cell-mediated immunity, specifically a depression in T-cell function and a shift toward TH2 T-cell phenotype. Upregulation of inducible nitric oxide synthase (iNOS) is well recognized after injury and contributes to the inflammatory response and organ damage early after trauma. However, it is unknown whether iNOS plays a role in adaptive immune dysfunction after trauma. This study utilized a murine model of severe peripheral tissue injury to show that iNOS is rapidly upregulated in macrophages and a (Gr-1-CD11b) myeloid-derived suppressor cell subpopulation in the spleen. Through the use of iNOS knockout mice, a specific iNOS inhibitor, and a nitric oxide (NO) scavenger, this study demonstrates that iNOS-derived NO is required for the depression in T-lymphocyte proliferation, interferon γ, and interleukin 2 production within the spleen at 48 h after trauma. These findings support the hypothesis that iNOS regulates immune suppression following trauma and suggest that targeting the sustained production of NO by iNOS may attenuate posttraumatic immune depression.

Memory T cells migrate to and reject vascularized cardiac allografts independent of the chemokine receptor CXCR3.

BACKGROUND: Memory T cells migrate to and reject transplanted organs without the need for priming in secondary lymphoid tissues, but the mechanisms by which they do so are not known. Here, we tested whether CXCR3, implicated in the homing of effector T cells to sites of infection, is critical for memory T-cell migration to vascularized allografts. METHODS: CD4 and CD8 memory T cells were sorted from alloimmunized CXCR3 and wildtype B6 mice and cotransferred to congenic B6 recipients of BALB/c heart allografts. Graft-infiltrating T cells were quantitated 20 and 72 hr later by flow cytometry. Migration and allograft survival were also studied in splenectomized alymphoplastic (aly/aly) recipients, which lack secondary lymphoid tissues. RESULTS: We found that polyclonal and antigen-specific memory T cells express high levels of CXCR3. No difference in migration of wildtype versus CXCR3 CD4 and CD8 memory T cells to allografts could be detected in wildtype or aly/aly hosts. In the latter, wildtype and CXCR3 memory T cells precipitated acute rejection at similar rates. Blocking CCR5, a chemokine receptor also upregulated on memory T cells, did not delay graft rejection mediated by CXCR3 memory T cells. CONCLUSIONS: CXCR3 is not critical for the migration of memory T cells to vascularized organ allografts. Blocking CXCR3 or CXCR3 and CCR5 does not delay acute rejection mediated by memory T cells. These findings suggest that the mechanisms of memory T cell-homing to transplanted organs may be distinct from those required for their migration to sites of infection.

A role for connexin43 in macrophage phagocytosis and host survival after bacterial peritoneal infection.

The pathways that lead to the internalization of pathogens via phagocytosis remain incompletely understood. We now demonstrate a previously unrecognized role for the gap junction protein connexin43 (Cx43) in the regulation of phagocytosis by macrophages and in the host response to bacterial infection of the peritoneal cavity. Primary and cultured macrophages were found to express Cx43, which localized to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation. Remarkably, mortality was significantly increased in a mouse model of bacterial peritonitis after Cx43 inhibition and in Cx43 heterozygous mice compared with untreated and wild-type counterparts. These findings reveal a novel role for Cx43 in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and they implicate Cx43 in the regulation of the host response to microbial infection.

Systemic inflammation and end organ damage following trauma involves functional TLR4 signaling in both bone marrow-derived cells and parenchymal cells.

Endogenous damage-associated molecular pattern (DAMP) molecules are released from cells during traumatic injury, allowing them to interact with pattern recognition receptors such as the toll-like receptors (TLRs) on other cells and subsequently, to stimulate inflammatory signaling. TLR4, in particular, plays a key role in systemic and remote organ responses to hemorrhagic shock (HS) and peripheral tissue injury in the form of bilateral femur fracture. TLR4 chimeric mice were generated to investigate the cell lineage in which functional TLR4 is needed to initiate the injury response to trauma. Chimeric mice were generated by adoptive bone marrow (BM) transfer, whereby donor marrow was given to an irradiated host using reciprocal combinations of TLR4 wild-type (WT; C3H/HeOuJ) and TLR4 mutant (Mu; C3H/HeJ) mice. After a period of engraftment, chimeric mice were then subjected to HS or bilateral femur fracture. Control groups, including TLR4-WT mice receiving WT BM and TLR4-Mu mice receiving Mu BM, responded to injury in a similar pattern to unaltered HeOuJ and HeJ mice, and protection was afforded to those mice lacking functional TLR4. In contrast, TLR4-WT mice receiving Mu BM and TLR4-Mu mice receiving WT BM demonstrated intermediate inflammatory and cellular damage profiles. These data demonstrate that functional TLR4 is required in BM-derived cells and parenchymal cells for an optimal inflammatory response to trauma.