Deletion of IL-6 Exacerbates Colitis and Induces Systemic Inflammation in IL-10-Deficient Mice.

BACKGROUND AND AIMS: Interleukin 6 [IL-6] or its receptor is currently a candidate for targeted biological therapy of inflammatory bowel disease [IBD]. Thus, a comprehensive understanding of the consequences of blocking IL-6 is imperative. We investigated this by evaluating the effects of IL-6 deletion on the spontaneous colitis of IL-10-deficient mice [IL-10-/-]. METHODS: IL-6/IL-10 double-deficient mice [IL-6-/-/IL-10-/-] were generated and analysed for intestinal inflammation, general phenotypes and molecular/biochemical changes in the colonic mucosa compared with wild-type and IL-10-/- mice. RESULTS: Unexpectedly, the IL-6-/-/IL-10-/- mice showed more pronounced gut inflammation and earlier disease onset than IL-10-/- mice, both locally [colon and small bowel] and systemically [splenomegaly, ulcerative dermatitis, leukocytosis, neutrophilia and monocytosis]. IL-6-/-/IL-10-/- mice exhibited elevations of multiple cytokines [IL-1ß, IL-4, IL-12, TNFα] and chemokines [MCP-1 and MIG], but not IFN-γ [Th1], IL-17A and IL-17G [Th17], or IL-22 [Th22]. FOXP3 and TGF-ß, two key factors for regulatory T [Treg] cell differentiation, were significantly down-regulated in the colonic mucosa, but not in the thymus or mesenteric lymph nodes, of IL-6-/-/IL-10-/- mice. CTLA-4 was diminished while iNOS was up-regulated in the colonic mucosa of IL-6-/-/IL-10-/- mice. CONCLUSION: In IL-10-/- mice, complete IL-6 blockade significantly aggravates gut inflammation, at least in part by suppressing Treg/CTLA-4 and promoting the IL-1ß/Th2 pathway. In addition, the double mutant exhibits signs of severe systemic inflammation. Our data define a new function of IL-6 and suggest that caution should be exercised when targeting IL-6 in IBD patients, particularly those with defects in IL-10 signalling.

Eradication of Hepatocellular Carcinoma by NKG2D-Based CAR-T Cells.

Despite the great success of chimeric antigen receptor T (CAR-T)-cell therapy in the treatment of hematologic malignancies, CAR-T-cell therapy is limited in solid tumors, including hepatocellular carcinoma (HCC). NK group 2 member D (NKG2D) ligands (NKG2DL) are generally absent on the surface of normal cells but are overexpressed on malignant cells, offering good targets for CAR-T therapy. Indeed, analysis of The Cancer Genome Atlas and HCC tumor samples showed that the expression of most NKG2DLs was elevated in tumors compared with normal tissues. Thus, we designed a novel NKG2D-based CAR comprising the extracellular domain of human NKG2D, 4-1BB, and CD3ζ signaling domains (BBz). NKG2D-BBz CAR-T cells efficiently killed the HCC cell lines SMMC-7721 and MHCC97H in vitro, which express high levels of NKG2DLs, whereas they less efficiently killed NKG2DL-silenced SMMC-7721 cells or NKG2DL-negative Hep3B cells. Overexpression of MICA or ULBP2 in Hep3B improved the killing capacity of NKG2D-BBz CAR-T cells. T cells expressing the NKG2D-BBz CAR effectively eradicated SMMC-7721 HCC xenografts. Collectively, these results suggested that NKG2D-BBz CAR-T cells could potently eliminate NKG2DL-high HCC cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for patients with NKG2DL-positive HCC.

Micromechanical Modeling of Damage Evolution and Mechanical Behaviors of CF/Al Composites under Transverse and Longitudinal Tensile Loadings.

This paper investigates the progressive damage and failure behavior of unidirectional graphite fiber-reinforced aluminum composites (CF/Al composites) under transverse and longitudinal tensile loadings. Micromechanical finite element analyses are carried out using different assumptions regarding fiber, matrix alloy, and interface properties. The validity of these numerical analyses is examined by comparing the predicted stress-strain curves with the experimental data measured under transverse and longitudinal tensile loadings. Assuming a perfect interface, the transverse tensile strength is overestimated by more than 180% and the transverse fracture induced by fiber failure is unrealistic based on the experimental observations. In fact, the simulation and experiment results indicate that the interface debonding arising from the matrix alloy failure dominates the transverse fracture, and the influence of matrix alloy properties on the mechanical behavior is inconspicuous. In the case of longitudinal tensile testing, however, the characteristic of interface bonding has no significant effect on the macroscopic mechanical response due to the low in-situ strength of the fibers. It is demonstrated that ultimate longitudinal fracture is mainly controlled by fiber failure mechanisms, which is confirmed by the fracture morphology of the tensile samples.

Tlr2 on Bone Marrow and Non-Bone Marrow Derived Cells Regulates Inflammation and Organ Injury in Cooperation with Tlr4 During Resuscitated Hemorrhagic Shock.

BACKGROUND: Although the role of TLR4 in driving inflammation and organ injury after hemorrhagic shock and resuscitation (H/R) is well established, the role of TLR2-another receptor for damage-associated molecular pattern (DAMP) molecules-is not. In this study, we used a combination of TLR2 and wild type (WT) mice treated with anti-TLR2 and anti-TLR4 neutralizing monoclonal antibodies (mAb) to discern the contribution of TLR2 relative to TLR4 to the systemic inflammatory response in murine H/R. MATERIAL AND METHODS: WT mice, TLR2, and WT mice receiving an anti-TLR2 or an anti-TLR4 mAB (given as a pretreatment) were sacrificed at 6 or 20 h post-H/R. Bone marrow TLR2/WT chimeric mice were created to assess the importance of immune and nonimmune cell-associated TLR2. RESULTS: TLR2 mice subjected to H/R exhibited significantly less liver damage and lower markers of systemic inflammation only at 20 h. Bone marrow chimeric mice using combinations of TLR2 mice and WT mice demonstrated that TLR2 on non-bone marrow derived cells played a dominant role in the differences at 20 h. Interestingly, WT mice treated with anti-TLR2 mAB demonstrated a reduction in organ damage and systemic inflammation at both 6 and 20 h following H/R. A combination of anti-TLR2 mAB and anti-TLR4 mAB showed that both receptors drive IP-10 and KC levels and that there is cooperation for increases in IL-6, MIG, and MCP-1 levels between TLR2 and TLR4. CONCLUSION: These data also support the conclusion that TLR2 and TLR4 act in concert as important receptors in the host immune response to H/R.

The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88.

Extracellular high-mobility group box 1 (HMGB1) (disulfide form), via activation of toll-like receptor 4 (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1-induced TLR4 activation, as assayed by the production of TNF-α by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334 or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver ischemia/reperfusion (I/R) models, both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4-driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside. Disulfide HMGB1 drives pathologic inflammation in many models by activating signaling through TLR4. Cell-based screening identified the mammalian protease cathepsin V as a novel therapeutic target to inhibit TLR4-mediated inflammation induced by extracellular HMGB1 (disulfide form). We identified two protease inhibitors (PIs) that block cathepsin V and thereby inhibit disulfide HMGB1-induced TLR4 activation: saquinavir (SQV), a first-generation PI targeting viral HIV protease and STO33438 (334), targeting mammalian proteases. We discovered that cathepsin V binds TLR4 under basal and HMGB1-stimulated conditions, but dissociates in the presence of SQV over time. Thus cathepsin V is a novel target for first-generation HIV PIs and represents a potential therapeutic target of pathologic inflammation.

Anti-HMGB1 monoclonal antibody ameliorates immunosuppression after peripheral tissue trauma: attenuated T-lymphocyte response and increased splenic CD11b (+) Gr-1 (+) myeloid-derived suppressor cells require HMGB1.

Although tissue-derived high mobility group box 1 (HMGB1) is involved in many aspects of inflammation and tissue injury after trauma, its role in trauma-induced immune suppression remains elusive. Using an established mouse model of peripheral tissue trauma, which includes soft tissue and fracture components, we report here that treatment with anti-HMGB1 monoclonal antibody ameliorated the trauma-induced attenuated T-cell responses and accumulation of CD11b(+)Gr-1(+) myeloid-derived suppressor cells in the spleens seen two days after injury. Our data suggest that HMGB1 released after tissue trauma contributes to signaling pathways that lead to attenuation of T-lymphocyte responses and enhancement of myeloid-derived suppressor cell expansion.

Benzyl alcohol attenuates acetaminophen-induced acute liver injury in a Toll-like receptor-4-dependent pattern in mice.

UNLABELLED: Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in industrialized countries. Understanding the mechanisms of APAP-induced liver injury as well as other forms of sterile liver injury is critical to improve the care of patients. Recent studies demonstrate that danger signaling and inflammasome activation play a role in APAP-induced injury. The aim of these investigations was to test the hypothesis that benzyl alcohol (BA) is a therapeutic agent that protects against APAP-induced liver injury by modulation of danger signaling. APAP-induced liver injury was dependent, in part, on Toll-like receptor (TLR)9 and receptor for advanced glycation endproducts (RAGE) signaling. BA limited liver injury over a dose range of 135-540 µg/g body weight or when delivered as a pre-, concurrent, or post-APAP therapeutic. Furthermore, BA abrogated APAP-induced cytokines and chemokines as well as high-mobility group box 1 release. Moreover, BA prevented APAP-induced inflammasome signaling as determined by interleukin (IL)-1ß, IL-18, and caspase-1 cleavage in liver tissues. Interestingly, the protective effects of BA on limiting liver injury and inflammasome activation were dependent on TLR4 signaling, but not TLR2 or CD14. Cell-type-specific knockouts of TLR4 were utilized to further determine the protective mechanisms of BA. These studies found that TLR4 expression specifically in myeloid cells (LyzCre-tlr4-/-) were necessary for the protective effects of BA. CONCLUSION: BA protects against APAP-induced acute liver injury and reduced inflammasome activation in a TLR4-dependent manner. BA may prove to be a useful adjunct in the treatment of APAP and other forms of sterile liver injury.

CD14 contributes to warm hepatic ischemia-reperfusion injury in mice.

INTRODUCTION: Ischemia/reperfusion (I/R) of the liver contributes to the pathobiology of liver injury in transplantation, liver surgery, and hemorrhagic shock. Ischemia/reperfusion induces an inflammatory response that is driven, in part, by Toll-like receptor 4 (TLR) signaling. CD14 is known to participate in the function of TLR4. We hypothesized that CD14 would be involved in the pathobiology of warm hepatic I/R. METHODS: Using a 70% liver inflow inclusion model, CD14 knockout and wild-type (WT) mice were subjected to 1-h warm ischemia followed by reperfusion. CD14 mRNA, circulating transaminase, interleukin 6, soluble CD14, and high-mobility group box 1 (HMGB1) levels were measured. CD14 neutralizing antibody or isotype control antibody was given before ischemia or reperfusion for CD14 blockade in WT mice. Recombinant HMGB1 was given before reperfusion in some experiments to test if liver injury worsens. RESULTS: There was an upregulation of CD14 mRNA in reperfused livers together with increased soluble CD14 levels in the circulation. Compared with WT control mice, CD14 knockout mice had much lower alanine aminotransferase and interleukin 6 levels at 6 and 24 h following I/R, and much less liver necrosis by histology. TUNEL (terminal deoxynucleotidyl-transferase dUTP nick end labeling) staining displayed less apoptosis at 24 h in the absence of CD14. CD14 blockage by neutralizing antibody also attenuated liver injury and the inflammatory response in C57BL/6 mice following I/R, but did not provide additional protection to TLR4 mutant C3H/Hej mice. CD14 deficiency did not change circulating HMGB1 levels following I/R (6 h). A dose of recombinant HMGB1, which worsened hepatic injury when given before reperfusion in WT mice, did not increase liver damage in CD14-deficient mice. CONCLUSIONS: CD14 is actively involved in hepatic I/R injury. Its deficiency or blockade ischemia attenuates liver injury and inflammatory response. CD14 mediates liver damage and inflammatory responses in the setting of warm hepatic I/R in mice.

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.

Wendan decoction improves learning and memory deficits in a rat model of schizophrenia.

An experimental model of schizophrenia was established using dizocilpine (MK-801). Rats were intragastrically administered with Wendan decoction or clozapine for 21 days prior to establishing the model. The results revealed that the latency of schizophrenia model rats to escape from the hidden platform in the Morris water maze was significantly shortened after administration of Wendan decoction or clozapine. In addition, the treated rats crossed the platform significantly more times than the untreated model rats. Moreover, the rate of successful long-term potentiation induction in the Wendan decoction group and clozapine group were also obviously increased compared with the model group, and the population spike peak latency was significantly shortened. These experimental findings suggest that Wendan decoction can improve the learning and memory ability of schizophrenic rats to the same extent as clozapine treatment.

APOBEC3G promotes liver metastasis in an orthotopic mouse model of colorectal cancer and predicts human hepatic metastasis.

Colorectal cancer is the second leading cause of death from cancer in the United States. Metastases in the liver, the most common metastatic site for colorectal cancer, are found in one-third of the patients who die of colorectal cancer. Currently, the genes and molecular mechanisms that are functionally critical in modulating colorectal cancer hepatic metastasis remain unclear. Here, we report our studies using functional selection in an orthotopic mouse model of colorectal cancer to identify a set of genes that play an important role in mediating colorectal cancer liver metastasis. These genes included APOBEC3G, CD133, LIPC, and S100P. Clinically, we found these genes to be highly expressed in a cohort of human hepatic metastasis and their primary colorectal tumors, suggesting that it might be possible to use these genes to predict the likelihood of hepatic metastasis. We have further revealed what we believe to be a novel mechanism in which APOBEC3G promotes colorectal cancer hepatic metastasis through inhibition of miR-29-mediated suppression of MMP2. Together, our data elucidate key factors and mechanisms involved in colorectal cancer liver metastasis, which could be potential targets for diagnosis and treatment.

Mast cells play a critical role in the systemic inflammatory response and end-organ injury resulting from trauma.

BACKGROUND: Much of the morbidity after trauma results from excessive activation of the innate immune system. This is manifested as a systemic inflammatory response and associated end-organ damage. Although mast cells are known to be important in many immune responses, their role in the systemic response to severe trauma is unknown. STUDY DESIGN: C57BL/6J-KitW-sh/BsmJ (mast cell deficient) and wild type mice were subjected to 1.5 hours of hemorrhagic shock plus bilateral femur fracture and soft tissue injury (HS/T), followed by resuscitation at 4.5 hours. Blood withdrawal volumes, mean arterial pressures, circulating cytokine, chemokine, high mobility group box-1 (HMGB-1), double strain DNA (dsDNA), transaminase levels, and histology in liver and lung were compared between groups. RESULTS: Mast cell deficient mice exhibited greater hemodynamic stability than wild type mice. At baseline, the mast cell deficient mice exhibited no difference in any of the organ injury or inflammatory markers measured. As expected, wild type mice subjected to HS/T exhibited end-organ damage manifested by marked increases in circulating alanine transaminase, aspartate aminotransferase, and dsDNA levels, as well as histologic evidence of tissue necrosis. In clear contrast, mast cell deficient mice exhibited almost no tissue damage. Similarly, the magnitude of increased circulating cytokine and chemokine induced by HS/T was much less in the mast cell deficient mice than in the wild type group. CONCLUSIONS: Mast cell deficiency resulted in a damped systemic inflammatory response, greatly attenuated multiple organ injury, and more stable hemodynamics in HS/T. So mast cells appear to be a critical component of the initial host response to severe injury.

Complement factor 3 deficiency attenuates hemorrhagic shock-related hepatic injury and systemic inflammatory response syndrome.

Although complement activation is known to occur in the setting of severe hemorrhagic shock and tissue trauma (HS/T), the extent to which complement drives the initial inflammatory response and end-organ damage is uncertain. In this study, complement factor 3-deficient (C3(-/-)) mice and wild-type control mice were subjected to 1.5-h hemorrhagic shock, bilateral femur fracture, and soft tissue injury, followed by 4.5-h resuscitation (HS/T). C57BL/6 mice were also given 15 U of cobra venom factor (CVF) or phosphate-buffered saline injected intraperitoneally, followed by HS/T 24 h later. The results showed that HS/T resulted in C3 consumption in wild-type mice and C3 deposition in injured livers. C3(-/-) mice had significantly lower serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and circulating DNA levels, together with much lower circulating interleukin (IL)-6, IL-10, and high-mobility group box 1 (HMGB1) levels. Temporary C3 depletion by CVF preconditioning also led to reduced transaminases and a blunted cytokine release. C3(-/-) mice displayed well-preserved hepatic structure. C3(-/-) mice subjected to HS/T had higher levels of heme oxygenase-1, which has been associated with tissue protection in HS models. Our data indicate that complement activation contributes to inflammatory pathways and liver damage in HS/T. This suggests that targeting complement activation in the setting of severe injury could be useful.

A critical role for IFN regulatory factor 1 in NKT cell-mediated liver injury induced by alpha-galactosylceramide.

NKT cells are remarkably abundant in mouse liver. Compelling experimental evidence has suggested that NKT cells are involved in the pathogenesis of many liver diseases. Activation of NKT cells with alpha-galactosylceramide (alpha-GalCer) causes liver injury through mechanisms that are not well understood. We undertook studies to characterize the key pathways involved in alpha-GalCer-induced liver injury. We found that expression of the transcription factor IFN regulatory factor 1 (IRF-1) in mouse liver was dramatically upregulated by alpha-GalCer treatment. Neutralization of either TNF-alpha or IFN-gamma inhibited alpha-GalCer-mediated IRF-1 upregulation. alpha-GalCer-induced liver injury was significantly suppressed in IRF-1 knockout mice or in wild-type C56BL/6 mice that received a microRNA specifically targeting IRF-1. In contrast, overexpression of IRF-1 greatly potentiated alpha-GalCer-induced liver injury. alpha-GalCer injection also induced a marked increase in hepatic inducible NO synthase expression in C56BL/6 mice, but not in IRF-1 knockout mice. Inducible NO synthase knockout mice exhibited significantly reduced liver injury following alpha-GalCer treatment. Finally, we demonstrated that both NKT cells and hepatocytes expressed IRF-1 in response to alpha-GalCer. However, it appeared that the hepatocyte-derived IRF-1 was mainly responsible for alpha-GalCer-induced liver injury, based on the observation that inhibition of IRF-1 by RNA interference did not affect alpha-GalCer-induced NKT cell activation. Our findings revealed a novel mechanism of NKT cell-mediated liver injury in mice, which has implications in the development of human liver diseases.