Pinhua Pan

Cisplatin prevents high mobility group box 1 release and is protective in a murine model of hepatic ischemia/reperfusion injury†

The nuclear protein high mobility group box 1 (HMGB1) is an important inflammatory mediator involved in the pathogenesis of liver ischemia/reperfusion (I/R) injury. Strategies aimed at preventing its release from stressed or damaged cells may be beneficial in preventing inflammation after I/R. Cisplatin is a member of the platinating chemotherapeutic agents and can induce DNA lesions that are capable of retaining high mobility group proteins inside the nucleus of cells. In vitro studies in primary cultured rat hepatocytes show that nontoxic concentrations of cisplatin can sequester HMGB1 inside the nucleus of hypoxic cells. Similarly, the in vivo administration of nontoxic doses of cisplatin prevents liver damage associated with a well‐established murine model of hepatic I/R as measured by lower circulating serum aminotransferase levels, lower hepatic inflammatory cytokine levels including tumor necrosis factor α and interleukin‐6, lower inducible NO synthase expression, and fewer I/R‐associated histopathologic changes. The mechanism of action in vivo appears to involve the capacity of cisplatin to prevent the I/R‐induced release of HMGB1 as well as to alter cell survival and stress signaling in the form of autophagy and mitogen‐activated protein kinase activation, respectively. Conclusion: Low, nontoxic doses of cisplatin can sequester HMGB1 inside the nucleus of redox‐stressed hepatocytes in vitro and prevent its release in vivo in a murine model of hepatic I/R. Furthermore, cell survival and stress signaling pathways are altered by low‐dose cisplatin. Therefore, platinating agents may provide a novel approach to mitigating the deleterious effects of I/R‐mediated disease processes. (HEPATOLOGY 2009.)

Interferon Regulatory Factor-1 Regulates the Autophagic Response in LPS-Stimulated Macrophages through Nitric Oxide

The pathogenesis of sepsis is complex and, unfortunately, poorly understood. The cellular process of autophagy is believed to play a protective role in sepsis; however, the mechanisms responsible for its regulation in this setting are ill defined. In the present study, interferon regulatory factor 1 (IRF-1) was found to regulate the autophagic response in lipopolysaccharide (LPS)-stimulated macrophages. In vivo, tissue macrophages obtained from LPS-stimulated IRF-1 knockout (KO) mice demonstrated increased autophagy and decreased apoptosis compared to those isolated from IRF-1 wild-type (WT) mice. In vitro, LPS-stimulated peritoneal macrophages obtained from IRF-1 KO mice experienced increased autophagy and decreased apoptosis. IRF-1 mediates the inhibition of autophagy by modulating the activation of the mammalian target of rapamycin (mTOR). LPS induced the activation of mTOR in WT peritoneal macrophages, but not in IRF-1 KO macrophages. In contrast, overexpression of IRF-1 alone increased the activation of mTOR and consequently decreased autophagic flux. Furthermore, the inhibitory effects of IRF-1 mTOR activity were mediated by nitric oxide (NO). Therefore, we propose a novel role for IRF-1 and NO in the regulation of macrophage autophagy during LPS stimulation in which IRF-1/NO inhibits autophagy through mTOR activation.

Low-dose cisplatin administration in murine cecal ligation and puncture prevents the systemic release of HMGB1 and attenuates lethality

Sepsis remains a major cause of morbidity and mortality worldwide. The systemic release of the nuclear protein HMGB1 is a late event in endotoxin‐related lethality in mice. The platinating chemotherapeutic Cis induces DNA lesions that sequester HMGB1 within the nucleus of cells. We sought to determine if low, nontoxic doses of Cis could be an effective strategy in ameliorating sepsis‐related mortality in a mouse model of CLP. In vitro studies with Cis prevented the LPS‐induced release of HMGB1 from RAW264.7 cells, limited MAPK signaling, but had no effect on NF‐κB activation or cytokine production. Low, nontoxic doses of Cis decreased mortality following CLP, whether delivered before or after puncture. Protection was associated with a decrease in the systemic release of HMGB1 and protection from end organ injury and in particular, less acute lung injury. Tissue‐specific iNOS expression was markedly reduced. Low, nontoxic doses of Cis sequester HMGB1 effectively inside of the nucleus of LPS‐stimulated immune cells and prevent its release in response to CLP. Platinating agents in general and Cis specifically may be a novel approach to the treatment of sepsis.

SPLENOCYTE APOPTOSIS AND AUTOPHAGY IS MEDIATED BY INTERFERON REGULATORY FACTOR-1 DURING MURINE ENDOTOXEMIA

Sepsis-induced lymphocyte and dendritic cell apoptosis contributes to immunosuppression, which results in an inability to eradicate the primary infection as well as a propensity to acquire new, secondary infections. Another cellular process, autophagy, is also activated in immune cells and plays a protective role. In the present study, we demonstrate that interferon regulatory factor 1 (IRF-1) regulates both immune cell apoptosis and autophagy in a murine endotoxemia model. Interferon regulatory factor 1 is activated at an early phase through a Toll-like receptor 4–dependent, myeloid differentiation primary response gene 88–independent manner in splenocytes. Furthermore, IRF-1 knockout (KO) mice are protected from a lethal endotoxemia model. This protection is associated with decreased apoptosis and increased autophagy in splenocytes. Interferon regulatory factor 1 KO mice experience decreased apoptotic cell loss, especially in CD4+ T lymphocytes and myeloid antigen-presenting cells. Meanwhile, IRF-1 KO mice demonstrate increased autophagy and improved mitochondrial integrity. This increased autophagy in KO mice is attributable, at least in part, to deactivation of mammalian target of rapamycin/P70S6 signaling—a main negative regulator of autophagy. Therefore, we propose a novel role for IRF-1 in regulating both apoptosis and autophagy in splenocytes in the setting of endotoxemia with IRF-1 promoting apoptosis and inhibiting autophagy. ABBREVIATION LPS—lipopolysaccharide; TLR4—Toll-like receptor 4; MyD88—myeloid differentiation primary response gene 88; TRIF—domain-containing adaptor inducing IFN-B; IRF-1—interferon regulatory factor 1; LC3—microtubule-associated protein 1 light chain 3; mTOR—mammalian target of rapamycin; WT—wild type; KO—knockout; APCs—antigen-presenting cells

Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury.

Neutrophil extracellular traps (NETs) facilitate the extracellular killing of pathogens. However, excessive NETs formation and poor degradation are associated with exacerbated immune responses and tissue injury. In this study, we investigated the role of NETs in lipopolysaccharide (LPS)-mediated acute lung injury (ALI) and assessed the use of DNase I, for the treatment of ALI. Additionally, we focused on the controversial issue of whether LPS directly induces NETs release in vitro. NETs formation was detected in murine ALI tissue in vivo and was associated with increased NETs markers, citrullinated-histone H3 tissue levels and NET-DNA levels in BALF. Treatment with DNase I significantly degraded NETs and reduced citrullinated-histone H3 levels, which protected against ALI and ameliorated pulmonary oedema and total protein in BALF. In addition, DNase I significantly reduced IL-6 and TNF-α levels in plasma and BALF. In vitro, LPS-activated platelets rather than LPS alone efficiently induced NETs release. In conclusion, NETs formed during LPS-induced ALI, caused organ damage and initiated the inflammatory response. NETs degradation by DNase I promoted NET-protein clearance and protected against ALI in mice; thus, DNase I may be a new potential adjuvant for ALI therapy. Specifically, LPS induced NETs formation in an indirect manner via platelets activation.

Interferon Regulatory Factor-1 Mediates Alveolar Macrophage Pyroptosis During LPS-Induced Acute Lung Injury in Mice.

ABSTRACT Previously, we demonstrated that pyroptosis in alveolar macrophages (AMs) plays an essential role in lipopolysaccharide (LPS)-induced acute lung injury. However, the underlying mechanism remains largely unclear. Here, we show that the absence of interferon regulatory factor 1 (IRF-1) in genetic knock-out mice strongly abrogates pyroptosis in AMs and alleviates the LPS-induced lung injury and systemic inflammation. Our study demonstrates that IRF-1 contributes to caspase-1 activation and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain pyroptosome formation in AMs and leads to downstream inflammatory cytokine release, including that of IL-1&bgr;, IL-18, and HMGB1. The nuclear translocation of IRF-1 is linked to the presence of toll-like receptor 4 (TLR4). Our findings suggest that pyroptosis and the downstream inflammatory response in AMs induced by LPS is a process that is dependent on TLR4-mediated up-regulation of IRF-1. In summary, IRF-1 plays a key role in controlling caspase-1-dependent pyroptosis and inflammation.

Protective role of cisplatin in ischemic liver injury through induction of autophagy

High mobility group box 1 (HMGB1) is a nuclear protein released from stressed or damaged cells that activates inflammatory cascades involved in the pathogenesis of liver ischemia reperfusion (I/R) injury. In efforts to develop strategies aimed at preventing its release from ischemic cells following I/R, we studied the use of cisplatin, a member of the platinating chemotherapeutic agents capable of inducing DNA lesions that have high binding affinities for high mobility group proteins inside the nucleus of cells. In addition to demonstrating that cisplatin can prevent liver damage associated with liver I/R by sequestering HMGB1 inside the nucleus of ischemic cells, cisplatin can also alter cell survival signaling through autophagy. Our results provide a potential approach involving the use of platinating agents and their effects on autophagy in mitigating the deleterious effects of ischemia reperfusion-mediated disease processes.

Novel chemokine-like activities of histones in tumor metastasis

Histones are intracellular nucleosomal components and extracellular damage-associated molecular pattern molecules that modulate chromatin remodeling, as well as the immune response. However, their extracellular roles in cell migration and invasion remain undefined. Here, we demonstrate that histones are novel regulators of tumor metastasis with chemokine-like activities. Indeed, exogenous histones promote both hepatocellular carcinoma (HCC) cell migration and invasion through toll-like receptor (TLR)4, but not TLR2 or the receptor for advanced glycosylation end product. TLR4-mediated activation of nuclear factor-κB (NF-κB) by extracellular signal-regulated kinase (ERK) is required for histone-induced chemokine (e.g., C-C motif ligand 9/10) production. Pharmacological and genetic inhibition of TLR4-ERK-NF-κB signaling impairs histone-induced chemokine production and HCC cell migration. Additionally, TLR4 depletion (by using TLR4−/− mice and TLR4-shRNA) or inhibition of histone release/activity (by administration of heparin and H3 neutralizing antibody) attenuates lung metastasis of HCC cells injected via the tail vein of mice. Thus, histones promote tumor metastasis of HCC cells through the TLR4-NF-κB pathway and represent novel targets for treating patients with HCC.

Neutrophil extracellular traps contribute to the pathogenesis of acid-aspiration-induced ALI/ARDS

Background Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a manifestation of systemic inflammation in the lungs, but the factors that trigger inflammation in ALI/ARDS are unclear. We hypothesized that neutrophil extracellular traps (NETs) contribute to the pathogenesis of acid aspiration-induced ALI/ARDS. Results Analysis of bronchial aspirates from ARDS patients showed that NETs were significantly correlated with the degree of ARDS (r = –0.5846, p = 0.0359). NETs in bronchoalveolar lavage fluid of acid-aspiration mice were significantly higher (141.6 ± 23.08) at 3 h after injury than those in the sham group (1234 ± 101.9; p = 0.003, n = 5 per group). Exogenous NETs aggravated lung injury, while alvelestat and DNase markedly attenuated the intensity of ARDS. Materials and Methods We investigated whether NETs are involved in the severity of gastric aspiration-induced ARDS. Then, a hydrochloric acid aspiration-induced ALI murine model was used to assess whether NETs are pathogenic and whether targeting NETs is protective. Exogenous NETs were administered to mice. Alvelestat can inhibit neutrophil elastase (NE), which serves an important role in NET formation, so we investigated whether alvelestat could protect against ALI in cell and mouse models. Conclusions NETs may contribute to ALI/ARDS by promoting tissue damage and systemic inflammation. Targeting NETs by alvelestat may be a potential therapeutic strategy.