The role of hydrogen sulfide, substance P and Kupffer cells on inflammation and liver sinusoidal endothelial cells in sepsis

Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection. The inflammatory response is an integral part of sepsis and leads to the systemic inflammatory response syndrome (SIRS) and multiple organ failure. The overall objective of this thesis was to investigate whether hydrogen sulfide (H2S), substance P (SP) and Kupffer cells modulate the inflammatory response, organ damage and liver sinusoidal endothelial cells (LSECs) fenestrations in sepsis. H2S is a key mediator of inflammation and recent studies have implicated H2S in the pathogenesis of sepsis. However, these studies have limitations due to the disadvantages of the H2S-synthesising enzyme inhibitor and H2S donors used to conduct the experiments. Gene deletion technology offers a definitive approach to investigate the role of H2S in sepsis. The aim of this thesis was to investigate the potential role of endogenous H2S synthesised through cystathionine-γ-lyase (CSE) using CSE knockout (CSE KO) mice in caecal-ligation and puncture (CLP)-induced sepsis. This thesis also aimed to examine the underlying mechanisms by which CSE-derived H2S regulates inflammation and to determine the interaction between H2S and SP in regulating the inflammatory response in sepsis. Kupffer cells are tissue-resident macrophages in the liver that play an important role in inflammation associated with infection. The studies described in this thesis investigated the potential roles of Kupffer cells on liver and lung injury, inflammation and the systemic inflammatory response in sepsis using gadolinium chloride (GdCl3) to inactivate these cells. LSECs are specialised fenestrated endothelial cells in the liver that undergo structural alteration during inflammation and infection. The structural alterations in LSEC fenestrae following CLP-induced sepsis were examined and the effect of GdCl3, CSE gene deletion and PPTA gene deletion (PPTA, a SP encoding gene) were determined. The final aim was to investigate the alteration of circulatory H2S and SP levels and their association with the inflammatory response in patients with sepsis compared to non-septic patients with similar disease severity and organ dysfunction admitted to the hospital Intensive Care Unit (ICU). Following CLP-induced sepsis in mice, increased expression of liver and lung CSE (liver: ~1.98 fold; lung: ~2.49 fold), increased liver H2S-synthesising activity (~1.27 fold) and plasma H2S levels (~1.45 fold) were observed. Mice deficient in the CSE gene showed significantly reduced sepsis-associated tissue (liver and lung) myeloperoxidase (MPO) activity, tissue (liver and lung) and circulatory levels of cytokines (TNF-α, IL-6 and IL-1β) and chemokines (MCP-1 and MIP-2α), and histological changes in the liver and lung. In addition, mechanistic studies revealed that the proinflammatory role of CSE-derived H2S was mediated by the activation of the ERK1/2-NF-B p65 signalling pathway. SP and NK-1R expression have been shown to play an essential role in sepsis-associated liver and lung injury. Mice with CSE gene deletion had significantly reduced tissue (liver and lung) and circulatory SP levels (liver: ~0.50 fold; lung: ~0.42 fold; plasma: ~0.61 fold) and tissue (liver and lung) NK-1R expression (liver: ~1.11 fold; lung: ~0.93 fold). This study showed that CSE-derived H2S in sepsis could upregulate SP and NK-1R expression, thereby contributing to liver and lung injury and inflammation. Examination of the effect of GdCl3 on the inflammatory response and organ injury following induction of sepsis showed there was protection against injury in the liver, as there was reduced MPO activity, cytokine (TNF-α, IL-6 and IL-1β) and chemokine (MCP-1 and MIP-2α) levels and histological changes in the liver. In contrast, administration of GdCl3 failed to reduce lung injury and inflammation (as there was no change in MPO activity, cytokine and chemokine levels and histological changes) and the systemic inflammatory response (as evidenced by no change in circulatory cytokines and chemokines) in sepsis. Study of LSEC fenestrae following induction of sepsis revealed that CLP-induced sepsis was associated with defenestration (decreased diameter, frequency and porosity) and gaps formation in LSEC fenestrae (~9 fold). Mice with CSE gene deletion, PPTA gene deletion and mice treated with GdCl3 showed less defenestration (increased diameter, frequency and porosity) and fewer gaps (~0.16 fold) in LSEC fenestrae following sepsis. Studies of septic patients admitted to the ICU showed higher circulatory levels of H2S and SP compared to non-septic patients, which correlated with the inflammatory response in septic patients. In conclusion, the results presented in this thesis have shown that the CSE-derived H2S, SP and Kupffer cells all play a key role in modulating inflammation, associated organ damage and LSEC fenestrae in experimental sepsis. This thesis has also shown that higher circulatory levels of H2S and SP are associated with inflammatory response in septic patients and are consistent with results from experimental sepsis, suggesting that CSE-derived H2S and SP play an important role in the inflammatory process of sepsis in both experimental and human sepsis. This study contributes to a better understanding of the pathogenesis of sepsis and highlights novel potential approaches to the treatment of sepsis

The role of hydrogen sulfide, substance P and Kupffer cells on inflammation and liver sinusoidal endothelial cells in sepsis

Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection. The inflammatory response is an integral part of sepsis and leads to the systemic inflammatory response syndrome (SIRS) and multiple organ failure. The overall objective of this thesis was to investigate whether hydrogen sulfide (H2S), substance P (SP) and Kupffer cells modulate the inflammatory response, organ damage and liver sinusoidal endothelial cells (LSECs) fenestrations in sepsis. H2S is a key mediator of inflammation and recent studies have implicated H2S in the pathogenesis of sepsis. However, these studies have limitations due to the disadvantages of the H2S-synthesising enzyme inhibitor and H2S donors used to conduct the experiments. Gene deletion technology offers a definitive approach to investigate the role of H2S in sepsis. The aim of this thesis was to investigate the potential role of endogenous H2S synthesised through cystathionine-γ-lyase (CSE) using CSE knockout (CSE KO) mice in caecal-ligation and puncture (CLP)-induced sepsis. This thesis also aimed to examine the underlying mechanisms by which CSE-derived H2S regulates inflammation and to determine the interaction between H2S and SP in regulating the inflammatory response in sepsis. Kupffer cells are tissue-resident macrophages in the liver that play an important role in inflammation associated with infection. The studies described in this thesis investigated the potential roles of Kupffer cells on liver and lung injury, inflammation and the systemic inflammatory response in sepsis using gadolinium chloride (GdCl3) to inactivate these cells. LSECs are specialised fenestrated endothelial cells in the liver that undergo structural alteration during inflammation and infection. The structural alterations in LSEC fenestrae following CLP-induced sepsis were examined and the effect of GdCl3, CSE gene deletion and PPTA gene deletion (PPTA, a SP encoding gene) were determined. The final aim was to investigate the alteration of circulatory H2S and SP levels and their association with the inflammatory response in patients with sepsis compared to non-septic patients with similar disease severity and organ dysfunction admitted to the hospital Intensive Care Unit (ICU). Following CLP-induced sepsis in mice, increased expression of liver and lung CSE (liver: ~1.98 fold; lung: ~2.49 fold), increased liver H2S-synthesising activity (~1.27 fold) and plasma H2S levels (~1.45 fold) were observed. Mice deficient in the CSE gene showed significantly reduced sepsis-associated tissue (liver and lung) myeloperoxidase (MPO) activity, tissue (liver and lung) and circulatory levels of cytokines (TNF-α, IL-6 and IL-1β) and chemokines (MCP-1 and MIP-2α), and histological changes in the liver and lung. In addition, mechanistic studies revealed that the proinflammatory role of CSE-derived H2S was mediated by the activation of the ERK1/2-NF-B p65 signalling pathway. SP and NK-1R expression have been shown to play an essential role in sepsis-associated liver and lung injury. Mice with CSE gene deletion had significantly reduced tissue (liver and lung) and circulatory SP levels (liver: ~0.50 fold; lung: ~0.42 fold; plasma: ~0.61 fold) and tissue (liver and lung) NK-1R expression (liver: ~1.11 fold; lung: ~0.93 fold). This study showed that CSE-derived H2S in sepsis could upregulate SP and NK-1R expression, thereby contributing to liver and lung injury and inflammation. Examination of the effect of GdCl3 on the inflammatory response and organ injury following induction of sepsis showed there was protection against injury in the liver, as there was reduced MPO activity, cytokine (TNF-α, IL-6 and IL-1β) and chemokine (MCP-1 and MIP-2α) levels and histological changes in the liver. In contrast, administration of GdCl3 failed to reduce lung injury and inflammation (as there was no change in MPO activity, cytokine and chemokine levels and histological changes) and the systemic inflammatory response (as evidenced by no change in circulatory cytokines and chemokines) in sepsis. Study of LSEC fenestrae following induction of sepsis revealed that CLP-induced sepsis was associated with defenestration (decreased diameter, frequency and porosity) and gaps formation in LSEC fenestrae (~9 fold). Mice with CSE gene deletion, PPTA gene deletion and mice treated with GdCl3 showed less defenestration (increased diameter, frequency and porosity) and fewer gaps (~0.16 fold) in LSEC fenestrae following sepsis. Studies of septic patients admitted to the ICU showed higher circulatory levels of H2S and SP compared to non-septic patients, which correlated with the inflammatory response in septic patients. In conclusion, the results presented in this thesis have shown that the CSE-derived H2S, SP and Kupffer cells all play a key role in modulating inflammation, associated organ damage and LSEC fenestrae in experimental sepsis. This thesis has also shown that higher circulatory levels of H2S and SP are associated with inflammatory response in septic patients and are consistent with results from experimental sepsis, suggesting that CSE-derived H2S and SP play an important role in the inflammatory process of sepsis in both experimental and human sepsis. This study contributes to a better understanding of the pathogenesis of sepsis and highlights novel potential approaches to the treatment of sepsis

The Challenges and Opportunities in the Development of MicroRNA Therapeutics: A Multidisciplinary Viewpoint

microRNAs (miRs) are emerging as attractive therapeutic targets because of their small size, specific targetability, and critical role in disease pathogenesis. However, <20 miR targeting molecules have entered clinical trials, and none progressed to phase III. The difficulties in miR target identification, the moderate efficacy of miR inhibitors, cell type-specific delivery, and adverse outcomes have impeded the development of miR therapeutics. These hurdles are rooted in the functional complexity of miR’s role in disease and sequence complementarity-dependent/-independent effects in nontarget tissues. The advances in understanding miR’s role in disease, the development of efficient miR inhibitors, and innovative delivery approaches have helped resolve some of these hurdles. In this review, we provide a multidisciplinary viewpoint on the challenges and opportunities in the development of miR therapeutics

Oxidative stress and immune cell activation quantification in sepsis and non-sepsis critical care patients by neopterin/7,8-dihydroneopterin analysis

Introduction: Neopterin and 7,8-dihydroneopterin are used as biomarkers of oxidative stress and inflammation, but the effect of kidney function on these measurements has not been extensively explored. We examine the levels of oxidative stress, inflammation and kidney function in intensive patients and compare them to equivalent patients without sepsis

Calculation of the pole-face windings compensation at injection

<div><p>Background</p><p>Hydrogen sulfide (H₂S), produced by the activity of cystathionine-gamma-lyase (CSE), is a key mediator of inflammation in sepsis. The liver sinusoidal endothelial cells (LSECs) are important target and mediator of sepsis. The aim of this study was to investigate the role of CSE-derived H₂S on inflammation and LSECs fenestrae in caecal-ligation and puncture (CLP)-induced sepsis using CSE KO mice.</p><p>Methods</p><p>Sepsis was induced by CLP, and mice (C57BL/6J, male) were sacrificed after 8 hours. Liver, lung, and blood were collected and processed to measure CSE expression, H₂S synthesis, MPO activity, NF-κB p65, ERK1/2, and cytokines/chemokines levels. Diameter, frequency, porosity and gap area of the liver sieve were calculated from scanning electron micrographs of the LSECs.</p><p>Results</p><p>An increased CSE expression and H₂S synthesizing activity in the liver and lung of wild-type mice following CLP-induced sepsis. This was associated with an increased liver and lung MPO activity, and increased liver and lung and plasma levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, and the chemokines MCP-1 and MIP-2α. Conversely, CSE KO mice had less liver and lung injury and reduced inflammation following CLP-induced sepsis as evidenced by decreased levels of H₂S synthesizing activity, MPO activity, and pro-inflammatory cytokines/chemokines production. Extracellular-regulated kinase (ERK1/2) and nuclear factor-κB p65 (NF-κB) became significantly activated after the CLP in WT mice but not in CSE KO mice. In addition, CLP-induced damage to the LSECs, as indicated by increased defenestration and gaps formation in the LSECs compared to WT sham control. CSE KO mice showed decreased defenestration and gaps formation following sepsis.</p><p>Conclusions</p><p>Mice with CSE (an H₂S synthesising enzyme) gene deletion are less susceptible to CLP-induced sepsis and associated inflammatory response through ERK1/2-NF-κB p65 pathway as evidenced by reduced inflammation, tissue damage, and LSECs defenestration and gaps formation.</p></div

CSE Protein Expression and H₂S-Synthesizing Activity Following CLP Induced Sepsis.

<p>(A-B) Liver CSE protein expression and (C-D) lung CSE protein expression. Liver and lung CSE protein expression was increased following CLP induced sepsis compared to sham control (liver: P<0.001 vs. sham control; lung P<0.01 vs. sham control) and no CSE protein expression was detected in CSE KO mice. Results were normalized with GAPDH and expressed as the relative fold increase of CSE protein expression compared with sham control. For western blot results, each lane represents a separate animal. The blots shown were representative of all animals in each group with similar results. (E) Liver H₂S synthesizing activity. H₂S synthesizing activity was increased following increased CSE protein expression in WT CLP induced sepsis mice compared to sham controls and CSE KO mice had significantly lower H₂S synthesizing activity compared to WT sepsis mice. Data represent the mean±standard deviation (n = 8). Data were analysed for Gaussian or Normal distribution using Shapiro-Wilk test. One-way ANOVA with post hoc Tukey’s test was performed to compare multiple groups. Statistical significance was assigned as *P<0.05; **P<0.01; ***P<0.001; and ****P<0.0001.</p

Effect of CSE Gene Deletion on Liver and Lung MPO Activity and Organ Injury in Mice Following CLP Induced Sepsis.

<p>(A) Liver MPO activity and (B) Lung MPO activity. Following CLP induced sepsis, liver and lung MPO activity levels were increased in WT sepsis mice compared to sham operation controls. CSE gene deletion decreased significantly liver and lung MPO activity following CLP induced sepsis compared to WT sepsis mice. Results were expressed as the relative fold increase of MPO activity compared with sham operation controls. (C) Representative images of the liver H&E sections revealed extensive capsular inflammation and lobular necrosis in CLP induced sepsis in the WT mice compared to sham control. The CSE KO mice showed a lower capsular inflammation and lobular necrosis. (D) Representative images of the lung H&E sections. Histological examination of the lung sections reveal marked leukocyte infiltration and alveolar thickening following CLP induced sepsis in the WT mice compared to sham operation controls. This effect was substantially reduced in the CLP induced CSE KO mice. Scale bar is 50 μm. Data represent the mean±standard deviation (n = 8). Data were analysed for Gaussian or Normal distribution using Shapiro-Wilk test. Liver and lung MPO activity data were analysed using One-way ANOVA with post hoc Tukey’s test whereas liver and lung histology scores were analysed with non-parametric Kruskal-Wallis test to compare multiple groups. Statistical significance was assigned as *P<0.05; and **P<0.01.</p