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

Author: Gaddam, Ravinder Reddy

Date: 2017

Publisher: University of Otago

Type: Thesis

Link to this item using this URL: http://hdl.handle.net/10523/7327

University of Otago

Abstract

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.

Subjects: Hydrogen sulfide, Substance P, Kupffer cells, Sepsis, Liver sinusoidal endothelial cells, Inflammation, Liver sieve

Citation: ["Gaddam, R. R. (2017). The role of hydrogen sulfide, substance P and Kupffer cells on inflammation and liver sinusoidal endothelial cells in sepsis (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/7327"]

Copyright: All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.