Introduction

LSECtin/Clec4g is a C-type lectin that recognizes pathogen patterns, and a T-cell ligand expressed by Kupffer Cells (KCs) and Liver Sinusoidal Endothelial Cells (LSECs). Its expression decreases during chronic liver disease progression, however, the mechanisms that regulate this process have not been fully characterised.

Aims & Methods

Given the relevance of epigenetic control in gene expression regulation during liver disease progression, our main aim was to characterise the epigenetic regulation of LSECtin regarding DNA methylation and Histone Posttranslational Modifications (HPTMs), main mechanisms of epigenetic gene regulation. For this purpose, immortalised KCs and LSECs lines were cultured and stimulated with selected epigenetic drugs: OTS186935 (Suv39h2 inhibitor), UNC0642 (G9a/Glp inhibitor), UNC1999 (EZH2/1 inhibitor), 5-Aza-2’-deoxycitidine (DNMT inhibitor) with or without IL-4, an LSECtin inductor. Simultaneously, probe-based methylation array and ChIP-Seq experiments were conducted in primary KCs and LSECs isolated from both control and an experimental chronic liver disease mice model, generated by a 12-week protocol of CCl₄ oral gavage.

Results

Different significant changes were observed in specific genomic loci, both in LSECs and Kupffer cells, these last particularly susceptible to methylation changes after CCl₄ treatment. We didn’t observe significant changes at general methylation levels in KCs and LSECs methylome from untreated vs CCl₄-treated mice. Beta-values of specific probes for Clec4g gene body and promoter region resulted to be statistically significantly different, especially in Kupffer cells. ChIP-Seq of KCs and LSECs from both untreated and CCl4-treated mice allowed the study of peak profile after immunoprecipitation with H3K9me3 and H3K27me3 antibodies. Both populations showed a differential peak profile at genomic level, but no differences at Clec4g gene body or proximal promoter. Pharmacological inhibition of histone-lysine N-methyltransferase enzymes for 48 h of treatment showed a downregulation in LSECtin expression. Consequently, LSECtin expression could be retrieved by IL-4+drug treatment, highlighting results obtained in KCs. Nevertheless, Ezh2/1 inhibition did not follow this trend, moderately increasing LSECtin expression after the treatment. In addition, LSECtin expression was upregulated by DNA passive demethylation due to DNMT inhibition, with a higher increase in Aza+IL-4 treated cells, thus supporting the potential role of DNA methylation in Clec4g regulation.

Conclusion

LSECtin expression upregulation by DNA passive demethylation together with methylation array analysis strongly points out DNA methylation as a regulatory mechanism of LSECtin expression. On the other hand, LSECtin expression modulation by epigenetic drugs, altogether with the absence of a specific peak profile at Clec4g suggests a more complex, indirect effect of HPTMs in the protein regulation.

Introduction

Platelet-Activating Factor Receptor (Ptafr /PAF-R) is hypomethylated in experimental cirrhosis. Its ligand, the Platelet Activating Factor (PAF) is a proinflammatory phospholipid with multiple biological activities, as aggregation of platelets and modulation of blood pressure^1,2. PAF/PAF-R interaction has been shown to be relevant in pulmonary³ and renal⁴ fibrosis progression. However, the contribution of this pathway to chronic liver disease (CLD) pathophysiology remains unknown.

Aims & Methods

To characterize the contribution of the PAF/PAF-R pathway in CLD progression. A methylation array was performed in hepatic macrophages sorted from control and cirrhotic mice by intragastric administration of CCl₄. Ptafr gene (qPCR) and protein (WB, IHC) expression was evaluated in the livers of cirrhotic and control mice and in primary non-parenchymal liver cells by flow cytometry; as well as in immortalized Kupffer cells (imKCs) in response to different proinflammatory stimuli by qPCR and WB. The presence of the PAF ligand was evaluated in serum, and the supernatant of immortalized hepatic sinusoidal endothelial cells (imLSECs) treated with proinflammatory stimuli by ELISA. The expression of enzymes involved in PAF synthesis and degradation was studied in liver samples. The consequences of treating imKCs with PAF in vitro were evaluated by qPCR and WB. The effect of PAF/PAF-R pathway hampering was tested both in vitro and in vivo by use of a PAF antagonist BN-52021.

Results

The Pafr promoter is hypomethylated in primary hepatic macrophages isolated from cirrhotic mice. We detected a significant increase in PAFR gene and protein expression in the liver of these animals. Liver cells that express PAFR during experimental cirrhosis are primarily hepatic macrophages. In vitro treatment of imKCs with the inflammatory ligands CpG and TNFα induces PAFR expression. Furthermore, stimulation of imKCs with PAF induces a polarization of imKCs toward a proinflammatory profile, increasing gene expression of proinflammatory cytokines (TNF, p≤0.01; IL1β, p≤0.01; TGFβ1, p≤0.01) and chemokines (CCL3, p≤0.05; CCL20, p≤0.0001; CXCL15, p≤0.05; and CXCL16, p≤0.01;), as well as reducing the expression of anti-inflammatory cytokines (IL4, p≤0.01; IL10, p≤.0.0001; and IL-13, p≤0.01 ). This polarization was blocked by the administration of BN-52021 to cell culture. We observed an increase in gene expression of Lpcat1, the enzyme responsible for PAF synthesis, and a decrease in Pafah2, the enzyme that degrades PAF, in liver samples from cirrhotic mice compared to controls, which explains the increase in PAF detected in the serum of cirrhotic mice. All inflammatory stimuli used (IL1b, TNFa, CpG, and LPS) induced PAF secretion by imLSECs in vitro. Cirrhotic mice treatment with BN-52102 resulted in fibrosis and liver damage amelioration (measured by sirius red, aSMA and Vimentin immunostaining), improvement of biochemical markers (hepatic transferases and bile acids) and a reduction of gene expression of proinflammatory and profibrogenic markers (Timp1, Acta2, Col1a1, IL-4, 13, 1b, Tnf, Tgfb).

Conclusion

We have detected the activation of the PAF/PAF-R pathway in experimental cirrhosis. PAF signalling through its receptor induces proinflammatory polarization of hepatic macrophages in vitro. The blockade of receptor activation with a ligand antagonist BN-52021 reduces liver damage and proinflammatory gene expression in cirrhotic mice. These data suggest the value of blocking the PAF/PAF-R pathway to reduce the progression of experimental cirrhosis and its potential application as a therapeutic target.

References

1- Yin et al. Diabetes Metab Syndr Obes. 2022 Jul 8:15:2003-30.
2- Yang et al. Gut. 2004 Jun; 53(6): 877-83.
3- Shahzad et al. J Clin Med. 2024 Apr 6;13(7):2126.
4- Jiang et al. Ren Fail. 2022; 44(1): 660-71.

Introduction

Liver Sinusoidal Endothelial C-type Lectin (LSECtin) is a type-C lectin expressed mainly by Liver Sinusoidal Endothelial cells (LSECs), involved in pathogen recognition but also capable of interacting with T lymphocytes inducing its inhibition. It is known to be downregulated during chronic liver disease while the proinflammatory hepatic response increases. Its loss compromises the protolerogenic activity promoted by LSECs antigenic receptors in favour of inflammation.

Aims & Methods

Our aim is to elucidate LSECs cytokine production in an in vitro model of LSECtin downregulation. For that Immortalized mouse LSECs (imLSECs), either wild type (wt) or LSECtin-Crispr (Crispr), were cultured with LSECtin ligands (CD44Fc and LAG3Fc at 1 ug/mL) or in combination with ligands of Toll-like Receptors (TLRs) (LPS, Pam3CSK4 y CpGs at 0.1 ug/mL). After 72 hours supernatants were collected. To study cytokine production multiplexed immunoassay kits based in fluorescent beads were used. Results were analyzed by flow cytometry. Experiments were done in triplicates. LSECtin downregulation and its downstream signaling was checked by Western Blot.

Results

LSECtin-Crispr LSECs exhibit a downregulation in LSECtin protein expression. Chemoattractant mediators (CCL5 or CXCL10) and proinflammatory cytokines (IL-6 or TNFalpha) secretion increased significantly upon cell stimulation with LSECtin and/or TLRs ligands in Crispr LSECs. On the contrary, IL-10 production was lower in LSECtin-Crispr LSECs than in wt cells upon stimulation.

Conclusion

LSECtin regulates the cytokine production of LSECs reducing the production of proinflammatory mediators and favoring Il-10 secretion. LSECtin loss may therefore contribute to the inflammatory microenvironment in cirrhosis.

Introduction

During chronic liver disease (CLD) the loss of the innate receptor C-type lectin LSECtin, expressed mainly by liver sinusoidal endothelial cells (LSECs), is associated to the dysregulation of hepatic immune response and Th17 expansion in the liver. CLD is also accompanied by intestinal dysbiosis, both promoting systemic inflammation and metabolic alterations that affect neurocognitive function through the liver-brain axis. As aging may worsen neurological damage and CLD and its complications are more prevalent in the aging population, we assessed the relationship of LSECtin with liver and neurocognitive damage in an aged model of cirrhosis.

Aims & Methods

CLD was induced by oral administration of carbon tetrachloride (CCl₄) for 12 weeks (w) in young mice (6 w old) and aged mice (40 w old). Liver damage was assessed through histology and molecular biology. Levels of ammonia in different tissues were examined, and a panel of behavioural assays was conducted to assess neurological impairment. Leukocyte populations from brain and liver were studied by flow cytometry (FC).

Results

Hepatic damage was more severe following the induction of CLD in aged mice compared to young ones, exhibiting significantly elevated levels of ALT and AST as well as higher levels of histological markers of fibrosis and leukocyte recruitment. This damage was accompanied by both, a loss of endothelial cells (CD31⁺ in FC) and a decrease in LSECtin expression, especially in old cirrhotic livers. Interestingly, we found a greater increase of Th17 (1.81 ± 0.58 vs 3.39 ± 0.63 % of CD4 cells, p = 0.003) and CD8 (12.90 ± 2.62 vs 19.22 ± 5.21 % of CD3 cells, p = 0.042) cells in the livers of aged cirrhotic mice as well. Additionally, ammonia concentrations were also higher in cirrhotic old mice than in young ones, in both serum (2.47 ± 0.74 vs 5.18 ± 2.36 nmol/uL, p = 0.015) and the brain (4.65 ± 0.41 vs 5.27 ± 0.86 nmol/uL, p = 0.114). Cirrhotic animals showed motor impairment and a tendency towards anxiety compared to healthy controls. Aged cirrhotic animals exhibited a significantly higher severity index in motor function than the young ones (p = 0.023). Interestingly, only ammonia levels in the brain correlated with results in the open field test (r: -0.544, p = 0.003) and motor evaluation test (r: 0.444, p = 0.018), suggesting a direct relationship between brain ammonia and neurological impairment. Interestingly, we have found higher levels of CD8 cells (12.90 ± 2.62 vs 19.22 ± 5.21 % of CD3 cells, p = 0.042) in old cirrhotic mice' brains compared to young ones.

Conclusion

Aged animals exhibit lower hepatic levels of LSECtin along with more severe liver damage during the experimental cirrhosis model, with higher levels of ammonia and CD8 cells in the brain, correlating with worse neurological function. Immune imbalance in the liver favoured by LSECtin downregulation might modulate immune populations in the brain, contributing to neurocognitive damage. Young cirrhotic animals show similar neurocognitive impairment than healthy aged animals, appointing to a direct contribution of liver damage to neurological impairment during experimental cirrhosis.

Introduction

Increased intestinal permeability is a central factor in the disruption of the gut-liver axis during cirrhosis. Innovative strategies focused on limiting intestinal inflammation are needed to restore the integrity of the mucosal, endothelial, and epithelial barriers in cirrhosis. Modulating the interaction between intestinal fibroblasts (Fib_INT) and cellular immunity in the lamina propria may help to restore intestinal homeostasis in cirrhosis.

Aims & Methods

A prospective experimental study was conducted using mice with advanced liver injury induced by oral administration of CCl₄ for 12 weeks (n=5). Control mice received olive oil as vehicle (n=5). Fib_INT activation, chemokine production, and immune recruitment were assesed in ileum samples through gene (RT-qPCR) and protein expression (immunofluorescence). Activation states of primary Fib_INT (CD45-CD326-CD90+) isolated from the small intestine and colon of control and cirrhotic mice were evaluated, as well as the expression of molecules involved in T-cell activation (MHC-II, CD40, CD80, CD86, OX-40L, PD-L1) by flow cytometry. Co-cultures of Fib_INT (cirrhosis) and T lymphocytes (control), and vice versa, were conducted to determine the modulatory effects of cell-to-cell interactions.

Results

An increase in gene (COL1A1, FAP, PDPN) and protein (⍺-SMA, Pdpn, Vim) expression of stromal cell activation markers was observed in the ileum during experimental cirrhosis. Augmented expression of chemokines (CCL2, CXCL10) in the ileum of cirrhotic mice correlated with increased leukocyte (CD45+) infiltration into the villi. Local increases in intestinal lymphocyte (CD3+) and macrophage (F4/80+) populations were detected. In cirrhosis, the percentages of primary Fib_INT from the small intestine expressing stromal cell activation markers (Vim and Pdpn) and T-cell stimulation molecules (CD40, CD80, CD86) were elevated compared to control mice. Isolated Fib_INT from cirrhotic mice interacted with more CD4+ T lymphocytes in vitro and were more effective at inducing their activation (CD25+) and differentiation towards a Th17 profile (IL-17+). Similarly, CD4+ intestinal T lymphocytes from cirrhotic mice could activate Fib_INT isolated from control animals (Vim+, CD86+, OX40L).

Conclusion

Fib_INT are activated in experimental cirrhosis and acquire the ability to induce an inflammatory Th17 response. These findings suggest that modulating the interaction between intestinal fibroblasts and T lymphocytes may recover intestinal homeostasis in cirrhosis.