Opposite regulation of intestinal and intrahepatic CD8 + T cells controls alcohol-associated liver disease progression

The gut-liver axis contributes importantly to the development of alcohol-associated liver disease.

“the gut-liver axis has been shown to play an important role in the development of ALD.5,6”

• cites [5] sourceSupported
  Experimental — accuracy not yet measured
• cites [6] sourceSupported
  Experimental — accuracy not yet measured

Human ALD patients show significant downregulation of gene sets related to T cell selection and adaptive immunity, with CD8A among the leading downregulated genes.

“Patients with ALD26 had a significant down-regulation of gene sets linked to T cell selection and adaptive immunity, with CD8A being one of the leading downregulated genes (Fig.4B, C, supplementary Table 3).”

• cites [26] sourceCould not verify

  The Results section describes a cohort of patients with alcohol-associated liver disease, but reports K18-M65 measurements and limited liver-tissue mRNA correlations, not gene-set analysis. Searches for CD8A, T-cell selection, and adaptive immunity found no passage in our copy reporting the claimed downregulation. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Intrahepatic CD8+ T cells negatively correlate with hepatic neutrophils and liver injury markers.

“our previous data revealed that intrahepatic CD8+ T cells negatively correlated with hepatic neutrophils and liver injury markers.17”

• cites [17] Ma, 2022, Distinct histopathological phenotypes of severe alcoholic hepatitis suggest different mechanisms driving liver injury and failure, J Clin Invest sourcePartly supported

  The Results section reports a negative correlation between parenchymal neutrophils and CD8+ T cells in fibrotic areas of livers from patients with severe alcoholic hepatitis. It also reports a negative correlation between those CD8+ T cells and MELD score, but the negative association with ALT was only a trend (P = 0.07). The claim overstates the evidence by describing a negative correlation with liver injury markers generally. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Duodenal CD8+ T cell measures in ALD patients are inversely associated with markers of bacterial translocation.

“duodenal CD8-related genes all inversely correlated with serum microbial translocation marker sCD14 (Fig.1E)”

The number of duodenal CD8+ T cells in ALD patients is inversely associated with serum markers of liver injury.

“duodenal CD8+ T cell number inversely correlated with serum ALT, AST, and K18-M65 (Fig. 1D).”

Within the same ALD patients, duodenal and intrahepatic CD8+ T cell numbers are inversely correlated.

“a negative correlation was observed between duodenal and intrahepatic CD8+ T cells from the same patients (Fig. 1B).”

In patients with alcohol-associated liver disease, CD8+ T cells in the duodenum are selectively decreased.

“In ALD patients, duodenal CD8+ T cells were selectively reduced and negatively correlated with liver injury and bacterial translocation markers, while intrahepatic CD8+ T cells were markedly increased.”

• Also stated in: § Opposite regulation of duodenal and intrahepatic CD8+ T cells in patients with ALD negatively correlates with ALD severity. · plain

  “duodenal CD8+ T cells were markedly reduced in ALD patients (Fig. 1A).”

In ALD patients, intrahepatic CD8+ T cells are markedly increased.

“ALD livers had markedly elevated CD8+ T cells (Fig. 1A).”

Serum ALT levels are similar between ethanol-fed WT and Cd8−/− mice in the 10d+1B model.

“Serum ALT levels were similar between ethanol-fed WT and Cd8−/− mice (Fig. 8A).”

BCL2 and CD8A expression are positively correlated in AUD patients.

“BCL2 and CD8A gene expression positively correlated in AUD patients (Fig. 6B and 6C).”

Liver fibrosis detected by Sirius Red staining is not elevated in Cd8−/− mice compared with WT mice.

“liver fibrosis as detected by Sirus Red staining was not elevated in Cd8−/− mice vs WT mice (Fig. 8C, 8D, supplemental Fig. S5C).”

After ethanol feeding, CD8Bcl-2 mice have lower serum LPS and ALT levels than WT mice.

“compared to WT mice, CD8Bcl-2 mice had lower levels of serum LPS and ALT (Fig. 7C)”

Chronic-plus-multiple binge ethanol feeding in mice selectively reduces duodenal CD8+ T cells.

“mice exposed to alcohol with the E8w+nB model had a specific diminution of CD8+ T cells in duodenum compared to pair-fed mice without affecting CD4+ T cells and IBA1+ macrophages (Fig. 3B, C).”

Intraperitoneal ethanol injection does not affect duodenal CD8+ T cells.

“without affecting duodenal CD8+ T cells (Fig. 5F, G).”

Serum endotoxin levels are higher in the E8w+nB mouse model than in the 10d+1B model.

“a much higher level of serum endotoxin in the E8w+nB model compared to the 10d+1B model (Fig. 3F).”

• AssumptionRests on an assumption: Serum endotoxin/LPS levels serve as a marker of intestinal dysfunction or bacterial translocation.

Ethanol treatment promotes mitochondrial depolarization in isolated duodenal CD8+ T cells.

“such ethanol treatment promoted mitochondria depolarization in isolated duodenal CD8+ T cells (supplemental Fig. S4B).”

Intraperitoneal ethanol injection produces similar ethanol concentrations in the duodenum and blood.

“Such treatment resulted in a similar ethanol concentration in duodenum and blood (Fig. 5E)”

Few intrahepatic CD8+ T cells are detected in the chronic-plus-single binge mouse model.

“we did not detect many intrahepatic CD8+ T cells on our chronic-plus-single binge model in mice.”

After E8w+nB ethanol challenges, CD8Bcl-2 mice have more intrahepatic CD8+ T cells than WT mice.

“higher number of intrahepatic CD8+ T cells in CD8Bcl-2 mice than that in WT mice post E8w+nB ethanol challenges (supplemental Fig. S4C, S4D).”

Cd8Bcl-2 transgenic mice have significantly more duodenal CD8+ T cells than WT mice in a three-binge model.

“Our data revealed significantly higher duodenal CD8+ T cells in Cd8Bcl-2 mice as compared with WT mice (Fig. 7B).”

In the scRNA-seq analysis, clusters 2, 4, and 8 show the highest expression of CD8A and CD8B.

“clusters 2, 4 and 8 had the highest expression of CD8A and CD8B (Fig. 2C).”

In vitro, 500 mM ethanol reduces Bcl2, but not Bcl2l1 or Mcl1, expression in primary duodenal CD8+ T cells.

“Ethanol treatment markedly reduced Bcl2, but not Bcl2l1 and Mcl1, expression in primary duodenal CD8+ T cells (Fig. 6F)”

Ethanol binge drinking produces very high duodenal ethanol concentrations (up to 500 mM), 10-fold higher than in systemic circulation or other small intestine segments.

“ethanol binge drinking resulted in very high concentrations of ethanol (up to 500 mM) in duodenum, which is 10-fold higher than levels found in the systemic circulation or in other segments of the small intestine”

Single-cell RNA sequencing of ALD patient livers identifies multiple CD8+ T cell populations that express activation, exhaustion, and survival genes, including Bcl2.

“ScRNA seq analysis of ALD patient livers revealed several populations of CD8+ T cells expressing activation, exhaustion, and survival genes (eg Bcl2).”

Serum LPS levels are comparable between Cd8−/− and WT mice treated with ethanol plus Ad-Cxcl1.

“serum LPS levels were comparable in Cd8−/− mice and WT mice treated with ethanol plus Ad-Cxcl1”

Tight junction-related genes are similarly downregulated in the duodenum of E8w+nB and E10d+1B mice.

“similar downregulation of tight junction-related genes in the E8w+nB and E10d+1B model (Fig. 3G).”

Seventy-two CD8+ T cell-related genes are shared between the mouse E8w+nB and human ALD duodenal downregulated gene sets.

“Seventy-two genes linked to CD8+ T cells were shared between the mouse and human gene sets (Fig. 4E, supplementary Table 4)”

Chronic-plus-multiple binge ethanol feeding does not alter CD8+ T cell numbers in mouse jejunum, ileum, colon, liver, or spleen.

“the number of CD8+ T cells was neither altered in jejunum, ileum, and colon (Fig. 3D) nor in the liver and spleen (Supplementary Fig. S2A).”

Activated intrahepatic CD38+CD8+ T cells are present in sAH patients.

“Immunohistochemistry further revealed the presence of activated intrahepatic CD38+CD8+ T cells in sAH patients (Fig. 2G).”

Ethanol alone induces similar elevation of hepatic neutrophils in Ad-Gfp control groups.

“ethanol alone induced similar elevation of hepatic neutrophils in Ad-Gfp control groups.”

Cd8−/− mice are more susceptible to Ad-Cxcl1- or Ad-Cxcl1 plus ethanol-mediated elevation of hepatic neutrophils.

“Cd8−/− mice were more susceptible to Ad-Cxcl1- or Ad-Cxcl1 plus ethanol-mediated elevation of hepatic neutrophils”

Baseline Bcl2 expression is similar in duodenal and liver CD8+ T cells isolated from the same mice.

“similar baseline Bcl2 gene expression in isolated duodenal and liver CD8+ T cells from the same mice (supplemental Fig. S4A)”

Despite higher blood acetaldehyde, Aldh2*1/*2 mice show duodenal CD8+ T cell loss similar to WT mice after ethanol exposure.

“despite higher blood acetaldehyde levels, Aldh2*1/*2 mice had similar duodenal CD8+ T cell loss as WT mice post ethanol exposure (Fig. 5B, C)”

Three consecutive daily ethanol binges significantly reduce duodenal CD8+ T cells in mice.

“three consecutive daily binges significantly reduced duodenal CD8+ T cells, indicating that multiple binges lead to CD8+ T cell diminution (Fig. 3E).”

Cd8−/− mice are more susceptible to Ad-Cxcl1 plus ethanol-mediated hepatic macrophage activation, oxidative stress, and impaired apoptosis.

“Cd8−/− mice were more susceptible to Ad-Cxcl1 plus ethanol-mediated activation of hepatic macrophages (cluster formation), oxidative stress (4-HNE staining), impaired apoptosis (cleaved caspase 3 staining)”

After oral ethanol intake, local duodenal ethanol concentrations (~500 mM) are 10-fold higher than blood concentrations (~50 mM).

“local duodenal ethanol concentrations (~500 mM) were 10-fold higher than those in the blood (~50 mM) after oral ethanol intake (Fig. 5D).”

Duodenal CD8+ T cell loss after ethanol exposure also occurs in female mice.

“Loss of duodenal CD8+ T cells was also observed in female mice post ethanol exposure (Supplementary Fig. S2B).”

Mouse duodenal transcriptomics after E8w+nB shows similar downregulation of CD8+ T cell-related gene sets.

“transcriptomics in mouse duodenum revealed similar down-regulation of CD8+ T cell-related gene sets (Fig. 4C, D).”

Liver CD8+ T cells in sAH express pro-survival, anti-inflammatory, and activation genes.

“These data revealed that sAH liver CD8+ T cells express pro-survival, anti-inflammatory, and activation genes.”

BCL2 is among the top six differentially expressed apoptosis-related genes in human duodenal RNA-Seq.

“We identified the top 6 differentially expressed genes directly linked to apoptosis (Fig. 6A) including BCL2”

E8w+nB alcohol feeding induces duodenal CD8+ T cell loss in WT mice but not in CD8Bcl-2 mice.

“using the E8w+nB model we found that such alcohol feeding induced duodenal CD8+ T cell loss in WT mice but not in CD8Bcl-2 mice (Fig. 7C).”

Intrahepatic CD8+ T cells from patients express higher PD-1 and CD38, but not TIM3 or Galectin9, than peripheral CD8+ T cells.

“intrahepatic CD8+ T cells expressed much higher PD-1 and CD38 (activation marker), but not TIM3 and Galectin9, than peripheral CD8+ T cells (Fig. 2F, Supplementary Fig. S1A).”

Aldh2*1/*2 knock-in mice have significantly higher blood acetaldehyde levels than WT mice after an ethanol binge.

“Aldh2*1/*2 knock-in mutant mice, which have significantly higher levels of blood acetaldehyde than WT mice after ethanol binge (Fig. 5A).”

BCL2 is highly expressed in CD8+ T cells.

“Immunofluorescence staining revealed high expression of BCL2 in CD8+ T cells (Fig. 6D).”

Intrahepatic CD8+ T cells protect against ALD in mice and humans.

“Together, these results indicate that intrahepatic CD8+ T cells protect against ALD in mice and humans.”

Increased liver CD8+ T cells play protective compensatory roles by limiting inflammation-induced liver injury.

“suggesting that increased liver CD8+ T cells play protective compensatory roles by limiting inflammation-induced liver injury.”

Acetaldehyde may not directly cause liver and gut cell death.

“Thus, acetaldehyde may not directly cause liver and gut cell death.”

Chronic-plus-multiple ethanol binges in mice mimic the duodenal CD8+ T cell reduction and enhanced bacterial translocation seen in ALD patients.

“chronic-plus-multiple ethanol binges in mice mimic human duodenal CD8+ T cell reduction and enhanced bacterial translocation seen in ALD patients.”

Intrahepatic CD8+ T cells may interact directly or indirectly with macrophages and/or neutrophils, blocking their activation and improving ALD.

“Intrahepatic CD8+ T cells may interact directly and/or indirectly with macrophages and/or neutrophils, blocking their activation thus improving ALD.”

Ethanol, rather than acetaldehyde, is involved in duodenal CD8+ T cell reduction.

“suggesting that ethanol, and not acetaldehyde, is involved in duodenal CD8+ T cell reduction.”

A conserved pathogenic mechanism underlies alcohol-induced duodenal CD8+ T cell depletion.

“indicating the presence of a conserved pathogenic mechanism involved in alcohol-induced duodenal CD8+ T cell depletion.”

Intrahepatic CD8+ T cells play protective compensatory roles in ALD by inhibiting macrophage and neutrophil activation, thereby reducing inflammation.

“Intrahepatic CD8+ T cells play protective compensatory roles in ALD by inhibiting macrophage and neutrophil activation, thus reducing inflammation.”

BCL2 is critical for duodenal CD8+ T cell survival in ALD.

“suggesting that BCL2 is critical for duodenal CD8+ T cell survival in ALD.”