Chronic Alcohol Ingestion Delays T Cell Activation and Effector Function in Sepsis.

Sepsis is the leading cause of death in intensive care units in the US, and it is known that chronic alcohol use is associated with higher incidence of sepsis, longer ICU stays, and higher mortality from sepsis. Both sepsis and chronic alcohol use are associated with immune deficits such as decreased lymphocyte numbers, impaired innate immunity, delayed-type hypersensitivity reactions, and susceptibility to infections; however, understanding of specific pathways of interaction or synergy between these two states of immune dysregulation is lacking. This study therefore sought to elucidate mechanisms underlying the immune dysregulation observed during sepsis in the setting of chronic alcohol exposure. Using a murine model of chronic ethanol ingestion followed by sepsis induction via cecal ligation and puncture, we determined that while CD4+ and CD8+ T cells isolated from alcohol fed mice eventually expressed the same cellular activation markers (CD44, CD69, and CD43) and effector molecules (IFN-γ, TNF) as their water fed counterparts, there was an overall delay in the acquisition of these phenotypes. This early lag in T cell activation was associated with significantly reduced IL-2 production at a later timepoint in both the CD4+ and CD8+ T cell compartments in alcohol sepsis, as well as with a reduced accumulation of CD8dim activated effectors. Taken together, these data suggest that delayed T cell activation may result in qualitative differences in the immune response to sepsis in the setting of chronic alcohol ingestion

Murine Lung Cancer Increases CD4+ T Cell Apoptosis and Decreases Gut Proliferative Capacity in Sepsis.

BACKGROUND:Mortality is significantly higher in septic patients with cancer than in septic patients without a history of cancer. We have previously described a model of pancreatic cancer followed by sepsis from Pseudomonas aeruginosa pneumonia in which cancer septic mice have higher mortality than previously healthy septic mice, associated with increased gut epithelial apoptosis and decreased T cell apoptosis. The purpose of this study was to determine whether this represents a common host response by creating a new model in which both the type of cancer and the model of sepsis are altered. METHODS:C57Bl/6 mice received an injection of 250,000 cells of the lung cancer line LLC-1 into their right thigh and were followed three weeks for development of palpable tumors. Mice with cancer and mice without cancer were then subjected to cecal ligation and puncture and sacrificed 24 hours after the onset of sepsis or followed 7 days for survival. RESULTS:Cancer septic mice had a higher mortality than previously healthy septic mice (60% vs. 18%, p = 0.003). Cancer septic mice had decreased number and frequency of splenic CD4+ lymphocytes secondary to increased apoptosis without changes in splenic CD8+ numbers. Intestinal proliferation was also decreased in cancer septic mice. Cancer septic mice had a higher bacterial burden in the peritoneal cavity, but this was not associated with alterations in local cytokine, neutrophil or dendritic cell responses. Cancer septic mice had biochemical evidence of worsened renal function, but there was no histologic evidence of renal injury. CONCLUSIONS:Animals with cancer have a significantly higher mortality than previously healthy animals following sepsis. The potential mechanisms associated with this elevated mortality differ significantly based upon the model of cancer and sepsis utilized. While lymphocyte apoptosis and intestinal integrity are both altered by the combination of cancer and sepsis, the patterns of these alterations vary greatly depending on the models used

Alcohol delays the kinetics of CD69 expression of naïve CD4+ T cells and prolongs CD69 expression on memory CD4+ T cells.

<p>A) At 24h, CD69 expression increased in the water septic group over water sham (11.6±1% vs 8.6±0.6%, p = 0.01). At 72h, CD69 increased due to sepsis in both water and alcohol-fed groups (H2O sham 10.1±0.9% vs H2O CLP 19.9±1.4%, p = 0.04; EtOH sham 9.4±1% vs EtOH CLP 23.6±1.7%, p = 0.004). B) In naïve CD4s at 24h, the water septic group exhibited increase in CD69 expression (10.2±1.1% vs 5.6±1.0%, p = 0.04), while the alcohol fed group did not. By 72h, both alcohol and water fed groups exhibit CD69 upregulation in sepsis (H2O sham 4.4±0.5% vs H2O CLP 10.9±0.9%, p = 0.04; EtOH sham 4.3±0.4% vs EtOH CLP 13.2±0.5%, p = 0.005). C) In memory CD4s at 24h, sepsis increased CD69 in both water and alcohol-fed groups (H2O sham 18.7±0.8% vs H2O CLP 33.6±2.6%, p = 0.006; EtOH sham 23.5±0.9% vs EtOH CLP 36.4±0.8%, p = 0.03). At 72h, CD69 remained increased in alcohol septic mice only (EtOH sham 27.2±1.7% vs and EtOH sepsis 55.6±4%, p = 0.0003). n = 6-9/group.</p

IL-2 (but not IFN-γ or TNF) production by CD4+ T cells is decreased 72h following CLP in alcohol-fed animals.

<p>Representative flow plot and summary data 72h following sepsis, demonstrating a trend toward decreased IL-2 production due to alcohol alone between sham groups which did not reach significance, but there was a significant decrease in IL-2 in alcohol septic animals compared with water septic (16.15±1.7% vs 26.7±1.74%, p = 0.004). There were no differences in the frequencies of IFN-γ or TNF- producing CD4+ T cells between any of the groups. n = 7-12/group.</p

Alcohol delays O-glycosylation of CD43 on memory CD4+ T cells in sepsis.

<p>A) At 24h, there was no significant increase in CD43 expression in total CD4 cells in either water or alcohol-fed mice due to sepsis. By 72h, both water sepsis and alcohol sepsis groups increased CD43 expression over shams (H2O sham 81.4±7.0 vs H2O CLP 334.5±27.6, p = 0.004; EtOH sham 96.6±3.7 vs EtOH CLP 420.0±65.0, p = 0.04). B) At 24h, there was no significant increase in CD43 expression in naïve CD4 cells. By 72h, both water sepsis and alcohol sepsis groups increased CD43 expression over shams (H2O sham 1.6±0.4% vs H2O CLP 12.9±1.6%, p = 0.01; EtOH sham 1.7±0.1% vs EtOH CLP 20.2±4.8%, p = 0.01). C) At 24h, water septic mice showed significant upregulation of CD43 on memory CD4s (H2O sham 27.8±0.5% vs H2O CLP 55.4±4.2%, p = 0.02), while alcohol septic mice did not. By 72h, CD43 expression is increased in both water and alcohol sepsis groups compared to sham (H2O sham 31.9±0.5% vs H2O CLP 56.7±3.3%, p = 0.03; EtOH sham 29.1±1.4% vs EtOH CLP 50.6±4.5%, p = 0.02).</p

CD4+ T cell frequencies and counts.

<p>A) Representative flow plots and gating strategy. B) There were no differences in CD4 frequencies or counts at 24h. C) At 72h, there were no relevant differences in CD4 frequency. A significant decrease in cell count was seen between alcohol sham and alcohol CLP (8.7x10⁷±1.3x10⁷ vs 4.1x10⁷±3.8x10⁶, p = 0.03). n = 6-9/group.</p

Alcohol decreases the frequency of CD8^dim cells in sepsis.

<p>A) Contour flow plot of the CD8^dim CD44^hi population of CD8+ T cells. B) At 72h after CLP, there is an increased frequency of CD8+ CD44^hi T cells with low expression of CD8 in water sepsis compared with alcohol sepsis (3.78±0.36% vs 2.71±0.07%, p = 0.01). n = 5-8/group.</p

Alcohol differently affects early activation of naïve and memory CD8+ T cells.

<p>A) At 24h after CLP, total CD69 expression increased in both the water and alcohol septic groups over sham controls (H2O sham 4.85±1.0% vs H2O CLP 10.65±0.83%, p = 0.03; EtOH sham 6.02±0.71% vs EtOH CLP 13.27±1.24%, p = 0.009). By 72h, the difference in CD69 expression had resolved in both groups. B) In naïve CD8s at 24h, the water septic group exhibited a trend toward increase in CD69 expression but did not reach significance (p = 0.09), while the alcohol fed group did significantly increase CD69 expression (EtOH sham 3.79±0.66% vs EtOH CLP 8.88±0.66%, p = 0.007). By 72h, neither alcohol nor water fed groups exhibit CD69 upregulation. C) In memory CD8s at 24h, sepsis increased CD69 in the water-fed group (H2O sham 6.95±0.57% vs H2O CLP 21.05±%, p = 0.01). The alcohol fed group increased similarly but did not reach significance. At 72h, CD69 remained increased in water septic group (H2O sham 8.93±1.3% vs H2O CLP 19.71±1.03%, p = 0.005) and the increase in the alcohol septic group also was significant (EtOH sham 11.48±0.5% vs and EtOH sepsis 26.2±3.09%, p = 0.02). n = 6-9/group.</p