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

Serum cytokine suggests Th2 skewing in EtOH-fed septic relative to water-fed septic animals.

<p>A) Chronic alcohol ingestion induced elevated baseline levels of serum IL1β over water-feeding in sham animals (906.5±187.7 vs 1883±444.6, p = 0.01). B) Alcohol ingestion increased baseline serum IL-4 concentration in sham animals (19322±473.2 vs 26584±2516, p = 0.02), with a concurrent strong trend toward increase in septic animals (19629±523 vs 24433±1686, p = 0.05). C) Chronic alcohol ingestion increased baseline serum IL12 concentration in sham animals (1073±421.5 vs 35.9±3.1, p = 0.04). D) Alcohol ingestion increased baseline serum TNF concentration in sham animals (1093±79.9 vs 823.8±34.7, p = 0.005). E) Serum IL-6 was increased in alcohol sepsis over water sepsis (58697±27081 vs 896.7±356, p = 0.02). F) Serum IL-10 concentration was increased is alcohol sepsis over water sepsis (10057±4412 vs 979.7±896.2, p = 0.01). n = 8/group.</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

CD8+ T cell frequencies and counts.

<p>A) There were no relevant differences in CD8 frequencies or counts specifically due to sepsis or ethanol alone at 24h. B) At 72h, there was a strong trend toward a decrease in absolute count due to sepsis in both the water and alcohol fed groups. 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

Alcohol delays increase in the CD69+CD43+ population in naïve and memory CD4+ T cells.

<p>A) Representative flow plot for frequency of CD69+CD43+ of CD4+ CD44^hi at 24h. B) Representative flow plot for frequency of CD69+CD43+ of CD4+ CD44^hi at 72h. C) By 24h, there was significant increase in CD69+ CD43+ population in naïve CD4s in water sepsis (H2O sham 0.4±0.04% vs H2O CLP 1.6±0.4%, p = 0.03). This population was not increased in alcohol sepsis. By 72h, both water septic and alcohol septic groups showed significant increase in the CD69+ CD43+ population (H2O sham 0.3±0.08% vs H2O CLP2.5±0.4%, p = 0.02; EtOH sham 0.3±0.05% vs EtOH CLP 3.5±0.5%, p = 0.01). D) In memory CD4s, at 24h there was a significant increase in CD69+ CD43+ population in water sepsis (H2O sham 6.2±0.7% vs H2O CLP 23.7±4.2%, p = 0.02) but not alcohol sepsis. By 72h, both water and alcohol septic groups show significant increase in this population above sham controls (H2O sham 11.4±-.2% vs H2O CLP 30.8±1.8%, p = 0.04; EtOH sham 10.3±0.9% vs EtOH CLP 32.9±2.6%, p = 0.005). n = 4-8/group.</p

Experimental design.

<p>A, 4–6 week old C57BL/6 mice were provided either ethanol or water for 12 weeks followed by sepsis induction via CLP or sham laparotomy. Spleens were harvested at 24 and 72 hours for flow cytometric analysis of lymphocytes. B, Body weights were not different between water-fed and alcohol-fed mice at the end of the 12-week period.</p