Chronic Ethanol Consumption Reduces Existing CD8 T Cell Memory and Is Associated with Lesions in Protection against Secondary Influenza A Virus Infections

Zeb R. Zacharias and Kevin L. Legge
J Immunol December 15, 2019, 203 (12) 3313-3324; DOI: https://doi.org/10.4049/jimmunol.1900770

Key Points

  • Chronic EtOH consumption alters the existing memory T cell compartment.

  • EtOH-induced changes in existing memory T cells increase susceptibility to IAV.

  • Chronic EtOH alters memory T cell–mediated killing and recruitment into the lungs.

Abstract

Chronic alcohol consumption is associated with an increased incidence of disease severity during pulmonary infections. Our previous work in a mouse model of chronic alcohol consumption has detailed that the primary influenza A virus (IAV)–specific CD8 T cell response in mice that consumed ethanol (EtOH) had a reduced proliferative capacity as well as the ability to kill IAV target cells. Interestingly, recent studies have highlighted that human alcoholics have an increased susceptibility to IAV infections, even though they likely possess pre-existing immunity to IAV. However, the effects of chronic alcohol consumption on pre-existing immune responses (i.e., memory) to IAV have not been explored. Our results presented in this study show that IAV-immune mice that then chronically consumed alcohol (X31→EtOH) exhibited increased morbidity and mortality following IAV re-exposure compared with IAV-immune mice that had consumed water (X31→H2O). This increased susceptibility in X31→EtOH mice was associated with reduced IAV-specific killing of target cells and a reduction in the number of IAV-specific CD8 T cells within the lungs. Furthermore, upon IAV challenge, recruitment of the remaining memory IAV-specific CD8 T cells into the lungs is reduced in X31→EtOH mice. This altered recruitment is associated with a reduced pulmonary expression of CXCL10 and CXCL11, which are chemokines that are important for T cell recruitment to the lungs. Overall, these results demonstrate that chronic alcohol consumption negatively affects the resting memory CD8 T cell response and reduces the ability of memory T cells to be recruited to the site of infection upon subsequent exposures, therein contributing to an enhanced susceptibility to IAV infections.

This article is featured in In This Issue, p.3089

Introduction

The abuse of alcohol by humans represents a significant health concern, causing 1 in 20 deaths worldwide (1). These deaths are due, in part, to the increased risk of severe pulmonary bacterial and viral infections that are associated with chronic alcohol consumption (2–6). A primary cause for the increased risk of severe pulmonary disease is the detrimental impacts that alcohol abuse has on the immune system. In humans that chronically abuse alcohol as well as following short periods of binge drinking, significant reductions in the numbers of T cells and B cells have been documented (2, 7, 8). This alcohol-induced lymphopenic state has been recapitulated in both rodent and nonhuman primate models of chronic ethanol (EtOH) consumption (2, 9–13). Although the overall reduced number in immune cell precursors likely contributes to disease severity following an infection, studies using animal models of chronic EtOH consumption have revealed impairments in the development and function of pathogen-specific adaptive immune responses. During influenza A virus (IAV) infection in mice that chronically consume EtOH, the few primary effector IAV-specific CD8 T cells that do develop have a significantly reduced proliferative capacity, decreased cytotoxicity, and decreased production of IFN-γ (14). This likely contributes to the increased magnitude and duration of a primary IAV infection observed in chronic EtOH-consuming mice (10).

Although studies have detailed the negative impacts of chronic alcohol consumption on the steady-state and developing immune responses during a primary infection, much remains unknown concerning the effects of chronic EtOH on pre-existing immune memory responses. Interestingly, heavy alcohol use in humans is associated with increases in the risk of severe IAV-associated disease (15). In part, this increase in disease may be related to the evidence discussed above of the lesions within the primary effector IAV-specific immune response; however, considering that the highest rates of alcohol dependence occur in adulthood (16), the issue becomes far more complex as these individuals likely had pre-existing T cell immunity. Studies show that there is a high prevalence of pre-existing T cell immune memory to IAV within the general population (17–20), including resident T cell memory within the lungs (21–24). Furthermore, studies examining immunity and protection during the 2009 IAV pandemic suggest that individuals that had pre-existing cross-reactive T cell immunity were more resistant to severe IAV-associated disease compared with those that did not (25–27). Thus, these studies indicate that chronic alcohol consumption may create additional lesions in pre-existing IAV-specific CD8 T cell immunity. Therefore, we determined the consequences of chronic alcohol consumption on existing memory CD8 T cell–mediated protection against IAV.

The composition of the CD8 T cell memory pool established following a primary infection consists of four known subsets. T central memory cells (TCM) express both CD62L and CCR7, allowing them to traffic into the lymph nodes where they undergo rapid proliferation upon pathogen re-exposure. Conversely, T effector memory cells (TEM) are absent from the lymph node because of their lack of expression of both CD62L and CCR7 and instead scan peripheral tissues and exhibit potent cytotoxicity (28–30). In recent studies, a third subset termed T peripheral memory cells (TPM) was identified that shares the proliferative capacity of TCM as well as the cytotoxic capabilities of TEM upon subsequent pathogen exposures. These three subsets are defined based on their expression of CX3CR1 and CD27, with TCM (CX3CR1loCD27hi), TEM (CX3CR1hiCD27lo), and TPM (CX3CR1intCD27hi) differentially expressing these surface markers (31). Although it is known that both TCM and TEM contribute to protection against IAV infection, the role that TPM play has not been determined. However, it is likely that TPM contribute to protection against IAV infection as they have been observed within the lung tissue (31). The fourth subset, known as T resident memory cells (TRM), are embedded within peripheral tissues and act as a first line of defense by rapidly responding upon pathogen re-exposure (32–35). This subset is distinguished from cells within the vasculature by intravascular Ab labeling and phenotypically characterized in the lungs by the expression of CD69 and CD103 (28, 36–38). Although originally characterized in animal models, highly proliferative and polyfunctional IAV-specific TRM have also been identified within the human lung (22, 39, 40). Studies reveal that TRM within the lung are important for mediating heterosubtypic protection as mice lacking lung TRM exhibit increased morbidity and mortality following heterologous IAV challenge (41, 42). Overall, these studies indicate that multiple T cell memory subsets contribute to protection against secondary IAV infection and could be negatively impacted by chronic alcohol consumption.

Our results demonstrate that chronic EtOH exposure of IAV-immune mice increased morbidity and mortality upon a secondary IAV infection compared with IAV-immune H2O control mice. Our findings further demonstrate alterations in the IAV-specific CD8 T cell memory response in IAV-immune EtOH mice compared with IAV-immune H2O control mice. Upon re-exposure with IAV, we observed that IAV-immune EtOH mice had a reduced accumulation of IAV-specific CD8 T cells within the lungs compared with IAV-immune H2O controls. This reduced accumulation was associated with lower levels of two important CD8 T cell chemoattractants (CXCL10 and CXCL11) within the lungs. Overall, our findings highlight that chronic EtOH consumption negatively impacts existing T cell immunity to IAV infection and is associated with increased morbidity and mortality during a secondary exposure to IAV.

Materials and Methods

Mice

Six- to eight-week-old wild-type C57BL/6 mice were originally obtained from the National Cancer Institute (Frederick, MD). B6.Cg-Tcratm1MomTg(TcrLCMV) 372Sdz CD90.1 (P14) mice whose T cells express a TCR specific for the GP33 epitope of lymphocytic choriomeningitis virus were a kind gift from Dr. J. T. Harty from the University of Iowa (Iowa City, IA). Mice were bred, housed, and maintained in the animal care facilities at the University of Iowa. All procedures performed were approved by and adhered to the regulatory standards and guidelines of the Institutional Animal Care and Use Committee of the University of Iowa.

IAV infection

Mouse-adapted laboratory strains of X31, A/Puerto Rico/8/34 (PR8), and PR8-GP33 (generated and kindly gifted by Dr. R. Langlois at the University of Minnesota, Minneapolis, MN) were prepared from stocks as previously described (43). X31-GP33 was a kind gift from Dr. S. Varga at the University of Iowa. Prior to intranasal (i.n.) infections, mice were anesthetized with isoflurane. The following doses of IAV strains were administered i.n. in 50 μl of Iscove medium: 0.1 LD50 (1.11 × 102 tissue culture infectious units [TCIU]) or 5 LD50 (5.54 × 103 TCIU) PR8; 1.20 × 103 TCIU X31; 1.37 × 104 TCIU X31-GP33; and 2 × 104 TCIU PR8-GP33.

EtOH administration

Mice were administered EtOH using the Meadows–Cook model (10, 13, 44–46). Briefly, mice were acclimated to pharmaceutical-grade EtOH supplemented in distilled water at the following concentrations: 10% for 2 d, 15% for 5 d, and 20% for 7 wk. These EtOH solutions were the only source of drinking fluid available to the EtOH group. Control animals received the same distilled water used for the EtOH solutions. Mice had free access to the appropriate drinking solution as well as laboratory chow during the treatment period. Once administered, mice were maintained on their respective drinking fluid through the remainder of the study (i.e., following IAV infection).

In vivo cytotoxicity assay

Procedures for the in vivo cytotoxicity assay were modified from the procedures previously described (47). Splenocytes from C57BL/6 mice were resuspended in 14% Nycodenz (Axis-Shield Diagnostics, Oslo, Norway) that was overlaid with an equal volume of Iscove medium. Splenocytes were centrifuged at 2200 × g for 15 min at 4°C without brake or start assist. Mononuclear cells were isolated from the interface and labeled with 2 μM PKH-26 (Sigma-Aldrich) at room temperature for 5 min. PKH-26–labeled mononuclear cells were split into two groups: one group was labeled with 1 μM CFSE (CFSElo) and the other was labeled with 3 μM CFSE (CFSEhi) at room temperature for 10 min. Labeling reactions were neutralized by adding equal volumes of FBS. CFSEhi and CFSElo cells were pulsed with 10 μM of OVA257 or 10 μM IAV nuclear capsid protein 366 (NP366) peptides for 30 min at 37°C, respectively. CFSEhi and CFSElo were mixed 1:1, and 50,000 cells were adoptively transferred i.n. into test mice. The ratio of CFSEhi:CFSElo cells was determined 8 h after transfer. The percentage of specific killing was determined by the equation 100 − (%CFSElo cells/%CFSEhi cells) and normalized to naive controls.

Intravascular cell labeling

Cells within the vasculature and tissue were distinguished using an intravascular stain as previously described (36). Briefly, mice received 1 μg of fluorophore-conjugated rat anti-mouse CD45.2 (clone 104; BioLegend, San Diego, CA) in 200 μl of PBS by retro-orbital i.v. injection 3 min prior to euthanasia.

MHC class I tetramers

MHC class I tetramers specific for the H-2D(b)–restricted IAV nucleocapsid protein epitope NP366–374/ASNENMETM were obtained from the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility (Atlanta, GA).

Flow cytometry

Lungs and lung draining lymph nodes (dLN) were harvested and digested for 30 min at 37°C in medium containing 1 mg/ml collagenase (Type 3; MP Biomedicals, Solon, OH) and 0.02 mg/ml DNase I (MP Biomedicals). Single cell suspensions were made, and 1 × 106 cells/well were plated in 96-well round bottom plates. Cells were blocked with 2% rat and hamster serum for 30 min at 4°C. After blocking, Ag-experienced (AgExp) CD8 T cells were identified as previously described (48). The following Abs were used to identify AgExp CD8 T cell memory subsets: rat anti-mouse CD8α (53-6.7; BioLegend), rat anti-mouse CD11a (M17/4; BD Biosciences, San Jose, CA), rat anti-mouse CD103 (M290; BD Biosciences), rat anti-mouse CD69 (H1.2F3; eBioscience), rat anti-mouse CD127 (A7R34; BD Biosciences), rat anti-mouse CD62L (MEL-14; BioLegend), hamster anti-mouse KLRG1 (2F1/KLRG1; BioLegend), mouse anti-mouse CX3CR1 (SA011F11; BioLegend), and hamster anti-mouse CD27 (LG.3A10; BioLegend). Cells were fixed with BD FACS Lysing Solution per the manufacturer’s instructions and resuspended in PBS. Data were acquired on an LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR).

Chemokine assay

Prior to euthanasia, blood was collected in nonheparinized capillary tubes (Thermo Fisher Scientific), and serum was collected as previously described (49). Lungs were homogenized using a Dounce tissue homogenizer, and homogenates were purified by centrifugation. Serum and lung lysates were stored at −80°C until analysis. Serum and lung lysate samples were analyzed using the Bio-Plex Pro Mouse Chemokine Panel 33-Plex (Bio-Rad Laboratories, Hercules, CA) per the manufacturer’s instructions. Data were acquired on a Bio-Plex 200 (Bio-Rad Laboratories).

Statistical analysis

Experiments were repeated at least twice unless noted otherwise. All statistical analysis listed below were performed using GraphPad Prism software (La Jolla, CA). Comparisons between two groups was performed with a two-tailed Student t test. Comparisons between more than two groups at different time points were analyzed using two-way ANOVA with Holm–Sidak multiple comparison post hoc test. Comparisons between more than two groups at a single time point were analyzed using a one-way ANOVA with a Tukey multiple comparison post hoc test. Survival comparisons were analyzed using a Mantel-Cox log-rank test. A p value ≤0.05 was considered significant.

Results

Chronic EtOH consumption increases disease severity following secondary IAV infection

To determine the effects of chronic alcohol consumption on memory CD8 T cell–mediated immune protection against a secondary IAV infection, mice that were previously infected with a sublethal dosage of IAV-X31 (H3N2) 8 wk prior were placed on 20% EtOH in distilled water (X31-immune→EtOH) using the Meadows–Cook protocol or distilled water alone (X31-immune→H2O) for 8 wk (Fig. 1A). IAV-X31 is a reassortant virus that contains the hemagglutinin and neuraminidase gene segments from an H3N2 virus, whereas the remaining six gene segments are on an IAV-PR8 (H1N1) backbone. This allowed us to subsequently challenge X31-immune mice with IAV-PR8, to avoid HA3– and NA2-mediated Ab neutralization of the challenge infection, and ascertain the contribution of CD8 T cells to immunity and protection. Following the PR8 challenge, we observed substantial weight loss in naive mice regardless of water or EtOH exposure (Fig. 1B). However, similar to previous studies (10, 14), we observed that naive EtOH-consuming mice (naive→EtOH) exhibited enhanced mortality compared with naive water consuming mice (naive→H2O) (Fig. 1C). As expected, disease-associated weight loss was significantly ameliorated in X31-immune→H2O compared with naive→H2O and naive→EtOH mice; however, X31-immune→EtOH mice showed increased weight loss compared with X31-immune→H2O mice (Fig. 1B). Although no differences in mortality were observed for X31-immune→H2O and X31-immune→EtOH mice challenged with a 0.1 LD50 exposure (Fig. 1C), the increased weight loss in X31-immune→EtOH mice suggests that EtOH-induced deficits have occurred that decrease the ability to control IAV reinfection. To probe this lesion further, we determined the capabilities of mice to control an infection with a high-dose challenge (5 LD50) of PR8, which mimics the severity of many pandemic IAV infections. The high pathogenicity of this dose of PR8 was apparent as all naive mice succumbed to the infection by day 10 (Fig. 1E). Although there were no differences in weight loss, 80% of X31-immune→EtOH mice succumbed to the high-dose PR8 challenge compared with 0% mortality in the X31-immune→H2O group (Fig. 1D, 1E). Given the known role in CD8 T cell–mediated protection against subsequent heterologous IAV exposures (41, 42), these data suggest that chronic EtOH consumption may negatively impact pre-existing IAV-specific immunity and increase susceptibility to disease-associated morbidity and mortality during subsequent IAV exposure.

FIGURE 1.
FIGURE 1.

Chronic EtOH consumption detrimentally impacts the susceptibility to IAV infection in IAV-immune mice. (A) C57BL/6 mice were infected with 1.20 × 103 TCIU of X31 (H3N2) and allowed to establish immune memory for 8 wk. At 8 wk after X31 infection, mice were placed on the Meadows–Cook EtOH protocol or distilled water for 8 wk. At 16 wk after X31 infection, mice were challenged with either a (B and C) 1.11 × 102 TCIU or a (D and E) 5.54 × 103 TCIU dose of heterologous PR8 (H1N1), and (B and D) weight loss and (C and E) survival were monitored. Error bars represent mean ± SEM. Data are from one independent experiment with n = 5 mice per group. X31→H2O versus naive→H2O: ‡p < 0.05; X31→H2O versus X31→EtOH: †p < 0.05; and X31→EtOH versus naive→H2O: *p < 0.05 (two-way ANOVA with Holm–Sidak multiple comparisons test).

Pre-existing IAV-specific CD8 T cell responses are reduced following chronic EtOH exposure

The increased susceptibility of X31-immune→EtOH mice to PR8 infection indicates deficiencies within the IAV-specific memory T cell response. Previous studies from our laboratory have associated defects in cytotoxic IAV-specific CD8 T cells with an increased susceptibility and duration of a primary IAV infection in EtOH-consuming mice (10, 14). As the challenge PR8 (H1N1) virus should evade the neutralizing X31-specific Ab response (i.e., H3N2) generated by the prior X31 infection, B cell responses are likely not contributing to the protection observed following PR8 infection of X31-immune mice. Thus, the deficiency in the IAV-specific memory response instead likely resides within the memory CD8 T cell compartment. To determine if limitations in cytotoxicity are present in the memory IAV-specific CD8 T cell immune response, we used an in vivo cytotoxicity assay. In both naive→H2O and naive→EtOH mice, we observed no killing of target cells as equal frequencies of IAV peptide-pulsed (CFSElo) and control OVA peptide-pulsed (CFSEhi) target cells were observed (Fig. 2A). This highlights, as expected in non–IAV-immune mice, the absence of a cytotoxic IAV-specific CD8 T cell response and that the lung environment of EtOH-consuming mice did not result in selective lysis of either target cell population. However, lysis of IAV-specific target cells within the lungs of X31-immune→H2O mice indicates the presence of a localized cytotoxic IAV-specific CD8 T cell response (Fig. 2A, 2B). Lysis of IAV-specific target cells was significantly lower in X31-immune→EtOH mice compared with X31-immune→H2O mice (Fig. 2A, 2B), which suggests a defective cytotoxic IAV-specific CD8 T cell response and is consistent with the increased morbidity and mortality observed in X31-immune→EtOH mice upon secondary IAV infection (Fig. 1). Because both groups of X31-immune mice should possess similar memory responses prior to EtOH or water exposure, these results suggest that chronic EtOH consumption negatively impacts the overall cytotoxic ability of the pre-existing CD8 T cell memory response.

FIGURE 2.
FIGURE 2.

Chronic EtOH consumption negatively impacts existing IAV-specific CD8 T cell cytotoxic responses. Mice were infected and then exposed to EtOH or water as in Fig. 1. At 16 wk after X31 infection, mice received 50,000 target cells i.n. from a 1:1 mixture of IAV NP366 (CFSElo) and OVA257 (CFSEhi) peptide-pulsed splenocytes. Eight hours after i.n. transfer, (A) the ratio of CFSEhi to CFSElo target cells within the lung were determined, and (B) the percentage of specific killing was calculated with the following equation: 100 − (%CFSElo cells/%CFSEhi cells). Data were normalized to the ratio of killing observed in naive→H2O and naive→EtOH controls. Error bars ± SEM. Data are representative of two combined experiments with n = 6–7 mice per group. The p values were determined by a two-tailed t test.

The inability of X31-immune→EtOH mice to clear IAV-specific target cells from the lungs suggests a lesion within the IAV-specific CD8 T cell memory compartment. Because total CD8 T cells are numerically lower within human alcoholics and EtOH-consuming animal models, we next determined if this numerical reduction extended into X31-immune mice. Consistent with prior findings (10), we observed a significant reduction in the total number of CD8 T cells in the lungs of naive→EtOH mice compared with naive→H2O mice (Fig. 3B). Consistent with the establishment of T cell memory prior to chronic EtOH consumption, total CD8 T cells were increased in the lungs of X31-immune→H2O mice compared with naive controls. However, the subsequent reduction from that memory CD8 T cell set point occurred in X31-immune→EtOH mice as these mice had reduced numbers of total CD8 T cells compared with X31-immune→H2O mice (Fig. 3B). This reduction led to the number of total pulmonary CD8 T cells present in X31-immune→EtOH to be similar to naive→H2O controls (Fig. 3B).

FIGURE 3.
FIGURE 3.

The numerical loss of existing CD8 T cell memory due to chronic EtOH consumption. Mice were infected and then exposed to EtOH or water as in Fig. 1. At 16 wk after ×31 infection, lungs were isolated and analyzed. (A) Representative gating strategies to enumerate (B) total CD8 T cells and (C) AgExp CD8 T cells (CD11ahiCD8αlo) within the lungs. (D and E) Lung-resident AgExp CD8 T cells (CD11ahiCD8αloCD45 intravascular Abneg) were determined by intravascular Ab labeling. Error bars represent mean ± SEM. Data are representative of three combined experiments with n = 8–15 mice per group. *p < 0.05, ****p < 0.0001 (one-way ANOVA with Tukey multiple comparisons test).

As EtOH consumption alters naive CD8 T cells within the lungs (10), it is possible that this reduction in total CD8 T cells in X31-immune→EtOH mice could be due to alterations in the naive CD8 T cell pool and/or the existing IAV-specific CD8 T cell memory pool within the lungs. Studies examining the IAV-specific CD8 T cell memory response have revealed that the epitope dominance is highly dynamic and changes over time (50). Therefore, to not bias our analysis and to encompass the entire IAV-specific CD8 T cell memory response, we used surrogate markers of activation to quantify the AgExp CD8 T cell memory pool (Fig. 3A) (48). As expected, AgExp CD8 T cells were significantly higher in X31-immune→H2O mice compared with naive controls, indicating that prior X31 infection induced a pulmonary IAV-specific CD8 T cell response (Fig. 3C). Interestingly, AgExp CD8 T cells were significantly reduced in X31-immune→EtOH mice compared with X31-immune→H2O mice, suggesting a numerical loss of the IAV-specific CD8 T cell memory response because of EtOH exposure (Fig. 3C). Furthermore, when examining the localization of these T cells within the lungs, our results demonstrate that although the frequency of lung-resident AgExp CD8 T cells was similar between X31-immune→H2O and X31-immune→EtOH mice (Fig. 3D), there was a significant reduction in lung-resident AgExp numbers in X31-immune→EtOH mice (Fig. 3E). There was also a significant reduction in AgExp CD8 T cells in naive→EtOH mice compared with naive→H2O mice, indicating that loss of AgExp CD8 T cells from the lungs after EtOH was not unique to the IAV-specific CD8 T cell response (Fig. 3C). However, the reduced frequency of lung-resident AgExp CD8 T cells in naive mice compared with X31-immune→H2O and X31-immune→EtOH mice suggests that IAV-specific CD8 T cell memory responses were negatively impacted by chronic EtOH exposure (Fig. 3D). The loss of IAV-specific CD8 T cell memory responses was confirmed by quantifying NP366-specific CD8 T cells in X31-immune→EtOH mice, which were reduced in numbers compared with X31-immune→H2O mice (Supplemental Fig. 1A). Further, when the IAV-specific CD8 T cell response was monitored using adoptively transferred donor P14 CD8 T cells (Supplemental Fig. 1B), we observed a significant reduction in IAV-specific CD8 T cells in X31gp33-immune→EtOH mice compared with X31gp33-immune→ H2O mice (Supplemental Fig. 1C). Altogether, these data verify that chronic EtOH consumption causes a numerical loss in the existing IAV-specific CD8 T cell memory pool within the lungs.

Chronic EtOH consumption causes a numerical reduction in CD8 T cell memory subsets

The memory CD8 T cell pool consists of a diverse population of cells that is established following a primary infection. CD8 T cell memory has traditionally been categorized into TCM and TEM. However, recent studies have expanded this dogma with the identification of TPM and TRM subsets (24, 31–35, 37, 38, 51). The representation of each of these subsets within the CD8 T cell memory pool changes over time following a primary infection. For the populations within the circulation, TEM predominate early in the CD8 T cell memory response, but the CD8 T cell pool eventually shifts to being dominated by TCM and TPM (31). Similarly, although TRM can be found within the lung tissue early during the CD8 T cell memory response (51), their numbers wane over time (52). To better understand the EtOH-induced loss of CD8 T cell memory, we next determined the effects of chronic EtOH consumption on the composition of the CD8 T cell memory pool by examining the representation of TRM (CD69posCD103pos), TCM (CD103negCD27hiCX3CR1lo), TEM (CD103negCD27loCX3CR1hi), and TPM (CD103negCD27hiCX3CR1int) within the AgExp CD8 T cell pool in the lungs prior to subsequent IAV exposure (Fig. 4A). Because prior studies have shown that TEM are largely excluded from the tissue and are primarily within the circulation (31), we began by analyzing the entire lung (i.e., circulation and tissue). As expected, X31-immune→H2O mice had significantly increased numbers of AgExp TCM, TEM, TRM, and TPM subsets in the lung compared with both naive controls (Fig. 4B). Interestingly, the numerical loss of AgExp CD8 T cells in X31-immune→EtOH mice was widespread as there were reductions among all CD8 T cell memory subsets examined compared with X31-immune→H2O mice (Fig. 4B). When we analyzed each memory subset for their tissue residence based on intravascular Ab labeling, we observed that TCM, TRM, and TPM, but not TEM, of X31-immune→H2O and X31-immune→EtOH mice had an increased frequency of lung-resident cells (intravascular Abneg) compared with naive controls (Fig. 4C). This observation agrees with previous studies that determined that TEM are devoid from most tissues and instead are within the circulation (31). Interestingly, we observed decreased frequencies of lung-resident TPM in X31-immune→EtOH mice compared with X31-immune→H2O mice, but frequencies of lung-resident TRM were similar (Fig. 4C). The current understanding is that whereas TPM survey peripheral sites by migrating in and out of peripheral tissues from the circulation (31), TRM remain embedded within tissue and do not egress into the circulation (32, 34, 37). Therefore, the decreased frequencies of lung-resident TPM may indicate that the ability of circulating memory to gain access into the lung tissue is altered, which would be consistent with the altered chemokine environment observed in chronic EtOH animal models (11, 12, 46, 53). Nevertheless, even though the frequency of lung-resident TRM were similar between X31-immune→H2O and X31-immune→EtOH mice, there was a significant reduction in the number of lung-resident TRM, as well as TCM and TPM, in X31-immune→EtOH mice (Fig. 4D). Overall, these data suggest that chronic EtOH consumption detrimentally impacts multiple IAV-specific CD8 T cell memory subsets within the lungs.

FIGURE 4.
FIGURE 4.

The numerical loss of existing IAV-specific CD8 T cell memory due to chronic EtOH consumption occurs across multiple T cell memory subsets in the lungs. Mice were infected and then exposed to EtOH or water as in Fig. 1. At 16 wk after ×31 infection, lungs were isolated and analyzed. (A) Representative gating strategies to (B) enumerate AgExp CD8 TRM(CD11ahiCD8αloCD69posCD103pos), TCM (CD11ahiCD8αloCD103negCX3CR1loCD27hi), TEM (CD11ahiCD8αloCD103negCX3CR1hiCD27lo), and TPM (CD11ahiCD8αloCD103negCX3CR1intCD27hi) within the lungs. (C) The frequency and (D) number of lung-resident (CD45 intravascular Abneg) memory was determined by intravascular Ab labeling. Error bars represent mean ± SEM. Data are representative of two combined experiments with n = 10–15 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey multiple comparisons test).

Previous studies have determined that the lung dLNs are an important site not only for the maintenance of the IAV-specific CD8 T cell memory response following the resolution of primary IAV infection (54) but also for their reactivation and expansion upon secondary IAV challenge (50). Therefore, existing AgExp CD8 T cell memory responses within the lung dLNs were quantified (Fig. 5). Similar to the trends observed in the lungs, total CD8 T cells and AgExp CD8 T cells in the lung dLNs of X31-immune→EtOH mice were significantly reduced compared with X31-immune→H2O mice (Fig. 5A, 5B). There were also reductions in the number of total IAV-specific CD8 T cells when responses were examined by an individual epitope specificity in the lung dLNs of X31-immune→EtOH mice compared with X31-immune→H2O mice (Supplemental Fig. 1A, 1C). When we analyzed the individual subsets that make up the complete AgExp CD8 T cell memory response, we observed reductions in TPM in X31-immune→EtOH mice compared with X31-immune→H2O mice, but TCM responses appeared unaltered (Fig. 5C, 5F). TEM and nonlymphoid organ TRM responses were not detected in large numbers within the lung dLNs as these subsets are generally not found in secondary lymphoid organs (SLO) (28, 31) (Fig. 5D, 5E). A recent study has reported on a SLO resident CD8 T cell subset (55); however, we observed no difference in the number of SLO resident CD8 T cells in X31-immune→EtOH mice compared with X31-immune→H2O mice (Fig. 5G). Altogether, these results suggest existing IAV-specific CD8 T cell memory responses in the lungs, and lung dLNs are negatively impacted by chronic EtOH consumption.

FIGURE 5.
FIGURE 5.

The numerical loss of existing IAV-specific CD8 T cell memory due to chronic EtOH consumption occurs across multiple T cell memory subsets in the lung dLNs. Mice were infected and then exposed to EtOH or water as in Fig. 1. At 16 wk after X31 infection, lung dLNs were isolated and analyzed. (A) Total CD8 T cells, (B) AgExp CD8 T cells (CD11ahiCD8αlo), (C) TCM (CD11ahiCD8αloCD103negCX3CR1loCD27hi), (D) TEM (CD11ahiCD8αloCD103negCX3CR1hiCD27lo), (E) nonlymphoid organ TRM (CD11ahiCD8αloCD69posCD103pos), (F) TPM (CD11ahiCD8αloCD103negCX3CR1intCD27hi), and (G) SLO TRM (CD11ahiCD8αloCD69posCD62Lneg) were enumerated within the lung dLNs. Error bars represent mean ± SEM. Data are representative of two combined experiments with n = 8–10 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA with Tukey multiple comparisons test).

IAV-specific CD8 T cell recall responses are altered in chronic EtOH-consuming mice

Following subsequent exposures of IAV, a critical component of protection is the early proliferation and accumulation of IAV-specific memory CD8 T cells within the lungs and lung dLNs (50). Interestingly, in mouse models of chronic EtOH consumption, the developing IAV-specific CD8 T cell effector response has a blunted proliferative capacity that ultimately contributes to increased disease severity (14). Considering we observed increases in disease severity in X31-immune→EtOH mice compared with X31-immune→H2O mice (Fig. 1), we next determined if EtOH consumption altered the expansion of existing IAV-specific CD8 T cell memory in X31-immune→EtOH mice following IAV re-exposure. To this end, X31-immune→H2O and X31-immune→EtOH mice were challenged with a 0.1 LD50 dose of PR8, and AgExp CD8 T cells were quantified in the entire lung, the lung parenchyma (CD45 intravascular Abneg), and the lung vasculature (CD45 intravascular Abpos) at day 3 postinfection (i.e., a time point preceding the arrival of de novo effector T cells in the lungs) (Fig. 6) (56–58). Within the total lungs (i.e., interstitium plus vasculature) of X31-immune→EtOH mice, the total AgExp CD8 T cell response was significantly lower in number 3 d after PR8 challenge compared with X31-immune→H2O mice (Fig. 6A); however, the fold expansion (i.e., accumulation) of total AgExp CD8 T cells was similar in X31-immune→EtOH mice and X31-immune→H2O mice based on the fold increase versus the numbers present in prechallenged mice (7.6× versus 7.7×, Fig. 6A). This translated into little to no defects in the fold increase of TCM, TEM, TRM, and TPM within the lungs of X31-immune→EtOH mice following PR8 challenge (Fig. 6B–E). Interestingly, when we examined the AgExp CD8 T cell responses within the lung vasculature (i.e., CD45 intravascular Abpos) of X31-immune→EtOH mice, the fold increase of total AgExp CD8s, as well as in TCM, TEM, and TPM, were higher compared with X31-immune→H2O mice (Fig. 6F–H, 6J). However, little to no difference was detected in the fold increase of lung-resident TRM in X31-immune→EtOH mice (Fig. 6I). Conversely, when we assessed cells within the lung tissue (i.e., CD45 intravascular Abneg), we observed substantial reductions in the fold increases of total AgExp CD8s, TCM, TEM, and TPM of X31-immune→EtOH mice compared with X31-immune→H2O mice (Fig. 6K–M, 6O) and little to no change in TRM (Fig. 6N). This reduced fold increase of TCM, TEM, and TPM within the lung tissue, when coupled to the increases observed within the lung vasculature of X31-immune→EtOH mice, suggests that EtOH consumption causes defects in the migration of these T cell subsets into the tissue from the vasculature and/or their proliferation once in the lung interstitium (Fig. 6).

FIGURE 6.
FIGURE 6.

Recall response of existing IAV-specific CD8 T cell memory are altered in the lungs of EtOH-consuming mice. Mice were infected and then exposed to EtOH or water as in Fig. 1. At 16 wk after X31 infection, mice were challenged with heterologous PR8 (H1N1), and lungs were isolated and analyzed at 3 d after PR8 challenge. (A, F, and K) AgExp CD8 T cells, (B, G, and L) TCM, (C, H, and M) TEM, (D, I, and N) TRM, and (E, J, and O) TPM were enumerated within the (A–E) total lung, (F–J) within the lung vasculature (i.e., CD45 intravascular Abpos), or (K–O) within the lung tissue (i.e., CD45 intravascular Abneg). Data were compared with prechallenge numbers from Fig. 5 (Day 0) to calculate the fold increase. Error bars represent mean ± SEM. Data are representative of two combined experiments with n = 4–5 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 (Student t test).

Previous studies show that effector IAV-specific CD8 T cells in chronic EtOH mice have a reduced proliferative capacity (14). Interestingly, based on Ki67 staining, we observed a significant reduction in the frequency of proliferating AgExp CD8 T cells within the lung tissue of X31-immune→EtOH mice prior to secondary challenge. However, no differences in the proliferation of AgExp memory CD8 T cells was observed within the lungs of X31-immune→EtOH mice and X31-immune→H2O mice at 3 d postchallenge (Supplemental Fig. 2). Furthermore, the fold increase in total AgExp CD8 T cells within the lung dLNs in X31-immune→EtOH mice was similar to X31-immune→H2O mice (Fig. 7A). TCM and TPM appear to equally contribute to this expansion as we observed similar fold increases in both subsets in X31-immune→EtOH mice and X31-immune→H2O mice (Fig. 7B, 7C). Overall, these data strongly suggest that the proliferation of memory CD8 T cells is not affected by EtOH consumption following secondary IAV challenge but that the defect in TCM, TPM, and TEM lies in their reduced initial numbers as well as their ability to migrate out of the blood and into the inflamed lung tissue in X31-immune→EtOH mice.

FIGURE 7.
FIGURE 7.

Recall response of IAV-specific CD8 T cell memory cells in the lung dLNs of EtOH-consuming mice. Mice were infected and then exposed to EtOH or water as in Fig. 1. At 16 wk after X31 infection, mice were challenged with PR8 (H1N1), and lung dLNs were isolated and analyzed at 3 d after PR8 challenge. (A) AgExp CD8 T cells, (B) AgExp TCM, and (C) AgExp TPM were enumerated within the lung dLNs. Data were compared with prechallenge numbers from Fig. 6 (Day 0) to calculate the fold increase. Error bars represent mean ± SEM. Data are representative of two combined experiments with n = 4–5 mice per group.

Chronic EtOH consumption significantly alters the chemokine milieu during secondary IAV infection

It is well established that chemokine and chemokine receptors have an essential role in controlling the migration patterns of immune cells, including T cells during the steady-state and during inflammation/infection (59–65). Studies show that following secondary respiratory virus exposure, CCR5 and CXCR3 play crucial roles in the trafficking of T cell memory to the lungs and lung airways (59, 61–65). Furthermore, it is known that the chemokine environment is significantly altered following chronic EtOH exposure in nonhuman primates (11, 12, 53) and that the ligands for CCR5 (CCL5) and CXCR3 (CXCL10, CXCL11) are significantly altered in the lungs of human alcoholics (66). Thus, we determined the expression of CCR5 and CXCR3 on AgExp CD8 T cells in the lungs of X31-immune→H2O and X31-immune→EtOH mice following a secondary IAV challenge. Unexpectedly, we observed little to no differences in the expression of CXCR3 or CCR5 on AgExp CD8 T cells from X31-immune→EtOH and X31-immune→H2O mice (Fig. 8A, 8C). We next determined the expression of the chemokine ligands for CXCR3 (CXCL10 and CXCL11) and CCR5 (CCL3, CCL4, and CCL5) within the lungs and serum of X31-immune→EtOH and X31-immune→H2O mice following secondary IAV challenge. Although there were no differences in chemokine levels prior to secondary challenge (Fig. 8B, 8D), we observed significant reductions in CXCL10 and CXCL11 within the lungs of X31-immune→EtOH mice at day 3 postchallenge (Fig. 8B). Conversely, we observed no difference in the level of CCR5 ligands within the lungs of X31-immune→EtOH mice compared with X31-immune→H2O mice (Fig. 8D). Interestingly, we observed similar levels of CXCL10, CXCL11, CCL4, and CCL5 within the serum of X31-immune→EtOH and X31-immune→H2O mice (Fig. 8B, 8D). Although we did observe a significant reduction in CCL3 within the serum of X31-immune→EtOH mice (Fig. 8D), the difference was marginal (i.e., only ∼4 pg/ml difference). These data indicate that chronic EtOH consumption causes reductions in CXCL10 and CXCL11 within the lungs following IAV re-exposure, which likely impacts the migration of AgExp CD8 T cell memory into the lungs from the vasculature because of reduced signaling through CXCR3.

FIGURE 8.
FIGURE 8.

Chronic EtOH consumption causes alterations in the chemokine milieu within the lungs of IAV-immune mice following secondary IAV challenge. Mice were infected and then exposed to EtOH or water as in Fig. 1. At 16 wk after X31 infection, mice were left unchallenged or challenged with heterologous PR8 (H1N1), and lungs were isolated and analyzed at 3 d after PR8 challenge. Chemokine receptor expression of (A) CXCR3 and (C) CCR5 was determined for CD45 intravascular Abpos and CD45 intravascular Abneg AgExp CD8 T cells within the lungs. Serum and lung levels of (B) CXCR3 ligands (CXCL10 and CXCL11) and (D) CCR5 ligands (CCL3, CCL4, and CCL5) were measured by Bio-Plex. Error bars represent mean ± SEM. Data are from one independent experiment with n = 3–4 mice per group. *p < 0.05 (Student t test).

Discussion

Chronic abuse of alcohol has a significant impact on the innate and adaptive branches of the immune system. Not only does chronic alcohol consumption result in an overall numerical reduction in several immune cell subsets, it has also been reported to alter immune cell function. During a primary IAV infection, IAV-specific effector CD8 T cell numbers are reduced in EtOH-consuming mice. Further, these cells have a reduced proliferative capacity and decreased cytotoxic ability (10, 14). This dysfunctional IAV-specific effector CD8 T cell response likely contributes to the increased burden of disease and mortality observed during primary IAV infection in chronic EtOH-consuming mice and humans (1–7, 10, 13, 67). Yet, unlike immunologically naive mouse models, humans likely have pre-existing IAV-specific memory responses prior to their chronic alcohol consumption. The data presented in this study demonstrate that mice with established IAV-specific CD8 T cell memory within the lungs that then chronically consumed EtOH (i.e., X31-immune→EtOH) exhibited increased morbidity and mortality following a subsequent IAV challenge (Fig. 1). The increase in IAV disease severity in X31-immune→EtOH mice was associated with reduced T cell cytotoxic activity within the lungs as X31-immune→EtOH mice had a reduced capacity to lyse IAV-specific target cells within the lungs compared with X31-immune→H2O (Fig. 2). This finding during memory is consistent with our previous studies examining the IAV-specific CD8 T cell response during a primary IAV infection, which determined that effector IAV-specific CD8 T cells from chronic EtOH mice had a reduced cytotoxic activity (14).

A consistent finding in our results is that the reduction in the number of existing IAV-specific CD8 T cells was observed across all T cell memory subsets within both the lungs (Figs. 3, 4, Supplemental Fig. 1) and the lung dLNs (Fig. 5, Supplemental Fig. 1) of X31-immune→EtOH mice compared with X31-immune→H2O mice. Interestingly, based on Ki67 staining, we did observe a reduction in the proliferation of IAV-specific CD8 T cells within the lungs of IAV-immune EtOH mice prior to PR8 challenge, which likely contributes to the reduced numbers we observed (Supplemental Fig. 2). Previous studies have determined that the CD8 T cell memory compartment is maintained by both Ag-dependent and Ag-independent mechanisms (24, 50, 54, 61, 68–76). In particular, the maintenance of the existing IAV-specific CD8 T cell memory compartment relies on both IL-15 (72) and Ag-bearing respiratory dendritic cells continually migrating out of the lungs and into the lung dLNs (54, 77). Interestingly, the number of IL-15–producing cells within the spleen is significantly reduced in chronic EtOH mice (78). Furthermore, recent studies from our laboratory have determined that the migration of CD11cpos dendritic cells out of the lung and into the lung dLNs is reduced in EtOH-consuming mice following IAV infection (79). Therefore, the reduction in the numbers of existing IAV-specific CD8 T cell memory in X31-immune→EtOH mice that we observed could be dictated by either the reduced expression of homeostatic cytokines, such as IL-7 and/or IL-15, or reduced access to Ag.

Our data further reveal that the negative impacts of chronic EtOH consumption not only affect resting CD8 T cell memory subsets but also alter the recall response following subsequent IAV challenge. Although the numbers of IAV-specific CD8 T cells in the lung interstitium of IAV-immune–consuming mice were reduced at day 3 postchallenge compared with IAV-immune water-consuming mice, the fold increase in the total lung (interstitium plus vasculature; Fig. 6) was similar between these two groups. Unlike what was observed for naive/effector CD8 T cells during a primary infection (14), this and our analysis of Ki67 (Supplemental Fig. 2) suggests that proliferation of IAV-specific memory CD8 T cells in IAV-immune EtOH-consuming mice is unaffected following secondary IAV challenge. However, we did observe a reduction in the fold increase of IAV-specific CD8 T cell subsets resident in the lung interstitium (Fig. 6K–O) and a higher fold increase of IAV-specific CD8 T cells within the lung vasculature (Fig. 6F–J) of IAV-immune EtOH-consuming mice compared with IAV-immune water controls. Altogether, these data suggest that trafficking of IAV-specific CD8 T cell memory from the blood into the lungs is reduced by chronic EtOH consumption.

Consistent with this concept of reduced migration, our results further demonstrate that the chemokine milieu within the lungs is significantly altered in IAV-immune mice following EtOH exposure and virus challenge. Although CCR5 and CXCR3 expression on IAV-specific memory CD8 T cells was not affected, we observed significant reductions in the levels of CXCL10 and CXCL11 within the lungs of IAV-immune EtOH-consuming mice on day 3 following secondary IAV challenge (Fig. 8). Interestingly, in addition to airway epithelial cells and alveolar macrophages, CD8 T cells have been observed to secrete CXCL10 upon TCR engagement and contribute to the CXCL10 pool within the lungs during IAV infection (80, 81). Furthermore, it is well known that CD8 TRM possess a “sensing and alarm function” that rapidly induces local chemokine expression, including CXCL10, by cells of the innate immune system (35, 82). Therefore, the reduced numbers of total and IAV-specific CD8 T cells within the lungs of X31-immune→EtOH mice (Figs. 3–6) likely contributes to the reduced levels of CXCL10 that we observed following secondary IAV exposure (Fig. 8). Although the specific role of CXCL10 and its receptor CXCR3 in the trafficking of CD8 T cell memory within the lungs following a secondary infection is not well defined, CXCR3 expression is important for the effective localization of CD8 T cell memory to the lung airways (62, 64). Interestingly, a recent study demonstrated CXCR3-deficient CD8 T cells have a reduced capacity to enter into areas of infection and have reduced interactions with virally infected cells within the skin (60). Thus, the reduced expression of CXCL10 and CXCL11 within the lungs of IAV-immune EtOH-consuming mice may deter the egress of IAV-specific CD8 T cells out of the blood and into the infected lung tissue, ultimately contributing to reduced virus control and increased disease severity.

Altogether the data presented in this study describe for the first time, to our knowledge, the negative impacts that EtOH consumption has on existing IAV-specific CD8 T cell memory responses. The consumption of EtOH led to an overall numerical reduction within the four known CD8 T cell memory subsets (i.e., TCM, TEM, TPM, and TRM) and was associated with increased disease severity upon secondary IAV challenge. Furthermore, the recall response following secondary IAV challenge was altered in X31-immune→EtOH mice, which had a reduced fold increase in IAV-specific CD8 T cells within the lung tissue that was associated with a significantly altered chemokine milieu within the lungs. These results support continued investigation into the effects of EtOH consumption on existing IAV-specific CD8 T cell memory pool and the impact it has on the protection against severe IAV-associated disease.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health Grant AA024860 (to K.L.L.). Z.R.Z. was supported by National Institutes of Health Grants T32AI007485 and T32AI007260.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AgExp
    Ag-experienced
    dLN
    draining lymph node
    EtOH
    ethanol
    IAV
    influenza A virus
    i.n.
    intranasal
    NP366
    nuclear capsid protein 366
    PR8
    A/Puerto Rico/8/34
    SLO
    secondary lymphoid organ
    TCIU
    tissue culture infectious unit
    TCM
    T central memory cell
    TEM
    T effector memory cell
    TPM
    T peripheral memory cell
    TRM
    T resident memory cell.

  • Received July 8, 2019.
  • Accepted October 16, 2019.

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