NK cells are required for indole’s protective effects on pulmonary bacterial burden.
Our previous data demonstrated that indole supplementation during alcohol-consumption alleviates alcohol-associated impairments in pulmonary host defense against bacterial pneumonia. Further, indole treatment restored pulmonary immune cell recruitment. However, it is unknown if indole works directly or indirectly on immune cells. Here we sought to investigate whether indole’s ability to mitigate the increased risk of alcohol-associated pneumonia was dependent on NK cells. Specifically, we selectively depleted NK cells with a monoclonal antibody against NK1.1, which lead to a greater than 95% depletion of pulmonary NK cells (Fig. 1A). Our representative gating strategy for NK cell depletion is shown in Fig. 1B. We first assessed the requirement of NK cells in indole treated mice on reducing the susceptibility to K. pneumoniae. Alcohol-fed mice had a higher pulmonary burden of K. pneumoniae 48 hrs. post infection compared to control mice (Fig. 1B). As seen previously, indole treatment reduced pulmonary K. pneumoniae burden in alcohol-fed mice (Fig. 1B). However, in alcohol-fed NK cell depleted mice indole treatment failed to mitigate pulmonary K. pneumoniae burden (Fig. 1B). Previously, we observed that indole treatment improves epithelial permeability in alcohol-fed mice yet is unknown if NK cells are involved in this process. We examined mucosal permeability following K. pneumoniae infection in alcohol-fed NK cell depleted mice treated with indole. Marked increases in both pulmonary and intestinal permeability were seen in alcohol-fed mice, as determined by increases in the circulating levels of surfactant protein-D (SDP-1; a biomarker of lung damage),8 and intestinal fatty-acid binding protein (IFABP; a biomarker of intestinal damage)9, 10, respectively (Fig. 1C and 1D). Alcohol-fed mice treated with indole exhibited reduced SDP-1 and IFABP levels and were indistinguishable from control fed mice after infection with K. pneumoniae (Fig. 1C and 1D). Interestingly, depletion of NK cells had no effect on the indole’s ability to mitigate alcohol-induced mucosal permeability (Fig. 1C and 1D). This data suggest that indole modulates host defense against bacterial pneumonia via NK cells, as loss of NK cells mitigates indoles protective effects against K. pneumoniae. Further, this data suggest that indole works both directly on NK cells, as well as other systems, as improvements in mucosal permeability are still preserved in NK cell depleted mice.
Alcohol impairs NK cell migratory capacity.
Our previous data demonstrated that indole supplementation during alcohol-consumption restored pulmonary NK cell recruitment. However, whether this effect was due to alterations in NK cell function or changes in chemokine production in the lungs is unknown. Here we sought to characterize the functional capacity of primary NK cells isolated from pair-fed mice, alcohol-fed mice, alcohol-fed mice treated with indole, as well as mice treated with the AhR inhibitor CH223191. NK cells were purified by negative selection, allowing us to examine functional characteristics including migration and bactericidal capacity. NK cells isolated from alcohol-fed mice have a significantly reduced ability to migrate in response to the chemokines CCL2 and CXCL12 (Fig. 2A). CCL2 and CXCL12 are both strong chemo-attractants for NK cells.11 Conversely, primary NK cells isolated from control animals retain the ability to migrate towards CCL2 and CXCL12. Furthermore, alcohol-associated impairments in migration can be overcome by the addition of the AhR agonist indole (Fig. 2A). The effect of indole is also mediated through the AhR receptor as the addition of the AhR antagonist CH223191 blocks the effects of indole (Fig. 2A).
Additionally, NK cells isolated from alcohol-fed mice have a significantly reduced ability to kill Klebsiella pneumoniae in co-culture experiments (Fig. 2B). Like the migration data, primary NK cells isolated from pair-fed animals retain the ability to kill K. pneumoniae. The alcohol-associated impairments in bactericidal capacity were also mitigated by the addition of the AhR agonist indole (Fig. 2B). The effect of indole was likewise dependent on AhR activation, as CH223191 blocks the effects of indole on NK cell bactericidal capacity (Fig. 2B).
Alcohol increases pulmonary and systemic TGFb levels.
The effects of alcohol on NK cell function suggests that alcohol consumption is associated with a highly immunosuppressive environment for NK cells. We sought to investigate systemic and pulmonary levels of TGF-β1, a potent immunosuppressive cytokine on NK cell function. Alcohol-fed mice exhibited marked increases in the levels of TGF-β1 in both serum and lung tissue (Fig. 3A and 3B, respectively), which is consistent with previous reports.12 The administration of indole suppresses the alcohol-associated increases in TGF-β1 levels which was, in part, dependent AhR activation. Specifically, in the presence of the AhR inhibitor CH223191, indole supplementation did not affect systemic or lung levels of TGF-β1 (Fig. 3A and 3B).
Additionally, we sought to evaluate the systemic and pulmonary levels of IL-22, a major cytokine downstream of AhR activation.13 IL-22 is also involved in wound healing and in the protection against microbial infections.13–16 Alcohol-fed mice exhibited marked decreases in the levels of IL-22 in both serum and lung tissue (Supplemental Fig. 1A and 1B, respectively). The administration of indole suppressed the alcohol-associated decrease in IL-22 levels and was dependent, in part, on AhR activation (Supplemental Fig. 1A and 1B).
Manipulation of TGFb or AhR signaling pathways alters pneumonia outcomes in alcohol-fed mice.
Divergent TGFb and AhR signaling associated with alcohol consumption suggests that alcohol use shift host defense pathways to an immunosuppressive phenotype (TGFb), while decreasing immune regulatory/stimulatory pathways (AhR). As such, we sought to pharmacologically manipulate both signaling pathways to alter the host response to bacterial infection. Specifically, cohorts of female mice were randomized into the following groups: 1) alcohol + vehicle, 2) alcohol + indole (20mg/kg), 3) alcohol + indole + CH-223191 (AhR inhibitor; 10 mg/kg), 4) alcohol + anti-TGF-b1 (10 mg/kg), 5) pair-fed + vehicle, and 6) pair-fed + TGF- b1 (0.5 µg/g) (Fig. 4A). We first assessed pulmonary bacterial burden 48 hrs post infection in all mice. Alcohol-fed mice exhibited a marked increase in pulmonary bacterial burden relative to pair-fed mice (Fig. 4B). Similarly, pair-fed mice treated with exogenous TGF-b1 or alcohol-fed mice treated with indole and the AhR inhibitor CH223191 exhibited a significant increase in pulmonary bacterial burden compared to pair-fed mice (Fig. 4B). Conversely administration of indole or the administration of anti-TGF-β1 monoclonal antibodies effectively reverses the detrimental effects of alcohol, by reducing the pulmonary bacterial burden to levels like those observed in pair-fed mice (Fig. 4B).
We also evaluated the pulmonary NK cell population following bacterial pneumonia. Our flow cytometry gating strategy for all NK cell populations is shown in Supplemental Fig. 2. Bacterial burden data for alcohol-fed mice, mice treated with the AhR inhibitor CH22319, and pair-fed mice treated with exogenous TGF-b1 exhibited a significant decrease in the percentage of pulmonary NK cells (Fig. 4C). However, indole or anti-TGF-β1 monoclonal antibody treatment effectively reverses the detrimental effects of alcohol, by increasing the percentage of pulmonary NK cells (Fig. 4C). We also found that the NK cells in the lungs of alcohol-fed mice, alcohol-fed mice treated plus indole and CH22319, as well as pair-fed mice treated with TGF-b1 expressed higher levels of TFGbR1 and lower levels of AhR (Fig. 4D, and 4E, respectively). These trends were reversed in pair-fed mice, as well as alcohol-fed mice treated with indole or the anti-TGF-β1 monoclonal antibody (Fig. 4D, and 4E).
NK cells were also evaluated based on the 4-stage model of maturation using CD27, and CD11b markers.17 Specifically, NK cells were grouped into CD11b-CD27-, CD11b-CD27+, CD11b + CD27+, and CD11b + CD27- where each step indicates the acquisition of NK cell effector functions and maturation. The percentage of stage 1, 2, or 3 NK cells were not significantly affected by alcohol consumption or by any of the exogenous treatments (Fig. 5A, 5B, and 5C). Conversely, marked effects were seen in the percentage of stage 4 NK cells in the lungs of mice post infection (Fig. 5D). Specifically, administration of either TGF-β1 or alcohol decreasing the number of stage 4 NK cells (Fig. 5D). These trends were reversed in pair-fed mice, as well as alcohol-fed mice treated with indole or the anti-TGF-β1 monoclonal antibody, as these mice exhibited a significant increase in the number of pulmonary stage 4 NK cells, compared to their respective controls (Fig. 5D).
Finally, we evaluated the number of pulmonary NK cells with nuclear AhR (active form) using imaging flow cytometry. Administration of either exogenous recombinant TGF-β1 to control animals or alcohol decreased the number of NK cells with nuclear AhR (Fig. 6). However, pair-fed mice, as well as alcohol-fed mice treated with indole or the anti-TGF-β1 monoclonal antibody, exhibited a significant increase in the number of NK cells with nuclear AhR, compared to their respective controls (Fig. 6). Further, as expected, the AhR receptor antagonist CH223191 completely abolishes translocation of the AhR complex to the nucleus (Fig. 6).
Alcohol and TGFb treatment increase pulmonary inflammation and epithelial leak.
Histopathologic findings were consistent with observations for pulmonary bacterial burden and immune infiltration (Fig. 7). Specifically, we observed a significant increase in inflammatory scores for mice treated with alcohol (p = 0.0091) and TGF-β1. Inflammation was alleviated to baseline by the addition of anti-TGF-β1 monoclonal antibody treatment, or indole supplementation. However, alleviation mediated by indole supplementation was not observed if indole was co-administered with the AhR inhibitor CH223191. Aggregation patterns mirrored inflammatory scores with noticeable aggregations noted for alcohol, TGF-β1 and alcohol/AhR inhibition treatments, however these findings did not reach significance (Fig. 7).
We observed that alcohol and TGF-β1 have several potentially detrimental effects on epithelial integrity. Consistent with our previous work, epithelial barrier function was impaired in the presence of either ethanol or TGF- β. Circulating levels of intestinal iFABP (Fig. 8A) and pulmonary SPD-1 (Fig. 8B) were increased, suggesting that barrier integrity was decreased by these treatments and contributing to immune burden via epithelial leakiness (Fig. 8). We further confirmed the effectiveness of recombinant TGF-β1 or anti-TGF-β1 monoclonal antibody treatment by measuring the levels of circulating TGF-β1. We confirmed that the exogenous administration of TGF-β1 utilized in our experiment increased TGF-β1 levels to a range like that observed in our alcohol treated animals (Fig. 8C). Interestingly, we also observed that the AhR agonist indole was able to decrease the circulating levels of TGF-β1, which could be due to improvements in epithelial integrity.
Alcohol and TGFb treatment significantly impair NK cell function.
We then tested the influence of alcohol and TGF-β1 on NK cell functions including NK cell trafficking, NK bactericidal capacity, and production of antibacterial products. Specifically, circulating and splenic NK cells were isolated from the following groups of mice: 1) alcohol + vehicle, 2) alcohol + indole (20mg/kg), 3) alcohol + indole + CH-223191 (AhR inhibitor; 10 mg/kg), 4) alcohol + anti-TGF-b1 (10 mg/kg), 5) pair-fed + vehicle, and 6) pair-fed + TGF- b1 (0.5 µg/g). Circulating and splenic NK cells from two mice per treatment group (n = 3 sets of NK cells per in vivo treatment group) were pooled to generate sufficient NK cells for ex-vivo testing. NK cells were isolated via negative-selection and following purification a 90–95% pure population of NK cells was obtained (Fig. 9A). We first investigated the migratory capacity of the isolated NK cells using a Transwell migration assay. Strikingly, we observed two distinct migration response profiles to common NK cell chemokines. NK cells isolated from pair-fed mice, as well as alcohol-fed mice treated with indole, or anti-TGF-b1 monoclonal antibodies readily migrated in response to CCL2, CXCL12 and CXC3CL1 (p < 0.0001 in all cases) but were relatively non-responsive to CXCR3 signals including CXCL9, CXCL10 and CXCL11 (Fig. 9B, 9E, 9F). Conversely, we found that NK cells obtained from alcohol-fed mice or pair-fed mice treated with recombinant TGF-β1 readily migrated in response to CXCR3 signals including CXCL9, CXCL10 and CXCL11, but were non-responsive to CCL2, CXCL12 and CXC3CL1cytokines (Fig. 9C, 9D, 9G). Suggesting that alcohol use alters the NK cell chemoattractant response and migratory capacity.
In addition to migratory impairment, bactericidal capacity was substantially impaired in NK cells isolated from alcohol-fed mice or pair-fed mice treated with TGF-β1. NK cells isolated from pair-fed mice, as well as alcohol-fed mice treated with indole, or anti-TGF-b1 monoclonal antibodies readily suppress bacterial viability to less than 25% of that observed for bacteria grown in media over the same timeframe (Fig. 10A). However, NK cells isolated from both alcohol and exogenous TGF-β1 treatment mice exhibit a near complete abolishment of bacterial killing (Fig. 10A). The loss of the NK bactericidal function appears to be partly attributable to dysfunction in alpha-defensin related pathways. Specifically, primary NK cells isolated from control animals were pretreated with various inhibitors prior to coculture with Klebsiella. NK cell treatment groups included: 1) Vehicle (0.1% DMSO), 2) Granzyme B inhibitor (10,000 ng/mL), 3) Concanamycin A (100 nM), 4) Granzyme B inhibitor and Concanamycin A, and 5) anti-DEFA1 (1µg/mL) for 1 hour prior to co-culture with Klebsiella. Inhibition of Granzyme B or perforin did not significantly impair the bactericidal capacity of NK cells, however the inhibition of alpha-defensin greatly limited the bactericidal capacity of primary NK cells (Fig. 10B). We further validated these results with an additional bacterial pathogen (Streptococcus pneumoniae), a gram-positive organism and leading cause of alcohol-associated bacterial pneumonia. NK cell-mediated killing of S. pneumoniae was significantly impaired by alcohol and TGF-b1 and was dependent on alpha-defensin (Supplemental Fig. 3). To complement the in vitro NK cell assays in vivo measurements of circulating alpha-defensin show that both alcohol and TGF-β1 suppress circulating levels of alpha-defensin (Fig. 11). In contrast, treatment of alcohol-fed mice with indole or anti-TGF-β1 restore circulating levels alpha-defensin, overcoming the suppressive effects of alcohol (Fig. 11).
Exogenous TGFb-1 does not exacerbate alcohol-associated pneumonia.
To further validate the role of TGF-b1 and AhR signaling in host defense against alcohol-associated bacterial pneumonia we repeated the previous experimental paradigm with several additional control groups. Specifically, an additional cohort of female mice were randomization into the following groups: 1) alcohol + vehicle, 2) alcohol + indole (20mg/kg), 3) alcohol + indole + CH-223191 (AhR inhibitor; 10 mg/kg), 4) alcohol + anti-TGF-b1 (10 mg/kg), 5) alcohol + TGF- b1 (0.5 µg/g), 6) pair-fed + vehicle, 7) pair-fed + TGF- b 1 (0.5 µg/g), 8) pair-fed + indole (20mg/kg), 9) pair-fed + indole + CH-223191 (10 mg/kg), and 10) pair-fed + anti-TGF- b1 (10 mg/kg). In the context of infection, we showed that effective clearance bacteria from the lungs of mice (Fig. 12A), as well as mitigation of bacterial dissemination (Fig. 12B) was dependent on a balance of increased AhR activity and decreased TGF-β1 signaling, in-line with the previous results. These data also demonstrated that exogenous TGF-β1 did not act synergistically or additively to alcohol-feeding alone.
Likewise, we isolated primary NK cells via negative selection from each of the corresponding treatment groups and assessed migratory capacity and bactericidal capacity. Similar to our previous study we found that NK cells isolated from pair-fed mice, as well as alcohol-fed mice treated with indole, or anti-TGF-b1 monoclonal antibodies readily migrated in response to CCL2, CXCL12 and CXC3CL1 but were relatively non-responsive to CXCR3 signals including CXCL9, CXCL10 and CXCL1. While the NK cells obtained from alcohol-fed mice or pair-fed mice treated with recombinant TGF-β1 readily migrated in response to CXCR3 signals including CXCL9, CXCL10 (Supplemental Fig. 4).
Finally, NK cells isolated from pair-fed mice, as well as alcohol-fed mice treated with indole, or anti-TGF-b1 monoclonal antibodies readily suppress bacterial viability, while NK cells isolated from both alcohol and exogenous TGF-β1 treatment mice exhibit abolished bacterial killing (Supplemental Fig. 5A). The loss of the NK bactericidal function appears to be partly attributable to dysfunction in alpha-defensin related pathways, as inhibition of Granzyme B or perforin did not significantly impair the bactericidal capacity of NK cells, however the inhibition of alpha-defensin greatly limited the bactericidal capacity of primary NK cells (Supplemental Fig. 5B).
Treatment of human NK cells with exogenous alcohol or TGFb significantly impair NK cell function.
We then tested the influence of exogenous alcohol and TGF-β1 on NK cell function using a human NK cell line (NK-92 cells). Specifically, NK-92 cells were pre-treated with 50 mM EtOH, 50 pg/mL of human TGF-b1, or with 20 µM indole and 50 mM EtOH for 24 hours. Following incubation NK cell migratory capacity was assessed using the Transwell migration assay. NK-92 cell treated with either EtOH alone or with TGF-b1failed to migrate in response to CCL2/CXCL12, compared to untreated NK-92 cells (Fig. 13A). Further, alcohol-treated NK-92 cells which also were supplemented with exogenous indole had improved migratory capacity in response to CCL2/CXCL12, compared to alcohol treated NK-92 cells (Fig. 13A).
In addition to migratory impairment, bactericidal capacity was substantially impaired in NK-92 cells pretreated with alcohol. Specifically, NK-92 cells treated with EtOH for 24 hours prior to co-culture with Klebsiella exhibited a dose dependent decrease in bactericidal capacity, compared to untreated NK-92 cells. Additionally, NK-92 cells pre-treated with 50 mM EtOH or 50 pg/mL of human TGF-b1, while alcohol-treated NK-92 cells also supplemented with exogenous indole had improved NK cells bactericidal capacity (Fig. 13C). Finally, the effects of TGF-b1 suppression of NK-92 cells bactericidal capacity could be mitigated by exogenous indole treatment in a dose dependent manner (Fig. 13D). However, the required dose of indole to mitigate TGF-b1 suppression was 100 times that required to overcome the effects of alcohol alone. Like our mouse ex-vivo data, loss of the NK-92 bactericidal function appears to be partly due to impaired alpha-defensin production, as the inhibition of Granzyme B or perforin did not significantly impair the bactericidal capacity of NK-92 cells, but the inhibition of alpha-defensin greatly limited the bactericidal capacity of NK-92 cells (Fig. 13E).