Regulation of Natural Killer Cell TGF-β and AhR Signaling Pathways Via the Intestinal Microbiota is Critical for Host Defense Against Alcohol-Associated Bacterial Pneumonia

Alcohol use is an independent risk factor for the development of bacterial pneumonia due, in part, to impaired mucus-facilitated clearance, macrophage phagocytosis, and recruitment of neutrophils. Alcohol consumption is also known to reduce peripheral natural killer (NK) cell numbers and compromises NK cell cytolytic activity, especially NK cells with a mature phenotype. However, the role of innate lymphocytes, such as NK cells during host defense against alcohol-associated bacterial pneumonia is essentially unknown. We have previously shown that indole supplementation mitigates increases in pulmonary bacterial burden and improves pulmonary NK cell recruitment in alcohol-fed mice, which were dependent of aryl hydrocarbon receptor (AhR) signaling. Employing a binge-on-chronic alcohol-feeding model we sought to define the role and interaction of indole and NK cells during pulmonary host defense against alcohol-associated pneumonia. We demonstrate that alcohol dysregulates NK cell effector function and pulmonary recruitment via alterations in two key signaling pathways. We found that alcohol increases transforming growth factor beta (TGF-β) signaling, while suppressing AhR signaling. We further demonstrated that NK cells isolated from alcohol-fed mice have a reduced ability to kill Klebsiella pneumoniae. NK cell migratory capacity to chemokines was also significantly altered by alcohol, as NK cells isolated from alcohol-fed mice exhibited preferential migration in response to CXCR3 chemokines but exhibited reduced migration in response to CCR2, CXCR4, and CX3CR1 chemokines. Together this data suggests that alcohol disrupts NK cell specific TGF-β and AhR signaling pathways leading to decreased pulmonary recruitment and cytolytic activity thereby increasing susceptibility to alcohol-associated bacterial pneumonia.


Introduction
Alcohol use disorder (AUD) is an established risk factor for bacterial pneumonia and alcohol misuse carries a ten-fold increase in the likelihood of pneumococcal pneumonia, as well as a four-fold increase in mortality. 1,2 lcohol ingestion is also associated with infection by other highly virulent respiratory pathogens including Klebsiella pneumoniae.Infection with K. pneumoniae carries a mortality almost double that of AUD patients infected by other pathogens and K. pneumoniae infections have historically been overrepresented in pneumonia patients with AUD. 3,4  variety of mechanisms contribute to the increased risk of pneumonia in subjects with an AUD including decreased mucus-facilitated clearance of invading pathogens and pulmonary immune respones.5 Though the above mechanisms are well established, our research has demonstrated the importance of other novel mechanisms, which derive from alterations to the composition and function of the gut-microbiota.Precisely, we demonstrated that alcohol-associated dysbiosis increases susceptibility Klebsiella pneumonia, independent of alcohol consumption.6 Marked changes in pulmonary host defense were associated with alcohol-dysbiotic mice, including an increase in pulmonary in ammatory cytokines and a decrease in the number of immune cells (CD4 + and CD8 + T-cells) in the lung cells following Klebsiella infection.However, contrary to the pulmonary host response, a marked increase in immune cell counts were seen within the intestinal tract, which suggests that GI T-cell sequestration or dysregulated immune cell tra cking may impair pulmonary host defense.6 Similarly, we found that prophylactic treatment with indole (a microbial speci c metabolite), or a cocktail of probiotics reduced both pulmonary and splenic bacterial burden, improved host immune responses, and facilitated pulmonary tra cking of immune cells.All of which were, in part, driven by aryl hydrocarbon receptors (AhR), as inhibition of AhR mitigated the protective effects.Further, indole increased the frequency of IL-22 immune cells in the lungs and small intestine, and enhanced pulmonary recruitment of CD45 + cells, particularly CD3 + T-cells and NK1.1 + natural killer (NK) cells.7 Here we demonstrate that alcohol dysregulates NK cell effector function and pulmonary recruitment via alterations in two key signaling pathways. W found that alcohol increases transforming growth factor beta (TGF-β1) signaling, while suppressing AhR signaling.We further demonstrate that NK cells isolated from alcohol-fed mice have a reduced ability to kill Klebsiella pneumoniae.NK cell migratory capacity to chemokines was also signi cantly altered by alcohol, as NK cells isolated from alcohol-fed mice exhibited preferential migration in response to CXCR3 chemokines but exhibited reduced migration in response to CCR2, CXCR4, and CX3CR1 chemokines.Together this data suggests that alcohol disrupts NK cell speci c TGF-β1 and AhR signaling pathways leading to decreased pulmonary recruitment and cytolytic activity thereby increasing susceptibility to alcohol-associated bacterial pneumonia.

Results
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.Speci cally, 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 rst 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 puri ed by negative selection, allowing us to examine functional characteristics including migration and bactericidal capacity.
NK cells isolated from alcohol-fed mice have a signi cantly 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. 11Conversely, 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 signi cantly 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. 12The administration of indole suppresses the alcohol-associated increases in TGF-β1 levels which was, in part, dependent AhR activation.Speci cally, 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. 134][15][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.Speci cally, 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 rst 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 signi cant 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 ow 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 signi cant 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. 17Speci cally, 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 signi cantly 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).Speci cally, 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 signi cant 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 ow 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 signi cant 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 in ammation and epithelial leak.
Histopathologic ndings were consistent with observations for pulmonary bacterial burden and immune in ltration (Fig. 7).Speci cally, we observed a signi cant increase in in ammatory scores for mice treated with alcohol (p = 0.0091) and TGF-β1.In ammation 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 in ammatory scores with noticeable aggregations noted for alcohol, TGF-β1 and alcohol/AhR inhibition treatments, however these ndings did not reach signi cance (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 con rmed the effectiveness of recombinant TGF-β1 or anti-TGF-β1 monoclonal antibody treatment by measuring the levels of circulating TGF-β1.We con rmed 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 signi cantly impair NK cell function.
We then tested the in uence of alcohol and TGF-β1 on NK cell functions including NK cell tra cking, NK bactericidal capacity, and production of antibacterial products.Speci cally, 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) pairfed + 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 su cient NK cells for ex-vivo testing.NK cells were isolated via negative-selection and following puri cation a 90-95% pure population of NK cells was obtained (Fig. 9A).We rst investigated the migratory capacity of the isolated NK cells using a Transwell migration assay.Strikingly, we observed two distinct migration response pro les 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.Speci cally, 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 signi cantly 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 signi cantly 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).
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 signi cantly 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 signi cantly impair NK cell function.
We then tested the in uence of exogenous alcohol and TGF-β1 on NK cell function using a human NK cell line (NK-92 cells).Speci cally, 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.Speci cally, 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 alcoholtreated 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 signi cantly 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).

Discussion
Numerous preclinical and clinical studies have demonstrated the critical role intestinal microbiota play in regulating and facilitating pulmonary host defense against bacterial and viral infections. 6, 18-24Mice devoid of GI microbiota (germ-free mice) or mice with signi cantly depleted microbial communities (antibiotic-treated mice) are highly susceptible to pulmonary infection with various bacterial and viral pathogens, including K. pneumoniae and S. pneumoniae. 18,19 everal mechanistic pathways have been identi ed that regulate the gut-lung axis.Speci cally, Nod-like receptor-stimulating bacteria present in the GI tract were shown to increase levels of interleukin-17A, which lead to the production of granulocytemacrophage colony-stimulating factor and killing of bacterial pathogen by alveolar macrophages. 19nhanced susceptibility to K. pneumoniae in germ-free mice was also associated increased levels of IL-10, decreased neutrophil pulmonary recruitment, and bacterial growth and dissemination. 18However, the rigor of prior research linking alcohol dysbiosis to bacterial pneumonia is limited.In fact, to our knowledge, we are the only group to have investigated the role of alcohol-associated intestinal dysbiosis on pulmonary infections. 6,25 n addition, nothing is known about how alcohol might in uence NK cell recruitment to the lungs.NK cells have been classically viewed as crucial for innate defense against viruses and intracellular bacteria, 26, 27 however emerging data indicate that NK cells also participate and are critical for optimal host defense against extracellular bacteria. 28For example, it has been shown in mouse models that NK cells are required for optimal pulmonary host defense against Pseudomonas aeruginosa, 30 and Staphylococcus aureus. 29Similarly, NK cell are also required to combat infection of the GI tract with the extracellular bacterium Citrobacter rodentium. 31In addition, lung NK cells promote host defense against respiratory infection by K. pneumoniae through the production of IL-22 and IFN-γ. 14,32 he mechanisms whereby NK cells protect against bacterial infections remain ill-de ned, but the production of cytokines, such as TNF-α, IFN-γ, IL-22, and IL-10, the recruitment of additional leukocytes, stimulation of macrophages, and direct bacterial killing are likely key mechanistic factors.Alcohol misuse is known to quantitatively and qualitatively alter NK cell function. 33Speci cally, alcohol reduces peripheral NK cell numbers, increases the number of IFN-γ producing NK cells, and compromises NK cell cytolytic activity by inhibiting the production of perforin, granzyme A, and granzyme B. [34][35][36] Recently, AhR signaling has emerged as a potential link between the intestinal microbiota and the host immune system, especially regarding host defense against pathogenic insult.For example, AhRdependent expression of IL-22 is shown to be critical for host defense against Candida albicans. 13dditionally, mice treated with antibiotics exhibited marked impairments in lung immunity to P. aeruginosa via decreased AhR expression in the intestinal tract and reduced peroyxnitrite production by AMs.Importantly, augmentation of AhR signaling by tryptophan supplementation (catabolized by the microbiota to AhR ligands) decreases P. aeruginosa bacterial counts in the lung and increases intestinal ROS production, as well as phagocytic activity of AMs. 37AhR signaling was rst demonstrated to be important for the development and differentiation of regulatory and Th17 T-cells. 38Since then, AhR expression has been reported in specialized innate lymphoid cells, 39 as well as in a number of other hematopoietic populations.[42] Interestingly, determinants of AhR activity, including AhR ligands found in the diet, were found to modulate the antitumor effector functions of NK cells. 41Similarly, the activity of NK cells is strongly affected by TGF-β.Speci cally, TGF-β is known to: 1) negatively regulate IFN-γ production, 43 2) decrease the surface level of the activating receptors NKG2D and NKp30, 44 3) decrease cytotoxicity, 45 and 4) decrease antitumor function of NK cells. 45TGF-β is also known to alter the surface expression of the chemokine receptors CXCR3, CXCR4, CX3CR1, which possibly impacts NK cell migration and recruitment. 44 propose that indole and TGF-b1 exert counter-regulatory effects through differential genetic expression regulated by AhR and SMAD2/3, respectively.Our working model is shown in Fig. 14.The experiments above establish that AhR and TGF-β1 interplay is relevant for a variety of immunological processes including circulating levels of alpha-defensins, NK cellular migration, and NK cytotoxicity, as well as epithelial barrier function.In addition, our data suggests that alcohol may likewise interfere with NK cell maturation through changes in AhR and SMAD2/3 signaling, although lineage tracking experiments are needed to con rm/validate these observations.Exposure to alcohol, through TGF-β signaling, alters the functional effectiveness and phenotypic characteristics of pulmonary NK cells.However, whether this represents a change in gene expression of circulating pools of NK cells or in the maturation of NK cells from stage 1 to stage 4 cells is unknown.We currently favor the later explanation and contend that SMAD2/3 and AhR signaling are opposing forces which control whether NK cells shift from a "normal" response pro le to an "alcohol-associated" response pro le.More precisely, following pulmonary infection, NK cells with a "normal" pro le robustly respond to chemokines, such as CCL12, CXCL12, CXC3L1, while the phenotype of alcohol exposed NK cells inverts to respond preferentially to CXCL9-11 chemokines.
This study focused on the intestinal microbiota from alcohol consuming mice to understand the relationship between alcohol, the microbiota, TGF-b and AhR signaling in NK cells, and host defense against bacterial pneumonia.Together this data suggests that alcohol causes disruption of NK cellspeci c TGF-β and AhR signaling leading to decreased pulmonary recruitment and cytolytic activity thereby increasing susceptibility to alcohol-associated bacterial pneumonia.Understanding the role of intestinal dysbiosis and TGF-β/AhR signaling pathways in the context of alcohol-associated pneumonia may foster novel prophylactic strategies (i.e., indole treatment) for the prevention of alcohol-associated pneumonia in high-risk individuals, which could be especially impactful for individuals with limited resources and access to alcohol treatment centers.

Methods
Mice.
Female 10-12-week-old C57BL/6 mice were obtained from Charles Rivers Breeding Laboratories (Wilmington, MA) and maintained in Comparative Medicine at UNMC.Food and water were provided ad libitum.All mice were housed in a SPF environment under standard social housing conditions with appropriate environmental enrichment.All protocols used in these studies were approved by the Institutional Animal Care and Use Committee of UNMC (IACUC# 19-120-11-EP).This research protocol is accordance with the NIH and O ce of Laboratory Animal Welfare (OLAW) guidelines.

Alcohol feeding model.
We utilized a binge-on-chronic alcohol feeding model as we have previously published. 6All animals were acclimated to liquid diet for 5 days using Lieber-DeCarli '82 Shake and Pour (Bioserv, Flemington, NJ.Cat.No. F1259).Groups of mice (n = 3 per cage) were randomized into ethanol-fed (Bioserv.Cat.No. F1258), ethanol-fed plus treatment, or pair-fed groups.Pair-fed mice were maintained on control-liquid diet adjusted daily according to the consumption of ethanol-fed mice.Mice were administered 4 g kg − 1 (31.5% vol/vol) ethanol by gavage following 5 and 10 days of alcohol-feeding.Pair-fed control mice were gavaged with 7 g kg − 1 (45% wt/vol) maltose dextrin on days 5 and 10 of control-diet feeding.dissolved in sterile corn oil.Mice were treated via IP injection with recombinant TGF-b1 or anti-TGF-b1 every 3 days with the rst dose coinciding with the initiation of alcohol-feeding.Mice were treated with mouse InVivoPlus anti-TGF-b1 monoclonal antibody (clone: 1D11.16.8) by IP injection (10 mg/kg/dose, of mouse anti-TGF-b1; BioXCell, Lebanon, NH.Cat.No. BP0057).Mice were treated with mouse recombinant TGF-b1 by IP injection (0.5 µg/g/dose of rTGF-b1: MedChemExpress.Cat.No. HY-P7117).All mice not receiving treatment via oral gavage were also gavaged daily with vehicle (PBS).Similarly, animals not receiving IP treatments were IP injected with a mouse IgG1 isotype control antibody (clone MOPC-21) in PBS (10 mg/kg/dose, of mouse IgG1 isotype control: BioXCell.Cat.No. BE0083).

Klebsiella pneumoniae infection.
Klebsiella pneumoniae infections and burden assessments were performed using standard methods. 6Brie y, K. pneumoniae (Strain 43816, serotype 2; American Type Culture Collection [ATCC], Manassas, VA.Cat.No. 43816) was grown in 100 mL tryptic soy broth (Becton Dickinson, Franklin Lakes, NJ) in a shaking incubator at 37 o C for 18 hours.Bacteria were then pelleted and resuspended in PBS at an estimated concentration of 1 x 10 3 colony-forming units (CFU)/mL.Mice were then infected with 100 µl of inoculum (1 x 10 2 CFU) via oropharyngeal aspiration.Mice were sacri ced 48 hrs.post infection, and pulmonary and splenic burden was determined via serial dilution and plating onto HiCrome Klebsiella Selective Agar plates.
Whole lungs were in ated with 10% formalin (ThermoFisher Scienti c) to maintain pulmonary structure.
The UNMC Tissue Sciences Core Facility processed all the lungs.Speci cally, lungs were para n embedded and 4-5 µm sectioned were mounted on glass slides and stained with hematoxylin and eosin.The Leica Aperio CS2 (Leica Biosystems, Deer Park, IL) imaging system and the Leica ImageScope software was used to obtain all images (20x).Lung in ammation was scored by a blinded pathologist using a previously published scoring system. 46,47 um Analysis.

Flow Cytometry.
Lungs immune cells were collected for ow cytometry analysis. 6Lung tissue of each animal were collected into C-tubes and immune cells were isolated using the mouse Lung Dissociation Kit (Miltenyi Biotec, Auburn, CA.Cat.No. 130-095-927) according to manufacturer's speci cations.Lung samples were homogenized using gentleMACS™ Octo Dissociator (Miltenyi Biotec).Following isolation any remaining red blood cells (RBCs) were lysed using RBC lysis buffer (BioLegend, San Diego, CA.Cat.No. 420301) Primary mouse NK cells.NK cell migration was assessed using Corning® Transwell® polycarbonate membrane cell culture inserts (Sigma, Cat.No. CLS3421-48EA).The number of viable Primary NK cells was determined following 24 hours of culture via trypan blue staining.NK cells were centrifuged at 300 x ~ g for 10 minutes and resuspended in fresh OptiMEM at a density of 1 x 10 7 viable cells/mL, and 100 µL of the NK cell suspension was added to the Transwell insert.viable cells/mL.100 µL of the primary NK cells were added to 900 µL (1 x 10 5 cells/well) of OptiMEM.In addition, a separate set of primary NK cells isolated from control mice was added to OptiMEM containing 10,000 ng/mL of Granzyme B inhibitor (Sigma, Cat.No. 368056), 100 nM of Concanamycin A (Sigma, Cat.No. C9705), or 1µg/mL of anti-DEFA1 (Biorbyt, Cambridge, United Kingdom.Cat.No. orb10527) for 1 hour prior to co-culture with bacteria.Klebsiella was grown in 100 mL tryptic soy broth (BD Biosciences) in a shaking incubator at 37 o C for 18 hours.Bacteria were then pelleted and resuspended in PBS at an estimated concentration of 1 x 10 7 colony-forming units (CFU)/mL.S. pneumoniae was grown in 100 mL Todd Hewitt Broth (BD Biosciences) at 37 o C in 5% CO 2 for 6 hours.Bacteria were then pelleted and resuspended in PBS at a concentration of 1 x 10 7 CFU/mL.Bactericidal activity was then assessed by adding 100 µL (MOI of 10) of either Klebsiella or Streptococcus to the wells containing NK cells and incubated for 3 hours.The number of viable Klebsiella and Streptococcus 3 hours post infection was determined via serial dilution and plating onto BD BBL™ Trypticase™ Soy Agar (TSA II™) with sheep blood (BD Biosciences).Percent killing was calculated as the total number of viable bacteria post 3 hrs.incubation divided by number of viable bacteria grown in OptiMEM without NK cells present.
Human NK-92 cells.NK-92 cell bactericidal activity was assessed as described for primary mouse NK cells, with the following modi cations.NK-92 cells were rst pretreated with 50 pg/mL of human recombinant TGFb1 (R&D Systems, Cat.No. 7754-BH-025/CF), 30 mM EtOH, 50 mM EtOH, 50 mM EtOH with 20 µM indole, and 50 pg/mL of human recombinant TGFb1 in the presence of 200, and 2000 µM indole for 24 hours.In addition, a separate set experiments with NK-92 cells, cells were pretreated with 10,000 ng/mL of Granzyme B inhibitor (Sigma, Cat.No. 368056), 100 nM of Concanamycin A (Sigma, Cat.No. C9705), or 1µg/mL of anti-human DEFA1-3 (Alpha Diagnostic International, San Antonio, TX.Cat.No. HDEFA11-A) for 1 hour prior to co-culture with bacteria.Bactericidal activity was then assessed as descried above.

Statistics and Reproducibility
Statistical analyses were performed using GraphPad Prism 10 (La Jolla, CA).Results are shown as the mean ± standard error of the mean.A p < 0.05 was deemed signi cant.Sample size and number of replicates is indicated in each respective gure legend.Statistical signi cance was assessed using a Mann-Whitney test for comparisons between two groups and a one-way analysis of variance (ANOVA) with Sidak's multiple comparison test for comparisons between three or more groups.

Declarations Figures
Exogenous treatments (Indole, CH223191, anti-TGF-b1, and TGF-b1 treatment).Mice were treated by oral gavage daily throughout the course of the experiments with indole or CH-223191.Mice received indole by gavage (20 mg/kg/dose indole: Sigma Aldrich, St. Louis, MO.Cat.No. BP0057) dissolved in sterile water warmed to 55°C.Mice received CH-223191 by gavage (10 mg/kg/dose or ~ 200 µL of 3 mM CH-223191: MedChemExpress, Monmouth Junction, NJ.Cat.No. HY-12684) 600 µL OptiMEM only, or OptiMEM containing 50 ng/mL of CCL2 (Sigma.Cat.No. 45-SRP3215-10UG), 100 ng/mL of CXCL12 (R&D Systems, Cat.No. 460-SD-050), 100 ng/mL of CXCL9 (R&D Systems, Cat.No. 492-MM-010), 100 ng/mL CXCL10 (R&D Systems, Cat.No. 466-CR-010), 100 ng/mL CXCL11 (R&D Systems, Cat.No. 572-MC-025), 300 ng/mL CX3CL1 (R&D Systems, Cat.No. 472-FF-025) was added to the bottom of the of the Transwell.Migration was then assessed by determining the number of viable cells in the bottom culture well 5 hours post incubation.Percent migration was calculated as the total number of viable NK cells in the bottom well divided by the total number of viable NK cells added to the Transwell insert.Human NK-92 cells.NK-92 cell migration was assessed as described for primary mouse NK cells, with the following modi cations.NK-92 cells were rst pretreated with 50 pg/mL of human recombinant TGFb1 (R&D Systems, Cat.No. 7754-BH-025/CF), 50 mM EtOH, or 50 mM EtOH with 20 µM indole for 24 hours.Following pre-treatment NK cells were centrifuged at 300 x ~ g for 10 minutes and resuspended in fresh OptiMEM at a density of 1 x 10 7 viable cells/mL, and 100 µL of the NK cell suspension was added to the Transwell insert.600 µL OptiMEM only, or OptiMEM containing 50 ng/mL of CCL2 (R&D Systems.Cat.No. 279-MC-050/CF) and 100 ng/mL of CXCL12 (R&D Systems, Cat.No. 350-NS-050/CF) was added to the bottom of the Transwell.Migration was then assessed by determining the number of viable cells in the bottom culture well 5 hours post incubation.NK cell bactericidal activity.Primary mouse NK cells.NK cell bactericidal capacity was assessed by co-culturing NK cells with Klebsiella pneumoniae (ATCC, Cat.No. 43816) or Streptococcus pneumoniae (Strain TIGR4 [JNR.7/87],serotype 4; ATCC, Cat.No. BAA0334).Primary NK cells isolated from each of the mouse treatment groups were centrifuged at 300 x ~ g for 10 minutes and resuspended in fresh OptiMEM at a density of 1 x 10 6

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