Alcohol Consumption Modulates Host Defense in Rhesus Macaques by Altering Expression in Circulating Leukocytes

This information is current as Tasha Barr, Thomas Girke, Suhas Sureshchandra, Christina of September 23, 2021. Nguyen, Kathleen Grant and Ilhem Messaoudi J Immunol published online 30 November 2015 http://www.jimmunol.org/content/early/2015/11/27/jimmun ol.1501527 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 30, 2015, doi:10.4049/jimmunol.1501527 The Journal of Immunology

Alcohol Consumption Modulates Host Defense in Rhesus Macaques by Altering in Circulating Leukocytes Tasha Barr,* Thomas Girke,† Suhas Sureshchandra,* Christina Nguyen,* Kathleen Grant,‡ and Ilhem Messaoudi*

Several lines of evidence indicate that chronic alcohol use disorder leads to increased susceptibility to several viral and bacterial infections, whereas moderate alcohol consumption decreases the incidence of colds and improves immune responses to some pathogens. In line with these observations, we recently showed that heavy ethanol intake (average blood ethanol concentrations > 80 mg/dl) suppressed, whereas moderate alcohol consumption (blood ethanol concentrations < 50 mg/dl) enhanced, T and B cell responses to modified vaccinia Ankara vaccination in a nonhuman primate model of voluntary ethanol consumption. To uncover the

molecular basis for impaired immunity with heavy alcohol consumption and enhanced immune response with moderate alcohol Downloaded from consumption, we performed a transcriptome analysis using PBMCs isolated on day 7 post–modified vaccinia Ankara vaccination, the earliest time point at which we detected differences in and Ab responses. Overall, chronic heavy alcohol consumption reduced the expression of immune involved in response to infection and wound healing and increased the expression of genes associated with the development of inflammatory disease and . In contrast, chronic moderate alcohol consumption upregulated the expression of genes involved in immune response and reduced the expression of genes involved in cancer. To uncover mechanisms

underlying the alterations in PBMC transcriptomes, we profiled the expression of microRNAs within the same samples. Chronic http://www.jimmunol.org/ heavy ethanol consumption altered the levels of several microRNAs involved in cancer and immunity and known to regulate the expression of mRNAs differentially expressed in our data set. The Journal of Immunology, 2016, 196: 000–000.

lcohol use disorder (AUD) results in a significant increase HIV (1–3). Similarly, chronic ethanol consumption in rodents in the incidence and severity of infections, such as results in increased pathogen burden and impaired ability to clear A bacterial pneumonia, tuberculosis, hepatitis C virus, and Listeria monocytogenes (4), Mycobacterium tuberculosis (5), and influenza virus (6). Likewise, rhesus macaques given ethanol via *Division of Biomedical Sciences, School of Medicine, University of California, Riverside, intragastric cannula show increased SIV replication compared Riverside, CA 92521; †Institute of Integrative Genome Biology, University of California, with controls (7). Increased vulnerability to infection in individ- by guest on September 23, 2021 ‡ Riverside, Riverside, CA 92521; and Division of Neurosciences, Oregon National uals with AUD is due to changes in barrier function, as well as Primate Research Center, Oregon Health and Science University, Beaverton, OR 97006 innate and adaptive immunity (8). Dysregulation of tight junction ORCIDs: 0000-0003-0710-3777 (T.G.); 0000-0002-0380-7219 (K.G.). in the and gut increases permeability, leading to Received for publication July 8, 2015. Accepted for publication October 30, 2015. bacterial translocation into the alveolar space and circulation, This work was supported by National Institute of Alcohol Abuse and Alcoholism respectively (2, 9). In addition, AUD results in the inhibition of Grants AA021947-02, U01 AA013510, and R24 AA109431 and by National Science Foundation Grant ABI-0957099. Sequencing was carried out by the University of phagocytic functions, reduction of chemotaxis and aberrant cy- California, Riverside Genomics Core, supported by National Institutes of Health tokine production, and diminished lymphocyte numbers and Ag- Grant 1S10RR028934-01. specific responses (10). The RNA-sequencing data and small RNA-sequencing data presented in this article In contrast, data from several studies support a beneficial role were submitted to the National Center for Biotechnology Information Sequence Read Archive under accession numbers SRP064253 and SRP064540. for moderate alcohol consumption in immunity. Moderate alco- Address correspondence and reprint request to Dr. Ilhem Messaoudi, Division of hol consumption is associated with a decreased incidence of the Biomedical Sciences, School of Medicine, University of California, Riverside, 900 common cold in humans (11–13), as well as improved bacterial University Avenue, Riverside, CA 92521. E-mail address: [email protected] clearance and increased delayed cutaneous hypersensitivity re- The online version of this article contains supplemental material. sponse following infection with Mycobacteria bovis in rats (14). Abbreviations used in this article: ADAM12, a disintegrin and metalloproteinase Recently, we showed, using a macaque model of ethanol self- domain-12; AHR, aryl hydrocarbon receptor; ALOX15, arachidonate 15-lipoxyge- nase; AUD, alcohol use disorder; BEC, blood ethanol concentration; C7, control administration (15), that moderate consumption resulted in a on day 7 post-MVA vaccination; C3AR1, complement component 3a receptor-1; more robust T cell and Ab vaccine response to modified vaccinia CELSR1, cadherin EGF LAG seven-pass G-type receptor-1; CLEC10A, C-type lec- Ankara (MVA), whereas heavy drinkers generated blunted T cell tin domain family-10A; DEG, differentially expressed gene; DLK1, d-like-1; ETS2, v-ets avian erythroblastosis virus oncogene homolog-2; FC, fold change; FRR, for- and Ab responses compared with controls (16). Moreover, we myl peptide receptor; GJA1, connexin 43; GO, ; H7, heavy drinker on showed that the dose-dependent effects of ethanol on the immune day 7 post-MVAvaccination; HBEGF, heparin-binding EGF-like growth factor; HGF, response to the MVA vaccine were independent of changes in the hepatocyte growth factor; IRS1, insulin receptor substrate-1; ITGA2B, integrin-a-2b; KLF, Kruppel-like factor; LEF1, lymphoid -binding factor-1; M7, moderate frequency of major immune cell subsets. Specifically, numbers of drinker on day 7 post-MVA vaccination; miRNA, microRNA; MVA, modified vac- circulating lymphocytes, monocytes, and neutrophils, as well as cinia Ankara; NOD2, nucleotide-binding oligomerization domain–containing-2; NRP1, neuropilin-1; OSM, oncostatin-M; PTX3, pentraxin-3; RGS, regulator of G- the frequency of CD4 T cells, CD8 T cells, and CD20 B cells (and signaling; RNA-Seq, RNA sequencing; SDC2, syndecan-2; SH2B3, SH2B adaptor-3; their naive and memory subsets), did not differ between control SLC, solute carrier; TNFRSF1A, TNFR superfamily-1A; TREM1, triggering receptor and ethanol-consuming animals (16). Instead, we detected expressed on myeloid cells-1; VEGF, vascular endothelial growth factor. changes in the expression of several microRNAs (miRNAs) as- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$30.00 sociated with the development and function of the immune sys-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501527 2 IMPACT OF ALCOHOL INTAKE ON GENE EXPRESSION tem, suggesting that ethanol dose–dependent modulation of im- was prepared with unique index primers for multiplexing and subjected to munity is mediated by changes in gene expression. Therefore, in single-end 50-bp sequencing on the HiSeq 2500 platform (Illumina). We were this study, we compared the transcriptomes of PBMCs isolated unsuccessful in generating one library from one of the heavy drinkers. from controls and moderate and heavy drinkers on day 7 post- RNA-sequencing analysis MVA vaccination. Data analysis was performed with the RNA-sequencing (RNA-Seq) Our results revealed that chronic heavy ethanol consumption workflow module of the systemPiperR package available on Bio- was associated with the significant downregulation of genes in- conductor (18, 19). Quality reports were generated with the seeFastq volved in immune response to infection and wound healing, as well function. RNA-Seq reads were mapped with the splice junction aware as upregulation of genes associated with the development of ob- short read alignment suite Bowtie2/Tophat2 (20, 21) against the Macaca mulatta genome sequence from Ensembl (22). For the alignments, we used structive lung disease and cancer. In contrast, chronic moderate default parameters of Tophat2 optimized for mammalian genomes. Raw alcohol consumption was associated with the reduced expression of expression values in the form of gene-level read counts were generated genes involved in neoplasia and the upregulation of genes involved with the summarizeOverlaps function (23). We counted only reads over- in host defense. To uncover mechanisms underlying the alterations lapping exonic regions of genes, discarding reads mapping to ambiguous in PBMC transcriptomes, we also examined changes in miRNA regions of exons from overlapping genes. Given the nonstranded nature of RNA-Seq libraries, the read counting was performed in a nonstrand- expression. Our analysis showed that chronic heavy ethanol specific manner. The RNA-Seq data were deposited in the National Cen- consumption altered the expression of several miRNAs whose ter for Biotechnology Information Sequence Read Archive under accession targets were differentially expressed in our data set and are in- number SRP064253 (http://www.ncbi.nlm.nih.gov/sra). Analysis of dif- volved in cancer progression and immune function. Overall, data ferentially expressed genes (DEGs) was performed with the generalized linear model method from the edgeR package (24, 25). DEGs were defined presented in this article provide novel insights into the mecha- as those with a fold change (FC) $ 2 and a false discovery rate # 0.05. Downloaded from nisms by which excessive alcohol consumption interferes with Enrichment analysis of functional annotations was performed to identify immune responses and exacerbates comorbidities, such as poor significant biological pathways, including gene ontology (GO) terms and wound healing, lung disease, and cancer, and moderate con- disease biomarkers using MetaCore software (GeneGo, Philadelphia, PA). sumption improves immunity. Small RNA-Seq analysis Adaptor contaminations were removed (trimmed) from the reads using Materials and Methods the preprocessReads function from the systemPipeR package. The pre- http://www.jimmunol.org/ Ethics statement processed reads were aligned with Bowtie2 (20, 21) against the M. mulatta genome sequence, with settings optimized for miRNA alignments, in- This study was performed in strict accordance with the recommendations cluding tolerance of multiple mappings. Reads overlapping with miRNA detailed in the Guide for the Care and Use of Laboratory Animals of the gene ranges were counted with the summarizeOverlaps function, as de- National Institutes of Health, the Office of Animal Welfare, and the U.S. scribed above, but in a strand-specific manner. The miRNA gene coordi- Department of Agriculture. All animal work was approved by the Oregon nates, required for this step, were downloaded from miRBase (release National Primate Research Center Institutional Animal Care and Use version 19). The small RNA-Seq data were deposited in the National Committee. Center for Biotechnology Information Sequence Read Archive under ac- cession number SRP064540 (http://www.ncbi.nlm.nih.gov/sra). Differen- Animal studies and sample description

tially expressed miRNA genes were identified with edgeR, as described by guest on September 23, 2021 The animal model and vaccination strategy were described previously (16). above. TargetScan was used to predict genes for each differentially Briefly, we used schedule-induced polydipsia to establish reliable self- expressed miRNA with a high context ratio of 0.95. These targets were administration of 4% (w/v) ethanol in eight male rhesus macaques (15). compared with our list of DEGs among the three groups of rhesus ma- Four animals served as controls, for a total of 12 animals. Following a caques. These combinations of differentially expressed mRNA and 4-mo induction period, animals were allowed a choice of 4% ethanol or miRNA were then segregated based on the directions of FCs. water for 22 h/d every day for 12 mo. In this nonhuman primate model of Gene validation via quantitative RT-PCR voluntary self-administration, animals segregate naturally into heavy and moderate drinkers within 2–3 mo, and these patterns remain stable for $12 cDNAwas synthesized from RNA, isolated as above, using a High Capacity mo (17). In this specific cohort, ethanol-drinking animals segregated into cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). two cohorts (n = 4 each), based on average blood ethanol concentration mRNA expression was determined by quantitative RT-PCR using TaqMan (BEC) values: moderate drinkers with average BEC of 22.3–48.8 mg/dl primer and probe kits specific for M. mulatta cDNA sequences and a and heavy drinkers with average BEC of 90–126 mg/dl (16). All 12 ani- StepOnePlus instrument (Life Technologies, Grand Island, NY). mRNA mals were vaccinated with MVA prior to the induction of ethanol and again expression levels of LYZ (Rh02902590), PTGS2 (Rh02787804), THBS1 after 7 mo of open access to ethanol. We used PBMCs isolated 7 d after (Rh00962902), Kruppel-like factor (KLF)4 (Rh02847953), TLR4 booster vaccination for RNA and miRNA expression analysis. Only three (Rh01060206), CD14 (Rh03648680), CD163 (Hs00174705), and FN1 animals from each group had sufficient numbers of PBMCs for RNA se- (Rh02621780) for each sample were calculated relative to control RPL32 quencing. mRNA expression using DCt calculations.

RNA isolation and mRNA library preparation Results Total RNA was isolated from PBMCs using the miRNeasy kit (QIAGEN, Heavy alcohol consumption leads to significant changes in Valencia, CA). One microgram of RNA was used to generate libraries using gene expression the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA). Poly(A)-enriched mRNA was fragmented, There were 514 DEGs between controls on day 7 post-MVA followed by cDNA synthesis with random hexamers. This product un- vaccination (C7) and heavy drinkers on day 7 post-MVA vacci- derwent end-repair, adapter ligation, and size selection using AMPure XP nation (H7; C7–H7), 479 of which were annotated, with 356 beads (Beckman Coulter, Brea, CA) to isolate cDNA templates of 320 nt that were amplified by PCR. Each library was prepared with unique index downregulated and 123 upregulated genes with heavy drinking primers for multiplexing and subjected to single-end 100-bp sequencing on (Supplemental Table I). We identified 368 DEGs between mod- the HiSeq 2500 platform (Illumina, San Diego, CA). erate drinkers on day 7 post-MVA vaccination (M7) and heavy Small RNA library preparation drinkers (M7–H7), 347 of which were annotated, with 290 downregulated and 57 upregulated genes with heavy drinking One microgram of total RNA, extracted as described above, underwent (Supplemental Table II). Finally, of the 60 DEGs between controls adapter ligation and primer hybridization prior to cDNA synthesis and PCR amplification using the NEBNext Small RNA Library Prep Set for Illumina and moderate drinkers (C7–M7), 47 were annotated, with 29 down- Kit (New England Biolabs). Size selection was performed with AMPure XP regulated and 18 upregulated with moderate ethanol consumption beads (Beckman Coulter) to isolate cDNA templates of 140 nt. Each library (Fig. 1A, Supplemental Table III). The Journal of Immunology 3

Heavy ethanol consumption was associated with the largest (FC = 5.6), CD14 (FC = 3.8), pyrin domain–containing-3 (FC = changes in gene expression compared with controls and moder- 3.3), TLR8 (FC = 2.9), and nucleotide-binding oligomerization ate consumption (Fig. 1A, 1B), with 171 downregulated DEGs domain–containing-2 (NOD2; FC = 2.4). Other DEGs encode (Fig. 1C) and 26 upregulated DEGs (Fig. 1D) compared with immune receptors, such as cadherin EGF LAG seven-pass G-type controls and moderate drinkers. The overwhelming majority of the receptor-1 (CELSR1; FC = 30.6), syndecan-2 (SDC2; FC = 7.8), downregulated (Fig. 1E) and upregulated (Fig. 1F) DEGs showed plasminogen activator receptor (FC = 6.5), macrophage scavenger a 2–4 FC in expression. To confirm the RNA-Seq results, eight receptor-1 (FC = 5.7), IL-1R type-1 (FC = 5.7), IL-13Ra-1 (FC = genes differentially expressed with chronic heavy ethanol con- 4.8), CCR1 (FC = 4.5), and neuropilin-1 (NRP1; FC = 3.4). Ad- sumption (LYZ, PTGS2, THBS1, KLF4, TLR4, CD14, CD163, ditional DEGs encode chemokines, cytokines, growth factors, and and FN1) were selected for confirmation using quantitative RT- antimicrobial peptides, including matrix metalloproteinase-1 PCR. Changes in the expression level of all eight DEGs were (FC = 8.6), CXCL8 (FC = 7.5), oncostatin-M (OSM; FC = 4.4), confirmed (Supplemental Fig. 1). To better understand the bio- IL-1b (FC = 3.7), vascular endothelial growth factor (VEGF)-A logical relevance of these gene-expression changes, we conducted (FC = 5.2), heparin-binding EGF-like growth factor (HBEGF; functional enrichment analysis using the MetaCore pathway- FC = 3.4), and S100 calcium binding protein-8/9 (FC = 3.6/2.9). mapping tool. Several genes listed above also mapped to “response to wounding” and “inflammatory process” and play a role in wound Heavy alcohol consumption downregulates genes that promote healing (Fig. 2B). For instance, CELSR1, SDC2, VEGFA, host defense compared with moderate drinkers and controls HBEGF, and NRP1 promote wound closure (26, 27), whereas

Of the 171 genes repressed in H7 compared with C7 and M7, 114 NOD2, pyrin domain–containing-3, CD14, coagulation factor Downloaded from mapped to the following GO terms: response to stress, response to plasminogen activator inhibitor-2 (FC = 17.6), complement com- wounding, inflammatory response, response to lipid, and positive ponent 5aR receptor-1 (FC = 4.0), complement component 3a regulation of response to external stimulus (Fig. 2A). Several receptor-1 (C3AR1; FC = 2.9), and IL-1b (which activates CXCL8, DEGs mapping to “response to stress” encode microbial sensors, FC = 7.5) promote chemotaxis and leukocyte extravasation into notably formyl peptide receptor (FPR)2 (FC = 128.2), TLR4 injury sites (28–31).

Additional analysis showed that 89 genes map to these disease http://www.jimmunol.org/ categories: obstructive lung diseases, pathologic processes, hy- persensitivity, bacterial infection and mycoses, and inflammation (Fig. 2C). Of the 52 genes mapping to “obstructive lung diseases” (Fig. 2D), 24 interact with each other (Fig. 2E) and are important for lung homeostasis, notably matrix metalloproteinase-1 [FC = 8.6, involved in lung alveolar epithelial cell migration (32)], VEGFA [important for alveolar structure (33)], cathelicidin anti- microbial peptide (LL37, FC = 2.8), and aryl hydrocarbon re- ceptor [AHR; FC = 2.7, regulates of lung epithelial by guest on September 23, 2021 cells (34)]. DEGs mapping to “pathological processes” include annexin-2 receptor (FC = 17.1), cysteine-rich secretory protein LCCL domain–containing-2 (FC = 9.7), epiregulin (FC = 9.2), triggering receptor expressed on myeloid cells-1 (TREM1; FC = 4.7), and HBEGF, which are involved in protection against cancer, sepsis, endotoxin shock, and necrotizing enterocolitis (35–39). Heavy drinking downregulates genes that promote wound healing and protect against chronic disease compared only with controls Of the 185 DEGs repressed in H7 compared with C7, 128 mapped to these GO terms: response to wounding, regulation of response to stimulus, positive regulation of response to stimulus, response to stimulus, and system development (Fig. 3A). The 37 genes map- ping to “response to wounding” play an important role in wound healing (Fig. 3B), including diacylglycerol [FC = 5.8, regulates fibroblast migration (40)], catenin a-1 [FC = 3.4, pro- motes wound repair in bronchial epithelial cells (41)], metal- lopeptidase inhibitor-2 [FC = 3.4, involved in wound closure (42)], solute carrier (SLC) family-11 [FC = 2.2, regulates mac- rophage activation in cutaneous wounds (43)], CSF1 [FC = 2.1, FIGURE 1. PBMC gene expression. (A) Bar graph showing the number involved in neoangiogenesis (44)], and hepatocyte growth factor of downregulated and upregulated genes for each comparison. (B) Venn [HGF; FC = 2.0, accelerates wound re-epithelialization (27)]. One diagram depicting the overlap of the annotated DEGs among controls highly downregulated gene mapping to “response to stimulus” is (C), moderate drinkers (M), and heavy drinkers (H) on day 7 after booster eosinophil peroxidase (FC = 22.6), a potent toxin for bacteria and vaccination (C7, M7, and H7 respectively). (C) Venn diagram depicting the overlap between genes that are downregulated with heavy alcohol con- parasites (45). sumption compared with controls and moderate drinkers. (D) Venn dia- Further analysis showed 106 genes mapped to these disease gram depicting the overlap between genes that are upregulated with heavy categories: obstructive lung diseases, pathological processes, im- alcohol consumption compared with controls and moderate drinkers. FC of mune system diseases, bronchial diseases, and immediate hyper- downregulated (E) and upregulated (F) genes with heavy drinking. sensitivity (Fig. 3C). Genes in “obstructive lung diseases” play an 4 IMPACT OF ALCOHOL INTAKE ON GENE EXPRESSION Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. Chronic heavy alcohol consumption downregulates genes that promote wound healing and contribute to obstructive lung diseases compared with controls and moderate drinkers. (A) Bar graph displaying the 10 most significant GO terms associated with the 170 genes downregulated with heavy ethanol consumption (H7) compared with controls and moderate drinkers (C7 and M7). Line represents the 2log(p value) associated with each GO term. (B) Heat map of DEGs between H7 and C7 in the “response to wounding” GO term. (C) The 10 most significant diseases by biomarkers associated with the 170 genes downregulated with heavy ethanol consumption (H7) compared with controls and moderate drinkers (C7 and M7). (D) Heat map of DEGs between H7 and C7 that mapped to “lung diseases-obstructive” category. (E) Network of DEGs that mapped to “obstructive lung diseases” showing direct interactions. important role in lung function (Fig. 3D). For instance, IL-1R– processes,” including C-type lectin domain family-10A like-1 (FC = 9.2), TLR5 (FC = 6.0), arachidonate 15-lipoxygenase (CLEC10A, FC = 4.8) and ATP-binding cassette subfamily-C-2/ (ALOX15, FC = 5.5), TREM2 (FC = 2.6), and TNFR superfamily- 3 (FC = 2.9/3.2), which play a role in Ag recognition and pre- 1A (TNFRSF1A, FC = 2.0) promote host defense against bacterial sentation (48, 49), as well as EGF-like module receptor-1 (FC = infection in the lungs and regulate lung inflammation, whereas 3.7) and IL-12Rb-2 (FC = 2.8), which are critical for host defense HGF (FC = 2.0) promotes lung regeneration after injury (27). (50). Genes unique to this category (Fig. 3E) regulate inflamma- Polymorphisms in myeloperoxidase (FC = 7.4) and A Disintegrin tion, such as leucine-rich repeat-containing-18 (FC = 104.5) and and Metalloproteinase domain-12 (ADAM12, FC = 4.5) modulate hemoglobin subunit g-2 [FC = 8.9 (51, 52)], as well as lymphocyte the development of lung cancer (46, 47). proliferation and differentiation, including SH2B adaptor-3 Several DEGs that mapped to “immune system diseases” also [SH2B3, FC = 2.0 (53)] and killer cell lectin-like receptor mapped to “obstructive lung diseases” and “pathological subfamily-G1 [FC = 2.4 (54)]. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. Alcohol abuse uniquely downregulates additional genes that promote wound healing and contribute to obstructive lung diseases. (A) Bar graph displaying the 10 most significant GO terms to which the 186 DEGs downregulated with heavy ethanol consumption (H7) compared with controls only (C7). Line represents the 2log(p value) for each GO term. (B) Heat map of DEGs between H7 and C7 mapping to the GO term “response to wounding.” (C) Bar graph displaying the 10 most significant Diseases by Biomarker to which the 186 DEGs downregulated with heavy drinking (H7) compared with controls (C7) only. Line represents the 2log (p value) for each disease category. (D) Heat map of the DEGs between H7 and C7 mapping to the “lung diseases-obstructive” disease category. (E) Heat map of the 21 DEGs between H7 and C7 mapping to “immune system diseases.” 6 IMPACT OF ALCOHOL INTAKE ON GENE EXPRESSION

Heavy alcohol consumption reduces expression of genes that (FC = 3.0), which regulates expression of genes that counter oxi- regulate the immune system compared with moderate dative stress, and ETS2, which is induced by shear stress to preserve consumption only the integrity of microvascular walls (64). Additional notable DEGs Of the 119 genes downregulated in H7 compared with M7, 66 that only mapped to “response to stress” included -g [FC = mapped to the following GO terms: immune system processes, 8.4, important in antitumor immune responses (65)] and BMX response to stress, immune response, defense response, and reg- nonreceptor tyrosine kinase [FC = 11.5, promotes tight junction ulation of immune system processes (Fig. 4A). In total, 47 mapped formation in epithelial cells during chronic hypoxia (66)]. to immune system–related GO terms, 20 of which interact with each other (Fig. 4B). DEGs mapping to these GO terms are in- Heavy drinking increases expression of genes associated with volved in lymphocyte activation and recruitment [CXCL10, FC = impaired wound healing, cardiovascular disease, and cancer 17.3; SLC16A1, FC = 3.2 (55); sterile a-motif-domain Src Within the 26 genes upregulated in H7 compared with C7 and homology-domain nuclear localization signals-1, FC = 3.7 (56); M7 (Fig. 5A), several encode transcription factors associated with ICAM1, FC = 5.5; CD83, FC = 3.0 (57)], antimicrobial response skin, colorectal, breast, and lymphoma , notably IFN-a- [TNFa, FC = 7.9; ficolin-2, FC = 6.5 (58); MD2, FC = 2.8], and inducible protein 27-like-1 [FC = 68.0 (67)], AXIN2 [FC = 2.2 regulation of gene expression [v-ets avian erythroblastosis virus (68)], lymphoid enhancer-binding factor-1 [LEF1; FC = 2.0 (69)], oncogene homolog-2 (ETS2), FC = 2.5; B cell lymphoma 2-re- Meis homeobox-1 [FC = 2.4 (70)], and four-and-a-half LIM lated protein-A1, FC = 5.4 (59); B cell CLL/lymphoma-3/6, FC = domains-1 [FC = 2.1 (71)]. Interestingly, increased expression 2.5/2.0 (60, 61); KLF10, FC = 3.0 (62); and NF of k light poly- of retinoid X receptor-g (FC = 5.2), which is associated with peptide gene enhancer in B cells inhibitor-a, FC = 2.9 (63)]. sensation seeking (72), a behavioral trait common among Downloaded from Several genes mapping to “immune system process” (Fig. 4C) also alcoholics, was detected. Another overexpressed gene was serum map to “response to stress,” notably hypoxia-inducible factor-1a deprivation protein response (FC = 2.8), which induces deformation http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 4. Heavy alcohol consumption down- regulates genes associated with regulation of the immune system. (A) Bar graph displaying the top 10 significant GO terms associated with the 122 genes downregulated with heavy ethanol con- sumption (H7) compared with moderate drinkers (M7) only. Line represents the 2log(p value) of each GO term. (B) Network of DEGs that mapped to the GO term “immune system processes” and directly interact with one another. (C)Heatmap of the DEGs between H7 and M7 that mapped to the GO term “immune system processes.” The Journal of Immunology 7 of plasma membrane invaginations, impairing endocytosis and, of neutrophils), CCL3 [FC = 17.1, recruits T cells (92)], and potentially, Ag presentation (73). Finally, expression of regulator CCL4L1/2 [FC = 8.1/5.5, trafficking of NK cells (93)]. Other of G-protein signaling (RGS)18 (FC = 3.3), growth factor- genes significantly upregulated in M7 include acute-phase protein independent 1B transcription repressor (FC = 2.3), and rho gua- pentraxin-3 [PTX3, FC = 15.3 (94)], ceruloplasmin (FC = 3.7), nine nucleotide exchange factor-4 (FC = 6.1), which play a role in and granzyme A (FC = 2.2), which are expressed primarily by NK megakaryocyte differentiation (74, 75), were also increased. cells and play a role in host defense (95, 96), as well as GJA1 Of the 97 genes upregulated in H7 compared with only C7, 26 (FC = 9.6), important for barrier function (97). mapped to the following GO terms: response to wounding, regu- Several of the 18 genes downregulated in M7 compared with C7 lation of body fluids, platelet activation, wound healing, and (Fig. 6B) are involved in cancer progression, including trans- platelet degranulation. Genes that mapped to “response to membrane protein-98 (FC = 286.9), serine-protease temperature wounding” (Fig. 5B) include connexin-43 (GJA1, FC = 11.0), a requirement-A1 (FC = 24.1), DNA nucleotidylexotransferase gap junction associated with impaired wound healing (76); IL-17F (FC = 5.7), MMP9 (FC = 3.5), and ten-eleven translocation-1 (FC = 6.7), which can delay wound closure (77); and P-selectin [FC = 3.0 (98–102)]. Interestingly, ABCA9 (FC = 6.9), retinoic (FC = 2.3), a glycoprotein that is highly expressed in wounds (78). acid-binding receptor-related orphan receptor-C (FC = 3.6), 29-59- Genes with roles in cardiovascular disease mapped to the disease oligoadenylate synthetase-2 (FC = 2.6), CD84 (FC = 2.2), and category “infarction,” including carbonic anhydrase III (FC = 5.2), myxovirus resistance protein-1 (FC = 2.2), which play a role in glycoprotein VI (FC = 2.9), phosphodiesterase-6H (FC = 2.6), and innate immunity, were downregulated in M7. integrin-a-2b [ITGA2B; FC = 2.6 (79–82)]. Furthermore, heavy Heavy ethanol consumption alters the expression of miRNAs ethanol consumption upregulated genes associated with cancer, Downloaded from involved in cancer and immune function notably transient receptor potential cation channel-M1 (FC = 14.9), tripartite motif containing-31 (FC = 9.8), RGS6 (FC = 7.6), To begin uncovering the mechanisms underlying changes in gene and CLDN5 [FC = 2.2 (83–85)]. expression regulation with moderate and heavy ethanol consump- A total of 21 of the 31 genes upregulated in H7 compared with tion, we compared the miRNA expression profiles of the same only M7 mapped to “neuro-ectodermal tumors” (Fig. 5C). These PBMCs isolated from controls, moderate, and heavy drinkers on ∼ genes are either expressed at high levels in cancer, including day 7 post-MVAvaccination. miRNAs are 22-nt-long endogenous http://www.jimmunol.org/ d-like-1 [DLK1, FC = 7.3 (86)] and insulin receptor substrate-1 RNAs that target mRNAs for translational repression or degrada- [IRS1; FC = 4.8 (87)] or are involved in progression of cancer, tion (103), and several reports indicate that ethanol can modulate such as phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor-2 [FC = 11.7 (88)], latent TGF-b–binding protein-2/3 [FC = 3.6/2.6 (89, 90)], and stromal Ag-3 [FC = 2.3 (91)]. Moderate drinking activates genes associated with immunity and represses genes associated with cancer compared with

controls by guest on September 23, 2021 Of the 29 annotated genes upregulated in M7 compared with C7 (Fig. 6A), 4 play a role in chemotaxis: CXCL3 (FC = 50.3, crit- ical for leukocyte chemotaxis), IL-1a (FC = 23.4, recruitment

FIGURE 5. Alcohol abuse upregulates genes that interfere with wound healing and contribute to cancer. Heat maps of the DEGs upregulated with FIGURE 6. Moderate ethanol consumption modulates genes associated heavy ethanol consumption (H7) compared with controls (C7) and mod- with immune response. (A) Heat map of the DEGs uniquely activated with erate (M7) drinkers (A), controls (C7) only and mapped to the GO term moderate drinking (M7) compared with controls (C7). (B) Heat map of the “response to wounding” (B), and moderate (M7) drinkers only and mapped DEGs uniquely repressed with moderate drinking (M7) compared with to the GO term “neuro-ectodermal tumors” (C). controls (C7). 8 IMPACT OF ALCOHOL INTAKE ON GENE EXPRESSION miRNA expression (104). As described for mRNA expression, the subfamily-G1 (54). In our previous study, excessive ethanol largest differences in miRNA expression were observed between consumption suppressed, whereas moderate ethanol consumption controls and heavy drinkers; only a few miRNAs were differen- enhanced, MVA-specific IgG responses (16). These defects in Ab tially expressed between controls and moderate drinkers. Interest- production could be explained, in part, by the decreased ex- ingly, no differentially expressed miRNAs were detected between pression of transcription factors BCL3 and BCL6, which are heavy and moderate drinkers. There were 79 differentially ex- important in germinal center formation, isotype class switching, pressed miRNAs between controls and heavy drinkers: 37 were and hypermutation (60, 61); SH2B3, which regulates B cell de- upregulated (Fig. 7A), and 42 were downregulated (Fig. 7B). velopment (53); and sterile a-motif-domain Src homology- Importantly, 53 of these miRNAs have mRNA targets within our domain nuclear localization signals-1, an adapter protein in- data set. Heavy drinking led to the upregulation of 29 miRNAs volved in immune cell signaling (56). Reduced expression of known to regulate 25 target mRNAs that were downregulated in our KLF10, which suppresses regulatory T cells by upregulating data set. A subset of these mRNA–miRNA pairs is shown in Fig. 7C; TGF-b (62), could further explain the reduction in T and B cell for a complete list, please refer to Table I. Some of the downregu- responses. lated mRNAs were targeted by several differentially expressed Our study also revealed insights into the mechanisms by which miRNAs. For instance, miR-16, miR-15b, miR-195, and miR-374b moderate alcohol consumption stimulates immunity. Animals that target VEGFA, which was downregulated .5-fold. Similarly, miR- drank moderate amounts of ethanol showed increased expression 30b and miR-30c target SH2B3, which was downregulated 2-fold, of chemokines CCL3 and CCL4L1, which signal through CCR5 to whereas miR-125a and miR-125b both target SCARB1, which was recruit memory T cells (92), and IL-1a and CXCL3, which recruit downregulated .3-fold. Of the 24 miRNAs downregulated with neutrophils. Although a vigorous innate immune response is Downloaded from heavy drinking, 7 were associated with an increase in their mRNA critical, it is equally important for the host to minimize damaging targets (Fig. 7C, Table I). For example, the downregulated miR-101 inflammation. PTX3, which plays a role in the resolution of in- targets GJA1, which was upregulated 11-fold with heavy drinking. flammation (94), and the NF-kB inhibitor, NF of k light poly- miR-144 targets AXIN2, which was upregulated 2-fold. miR-183 peptide gene enhancer in B-cells inhibitor-a, were significantly and miR-202 both target ROBO2, which was upregulated .3-fold. upregulated with moderate drinking. These observations are in

Finally, miR-29a, miR-29b, and miR-29c all target SH3PXD2A, line with previous studies that showed that moderate alcohol http://www.jimmunol.org/ which was upregulated 4-fold in our data set. consumption in humans significantly alters genes involved in B cell, T cell, and IL-15 signaling pathways, as well as attenuates NF-kB signaling pathways in leukocytes (111). Discussion Several of the genes that were differentially expressed with Using a macaque model of ethanol self-administration, we re- ethanol consumption were previously described as being impor- cently showed that heavy ethanol consumption suppresses, whereas tant for mounting immune responses to vaccination. For instance, moderate ethanol consumption enhances, T and B cell responses to studies that investigated yellow fever vaccine (YF-17D)-induced MVA (15). The goal of this study was to uncover the molecular signatures in blood of healthy adults reported significant in- mechanisms underlying this dose-dependent effect. We used RNA- creases in the expression of proinflammatory mediators CXCL10 by guest on September 23, 2021 Seq to identify changes in gene expression on day 7 following and IL-1a and complement gene C3AR1, which were found to be vaccination with MVA, the earliest time point at which we de- predictive of robust vaccine responses (112). Another study tected differences in Ab and T cell responses (15). We also se- identified ETS2 as an additional key regulator of the early innate quenced small RNA molecules to gain insight into mechanisms immune response to YF-17D (113). In our study, we observed a underlying the changes in gene regulation. Overall, our study 17-fold downregulation of CXCL10, a 3-fold downregulation of revealed robust changes in gene and miRNA expression among C3AR1, and a 2.5-fold downregulation of ETS2 with excessive controls and moderate and heavy drinkers, with fewer changes ethanol consumption, which could explain the suppression of between moderate drinkers and controls compared with either vaccine responses in this cohort. In contrast, we detected a 23-fold group versus heavy drinking status. upregulation of IL-1a in moderate drinkers compared with con- Thechangesingeneexpressionreportedinthisarticleprovide trols, reinforcing the association of this marker with successful novel insights into the reduced immune response to vaccination immune responses. and the increased vulnerability to infection seen in humans with Our gene-expression analysis is also in line with clinical ob- AUD. Specifically, we detected large decreases in the expression of servations linking alcohol abuse with impaired wound healing microbial sensors that are critical in the detection of bacterial (114), increased susceptibility to wound infections (115), and peptides [FPR2/3 (105)], LPS (MD2, CD14, TLR4), bacterial delay of wound closure (116). These defects have significant flagellin (TLR5), and certain helminths and filoviruses [CLEC10A clinical ramifications because half of emergency room trauma (48)]. There was also decreased expression of genes important for cases involve alcohol exposure (117). Previous studies showed Ag presentation [ATP-binding cassette subfamily-C-2/3, that ethanol exposure at the time of traumatic injury impairs CLEC10A (48, 49)], recruitment of immune cells [C3AR1, wound closure via decreased proinflammatory cytokine release, complement component 5aR receptor-1, CCR2, CXCL3, CXCL8, neutrophil recruitment, and phagocytic function (114). Our CXCL10, CCL3, CCL4L1/2, ICAM1 (30, 92, 106–108)], and gene-expression data support and extend these earlier observa- soluble mediators that play a role in response to infection [TNF-a, tions. We detected significantly decreased expression of multiple TNFRSF1A, IFN-g, IFNGR1, IL-1R, IL-1R1, IL-1RL1, IL-1b, components of the innate immune system that play a critical role S100 calcium binding protein-8/9, LL37, eosinophil peroxidase, in the prevention of wound infections, including pattern recogni- and granzyme A (45, 109)]. As previously described (110), ex- tion receptors (FPR2/3, NALP3, NOD2, MD2, CD14, TLR4, pression of IFN signaling IFN receptor-2 was downregulated in TLR5, TLR8, CLEC10A, SCARB1), proinflammatory cyto- heavy drinkers, which would contribute to deficits in both innate kines and their receptors (TNFRSF1A, IFN-g, IFNgg1, IL-1R, and adaptive immunity. IL-1R1, IL-1RL1, IL-1b, IL-13Ra-1), and chemokines and their We also found decreased expression of lymphocyte activation receptors (CXCL2, CXCL8, CXCL10, CCR1, CCR2, OSM, markers, including CD83 and killer cell lectin-like receptor CSF3R, and CSF1). The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ FIGURE 7. Heavy ethanol consumption changes expres- sion of several miRNAs. Heat maps of the upregulated (A) and downregulated (B) miRNAs with heavy drinking (H7) compared with controls (C7). (C) Network of a subset of the differentially expressed miRNAs and their mRNA targets that were both differentially expressed in our study. by guest on September 23, 2021

Earlier studies suggested that the disruption of VEGF signaling associates with the VEGF receptor to promote angiogenesis and and reduced expression of hypoxia-inducible factor-1a in endo- wound repair (118). We also report decreased expression of thelial cells with chronic alcohol consumption interferes with CELSR1 and SDC2, which were shown to promote effective wound closure (116). Our gene-expression data also show a sig- wound repair (26, 119), as well as secreted proteins, such as fi- nificant decrease in the expression of both of those genes with bronectin and MMP-1, which promote platelet aggregation, an- excessive ethanol consumption. Moreover, we detected fewer giogenesis, and tissue remodeling (120, 121). The expression of transcripts of NRP1, which is expressed by endothelial cells and additional growth factors, HGF and HB-EGF, which promote 10 IMPACT OF ALCOHOL INTAKE ON GENE EXPRESSION

Table I. mRNA–miRNA pairs in our data set naling molecules (IL-1R, IL-1b, CXCL8, CD14, CD163, G-CSF, TNFR1, TLR5, ALOX15, ADAM12), transporters (SLC11A1/ Differentially Differentially 16A1), and growth factors (VEGF, HBEGF, HGF). AHR and Expressed Expressed miRNAs mRNA Targets RORg play a role in suppressing lung inflammation (34, 129), whereas decreased expression is associated with hypoxia- Downregulated Upregulated induced lung disease (130). In addition, transcripts associated miR-144 AXIN2 miR-203 AFAP1L2, ROBO2 with immune genes that promote host defense against pulmonary miR-183 ROBO2 infection (ALOX15, CD14, G-CSF, TREM1, TLR5, NRAMP1, miR-29c SH3PXD2A TNFR1) were reduced with heavy drinking (131–134). Critical miR-29a SH3PXD2A growth factors important for repairing lung injury were also miR-101 GJA1 downregulated. Decreased levels of VEGF correlate with loss of miR-29b SH3PXD2A alveolar structure in emphysema patients (33) and a compro- Upregulated Downregulated miR-26a HGF, PTGS2, NTN4, RP2, RAB3IP, mised integrity of the alveolar–capillary barrier (135). Finally, CHAC1, PFKB3, DAPK1 HGF is important for lung development and promotes regener- miR-25 GRHL1, KLF4, FAM20C ation after lung injury in animals (27). miR-142-5p GAS7, BVES, ATP13A3, PRRG4, RTN1, Chronic alcohol consumption is associated with an increased risk LPP, PRDM8, NRG1 for cardiovascular disease (136) and stroke (137). Our analysis miR-374b GAS7, VEGFA, ATXN1, DUSP6, DUSP8 miR-125b SCARB1, CCR2, FAM129B, C19orf39 revealed increased expression of genes implicated in disease, miR-410 RORA, SMAD7, RAPGEF2, SASH1, including CLDN5, VWF, phosphodiesterase-6H, ITGA2B, and Downloaded from SNAI1, PTX3 carbonic anhydrase III (79, 81, 82, 138, 139), as well as megakar- miR-485-3p BAIAP2 yocyte differentiation [RGS18, growth factor-independent 1B miR-16 SMAD7, TNFSF13B, VEGFA, ATP13A3, RASGEF1B transcription repressor, and rho guanine nucleotide exchange factor- miR-425 FSCN1 4 (74, 75)]. ITGA2B is used as a biomarker for myocardial in- miR-30c SH2B3, SNAI1, RUNX2, HLX, EAF1, farction risk, and therapeutic modulation of phosphodiesterases is

RRAD one strategy for treating cardiovascular disease. Interestingly, http://www.jimmunol.org/ miR-106a LIMA1, NTN4, OSM, WFS1, EGR2, EREG, claudin-5 levels are reduced in human and mice models of car- DOCK4, FAM129A, RORC, IL8 miR-342-3p ZAK, NEURL1B diomyopathy; therefore, its increased expression in this study might miR-494 IRAK3, ZFHX3 be a compensatory mechanism. miR-125a-5p SCARB1, CCR2, FAM129B, C19orf38 Finally, heavy alcohol use is a major risk factor for , head miR-148a NRP1, B4GALT5, SESTD1 and neck, and colorectal cancers (140–142). Our gene-expression miR-15b SMAD7, TNFSF13B, VEGFA, ATP13A3, RASGEF1B, AATK analysis showed increased expression of several genes that pro- miR-221 BMF, NRG1 mote cancer progression with chronic heavy alcohol consumption miR-365 KCNQ1 (latent TGF-b–binding protein-2/3, IRS1, SFRP5, LEF1, DLK1, miR-454 ZAK, ATXN1, RTN1, ADAM12, ACSL1, stromal Ag-3, and H2B). Higher levels of IRS1 are found in he- by guest on September 23, 2021 WDFY3, MB21D2, TMEM170B patocellular carcinoma and breast, ovarian, and colorectal cancers miR-195 SMAD7, TNFSF13B, VEGFA, ATP13A3, RASGEF1B, AATK (143–146). Increased expression of LEF1 is associated with hu- miR-329 ATXN1 man endometrial tumors and prostate and colon cancer (147–149). miR-34a CLEC10A, REPS2 DLK1 is also expressed at higher levels in colon adenocarci- miR-30b SH2B3, SNAI1, RUNX2, HLX, EAF1, nomas, pancreatic islet carcinomas, and small cell lung carcino- RRAD miR-194 HBEGF, THBS1, ZFHX3 mas (86). In contrast, moderate alcohol consumption is associated miR-223 OLFM1, SLC8A1 with a reduced risk for developing cancer (150), Hodgkin’s lymphoma (151), and thyroid cancer (152). Our study revealed Downregulated miRNAs that target upregulated mRNAs and the upregulated miRNAs that target downregulated mRNAs in our data sets. that moderate drinking repressed genes associated with reduced cancer incidence, including transmembrane protein-98 (101), serine-protease temperature requirement-A1 (100), DNA nucleo- angiogenesis and tissue regeneration, was also significantly de- tidylexotransferase (98), ten-eleven translocation-1 (99), and creased (27). Furthermore, heavy alcohol abuse increased ex- MMP9 (102). pression of additional genes known to interfere with wound We also investigated differences in miRNA expression levels healing [PSEL, VWF, CX43, IL-17 (77, 78, 122)]. among the three experimental groups. miRNAs can modulate gene Additionally, our data are in line with clinical observations that expression through translational repression or degradation of target heavy alcohol consumption is associated with increased incidence mRNAs and play a critical role in regulating immune function of chronic obstructive pulmonary disease (123, 124), lung injury in (153). We identified several differentially expressed miRNAs with response to inflammatory insults (125), acute respiratory distress validated mRNA targets present in our RNA-Seq data set. Inter- syndrome (126), and risk for mortality in acute lung injury patients estingly, and as described for mRNA, several of these upregulated (127). Impaired immunity and increased oxidative stress, both miRNAs have also been implicated in the development and pro- consequences of AUD, are considered risk factors for chronic gression of cancer. For instance, miR-494 was shown to be up- obstructive pulmonary disease and acute respiratory distress syn- regulated in hepatocellular carcinomas and promote proliferation drome (128). Our transcriptome analysis provides new insights in tumor cells (154), whereas miR-106a is upregulated in gastric, into the mechanisms that underlie increased susceptibility and colorectal, and pancreatic cancers in humans (155). As described severity of lung injury and chronic lung inflammatory diseases. previously in human hepatocytes and cholangiocytes treated with Heavy ethanol consumption was associated with downregulation ethanol (156), miR-34a was upregulated .5-fold in our data set. of several genes important for maintaining lung homeostasis that Furthermore, ethanol-induced hypomethylation of the miR-34a can be categorized as transcription factors (AHR, P21, RORg, , which results in increased expression of this miRNA, ATF/CREB), receptors (TREM1, FCgR, NOR1), immune sig- plays a role in the development of alcoholic liver disease (156). The Journal of Immunology 11

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