Alopecia areata (AA) is a highly prevalent autoimmune disease (AD) leading to hair loss in affected individuals, in which both genetic and environmental factors likely play a role. Associations between changes in the microbiome composition and many ADs have been reported, however, a characterization of the role of gut microbiota in the development of AA has not been undertaken. To investigate the functional relevance of gut microbiota to AA development in vivo, we depleted the gut microbiome in the C3H/HeJ mouse model of AA with broad-spectrum antibiotics. We found that antibiotic-treated mice were protected from AA induction. Additionally, using 16S rRNA gene sequencing (16SrRNA Seq) we found that AA-affected mice show an overrepresentation of a single species of Ligilactobacillus, Ligilactobacillus murinus (L. murinus), that preceded the development of the first clinical signs of AA. Further, using L. murinus specific primers, we showed that co-housed C3H/HeJ mice protected from AA show reduced levels of L. murinus in comparison to mice affected with AA, suggesting that L. murinus is associated with the development of AA. Examining the effects of the microbiome on T cell composition, we found that gut microbiota-depleted mice had a decreased percentage of effector CD8+ T cells in skin draining lymph nodes, which we previously showed to be required to drive the pathogenesis of AA. To establish the relevance of these observations to human AA, we performed 16SrRNA Seq sequencing and metagenomics and compared gut microbiome composition in stool samples between AA patients and healthy controls (HCs). We discovered a striking gut dysbiosis in AA patients compared to HCs and identified species of Lachnospiraceae and Bacteroidaceae to be overrepresented in AA. Taken together, these findings suggest that gut microbiome dysbiosis contributes to the pathogenesis of AA and offer new avenues of treatment for this disease.

Primary cicatricial alopecia (PCA), often referred as scarring alopecia, is a group of disorders that result in permanent hair loss, and for which no FDA approved treatments exist. Common forms of PCA include Lichen Planopilaris (LPP), Frontal Fibrosing Alopecia (FFA) and Central Centrifugal Cicatricial Alopecia (CCCA), with LPP predominating in middle aged white women, FFA in the postmenopausal group, and CCCA in women of African descent. FFA is considered to be a variant of LPP, and CCCA shares several pathological features with advanced LPP, however, whether PCAs are separate diseases or share common pathomechanisms remains an unresolved question. To determine if PCAs are molecularly distinct, we performed RNAseq of scalp biopsies from 28 LPP, 30 FFA, and 9 CCCA patients compared to 12 normal controls. This revealed a core set of dysregulated pathways shared among all PCA subtypes: 1) downregulated cholesterogenic genes (CYP51A1, NSDHL), 2) upregulated fibrosis and scarring genes (COL1A1, BMP10, CLDN5), and 3) a striking enrichment of mast cell genes (TPSAB1, MS4A2, CMA1). Notably, positive staining for mast cells (MC tryptase) was detected near the sebaceous gland of PCAs and not in controls. We also found that each subtype was associated with unique pathways, such as JAK/STAT signaling in CCCA, indicating potential molecular signatures within each subtype. To investigate the clinical significance of mast cells in PCAs, we treated 37 patients with the oral mast cell stabilizer drug, cromolyn. Five patients reported improvement within 6 weeks and 3 continued to report transient improvement of symptoms after 3 months. While clinical improvement was transient, optimization in formulation and delivery may improve durability, and many additional drugs to target mast cells are available for investigation. Our findings revealed common gene expression pathways among PCAs, and demonstrated targeting of mast cells as a novel treatment strategy.

Alopecia Areata (AA) is a highly prevalent autoimmune disease leading to hair loss in affected individuals. We and others have demonstrated a strong genetic component in the development of AA, however, emerging evidence suggests that environmental factors clearly contribute to AA pathogenesis. To determine the role of the microbiome as an environmental factor in AA, we performed 16S sequencing of skin, hair follicle (HF), and feces from 34 AA patients and 12 healthy controls (HCs). We found no significant differences in the composition of the skin or HF microbiome, but we discovered striking gut dysbiosis in AA patients compared with HCs. We detected a pronounced dysbiosis characterized by an increased relative abundance of members of the Firmicutes phylum and decreased relative abundance of the Bacteroides phylum in the gut microbiome of AA patients compared with HCs. Utilizing metagenomics sequencing, we identified perturbations in the relative abundance of different microorganisms of the Firmicutes and Bacteroides phyla, such as Ruminococcus sp., Alistipes shahii, and some lactobacillales in the gut microbiome of AA patients. To investigate the causal role of the gut microbiome in AA development, we conducted antibiotic-mediated depletion of the gut microbiome in C3H/HeJ mice, and found that treated mice were largely protected from hair loss, concomitant with decreased numbers of CD8+ T cells and an increase in the Treg/CD8+ ratio. Taken together with recent reports of reversal of AA in patients following fecal microbiota transplant (FMT), our findings suggest that restoring homeostasis of the gut microbiome may represent a new therapeutic approach for AA. We recently initiated a clinical trial of FMT for treatment of AA, in which we will monitor the normalization of gut microbiome dysbiosis relative to the restoration of hair growth.

Alopecia Areata (AA), one of the most prevalent autoimmune diseases with a lifetime risk of 2.1%, is a complex genetic disease characterized by autoimmune-directed hair loss of the scalp and body. In AA, pigmented hairs are preferentially attacked, suggesting a role for hair follicle (HF) melanocytes, as cellular targets harboring AA antigens. Our previous genome-wide association study (GWAS) and meta-analysis identified an AA-risk locus that contains Syntaxin17 (STX17), an autophagy-related gene with a reported role in hair pigmentation. To define the role of STX17 variants in AA, we performed targeted genomic sequencing across the 550 kb GWAS locus containing STX17 in 849 AA patients. We defined a risk haplotype that carried the GWAS risk allele together with 34 additional variants associated with AA. Using in silico and multi-dimensional patient data, we discovered that 33 of these variants were eQTLs associated with decreased expression of STX17 in AA scalp skin. Additionally, AA HFs revealed dysregulated expression of STX17 proximal to dysmorphic follicular melanocytes. We identified an autophagy-independent role for STX17 in melanogenesis, and found that knockdown of STX17 resulted in accumulation of the melanocyte antigen, MART1. In this study, we used functional genomics to identify functional variants on a common AA risk haplotype that significantly downregulated skin expression in AA patients, and found that reduced levels of STX17 in melanocytes resulted in increased antigen expression, our findings provide a mechanistic link between genetic susceptibility to AA and the preferential attack of pigmented hair follicles in disease.

Alopecia Areata (AA) is a complex genetic disease, in which we previously identified common variation at 14 loci contributing to disease risk using GWAS. However, rare variants with moderate effect size are becoming increasingly important determinants in the genetic landscape of complex diseases. In order to identify novel rare variants contribute to AA risk burden, we performed whole exome sequencing (WES) on 849 AA patients compared to 15,640 controls using an unbiased genome-wide approach. Gene-level burden analyses (genome wide signficance, p=2E-7) identified a novel gene, KRT82, as the gene containing the most variants in AA patients compared to controls. The allelic series of KRT82 variants included 2 nonsense mutations (18 AA patients), 1 frameshift (1 patient), 7 missense (28 patients), and 1 splicing mutation (4 patients), for a total of 51 out of 849 AA patients carrying rare damaging heterozygous mutations in KRT82 (6.01%). The most common variant among our AA cohort (15 AA patients; 1.77%) was a nonsense mutation at arginine position 47 (R47X), a hotspot mutation that arose independently on several haplotypes, likely via spontaneous deamination of C to T. Interestingly, KRT82 is a type II hair keratin that is exclusively expressed in the hair shaft cuticle during anagen phase, when AA attack on the HF occurs. Using gene expression analysis and immunofluorescence, we found that AA patient scalp exhibits decreased expression of KRT82 in the skin and HF. Loss of KRT82 in the hair shaft cuticle may prevent dimerization of KRT82 with its potential partners (KRT32/39/40), and previous SEM studies showed morphological defects in the hair shaft cuticle in AA patients. Our WES data identified rare pathogenic variants in KRT82 as a novel mechanism implicating loss of structural integrity of the hair shaft in the pathogenesis of AA.

The presence of commensal and potential pathogenic microorganisms in the gastrointestinal system requires a specialized epithelial barrier function to block the entry of microbes, antigens and toxins into the gut, while allowing the absorption of nutrients. Loss of integrity of the intestinal epithelium plays a key pathogenic role in autoimmune diseases and leads to the "leaky gut" phenotype. However, this may be disrupted by different factors including microbiota dysbiosis, or reactions to microbes in the intestinal defense mechanisms, resulting in a leaky gut, capable of promoting local and systemic immune responses. A leaky gut has been associated with multiple autoimmune diseases as inflammatory bowel disease, celiac disease, and type 1 diabetes. Recently, we identified the gut microbiome as a potential trigger for the development of Alopecia Areata (AA). To investigate this finding, we induced a leaky gut in mice of AA using Dextran Sulfate Sodium (DSS), and observed a depletion of TJs Occludin, ZO-1, and Claudin-1 in large and small intestine of C3H and C57/B6 mice. We measured the amount of fluorescent marker (FITC-Dextran) in the sera to determine permeability of the gut, and found that C57/B6 mice with an experimentally induced leaky gut had an approximately 2.59-fold increase in gut leakiness compared to control mouse sera. Recent studies have shown that the normal microbiota may be mediated in part by oxidative stress or Reactive Oxygen Species (ROS)-dependent mechanisms. One candidate susceptibility gene in AA is peroxiredoxin 5 (PRDX5), which is also a candidate gene in Crohn's Disease and Psoriasis. We found decreased expression of PRDX5 in mouse intestine treated with DSS to induce intestinal damage. Our findings invite further investigation into dysbiosis-induced disruption of the gut epithelial barrier function and the systemic immune response as potential upstream triggers of AA.

Alopecia Areata (AA) is one of the most prevalent autoimmune disorders in humans leading to patchy or total loss hair with about a 2% lifetime prevalence. AA has a significant impact on patients’ quality of life, and associations with other autoimmune diseases. The development of AA is influenced by genetic, immunological, and environmental factors, though these are not completely defined. The gut microbiome has an immunomodulatory effect capable of eliciting pathologic immune responses beyond the gut. Our recent studies in the C3H/HeJ mouse model of AA showed that oral broad spectrum antibiotics prevented onset of AA, suggesting the gut microbiota is required for AA onset. Thus, to determine the microbiome composition of patients with AA, we collected skin swabs, hair follicle samples, and stool samples from a cohort of 26 AA patients. Analysis of 16S rRNA sequencing on stool samples revealed significant differential representation of bacterial taxa, between AA patients and healthy subject specifically, members of the firmicutes and bacteroides phyla, similar to our mouse model findings. When AA patients were compared to healthy controls we found under representation the bacteroides phyla and over representation of the firmicutes phyla in AA gut microbiome, similar to changes reported in other autoimmune disorders. Importantly, there was no difference in the skin or hair follicle microbiome in AA patients as compared to healthy controls, underscoring the importance of gut microbiota dysbiosis in AA patients. The presence of gut microbiota dysbiosis in human AA patients provides a rationale for the development of novel therapeutic strategies for AA patients, including Fecal Microbiota Transfer (FMT) and targeted microbial therapy, since restoring the gut microbiota composition to a healthy state has been suggested as an approach to improve the course of autoimmune diseases, such as AA.

We previously published a genome-wide association study (GWAS) and meta-analysis to search for common alleles that contribute to risk of AA, and identified several genomic regions harboring potential susceptibility genes. One candidate susceptibility gene expressed in the hair follicle (HF) in AA is peroxiredoxin 5 (PRDX5) (p= of 8.7*10-14), which is also a GWAS gene in Crohn’s disease, sarcoidosis, and psoriasis. PRDX5 is a member of the family of antioxidant enzymes that are crucial for regulating oxidative stress. Our lab has completed sequencing of 897 whole exomes with a custom capture region of genomic sequencing. Using a chi-square or ‘goodness of fit’ test of variant enrichment we identified variants that are significant in both our GWAS and exome studies, thus likely candidate causal variants. Using Bayesian fine mapping, we found a GWAS and exome significant variant, rs574087, is expected to be a causal variant in keratinocytes and melanocytes (with a posterior inclusion probability index or PIP score greater than 0.1, lending to high likelihood of causality). To functionally validate our in silico studies, we immunostained healthy human HF and AA affected HF and found PRDX5 is upregulated AA human HF. PRDX5 is expressed in cultured melanocytes by immunostaining, which also was expected since melanocytes are known to have high levels of oxidative stress. We postulate that PRDX5 is crucial for protection from oxidative stress and its dysregulation can ultimately lead to autoimmunity. Our findings establish a connection between PRDX5 and causal variants, which provides a functional framework for further fine mapping define the role of PRDX5 in AA disease pathogenesis.

Research into the genetic architecture of chronic disease has unequivocally demonstrated etiological contributions from both common (polymorphisms; SNPs) and rare (mutations) genetic variants. For alopecia areata, our GWAS successfully identified SNPs that increase disease risk, however, mutations remain to be identified. While exome sequencing permits genome-wide investigation of disease mutations, challenges arise because human genomes are riddled with mutations, most of which exert no effect on health, and because low allele frequencies limit power to detect associations. Therefore, new methods are needed to identify causal mutations in chronic disease. Here, we developed a novel analytic pipeline to identify mutations that contribute to alopecia areata etiology. First, genome-wide tagSNPs were used to perform linkage analysis and family-based association tests, which identified 20 cosegregating regions (4>LOD>1) that contain 373 associated tagSNPs, capturing variation in 6,688 SNPs and implicating 178 genes. Next, we performed exome sequencing on a subset of probands and extracted co-segregating mutations that alter protein sequence in these 178 genes, reducing the number of candidate genes to 58. Pathway analysis of these 58 genes indicated that 12 genes from 5 genomic regions contribute to extracellular matrix (ECM) structure, organization and/or signaling (0.016

The gut microbiome has emerged as a potential immunomodulatory factor capable of eliciting pathologic immune responses beyond the gut. Here, we studied the composition of the gut microbiome and its role in alopecia areata (AA) development in the C3H/HeJ mouse model. 8-wk old female C3H/HeJ mice were grafted with skin from AA mice and treated with a broad-spectrum antibiotic cocktail (ampicillin, metronidazole, neomycin and vancomycin), and followed for development of AA. We also tested antibiotic pretreatment in young animals (3wk old) prior to grafting. Strikingly, antibiotic treatment resulted in significant protection from hair loss. To determine the characteristics of the microbiome associated with the development of AA in the C3H mouse model, DNA was extracted from skin swabs and fecal samples and the 16S-rRNA was sequenced. Microbial diversity in the gut was significantly reduced in antibiotic treated mice, but no changes in the skin microbiome were observed. Principal component analysis of the gut microbiome showed distinct pretreatment, untreated and treated clusters, with dysbiosis characterized by over representation of Lactobacillus and under representation of Bacteroides in AA animals, similar to our findings in human AA. In C3H mice that spontaneously developed AA, the levels of butyric acid were significantly reduced when compared with control mice. Flow cytometry analysis of gut- and skin-draining lymph nodes showed a population of pathogenic T cells in the gut and a decrease in these cells infiltrating the skin in antibiotic treated animals. AMNV treated mice showed an increase in the Treg/CD8+ ratio and a decrease in CD4+ T cells in the spleen, as well as reduced numbers of CD4 and CD8 cells in the Peyer's patches. Together, these data indicate a potential role of the gut microbiome in T cell priming in AA and offer new therapeutic opportunities in AA focused on restoring gut microbial homeostasis.

In human skin, Langerhans cells (LC) constitutively express high levels of CD1a, an antigen presenting molecule specifically equipped to present lipid antigens to T cells. The abundance of CD1a in normal skin supports a role for lipid antigen presentation in skin immunity and homeostasis, yet the exact physiological functions of lipid-specific T cells remain unclear. Previously, we described the presence of CD1a-autoreactive T cells in normal skin, and identified several self-lipids that can function as antigens for these T cells. More recent studies have suggested a role for CD1a in the pathogenesis of inflammatory skin diseases. Whereas we previously focused on self-lipid antigens, here we investigated if CD1a-restricted T cells also recognize foreign bacterial lipids found in common skin pathogens, including S.aureus. For this, we developed fluorescently-labeled CD1a tetramers loaded with a candidate bacterial phospholipid (BPL), as well as unloaded control CD1a tetramers. Preliminary data revealed that BPL-loaded CD1a tetramers, but not unloaded tetramers, stained a population of Vδ1 T cells within polyclonal skin T cells. BPL-loaded CD1a tetramers also identified a low frequency T cell population in peripheral blood of multiple individuals. These T cells expressed αβ rather than γδ T cell receptors, indicating that, despite similar specificities, the skin and blood CD1a-restricted T cells belong to different T cell subsets, and potentially have distinct roles. We successfully purified and expanded tetramer+ T cell populations for gene expression analysis, and for investigating the functions of these T cells in the context of infections, by analyzing their response to S.aureus-treated LC. Given the association between atopic dermatitis (AD) and S.aureus skin colonization, follow up studies include phenotypic and functional analysis of CD1a-BPL specific T cells in the skin and blood of AD patients and controls.

Alopecia areata (AA) is an autoimmune disease that causes hair loss by T-cell autoimmune reaction against the hair follicles. We previously published a GWAS and meta-analysis to search for common alleles contributing to AA risk and identified 14 genomic regions harboring potential susceptibility genes. In this study, we conducted targeted resequencing on several GWAS regions in 122 cases and focused on the functionally relevant IL2RA locus. Interleukin-2 (IL2) is involved in T-cell proliferation and survival making it essential for maintaining immune tolerance. Various IL2RA haplotypes have been shown to regulate IL2 Receptor expression on CD4+ T cells inducing Tregs. Several polymorphisms surrounding the IL2RA locus have been reported to be associated with type 1 diabetes (T1D), rheumatoid arthritis, vitiligo, and AA. Haplotype-dependent gene expression can confer either risk or protection from autoimmunity. For example, rs4147359 (haplotype07) contains an A risk allele in AA and T1D. We identified novel variants in the IL2RA region that show rare enrichment in AA and were replicated in our whole exome sequencing, thus prioritizing these variants as candidates for functional studies. We discovered 458 variants, 65 of these we classified as rare enriched defined as a variant present in less than 1% of population in population databases and in 3 or more patients in our cohort. These rare enriched variants fall mainly within introns, intergenically or downstream of IL2RA. Using an algorithm (FUN-LDA) that predicts functional effects of certain non-coding genetic variants in tissue types, we localized the majority of these 65 rare enriched variants in the context of cell types and found they mostly fall in the immune cell cluster. Overall, this analytical pipeline helps discover and place rare enriched disease variants in the context of specific cell types for future functional studies.

Sudden whitening of the hair is a clinical observation in Alopecia Areata (AA) that is known as 'canities subita', a phenomenon in which scalp hair appears to turn completely white due to the rapid and preferential attack of pigmented hair follicles (HF). This observation led to the hypothesis that HF melanocyte-specific antigens play a key role in AA disease onset. Recently, essential autophagy proteins have been found to have pleiotropic roles in the regulation of melanin production and melanosome formation in the melanogenesis pathway. Interestingly, our previous and meta-analysis (2489 cases and 5297 controls) uncovered two AA risk genes, STX17 (P=3.6x 10-7) and BIM (P=1.5x10-8), which are known to play a role in autophagy. STX17 is involved in hair pigmentation of gray horses, in which an intronic mutation was identified as the cause of the premature loss of pigmentation. We found that STX17 exhibits a 1.5-fold reduction in expression in AA patient skin compared to unaffected controls. Moreover, we identified the most significantly associated SNP in the STX17 region, rs10760706, as a potential eQTL due to the finding that individuals with risk allele C expressed significantly reduced levels of STX17 (P=0.0152). We found that STX17 is expressed in hair follicle melanocytes and that STX17 subcellular localization changes in response to melanogenesis stimulation as STX17 appears to migrate distally towards the dendritic tips. Knockdown of STX17 led to inhibition of αMSH-stimulated melanin production and increased expression of MART1 and Tyrosinase, two antigens capable of eliciting T-cell responses in human patients with vitiligo and AA. Our findings suggest that STX17 plays a role in melanogenesis and that disruption of this pathway may serve as a catalyst for AA pathogenesis due to subsequent upregulation of antigens capable of initiating the autoimmune attack on the HF.

Our lab previously published a genome-wide association study (GWAS) and meta-analysis to search for common alleles that contribute to risk of Alopecia Areata (AA), and identified genomic regions harboring potential susceptibility genes. One candidate susceptibility gene expressed in the hair follicle (HF) in AA is peroxiredoxin 5 (PRDX5) (p= 8.7*10-14), which is also a susceptibility gene in Crohn’s disease, sarcoidosis, and psoriasis. PRDX5 is a member of the peroxiredoxin family of antioxidant enzymes crucial for regulating oxidative stress, which causes disruption in redox potentials that extend to the endoplasmic reticulum (ER), causing accumulation of misfolded proteins. The accumulation of unfolded proteins in the ER lumen is sufficient to produce reactive oxygen species (ROS), which in turn activate the unfolded protein response (UPR) and lead to apoptosis. In Crohn’s, induction of the UPR has been shown to play a key role in disease pathogenesis in the intestinal epithelium. We postulate that dysregulation of PRDX5 may induce the UPR response in HF epithelium and lead to upregulation of danger signals, like NKG2D ligands. To test this hypothesis, human HF in organ culture were treated with tunicamycin to induce UPR response and using qPCR we measured the induction of all 8 NKG2D ligands from mRNA isolated from stressed human HF. We found a marked upregulation of ULBP 1, 4, and 6 in response to induction of the UPR in HF. This response was selective, since we observed very little change in MICA, MICB, and ULBP 2 and 3 in this assay. Our findings establish a connection between the UPR response and upregulation of NKG2D ligands on stressed HFs, providing a functional framework to further define the role of PRDX5 in AA disease pathogenesis.

During the onset of Alopecia Areata, a phenomenon can occur in which patients' hair suddenly becomes white (canities subita). It is hypothesized that this occurs as a result of pigmented hairs being preferentially targeted and attacked, leaving only white, non-pigmented hairs remaining. This lead to the longstanding hypothesis that hair follicle melanocytes play a role in the pathogenesis and progression of AA. Syntaxin17, an autophagasomal protein, was identified as an AA-associated locus in our initial GWAS (OR=1.33, P= 3.6x10-7) and has been shown to play a role in pigmentation. We have shown that Stx17 is expressed in the hair shaft cortex and inner root sheath of the hair follicle. Additionally, we found that Stx17 exhibits a 1.5-fold expression reduction in AT/AU patient scalp biopsies compared to unaffected controls. In horses, a mutation in the Stx17 gene has shown to cause a phenotype in which the horses prematurely gray as a result of hair pigmentation loss. This mutation resulted in a duplication of MITF-binding sites, which are crucial in regulating pigment cell-specific transcription of genes essential for melanogenesis. In addition to its role in the pigmentation process, Stx17 was recently defined as a SNARE protein involved in autophagy. Dysregulation of the autophagy process has been implicated in neurodegenerative diseases, tumorgenesis, and other autoimmune diseases, such as IBD, SLE, MS, and RA. Preliminary Stx17 exon sequencing in 113 patients did not reveal any causal variants; however whole genome sequencing of 500 patients is currently underway to identify possible regulatory variants contributing to the disease phenotype. Given the important role that the autophagy process plays in pigmentation and melanin production, as shown in vitiligo, we will define the roles of Stx17, autophagy, and pigmentation in AA pathogenesis and disease progression.

There is emerging evidence that host microbiomes, which have co-evolved with humans, play a significant role in human health. Recent research has highlighted a potential role for the gut microbiome in the pathogenesis of autoimmune diseases. The bacterial load and composition of gut microbiomes has been linked to susceptibility in rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, and type I diabetes. Here, we evaluated the role of the gut microbiome in host susceptibility to Alopecia Areata (AA), an autoimmune disease characterized by the infiltration of NKG2D+, CD8+ T cells into the hair follicle. The C3H/HeJ mouse model spontaneously develops AA, and these mice can be used to induce AA pathology in unaffected C3H/HeJ mice via skin grafting. Strikingly, we found that treating unaffected mice with an oral antibiotic cocktail prior to grafting completely prevented the develpment AA. In addition to the absence of alopecia, we noted a reduction in activated T cell infiltrating the skin, though the T cells were present in the blood and lymph nodes. To rule out contributions from the skin microbiome, we performed 16S sequencing of skin samples after 20 weeks of treatment. The overall bacterial load in the skin samples was not significantly altered and no clusters of bacterial Operational Taxonomic Units (OTUs) segregated the treated and untreated mice. These results indicate that the bacterial effectors contributing to the development of AA reside in the gut microbiome, and not in the skin. We are characterizing the minimum gut bacteria that mediate the differences in AA disease susceptibility using combinatorial antibiotic treatments and fecal transplants. The identification of pathogenic contributors in the gut microbiome will have significant implications on both our understanding of AA susceptibility, as well as actionable therapeutic targets for treatment.