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Plasmodium parasites caused 241 million cases of malaria and over 600,000 deaths in 2020. Both P. falciparum and P. ovale are endemic to Mali and cause malaria illness, with P. falciparum infections typically being more severe. Here, we sequenced RNA from nine pediatric blood samples collected during uncomplicated, symptomatic infections with either P. falciparum or P. ovale and characterized the host and parasite gene expression profiles. We found that human gene expression varies more between individuals than according to the parasite species causing the infection, while parasite gene expression profiles cluster by species. Additionally, we characterized DNA polymorphisms of the parasites directly from the RNA-seq reads and found comparable levels of genetic diversity in both species despite dramatic differences in prevalence. Our results provide unique insights into host-pathogen interactions during malaria infections and their variations according to the infecting Plasmodium species, which will be critical to develop better elimination strategies against all human Plasmodium parasites.Author SummaryMultiple species of Plasmodium parasites can cause human malaria. Most studies and elimination efforts target P. falciparum, the most common cause of malaria worldwide and the species responsible for the vast majority of the mortality. Other Plasmodium species, such as P. ovale, typically lead to less severe forms of the disease but little is known about the molecular mechanisms at play during malaria infections with different parasites. We analyzed host and parasite gene expression from children successively infected with P. ovale and P. falciparum and found that, while the parasite gene expression differed significantly, the transcriptional profiles of the host immune cells were similar in P. ovale or P. falciparum infections. This suggests that infected individuals respond to uncomplicated malaria infections similarly, regardless of the Plasmodium species causing the infection, and that alternative immune processes may become important during the progression to severe P. falciparum malaria (rather than being inherent features of P. falciparum infections). Additionally, we observed similar levels of genetic diversity among P. ovale and P. falciparum parasites, suggesting that the P. ovale population might be larger than currently thought, possibly due to extensive misdiagnosis or the existence of hidden reservoirs of parasites.

Plasmodium falciparumerythrocyte membrane protein-1s (PfEMP1s), diverse malaria proteins expressed on the infected erythrocyte surface, play an important role in pathogenesis, mediating adhesion to host vascular endothelium. Antibodies to particular non-CD36-binding PfEMP1s are associated with protection against severe disease. We hypothesized that given lifelongP. falciparumexposure, Malian adults would have broad PfEMP1 serorecognition and high seroreactivity levels during follow-up, particularly to non-CD36-binding PfEMP1s such as those that attach to endothelial protein C receptor (EPCR) and intercellular adhesion molecule-1 (ICAM-1). Using a protein microarray, we determined serologic responses to 166 reference PfEMP1 fragments during a dry and subsequent malaria transmission season in Malian adults. Malian adult sera had PfEMP1 serologic responses throughout the year, with decreased reactivity to a small subset of PfEMP1 fragments during the dry season and increases in reactivity to a different subset of PfEMP1 fragments during the subsequent peak malaria transmission season, especially for intracellular PfEMP1 domains. For some individuals, PfEMP1 serologic responses increased after the dry season, suggesting antigenic switching during asymptomatic infection. Adults were more likely to experience variable serorecognition of CD36-binding PfEMP1s than non-CD36-binding PfEMP1s that bind EPCR or ICAM-1, which remained serorecognized throughout the year. Sustained seroreactivity to non-CD36-binding PfEMP1s throughout adulthood amid seasonal fluctuation patterns may reflect underlying protective severe malaria immunity and merits further investigation.

Enzyme-linked immunosorbent assays (ELISAs) remain the gold standard for measuring antibodies, but are time-consuming and use significant amounts of precious sample and reagents. Protein microarrays represent an appealing alternative, particularly for studies focused on large gene families such as those encoding variant surface antigens in the malaria parasite Plasmodium falciparum. Such microarrays represent an ideal high-throughput platform to study antibody responses to hundreds of malaria parasite variant surface antigens at once, providing critical insights into the development of natural immunity to malaria. We describe the essential background and approach to run an assay using a P. falciparum microarray populated with variant surface antigens. This allows the user to define serologic profiles and identify serodominant antigens that represent promising targets for vaccine or therapeutic development.

Background Owing to the large amount of host DNA in clinical samples, generation of high-quality Plasmodium falciparum whole genome sequencing (WGS) data requires enrichment for parasite DNA. Enrichment is often achieved by leukocyte depletion of infected blood prior to storage. However, leukocyte depletion is difficult in low-resource settings and limits analysis to prospectively-collected samples. As a result, approaches such as selective whole genome amplification (sWGA) are being used to enrich for parasite DNA. However, sWGA has had limited success in generating reliable sequencing data from low parasitaemia samples. In this study, enzymatic digestion with MspJI prior to sWGA and whole genome sequencing was evaluated to determine whether this approach improved genome coverage compared to sWGA alone. The potential of sWGA to cause amplification bias in polyclonal infections was also examined. Methods DNA extracted from laboratory-created dried blood spots was treated with a modification-dependent restriction endonuclease, MspJI, and filtered via vacuum filtration. Samples were then selectively amplified using a previously reported sWGA protocol and subjected to WGS. Genome coverage statistics were compared between the optimized sWGA approach and the previously reported sWGA approach performed in parallel. Differential amplification by sWGA was assessed by comparing WGS data generated from lab-created mixtures of parasite isolates, from the same geographical region, generated with or without sWGA. Results MspJI digestion did not enrich for parasite DNA. Samples that underwent vacuum filtration (without MspJI digestion) prior to sWGA had the highest parasite DNA concentration and displayed greater genome coverage compared to MspJI + sWGA and sWGA alone, particularly for low parasitaemia samples. The optimized sWGA (filtration + sWGA) approach was successfully used to generate WGS data from 218 non-leukocyte depleted field samples from Malawi. Sequences from lab-created mixtures of parasites did not show evidence of differential amplification of parasite strains compared to directly sequenced samples. Conclusion This optimized sWGA approach is a reliable method to obtain WGS data from non-leukocyte depleted, low parasitaemia samples. The absence of amplification bias in data generated from mixtures of isolates from the same geographic region suggests that this approach can be appropriately used for molecular epidemiological studies.

var genes encode Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens. These highly diverse antigens are displayed on the surface of infected erythrocytes and play a critical role in immune evasion and sequestration of infected erythrocytes. Studies of var expression using non-leukocyte-depleted blood are challenging because of the predominance of host genetic material and lack of conserved var segments. Our goal was to enrich for parasite RNA, allowing de novo assembly of var genes and detection of expressed novel variants. We used two overall approaches: (i) enriching for total mRNA in the sequencing library preparations and (ii) enriching for parasite RNA with a custom capture array based on Roche’s SeqCap EZ enrichment system. The capture array was designed with probes based on the whole 3D7 reference genome and an additional >4,000 full-length var gene sequences from other P. falciparum strains. We tested each method on RNA samples from Malian children with severe or uncomplicated malaria infections. All reads mapping to the human genome were removed, the remaining reads were assembled de novo into transcripts, and from these, var-like transcripts were identified and annotated. The capture array produced the longest maximum length and largest numbers of var gene transcripts in each sample, particularly in samples with low parasitemia. Identifying the most-expressed var gene sequences in whole-blood clinical samples without the need for extensive processing or generating sample-specific reference genome data is critical for understanding the role of PfEMP1s in malaria pathogenesis. IMPORTANCE Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system. Identifying these expressed antigens is critical to understanding the development of severe malarial disease. However, clinical samples contain limited amounts of parasite genetic material, a challenge for sequencing efforts further compounded by the extreme diversity of the parasite surface antigens. We present a method that enriches for these antigen sequences in clinical samples using a custom capture array, requiring minimal processing in the field. While our results are focused on the malaria parasite Plasmodium falciparum, this approach has broad applicability to other highly diverse antigens from other parasites and pathogens such as those that cause giardiasis and leishmaniasis.

Background Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α “tag”). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or “parent” domain) coupled with the succeeding cysteine-rich interdomain region (CIDR). Methods A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season. Results Malian serologic responses to a PfEMP1’s DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure. Conclusions Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure. Electronic supplementary material The online version of this article (10.1186/s12936-019-2905-9) contains supplementary material, which is available to authorized users.

Objective:To evaluate the effect of the burden of Staphylococcus aureus colonization of nursing home residents on the risk of S. aureus transmission to healthcare worker (HCW) gowns and gloves.Design:Multicenter prospective cohort study.Setting and participants:Residents and HCWs from 13 community-based nursing homes in Maryland and Michigan.Methods:Residents were cultured for S. aureus at the anterior nares and perianal skin. The S. aureus burden was estimated by quantitative polymerase chain reaction detecting the nuc gene. HCWs wore gowns and gloves during usual care activities; gowns and gloves were swabbed and then cultured for the presence of S. aureus.Results:In total, 403 residents were enrolled; 169 were colonized with methicillin-resistant S. aureus (MRSA) or methicillin-sensitive S. aureus (MSSA) and comprised the study population; 232 were not colonized and thus were excluded from this analysis; and 2 were withdrawn prior to being swabbed. After multivariable analysis, perianal colonization with S. aureus conferred the greatest odds for transmission to HCW gowns and gloves, and the odds increased with increasing burden of colonization: adjusted odds ratio (aOR), 2.1 (95% CI, 1.3–3.5) for low-level colonization and aOR 5.2 (95% CI, 3.1–8.7) for high level colonization.Conclusions:Among nursing home patients colonized with S. aureus, the risk of transmission to HCW gowns and gloves was greater from those colonized with greater quantities of S. aureus on the perianal skin. Our findings inform future infection control practices for both MRSA and MSSA in nursing homes.

BackgroundPlasmodium falciparum(Pf) whole-organism sporozoite vaccines have provided excellent protection against controlled human malaria infection (CHMI) and naturally transmitted heterogeneous Pf in the field. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy (VE) have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions.MethodsWhole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generatede novogenome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequence polymorphisms and structural variants in each strain relative to the reference Pf 3D7 (a clone of NF54) genome. Strains were compared to each other and to clinical isolates from South America, Sub-Saharan Africa, and Southeast Asia.ResultsWhile few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains both genome-wide and within regulatory and immunologically important regions, including in pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contribute to diversity in immunologically important regions of the genomes as detected throughin silicoCD8+T cell epitope predictions. Of all heterologous strains, NF135.C10 consistently had the highest number of unique predicted epitope sequences when compared to NF54, while NF166.C8 had the lowest. Comparison to global clinical isolates revealed that these four strains are representative of their geographic region of origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently-formed drug resistant subpopulations present in the Greater Mekong Sub-region.ConclusionsThese results are assisting the interpretation of VE of whole-organism vaccines against homologous and heterologous CHMI, and may be useful in informing the choice of strains for inclusion in region-specific or multi-strain vaccines.

Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs., The repetitive interspersed family (RIFIN) and the subtelomeric variable open reading frame (STEVOR) family represent two of three major Plasmodium falciparum variant surface antigen families involved in malaria pathogenesis and immune evasion and are potential targets in the development of natural immunity. Protein and peptide microarrays populated with RIFINs and STEVORs associated with severe malaria vulnerability in Malian children were probed with adult and pediatric sera to identify epitopes that reflect malaria exposure. Adult sera recognized and reacted with greater intensity to all STEVOR proteins than pediatric sera did. Serorecognition of and seroreactivity to peptides within the semiconserved domain of STEVORs increased with age and seasonal malaria exposure, while serorecognition and seroreactivity increased for the semiconserved and second hypervariable domains of RIFINs only with age. Serologic responses to RIFIN and STEVOR peptides within the semiconserved domains may play a role in natural immunity to severe malaria. IMPORTANCE Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs.

Eosinophilic esophagitis (EoE) is a Th2 cytokine–associated disease characterized by eosinophil infiltration, epithelial cell hyperplasia, and tissue remodeling. Recent studies highlighted a major contribution for IL-13 in EoE pathogenesis. Paired Ig-like receptor B is a cell surface immune-inhibitory receptor that is expressed by eosinophils and postulated to regulate eosinophil development and migration. We report that Pirb is upregulated in the esophagus after inducible overexpression of IL-13 (CC10-Il13Tg mice) and is overexpressed by esophageal eosinophils. CC10-Il13Tg/Pirb−/− mice displayed increased esophageal eosinophilia and EoE pathology, including epithelial cell thickening, fibrosis, and angiogenesis, compared with CC10-Il13Tg/Pirb+/+ mice. Transcriptome analysis of primary Pirb+/+ and Pirb−/− esophageal eosinophils revealed increased expression of transcripts associated with promoting tissue remodeling in Pirb−/− eosinophils, including profibrotic genes, genes promoting epithelial-to-mesenchymal transition, and genes associated with epithelial growth. These data identify paired Ig-like receptor B as a molecular checkpoint in IL-13–induced eosinophil accumulation and activation, which may serve as a novel target for future therapy in EoE.

Eosinophilic esophagitis (EoE) is an allergic inflammatory esophageal disorder with a complex underlying genetic etiology often associated with other comorbidities. Using whole-exome sequencing (WES) of 63 patients with EoE and 60 unaffected family members and family-based trio analysis, we sought to uncover rare coding variants. WES analysis identified 5 rare, damaging variants in dehydrogenase E1 and transketolase domain–containing 1 (DHTKD1). Rare variant burden analysis revealed an overabundance of putative, potentially damaging DHTKD1 mutations in EoE (P = 0.01). Interestingly, we also identified 7 variants in the DHTKD1 homolog oxoglutarate dehydrogenase-like (OGDHL). Using shRNA-transduced esophageal epithelial cells and/or patient fibroblasts, we further showed that disruption of normal DHTKD1 or OGDHL expression blunts mitochondrial function. Finally, we demonstrated that the loss of DHTKD1 expression increased ROS production and induced the expression of viperin, a gene previously shown to be involved in production of Th2 cytokines in T cells. Viperin had increased expression in esophageal biopsies of EoE patients compared with control individuals and was upregulated by IL-13 in esophageal epithelial cells. These data identify a series of rare genetic variants implicating DHTKD1 and OGDHL in the genetic etiology of EoE and underscore a potential pathogenic role for mitochondrial dysfunction in EoE.

Eosinophilic esophagitis (EoE) is a chronic inflammatory disease of the esophagus triggered by immune hypersensitivity to food. Herein, we tested whether genetic risk factors for known, non-allergic, immune-mediated diseases, particularly those involving autoimmunity, were associated with EoE risk. We used the high-density Immunochip platform, encoding 200,000 genetic variants for major auto-immune disease. Accordingly, 1,214 subjects with EoE of European ancestry and 3,734 population controls were genotyped and assessed using data directly generated or imputed from the previously published GWAS. We found lack of association of EoE with the genetic variants in the major histocompatibility complex (MHC) class I, II, and III genes and nearly all other loci using a highly powered study design with dense genotyping throughout the locus. Importantly, we identified an EoE risk locus at 16p13 with genome-wide significance (Pcombined = 2.05 × 10−9, odds ratio = 0.76–0.81). This region is known to encode for the genes CLEC16A, DEXI, and CIITI, which are expressed in immune cells and esophageal epithelial cells. Suggestive EoE risk were also seen 5q23 (intergenic) and 7p15 (JAZF1). Overall, we have identified an additional EoE risk locus at 16p13 and highlight a shared and unique genetic etiology of EoE with a spectrum of immune-associated diseases.

Eosinophilic esophagitis (EoE) requires a peak count of 15 eosinophils per high-power field (hpf). Herein, the peak eosinophil count specified by a pathologist was compared with the second review of a research assistant. Of 477 biopsies, 106 had a peak count between 1 and 14 eosinophils/hpf cited in the pathology report, and 23/106 (22%) had ≥15 eosinophils/hpf on second review. The pathology report detected potential EoE with 99% specificity, but 80% sensitivity. As such, an additional review of esophageal biopsies yields higher eosinophil counts in ∼5% of cases. We propose that biopsies with a count between 1 and 14 eosinophils/hpf require further investigation because ∼22% may yield a potential EoE diagnosis.

Background The definition of eosinophilic gastritis (EG) is currently limited to histologic EG based on the tissue eosinophil count. Objective We aimed to provide additional fundamental information about the molecular, histopathologic, and clinical characteristics of EG. Methods Genome-wide transcript profiles and histologic features of gastric biopsy specimens, as well as blood eosinophil counts, were analyzed in patients with EG and control subjects (n = 15 each). Results The peak gastric antrum eosinophil count was 283 ± 164 eosinophils/×400 high-power field in patients with EG and 11 ± 9 eosinophils/×400 high-power field in control subjects ( P = 6.1 × 10 −7 ). Patients with EG (87%) had coexisting eosinophilic inflammation in multiple gastrointestinal segments; the esophagus represented the most common secondary site. Increased peripheral blood eosinophil counts (patients with EG: 1.09 ± 0.88 × 10 3 /μL vs control subjects: 0.09 ± 0.08 10 3 /μL, P = .0027) positively correlated with peak gastric eosinophil counts (Pearson r 2 = .8102, P + (proliferating), CD117 + (mast cells), and FOXP3 + (regulatory T cells, activated T cells, or both) cell counts were increased in patients with EG. Transcript profiling revealed changes in 8% of the genome in gastric tissue from patients with EG. Only 7% of this EG transcriptome overlapped with the eosinophilic esophagitis transcriptome. Significantly increased IL4 , IL5 , IL13 , IL17 , CCL26 , and mast cell–specific transcripts and decreased IL33 transcripts were observed. Conclusion EG is a systemic disorder involving profound blood and gastrointestinal tract eosinophilia, T H 2 immunity, and a conserved gastric transcriptome markedly distinct from the eosinophilic esophagitis transcriptome. The data herein define germane cellular and molecular pathways of EG and provide a basis for improving diagnosis and treatment.

Cadherins (CDH) mediate diverse processes critical in inflammation, including cell adhesion, migration, and differentiation. Herein, we report that the uncharacterized cadherin 26 (CDH26) is highly expressed by epithelial cells in human allergic gastrointestinal tissue. In vitro, CDH26 promotes calcium-dependent cellular adhesion of cells lacking endogenous CDHs by a mechanism involving homotypic binding and interaction with catenin family members (alpha, beta, and p120), as assessed by biochemical assays. Additionally, CDH26 enhances cellular adhesion to recombinant integrin α4β7 in vitro; conversely, recombinant CDH26 binds αE and α4 integrins in biochemical and cellular functional assays, respectively. Interestingly, CDH26-Fc inhibits activation of human CD4(+) T cells in vitro including secretion of IL-2. Taken together, we have identified a novel functional CDH regulated during allergic responses with unique immunomodulatory properties, as it binds α4 and αE integrins and regulates leukocyte adhesion and activation, and may thus represent a novel checkpoint for immune regulation and therapy via CDH26-Fc.

Eosinophilic esophagitis (EoE) is an allergic inflammatory disorder of the esophagus that is compounded by genetic predisposition and hypersensitivity to environmental antigens. Using high-density oligonucleotide expression chips, a disease-specific esophageal transcript signature was identified and was shown to be largely reversible with therapy. In an effort to expand the molecular signature of EoE, we performed RNA sequencing on esophageal biopsies from healthy controls and patients with active EoE and identified a total of 1607 significantly dysregulated transcripts (1096 upregulated, 511 downregulated). When clustered by raw expression levels, an abundance of immune cell-specific transcripts are highly induced in EoE but expressed at low (or undetectable) levels in healthy controls. Moreover, 66% of the gene signature identified by RNA sequencing was previously unrecognized in the EoE transcript signature by microarray-based expression profiling and included several long non-coding RNAs (lncRNA), an emerging class of transcriptional regulators. The lncRNA BRAF-activated non-protein coding RNA (BANCR) was upregulated in EoE and induced in interleukin-13 (IL-13)-treated primary esophageal epithelial cells. Repression of BANCR significantly altered the expression of IL-13-induced proinflammatory genes. Together, these data comprise new potential biomarkers of EoE and demonstrate a novel role for lncRNAs in EoE and IL-13-associated responses.

The desmosomal cadherin desmoglein-1 (DSG1) is an essential intercellular adhesion molecule that is altered in various human cutaneous disorders; however, its regulation and function in allergic disease remains unexplored. Herein, we demonstrate a specific reduction in DSG1 in esophageal biopsies from patients with eosinophilic esophagitis (EoE), an emerging allergic disorder characterized by chronic inflammation within the esophageal mucosa. Further, we show that DSG1 gene silencing weakens esophageal epithelial integrity, and induces cell separation and impaired barrier function (IBF) despite high levels of desmoglein-3 (DSG3). Moreover, DSG1 deficiency induces transcriptional changes that partially overlap with the transcriptome of inflamed esophageal mucosa; notably, periostin, a multipotent pro-inflammatory extracellular matrix molecule, is the top induced overlapping gene. We further demonstrate that IBF is a pathological feature in EoE, which can be partially induced through the downregulation of DSG1 by interleukin-13 (IL-13). Taken together, these data identify a functional role for DSG1 and its dysregulation by IL-13 in the pathophysiology of EoE and suggest that the loss of DSG1 may potentiate allergic inflammation through the induction of pro-inflammatory mediators such as periostin.

Background Eosinophilic esophagitis (EoE) is an emerging chronic inflammatory disease mediated by immune hypersensitization to multiple foods and strongly associated with atopy and esophageal remodeling. Objective We provide clinical and molecular evidence indicating a high prevalence of EoE in patients with inherited connective tissue disorders (CTDs). Methods We examined the rate of EoE among patients with CTDs and subsequently analyzed esophageal mRNA transcript profiles in patients with EoE with or without CTD features. Results We report a cohort of 42 patients with EoE with a CTD-like syndrome, representing 0.8% of patients with CTDs and 1.3% of patients with EoE within our hospital-wide electronic medical record database and our EoE research registry, respectively. An 8-fold risk of EoE in patients with CTDs (relative risk, 8.1; 95% confidence limit, 5.1-12.9; χ 2 1 = 112.0; P −3 ) was present compared with the general population. Esophageal transcript profiling identified a distinct subset of genes, including COL8A2 , in patients with EoE and CTDs. Conclusion There is a remarkable association of EoE with CTDs and evidence for a differential expression of genes involved in connective tissue repair in this cohort. Thus, we propose stratification of patients with EoE and CTDs into a subset referred to as EoE-CTD.

We recently identified a genome-wide genetic association of eosinophilic esophagitis (EoE) at 2p23 spanning the calpain 14 (CAPN14) gene, yet the causal mechanism has not been elucidated. We now show that recombinant CAPN14 cleaves a calpain-specific substrate and is inhibited by 4 classical calpain inhibitors: MDL-28170, acetyl-calpastatin, E-64, and PD151746. CAPN14 is specifically induced (>100-fold) in esophageal epithelium after IL-13 treatment. Epithelial cells overexpressing CAPN14 display impaired epithelial architecture, characterized by acantholysis, epidermal clefting, and epidermolysis. CAPN14 overexpression impairs epithelial barrier function, as demonstrated by decreased transepithelial resistance (2.1-fold) and increased FITC-dextran flux (2.6-fold). Epithelium with gene-silenced CAPN14 demonstrates increased dilated intercellular spaces (5.5-fold) and less organized basal cell layering (1.5-fold) following IL-13 treatment. Finally, CAPN14 overexpression results in loss of desmoglein 1 (DSG1) expression, whereas the IL-13-induced loss of DSG1 is normalized by CAPN14 gene silencing. Importantly, these findings were specific to CAPN14, as they were not observed with modulation of CAPN1 expression. These results, along with the potent induction of CAPN14 by IL-13 and genetic linkage of EoE to the CAPN14 gene locus, demonstrate a molecular and cellular pathway that contributes to T helper type 2 responses in mucosal epithelium.

We have previously proposed that the pathogenesis of eosinophilic esophagitis (EE) is mediated by an IL-13–driven epithelial cell response associated with marked gene dysregulation including eotaxin-3 overproduction. In this study, we compared epithelial responses between healthy patients and those with EE, aiming to uncover molecular explanations for EE pathogenesis. Esophageal epithelial cells could be maintained for up to five passages, with 67% and 62% of cell lines reaching confluence in healthy controls and EE cases, respectively. Both sets of epithelial cells avidly responded to IL-13 at similar levels as assessed by eotaxin-3 production. Acidic pH increased cellular release of eotaxin-3 (4.6 ± 1.98 ng/ml versus 12.46 ± 2.90 ng/ml at pH 7.4 and 4, respectively; p < 0.05). Numerous epidermal differentiation complex (EDC) genes, such as filaggrin and SPRR3, were downregulated both in IL-13–stimulated esophageal epithelial cells and in EE biopsies specimens compared with healthy controls. Whereas the filaggrin loss of function mutation 2282del4 was overrepresented in EE compared with control individuals (6.1% versus 1.3% respectively; p = 0.0172), the decreased filaggrin expression was uniformly seen in all EE cases in vivo. Indeed, expression of the EDC genes filaggrin and involucrin was strongly decreased directly by IL-13. These results establish that the epithelial response in EE involves a cooperative interaction between IL-13 and expression of EDC genes.

Although interleukin (IL)-13 and neurotrophins are functionally important for the pathogenesis of immune responses, the interaction of these pathways has not been explored. Herein, by interrogating IL-13-induced responses in human epithelial cells we show that neurotrophic tyrosine kinase receptor, type 1 (NTRK1), a cognate, high-affinity receptor for nerve growth factor (NGF), is an early transcriptional IL-13 target. Induction of NTRK1 was accompanied by accumulation of activating epigenetic marks in the promoter; transcriptional and epigenetic changes were signal transducer and activator of transcription 6 dependent. Using eosinophilic esophagitis as a model for human allergic inflammation, we found that NTRK1 was increased in inflamed tissue and dynamically expressed as a function of disease activity and that the downstream mediator of NTRK1 signaling early growth response 1 protein was elevated in allergic inflammatory tissue compared with control tissue. Unlike NTRK1, its ligand NGF was constitutively expressed in control and disease states, indicating that IL-13-stimulated NTRK1 induction is a limiting factor in pathway activation. In epithelial cells, NGF and IL-13 synergistically induced several target genes, including chemokine (C-C motif) ligand 26 (eotaxin-3). In summary, we have demonstrated that IL-13 confers epithelial cell responsiveness to NGF by regulating NTRK1 levels by a transcriptional and epigenetic mechanism and that this process likely contributes to allergic inflammation.

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder associated with allergic hypersensitivity to food. We interrogated1.5 million genetic variants in EoE cases of European ancestry and subsequently in a multi-site cohort with local and out-of-study control subjects. In addition to replicating association of the 5q22 locus (meta-analysis P=1.9×10(-16)), we identified an association at 2p23 spanning CAPN14 (P=2.5×10(-10)). CAPN14 was specifically expressed in the esophagus, was dynamically upregulated as a function of disease activity and genetic haplotype and after exposure of epithelial cells to interleukin (IL)-13, and was located in an epigenetic hotspot modified by IL-13. Genes neighboring the top 208 EoE-associated sequence variants were enriched for esophageal expression, and multiple loci for allergic sensitization were associated with EoE susceptibility (4.8×10(-2)P5.1×10(-11)). We propose a model to explain the tissue-specific nature of EoE that involves the interplay of allergic sensitization with an EoE-specific, IL-13-inducible esophageal response involving CAPN14.

Eosinophils are major effector cells in type 2 inflammatory responses and become activated in response to IL-4 and IL-33, yet the molecular mechanisms and cooperative interaction between these cytokines remain unclear. Our objective was to investigate the molecular mechanism and cooperation of IL-4 and IL-33 in eosinophil activation. Eosinophils derived from bone marrow or isolated from Il5-transgenic mice were activated in the presence of IL-4 or IL-33 for 1 or 4 h, and the transcriptome was analyzed by RNA sequencing. The candidate genes were validated by quantitative PCR and ELISA. We demonstrated that murine-cultured eosinophils respond to IL-4 and IL-33 by phosphorylation of STAT-6 and NF-κB, respectively. RNA sequence analysis of murine-cultured eosinophils indicated that IL-33 induced 519 genes, whereas IL-4 induced only 28 genes, including 19 IL-33–regulated genes. Interestingly, IL-33 induced eosinophil activation via two distinct mechanisms, IL-4 independent and IL-4 secretion/autostimulation dependent. Anti–IL-4 or anti–IL-4Rα Ab-treated cultured and mature eosinophils, as well as Il4- or Stat6-deficient cultured eosinophils, had attenuated protein secretion of a subset of IL-33–induced genes, including Retnla and Ccl17. Additionally, IL-33 induced the rapid release of preformed IL-4 protein from eosinophils by a NF-κB–dependent mechanism. However, the induction of most IL-33–regulated transcripts (e.g., Il6 and Il13) was IL-4 independent and blocked by NF-κB inhibition. In conclusion, we have identified a novel activation pathway in murine eosinophils that is induced by IL-33 and differentially dependent upon an IL-4 auto-amplification loop.

Background & Aims Gene expression profiling provides an opportunity for definitive diagnosis but has not yet been well applied to inflammatory diseases. Here we describe an approach for diagnosis of an emerging form of esophagitis, eosinophilic esophagitis (EoE), which is currently diagnosed by histology and clinical symptoms. Methods We developed an EoE diagnostic panel (EDP) comprising a 96-gene quantitative polymerase chain reaction array and an associated dual-algorithm that uses cluster analysis and dimensionality reduction using a cohort of randomly selected esophageal biopsy samples from pediatric patients with EoE (n = 15) or without EoE (non-EoE controls, n=14) and subsequently vetted the EDP using a separate cohort of 194 pediatric and adult patient samples derived from both fresh or formalin-fixed, paraffin-embedded tissue: active EoE (n = 91), control (non-EoE and EoE remission, n=57), histologically ambiguous (n = 34), and reflux (n = 12) samples. Results The EDP identified adult and pediatric patients with EoE with approximately 96% sensitivity and approximately 98% specificity, and distinguished patients with EoE in remission from controls, as well as identified patients exposed to swallowed glucorticoids. The EDP could be used with formalin-fixed, paraffin-embedded tissue RNA and distinguished patients with EoE from those with reflux esophagitis, identified by pH-impedance testing. Preliminary evidence showed that the EDP could identify patients likely to have disease relapse after treatment. Conclusions We developed a molecular diagnostic test (referred to as the EDP) that identifies patients with esophagitis in a fast, objective, and mechanistic manner, offering an opportunity to improve diagnosis and treatment, and a platform approach for other inflammatory diseases.

Background Eosinophilic esophagitis (EE) is an emerging worldwide disease that mimics gastroesophageal reflux disease. Objective Early studies have suggested that esophageal eosinophilia occurs in association with T H 2 allergic responses, yet the local and systemic expression of relevant cytokines has not been well characterized. Methods A human inflammatory cytokine and receptor PCR array containing 84 genes followed by PCR validation and multiplex arrays were used to quantify cytokine mRNA in esophageal biopsies and blood samples. Results Esophageal transcripts of numerous chemokines (eg, chemokine [C-C motif] ligand [CCL] 1, CCL1, CCL23, CCL26 [eotaxin-3], chemokine [C-X-C motif] ligand [CXCL] 1, and CXCL2), cytokines (eg, IL13 and ATP-binding cassette, subfamily F, member 1), and cytokine receptors (eg, IL5 receptor, alpha) were induced at least 4-fold in individuals with EE. Analysis of esophageal biopsies (n = 288) revealed that eotaxin-3 mRNA level alone had 89% sensitivity for distinguishing individuals with and without EE. The presence of allergy was associated with significantly increased esophageal expression of IL4 and IL5 mRNA in patients with active EE. We identified 8 cytokines (IL-4, IL-13, IL-5, IL-6, IL-12p70, CD40 ligand, IL-1α, and IL-17) whose blood levels retrospectively distinguished 12 patients without EE from 13 patients with EE with 100% specificity and 100% sensitivity. When applied to a blind, prospectively recruited group of 36 patients, the cytokine panel scoring system had a 79% positive predictive value, 68% negative predictive value, 61% sensitivity, and 83% specificity for identifying EE. Conclusion Evidence is presented that IL13 and IL5 associate with eosinophil and eotaxin-3 levels, indicating the key role of adaptive T H 2 immunity in regulating eotaxin-3–driven esophageal eosinophilia in the absence of a consistent systemic change in cytokines.