Critical Role for the Microbiota in CX3CR1+ Intestinal Mononuclear Phagocyte Regulation of Intestinal T Cell Responses.

The intestinal barrier is vulnerable to damage by microbiota-induced inflammation that is normally restrained through mechanisms promoting homeostasis. Such disruptions contribute to autoimmune and inflammatory diseases including inflammatory bowel disease. We identified a regulatory loop whereby, in the presence of the normal microbiota, intestinal antigen-presenting cells (APCs) expressing the chemokine receptor CX3CR1 reduced expansion of intestinal microbe-specific T helper 1 (Th1) cells and promoted generation of regulatory T cells responsive to food antigens and the microbiota itself. We identified that disruption of the microbiota resulted in CX3CR1+ APC-dependent inflammatory Th1 cell responses with increased pathology after pathogen infection. Colonization with microbes that can adhere to the epithelium was able to compensate for intestinal microbiota loss, indicating that although microbial interactions with the epithelium can be pathogenic, they can also activate homeostatic regulatory mechanisms. Our results identify a cellular mechanism by which the microbiota limits intestinal inflammation and promotes tissue homeostasis.

CAR directs T cell adaptation to bile acids in the small intestine.

Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.

Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

Increased number of children in households may protect against inflammatory bowel disease.

BACKGROUND: The increasing incidence of inflammatory bowel disease (IBD: Crohn's disease and ulcerative colitis) around the world has coincided with a wide array of environmental and epidemiologic changes. The relationship between IBD incidence and household or family size decline, however, has not been examined before. Our background epidemiological analyses suggested an inverse association between household size and IBD incidence. We aimed to examine this further in a murine model. METHODS: We designed a unique two-generation cohousing model of family size and IBD susceptibility in C57BL/6J mice. Serial fecal microbiomes during cohousing were examined by high-throughput 16S rRNA sequencing. After cohousing for 10 days, mice were exposed to dextran sulfate sodium (DSS) to induce acute colitis. Body weight as a significant correlate of colitis severity was measured. RESULTS: Mice in a large household arrangement demonstrated less weight loss than mice in the small household arrangement in the DSS model. Age- and housing-dependent microbiome shifts were found. CONCLUSIONS: Larger households may be protective against intestinal inflammation through intergenerational microbiome modulation. Our observations may set the foundation for age-dependent, microbiome-directed future prevention against IBD. IMPACT: Epidemiological analyses in this study suggested that IBD incidence may inversely correlate with household size (an indicator of family size/children per family), which has not been examined before. A uniquely designed two-generation cohousing model of family size and IBD susceptibility in mice supported our epidemiologic observations. Microbiome changes in our cohousing model may set the foundation for age-dependent, microbiome-directed prevention against IBD.

Bacteroides ovatus Promotes IL-22 Production and Reduces Trinitrobenzene Sulfonic Acid-Driven Colonic Inflammation.

The intestinal microbiota influences the development and function of the mucosal immune system. However, the exact mechanisms by which commensal microbes modulate immunity is not clear. We previously demonstrated that commensal Bacteroides ovatus ATCC 8384 reduces mucosal inflammation. Herein, we aimed to identify immunomodulatory pathways employed by B. ovatus. In germ-free mice, mono-association with B. ovatus shifted the CD11b+/CD11c+ and CD103+/CD11c+ dendritic cell populations. Because indole compounds are known to modulate dendritic cells, B. ovatus cell-free supernatant was screened for tryptophan metabolites by liquid chromatography-tandem mass spectrometry and larger quantities of indole-3-acetic acid were detected. Analysis of cecal and fecal samples from germ-free and B. ovatus mono-associated mice confirmed that B. ovatus could elevate indole-3-acetic acid concentrations in vivo. Indole metabolites have previously been shown to stimulate immune cells to secrete the reparative cytokine IL-22. Addition of B. ovatus cell-free supernatant to immature bone marrow-derived dendritic cells stimulated IL-22 secretion. The ability of IL-22 to drive repair in the intestinal epithelium was confirmed using a physiologically relevant human intestinal enteroid model. Finally, B. ovatus shifted the immune cell populations in trinitrobenzene sulfonic acid-treated mice and up-regulated colonic IL-22 expression, effects that correlated with decreased inflammation. Our data suggest that B. ovatus-produced indole-3-acetic acid promotes IL-22 production by immune cells, yielding beneficial effects on colitis.

Routine Histology-Based Diagnosis of CMV Colitis Was Rare in Pediatric Patients.

Cytomegalovirus (CMV) induced or complicated colitis is important to identify, yet its incidence is unknown among pediatric patients. We aimed to establish the incidence of routine histology-confirmed CMV colitis among pediatric patients with colitis. Pathology reports at Texas Children's Hospital (TCH) between January 1, 2011 and November 6, 2019 were reviewed. Of 1801 cases of histologic colitis, 11 patients had CMV found by histology (mean age 15.4, 72.7% female), with an incidence of 0.6%. Nine out of these 11 (81.8%) patients were immunocompromised and 4 (36.4%) had inflammatory bowel disease (IBD) as an underlying diagnosis of whom 2 had new-onset ulcerative colitis. At an average follow-up of 3.7 years, none of the CMV colitis cases experienced recurrence or severe complications (such as colectomy). An independent analysis of 54 consecutive IBD-associated colectomy cases at TCH showed no histologic evidence of CMV. We conclude that routine histology proven CMV-associated colitis in pediatric patients and IBD-colon explants was rare.Key Words: cytomegalovirus; colitis; children; histopathology; ulcerative colitis.

Constitutive activation of mitogen-activated protein kinase kinase (MEK1) in ileal enterocytes leads to dysplasia and a predisposition to cancer.

Activation of mitogen-activated protein kinases (MAPKs) is a key factor in the pathogenesis of cancer, although the specific role of mitogen-activated protein kinase kinase (MEK1) is not well understood. Villin promoter-driven Cre expression was used to excise a floxed stop cassette from a phosphomimetically constitutively activated MEK1 (caMEK1) expression construct in the intestine of C57BL/6 mice. Zygosity status of caMEK1 afforded assessment of the dose dependence of the effect. The expected mendelian distribution of genotypes and sex was observed in 443 progenies. Between 21 and 63 days of life, caMEK1 had no effect on body weight in male mice, but reduced body weight in female mice homozygous for caMEK1. At 10 wk of age, the ileum of caMEK1-expressing mice was characterized by the finding of dysplasia and profound changes in overall architecture. Paneth cells were nearly absent in caMEK1 homozygotes. Targeted proteomic profiling via reverse phase protein array analyses with confirmatory Western blotting revealed significant changes in protein and phosphoprotein expression, including upregulation of proteins downstream of MEK1, associated with enhanced markers of proliferation, diminished apoptosis, alterations in cell-fate determination, cell-cell interactions, and tight junctions. Long-term viability of caMEK1 homozygous mice was reduced with no survival beyond 1 yr. Invasive adenocarcinoma developed in three of ten older mice [15 wk (homozygous), 26 wk (homozygous), and 35 wk (heterozygous) of age]. Expression of caMEK1 in enterocytes leads to marked derangements in the intestinal epithelium, which is associated with a predisposition to the development of invasive cancer.NEW & NOTEWORTHY The ileum of mice with constitutive expression of activated MEK1 (via phosphomimetic changes) in enterocytes is markedly abnormal with architectural distortion and cytologic atypia, which evolves into an adenoma invasive carcinoma sequence. Phosphoproteomic analysis reveals upregulation of proteins downstream of MEK1, associated with enhanced markers of proliferation, diminished apoptosis, alterations in cell-fate determination, cell-cell interactions, and tight junctions. This novel model provides new insights into intestinal homeostasis and carcinogenesis.

Heterozygous Truncating Variants in POMP Escape Nonsense-Mediated Decay and Cause a Unique Immune Dysregulatory Syndrome.

The proteasome processes proteins to facilitate immune recognition and host defense. When inherently defective, it can lead to aberrant immunity resulting in a dysregulated response that can cause autoimmunity and/or autoinflammation. Biallelic or digenic loss-of-function variants in some of the proteasome subunits have been described as causing a primary immunodeficiency disease that manifests as a severe dysregulatory syndrome: chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE). Proteasome maturation protein (POMP) is a chaperone for proteasome assembly and is critical for the incorporation of catalytic subunits into the proteasome. Here, we characterize and describe POMP-related autoinflammation and immune dysregulation disease (PRAID) discovered in two unrelated individuals with a unique constellation of early-onset combined immunodeficiency, inflammatory neutrophilic dermatosis, and autoimmunity. We also begin to delineate a complex genetic mechanism whereby de novo heterozygous frameshift variants in the penultimate exon of POMP escape nonsense-mediated mRNA decay (NMD) and result in a truncated protein that perturbs proteasome assembly by a dominant-negative mechanism. To our knowledge, this mechanism has not been reported in any primary immunodeficiencies, autoinflammatory syndromes, or autoimmune diseases. Here, we define a unique hypo- and hyper-immune phenotype and report an immune dysregulation syndrome caused by frameshift mutations that escape NMD.

Approaches to Research Determination of Late Acute Cellular Rejection in Pediatric Liver Transplant Recipients.

A central pathology or site reading of biopsy slides is used in liver transplant clinical trials to determine rejection. We evaluated interrater reliability of readings of "rejection or not" using digitized slides from the Medication Adherence in Children who had a Liver Transplant (MALT) study. Four masked experienced pathologists read the digitized slides and then reread them after a study-specific histologic endpoint development program. Agreement was expressed throughout as a Kappa or Fleiss Kappa statistic (Ò¡). A Ò¡ > 0.6 was predefined as desirable. Readings were correlated with immunosuppressant adherence (the Medication Level Variability Index, [MLVI]), and maximal liver enzyme levels during the study period. Interrater agreement between site and central review in MALT, and between 4 pathologists later on, was low (Ò¡ = 0.44, Fleiss Ò¡ = 0.41, respectively). Following the endpoint development program, agreement improved and became acceptable (Ò¡ = 0.71). The final reading was better-aligned with maximal gamma-glutamyl transferase levels and MLVI as compared with the original central reading. We found substantial disagreement between experienced pathologists reading the same slides. A unique study-specific procedure improved interrater reliability to the point it was acceptable. Such a procedure may be indicated to increase reliability of histopathologic determinations in future research, and perhaps also clinically.

Biomarkers for liver disease in urea cycle disorders.

BACKGROUND: Urea cycle disorders (UCDs) are among the most common inborn errors of liver metabolism. As therapies for hyperammonemia associated with urea cycle dysfunction have improved, chronic complications, such as liver disease, have become increasingly apparent in individuals with UCDs. Liver disease in UCDs may be associated with hepatic inflammation, hepatic fibrosis, portal hypertension, liver cancer and even liver failure. However, except for monitoring serum aminotransferases, there are no clear guidelines for screening and/or monitoring individuals with UCDs for liver disease. Thus, we systematically evaluated the potential utility of several non-invasive biomarkers for liver fibrosis in UCDs. METHODS: We evaluated grey-scale ultrasonography, liver stiffness obtained from shear wave elastography (SWE), and various serum biomarkers for hepatic fibrosis and necroinflammation, in a cohort of 28 children and adults with various UCDs. RESULTS: Overall, we demonstrate a high burden of liver disease in our participants with 46% of participants having abnormal grey-scale ultrasound pattern of the liver parenchyma, and 52% of individuals having increased liver stiffness. The analysis of serum biomarkers revealed that 32% of participants had elevated FibroTest™ score, a marker for hepatic fibrosis, and 25% of participants had increased ActiTest™ score, a marker for necroinflammation. Interestingly, liver stiffness did not correlate with ultrasound appearance or FibroTest™. CONCLUSION: Overall, our results demonstrate the high overall burden of liver disease in UCDs and highlights the need for further studies exploring new tools for identifying and monitoring individuals with UCDs who are at risk for this complication. TRIAL REGISTRATION: This study has been registered in ClinicalTrials.gov (NCT03721367).

Streptococcus gallolyticus subsp. gallolyticus promotes colorectal tumor development.

Streptococcus gallolyticus subsp. gallolyticus (Sg) has long been known to have a strong association with colorectal cancer (CRC). This knowledge has important clinical implications, and yet little is known about the role of Sg in the development of CRC. Here we demonstrate that Sg promotes human colon cancer cell proliferation in a manner that depends on cell context, bacterial growth phase and direct contact between bacteria and colon cancer cells. In addition, we observed increased level of ß-catenin, c-Myc and PCNA in colon cancer cells following incubation with Sg. Knockdown or inhibition of ß-catenin abolished the effect of Sg. Furthermore, mice administered with Sg had significantly more tumors, higher tumor burden and dysplasia grade, and increased cell proliferation and ß-catenin staining in colonic crypts compared to mice receiving control bacteria. Finally, we showed that Sg is present in the majority of CRC patients and is preferentially associated with tumor compared to normal tissues obtained from CRC patients. These results taken together establish for the first time a tumor-promoting role of Sg that involves specific bacterial and host factors and have important clinical implications.

Protective role of commensal bacteria in Sjögren Syndrome.

CD25 knock-out (CD25KO) mice spontaneously develop Sjögren Syndrome (SS)-like inflammation. We investigated the role of commensal bacteria by comparing CD25KO mice housed in conventional or germ-free conditions. Germ-free CD25KO mice have greater corneal barrier dysfunction, lower goblet cell density, increased total lymphocytic infiltration score, increased expression of IFN-γ, IL-12 and higher a frequency of CD4+IFN-γ+ cells than conventional mice. CD4+ T cells isolated from female germ-free CD25KO mice adoptively transferred to naive immunodeficient RAG1KO recipients caused more severe Sjögren-like disease than CD4+ T cells transferred from conventional CD25KO mice. Fecal transplant in germ-free CD25KO mice reversed the spontaneous dry eye phenotype and decreased the generation of pathogenic CD4+IFN-γ+ cells. Our studies indicate that lack of commensal bacteria accelerates the onset and severity of dacryoadenitis and generates autoreactive CD4+T cells with greater pathogenicity in the CD25KO model, suggesting that the commensal bacteria or their metabolites products have immunoregulatory properties that protect exocrine glands in the CD25KO SS model.

Granulomatous Upper Gastrointestinal Inflammation in Pediatric Ulcerative Colitis.

OBJECTIVES: Differentiating ulcerative colitis (UC) and Crohn disease (CD) can be clinically challenging, especially in children. Granulomatous inflammation has traditionally been attributed to CD. Crypt-associated giant cells and granulomas, however, have been observed in colonic biopsies of patients with UC. This phenomenon has not been described in the upper gastrointestinal (UGI) tract with UC. METHODS: Seven pediatric patients with UC with granulomatous UGI (gUGI) lesions were identified. Diagnosis of UC was based on symptoms, clinical course, laboratory results, imaging, and endoscopy. We compared the gUGI patients to a large cohort of pediatric patients with UC (n = 149). RESULTS: All fully evaluated cases were associated with bloody diarrhea and moderate to severe pancolitis. Gastric and/or duodenal biopsies demonstrated giant cells or granulomas near gland destruction. Small bowel imaging did not reveal any involvement. The majority of cases responded to standard medical therapies, except for 2 patients (28.6%) who required total colectomy. Acute severe, refractory colitis (ie, colectomy within 1 month of presentation) was significantly more common in the gUGI group than the large pediatric UC group (28.6% vs 1.3%, Fisher exact P = 0.01). CONCLUSIONS: This is the first report of pediatric UC-associated granulomatous inflammation in the UGI tract. We speculate that these lesions represent extracolonic manifestations of intense colonic disease. These atypical findings expand the diagnostic considerations that should be incorporated during the differentiation between UC and CD in the pediatric age group.

Fatal disseminated Trichosporon asahii fungemia in a child with acute lymphoblastic leukemia and a morbilliform eruption.

Trichosporonosis is a rare, life-threatening, opportunistic fungal infection that affects immunocompromised individuals with neutropenia, particularly those with underlying hematologic malignancies. We present the case of a 10-year-old boy with acute lymphoblastic leukemia who developed a diffuse, morbilliform eruption in the setting of fever and pancytopenia. He was found to have Trichosporon asahii fungemia with widespread visceral dissemination, and his condition rapidly deteriorated despite treatment. It is important to consider trichosporonosis in the evaluation of a critically ill individual with neutropena and a rash, because the initial cutaneous presentation may appear benign and delayed therapy results in death.

Efficacy of a 4-Food Elimination Diet for Children With Eosinophilic Esophagitis.

BACKGROUND & AIMS: A 6-food elimination diet induces remission in most children and adults with eosinophilic esophagitis (EoE). The effectiveness of empiric elimination of only 4 foods has not been studied in children. We performed a prospective observational outcome study in children with EoE treated with dietary exclusion of cow's milk, wheat, egg, and soy. The objective was to assess the clinical, endoscopic, and histologic efficacy of this treatment in EoE. METHODS: We recruited children (1-18 years old, diagnosed per consensus guidelines) from 4 medical centers. Study participants (n = 78) were given a proton pump inhibitor twice daily and underwent a baseline esophagogastroduodenoscopy. Subjects were instructed on dietary exclusion of cow's milk, wheat, egg, and soy. Clinical, endoscopic, and histologic assessments were made after 8 weeks. Responders had single foods reintroduced for 8 weeks, with repeat endoscopy to assess for recurrence of active disease. The primary endpoint was histologic remission (fewer than 15 eosinophils per high-powered field). Secondary endpoints included symptom and endoscopic improvements and identification of foods associated with active histologic disease. RESULTS: After 8 weeks on 4-food elimination diet, 50 subjects were in histologic remission (64%). The subjects' mean baseline clinical symptoms score was 4.5, which decreased to 2.3 after 8 weeks of 4-food elimination diet (P < .001). The mean endoscopic baseline score was 2.1, which decreased to 1.3 (P < .001). After food reintroduction, the most common food triggers that induced histologic inflammation were cow's milk (85%), egg (35%), wheat (33%), and soy (19%). One food trigger that induced recurrence of esophageal inflammation was identified in 62% of patients and cow's milk-induced EoE was present in 88% of these patients. CONCLUSIONS: In a prospective study of children with EoE, 8 weeks of 4-food elimination diet induced clinical, endoscopic, and histologic remission in more than 60% of children with EoE. Although less restrictive than 6-food elimination diet, 4-food elimination diet was nearly as effective, and can be recommended as a treatment for children with EoE.

Lung Transplantation for FLNA-Associated Progressive Lung Disease.

OBJECTIVE: To describe a series of patients with pathogenic variants in FLNA and progressive lung disease necessitating lung transplantation. STUDY DESIGN: We conducted a retrospective chart review of 6 female infants with heterozygous presumed loss-of-function pathogenic variants in FLNA whose initial presentation was early and progressive respiratory failure. RESULTS: Each patient received lung transplantation at an average age of 11 months (range, 5-15 months). All patients had pulmonary arterial hypertension and chronic respiratory failure requiring tracheostomy and escalating levels of ventilator support before transplantation. All 6 patients survived initial lung transplantation; however, 1 patient died after a subsequent heart-lung transplant. The remaining 5 patients are living unrestricted lives on chronic immunosuppression at most recent follow-up (range, 19 months to 11.3 years post-transplantation). However, in all patients, severe ascending aortic dilation has been observed with aortic regurgitation. CONCLUSIONS: Respiratory failure secondary to progressive obstructive lung disease during infancy may be the presenting phenotype of FLNA-associated periventricular nodular heterotopia. We describe a cohort of patients with progressive respiratory failure related to a pathogenic variant in FLNA and present lung transplantation as a viable therapeutic option for this group of patients.

Hemolytic uremic syndrome as the presenting manifestation of WT1 mutation and Denys-Drash syndrome: a case report.

BACKGROUND: Hemolytic uremic syndrome (HUS) can occur as a primary process due to mutations in complement genes or secondary to another underlying disease. HUS sometimes occurs in the setting of glomerular diseases, and it has been described in association with Denys-Drash syndrome (DDS), which is characterized by the triad of abnormal genitourinary development; a pathognomonic glomerulopathy, diffuse mesangial sclerosis; and the development of Wilms tumor. CASE PRESENTATION: We report the case of a 46, XX female infant who presented with HUS and biopsy-proven thrombotic microangiopathy. Next generation sequencing of genes with known mutations causative of atypical HUS found that she was homozygous for the Complement Factor H H3 haplotype and heterozygous for a variant of unknown significance in the DGKE gene. Whole exome sequencing identified a de novo heterozygous WT1 c.1384C > T; p.R394W mutation, which is classically associated with Denys-Drash syndrome (DDS). At the time of bilateral nephrectomy five months after her initial biopsy, she had diffuse mesangial sclerosis, typical of Denys-Drash syndrome, without evidence of thrombotic microangiopathy. CONCLUSION: This unique case highlights HUS as a rare but important manifestation of WT1 mutation and provides new insight into the genetics underlying this association.

Intramural esophageal bronchogenic cyst mimicking achalasia in a toddler.

Bronchogenic cysts are congenital malformations of the tracheobronchial tree. We describe a 20-month-old male who presented with persistent non-bilious emesis; manometry and imaging were consistent with esophageal achalasia. During a planned laparoscopic Heller myotomy, an intramural bronchogenic cyst was discovered in the anterior esophagus at the level of the gastroesophageal junction and successfully resected with resolution of his symptoms.

Extratesticular masses in children: taking ultrasound beyond paratesticular rhabdomyosarcoma.

Extratesticular cystic and solid scrotal masses are commonly encountered in pediatrics. The most common extratesticular malignancy is paratesticular rhabdomyosarcoma. The remainder of the common pathologies encountered -- appendage torsion, epididymitis and varicoceles -- are mostly benign. These frequently encountered benign lesions are confidently differentiated from paratesticular rhadbomyosarcoma using high-frequency scrotal sonography in combination with clinical features. Less commonly encountered extratesticular masses may not be as easily classified; however, these also have distinguishing features that can enable differentiation from malignancy. This review discusses the sonographic findings, and relevant clinical and pathological manifestations of the more unusual extratesticular masses encountered in two tertiary pediatric institutions during a 10-year period. While these extratesticular pathologies are encountered relatively infrequently, recognition of their manifestations enables appropriate management.