Ramos, Guilherme Piovezani

Abstract Background Immunosuppression is a recognized risk factor for severe and disseminated coccidioidomycosis; however, data describing outcomes in patients with inflammatory bowel disease (IBD) receiving advanced therapy, including biologics and small molecules, remain limited. We aim to describe the prevalence, clinical presentation, treatment patterns, and outcomes of coccidioidomycosis in IBD patients receiving advanced therapy at an endemic tertiary care center. Methods We performed a retrospective observational study of adult patients with IBD at Mayo Clinic Arizona diagnosed with coccidioidomycosis between 2023 and 2026. Patients were included if IBD diagnosis preceded coccidioidomycosis diagnosis and advanced therapy had been active for at least 4 weeks before infection. Cases were classified as proven, probable, or possible based on microbiologic, clinical, radiographic, and serologic criteria. Clinical outcomes included antifungal treatment duration, hospitalization related to coccidioidomycosis or IBD, IBD treatment modification after diagnosis, re-emergence of coccidioidomycosis symptoms, and IBD-related surgery. Results A total of 48 patients met inclusion criteria. Among 1083 tested IBD patients receiving advanced therapy, coccidioidomycosis positivity prevalence was 4.4% when including all serologically positive patients and 1.9% when restricted to proven or probable cases. The median age at coccidioidomycosis diagnosis was 40 years (range 19-87 years), and 30 (62.5%) of 48 had Crohn’s disease. Proven or probable infection was identified in 21 (43.8%) of 48 patients, while 14 (29.2%) of 48 patients were classified as possible. Antifungal therapy was prescribed in 32 (66.7%) of 48 patients, including all proven cases, 15 (88.2%) of 17 probable cases, 9 (64.3%) of 14 possible cases, and 3 (25.0%) of 12 nondiagnostic cases. Among proven/probable/possible cases, the median antifungal treatment duration was 174 days (range 8-831 days). Advanced IBD therapy was continued in 3 (75.0%) of 4 proven cases, 9 (52.9%) of 17 probable cases, 12 (85.7%) of 14 possible cases, and all nondiagnostic cases. Hospitalization occurred in 6 (12.5%) of 48 of the cohort, all of which were proven/probable cases. Most patients remained on advanced therapy after diagnosis, although tumor necrosis factor α inhibitors were the most interrupted or discontinued agents. Conclusions In this endemic tertiary care cohort, management decisions varied according to diagnostic certainty. Patients with proven or probable coccidioidomycosis were more likely to receive antifungal therapy and undergo advanced IBD therapy modification, whereas most patients with possible or nondiagnostic serologic findings remained on advanced therapy without major IBD-related complications. Prolonged antifungal treatment was common, highlighting the complexity of managing coccidioidomycosis in immunosuppressed patients with IBD.

ABSTRACT Enteroendocrine cells (EECs) are specialized sensors of the gastrointestinal (GI) epithelium that regulate gut function and systemic metabolism through hormone secretion. The molecular pathways directing intestinal stem cell (ISC) differentiation into EECs are incompletely understood due, in part, to their rarity. We sought to identify novel regulators of human EEC differentiation using a high-throughput screen of FDA-approved drugs and human duodenal organoids. Two epidermal growth factor receptor inhibitors (EGFRi) commonly used in cancer therapy and known to cause GI side effects, erlotinib and lapatinib, emerged as strong inducers of EEC differentiation, dramatically increasing chromogranin A (CHGA) expression compared to controls, while maintaining ISC function and organoid growth. EGFRi-treated organoids revealed robust and broad upregulation of EEC hormones, including serotonin (5HT), motilin (MLN), and somatostatin (SST), among others. In agreement with these findings, analysis of a patient cohort with lung cancer revealed an association with erlotinib use and increased circulating levels of the above EEC hormones compared to matched controls. Supporting a direct effect of EGFRi on EEC differentiation, mice treated with erlotinib demonstrated increased EEC numbers and hormones and showed EGFRi-associated diarrhea (EAD), a limiting side effect of these medications. Mechanistically, EGFRi induced upregulation of interferon (IFN) signaling targets during early ISC-to-EEC differentiation. Consistent with this, inhibition of Signal Transducer and Activator of Transcription 1 (STAT1) attenuated EGFRi-induced EEC differentiation. These findings provide important insight into EEC differentiation that could inform treatment strategies for EAD, metabolic diseases, and GI diseases. Brief Summary Inhibition of EGFR signaling promotes human ISC-to-EEC differentiation through activation of STAT1 signaling.

Abstract IBD affects roughly 1.6 million US adults, with its prevalence increasing since the late 1990s. Characterized by unchecked intestinal inflammation, multiple proinflammatory cytokines including IL6 have been implicated as critical factors in this pathology. It has been established that EZH2 is a gene involved in the production of specific histone methyltransferase enzymes, as well as in the differentiation of Treg cells. The association between the Treg master transcription factor, Foxp3 and EZH2 results in the suppressive function of Tregs, with our previous work revealing impairment of this interaction under the influence of IL6 signaling in an inflamed intestinal setting. We investigated this further in a murine preventative T cell transfer model of colitis that involved injecting donor Treg cells that lacked IL6Ra, into immunocompromised mice. Histological scoring of the mice receiving the IL6Rko Tregs revealed a resolved inflammatory milieu and flow cytometric analysis of cells isolated from the spleens and MLNs showed increased Foxp3+ expression when compared to mice that received WT Tregs. Foxp3IL6Rako mice were subjected to a DSS model of colitis to further characterize the absence of the IL6R on Treg function in the intestinal milieu. Exacerbated signs of colitis were observed through histology and colon shortening of the conditional knockout mice when compared to their WT counterparts. We postulate that Tregs could be acting as a donor of sILR to promote intestinal integrity and healing. Further experiments will be conducted to define a role for the IL6R in the development of Foxp3+ cells, along with outlining the instrumental process of sIL6R donation in the intestinal milieu. Supported by Mayo Clinic Graduate School for Biomedical Sciences

Abstract Background Significant proportion of inflammatory bowel disease (IBD) patients respond inconsistently to therapies, underscoring disease complexity. Interleukin 21 (IL21) is highly expressed during IBD. In addition to T helper (Th) cells, inflammatory regulatory T cells (Tregs) have been linked to refractory IBD. Thus, we explored the metabolic role of IL21 in inducing Treg dysfunction and IBD. Methods Human Tregs and Th cells were subjected to transcriptional profiling and metabolic phenotyping. Colitis was induced in Rag1−/− mice by naïve CD4 T cell adoptive transfer. Results IL21 stimulation of human Tregs induced glycolysis and OXPHOS. In agreement, IL21 enhanced the expression of genes associated with glycolysis, anabolism, and OXPHOS, leading to inflammatory cytokine production. Mechanistically, we found disruption to mitochondrial integrity with concomitant activation of glycogen synthase kinase 3 (GSK3) β, a kinase known to prevent mitochondrial pyruvate metabolism. IL21-induced GSK3β activation was accompanied by pyruvate and lactate buildup. Notably, GSK3 inhibition or supplementation with membrane-permeable methyl pyruvate abrogated metabolic wiring of and inflammatory responses by IL21-stimulated Tregs and effector Th cells. These results suggest that impaired mitochondrial pyruvate metabolism is a feature of inflammatory CD4 T cells. Lastly, GSK3 inhibition prevented pathogenic CD4 T cell-induced colitis in mice, as evidenced by reduced Mouse Colon Histology Index and serum inflammatory cytokines. Conclusions IL21 engages Tregs in a hypermetabolic state that augments inflammatory cytokine production. Therefore, desensitizing CD4 T cells to IL21 may also augment Treg function during IBD. Supported by NIDDK award K01DK124358, the Center for Cell Signaling in Gastroenterology (P30DK084567), and the Mayo Clinic Center for Biomedical Discovery.

Abstract Background Upadacitinib is a selective Janus kinase inhibitor approved for the management of ulcerative colitis and is under evaluation for the management of Crohn’s disease [CD] in Phase 3 clinical trials. Aims Our goal was to describe our real-world experience with upadacitinib in CD. Methods This is a two-centre retrospective cohort study of adult patients with moderate to severe CD on upadacitinib. The primary outcome was clinical response and remission as determined by stool frequency and abdominal pain scores. Secondary endpoints included endoscopic response and remission as determined by change in Simple Endoscopic Score for CD. Outcomes were assessed at 3 months after starting upadacitinib and at the patient’s most recent follow-up. We further evaluated adverse events and dose-related response. Results A total of 45 CD patients received upadacitinib and were included in the safety analysis. Thirty-six patients received upadacitinib for CD, whereas nine received it for inflammatory arthritis [n = 8] or pyoderma [n = 1]. Thirty-three patients received upadacitinib for 3 months or longer and were included in the efficacy analysis. At the 3-month follow-up, 21 patients achieved clinical response [63.6%] and nine achieved clinical remission [27.2%]. At time of last follow-up, 23 patients had clinical response [69.7%], ten achieved clinical remission [30.3%] and four [28.6%] achieved endoscopic remission. Adverse events occurred in 12 patients [26.7%]. Two patients had a serious adverse event [4.5%] without associated mortality. Conclusion In this real-world cohort of highly refractory CD patients, upadacitinib was effective in inducing remission and had an acceptable safety profile.

Background Significant proportion of inflammatory bowel disease (IBD) patients continue to respond inconsistently to therapies, underscoring disease complexity and the need for efficacious treatment. Interleukin 21 (IL‐21), which is known to support T helper (Th) cell function, is highly expressed within inflamed intestinal tissues of IBD patients compared to healthy controls. In addition, inflammatory regulatory T cells (Tregs) have been linked to refractory human IBD. Given that healthy Tregs are critical for self‐tolerance and prevention of IBD, we investigated the metabolic role of IL‐21 in instigating Treg dysfunction and the therapeutic ramifications of targeting metabolism pathways during IBD pathogenesis. Methods Human Tregs as well as relevant control effector Th cells were generated from naïve CD4 + T cells isolated from healthy blood donors. Microarray analysis was utilized for targeted metabolic transcriptional profiling. Immune phenotyping was assessed by fluorescence‐activated cell sorting. Metabolic phenotyping of cells was assessed by Seahorse flux analysis and mass spectrometry‐based metabolomics. Ultrastructural analysis of mitochondria was performed by confocal and transmission electron microscopy. Intestinal inflammation was induced in Rag1 ‐/‐ (T and B cell deficient) mice by the adoptive transfer of pathogenic naïve CD4 + T cells. Results Acute IL‐21 stimulation of human Tregs induced glycolysis and fluctuations in mitochondrial respiration (i.e. oxidative phosphorylation – OXPHOS), as assessed by Seahorse flux analysis. In agreement, microarray analysis, validated by qPCR, revealed an IL‐21‐mediated increase in the expression of genes associated with glycolysis and pathways known to support anabolic and OXPHOS metabolism, thus resembling a hypermetabolic state. Furthermore, IL‐21 stimulation rendered Tregs susceptible to inflammatory response, as evidenced by the production of effector Th cell‐associated cytokines such as interferon γ, tumor necrosis factor, IL‐17A, and IL‐17F. Exploring the mechanisms underlying IL‐21‐induced effects, we found significant disruption of mitochondrial integrity with concomitant activation of glycogen synthase kinase 3 (GSK3) β, a kinase known to prevent pyruvate entry into the mitochondria. IL‐21‐induced GSK3β activation was accompanied by a marked increase in intracellular and extracellular metabolites such as pyruvate and lactate, as assessed by metabolomics. Importantly, GSK3 inhibition or supplementation with mitochondrial membrane‐permeable methyl pyruvate broadly abrogated metabolic wiring of and inflammatory responses by IL‐21‐stimulated Tregs and effector Th cells. Collectively, these results suggest that impaired mitochondrial pyruvate metabolism is a feature of inflammatory CD4 + T cells. Lastly, GSK3 inhibition prevented pathogenic CD4 + T cell‐induced colitis in mice as evidenced by reduced Disease Activity Index, Mouse Colon Histology Index, and serum inflammatory cytokines. Conclusions IL‐21 potently engages human Tregs in a hypermetabolic state that augments inflammatory cytokine production via induction of mitochondrial dysfunction. Therefore desensitizing CD4 + T cells to detrimental cues, such as IL‐21, may also augment Treg function during human IBD.

Abstract Background Microscopic colitis (MC) is suspected to result from increased immune activity in gut mucosa. Immune checkpoint inhibitors (ICIs) treat cancer by activating the immune system, and further investigation is needed regarding their role in the development of MC. Methods A retrospective case series investigated cases of endoscopically and histologically confirmed MC developing after administration of ICIs. Clinical notes and medication administration records were reviewed for demographics, symptom duration, and treatment response. Results Nineteen cases of de novo MC were identified, with 95% of cases requiring steroid treatment, 53% presenting with hospitalization, and colitis-related mortality in 1 individual. Symptom onset occurred a median of 160 days after initiation of ICI therapy and 53 days after their most recent dose of therapy. Patients had a median of 125 days of symptoms, and ICI therapy was held in 70% of individuals due for treatment. Conclusions MC can develop after ICI administration, and presents with severe symptoms, often requiring hospitalization and steroid treatment. In certain individuals this can require a prolonged treatment course of steroid therapy or immunomodulators. Individuals developing diarrhea after ICI therapy warrant thorough workup including endoscopy and rapid treatment initiation given the disease severity observed in this series.

LINKED CONTENT This article is linked to Meserve et al paper. To view this article, visit https://doi.org/10.1111/apt.16217