Probes for INS

Detection of individual cytokines in routine biopsies from patients with inflammatory skin diseases has the potential to personalize diagnosis and treatment selection, but this approach has been limited by technical feasibility. We evaluate whether a chromogen-based RNA in situ hybridization approach can be used to detect druggable cytokines in psoriasis and atopic dermatitis. A series of psoriasis (n = 20) and atopic dermatitis (n = 26) biopsies were stained using RNA in situ hybridization for IL4, IL12B (IL-12/23 p40), IL13, IL17A, IL17F, IL22, IL23A (IL-23 p19), IL31, and TNF (TNF-α). NOS2 and IFNG, canonical psoriasis biomarkers, were also included. All 20 of the psoriasis cases were positive for IL17A, which tended to be the predominant cytokine, although some cases had relatively higher levels of IL12B, IL17F, or IL23A. The majority of cytokine expression in psoriasis was epidermal. A total of 22 of 26 atopic dermatitis cases were positive for IL13, also at varying levels; a subset of cases had significant IL4, IL22, or IL31 expression. Patterns were validated in independent bulk RNA-sequencing and single-cell RNA-sequencing datasets. Overall, RNA in situ hybridization for cytokines appears highly specific with virtually no background staining and may allow for individualized evaluation of treatment-relevant cytokine targets in biopsies from patients with inflammatory skin disorders.

Tumour necrosis factor-α (TNF) antagonists have improved the management of inflammatory bowel disease (IBD), however, their usage and administration persist to be suboptimal. Here, we examined the relationship between tissue-specific TNF mRNA expression in mucosal biopsies from IBD patients and anti-TNF treatment response.Archived tissue samples from patients with luminal IBD that had all been or were in treatment with anti-TNF were included (18 adults and 24 paediatric patients). Patients were stratified into three groups according to anti-TNF response: responders, primary non-responders (PNR) and secondary loss of response (SLOR). TNF mRNA was detected using RNAscope in situ hybridisation (ISH) and the expression was quantified using image analysis.The ISH analysis showed varying occurrence of TNF mRNA positive cells located in lamina propria and often with increased density in lymphoid follicles (LF). Consequently, expression estimates were obtained in whole tissue areas with and without LF. Significantly higher TNF mRNA expression levels were measured in adults compared to paediatric patients in both the analyses with and without LF (p = .015 and p = .016, respectively). Considering the relation to response, the adult and paediatric patients were evaluated separately. In adults, the TNF expression estimates were higher in PNRs compared to responders with and without LF (p = .017 and p = .024, respectively).Our data indicate that adult PNR have significantly higher TNF mRNA levels than responders. This suggests that higher anti-TNF dose may be considered for IBD patients with high TNF mRNA expression estimates from the start of treatment.

Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA damage response (DDR) in vitro. These toxins are produced by Gram-negative bacteria, enriched in the microbiota of inflammatory bowel disease (IBD) and colorectal cancer (CRC) patients. However, their role in infection remains poorly characterized. We address the role of typhoid toxin in modulation of the host-microbial interaction in health and disease. Infection with a genotoxigenic Salmonella protects mice from intestinal inflammation. We show that the presence of an active genotoxin promotes DNA fragmentation and senescence in vivo, which is uncoupled from an inflammatory response and unexpectedly associated with induction of an anti-inflammatory environment. The anti-inflammatory response is lost when infection occurs in mice with acute colitis. These data highlight a complex context-dependent crosstalk between bacterial-genotoxin-induced DDR and the host immune response, underlining an unexpected role for bacterial genotoxins.

Abstract BACKGROUND: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4 T-cell function. METHODS: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4 T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. RESULTS: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4 T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1β leads to enhanced production of IL17A. CONCLUSIONS: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

Tumor-elicited inflammation confers tumorigenic properties, including cell death resistance, proliferation, or immune evasion. To focus on inflammatory signaling in tumors, we investigated linear ubiquitination, which enhances the nuclear factor-κB signaling pathway and prevents extrinsic programmed cell death under inflammatory environments. Here, we showed that linear ubiquitination was augmented especially in tumor cells around a necrotic core. Linear ubiquitination allowed melanomas to tolerate the hostile tumor microenvironment and to extend a necrosis-containing morphology. Loss of linear ubiquitination resulted in few necrotic lesions and growth regression, further leading to repression of innate anti-PD-1 therapy resistance signatures in melanoma as well as activation of interferon responses and antigen presentation that promote immune-mediated tumor eradication. Collectively, linear ubiquitination promotes tumor-specific tissue remodeling and the ensuing immune evasion.

Regulatory T cells (Tregs) are key modulators of inflammation and are important for the maintenance of peripheral tolerance. Adoptive immunotherapy with polyclonal Tregs holds promise in organ transplantation, graft-versus-host disease, and autoimmune diseases, but may be enhanced by antigen-specific, long-lived Treg cells. We modified primary human Tregs with chimeric antigen-receptors (CARs) bearing different costimulatory domains and performed in vitro analyses of their phenotype and function. While neither the presence of a CAR nor the type of costimulation domain influenced Foxp3 expression in Tregs, the costimulation domain of the CARs affected CAR Treg surface phenotype and functions such as cytokine production. Furthermore, signaling from the CD28 costimulation domain maintained CAR Treg suppressor function, whereas 4-1B costimulation did not. In vivo, CAR Tregs accumulated at sites expressing target antigen, and suppressed antigen specific effector T cell responses; however, only CAR Tregs with CD28 signaling domains were potent inhibitors of effector T cell mediated graft rejection in vivo. Our findings support the use of CD28 based CAR-Tregs for tissue specific immune suppression in the clinic.

Microglial-derived inflammation has been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including amyotrophic lateral sclerosis (ALS). Using single-cell RNA sequencing, a class of Disease-Associated Microglia (DAMs) have been characterized in neurodegeneration. However, the DAM phenotype alone is insufficient to explain the functional complexity of microglia, particularly with regard to regulating inflammation that is a hallmark of many neurodegenerative diseases. Here, we identify a subclass of microglia in mouse models of ALS which we term RIPK1-Regulated Inflammatory Microglia (RRIMs). RRIMs show significant up-regulation of classical proinflammatory pathways, including increased levels of Tnf and Il1b RNA and protein. We find that RRIMs are highly regulated by TNFα signaling and that the prevalence of these microglia can be suppressed by inhibiting receptor-interacting protein kinase 1 (RIPK1) activity downstream of the TNF receptor 1. These findings help to elucidate a mechanism by which RIPK1 kinase inhibition has been shown to provide therapeutic benefit in mouse models of ALS and may provide an additional biomarker for analysis in ongoing phase 2 clinical trials of RIPK1 inhibitors in ALS.

METHODS : Retinal, optic nerve head (ONH) and distal optic nerve (ON) tissues from 8 juvenile 10-12 week-old cats (4 males and 4 females) with feline congenital glaucoma (FCG) and 5 age-matched normal control cats (3 males and 2 females) were used. Data for weekly intraocular pressure (IOP) and optic nerve axon counts were available for all subjects. Protein and gene expression in tissue cryosections were examined by immunofluorescence labeling (IF) and RNAscope in situ hybridization (ISH), respectively. Retinal tissue was IF labeled for myeloid cell marker, IBA-1 and flat-mounted. ISH for markers of infiltrating monocytes/macrophages (_CCR2_) and proinflammatory cytokines (_IL1A_, _C1QA_, _TNF_) was performed. Microglia were identified by IF of homeostatic microglial marker, P2RY12. Microscopy images wereanalyzed using Image J, QuPath and Imaris. Two-tailed unpaired t-test or Mann-Whitney test or ANOVA were used for between-group comparisons (p