Cytokine RNA In Situ Hybridization Permits Individualized Molecular Phenotyping in Biopsies of Psoriasis and Atopic Dermatitis

JID Innovations

2021 Jun 01

Wang, A;Fogel, A;Murphy, M;Panse, G;McGeary, M;McNiff, J;Bosenberg, M;Vesely, M;Cohen, J;Ko, C;King, B;Damsky, W;
| DOI: 10.1016/j.xjidi.2021.100021

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.

Estimating tissue-specific TNF mRNA levels prior to anti-TNFα treatment may support therapeutic optimisation in IBD patients

Scandinavian journal of gastroenterology

2023 May 28

James, JP;Nielsen, BS;Langholz, E;Malham, M;Høgdall, E;Riis, LB;
PMID: 37246424 | DOI: 10.1080/00365521.2023.2217313

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.

Complement C3 mediates early hippocampal neurodegeneration and memory impairment in experimental multiple sclerosis

Neurobiology of disease

2021 Oct 19

Bourel, J;Planche, V;Dubourdieu, N;Oliveira, A;Séré, A;Ducourneau, EG;Tible, M;Maitre, M;Lesté-Lasserre, T;Nadjar, A;Desmedt, A;Ciofi, P;Oliet, SH;Panatier, A;Tourdias, T;
PMID: 34673149 | DOI: 10.1016/j.nbd.2021.105533

Memory impairment is one of the disabling manifestations of multiple sclerosis (MS) possibly present from the early stages of the disease and for which there is no specific treatment. Hippocampal synaptic dysfunction and dendritic loss, associated with microglial activation, can underlie memory deficits, yet the molecular mechanisms driving such hippocampal neurodegeneration need to be elucidated. In early-stage experimental autoimmune encephalomyelitis (EAE) female mice, we assessed the expression level of molecules involved in microglia-neuron interactions within the dentate gyrus and found overexpression of genes of the complement pathway. Compared to sham immunized mice, the central element of the complement cascade, C3, showed the strongest and 10-fold upregulation, while there was no increase of downstream factors such as the terminal component C5. The combination of in situ hybridization with immunofluorescence showed that C3 transcripts were essentially produced by activated microglia. Pharmacological inhibition of C3 activity, by daily administration of rosmarinic acid, was sufficient to prevent early dendritic loss, microglia-mediated phagocytosis of synapses in the dentate gyrus, and memory impairment in EAE mice, while morphological markers of microglial activation were still observed. In line, when EAE was induced in C3 deficient mice (C3KO), dendrites and spines of the dentate gyrus as well as memory abilities were preserved. Altogether, these data highlight the central role of microglial C3 in early hippocampal neurodegeneration and memory impairment in EAE and, therefore, pave the way toward new neuroprotective strategies in MS to prevent cognitive deficit using complement inhibitors.

Linear ubiquitination-induced necrotic tumor remodeling elicits immune evasion

FEBS letters

2023 Apr 15

Sasaki, K;Hayamizu, Y;Murakami, R;Toi, M;Iwai, K;
PMID: 37060248 | DOI: 10.1002/1873-3468.14623

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.

A RIPK1-regulated inflammatory microglial state in amyotrophic lateral sclerosis

Proceedings of the National Academy of Sciences of the United States of America

2021 Mar 30

Mifflin, L;Hu, Z;Dufort, C;Hession, CC;Walker, AJ;Niu, K;Zhu, H;Liu, N;Liu, JS;Levin, JZ;Stevens, B;Yuan, J;Zou, C;
PMID: 33766915 | DOI: 10.1073/pnas.2025102118

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.

Early neuroinflammatory responses in the visual pathway in a feline inherited glaucoma model

Investigative Ophthalmology & Visual Science

2022 Jan 01

Oikawa, K;Kiland, J;Mathu, V;Torne, O;

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

The complement system drives local inflammatory tissue priming by metabolic reprogramming of synovial fibroblasts

Immunity

2021 Mar 19

Friščić, J;Böttcher, M;Reinwald, C;Bruns, H;Wirth, B;Popp, SJ;Walker, KI;Ackermann, JA;Chen, X;Turner, J;Zhu, H;Seyler, L;Euler, M;Kirchner, P;Krüger, R;Ekici, AB;Major, T;Aust, O;Weidner, D;Fischer, A;Andes, FT;Stanojevic, Z;Trajkovic, V;Herrmann, M;Korb-Pap, A;Wank, I;Hess, A;Winter, J;Wixler, V;Distler, J;Steiner, G;Kiener, HP;Frey, B;Kling, L;Raza, K;Frey, S;Kleyer, A;Bäuerle, T;Hughes, TR;Grüneboom, A;Steffen, U;Krönke, G;Croft, AP;Filer, A;Köhl, J;Klein, K;Buckley, CD;Schett, G;Mougiakakos, D;Hoffmann, MH;
PMID: 33761330 | DOI: 10.1016/j.immuni.2021.03.003

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.

Defining the pig microglial transcriptome reveals its core signature, regional heterogeneity, and similarity with human and rodent microglia

Glia

2022 Sep 19

Shih, BB;Brown, SM;Barrington, J;Lefevre, L;Mabbott, NA;Priller, J;Thompson, G;Lawrence, AB;McColl, BW;
PMID: 36120803 | DOI: 10.1002/glia.24274

Microglia play key roles in brain homeostasis as well as responses to neurodegeneration and neuroinflammatory processes caused by physical disease and psychosocial stress. The pig is a physiologically relevant model species for studying human neurological disorders, many of which are associated with microglial dysfunction. Furthermore, pigs are an important agricultural species, and there is a need to understand how microglial function affects their welfare. As a basis for improved understanding to enhance biomedical and agricultural research, we sought to characterize pig microglial identity at genome-wide scale and conduct inter-species comparisons. We isolated pig hippocampal tissue and microglia from frontal cortex, hippocampus, and cerebellum, as well as alveolar macrophages from the lungs and conducted RNA-sequencing (RNAseq). By comparing the transcriptomic profiles between microglia, macrophages, and hippocampal tissue, we derived a set of 239 highly enriched genes defining the porcine core microglial signature. We found brain regional heterogeneity based on 150 genes showing significant (adjusted p < 0.01) regional variations and that cerebellar microglia were most distinct. We compared normalized gene expression for microglia from human, mice and pigs using microglia signature gene lists derived from each species and demonstrated that a core microglial marker gene signature is conserved across species, but that species-specific expression subsets also exist. Our data provide a valuable resource defining the pig microglial transcriptome signature that validates and highlights pigs as a useful large animal species bridging between rodents and humans in which to study the role of microglia during homeostasis and disease.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

Search form

Probes for INS

Content for comparison

Gene

Product

Research area

Category

Advanced Cell Diagnostics

Bio-Techne

Advanced Cell Diagnostics China