Eoin N. McNamee’s research while affiliated with National University of Ireland, Maynooth and other places

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Publications (6)


Fig. 1. HDM primes BMDMs from CATT 7 mice, increasing their relative M1 gene expression. (A) Model of allergic airway inflammation to investigate HDM-induced trained immunity. Bone marrow was isolated from naive or HDM-challenged WT and CATT 7 mice 4 h after last challenge and differentiated with M-CSF over 7 days. (BÀF) M1 and (G,H) M2 macrophage marker expression in BMDMs polarized with LPS or IL-4 on day 7 for 24 h was measured by PCR. Each data point represents a single animal. Data are presented as mean § SEM (n = 3 mice per group). ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. PCR, polymerase chain reaction; SEM, standard error of the mean; i.n., intranasal. (Color version of figure is available online.)
Fig. 2. hBM-MSCs block HDM-induced priming effect in CATT 7 mice, decreasing M1 macrophage gene expression. (A) Bone marrow cells containing HSPCs were isolated from HDM-challenged WT and CATT 7 mice 4 h after last challenge and differentiated with M-CSF over 7 days. On day 7, hBM-MSCs were co-cultured with BMDMs using 0.4-mM transwells at time of polarization with LPS or IL-4 for 24 h. (BÀF) Gene expression and protein production of M1 macrophage markers in BMDMs measured by PCR or ELISA. (G,H) Gene expression of M2 markers arg1 and retnla. (I,J) To investigate the role of COX-2 in the ability of MSCs to suppress trained immunity in CATT 7 BMDMs, MSCs were pre-exposed to the COX-2 inhibitor NS-398 or vehicle control for 24 h prior to co-culture with BMDMs in transwells. TNF-a and IL-6 protein production in supernatants from CATT 7 BMDMs co-cultured with MSCs § NS-398 or vehicle control was measured by ELISA. Each data point represents a single animal. Data are presented as mean § SEM (n = 3 mice per group). ns, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. PCR, polymerase chain reaction; SEM, standard error of the mean; i.n., intranasal. (Color version of figure is available online.)
Mesenchymal stromal cells dampen trained immunity in house dust mite-primed macrophages expressing human MIF polymorphism
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May 2024

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Cytotherapy

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Characterization of WT and CATT7 mice after HDM exposure. (A) Preclinical mouse model of acute allergic airway inflammation. Mice received 25 μg of house dust mite or PBS control intranasally three times for 3 weeks. BALF and lung homogenates were collected on Day 18, 4 h after last challenge, or lungs were taken for histology on Day 21. (B) Cells infiltrated into the BALF were analyzed using differential stained cytospin slides. 300 cells were counted and identified as eosinophils, macrophages, lymphocytes or neutrophils based on their morphology, and cell numbers were normalized to the volume of BALF collected. (C) Representative images of lung tissue from WT and CATT7 mice after HDM or PBS challenge (I.N.). Tissue sections were stained with Periodic Acid Schiff (Goblet cell hyperplasia) at 20× magnification or Masson's Trichrome (subepithelial fibrosis) at 4× magnification. Scale bar = 20 μm. (D) 24 h after last HDM challenge, the lung function of WT and CATT7 mice was measured using a FlexiVent® instrument (SCIREQ) in response to PBS or methacholine (25 mg/mL). Parameters include airway resistance (RN), tissue damping (G), and tissue elasticity (H). Data are presented as peak response normalized to the baseline and expressed as % increase over baseline. (E) Average CT values for M1 and M2 macrophage markers from WT and CATT7 lung homogenate after HDM challenge. (F) Pro‐inflammatory status of lung homogenates from WT and CATT7 mice after HDM or PBS challenge were analyzed by ELISA. Th2 markers include IL‐4, IL‐5, IL‐13, and CCL17. Th1 markers include TNFα, IL‐6, and IL‐1β. Data are presented as mean ± SEM; N = 3–6 mice per group. ns, nonsignificant, *p < .05, **p < .01, ***p < .001, ****p < .0001.
Polarization of BMDMs from naive CATT7 mice and WT mice are similar under steady state conditions. HSPCs are isolated from WT and CATT7 mice and differentiated by culturing in M‐CSF supplemented media for 7 days. BMDMs from naive WT and CATT7 mice were stimulated with polarizing conditions; LPS or IL‐4 for 24 h. Gene expression and protein production of M1 macrophage markers A tnfα, B il‐6, C il‐1β, D nos2, E il‐10 or M2 markers F arg1 and G retnla were analyzed using RT‐PCR. Data are presented as mean ± SEM; N = 3 mice per group. ns, nonsignificant, *p < .05, **p < .01, ****p < .0001.
HDM challenge in vivo significantly increases human MIF production by BMDMs from CATT7 mice. Bone marrow cells were isolated from naive or HDM challenged (25 μg of house dust mite intranasally three times for 3 weeks) WT and CATT7 mice. BMDMs were generated and the concentration of human MIF released into the supernatant from A naive or B CATT7 BMDMs was measured by ELISA. Data are presented as mean ± SEM; N = 4–6 per group. ND, not detected, **p < .01; ****p < .0001.
Enhanced pro‐inflammatory priming in BMDMs from humanized MIF mice following HDM challenge is MIF specific (Blockade via SCD‐19). (A) MIF antagonist SCD‐19 is administered intraperitoneally twice a week for three weeks in a HDM‐induced model of allergic asthma. Bone marrow cells containing HSPCs were isolated from WT and CATT7 on Day 18, 4 h after last challenge. BMDMs were generated by culturing cells in M‐CSF supplemented cRPMI for 7 days. After being stimulated with polarizing conditions; LPS (M1) or IL‐4 (M2) for 24 h, (B–F) M1 and (G,H) M2 gene expression and/or protein production was analyzed by RT‐PCR and ELISA. Data are presented as mean ± SEM; N = 3 mice per group. ns, nonsignificant, *p < .05, **p < .01, ***p < .001, ****p < .0001.
Methyltransferase inhibition blocks HDM‐induced innate immune training effect in HSPCs from CATT7 mice. (A) Experimental design. After isolation from the bone marrow of CATT7 mice, bone marrow cells containing HSPCs were exposed to MTA or vehicle control 30 min prior to HDM challenge on day 0. HDM was washed out after 24 h. Cells were allowed a rest period from Days 6 to 10, before being stimulated with LPS for 24 h. (B) TNFα and (C) IL‐6 protein production in differentiated BMDM supernatants were analyzed by ELISA. Data are presented as mean ± SEM; N = 3–6 mice per group. ns, nonsignificant, *p < .05, ***p < .001.
The human MIF polymorphism CATT7 enhances pro‐inflammatory macrophage polarization in a clinically relevant model of allergic airway inflammation

March 2024

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1 Citation

High level expression of the pro‐inflammatory cytokine macrophage migration inhibitory factor (MIF) has been associated with severe asthma. The role of MIF and its functional promotor polymorphism in innate immune training is currently unknown. Using novel humanized CATT7 MIF mice, this study is the first to investigate the effect of MIF on bone marrow‐derived macrophage (BMDM) memory after house dust mite (HDM) challenge. CATT7 BMDMs demonstrated a significant primed increase in M1 markers following HDM and LPS stimulation, compared to naive mice. This M1 signature was found to be MIF‐dependent, as administration of a small molecule MIF inhibitor, SCD‐19, blocked the induction of this pro‐inflammatory M1‐like phenotype in BMDMs from CATT7 mice challenged with HDM. Training naive BMDMs in vitro with HDM for 24 h followed by a rest period and subsequent stimulation with LPS led to significantly increased production of the pro‐inflammatory cytokine TNFα in BMDMs from CATT7 mice but not WT mice. Addition of the pan methyltransferase inhibitor MTA before HDM training significantly abrogated this effect in BMDMs from CATT7 mice, suggesting that HDM‐induced training is associated with epigenetic remodelling. These findings suggest that trained immunity induced by HDM is under genetic control, playing an important role in asthma patients with the high MIF genotypes (CATT6/7/8).


Epithelial miR‐155 is elevated in active EoE and is highest in immature esophageal epithelial cells at 3D‐ALI. (A) Representative images of miR‐155 in situ hybridization on biopsies from uninflamed controls, active EoE, and inactive EoE. (B) Compartmental analysis on the individual esophageal layers shows that miR‐155 was highest within the basal and suprabasal regions in active EoE and further reduced through therapeutic intervention in inactive EoE. (C) Total epithelial miR‐155 was also evaluated by merging the data of the three constituent esophageal layers and found that EoE patients display significantly higher miR‐155 than uninflamed controls and inactive EoE individuals. (D) EPC2‐hTERTs were grown in organotypic 3D‐ALI culture and harvested on days 2, 7, and 11. The expression of miR‐155 was elevated during immature epithelial developmental time points on (E) days 2 and 7. Predicted miR‐155 targets CLDN1, TJP1, OCLN, and PTEN were investigated by qRT‐PCR and found to display inverse kinetics to the expression of miR‐155 at 3D‐ALI. Statistical significance of ISH data was assessed using a Kruskal–Wallis test with a Dunn's multiple comparisons test. 3D‐ALI data were assessed for significance using a one‐way ANOVA with Tukey's multiple comparisons test. *p ≤ .05, **p ≤ .01, ***p ≤ .001. Data are presented as means ± SEM and represent three experimental repeats.
Extended experimental hypoxia induces miR‐155 and differentially regulates its targets. (A) Complimentary binding of the HIF1A 3′UTR to that of the miR‐155 seed region. (B) HIF1A and miR‐155 expression was assessed by qRT‐PCR from esophageal epithelial cells exposed to a time course (0, 4, 24, 48, and 72 h) of experimental hypoxia compared with time‐matched normoxic controls. Inverse linear regression analysis was carried out to show the significantly inverse relationship between the expression of miR‐155 and HIF1A in experimental hypoxia. (C) HIF‐1α expression was assessed by Western blotting of nuclear isolates from esophageal epithelial cells exposed to a time course (0, 4, 24, 48, and 72 h) of experimental hypoxia compared to time‐matched normoxic controls. (D and E) qRT‐PCR analysis of HIF2A and HIF1B (D), and miRNA processing genes DROSHA, DICER, and AGO2 (E). For Western blots, a representative for a single time course is presented. Statistical significance was assessed using a Students' t‐test comparing time point–matched normoxic controls with hypoxic samples. *p < .05, **p < .01, ***p < .001 (n = 3). Data are presented as means ± SEM and represent three experimental repeats.
Stable miR‐155‐5p overexpression reduces HIF‐1α signaling and induces a deficient epithelial barrier. (A) Submerged monolayer cultures of miR‐155OE cells show a significant elevation in miR‐155. qRT‐PCR of miR‐155 targets showed attenuation of HIF1A in miR‐155OE cells and a significant decrease in OCLN. We did not observe significant differential expression in the remaining targets CLDN1, TJP1, PTEN, or WEE1 in submerged culture. (B) Using stratified squamous 3‐dimensional air–liquid interface (3D‐ALI) cultures, miR‐155 overexpression diminished barrier as measured by transepithelial electrical resistance (TEER) (n = 4). (C) H&E‐stained sections from miR‐155OE cells cultured at 3D‐ALI indicate perturbed stratification. Black scale bar represents 60 μm. These data suggest that in the setting of miR‐155OE, epithelial defects exist. (D) qRT‐PCR of barrier‐forming molecules showed a significant decrease in the tight junction CLDN7. (E) Whole‐cell protein was analyzed by Western blot whereby claudin‐7 was shown to be attenuated in miR‐155OE cells in 3D‐ALI. Submerged culture data were analyzed for significance using a Students' t‐test comparing time point–matched controls. *p < .05, **p < .01, ***p < .001. Data are presented as means ± SEM and represent a minimum of three experimental repeats.
MiR‐155 overexpression dysregulates esophageal epithelial function. RNA sequencing of miR‐155OE and control cells cultured at 3D‐ALI shows impaired epithelial function. (A) Significance was attributed to differentially expressed genes with an adjusted p‐value of ≤0.1, and yielded 627 downregulated genes and 1026 upregulated genes. A selection of genes was selected which have functions within cytoskeleton and epithelial organization or have been validated in the literature to contribute to EoE pathophysiology. (B) GO analysis for pathway enrichment for upregulated and downregulated pathways in miR‐155OE cells. (C) Venn diagram showing the number of genes differentially expressed as identified by publicly available RNA‐seq of patients with inflammatory EoE as compared to controls (cut‐off of fold change >2 or <0.5, adjusted p‐value <.05) and in miR‐155OE cells in 3D‐ALI cultures for 11 days. Genes overlapping between these two data sets were identified (167 genes) and a selection with functions relevant to EoE pathophysiology was graphed into a heatmap. GO analyses of the miR‐155OE‐EoE overlap gene set depicted localization of cellular components and enrichment of diseases based on the 167 gene overlap based on adjusted p‐value (−log10) showed a selection of diseases with perturbed epithelial function including Clouston syndrome, Netherton syndrome, palmoplantar keratosis, and eosinophilic esophagitis. GO analysis of the 167 intersected genes' enriched pathways involved “intermediate filament,” “keratin filament,” and “intermediate filament cytoskeleton” which shared five genes. These pathways reveal a dysregulation in cytoskeletal arrangement of miR‐155OE cells. (D) Immunofluorescent‐stained miR‐155OE organoids show no difference in basal KRT14, however, miR‐155OE organoids display less intense and mislocalized KRT4. (E) qRT‐PCR of miR‐155OE showing differential expression of an array of basal and suprabasal keratins.
MiR‐155‐5p overexpression attenuates CLDN7 expression and localization in esophageal epithelial organoids. (A) Schematic showing normal esophageal organoid culture development. (B) qRT‐PCR of the key tight junction molecules of the esophagus showed a significant decrease in OCLN and CLDN7. Western blots of whole‐cell protein isolated from miR‐155OE organoids displayed (C) attenuated claudin‐7 expression. (D) H&E staining shows dilated intracellular‐like spaces evident within the basal region of miR‐155OE organoids. Immunofluorescent staining shows perturbed claudin‐7 localization in miR‐155OE organoids, note the attenuated perimembranous staining of claudin‐7 within the miR‐155OE organoids compared to controls. (E) Representative H&E staining of claudin‐7KD and shRNA control organoid showing dilated spaces in claudin‐7KD compared to controls. (F) Claudin‐7 promoter showing putative binding sites for transcription actors HNF4α and PU.1 of which are predicted miR‐155 targets. Data were analyzed for significance using a Students' t‐test comparing controls. *p < .05, **p < .01, ***p < .001. Data are presented as means ± SEM and represent three experimental repeats.
Hypoxia‐inducible microRNA‐155 negatively regulates epithelial barrier in eosinophilic esophagitis by suppressing tight junction claudin‐7

December 2023

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19 Reads

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3 Citations

MicroRNA (miRNA)‐mediated mRNA regulation directs many homeostatic and pathological processes, but how miRNAs coordinate aberrant esophageal inflammation during eosinophilic esophagitis (EoE) is poorly understood. Here, we report a deregulatory axis where microRNA‐155 (miR‐155) regulates epithelial barrier dysfunction by selectively constraining tight junction CLDN7 (claudin‐7). MiR‐155 is elevated in the esophageal epithelium of biopsies from patients with active EoE and in cell culture models. MiR‐155 localization using in situ hybridization (ISH) in patient biopsies and intra‐epithelial compartmentalization of miR‐155 show expression predominantly within the basal epithelia. Epithelial miR‐155 activity was evident through diminished target gene expression in 3D organotypic cultures, particularly in relatively undifferentiated basal cell states. Mechanistically, generation of a novel cell line with enhanced epithelial miR‐155 stable overexpression induced a functionally deficient epithelial barrier in 3D air–liquid interface epithelial cultures measured by transepithelial electrical resistance (TEER). Histological assessment of 3D esophageal organoid cultures overexpressing miR‐155 showed notable dilated intra‐epithelial spaces. Unbiased RNA‐sequencing analysis and immunofluorescence determined a defect in epithelial barrier tight junctions and revealed a selective reduction in the expression of critical esophageal tight junction molecule, claudin‐7. Together, our data reveal a previously unappreciated role for miR‐155 in mediating epithelial barrier dysfunction in esophageal inflammation.


MicroRNA Dysregulation and Therapeutic Opportunities in Esophageal Diseases

May 2023

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4 Citations

AJP Gastrointestinal and Liver Physiology

MicroRNAs (miRNAs) are a class of small endogenous RNA molecules between 18 to 25 nucleotides long. The primary function of miRNAs is in the posttranscriptional regulation of mRNA targets through RNA interference culminating in mRNA degradation or translational repression. MiRNAs are fundamental in physiological and pathological processes such as, cell proliferation, differentiation, apoptosis, and inflammation. Among this includes the uncovered potential of miRNAs in overall esophageal disease with a focus on the clinicopathologic allergic disease eosinophilic esophagitis (EoE), gastroesophageal reflux disease (GERD) and the tumorigenic continuum from Barrett's esophagus towards esophageal adenocarcinoma (EAC). Although these pathologies are distinct from one another, they share pathophysiological elements such as an intense inflammatory milieu, esophageal dysfunction, and, as presented in this review, an overlap in miRNA expression which contributes to overall esophageal disease. The overlap in the dysregulated miRNA transcriptome of these pathologies highlights the key role miRNAs play in contributing to esophageal disease progression. Owing to this notable dysregulation, there is an attractive utility for miRNAs as less-invasive diagnostic and prognostic biomarkers in esophageal diseases which already require invasive endoscopies and biopsy retrieval. In this review, miRNAs within EoE, GERD, BE, EAC and esophageal achalasia are discussed, as well as reviewing a core set of miRNAs shared in the disease progression among some of these pathologies, as well as the potential utility of targeting miRNAs as therapeutic options in overall esophageal disease.



Mucosal associated invariant T cells are altered in patients with Hidradenitis Suppurativa and contribute to the inflammatory milieu

January 2022

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108 Reads

Mucosal Associated Invariant T cells are a population of innate T cells, which express the invariant T cell receptor (TCR) alpha chain Va7.2 and are capable of robust rapid cytokine secretion, producing a milieu of cytokines including IFN-gamma; and IL-17. MAIT cells have been reported in multiple human tissues including the gut, periphery and skin. On-going research has highlighted their involvement in numerous inflammatory diseases ranging from rheumatoid arthritis and obesity to psoriasis. Hidradenitis Suppurativa (H.S) is a chronic inflammatory disease of the hair follicles, resulting in painful lesions of apocrine-bearing skin. Several inflammatory cytokines have been implicated in the pathogenesis of H.S including IL-17. The role of MAIT cells in H.S is currently unknown. In this study we show for the first time, that MAIT cells are altered in the peripheral blood of patients with H.S, with reduced frequencies and an IL-17 cytokine bias. We show that CCL20 expression is elevated in lesions of patients with H.S, and MAIT cells can actively traffic towards lesions via CCL20. We show that MAIT cells can accumulate in the lesions from patients with H.S. when compared to adjacent skin, with an IL-17 bias. We show that elevated IL-17, can be linked to the activation of dermal fibroblasts, promoting the expression of chemotactic signals including CCL20 and CXCL1. Finally, we show that targeting the IL-17A transcription factor RORyt robustly reduces IL-17 production by MAIT cells from patients with H.S. Collectively our data details IL-17 producing MAIT cells as a novel player in the pathogenesis of H.S and highlights the potential of RORyt inhibition as a novel therapeutic strategy.

Citations (4)


... We have demonstrated exacerbated HDM-induced airway inflammation in mice expressing the human MIF CATT 7 allele [4,6]. Recently, using in vitro assays and an in vivo model of trained immunity, we identified a novel role for the high-expression MIF CATT 7 allele in significantly enhancing HDM-induced trained immunity in mouse bone marrow-derived macrophages (BMDMs) [5]. ...

Reference:

Mesenchymal stromal cells dampen trained immunity in house dust mite-primed macrophages expressing human MIF polymorphism
The human MIF polymorphism CATT7 enhances pro‐inflammatory macrophage polarization in a clinically relevant model of allergic airway inflammation

... MiRs are a short RNA fragment that control biologic processes including allergic responses. Mechanistically, these molecules may participate in the generation of the associated pathogenetic immune response or healing in EoE [45][46][47][48]. In a study of 22 adults, measurements of MiR-4688 was found to associate with the diagnosis of EoE [49]. ...

Hypoxia‐inducible microRNA‐155 negatively regulates epithelial barrier in eosinophilic esophagitis by suppressing tight junction claudin‐7

... MiRNA is non-coding RNA that can regulate the transcription or post-transcriptional stability of mRNA, and participate in a series of physiological activities of cells, including metabolism, cell differentiation, etc. [24][25][26]. MiRNAs play critical roles in guiding changes in your cell's transcripts and subsequently its form and functions during EMT [27][28][29]. This study aims to further clarify the biological effects of miR-1199-5p targeting SRD5A2 on the regulation of hypospadias cells and the molecular mechanism of EMT transformation. ...

MicroRNA Dysregulation and Therapeutic Opportunities in Esophageal Diseases
  • Citing Article
  • May 2023

AJP Gastrointestinal and Liver Physiology

... A similar finding was published in the case of patients with a chronic HIV-1 infection, where Vα7.2 + /CD161 − accumulated and MAIT cells decreased compared to in uninfected healthy controls, despite an unchanged total T cell number [13]. The active roles of MAIT cell subsets were identified in the pathogenesis of skin diseases, such as psoriasis and hidradenitis suppurativa, where they were found to be another alternative source of IL-17, increasing the severity of disease [14,15]. ...

Mucosal-Associated Invariant T Cells Are Altered in Patients with Hidradenitis Suppurativa and Contribute to the Inflammatory Milieu
  • Citing Article
  • December 2022

Journal of Investigative Dermatology