bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of myeloid derived suppressor cells in regulating herpes stromal keratitis
Authors: Sarkar Roman , Sehrawat Sharvan
Published: January 2023

Myeloid derived suppressor cells (MDSCs) are heterogeneous subsets of immature myeloid cells. Phenotypically such cells are identified as CD11b+Gr1lo-int cells in mice and CD33+CD11+LIN−HLA-DR− cells in humans. That MDSCs expand and modify the disease outcome in cancers and some infections is known. In this study, we investigated the role of MDSCs during HSV1 induced herpes stromal keratitis (HSK), an immunoblinding disease of the cornea. MDSCs are expanded during the progression of HSK and effected immune suppression. MDSCs generated from bone marrow precursor cells using IL-6, IL-4 and GM-CSF inhibited CD4+ T cell activation in a contact dependent manner. Adoptive transfer of in vitro generated MDSCs in mice reduced the severity of HSK and recipient mice expressed reduction in PMNs and T effector cells in cornea. CD4+ T cells isolated from lymphoid organs of MDSCs recipient mice expressed higher levels of CD62L suggesting their hampered emigration. Pro inflammatory TH17 cells are also reduced in MDSCs recipient animals but the magnitude of regulatory T cells were increased. The promotion of foxp3+ regulatory T cells was achieved by both the conversion of non-Tregs into Tregs and stabilization of foxp3 in already committed regulatory T cells. Insights into the molecular mechanisms obtained by transcriptomic profile of two populations will be discussed.
The growth regulator human proliferation-associated 2G4 (PA2G4) protein interacts with NLRP7 which is involved in recurrent hydatidiform molar pregnancy
Authors: So Matthew , Lupfer Christopher R
Published: January 2023

Recurrent hydatidiform molar pregnancies are characterized by uncontrolled proliferation of trophoblasts resulting in the failure of the embryo and placenta to develop properly. Previous studies genetically mapped a mutation in NLRP7 as a cause for recurrent molar pregnancies. The goal of this study is to identify human proteins that interact with NLRP7, especially those that are involved in DNA acetylation and methylation, which could lead to the development of molar pregnancies. A yeast two-hybrid system using a human cDNA library was used to screen for interacting proteins, and protein interactions were verified by co-immunoprecipitation in HEK 239T cells. Additional experiments are planned to determine the functional significance of such interactions. Yeast screening results indicated several proteins that interact with NLRP7, however further testing will be focused on the interaction with Human proliferation-associated 2G4 (PA2G4), a human protein involved in growth regulation. Having potentially identified a protein involved in development through acetylation/deacetylation that interacts with NLRP7, we hope to shed more light on the molecular pathways that lead to the formation of recurrent molar pregnancies.
Connecting GWAS SNPs to Target Genes in primary human immune cell types
Authors: Chandra Vivek , Bhattacharyya Sourya , Schmiedel Benjamin J. , Madrigal Ariel , Gonzalez-Colin Cristian , Fotsing Stephanie , Kronenberg Mitchell , Peters Bjoern , Ay Ferhat , Vijayanand Pandurangan
Published: January 2023

Expression quantitative trait loci (eQTLs) studies have provided associations with gene expression for many non-coding genetic variants associated with human disease and evolution. However, they fail to pinpoint the functionally important eQTLs or provide mechanistic insights. Also, most eQTL discovery studies restrict analysis to SNPs within ±1 Mb of the TSS of the gene tested to minimize corrections for multiple association tests. Using proximity ligation-assisted H3K27ac ChIP-seq (HiChIP) assays, here we mapped eQTLs that overlap active cis-regulatory elements and interact with their target gene promoters (promoter-interacting eQTLs, pieQTLs) in five common immune cell types. This allowed us to narrow down functionally important eQTLs and show mechanisms that explain their cell type restriction. A subset of the identified pieQTLs overlapped non-transcribed promoters, which interact with their target gene promoters and function as enhancers as validated by CRISPRi. In addition, we devised a novel approach to eQTL discovery that relied on HiChIP-based promoter interaction maps as a structural framework for deciding what SNPs to test for association with gene expression. We discovered ultralong distance pieQTLs (>1Mb from target gene), including several disease-risk variants and new eGenes such as FHIT for which we validated the role of an ultralong distance pieQTL by CRISPRi. Our study provides insights into cell-specific gene regulatory mechanisms and highlights the potentially functional non-coding variants (pieQTLs) linked to human diseases.
Regulatory T cells suppress novel skeletal muscle macrophages identified by single-cell RNA sequencing
Authors: COULIS Gerald , Kastenschmidt Jenna , Mannaa Ali , Nguyen Quy , Pervolarakis Nicholas , Duarte Jorge , Emami Melissa , Yang Catherine , Kessenbrock Kai , VILLALTA Armando
Published: January 2023

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder that results from mutations in the dystrophin gene causing necrosis, inflammation and ultimately fibrosis. In DMD, muscle macrophages are multifaceted effector immune cells that regulate multiple pathogenic processes, including myofiber injury, inflammation and fibrosis, but also promote regeneration. Previously, we demonstrated that regulatory T cells (Tregs) ameliorated the severity of muscular dystrophy by regulating M1-like and M2-like macrophage activation, and more recently, a subset of undefined macrophages. Herein, we used single-cell RNA sequencing (scRNAseq) approaches to define the phenotypic complexity and transcriptional profile of muscle macrophages in healthy and dystrophic muscle. A T-distributed Stochastic Neighbor Embedding (t-SNE) analysis of gene expression data revealed that muscle macrophages clustered into six novel populations defined by unique transcriptional programs. We observed that a population expressing high levels of galectin-3 was absent in wildtype and dystrophic muscle before the onset of disease, but was increased at the onset of acute pathology and further expanded when Tregs were depleted in the mdx mouse model of DMD. In light of emerging evidence in the literature suggesting a role for galectin-3 and Tregs in fibrosis, we performed in vivo functional assays to underscore the role of the Treg-Macrophages axis in the control of fibrosis in DMD. Our data support a new paradigm, in which unique transcriptional programs define novel macrophage populations likely adapted to the diseased muscle milieu, and suggest that therapies focused on augmenting Tregs function in dystrophic muscle may be beneficial to treat fibrosis.
Functional studies to understand class II MHC as a gene modifier in cystic fibrosis
Authors: Lu Shiping , Song Kejing , Kolls Jay K.
Published: January 2023

Cystic fibrosis (CF) is an inherited life-threatening disease caused by mutated chlorine channel cystic fibrosis transmembrane conductance regulator(CFTR). Around 60% of CF patients have homozygous F508del mutation. Along with chronic bacterial infection, the integrity and function of pulmonary epithelium were compromised. Pseudomonas aeruginosa (P. a) is the most predominant pathogen, leading to decreased lung function and increased mortality in CF patients. So, prevention/eradication of airway P.a colonization is crucial to maintain lung function. Genome-wide association studies revealed human leukocyte antigen class II(HLAII) within the F508del population are associated with the age of P.a colonization and lung disease severity. However, the role of HLA class II in CF progress is not fully defined. We hypothesized that there may exist differences in antibody isotypes, titer, specificity or affinity that influence/determine lung function of CF patients. Therefore, we recruited a cohort of non-progressive F508del CF patients with confirmed P. a colonization but different lung function. Our results showed sera of CF patients have significantly higher specific IgG and IgG1 titers and stronger bacterial surface binding against mucoid P.a compared of non-CF controls. To assess antigen specificity, we performed immunoprecipitation followed by proteomics, that antibodies from CF sera have a common target, outer membrane protein OprI. Besides, CF sera inhibited bacterial biofilm formation time-dependently compared to non-CF serum. Taken together these studies may shed light on the role of HLA class II as a gene modifier in CF.
A Type I interferon response is associated with increased mTOR activation in idiopathic multicentric Castleman disease
Authors: Pai Ruth-Anne Langan , Japp Alberto Sada , Gonzalez Michael , Rasheed Rozena F , Okumura Mariko , Arenas Daniel , Pierson Sheila K , Layman Awo Akosua Kesewa , Kao Charlly , Hakonarson Hakon , van Rhee Frits , Betts Michael R , Kambayashi Taku , Fajgenbaum David C
Published: January 2023

Idiopathic multicentric Castleman disease (iMCD) is a deadly hematologic illness of unknown etiology involving episodic disease flares characterized by systemic inflammation and multiple organ system dysfunction. We previously showed that the mTOR signaling pathway is elevated in lymph nodes of iMCD patients and that an mTOR inhibitor is effective in preventing disease flares in a small cohort of patients. However, the upstream mechanisms and the cell types and mediators involved in disease pathogenesis remain unknown. Here, we developed a targeted approach to identify candidate cellular drivers and mechanisms in iMCD through flow cytometric and single-cell transcriptomic analysis. Using paired PBMC samples collected from ten iMCD patients during flare and later following resolution of flare (remission), we identified increased T cell activation and alterations in NK cell and monocyte subset frequencies during iMCD flare. These changes were associated with increased Type I Interferon (IFN-I) response gene signatures across CD8+ T cells, NK cells, and monocytes as well as mTOR signaling gene signatures within monocytes. Finally, we demonstrated that increased mTOR activation in iMCD may occur downstream of IFN-I signaling as we observed significantly greater induction of pS6, a readout of mTOR activation, in monocytes and T cells from iMCD remission samples compared to healthy donors. Importantly, this IFN-I-mediated mTOR activation can be abrogated through treatment with the JAKi, ruxolitinib. The data presented here corroborate the notion of widespread systemic inflammation during iMCD disease flare and suggest a role for hyperresponsiveness to IFN-I as a mechanistic driver of increased mTOR signaling in iMCD.
Epigenetic Regulation of the COX-2 Pathway Modulates Macrophage Inflammation in Diabetic Wound Repair
Authors: Davis Frank M , Dendekker Aaron , Joshi Amrita , Wilke Carol , Deng Hongping , Smith Andrew , Obi Andrea , Huang Steve , Robinson Scott , Melvin William J , Fuentes Jaime , Kunkel Steven L , Moore Bethany B , Gallagher Katherine
Published: January 2023

Macrophage plasticity is critical for normal tissue repair to ensure transition from the inflammatory to proliferative phase of healing. In pathologic conditions, such as type 2 diabetes (T2D), wounds fail to heal due to impaired resolution of inflammation. There is increasing evidence that epigenetic-based mechanisms control macrophage function. The cyclooxygenase (COX)-2/Prostaglandin E2 (PGE2) axis, as well as upstream pathways including cytosolic phospholipase A2 (c(PL)A2), have been associated with chronic inflammation, however the underlying mechanisms of COX-2/PGE2 regulation in wound repair have not been examined. Here, we find in human and murine diabetic wounds, that PGE2 production is elevated in wound macrophages and regulates inflammation. Further, epigenetic modification of the COX-2/PGE2 pathway was regulated by MLL1, a methyltransferase, that increased H3K4 trimethylation on the cPLA2 promoter in diabetic wound macrophages and human T2D monocytes resulting in increased cPLA2 expression. Myeloid-specific deletion of MLL1 restored basal cPLA2 levels. Separately, increased TGFβ1 in diabetic wounds augmented miR-29b and DNMT 3a/3b in wound macrophages, resulting in hypomethylation of the Cox-2 gene promoter and overexpression of COX-2/PGE2. Inhibition of the PGE2 pathway, with a macrophage-specific nanocarrier containing a COX-2 inhibitor improved diabetic healing. Taken together, our results indicate the COX-2/PGE2 pathway is a critical regulator of macrophage-mediated inflammation in diabetic wounds and therapeutic manipulation of this pathway improves pathologic wound repair.
Wildfire smoke exposure is associated with decreased methylation of the PDL2 gene
Authors: Prunicki Mary , Miller Sarah , Hopkins Austin , Poulin Matthew , Movassagh Hesam , Yan Liying , Nadeau Kari Christine
Published: January 2023

Objective

To investigate the association between wildfire smoke exposure and DNA methylation of Foxp3-related genes using targeted bisulfite specific methylation sequencing assays recently developed by EpigenDx.

Methods

Subjects (n=16) from the San Francisco Bay area were recruited and gave blood samples during wildfire smoke exposure from the Paradise, CA wildfire 165 miles away. Subjects had their blood re-drawn 3 months later when there was no smoke exposure. An immunology/Foxp3 methylation panel was developed for 20 genes covering 150 CpG sites, targeting genes known to exhibit distinctive methylation profiles in regulatory T cells. Multiplex PCR amplified the regions of interest, using minimal sample DNA and generates inserts ready for NGS adaptor ligation. By initially amplifying specific regions of bisulfite treated DNA, library preparation is enriched with a population of molecules that have potential as indicators or affecters of gene regulation. Using our EpigenDx Ion Torrent library preparation kit, we can ligate these amplified regions to barcoded adaptors in one step.

Results

Paired t-tests revealed less methylation of the PDL2 gene during wildfire exposure at sites immediately upstream of the transcriptional start sites (CpG #-85: p=.002; χ̄ wildfire= 26.3 +14.0; χ̄ no wildfire= 30.8+15.9 and CpG #-86:p=.004; χ̄ wildfire = 22.2+13.4; χ̄ no wildfire= 25.7+15.6), but not in the 5′UTR region (CpG #-84; p=.53). No significant differences were found for the other genes.

Conclusion

Wildfire smoke is associated with decreased PDL2 gene methylation. PDL2, a programmed death ligand, downregulates T cell responses and has previously been associated with airway inflammation and cancer, but not pollution exposure.
Chronic Colitis Impairs Heart Function by Inducing Epigenetic Remodeling in the Adult Heart
Authors: Li Qingjie , Kline Kevin T , Zhong Xiaoying S , Lian Haifeng , Peng Jun , Powell Don W
Published: January 2023

Background and Aims

Inflammatory bowel disease (IBD) represents a spectrum of chronic inflammatory mediated conditions with known extraintestinal manifestations. The interplay between heart and gut in IBD has previously been noted, but the mechanisms remain elusive. The aim of this study was to identify epigenetic mechanisms by which chronic colitis impairs cardiac function.

Methods

To induce chronic colitis, dextran sodium sulfate (DSS, 5% in drinking water) was given to adult rats for 5 days followed by 9 days with normal drinking water for 4 cycles over 8 weeks. Transthoracic echocardiography (TTE) was performed to evaluate heart function.

Results

DSS-induced chronic colitis led to a significant decrease in left ventricular ejection fraction on TTE (68% vs 49%, P<0.05), increased left ventricular mass (107 vs 148, P<0.05), and increased B-type natriuretic protein (222 vs 487, P<0.05). Cardiac and circulating BDNF, as well as GSK-3β, BCL2 and pAKT(S133), was markedly decreased. BDNF was also decreased in colitis induced by trinitrobenzene sulfonic acid. MiRNA profiling showed a total of 52 miRNAs significantly increased in DSS colitis, 7 of which are predicted to target BDNF. RT-qPCR subsequently validated the increase of miR-1b, Let-7d, and miR-155-5p in the adult heart by colitis. Transient transfection demonstrated that miR-1b, Let-7d, and miR-155-5p suppress BDNF in H9c2 rat myoblasts. BDNF overexpression mitigated H2O2-induced oxidative damage n H9c2 cells. Cardiac-specific conditional knockout of BDNF enlarged the heart size and impaired cardiac function.

Conclusions

Chronic colitis suppresses cardiac BDNF by elevating miRNAs; this was accompanied by biochemical and TTE findings suggestive of heart failure.
The hematopoietic transcription factor GATA2 is a novel regulator of apoptotic cell clearance by macrophages in atherosclerosis
Authors: Yin Charles , Vrieze Angela M , Akingbasote James , Pawlak Emily N , Jacob Rajesh A , Hu Jonathan , Sharma Neha , Blackler Garth , Dikeakos Jimmy D , Barra Lillian , Nagpal Dave , Heit Bryan
Published: January 2023

Atherosclerosis is an inflammatory disease involving formation of lipid-rich lesions within the arteries. Macrophages clear apoptotic cells that accumulate within these lesions. Defective apoptotic cell clearance is a hallmark of advanced atherosclerotic disease, yet mechanisms that drive this defect are poorly understood. In this study, we identify the hematopoietic transcription factor GATA2 as a novel regulator of macrophage-mediated apoptotic cell clearance.

Macrophages were isolated by laser capture microdissection from atherosclerotic lesions in aortic tissue obtained from patients undergoing open heart surgery. Gene expression profiling was performed on these macrophages by microarray, with macrophages derived from peripheral blood monocytes used as a control.

We found approximately 3,000 genes to be differentially expressed in aortic punch macrophages, with enrichment in pathways involved in apoptotic cell clearance. In particular, we identified upregulation of GATA2. Mutations in GATA2 have previously been associated with increased risk of coronary artery disease and we found that overexpression of GATA2 in vitro resulted in decreased ability of macrophages to both internalize and degrade apoptotic cells. Conversely, GATA2 downregulation is sufficient to abrogate oxLDL-induced defective apoptotic cell clearance.

Atherosclerotic macrophages exhibit dysregulated expression of genes involved in apoptotic cell clearance and upregulation of GATA2. Recapitulation of GATA2 overexpression is sufficient to impair apoptotic cell clearance in vitro. To our knowledge, we are the first to identify a potential role for GATA2 in driving defective apoptotic cell clearance in atherosclerosis.
Aerosolized Toll-like Receptor Agonists Suppress Allergic Inflammation
Authors: Goldblatt David , Fouts Adrienne , Wali Shradha M , Valverde-Ha Gabriella , Moczygemba-Martinez Margarita , Huston David P , Tuvim Michael J , Dickey Burton , Evans Scott
Published: January 2023

Recent studies have linked exposure to microbes and a reduction of allergic diseases. We sought to determine whether activation of innate immunity by aerosolized Toll-like receptor (TLR) agonists could attenuate the development of allergic inflammation in mice.

BALB/cJ mice were sensitized and challenged with House Dust Mite (HDM). Some mice were treated by aerosol with ligands for TLR9 (ODNm362) and TLR2/6 (Pam2CSK4) in combination (Pam2-ODN). Allergic inflammation was assessed by quantification of leukocytes in bronchoalveolar lavage fluid (BALF) and epithelial mucin content with periodic acid fluorescent Schiff stain (PAFS). Pam2-ODN-treated mice exhibited a 94% reduction in eosinophils, 90% reduction in lymphocytes, and a 44% reduction in epithelial mucin content. Lymphocyte subset analysis showed a 50% reduction in TH2 cells without a detectable change in any other TH subset. Using an alternate allergic inflammation model with Ovalbumin (OVA) Pam2-ODN-treated mice showed greater than 50% reductions in total and OVA-specific IgE serum concentrations. IgG2a concentrations, which are reduced in OVA-sensitized mice, returned to baseline with Pam2-ODN treatment.

There is a stark contrast of the efficacy in mice treated before and after sensitization that suggests Pam2-ODN may modulate the tendency of sensitization when exposed to aeroallergens. Previous studies have shown that lung epithelial cells are required for Pam2-ODN lung responses, and taken together with these new data, Pam2-ODN may reprogram airway epithelial cells to be tolerogenic to aeroallergens, but additional investigation is required to understand the precise molecular mechanism of Pam2-ODN in this context.
Reduced BDCA3+ dendritic cells in nasal mucosa induce severe inflammation in patients with allergic rhinitis and chronic rhinosinusitis
Authors: Park Sang Chul , Kim Tae-Gyun , Cho Hyung-Ju , Kim Chang-Hoon , Yoon Joo-Heon
Published: January 2023

Dendritic cells (DCs) are specialized antigen-presenting cells that play a crucial role in T helper (Th) cell differentiation. DCs of human airway comprise conventional DCs (cDCs) and plasmacytoid DCs (pDCs), of which the cDCs lineage consists of cDC type 1 (cDC1) and cDC type 2 (cDC2). These DCs subsets differ in ontogeny and functional properties in immune response. Thus, we aimed to evaluate the expression of different dendritic cell subsets in nasal mucosa of patients with allergic rhinitis (AR) and chronic rhinosinusitis (CRS). Nasal polyp tissues or ethmoid mucosa from patients undergoing endoscopic sinus surgery or septoplasty were collected. The expression of DCs was investigated by flow cytometry via surface markers including BDCA-1 (CD1c) for cDC1, BDCA-3 (CD141) for cDC2, and BDCA-2 (CD303) for pDC. In subjects with AR, BDCA-3+cDC levels significantly decreased in patients with accompanying CRS, more reduced in eosinophilic CRS (ECRS) than in non-eosinophilic CRS (NECRS), compared to non-CRS patients. In subjects with CRS, the expression of BDCA-3+cDC decreased in patients with accompanying AR; more reduced in multiple allergens sensitization. Consequently, the IF result showed CD4+ CD25+ Foxp3+ regulatory T cell expression was significantly decreased in CRS patients with AR compared to those without AR. Furthermore, the level of BDCA-3+cDC was also inversely related with preoperative CT scores, serum eosinophils and IgE levels. We suggest that BDCA-3+cDC may play an essential role in immunoregulation and induction of clinical tolerance. Reduced frequency of BDCA-3+cDC followed by impaired Treg expression in CRS patients with AR might reflect the mechanism of severe inflammation in patients of CRS combined with AR.
Hyperglycemia impairs tolerance and promotes allergic inflammation in mouse models of asthma
Authors: Queener Ashley , Jeong Brian M , Coden Mackenzie , Doan Ton , Walker Matthew , Nagasaka Reina , Berdnikovs Sergejs
Published: January 2023

Glucose is a key source of energy in systemic and cellular metabolism and has known pro-inflammatory properties. Metabolic disorders (diabetes, insulin resistance) associate with asthma and other allergic diseases. This study sought to understand the role of hyperglycemia in the allergic response. First, we injected fasted Balbc/J mice intraperitoneally (i.p.) with 2g/kg dextrose to test whether hyperglycemia promotes antigen sensitization. Within one hour of injection, we detected a rapid increase in blood glucose levels followed by an increase in expression of inflammatory markers Il1β, Tslp, and Cxcl9 in peritoneal tissue. Concurrently, MHCII+ cells infiltrated the peritoneum. Control experiments showed that inflammatory responses were not due to hyperosmotic effects. To determine whether glucose promotes allergic response, we sensitized mice to chicken egg ovalbumin (OVA) using either alum (standard adjuvant), dextrose, or a vehicle control. Both alum/OVA and dextrose/OVA treated mice mounted lung allergic inflammation in response to inhaled OVA antigen. Dextrose/OVA mice had OVA-specific IgE production similar to alum/OVA group, demonstrating glucose’s ability to promote sensitization. Total cellular infiltrates, bronchoalveolar lavage (BAL) eosinophils, and lung expression of IL-4, IL-13 and IL-33 in dextrose-sensitized mice were equal to or surpassing allergic inflammatory responses in mice given standard adjuvant. Finally, in a separate mouse tolerance model we demonstrated that hyperglycemia impairs development of tolerogenic response to innocuous OVA antigen. These results demonstrate a potentially critical role for glucose dysregulation in loss of tolerance and promotion of allergy.
E3 Ubiquitin Ligase Cbl-b Inhibits Type 2 Innate Lymphoid Cell Development by Targeting ST2 for Ubiquitination
Authors: Guo Hui , Huang Ling , Tang Na , Waldstein Kody , Varga Steven M , Zhang Jian
Published: January 2023

Type 2 innate lymphoid cells (ILC2) play a crucial role in driving allergic airway inflammation, but the regulation of ILC2 development is not fully understood. Previously we have shown that E3 ubiquitin ligase Cbl-b negatively regulates Th2 response, but whether Cbl-b also regulates ILC2-mediated type 2 immunity remains to be defined. In this study, we found that Rag1−/−Cblb−/− mice are hypersensitive to inhalation of A. Fumigatus, a fungus that causes allergic asthma, as revealed by heightened airway hyperresponsiveness (AHR), increased airway inflammation, and mucus production. This hypersensitivity is associated with increased frequencies of ILC2 and eosinophils, but reduced frequency of alveolar macrophages in the lungs. Consistent with this, type 2 cytokines are also increased in the lung homogenates of Rag1−/−Cblb−/− mice. At the molecular levels, ST2 undergoes ubiquitination and proteasome-mediated degradation upon IL-33 stimulation, which is abrogated in cells expressing Cbl-b C373A mutant. Therefore, our data indicate that Cbl-b inhibits the development of ILC2 and ILC2-mediated airway inflammation by targeting ST2 for ubiquitination.
Autophagy is critical for group 2 innate lymphoid cell metabolic homeostasis and effector function
Authors: Galle Lauriane , Hurrell Benjamin P , Lewis Gavin , Howard Emily , Jahani Pedram Shafiei , Banie Homayon , Razani Babak , Soroosh Pejman , Akbari Omid
Published: January 2023

Allergic asthma is a chronic inflammatory disorder that is characterized with airway hyperreactivity (AHR) and driven by Th2 cytokine production. Group 2 innate lymphoid cells (ILC2s) secrete high amount of Th2 cytokines and contribute to the development of AHR. Autophagy is a cellular degradation pathway that recycles cytoplasmic content. However, the role of autophagy in ILC2s remains to be fully elucidated. We characterized the effects of autophagy deficiency on ILC2 effector functions and metabolic balance. ILC2s from autophagy deficient mice were isolated to evaluate homeostasis, cytokine secretion, gene expression and cell metabolism. Also, autophagy deficient ILC2s were adoptively transferred into Rag−/−GC−/− mice, which were them challenged with IL-33 and assessed for AHR and lung inflammation. We demonstrate that autophagy is extensively used by activated ILC2s to maintain their homeostasis and effector functions. Deletion of the critical autophagy gene Atg5 resulted in decreased cytokine secretion and increased apoptosis. Moreover, lack of autophagy among ILC2s impaired their ability to utilize fatty acid oxidation and promoted glycolysis as evidenced by our transcriptomic and metabolite analyses. This shift of fuel dependency led to impaired homeostasis and Th2 cytokine production, thus inhibiting the development of ILC2-mediated AHR. Notably, this metabolic reprogramming was also associated with an accumulation of dysfunctional mitochondria producing excessive reactive oxygen species. These findings provide new insights into the metabolic profile of ILC2s and suggest that modulation of fuel dependency by autophagy is a potentially new therapeutic approach to target ILC2 dependent inflammation.
o-Coumaric acid attenuates atopic dermatitis-like skin inflammation
Authors: Kim Hyemi , Kim Sang-Hyun
Published: January 2023

Background

Hydroxycinnamic acids (coumaric acid; CA) and their derivatives display anti-oxidant and anti-inflammatory effects. CA has three isomers including o-CA, m-CA, and p-CA. Among them, recent report showed the effectiveness of p-CA on skin inflammation.

Objective

In this study, we investigated the role of o-CA on atopic dermatitis (AD) comparing with p-CA.

Methods

For the induction of atopic dermatitis (AD)-like skin inflammation, 1,4-dinitrochlorobenzene (DNCB) and house dust mite extract (Dermatophagoides farinae extract, DFE) were applicated repeatedly, and o-CA were orally administered. For mechanism study, tumor necrosis factor (TNF)-α and interferon (IFN)-γ stimulated human keratinocyte cell line (HaCaT) was used.

Results

In AD-like skin inflammation, the clinical symptoms including the redness, dermal/epidermal thickness, and infiltration of mast cells were significantly reduced by the oral treatment of o-CA. Local inflammatory cytokines expression and systemic production of IgE and IgG2a were also suppressed by the o-CA. In addition, o-CA inhibited the expression of interleukin (IL)-1β, IL-6 and CC-chemokine ligand 17 in TNF-α/IFN-γ stimulated HaCaT cells by the reduction of nuclear translocation of nuclear factor-κB. In all cases, o-CA showed more effectiveness than p-CA.

Conclusions

These results showed that o-CA could be a potential candidate for AD treatment.
Immune Sensing of Aeroallergen-Associated Double-Stranded RNA Triggers an IFN Response and Modulates Type 2 Lung Inflammation
Authors: Alanazi Hamad , She Li , Yan Liping , Sun Yilun , Zou Yi , Dube Peter , Brooks Edward , Barrera Gema , Lai Zhao , Chen Yidong , Liu Yong , zhang Xin , Li Xiao-Dong
Published: January 2023

Over 20 million Americans are affected by allergies such as asthma and allergic rhinitis, which can be caused by common allergens like house dust mites (HDM) or their associated components. The exacerbation of allergies can be fatal in patients with pre-existing chronic lung inflammation. Until now, there is no cure for asthma and type 2 allergies and the only available treatments aim to reduce the disease symptoms. Thus, there is an urgent need to understand the pathogenic mechanism of HDM-induced allergic responses. Since HDM play a major role in triggering allergic reactions, several studies focused on identifying their constituents. Although it is important to study HDM protein components that elicit adaptive type 2 inflammation (e.g. Der p 1), the role of innate sensors such as Toll-like receptors (TLRs) is unknown. A number of studies investigated presence of common adjuvants that may activate host immune receptors and modulate allergic diseases, for example, lipopolysaccharide (LPS) contaminating HDM triggered asthma in mice through acting on TLR4. In addition, single and double-stranded nucleic acids from bacteria or viruses can also act as adjuvants that stimulate innate immune receptors. Clearly, diverse allergen-associated components play an essential role in modulating type 2 lung inflammation in allergic patients. Recently, we have shown that HDM contain immunogenic RNA capable of modulating type 2 lung inflammation by inducing protective interferon responses. Using RNA-seq analysis, we show that HDM contain previously uncharacterized double stranded RNA sequences derived from novel RNA-viruses. Our findings suggest that other allergens may also have immunostimulatory RNA that can regulate type 2 lung inflammation.
IL-33 and allergen exposure promote development of “memory-like” ILC2s in the Airways
Authors: Ferrini Maria M , Jaffar Zeina , Roberts Kevan
Published: January 2023

The alarmin, IL-33 plays a critical role in the inception of lung inflammation and the exacerbation of allergic airway disease. Inhalation of environmental allergens and particulates is known to promote IL-33 release, and the cells responsive to this cytokine include group 2 innate lymphoid cells (ILC2s), dendritic cells and natural killer (NK) cells. In this study, we evaluated the effect of IL-33 and house dust mite allergen inhalation on the properties of lung ILC2s. Instillation of IL-33 into the airways of mice resulted in a marked elevation in number of ILC2s and eosinophils. Importantly, a notable feature of the inflammatory response was an increase in CD103 expression by ILC2 (from 3% to 45%). These CD103+ ILC2s appeared to represent memory-like ILC2s since they were highly responsive to IL-25 and produced significant levels of IL-9 and IL-13. Critically, inhalation of house dust mite allergen also increased CD103 expression by ILC2s. Given that CD103 expression can be induced by TGF-β, we next determined whether active TGF-β1 is released following IL-33-driven lung inflammation. Remarkably, the levels of active TGF-β1 present in the airways were markedly raised following IL-33 instillation, and this was responsible for a reduction in IFN-γ-producing NK cells and raised CD226 (DNAM-1) expression. Collectively, these findings reveal that IL-33 and allergen promote the accumulation of CD103+ memory-like ILC2s in the airways. A role for TGF-β in promoting CD103 expression while suppressing NK cell-mediated regulation of the inflammatory process is proposed.
The emerging roles of neutrophils and macrophages during experimental acute pyelonephritis
Authors: de Dios Ruiz Rosado Juan , Robledo-Avila Frank , Becknell Brian , Partida-Sanchez Santiago
Published: January 2023

Every year, pediatric urinary tract infections (UTI) account for 1.5 million clinician visits in the US alone. Up to 50% of infants with UTI develop a kidney infection (acute pyelonephritis, APN). Despite antibiotic therapy, 15% of children with APN develop renal scarring, which is associated with deterioration in kidney function. Here, we determined the contribution of macrophages and neutrophils to bacterial clearance and the development of kidney fibrosis during experimental APN.

Female C3H/HeOuJ mice were treated, via retro-orbital, with one of the following antibodies: isotype IgG (control), anti-Ly6G (depletes neutrophils), anti-CD115 (depletes monocytes), and anti-GR1 (depletes monocytes and neutrophils). After cell depletion, the animals were transurethrally infected with UPEC. Ascending UTI was evaluated via biophotonic imaging. The dynamics of monocytes and neutrophils were determined by flow cytometry. Histopathological scores were performed by H&E and Sirius-Red stainings. Transcriptomic analyses of kidneys were performed using TaqMan Arrays.

Our data indicate that neutrophils are required for bacterial clearance, while macrophages promote inflammation in the infected urinary tract. However, the absence of neutrophils results in increased macrophage infiltration and exaggerated macrophage-dependent inflammatory responses in the bladder and kidney. Also, our results demonstrate that macrophage-dependent inflammation during APN, leads to renal scarring and renal functional impairment. These findings uncover a role for the neutrophil-macrophage imbalance in promoting kidney fibrosis during APN. This knowledge will be helpful to develop novel therapies to prevent post-APN scarring in children.
Enhancement of vitamin C on PMN function in healthy individuals
Authors: Ruknarong Laongthip , Nonsa-ard Rujira , Chuangchot Nisa , Jumnainsong Amonrat , Leelayuwat Naruemon , Jusakul Apinya , Leelayuwat Chanvit
Published: January 2023

Vitamin C (ascorbic acid, ascorbate) promotes immune cell activities by supporting various cellular functions especially, in neutrophils or polymorphonuclear (PMN) leukocytes, which play an important role in bacterial infection and acute inflammation. Little data have been reported in impact of vitamin C on PMN function during bacterial phagocytosis in healthy individuals. Here, we have elucidated the effect of vitamin C on PMN phagocytosis and oxidative burst to Staphylococcus aureus by whole blood assay and flow cytometry after pre-incubation without and with vitamin C in vary concentrations. The results showed that vitamin C concentration at 20 mM induced a significant increase phagocytosis (p-value=0.03) and had trend to significant increase oxidative burst (p-value=0.06) while vitamin C concentration of 30 mM showed significant increase both phagocytosis and oxidative burst (p-value=0.04 and 0.02, respectively) at only 15 min of exposure to the bacteria. As a minimal concentration that can increase phagocytosis, we selected vitamin C at 20 mM as optimal concentration to investigate more data by enrollment of healthy volunteers (n=17). The results revealed significant difference both phagocytosis and oxidative burst after 20 mM vitamin C pre-incubation (p<0.0001). In conclusion, we showed that vitamin C at 20 mM can boost phagocytosis and oxidative burst of neutrophils in healthy adults within 15 min after bacterial exposure. This rapid activation may be applied to fight bacterial infection. Nevertheless, further research in clinical trial and underlying mechanisms await further studies.
Identification of N-formylated peptides as the neutrophilic chemotactic factors of Mycobacterium tuberculosis
Authors: Dean Kristin T , Jung Bockgie , Samten Buka
Published: January 2023

Since tuberculosis (TB) remains a major cause of human death, it is urgent to understand the pathology of TB. Lung neutrophil infiltration initiates granuloma formation and is associated with disease severity. Though several cytokines and chemokines are associated with neutrophil infiltration in chronic inflammation, neutrophil chemotactic factors of Mycobacterium tuberculosis (Mtb) remain unexplored. Thus, we performed neutrophil chemotactic assays by culturing neutrophils of healthy human blood samples and mouse bone marrow in the upper chamber of a transwell plate with 5–8 μm pores with the culture filtrates of Mtb H37Rv (CF) in the lower chamber using N-formyl-met-leu-phe (fMLF), a bacterial neutrophil chemotactic tripeptide, as a positive control. The cell numbers in the lower chamber were determined by flow cytometry as a chemotactic indicator. Our results show that CF induced chemotaxis of both human and mouse neutrophils in an Mtb culture period dependent manner. Testing CF of 11 clinical isolates of Mtb also showed neutrophil chemotactic activity. Sulfasalazine, a formylated peptide receptor inhibitor, blocked chemotaxis of neutrophils, indicating the involvement of fPRs in CF induces chemotaxis of neutrophils. Mass spectrometry analysis of CF identified three candidate N-formylated heptapeptides. Authentication of the chemotactic activities of the identified peptides with synthetic mimetics confirmed their neutrophil chemotactic activities and indicated the requirement of N-formylation for their chemotactic activities. Thus, we have identified novel formylated peptides of Mtb with neutrophil chemotactic activities, and further studies are in progress to determine their significance in TB infection.
The TRPM2 ion channel regulates the inflammatory response of neutrophils during Listeria monocytogenes infection
Authors: Robledo-Avila Frank Hanz , de Dios Ruiz Rosado Juan , Brockman Kenneth L , Partida-Sanchez Santiago
Published: January 2023

Calcium signaling is required for most antimicrobial functions of neutrophils. The transient receptor potential melastatin-2 (TRPM2) cation channel has been proposed to play important roles in modulating calcium mobilization, cell migration and oxidative stress in neutrophils. However, the precise mechanism by which TRPM2 activation may exert a pro- or anti-inflammatory function remains unclear. In this study, we used a mouse model of Listeria monocytogenes infection to define the role of TRPM2 in the regulation of neutrophils’ function. We found that infected Trpm2−/− mice were associated with high bacterial burden in spleen and liver indicating increased susceptibility to Listeria infection. Pronounced migration rates of neutrophils and monocytes to the liver and spleen characterized the inflammatory response to Listeria during the first 18h. In the Trpm2−/− mice, the acute phase infection resulted in septic shock, defined by increased serum levels of TNF-α, IL-6, and IL-10, but no decreased levels of IFN-γ or IL-12. Furthermore, depletion of neutrophils demonstrated the critical role of these cells in regulating acute inflammation by conferring resistance of Trpm2−/− mice to Listeria infection. Gene expression and inflammatory cytokine analyses of infected tissues further confirmed the hyperinflammatory profile of Trpm2−/− neutrophils. Finally, the increased inflammatory properties of Trpm2−/− neutrophils correlated with cellular calcium overloading and potentiated membrane depolarization, in response to Listeria infection. In conclusion, our findings suggest that TRPM2 channel plays critical roles in the regulation of the inflammatory properties of neutrophils during Listeria monocytogenes infection.
Role of invariant nature killer T cell in dysbiosis of gut microbiota among Mycobacterium tuberculosis infected patients
Authors: Huang Shiang-Fen , Su Wei-Juin
Published: January 2023

Background

In Mycobacterium tuberculosis (MTB)-infected patients, the dysbiosis in gut microbiota was associated with systemic proinflammation. The invariant nature killer T (iNKT) cell was suggested the T-cell involved in immune modulation activated by metabolites or structural molecules of microbial in gut lumen. Herein we investigated how the gut microbiota influence on the systemic immunity by study the iNKT cells in peripheral blood in human.

Method

Patients with active TB, latent TB infection (LTBI), and healthy controls (HC) were enrolled. Interleukin (IL)-10, IL-4, IL-6, and IL-1B levels, the CD3+, CD4+, CD8+ cells, iNKT (CD3+6B11+) cells counts in blood, proliferation index (PI) of iNKT cells by α-galactosylceramide (α-GalCer) stimulation in vitro, and the interferon-gamma (IFN-γ) concentration by IFN-r-releasing-assay (IGRA) were measured.

Results

In active TB, both the cell counts and PI of iNKT were decreased. In LTBI, the PI of iNKT cells and the stimulated IFN-γ by MTB-Antigen divided to the iNKT cell counts (IFN-TBAg/iNKT) were positively associated with the relative abundance of Firmicutes. In active TB, a higher iNKT cell counts were also associated with higher relative abundance of Firmicutes. After adjusted by iNTK cell counts, the proinflammatory cytokines IL-10, IL-1B, and IL-6 were positively associated with the relative abundance of Bacteroidaceae in the gut microbiota.

Conclusion

In LTBI, Firmicutes in the gut microbiota determined the activities of iNKT cell and the IFN-TBAg level in peripheral blood. In active TB, the iNKT cell was related to the pro-inflammation and the relative abundances of Bacteroidaceae in gut microbiota.
CALCIUM MOBILIZATION AND EXPRESION OF TRPM2 AND CRAC CHANNELS ARE ALTERED IN HUMAN NEUTROPHILS WITH AGING
Authors: Sumoza-Toledo Adriana , de los Ángeles Vázquez-Prieto María , Lascurais-Santamaría Nallely , Fernández-Eufrasio Nilda Belén , Montiel-Condado Dvorak , Garibay-Escobar Adriana
Published: January 2023

Elderly susceptibility to infectious diseases may be a result of a decline in the function of neutrophils. We analyzed the number and percentage of neutrophils in peripheral blood of healthy elderly (60–85 years old) and young adults (20–35 years old), as well as the effect of aging on store-operated Ca+2 entry (SOCE), Ca+2 mobilization towards 100 nM or 1μM fMLP, 300 nM IL-8, 10 nM C5a, and 25 μg/ml LPS, by flow cytometry; and its relation to the expression of CD88, CXCR2, FPLR1, TRL4 and TRPM2 and CRAC channels. To examine Ca+2 mobilization, cells were loaded with 1 μM Fluo-4-AM. Baseline Ca+2 level was recorded for 60 s before the addition of the stimulus; response was recorded up to 10 min. Subcellular expression of CRAC and TRPM2 was investigated by confocal microscopy and their mRNA quantified by real time RT-PCR. Finally, migration towards 10 nM fMLP, C5a or IL-8 was measured by transwell chemotaxis assay. The results showed increased numbers and percentage of neutrophils in elderly, mainly in females. However, these cells presented lower Ca+2 entry in response to C5a, IL-8 and fMLP, and significantly lower SOCE. We did not find difference in the Ca+2 response to LPS or in the expression of the receptors of the stimuli (CD88, CXCR2, FPLR1 and TRL4). A punctuated intracellular pattern of TRPM2 and CRAC was observed in the neutrophils. TRPM2 was located in late endosomes and presented changes in its distribution with age. It was also detected a decrease in the mRNA of the TRPM2 and CRAC channels, mainly in women. Finally, we found that neutrophils from elderly migrated less towards fMLP, IL-8 and C5a. Our data suggest that the mechanisms that underlie the decrease on neutrophil functions with aging might be alterations in Ca+2 signals.
Human influenza-specific adaptive NK cells mediate potent HLA-E-restricted responses against antigenically distinct influenza strains
Authors: Jost Stephanie , Lucar Olivier , Yoder Taylor , Werner Alexandra , Ghofrani Joshua , Reeves R. Keith
Published: January 2023

Novel influenza vaccine approaches to achieve cross-protection against a broad range of antigenically distinct strains are needed. Beyond their ability to eliminate infected cells without the need for prior sensitization, natural killer (NK) cells have been shown to mediate virus-specific recall responses. Notably, exposure to influenza antigens induces protective influenza-specific memory NK cells in mice, but evidence of influenza-specific memory NK cells in humans is still lacking.

Using intracellular cytokine staining, we found that NK cells from 45% of adults can robustly respond to nucleoprotein (NP) from at least 2 heterosubtypic influenza strains completely independent of T cell help. To further determine if a subset of NK cells can specifically react to NP peptide pools from distinct strains, we clonally expanded individual NK cells (NKCL) and measured H1N1, H2N2 and H3N2 NP-specific killing by 33 NKCL from 6 donors. 63% of NKCL showed positive responses against the 2009 H1N1 pandemic strain (2%–57% specific lysis). Strikingly, half of H1N1 NP-specific NKCL also displayed cytotoxicity against either H2N2 or H3N2 (9%–100% specific lysis) or against both H2N2 and H3N2 strains (5%–11% specific lysis). Potent killing by NKCLs was associated with high cell surface expression of the activating NKG2C receptor, a ligand for HLA-E. Accordingly, we identified an HLA-E-restricted NP epitope that triggers activation of influenza-specific NK cells.

Collectively, these data provide the first mechanistic evidence of influenza-specific memory NK cells in humans and suggest influenza-specific responses mediated by NK cells may have the potential to be harnessed to enhance influenza vaccine-induced cross-protection.