bluuebunny/crossref_metadata_2025 huggingface dataset:
The P2Y purinoceptor, P2Y14R, promotes AHR in an animal model of asthma
Authors: Karcz Tadeusz , Nakano Hideki , Jacobson Kenneth , Cook Donald
Published: January 2023

The initiation and propagation of immune responses in the lung are mediated in part by so-called ‘alarmins’ that include nucleotides and nucleotide-sugars. Uridine diphosphate (UDP), and UDP-sugars, such as UDP-glucose (UDP-Glc) can be released by airway epithelial cells and bind to the G-protein coupled receptor, P2Y14R on leukocytes. However, the role of these molecules in allergic airway inflammation remains unclear. Our analysis of flow cytometry sorted cells from the lung revealed that P2ry14, the gene encoding P2Y14R, is primarily expressed in alveolar macrophages and in CD103+ dendritic cells. Using P2Y14R-specific agonists and antagonists, we tested whether this receptor has a role in allergic airway disease. Although we found no evidence that UDP-glucose or P2Y14R were involved in allergic sensitization through the airway, administration of UDP-Glc or agonists of P2Y14R during the allergen challenge phase acted synergistically with the inhaled allergen to promote neutrophilic inflammation. Conversely, administration of the P2Y14R antagonist, PPTN, during the challenge phase attenuated inflammation. To exclude the possibility that these results stemmed from non-specific effects or cell toxicity, we studied P2ry14-deficient mice. Compared to WT animals, P2ry14-deficient mice displayed significantly less airway hyperresponsiveness following allergen challenge. Together, these results suggest that existing antagonists of P2Y14R, or their derivatives, might be useful to control asthma exacerbations in humans.
IL33 signaling contributes to diesel exhaust particles-induced asthma exacerbations by promoting innate and adaptive type 2 and type 17 responses
Authors: Brandt Eric B. , Bolcas Paige E. , Ruff Brandy P. , Khurana Hershey Gurjit K.
Published: January 2023

Rationale

Exposure to traffic pollution, notably diesel exhaust particles (DEP), promote asthma exacerbations and a mixed Th2/Th17 response. The contribution of TSLP and IL33, generated by sressed lung epithelial cells, to DEP-induced asthma exacerbations remains poorly understood.

Method

We used TSLP receptor deficient mice, mice lacking the IL33 receptor (ST2) and Balb/c control mice and exposed them 9 times over a 3-week period to saline, house dust mite extract (HDM; 10ug) and/or DEP (100ug). Seven weeks later some mice received a single HDM challenge to assess memory responses. Airway hyper-responsiveness (AHR), BALF inflammation and lung T-cells and ILC subsets were assessed after primary and recall responses.

Results

DEP co-exposure with HDM exacerbates HDM-induced AHR and Th2 responses. AHR was similar between TSLPR deficient mice and control mice exposed to HDM+DEP. In contrast, AHR was significantly decreased in ST2-deficient mice compared to wild type mice. The impact of ST2 deficiency on lung accumulation of ILC2 and Th2 cells was modest. Following HDM+DEP co-exposures, decreases in ILC3, γδ T-cells and Th17 cells were observed in the lungs of ST2 deficient mice compared to control mice. The in vivo HDM recall response recapitulated the impaired AHR associated with decreases in IL17A secreting cells observed after the primary response in HDM+DEP exposed ST2 deficient mice. Finally, a decrease in lung levels of pathogenic IL5/IL13/IL17A producing CD4+ T-cells was observed after the primary and recall responses.

Conclusions

IL33 but not TSLP contributes to DEP-induced asthma exacerbations by affecting the accumulation of innate and adaptive type 2 and type 17 cells, notably pathogenic Th2/Th17 cells.
miR-396 ameliorate allergic inflammation in a mouse model of asthma
Authors: Shen Chao-Bin , Yu Lan , Wang Song
Published: January 2023

Aim of the Study

Astragalus root has good properties in enhancing immune function as well as preventing and treating disease. In previous ours study, Next-generation sequencing identified miR-396 as the most abundant plant microRNA expressed in Astragalus root. We investigated the effects of an synthetic miR-396 mimics and its mechanism of action in a mouse model of acute bronchial asthma.

Materials and Methods

BALB/c mice were sensitized and challenged with ovalbumin (OVA). The functional role of miR-396 was assessed by using in silico analysis. The miR-396 mimics was administered by intranasal instillation from the day of challenges every 2 days. HE staining was used to observe the pathological changes of the lung tissue, and expression levels of miR-396 mimics in lung tissues, serums, and spleen were analyzed by RT-PCR. The transcription factors mRNA were determined by RT-PCR in mouse model with asthma and control groups in splenocytes.

Results

GATA-3 of mRNA was significantly down-regulated in asthma model of mice treated with miR-396 mimics in splenocytes. The miR-396 mimics also suppressed STAT-6 and ROR-γt of mRNA compared with that of the OVA-challenged groups. The levels of transcription factors involved in the Th2-signaling pathway were reduced in lung tissues on treatment with the miR-396 mimics.

Conclusions

Our data suggest that miR-396 acts in a negative feedback loop to control the production of IL-5/IL-13 T cells and might thus impact the T-cell responses in asthma. Our results suggest for the first time that plant miR-396 from astragalus root of Chinese herbs plays a key role in regulating GATA-3 expression and could be useful in developing better therapeutic strategies for alleviating lung inflammations.
Scavenger receptor BI attenuates IL-17-dependent neutrophilic inflammation in asthma.
Authors: Reece Sky W. , Kilburg-Basnyat Brita , Gilliard Andrea , Hodge Myles , Luo Bin , Madenspacher Jennifer , Cook Donald , Fessler Michael , Gowdy Kymberly M.
Published: January 2023

Asthma impacts over 24 million people and costs about $56 billion annually in the United States. Airway neutrophil accumulation and high levels of IL-17 are characteristic of severe asthma. However, mechanisms underlying IL-17/neutrophilic inflammation in severe asthma remain poorly defined. Scavenger receptor class B type I (SR-BI) is a multi-recognition receptor that regulates cholesterol trafficking. We previously reported SR-BI dampens pulmonary innate immune responses during bacterial pneumonia, but the role of SR-BI in asthma is unknown. We hypothesize pulmonary SR-BI expression is protective against house dust mite (HDM)-induced IL-17-dependent neutrophilic asthma. SR-BI+/+ and SR-BI−/− mice were sensitized with 10 μg HDM by oropharyngeal aspiration on day 0 and 7, and challenged with 2 μg HDM on day 14, 15, 16. Airway inflammation and cytokine production were quantified on day 17. SR-BI−/− mice had increased neutrophils but decreased eosinophils in bronchoalveolar lavage (BAL) after HDM challenge compared to SR-BI+/+ mice. BAL levels of neutrophil chemoattractants CXCL1, CXCL2 and CXCL5 were not significantly different between SR-BI+/+ and SR-BI−/− mice. SR-BI−/− mice had increased pulmonary IL-17A and GM-CSF production and decreased IL-5 production. To identify cellular sources of airspace IL-17, single cells isolated from lung were labeled with antibodies for flow cytometry. IL-17A increased in neutrophils (CD45+Ly6G+) and macrophages (CD45+CD64+CD11b−CD11c+) but not T cells (CD45+CD4+) of asthmatic SR-BI−/− mice suggesting myeloid cells rather than Th17 cells as sources of IL-17. Overall, SR-BI expression in the lung suppresses neutrophilic inflammation and IL-17 production in HDM-induced asthma model.
PKCλ/ι regulates Th17 cells and house dust mite-induced allergic airway inflammation
Authors: Yang Jun-Qi , Dong Panpan , Yang Yingying , Zhou Wei , Mei Congjin , Guo Fukun , Zheng Yi
Published: January 2023

Asthma is a chronic airway inflammatory disease affecting millions of people in the world. CD4+ T helper cells play central roles in its pathogenesis. We have reported that atypical protein kinase PKCλ/ι regulates Th2 differentiation and function. Recently, we found that PKCλ/ι was involved in the regulation of Th17 differentiation and function. PKCλ/ι-deficient CD4+ T cells showed impaired ability to differentiate into Th17 cells in vitro under Th17-polarizing culture conditions. The secretion of IL-17, IL-17F, IL21 and IL-22, were inhibited from PKCλ/ι-deficient T cells under non-polarizing and Th17-polarizing culture conditions. Moreover, the impaired Th17 differentiation and function by the PKCλ/ι-deficiency was associated with the downregulation of Stat3 and Rorgt, key Th17 transcription factors. On the other hand, PKCλ/ι did not affect the development and induction of regulatory T (Treg) cells. In order to study the in vivo roles of PKCλ/ι-Th17 axis, we applied a murine model of house dust mites (HDM)-induced allergic airway inflammation, in which Th17 cells and neutrophils are dominant responder among others. The infiltrating cells in the lungs and bronchoalveolar lavage fluids were significantly reduced in conditional PKCλ/ι-deficient mice. Th17 and Th2 effector cytokines, such as IL-17, IL-4, IL-5 and IL-13 were inhibited in the bronchoalveolar lavage fluids. Their mRNA levels in the lungs were also significantly downregulated. Thus, PKCλ/ι emerges as an important regulator of Th17 differentiation and allergic airway inflammation. Further investigations are underway to explore the underlying molecular mechanisms.
Resveratrol treatment alters lung microbiome in the murine ovalbumin-induced asthma model by increasing mucus-degrading Akkermansia muciniphila.
Authors: Alharris Esraah Sattar , Alghetaa Hasan F. , Busbee Philip Brandon , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Asthma is a chronic inflammatory disease that involves the narrowing of the lung airways and excessive mucus production. Resveratrol (RES), a polyphenolic stilbene, is known to control asthmatic attacks via different molecular mechanisms. However, no studies have examined the effect of resveratrol on the microbiome in the ovalbumin (OVA)-induced asthma mouse model. In this study, we induced asthma in BALB/c mice by injecting OVA followed by 7 days treatment with RES. Plethysmography showed that the expiratory resistance in the lung tissue was significantly reduced in the RES treated group, while mean volume, peak expiratory flow, and frequency of respiration was increased. Histopathological examination of the lungs of the RES-treated group showed significant reduction in inflammatory cell infiltration and led to restoration of normal lung tissue architecture. In addition, there were significant increases in the expression of the genes encoding tight-junction molecules (claudin-1 and cadherin-18) in the RES-treated group. We performed 16S rRNA microbial analysis of cecal flushes and pulmonary tissues, which showed that RES treatment alters the gut microbiome by significantly increasing the level of Bacteroides acidifaciens spp. compared to disease controls. In addition, there was a significant increase in Akkermansia muciniphila (AM) species within the lungs after RES treatment. AM is a gram-negative, non-spore-forming bacterium known to induce mucus degradation. Since asthma is characterized by an increase in mucus in the lungs, we concluded that RES improves asthma in OVA-induced mouse model by significantly increasing AM and preventing mucus build-up in the lungs.
IκB kinase β inhibitor, BMS-345541, suppresses the dendritic cell response in a murine model of allergic airway inflammation
Authors: Gregorczyk Izabela , Maślanka Tomasz
Published: January 2023

Pharmacological IκB kinase (IKK) inhibition could be an effective therapeutic strategy for the prevention and treatment of allergic asthma. Recently, we have demonstrated that a selective inhibitor of IKKβ prevented allergic asthma in a mouse model through inhibition of CD4+ effector T cell responses in the lung-draining mediastinal lymph nodes (MLNs). However, the precise mechanism responsible for this effect was not been fully elucidated. The migration of dendritic cells (DCs) from the lungs to the regional lymph nodes is necessary for the development of allergic airway inflammation (AAI). The study attempts to shed light on the involvement of DCs in the anti-asthmatic action of BMS-345541 (BMS), i.e. a selective inhibitor of IKKβ. We studied the effect of a 7-day treatment with BMS on the development of AAI and the absolute count of CD11chighCD11b+CD103− (CD11b+) and CD11chighCD11b−CD103+(CD103+) DCs in the MLNs and lungs of OVA-sensitized/challenged mice. The absolute count of CD11b+ and CD103+ DCs was increased in the MLNs of untreated OVA-immunized mice. Moreover, the absolute number of CD11b+, but not CD103+, DCs was increased in the lungs of these mice. BMS reduced, but did not fully prevent, the OVA-induced increase in the count of migratory CD11b+ DCs in the MLNs. In turn, administration of this agent prevented the OVA-induced increase in absolute counts of CD103+ DCs in the MLNs and CD11b+ DCs in the lungs. Treatment with BMS protected the lungs from OVA-induced AAI. These results strongly suggest that one of the mechanisms, and probably a key one, underlying the anti-asthmatic action of IKKβ inhibitors is the suppression of the formation of inflammatory DCs at the inflammatory site and their accumulation in the draining lymph nodes.
Intratracheal ovalbumin administration induces colitis through the IFN-γ
Authors: SHIN DA SOM , Jung Kyoung-Hwa , Bae Hyunsu
Published: January 2023

Recent studies it has reported an increased incidence of inflammatory bowel disease (IBD) in patients with pulmonary diseases. Despite clinical and epidemiological studies of the interplay between colitis and asthma, the diseases’ related underlying echanisms remain unclear. In this study, we evaluated the development of colitis in a model of allergic airway inflammation. We revealed that intratracheal chronic ovalubmin (OVA) exposure induces colitis and allergic airway inflammation. Interestingly, induction of colitis was largely regulated by Th1, rather than Th2 responses, whereas allergic airway inflammation was primarily mediated by Th2 responses. Experiments T-bet−/− and Ifng−/− mice deficient mice confirmed that IFN-γ is a major mediator involved in OVA-induced colitis. These findings broaden current understanding of allergen induced colitis pathology and could play a role in the development of novel clinical treatment strategies for asthmatic patients who are at risk of developing colitis.
Lupus susceptibility gene Esrrg regulates Treg development and suppressive function through mitochondrial metabolism
Authors: Li Wei , Choi Seung-Chul , Titov Anton , Abboud Georges , Morel Laurence
Published: January 2023

Estrogen-Related Receptor Gamma (Esrrg) is the candidate gene for the Sle1c2 susceptibility locus, which encodes the orphan nuclear receptor ERR-g, in the NZM2410 mouse model of Systemic Lupus Erythematosus (SLE). Congenic C57BL/6 (B6) mice expressing this locus have an increased IFN-g production and dysregulated metabolism in their CD4 T cells, but the role of ERR-g in CD4 T cells is still unclear. In muscle, heart, iPSCs, pancreatic β cells and neurons, Esrrg has been identified as a major regulator of mitochondrial metabolism. We generated Treg conditional Esrrg knockout mice Foxp3cre Esrrgf/f (Esrrg-KO) and compared CD4+ T cells between Esrrg-KO and B6 mice. Tregs from Esrrg-KO mice showed a reduced expression of Foxp3 and CD25, and other markers associated with Treg suppressive activity. Esrrg-KO mice demonstrated non-Treg global CD4 T cell activation, including the increased frequency and number of CD69 and T effector memory cells, as well as increased spontaneous T follicular helper (Tfh) cells and decreased T follicular regulatory (Tfr) cell frequencies. In the T-dependent NP-KLH immunization, Esrrg-KO mice produced increased Tfh and germinal center B cells and less Tfr, which led to high affinity anti-NP IgG. Esrrg-deficiency in Tregs significantly altered the expression of genes and metabolites, ATP, NAD+ and ROS, involved in mitochondria function. Esrrg-KO mice also exhibited a high expression of mTORC1 and Hif1-a, a pathway that is known to destabilize Foxp3 expression. Finally, Esrrg-deficiency decreased both respiration and glycolysis in Tregs. These results demonstrate that Esrrg-deficiency results in defective mitochondrial function in Tregs, which leads to defective Tregs and global CD4 T cell hyperactivation.
Immunological and electrophysiological characterization of T-cell subsets in inflammatory bowel diseases
Authors: Tajti Gabor , Palatka Karoly , Hajdu Peter , Szanto Tibor Gabor , Panyi Gyorgy
Published: January 2023

Crohn’s Disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) characteristic of massive T-cell infiltration of the affected GI tract. CD is Th1-Th17 dominant whilst UC is considered as a Th2-Th17 prevailing disease.

It is also known that ion channels play a crucial role in T-cell physiology and the expression profile is different among T-cell subsets. This differential expression allows ion channel-specific targeting of autoimmune diseases (e.g. psoriasis).

Motivated by these findings we set out experiments to characterize the infiltration of inflamed GI tract by various T-cell subsets along with the subtype specific expression of ion channels in these cells.

We isolated T-cells from biopsy specimens and blood samples of therapy naïve IBD patients (under the ethical approval of the Medical Research Council of Hungary). Following enzymatic (0.5 mg/ml collagenase IV and 250 IU/ml DNAse I) and mechanical disruption, T-cell subsets were separated using flow cytometry. Preliminary results indicate the following proportions of subsets in biopsy samples (% of CD4+ cells): 22.3% Th1, 16.0% Th2 and 25.1% Th17 in CD, while 17.0% Th1, 19.1% Th2, and 16.4% Th17 in UC. We developed electrophysiological techniques to monitor the activity of Kv1.3 and KCa3.1 channels whereas intracellular calcium imaging is used to characterize the functional expression of CRAC, TRPA1 and TRPV1 channels.

Patients included in the study will be monitored for therapy follow-up: changes of T-cell subset distribution and ion channel function of subsets will be determined. Our main goal is to identify novel targets for IBD therapy that is of great importance if we consider that even the modern biological therapies often turn into ineffectiveness.
Selective inhibition of Tfh cells by a small molecule inhibitor abrogates progression of experimental inflammatory arthritis
Authors: Zhang Xin , Migliore Frank , Hellmers Linh , Bradley Sedrick , Zakem Jerald , Davis William , You Zongbing , Quinet Robert
Published: January 2023

Background

Rheumatoidarthritis (RA) is an inflammatory autoimmune disease characterized by T cell infiltration in the joints and autoantibody production. T follicular helper (Tfh) cells are a unique subset of CD4+ T cells regulating antibody production in B cell follicle. Our previous studies have showed that increased circulating Tfh cells were correlated with anti-CCP antibody titer and disease activity in active RA patients, indicating that Tfh cells may play an important role in RA pathogenesis. Here we investigate the therapeutic potential of a small molecule inhibitor targeting Tfh cells (SMI-Tfh) in mice with collagen-induced arthritis (CIA).

Methods

CIA was induced in DBA/1 mice by immunization with chicken type II collagen. Following the onset of clinical arthritis, mice were treated with SMI-Tfh (50mg/kg/day) for 10 days. Arthritis progression was monitored daily and recorded by the paw swollenness. Blood, spleen, and affected paws were collected at the end of the study. Tfh cells were examined and defined by CD4+CXCR5+ICOS+via flow cytometry and further confirmed by immunohistochemistry staining in spleen.

Results

Mice developed arthritis four weeks after immunization with type II collagen. Treatment with SMI-Tfh significantly reduced the disease progression/activity in mice with CIA. SMI-Tfh significantly inhibited the frequency of Tfh cells in spleen (P<0.01), but not the frequency of circulating Tfh cells in CIA mice (P>0.05).

Conclusion

The small molecule inhibitor SMI-Tfh selectively inhibits Tfh cells and abrogates progression/activity of inflammatory arthritis in CIA mouse model. Treatment with SMI-Tfh in CIA mice provides a potential strategy for joint protection and may be beneficial in RA patients.
Autoimmune regulator gene supports maternal-fetal immune tolerance during early pregnancy
Authors: Gillis-Buck Eva Mae , Bautista Jhoanne L. , Anderson Mark S. , MacKenzie Tippi C.
Published: January 2023

Numerous mechanisms protect against immune responses between the mother and her fetus in order to maintain a healthy pregnancy. The autoimmune regulator gene (AIRE) is a crucial component of tolerance to self-antigens, but its relevance for maternal-fetal tolerance has not yet been explored. AIRE contributes to both central and peripheral tolerance via the presentation of tissue specific antigens (TSAs), leading to clonal deletion of self-reactive T cells. These TSAs include placental antigens, which we call “distant self” antigens, since they are encoded in the maternal genome, but have not been expressed since the mother was herself an embryo with a placenta. We hypothesize that maternal tolerance of “distant self” placental TSAs are critical for successful embryo implantation and development. To test this, we ablated AIRE-expressing cells during the first nine days of mouse pregnancy using an AIRE-diphtheria toxin receptor (DTR) model. We found significantly smaller litter sizes (p<0.0001) and smaller embryos in a subset of AIRE-DTR pregnancies (30% of N=10), compared to wildtype (WT) pregnancies given DT (N=11). Flow cytometry of maternal tissues showed AIRE-DTR pregnancies had significantly more CD4+ T cells and fewer FoxP3+ T regs in the uterus, and more CD25+CD4+ T cells in maternal peripheral lymph nodes. AIRE-DTR mice were also less likely to be pregnant after a confirmed plug (rate of pregnancy on embryonic day 9.5: 67% AIRE-DTR vs 92% WT), suggesting decreased fertility or early pregnancy loss. Thus, AIRE-expressing cells may provide an additional mechanism of maternal-fetal tolerance by deleting reactive maternal T cells and promoting maternal T reg differentiation at the maternal-fetal interface.
The Benzathine Penicillin G (BPG) Reformulation Preferences Study - Samoa
Authors: Sika-Paotonu Dianne , Viali Satupa’itea , Naseri Leausa Toleafoa Take K. , So’o Fui Le’apai Tu’ua Ilaoa Asofou , Lameko Toleafoa Viali , Punivalu Monalisa , Maiava Fuatai , Ah-Ching Salausa John , Tiatia Ramona , Sung Yun K. , Zonneveld Rebecca , Poot Betty , Norris Pauline , Betty Bryan , Thornley Craig , Eddie Barbara , Wineera-Parai Ranei , Baker Michael , Carapetis Jonathan
Published: January 2023

Acute Rheumatic Fever (ARF) is an autoimmune condition caused by untreated group A streptococcal (GAS) infection of the upper respiratory tract (and possibly skin). Multiple or severe attacks of ARF can cause cardiac damage known as rheumatic heart disease (RHD). In Australia, New Zealand (NZ) and the Pacific Region, the disease burden of ARF and RHD amongst Indigenous and Pacific communities is one of the highest in the world, usually affecting children and young adults. The most effective recommended management for ARF requires monthly intramuscular injections of 1.2 million units of Benzathine Penicillin G (BPG) known as secondary prophylaxis (SP) for 10 years or more. The goal of SP is to prevent GAS infections that may lead to the recurrence of ARF. Even with these monthly BPG injections, adherence to SP schedules are usually low due to the frequency and duration of injection, pain and access to proper and timely healthcare. A less painful and longer acting BPG formulation would ideally help prevent recurrence of ARF and improve compliance rates to this schedule with improved understanding of barriers and novel approaches to BPG delivery urgently needed. To better understand the BPG reformulation preferences of children/teens currently receiving monthly BPG intramuscular injections, and that of their families and healthcare providers who administer BPG, three software applications will be developed from pre-existing applications that have been optimized for use in target populations in New Zealand. This will be the first time software applications have been used to collect qualitative and quantitative data on individual preferences for BPG formulations and dosing regimens in Samoa.
T cell receptor mining of RNA-Seq data to gain insight into inflammatory responses: correlative studies compare the αβ to the γδ T cell infiltrate in psoriasis
Authors: Marusina Alina I. , Merleev Alexander A. , Shimoda Michiko , Maverakis Emanual
Published: January 2023

γδ T cells have been proposed to play a major role in the pathophysiology of psoriasis and other immune-mediated skin diseases. T cell repertoire analysis is one method of characterizing the T cell component of an immune response. Although traditionally conducted on T cell receptor (TCR)-dedicated PCR products, with the appropriate bioinformatics pipeline and sequencing depth, T cell repertoire analysis can also be performed directly on RNA-Seq data sets, allowing for informative correlative studies to be performed in parallel. To compare the role of γδ versus αβ T cells in psoriasis, we have chosen to mine psoriasis and healthy control skin RNA-Seq data sets for TCR receptor sequences. Unlike prior studies, our results are validated in four independently collected datasets.

We demonstrate that in the setting of psoriasis, TCRγ and TCR δ-mapped reads are only a small fraction of their TCR α and TCR β counterparts. Although we identified several TCR sequences that were found in multiple skin samples, none were associated with HLA-Cw6. We also observed an increase in TCR repertoire diversity in psoriatic biopsies, demonstrating the polyclonal nature of the T cell infiltrate in psoriasis. Analysis of CDR3 sequences were also conducted revealing no psoriasis-associated public CDR3 sequences. A meta-analysis revealed a statistically significant link between the of TRAJ23 usage in psoriasis and the psoriasis-associated cytokine IL17A. TRGV5, a TCR γ segment, was also associated with psoriasis but correlated instead with IL36A.

This work highlights novel strategies for characterizing tissue-infiltrating T cells from RNA-Seq data sets, an approach with broad applications in the fields of autoimmunity and cancer immunotherapy.
Regulation of NKT cell function by a 0.1 Mbp interval on chromosome 1 encompassing Fcgr3
Authors: DeVault Victoria L. , Malagic Murisa , Mei Linda , Dienz Oliver , Lilley Graham W.J. , Benoit Patrick , Ather Jennifer L. , Poynter Matthew E. , Boyson Jonathan E.
Published: January 2023

NKT cells play a critical role in tissue-specific immune responses by rapidly producing cytokines in response to glycolipid stimulation. We previously reported that host genetic background played a substantial role in the NKT cell response to the agonist glycolipid α-galactosylceramide (αGalCer). A 6.6 Mbp region on chromosome 1 was identified as a major regulator of the NKT cell cytokine response between C57BL/6 (B6) and 129X1/SvJ mice. To fine-map this locus, we generated several B6.129 congenic lines with overlapping 129X1/SvJ intervals from 0.1 to 1.1 Mbp, and assessed the response of NKT cells to αGalCer. An analysis of serum and intracellular cytokine production after αGalCer challenge revealed significantly reduced levels of all cytokines tested in the B6.129c3 (c3) congenic strain that possessed the minimal 129 interval containing 5 genes. In vitro analysis of sorted splenic NKT cells indicated that the reduced cytokine production was NKT cell-intrinsic. In addition, we observed significantly fewer thymic NKT cells in the B6.129c3 congenic mice suggesting that the c3 interval may also regulate NKT cell development. Analysis of candidate genes within the c3 interval revealed a 5-fold increase in Fcgr3 gene expression on c3 NKT cells, which was associated with increased expression of FcγR3 protein on B6.129c3 NKT, NK and neutrophils. Taken together, these data identify the c3 interval as a novel locus regulating the response of NKT cells to glycosphingolipid, and reveal a link between this phenotype and an expression polymorphism that regulates Fcgr3 expression.
Tart cherry extract improves skeletal muscle recovery by increasing type II MHC expression, MCP-1 and HGF secretion, and attenuating ROS production by neutrophils
Authors: Pelletier Margery G.H. , Szymczak Klaudia , Barbeau Anna M. , Bishop Erin J. , Johnson Benjamin B. , Lee Andrew J. , O’Fallon Kevin S. , Gaines Peter
Published: January 2023

Ingestion of tart cherry extract (TCE), a rich source of polyphenolic anti-oxidant/anti-inflammatory compounds, improves skeletal muscle (SM) recovery following exercise-induced muscle damage. This response may be due in part to attenuated oxidative/pro-inflammatory activities of recruited neutrophils and macrophages during SM regeneration. How TCE promotes the molecular mechanisms regulating SM regeneration or attenuates those controlling myeloid cell pro-inflammatory responses is unknown. Herein, we describe how TCE affects two murine cell models optimized to recapitulate in vivo interactions between regenerating SM and recruited pro-inflammatory myeloid cells: 1) strained skeletal muscle tissue derived from C2C12 myoblasts, and 2) mature myeloid lineages derived from ex vivo-cultured primary bone marrow. We show that TCE treatment may improve SM regeneration by increasing MHC protein expression plus MCP-1 and HGF secretion. Importantly, TCE treatment modestly reduced myoblast proliferation but without affecting viability, and did not affect proliferation or differentiation-specific protein expression profiles (via imaging flow cytometry) of myeloblasts or derived progenitors. Moreover, TCE treatment reduced levels of reactive oxygen species (ROS) released by activated neutrophils. Our continued analyses will identify how TCE either independently affects damaged SM regeneration or myeloid cell functional responses, or dependently affects the interactions between these disparate cell types upon muscle injury. Our studies may reveal that this natural compound not only improves muscle recovery following damage but also attenuates pro-inflammatory responses of recruited myeloid cells.
RNA-binding protein HuR is required for suppressive function but not development of Foxp3+ CD4+ Tregs
Authors: Atasoy Ulus , Ellis Jason S. , Taylor Tiffany , Ridenhour Suzanne
Published: January 2023

The RNA-binding protein (RBP) HuR (Elavl1) controls differentiation of CD4+ T cells into multiple helper subsets through regulating transcript stability of multiple T helper subset-specific targets. We generated dLck-Cre HuRfl/fl mice, in which HuR is conditionally knocked out in T cells late in thymic development. There was a 5-fold skewing toward HuR sufficient regulatory T cells (Treg) in these mice. HuR KO naïve CD4+ T cells from these mice are also less susceptible to Treg polarization than controls and fail to upregulate Foxp3 following activation. We conditionally ablated HuR in Tregs (Foxp3YFP-Cre HuRfl/fl mouse) (HuR KO). Heterozygous Foxp3YFP-Cre/WT HuRfl/fl females have normal weight gain over time. In stark contrast, both hemizygous Foxp3YFP-CreHuRfl/fl males (39% weight reduction) and homozygous Foxp3YFP-Cre/YFP-Cre HuRfl/fl females (30% reduction) have significant failure to thrive. Heterozygous HuR KO females also have a 5:1 shift in the ratio of HuR− Treg to HuR+ Treg indicating a possible survival bias toward Treg expressing WT levels of HuR. HuR KO mice have features of scurfy mice, including alopecia and stippled tails, as well as multi-organ inflammation which includes lung, skin, stomach and liver. Additionally, naïve HuR KO mice have significantly reduced peripheral Tregs and increases in activated Foxp3− effector T cells. In vitro suppression assays showed reduced suppressive capacity of HuR KO Tregs relative to WT Tregs. Together, these data suggest that HuR is critical for effective Treg function, but is dispensable for the development of Foxp3+ CD4+ Tregs.
The NINJA mouse: a novel model to study tissue-specific peripheral T cell tolerance
Authors: Damo Martina , Joshi Nikhil S.
Published: January 2023

T cell inhibitory pathways induce peripheral tolerance and have evolved to avoid pathogenic autoimmunity. Generally, peripheral tolerance either prevents T cell activation or inhibits T cells during chronic responses, but it is not clear whether these functions are orchestrated by the same mechanisms. Moreover, it is not known if these mechanisms have a tissue-specific usage. Persistent viral infections and cancer leverage the T cell inhibitory pathways involved in peripheral tolerance, thus providing translational relevance to the role of these pathways in disease. However, many of the mechanisms of peripheral tolerance remain understudied, in part because few animal models can distinguish these mechanisms from those driving central tolerance. We have generated a novel mouse model (iNversion INduced Joined neoAntigen, NINJA) that bypasses central and peripheral tolerance by using genetic recombination to create a neoantigen-coding gene for de novo neoantigen expression in any tissue of choice. NINJA expresses LCMV-derived GP33–41 and GP61–80 after induction, enabling investigation of tolerance using well established immunologic tools. Prior to recombination, NINJA mice respond robustly to LCMV-Arm infection, indicating absence of tolerance. However, after recombination, LCMV GP-specific responses are significantly impaired, although mice retain the capacity of developing immunity against unrelated LCMV epitopes. PD-1 is upregulated by tolerized endogenous GP33–41-specific CD8+ T cells after hepatocyte-specific NINJA activation, thus suggesting its involvement in hepatic peripheral tolerance. We therefore propose NINJA as a model to investigate tissue-specific mechanisms of peripheral T cell tolerance.
The unfolded protein response, microRNA-214, and expression of the transcription factor XBP1
Authors: Brewer Joseph W. , Jackson Kevin P. , Lee Eleanor H. , Smith Kelsie M.
Published: January 2023

The transcription factor X-box binding protein 1 (XBP1) plays critical roles in the immune system. For example, XBP1 is required for plasma cell development and can disrupt proper function of tumor-associated dendritic cells. A number of post-transcriptional mechanisms regulate XBP1, but a complete understanding of the processes that govern its expression and activity in distinct situations is lacking. The active form of XBP1, termed XBP1s, is dependent upon cytoplasmic splicing of Xbp1 mRNA by IRE1, a transmembrane endoribonuclease/kinase positioned in the endoplasmic reticulum (ER) membrane. IRE1 is a key signal transducer in the cellular response to ER stress, also known as the unfolded protein response (UPR). The IRE1-XBP1 pathway coordinates a broad program of gene transcription that promotes functions of the secretory pathway and alters various aspects of lipid homeostasis under conditions that perturb the ER environment and/or increase demand on its protein folding capacity. ER stress also activates the UPR signal transducer PERK, an ER transmembrane kinase that mediates efficient repression of protein synthesis. Previous studies have shown that microRNA(miR)-214 can negatively regulate XBP1 expression and is down-regulated during ER stress. Using PERK-deficient mouse embryo fibroblasts, we have found that ER stress-mediated diminishment of miR-214 is partially dependent on the PERK pathway, suggesting a novel mechanism of regulatory crosstalk between the PERK and IRE1-XBP1 branches of the UPR. The significance of miR-214 in the cellular response to ER stress and to XBP1-mediated events is under investigation.
Differential regulation of B cell responses to acute and chronic infection by histone-modifying complexes
Authors: Good-Jacobson Kim , Di Pietro Andrea , Yiannis Callisthenis , Farighi Rohia
Published: January 2023

Molecular regulation of B cell differentiation is critical for formation of immune memory and becomes dysregulated in B cell lymphomas. Alterations in the genomic landscape that allow or restrict access of transcription factors are critical to enact gene expression profiles that in turn drive cell differentiation. Chromatin accessibility can be modulated by enzymes that modify the N-terminal tail of histones. The Trithorax group protein MLL1 forms histone-modifying complexes to permit transcription, whereas the two Polycomb repressor complexes (PRC) work together to repress gene expression. The dynamic regulation of these complexes can modulate B cell fate and function during immune responses. Yet, little is known about the temporal and spatial requirements of MLL1, canonical PRC1 and PRC2 during acute and chronic immune responses. Deletion of the PRC2 member EED (Eedf/fCd23Cre/+ mice) resulted in a complete absence of GC and B cell memory following immunization or influenza infection. Mll1f/fCd23Cre/+ mice also could not mount an effective GC response to immunization, yet their plasmablast response remained intact. In contrast, the canonical PRC1 member BMI-1 was required for plasmablast but not GC responses. Surprisingly, the requirement for EED and MLL1 in a chronic persisting infection differed from acute responses. In contrast to acute responses, Eedf/fCd23Cre/+ mice infected with LCMV-Docile or P. chabaudi had normal GC formation, whereas Mll1f/fCd23Cre/+ mice formed GCs to the acute LCMV strain but not in chronic infection. Together, our data demonstrate the differential requirements of these complexes in B cell fate decisions, as well as suggest epigenetic regulatory checks are subverted in persisting infections.
Hypoxia-Inducible Factors regulate metabolism and germinal center response.
Authors: Cho Sung Hoon , Raybuck Ariel , Kemboi Edna , Boothby Mark R.
Published: January 2023

Germinal centers (GCs) are micronantomic structures that B cells proliferate, are selected for affinity maturation, undergo antibody (Ab) class-switch recombination and differentiate into Ab-secreting plasma cells and memory B cells. Both cell-autonomous and extrinsic metabolic reprogramming have emerged as modulators of T cellmediated immunity. We have shown that GC light zone (LZ) are hypoxic, and the low oxygen tension or persistent stabilization of Hypoxia-Inducible Factor (HIF)-1a and 2a alters B cell physiology and function. Depletion of HIF-1a and HIF-2a in CD4 T cells caused deficiencies of the antibody response due to HIF functions in both B lineage cells and the follicular CD4 subset. Among CD4 T cells, HIF restrained the FoxP3-positive T follicular regulatory (TFR) population while promoting CD40L expression. In the B lineage, HIF promoted humoral responses both through their capacity to promote effective proliferation and via enhancement of plasma cell differentiation. Collectively, these results provide evidence that HIF transcription factors regulate humoral responses via vital functions of B and Tfh cells. We propose that restriction of oxygen in lymphoid structure and organs, which can be altered in pathophysiological status, modulates humoral immune response and memory.
Activation-induced cytidine deaminase mutator activity targeting through phosphorylation
Authors: McBride Kevin M. , Mu Yunxiang , Zelazowska Monika Anna
Published: January 2023

Activation induced cytidine deaminase (AID) initiates immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) by deaminating transcribed double stranded DNA to produce U:G mismatches. Subsequent processing of lesions complete SHM or CSR. AID mutator activity predominantly targets the immunoglobulin locus, although “off-target” association also occurs at transcribed regions. The promiscuous targeting has the potential to cause oncogenic mutations, DSBs and chromosome rearrangements and so AID activity must be carefully regulated. How this is accomplished is not completely understood as AID occupancy does not directly correlate with DNA damage, suggesting factors beyond AID association contribute to mutation targeting. It is known that post-translational modifications, in particular AID serine 38 phosphorylation (pS38) regulate AID activity. AID with the inability to be phosphorylated results in deficient CSR and SHM. However, the regulation of pS38 is poorly understood and the contribution of pS38 phosphorylation to off-target AID activity is unknown. Through evaluation of pharmacological inhibitors and activators we identify several that impact AID phosphorylation. We determined that a FDA approved drug, lithium, enhanced AID S38 phosphorylation. Using wild-type B cells and those expressing AID with a S38A mutation, we evaluated the effect of enhanced AID phosphorylation. We find increased off-target mutations, DSBs, and chromosome translocations without an increase in CSR. These studies demonstrate how AID activity can be differentially targeted by phosphorylation to diversify immunoglobulin genes or induce oncogenic lesions.
Targeting colorectal cancer metastasis-initiating cells through hepatic iNKT cells
Authors: Feng Hao , Lu Ai-guo , Wang Jian
Published: January 2023

Aims

To investigate the immune microenvironment modulation of iNKT cells for recognition and elimination of colorectal metastasis-initiating cell (MIC).

Methods

Different subtypes of human colorectal cancer cells and iNKT cells were analyzed by BD LSRFortessa™, TMA, and immunofluorescence(n=138). Microfluidic circulating tumor cell enrichment, Ex vivo 3D co-culture, Quansys Multiplex array, survival data analysis, and Elisa array were performed.

Results

There is a subtype of colorectal cancer stem cell referred as a metastasis-initiating cell (MIC), which do not express upregulated mesenchymal genes but express elevated levels of CD36 (the fatty acid receptor) and other lipid metabolism genes. Intriguingly, those cells overexpress NKG2DL, Various immune cells express NKG2D receptors, including natural killer (NK) cells, CD8+ T cells, and human iNKT cells. Concerning the NKT cells, there is an increase of NKG2D expression on liver resident iNKT cells (P < 0.05). We further observed that hepatic iNKT cells located at the invasive margin had been activated as shown by increased IFN-γ, TNF-α, granzyme B and Ki-67 expression. Furthermore, transmembrane CD1d density and intra/tmCXCL16 expression were also elevated in CD36+colorectal cancer stem cells, those cells showed significantly elevated α-GalCer: CD1d complex formation than other cancer stem cells in an ex vivo iNKT cells activation assay. CD36+cancer stem cells show susceptibility to iNKT cells induced cytotoxicity.
A novel methodology to modulate immune environment using synthetic mRNA
Authors: Xu Yitian , Huang Lu , Kirschman Johnathan L. , Vanover Daryll , Tiwari Pooja M. , Santangelo Philip J. , Shen Xiling , Russell David G.
Published: January 2023

As the next generation of gene therapy, synthetic mRNA therapy is known for its intrinsic advantages to avoid genomic integration that associated with DNA-based therapy, and its prolonged production of target proteins over traditional recombinant protein therapy. Synthetic mRNA therapy has been studied in various disease models including cancer, infectious disease and cardiovascular disease. However, these studies focus mainly on expressing proteins with direct therapeutic effect or antigens to elicit an immune response. It is not well established that whether synthetic mRNA can be taken advantage in cell based therapies in vivo to recruit cells of interest and modify them locally. Here we reported a synthetic mRNA based immune-modulation method, exampled by using Ccl2 and Ccl3 synthetic mRNA to recruit a certain population of monocyte in a non-inflamed manner. These recruited monocytes exhibit neither bactericidal or tissue-repairing behaviors, but stay in a neutral, non-programmed state. With additional Ifn-g mRNA and Il-4 mRNA, these cells can be polarized to different phenotypes. Furthermore, the monocytes recruited by Ccl3 and Ifn-g mRNA are able to launch the most rapid and strong superoxide burst, which demonstrates their active functionality upon proper signals. In summary, our data established a synthetic mRNA based immune-modulation system, which allows recruitment and modification of specific immune cell populations in a precise and localized manner, and more importantly, proves the concept that synthetic mRNA can be utilized in cell based therapies to replace the time-consuming and labor-demanding process of modifying patients’ own cells ex vivo and transferring back to patients afterwards.
Sex differences in the acute neuroinflammatory events after experimental traumatic brain injury
Authors: Doran Sarah J. , Ritzel Rodney , Glaser Ethan , Henry Rebecca , Faden Alan , Loane David
Published: January 2023

Traumatic brain injury (TBI) is sexually dimorphic, impacting males 3 times more than females. Neuroinflammation is a key secondary response activated following TBI, where it has recently been shown that TBI leads to an exacerbated neuroinflammatory response in males versus female mice. The aim of this study was to investigate potential differences in microglial activation at baseline and acute time points after experimental TBI. Age matched male and female C57BL/6 mice underwent moderate-to-severe controlled cortical injury (CCI) and brain samples were collected for flow cytometry analysis at 1, 3, and 7 days post-injury (dpi). In addition, animals underwent motor and cognitive behavioral tasks. Baseline microglia function in females showed significantly higher production of pro-inflammatory cytokines (TNF-α, IL-1β), whereas male microglia exhibited increased levels of reactive oxygen species (ROS) and greater phagocytic activity. On flow cytometry analysis, in both sexes pro-inflammatory cytokines, ROS, and phagocytic activity all increase 1dpi, remain elevated at 3dpi, returning to near baseline levels by 7dpi. At 1, 3, and 7 dpi leukocytes infiltrating the brain after injury in males and females produced more TNF-α, IL-1β, TGF-β, and ROS and showed greater phagocytic activity than microglia. However, at 1 dpi females have significantly less infiltrating peripheral immune cells perhaps indicating that peripheral cells are driving acute inflammation after TBI, leading to an early sex difference in behavior. This data indicates that while microglia in both sexes may respond similarly after TBI, there are significant sex differences in peripheral immune activation and infiltration after TBI that may affect acute outcomes.