bluuebunny/crossref_metadata_2025 huggingface dataset:
Defective Microglia-Neuronal Communication during Demyelinating Disease Correlates with Altered Neurogenesis
Authors: Cardona Astrid E. , Church Kaira , Mendiola Andrew , Cardona Sandra M. , Vanegas Difernando , Garcia Charles , Luera Dezzarae , Lira Sergio A. , Kokovay Erzsebet , Lin Chi-Hsing Annie
Published: January 2023

Inflammatory demyelination is a hallmark of the pathology of multiple sclerosis (MS) and microglia are considered responsible for exacerbating myelin loss. The link between inflammation and neural stem cell (NSC) development, key events during the remyelination process, is being increasingly recognized. However, the mechanisms that connect microglia, myelination and inflammatory-mediated oxidative damage are unclear. Fractalkine (FKN), a neuronal derived chemokine, and its unique receptor, CX3CR1, expressed by microglia, are highly abundant in the healthy brain, and our laboratory has reported severe CNS demyelination and neuronal damage in EAE-induced CX3CR1-KO mice. Our hypothesis is that CX3CR1-defective signaling promotes inflammatory-mediated damage to neuron and myelin-forming cells. We found that expression of the adhesive defective human variant hCX3CR1-M280 caused enhanced disease severity, increased demyelination and exacerbated neuronal loss, mirroring the findings we observed in CX3CR1KO mice. Also, enhanced demyelination was observed after 4 wk of cuprizone treatment. Densities of NG2+ glia were comparable between WT and CX3CR1KO mice, but a significant reduction in DCX+ cells in the subgranular zone of the hippocampus was observed. In addition, lipocalin-2 an acute phase protein, linked to resolution of inflammation by reducing iron-induced toxicity, was found upregulated in cuprizone-induced demyelinated lesions of WT but not CX3CR1KO or hCX3CR1M280 mice. Thus, the FKN/CX3CR1 signaling pathway in microglia plays an under-appreciated role in MS, perhaps via anti-inflammatory processes that enhance neurogenesis through regulation of oxidative damage.
IL7R regulates fetal tissue resident macrophage development
Authors: Leung Gabriel , McCann Taylor , Forsberg Camilla , Beaudin Anna
Published: January 2023

Tissue-resident macrophages play critical roles in tissue homeostasis and disease. Many populations of tissue-resident macrophages derive from fetal progenitors and self-maintain across the lifespan through in situ proliferation, independent of bone marrow hematopoiesis. However, the developmental mechanisms that specify fetal-derived tissue-resident macrophages are poorly understood. Here, we have identified a novel cytokine regulating tissue-resident macrophage development using an IL7Ra-cre lineage tracing model. Adult tissue resident macrophages in the brain, skin, liver, and lung were extensively labeled by IL7ra-cre, in the absence of IL7ra message or protein expression. To gain insight into developmental expression of IL7ra, we profiled IL7Ra surface expression, mRNA expression, and IL7ra-cre-driven labeling across fetal myeloid development. We observed rapid upregulation of IL7ra surface expression during a limited window of tissue macrophage establishment, and dynamic regulation of IL7ra message and protein levels in monocyte precursors, suggesting that IL7ra regulated the transition from fetal liver monocytes into tissue macrophages. Blockade of the IL7R using a monoclonal antibody decreased cellularity of liver, lung and skin tissue resident macrophages at birth, and increased cellularity of fetal liver monocytes. These data suggest that late in gestation, IL7Ra is upregulated as fetal monocytes exit the fetal liver and differentiate into macrophages, and provides evidence that IL-7R signaling regulates fetal tissue-resident macrophage development. Ongoing work addresses the function of IL-7 signaling in myeloid development, and the function of IL-7R marked macrophages in adult immunity.
Assessing the disease specificity of urinary PF4 for active lupus nephritis
Authors: Gokaraju Sirisha , Haque Anam , Vanarsa Kamala , Soliman Samar , Saxena Ramesh , Mohan Chandra
Published: January 2023

Background

The use of urinary biomarkers is particularly helpful in avoiding invasive procedures such as renal biopsy. We recently observed that urinary Platelet factor 4 (PF4) was elevated in patients with active lupus nephritis compared to patients with inactive lupus or healthy controls. Objective of this study is to assess the specificity of urinary PF4 for lupus nephritis (LN). PF4 is a small cytokine, also known as CXCL-4, known to play a role in inflammation.

Methods

Urinary PF4 level was measured using ELISA in urine samples obtained from 50 chronic kidney disease patient samples comprising of DN (Diabetic Nephropathy) (N = 23), HTN Nephropathy (Hypertensive nephropathy) (N = 6), combination of DN and HTN nephropathy (N = 4), IgA nephropathy (N = 4), FSGS (Focal segmental glomerular sclerosis) (N = 13). As a negative control, urine samples were included from healthy controls, while urine samples from patients with active lupus nephritis were included as a positive control. All results were normalized to urinary creatinine. The urinary PF4 levels in various CKD groups were compared to both negative and positive controls.

Results

Urinary PF4 was significantly elevated (P<0.05) in samples from DN (678±285 pg/mg), FSGS (423±208 pg/mg), and LN (1438±901 pg/mg), compared to healthy controls (11±10 pg/mg). Urine PF4 levels in IgA nephropathy (24±15 pg/mg), HTN nephropathy (38±33), and DN/HTN nephropathy (463±410 pg/mg) were not significantly elevated compared to healthy controls.

Conclusion

PF4, as a urinary biomarker, exhibited significant elevation in patients with DN, FSGS as well as LN when compared to other renal diseases like IgA nephropathy and HTN nephropathy. Hence PF4 may aid in the noninvasive monitoring of DN, FSGS, and LN.
Reducing airway constriction and inflammation through GABAA receptors in the lung with a novel, orally available drug candidate.
Authors: Nieman Amanda N. , Forkuo Gloria S. , Zahn Nicolas , Kodali Revathi , Li Guanguan , Roni M.S. Rashid , Stephen Michael Rajesh , Harris Ted , Jahan Rajwana , Guthrie Margaret , Yu Olivia , Fisher Janet , Yocum Gene , Emala Charles , Steeber Douglas A. , Stafford Douglas C. , Cook James , Arnold Leggy A.
Published: January 2023

MIDD0301 is a new lead compound designed for the oral treatment of asthma. It was developed from previously described α5β3γ2 selective GABAA receptor (GABAAR) modulators. MIDD0301 showed significant relaxation of Substance P contracted guinea pig tracheal rings ex vivo at low micromolar concentrations. In vivo, MIDD0301 decreased airway hyperresponsiveness in asthmatic ovalbumin-sensitized and challenged mice after five day b.i.d. oral administration. MIDD0301 reduced eosinophil, macrophage, and CD4+T-lymphocyte cell counts in broncoalveolar lavage fluid without stimulating mucus hyperplasia. Pro-inflammatory cytokines (IL-17A, IL-4, TNFα) were decreased in the lung while the anti-inflammatory cytokine IL-10 remained unchanged. Pharmacokinetic studies showed MIDD0301 has a half-life of approximately 14 hours and negligible brain distribution. The lack of adverse CNS side effects was confirmed with rotarod studies. CD4+ T-lymphocytes isolated from ova s/c mice showed a dose dependent change in transmembrane current with GABA and MIDD0301, confirming that MIDD0301 is acting via GABAAR. Overall, MIDD0301 represents a novel, orally available, and safe alternative for the treatment of asthma through airway smooth muscle relaxation and attenuation of airway inflammation.
Unexpected role for androgen and androgen receptor as enhancers of M2 macrophage polarization
Authors: Becerra-Diaz Mireya , Strickland Ashley B. , Keselman Aleksander M. , Heller Nicola M.
Published: January 2023

Allergic asthma is an inappropriate Th2 immune response in the lungs to innocuous inhaled antigens. This results in M2 polarization of alveolar macrophages (AM), which number correlates with asthma severity in humans. Sex differences in asthma suggest that sex hormones modify lung inflammation and macrophage polarization. Since estrogen enhances M2 polarization and androgens have been used as experimental asthma therapy, we hypothesized that androgens [testosterone and dihydrotestosterone (DHT)] and androgen receptor (AR) suppress M2 macrophage polarization.

Here, we demonstrate that although androgen reconstitution in castrated mice reduced inflammation of the bronchoalveolar space in a mouse model of allergic lung inflammation, it enhanced the production of the eosinophil-recruiter and M2 marker YM1 in AM, indicating a cell-specific role for androgens. DHT also enhanced IL-4-induced M2 macrophage polarization in vitro. Using mice lacking the AR in monocytes/macrophages (ARfloxLysMCre), we found that ARfloxLysMCre males had less eosinophil recruitment and lung inflammation in our model of allergic lung inflammation. No differences were found in females. This decreased inflammation was due to impaired M2 polarization and diminished production of eosinophil-recruiting chemokines by AM rather to a regulatory profile of AM lacking AR. Furthermore, macrophages lacking AR did not show an increase in IL-4-induced M2 polarization in vitro with DHT treatment.

These data reveal an unexpected role for androgen/AR in promoting M2 macrophage polarization, and suggest possible mechanisms to counteract diseases that are promoted by M2 macrophages and affect mainly men, such as eosinophilic esophagitis and prostate cancer.
A comparison of IL-5 and IL-10-producing antigen-specific clonal T cell repertoires
Authors: Schulten Véronique MJ. , Westernberg Luise , Peters Bjoern
Published: January 2023

Allergic rhinitis affects up to 40% of North Americans, and is associated with potentially debilitating symptoms. Allergen-specific immunotherapy (AIT) is a disease-modifying treatment associated with Treg induction and elevated IL-10 production. To date, it is unknown if IL-10-producing T cells are induced de novo or if AIT modulates allergen-specific Th2 cells to become tolerogenic. To investigate whether IL-5 and IL-10-producing antigen-specific T cells share a common T cell repertoire, we performed a longitudinal TCRβ sequence analysis of Timothy grass (TG)-specific T cells from donors who received AIT for 15 months. As a control, TCRβ sequencing was also performed on TG-specific T cells from an allergic donor. TG-specific cells were expanded by 14-day in vitro culture with a pool of immuno-dominant TG-derived T cells epitopes (TG pool). A pertussis epitope pool was used in parallel as a control. Subsequently, cells were restimulated with the same pool and allergen-specific cells were isolated by cytokine capture with column enrichment, sorting IL-5 and IL-10 -producing cells. The TCRβ repertoire was sequence from DNA isolated from sorted cells. Preliminary results revealed that in the TG pool-specific cells, some clonal overlap was observed between IL-10+ and IL-5+ cells, suggesting that a fraction of IL-10+ cells may originate from IL-5+ cells, but some are generated de novo. Longitudinal analysis of the allergen-specific TCR repertoire is a reliable method to determine if tolerogenic T cells are induced de novo or if they are modulated T cells that originate from the same repertoire as allergen-specific Th2 cells.
GATA3, SATB1, IRF4 complex binds to the Th2 cytokine locus through cis-acting element RHS6, regulating allergic airway inflammation.
Authors: Jang Sung Woong , Lee Gap Ryol
Published: January 2023

Rad50 hypersensitive site 6 (RHS6) is a cis-acting element in the Th2 locus control region which is essential for the expression of Th2 cytokine genes. However, molecular mechanisms how it regulates the genes are poorly understood. We found that RHS6 recruits transcription factors GATA3, SATB1, and IRF4 during Th2 cell differentiation. GATA3, SATB1, and IRF4 have a crucial role in regulating Th2 cytokine genes. Knock-out or knock-down of each of GATA3, SATB1, and IRF4 genes significantly decrease the expression of Th2 cytokine genes. Mechanistically RHS6 mediates the transcription factor-induced regulation of Th2 cytokine genes. GATA3, SATB1, and IRF4 physically associates with each other, and forms a protein complex at RHS6 sequence. They directly bind to the many regulatory elements throughout the Th2 cytokine locus, RHS6 dependent manner. Furthermore, deletion of RHS6 caused a dramatic resistance to airway inflammation in ovalbumin-induced allergic inflammation model. Together, RHS6 recruits GATA3, SATB1, and IRF4 coordinating their regulation of Th2 cytokine genes, so is essential for Th2-mediated allergic airway inflammation.
dsRNA induced intestinal epithelial cell activation of immune dysfunction in type 1 diabetes
Authors: Wallet Shannon , Golas Gavin , Tyler Melissa , Sorenson Heather
Published: January 2023

Environmental factors contribute to the initiation, progression, and maintenance of many autoimmune diseases including type 1 diabetes (T1D). We propose the context in which an environmental signal is received by the intestinal epithelial cell (IEC) may be more important to disease than the specific identity of the environmental factor. Viral infections are proposed to be one such trigger. IEC-specific responses to double-stranded RNA, a common intermediate in many viral life cycles, was evaluated by comparing and contrasting the expression of soluble immune mediators from T1D and non-T1D derived primary (murine and human) IECs. The effect of the soluble mediator milieu on innate and adaptive immune cell phenotype and function was also assessed. While poly I:C induces a robust induction of IFN-I in primary IEC derived from non-T1D sources, a significantly less robust IFN-I response was observed in T1D-derived IECs. Similarly, a significantly less robust induction of IL-18, TSLP, and IL-17C mRNA was observed in T1D-derived IECs cultures whereby this hyporesponsive phenotype became more apparent in T1D-IEC derived at later stages of disease. Supernatants from T1D-derived IECs induced an M1 macrophage phenotype and directly enhanced CD3/CD28 induced T cell proliferation. T cells induced to proliferate were polarized to a more Th(c)1 or Th(c)1/Th(c)7 phenotype at the expense of a Th(c)17 phenotype. Supernatants from T1D-derived IECs also enhanced β-cell killing by transgenic T cells. These data support our hypothesis that IECs are key players in driving innate and adaptive immune dysfunction observed in the gastrointestinal track of individuals with T1D, as well as capable of supporting and expanding an autoreactive T cell repertoire.
Integration of genome-wide transcriptome and DNA methylome uncovered aberrant methylation-regulated genes and pathways in the peripheral blood mononuclear cells of systemic sclerosis
Authors: Li Quanzhen , Zhu Honglin , Zhu Chengsong , Mi Wentao , Zuo Xiaoxia , Luo Hui
Published: January 2023

Objectives

To delineate the interaction network between gene transcription and DNA methylation in PBMC of systemic sclerosis (SSc) patients and to identify methylation-regulated genes which are involved in the pathogenesis of SSc.

Methods

Genome-wide transcriptome and DNA methylome were profiled on PBMC from 30 SSc patients and 30 matched normal controls (NC). Differential expressed genes (DEGs) and differential methylated positions (DMPs) were integratively analyzed to identify methylation-regulated genes and associated molecular pathways.

Results

Transcriptome profiling distinguished 453 DEGs in SSc. Global DNA methylation analysis identified 925 DMPs located on 618 genes. Integration of DEGs and DMPs revealed 20 DEGs containing inversely associated DMPs. These 20 potential methylation-regulated DEGs (MeDEGs), including 12 up-regulated genes (ELANE, CTSG, LTBR, C3AR1, CSTA, SPI1, ODF3B, SAMD4A, PLAUR, NFE2, ZYX and CTSZ) and 8 down-regulated genes (RUNX3, PRF1, PRKCH, PAG1, RASSF5, FYN, CXCR6 and F2R), are predominantly involved in the migration, proliferation, activation and inflammation of immune cells. Unsupervised cluster analysis of the top MeDEGs distinguished SSc from NC with 90% sensitivity and 87% specificity. 4 MeDEGs, F2R, FYN, PAG1 and PRKCH, exhibited significantly progressive decrease in SSc with interstitial lung disease (ILD) compared with SSc without ILD.

Conclusion

By integration of genome-wide transcriptome and DNA methylome, we identified differential expressed genes associated with aberrant DNA methylation in the PBMC of SSc. The epigenetically dysregulated genes may lead to the abnormal activation of immune regulatory pathways which may contribute to the pathogenesis of SSc.
Pan-Immunome Initiative: Building an integrated T and B cell receptor repertoire map for immune-related diseases
Authors: Liu Xiao , Zhang Wei , Wu Jinghua , Lin Liya , Nie Jianzheng , Zhao Ming , Lu Qianjin
Published: January 2023

The human adaptive immune system provides protection which is directly triggered by the receptors on the surfaces of T cells (TCRs) and B cells (BCRs). However, up till now, there are still difficulties in precise diagnosis for some immune related diseases. Here, we proposed a pan-immunome initiative, building an integrated T and B cell receptor repertoire map for immune related disease. As the early stage of this project, we have established a pan-immune-repertoire database (PIRD, https://db.cngb.org/pird/). We have collected more than 15,000 samples comprising 15 sorts of diseases. All samples have been amplified TCR b chain repertoire and sequenced using high throughput sequencer. We took systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) as examples for in-depth analysis. Significant differences of V, J and V-J pairing usage between SLE or RA and control groups were found and could be utilized as classification variables to discriminate the three groups (V: AUC>0.99). 228 SLE-associated and 169 RA-associated TCRs were identified using statistical test. A statistical model was generated to diagnose diseases using those disease-associated TCRs, with high specificity and sensitivity. Interestingly, lower overall diversities and massive expanded clones were observed in both SLE and RA samples, and higher TCR heterogeneity in SLE was found than in RA. However, clones related to specific organ damage in SLE were not observed. Besides, we have also been able to classify the TRB repertoire data of multiple diseases using an ensemble decision tree model. Thus, the immune repertoire data could reflect the individual immune status and serve as a powerful biomarker for molecular classification of immune related diseases.
Mast cell accumulation drives allergy-driven chronic pain responses in outbred mice
Authors: Chatterjea Devavani , Arriaga-Gomez Erica , Neamonitaki Nefeli , Kline Jaclyn , Boo Beebie
Published: January 2023

We are building novel outbred mouse models of a poorly understood yet prevalent vulvar pain condition – localized, provoked vulvodynia – that affects a substantial proportion of 18–60 year old women. Epidemiologic studies have shown a surprising and increased risk for developing this condition in women with a history of skin and airway allergies. We show that repeated exposures to low doses of the common contact irritant haptens oxazolone or dinitrofluorobenzene on the genital skin of mice produce sustained painful sensitivity to touch as well as a non-normative accumulation of mast cells and an overgrowth of cutaneous nerves in the affected tissue – changes that are also seen in vulvar biopsies taken from women diagnosed with vulvodynia providing the first evidence of biological plausibility for the immunological underpinnings of this chronic pain condition. In addition to laboratory haptens, we show that the hapten methylisothiazolinone (a common cosmetic preservative found in soaps, shampoos, and feminine hygiene products) can induce these responses suggesting that this ubiquitous environmental exposure might be a possible immunological trigger of hitherto unexplained genital pain conditions that affect many women.

We have further characterized infiltrating T cells at the allergic site to show that FoxP3+ regulatory CD4 T cells and CD103+ resident memory CD8 T accumulate at these sites as well. Our mouse models provide new tools to both dissect allergy driven pain conditions as well as test novel therapies targeting immune cells and mechanisms for the management and treatment of these debilitating disorders.
A gene expression module that identifies low-density granulocytes in the blood of patients with systemic lupus erythematosus is associated with low complement, elevated anti-dsDNA titer and both the interferon and TNF signatures
Authors: Kegerreis Brian , Catalina Michelle , Geraci Nicholas S. , Bachali Prathyusha , Lipsky Peter , Grammer Amrie
Published: January 2023

SLE is an autoimmune disease characterized by the presence of abnormal low-density granulocytes (LDG) with a heightened capacity for spontaneous NETosis. To characterize LDGs in SLE, gene expression profiles derived from isolated LDGs were characterized by Weighted Gene Co-expression Network Analysis, and a 92 gene module was identified. This module contains 30 genes with the Neutrophil Degranulation GO designation and many genes associated with DNA synthesis and proliferation. Comparison with gene expression by bone marrow neutrophil precursors demonstrated that the module of genes was most similar to that expressed by promyelocytes/myelocytes. The LDG gene signature was assessed in gene expression data sets from SLE whole blood (WB), PBMC, kidney, synovium, and skin. The LDG module was enriched consistently in blood but inconsistently in tissues. Lack of granule protein transcripts in tissues suggests that LDGs are not enriched in SLE affected organs. The LDG gene signature was also analyzed in WB and PBMC of 1,417 SLE patients by Gene Set Variation Analysis (GSVA). 35% of inactive patients and 50% of active patients expressed an LDG signature, which showed a significant but modest correlation with disease activity as measured by the SLE Disease Activity Index (r = 0.16, p = 4.1E-9). The LDG signature was also associated with the presence of anti-dsDNA (p < 1E-6), decreased levels of C3 and C4 (p < 1E-6), the type 1 interferon signature (p < 1E-11) and a module of genes induced by TNF (p < 1E-15, all by Fisher’s exact test). These findings suggest that LDGs are a population of immature neutrophils over-represented in the blood of SLE patients in association with cytokine stimulation, the presence of autoantibodies, and complement consumption.
Periodontal pathogen exposure facilitates disease activity in Systemic Lupus Erythematosus
Authors: Bagavant Harini , Dunkleberger Micah L. , Wolska Nina , Rybakowska Paulina , Sroka Magdalena , Rasmussen Astrid , Adrianto Indra , Montogomery Courtney , Sivils Kathy , Guthridge Joel M. , James Judith A. , Merrill Joan T. , Deshmukh Umesh S.
Published: January 2023

This study was undertaken to evaluate whether exposure to subgingival plaque bacteria and periodontal dysbiosis influences disease parameters in systemic lupus erythematosus (SLE).

Circulating antibodies to periodontal bacteria are surrogate markers to determine an ongoing bacterial burden, or are indicators of past exposure. SLE patient sera in the Oklahoma Lupus Cohort (n=303) were used to measure antibody titers against periodontal pathogens (A. actinomycetemcomitans, P. gingivalis, and T. denticola) and commensals (C. ochracea and S. gordonii) by ELISA. Correlations between anti-bacterial antibodies and clinical parameters of SLE including autoantibodies, complement, and disease activity (SLEDAI and BILAG) were studied. The effect of oral infection with A. actinomycetemcomitans on SLE in NZM2328 lupus mice was evaluated.

SLE patients had varying amounts of antibodies to different oral bacteria. The antibody titers against A. actinomycetemcomitans, P. gingivalis, T. denticola, and C. ochracea were higher in patients positive for anti-dsDNA, and in patients with low complement. Only antibodies to A. actinomycetemcomitans and P. gingivalis, but not T. denticola, were associated with higher disease activity. A. actinomycetemcomitans infected NZM2328 mice developed an increase in histone reactive T cells and an accelerated onset of proteinuria, and lupus nephritis.

Our results indicate that exposure to only specific pathogenic periodontal bacteria influences disease activity in SLE patients. The results from lupus mice suggest that this may be due to the amplification of local autoimmunity in response to infection. These findings provide a rationale for assessing and improving periodontal health in SLE patients.
2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD)-induced myeloid derived suppressor cells (MDSC) have heightened metabolic activity which correlates with microRNA and gene expression involved in immunosuppression
Authors: Neamah Wurood Hantoosh , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are bone marrow (BM)-derived cells consisting of monocytic (M-MDSC) or granulocytic (G-MDSC) subtypes with immunosuppressive activity. In the current study, we examined the metabolism and functions of MDSCs induced by TCDD a highly toxic environmental pollutant. Inasmuch, we injected mice with either vehicle or 10μg/kg TCDD interpersonally and found that TCDD increased both M-MDSCs and G-MDSCs in the peritoneal exudate when compared to controls. We determined the cell bioenergetics in TCDD-induced MDSCs when compared to naïve BM-MDSCs. For this purpose, we examined the mitochondrial oxygen consumption rate (OCR) and found that TCDD-induced MDSCs had heightened basal respiration, ATP-linked respiration, proton (H+) leak, maximal respiration, spare respiratory capacity as well as total proton efflux rate (PER) which means more suppressive function when compared to BM-MDSCs. We next examined whether the heightened metabolic potential in TCDD-induced MDSCs correlated with the microRNA and gene expression. MiRNA array analysis demonstrated that in TCDD-induced MDSCs, certain miRNAs (e.g. mir-543-3p, mir-150-5p) were downregulated which target immunosuppressive genes involved in MDSC functions, such as ARG2, IL-10, STAT-3, and PIM1. Next, we investigated the role of miRNA-150-5p in IL-10, PIM1, and STAT3 expression as well as miRNA-543-3p effects on ARG2 using transfection experiments. We found that MDSCs transfected with mature miRNA-150-5p and miRNA-543-3p were downregulated whereas inhibitors upregulated the expression of respective targets. In summary, our data revealed that TCDD modulates miRNA expression which can impact gene expression and metabolism in MDSC.
Use of fluorescent Fab/Ab complexes and IncuCyte live-cell analysis to dynamically track cell surface markers and cell populations in mixed cultures
Authors: Lovell Gillian F. , Bevan Nicola , Campwala Hinnah , Blancheteau Vincent , Dale Tim , Dana Nicholas , Holtz Nevine , Endlsey Eric , Trezise D. J.
Published: January 2023

Fluorescently-labeled antibodies are widely used for visualising cellular protein expression/distribution (e.g. immunocytochemistry) and immuno-phenotyping (e.g. flow cytometry). However, their applications are largely confined to end-point or short term (min-h) detection, and the cell processing and labeling steps that are required often perturb the biology of interest. To enable longer-term, fully kinetic applications of cell surface protein markers in living cells, we have developed a novel strategy based on fluorescently-labeled antibody fragments (Fabs) and IncuCyte live-cell analysis. An Fc-targeted anti-mouse Fab fragment conjugated to a green-emitting fluoroprobe (IncuCyte FabFluor-488) was used to tag Abs to surface markers (e.g. CD4, CD20, Her2) via a simple one-step, no-wash protocol. Addition of the corresponding FabFluor-488-Ab complex to living cells (e.g. Jurkats, RAMOS, SKOV-3) produced long lasting (up to 5d), specific and stable cellular fluorescence at relatively low Ab concentrations (1mg ml−1) that did not perturb cell morphology or growth (IncuCyte S3). To illustrate the application of this approach the method was used to (a) quantify upregulation of the checkpoint protein PDL-1 over 48h in IFN-gamma-treated MDA-MB231 and SKOV-3 and tumor cells, (b) identify cellular subsets in PBMCs, and (c) observe proximity and engagement of A549 target cells by CD-8 positive PBMCs in immune-cell killing experimentsThis FabFluor-488/IncuCyte method enables long-term tracking and quantification of cell surface protein expression and the ability to identify cell subsets in living cultures over time. This method should prove powerful in analyses on complex and advanced heterogeneous cell models.
A novel kynurenine-dependent circuit in DC1 promote IDO1 expression in DC2 leading to experimental autoimmune encephalomyelitis suppression
Authors: Gargaro Marco , Scalisi Giulia , Briseño Carlos G. , Manni Giorgia , Durai Vivek , Bagadia Prachi , Puccetti Paolo , Murphy Theresa L. , Murphy Kenneth M. , Fallarino Francesca
Published: January 2023

Classical dendritic cells (cDCs) are professional antigen-presenting cells that play a key role in shaping appropriate immune responses. DCs are a potent T cell activators but they are also involved in maintaining immune homeostasis and self-tolerance. DCs can be classified into three major types: pDC, DC1 and DC2. One mechanism by which DCs regulate tolerance involves indoleamine 2,3-dioxygenase 1 (IDO1) a tryptophan (Trp) metabolizing enzyme. In this study, we analyzed the ability of L-Kyn to induce tolerogenic IDO1 pathway in different DCs subsets in vitro and in vivo model of experimental autoimmune encephalomyelitis (EAE). We show that inflammatory stimuli, like LPS, was able to induce IDO1 only in DC1, but not in DC2 or pDC, when DCs were treated as isolated cultures. In contrast, when LPS was added to cultures containing all three DC subsets, LPS could also induce IDO1 expression in DC2, which acquired tolerogenic function. Induction of IDO1 in DC2 involved a novel DC1-DC2 communication pathway mediated by a Kyn-AhR-RelB axis. Kynurenine produced by DC1 activates AhR in DC2 inducing IDO1 in a RelB-dependent manner. In vitro L-Kyn treatment impaired DC2 T cells priming ability causing suppression of MOG-specific reactivity with an increment of Foxp3+ CD4+ T cells. In vivo, oral administration of L-Kyn induces functional Treg cells that suppress EAE and this effect is completely abrogated in Ahrflox/floxCD11C Cre+ mice. These data suggest that in specific microenvironments, small numbers of IDO1-expressing DC1 may spread tolerogenic activity to DC2 cells through a kynurenine-AhR axis and L-Kyn could constituting a unique endogenous molecule for therapeutic immunomodulation of inflammatory and autoimmune diseases.
Regulation of necroptotic factors in gastric cancer cells through miR-93-5p and EBV-miR-BART 18-3p.
Authors: Kim Seon Hyun , CHOI DABIN , HUR DAEYOUNG , KIM YEONGSEOK
Published: January 2023

Epstein-Barr virus (EBV), as well as Helicobacter pylori, has recently been admitted as an infective agent causing gastric carcinoma(GC). EBV encodes multiple non-coding RNAs during all types of latency, 44 mature miRNAs have been identified. EBV-encoded microRNAs showed that these small molecules function as post-transcriptional gene regulators and may play a role in the carcinogenesis process. MiR-BARTs produced by EBV can regulate cellular genes for preventing apoptosis and escaping the host immune system. We examined interactions of microRNAs with necroptosis factors in EBV positive/negative gastric cancer cells. RIPKs(receptor-interacting serine/threonine-protein kinase) were little expressed in EBV positive cells. Incubation of EBV negative gastric cells with EBV particles or exosome purified from EBV positive cells revealed that MLKL as a regulator of necroptosis were decreased. We investigated expression profiles for viral and cellular miRNAs in EBV positive/negative gastric cancer cells. To identify possible miRNAs targeting necrotic factors, we searched miRNA candidates that could bind to sequence in the 3′UTR of necrosis factors using an online miRNA database. We found the putative target binding sites of EBV-miR-BART18-3p (BART18-3p) within MLKL 3′UTR and miR-93-5p within RIPK3 3′UTR. Expectedly, we observed that EBV negative KATOIII transfected with mimics for BART18-3p diminished expression of p-MLKL, inhibitor for BART18-3p increased p-MLKL in EBV positive SNU719. However, mimics for miR-93-5p decreased RIPK1 rather than RIPK3 in EBV negative KATOIII. Taken together, we suggested that EBV might regulate necroptosis in gastric cancer cells through miRNAs including BART18-3p and miR-93-5p.
Non-canonical autophagy mediates immunosuppression during challenge
Authors: Martinez Jennifer
Published: January 2023

Autophagy is an evolutionarily conserved, catabolic pathway that lies at the intersection of homeostasis and host immune response. The autophagy machinery functions in a multitude of biological pathways, including the recycling of intracellular nutrients during starvation or stress (macroautophagy), the quality control removal of damaged proteins or organelles (aggrephagy, mitophagy), or the degradation of and host defense to extracellular threats, such as pathogens or dying cells. However, the non-canonical roles of the autophagy machinery are a critical node in the immune response to invasive pathogens, as well as prevention of autoimmunity. We have identified a form of non-canonical autophagy termed LC3-associated phagocytosis (LAP), a phenomenon in which phagosomes containing engulfed particles, including pathogens and dying cells, recruit certain elements of the autophagy pathway to facilitate phagosome maturation, digestion of cargo, and modulation of innate immunity. Importantly we have characterized Rubicon as a LAP-specific, autophagy-independent molecule, allowing the study of the unique role of LAP. We recently demonstrated that defects in LAP, but not canonical autophagy, result in a lupus-like disease with age. Using Rubicon−/− mice, we are now investigating the role of LAP in other autoimmune disease onset and progression. Our evidence clearly indicates that LAP is critical in the maintenance of immunotolerance in a variety of inflammatory diseases, including type 1 diabetes, allergic asthma, and atopic dermatitis. Mechanistically, we have begun to dissect the molecular pathways, such as metabolism, that LAP modulates in order to achieve immunosuppression.

This research was supported by the NIEHS.
The Effect of Exogenously added and Endogenously Produced Cytokines on the Regulation of Inflammatory Mediators and SOCS Proteins in Mouse Macrophages Exposed to Chlamydia muridarum Major Outer Membrane Protein
Authors: Duncan Skyla A. , Sahu Rajnish , Singh Shree R. , Dennis Vida
Published: January 2023

The major outer membrane protein (MOMP) of Chlamydia is a candidate for vaccine developmental efforts due to its high immunogenic potential. We previously reported that IL-10 can regulate Chlamydia inflammation by inhibiting its associated-induced inflammatory mediators possibly via induction of the Suppressor of Cytokine Signaling (SOCS) proteins. In this study, we determined the effect of exogenously added and endogenously produced cytokines on the regulation of inflammatory mediators and SOCS proteins as induced in mouse J774 macrophages exposed to C. muridarum (Cm) recombinant MOMP. Macrophages were stimulated with rMOMP (10 ug/mL) in the presence and absence of exogenous IL-10, TNF and IL-6 (each at 10 ng/mL). Cytokine and chemokine specific ELISAs revealed that exogenously added IL-6 and TNF did not alter the levels of inflammatory mediators (CCL5, CXCL10, IL-12) induced by rMOMP; whereas IL-10 downregulated all mediators. Neutralization of endogenously produced IL-6 and TNF, respectively by anti-IL-6 or anti-TNF antibodies (each at 25 μg/mL) did not significantly perturb the expression of rMOMP-induced inflammatory mediators. On the contrary, neutralization of IL-10 by an anti-IL-10 antibody increased the production of all mediators, thus confirming that IL-10 regulates Cm-induced inflammation. Removal of endogenously produced IL-10 increased SOCS1 expression, which suggest an important role of IL-10 in the regulation of SOCS1 expression to potentially control Cm-induced inflammatory responses. Collectively, our data sheds more light on the potential roles of these cytokines implicated during chlamydial infections, but more importantly, how IL-10 regulates Chlamydia inflammation via SOCS proteins.
Hrd1 protects regulatory T cells from ER stress-induced instability and dysfunction
Authors: Xu Yuanming , Cardenas Sindey Johanna Melo , Jin Hongjian , voss Jessica heinrichs , Gau Isabella , Wei Juncheng , Montauti Elena , Zhang Yusi , Gao Beixue , Mathew James , Fang Deyu
Published: January 2023

T regulatory cells (Treg)differentiation, maintenance, and function are controlled by the transcription factor FoxP3, which can be destabilized under inflammatory or other pathological conditions. However, the microenvironmental factors that compromise Treg identity and the underlying mechanisms remain unclear. Here, we show that ER stress destabilizes both human and mouse Treg by suppressing FoxP3 expression, suggesting a critical role of the ER stress response in maintaining Treg properties. We found that genetic deletion of Hrd1, an E3 ligase critical in suppressing the ER stress response, leads to elevated expression of ER stress-responsive genes in Treg. Importantly, mice with Treg-specific ablation of Hrd1 displayed massive multi-organ lymphocyte infiltration, body weight loss, and the development of severe small intestine inflammation with aging. At the molecular level, the deletion of Hrd1 led to upregulation of the ER stress response, in particular, the activation of the ER stress sensor IRE1. Impaired FoxP3 expression caused by Hrd1 deletion was largely rescued by an IRE1 kinase inhibitor. Taken together, these findings indicate that Hrd1 is crucial for maintaining the expression and suppressive function of Treg through suppression of the IRE1-mediated ER stress response. The study is significant because it will help to fill fundamental gaps in our knowledge of the role of Hrd1 in T regulatory cells, which will inform the design of future immunotherapies for cancer patients or immune dysregulated diseases.
Fetal naive T cells are primed for preferential regulatory T cell differentiation through increased chromatin accessibility and expression at the Helios locus.
Authors: Ng Melissa , Burt Trevor D.
Published: January 2023

Human fetal CD4+ naïve T cells are primed to differentiate into CD25hiFOXP3hi regulatory T (Treg) cells upon T cell receptor (TCR) stimulation without TGFβ supplementation, thus contributing to the generation of immunotolerance in the developing human fetus. In addition to expression of the lineage-determining factor FOXP3, commitment to the Treg cell fate is preceded by the acquisition of permissive epigenetic modifications at Treg-specific enhancers associated with the transcriptional control of Treg signature genes. Through ATACseq (Assay for Transposase-Accessible Chromatin Sequencing) and H3K27ac ChIPseq (Chromatin Immunoprecipitation Sequencing), we reveal that this intrinsic predisposition for Treg differentiation in fetal naïve T cells is correlated with increased chromatin accessibility and H3K27ac enrichment at 906 and 109 Treg-specific enhancers respectively. We show by RNA sequencing that fetal naïve T cells subsequently have increased transcription of the underlying Treg-associated genes such as Helios (IKZF2). Using flow cytometry, we confirmed that Helios expression is higher in fetal naïve T cells relative to adult naïve T cells. CRISPR (clustered regular interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) mediated knockdown of Helios expression in primary human fetal naïve T cells reduced induction of CD25hiFOXP3hi Treg cells in response to TCR stimulation alone, indicating that high baseline Helios expression contributes to the priming of Treg differentiation in fetal naïve T cells. Overall, a novel Treg-associated epigenome and transcriptome within fetal naïve T cells establishes preferential Treg differentiation as a mechanism to maintain immunotolerance in utero.
GDF15 and BMP4 co-regulate stress erythropoiesis cmaintain erythroid homeostasis and resolve inflammation during infection.
Authors: Fraser James William , Dey Adwitia , Nettleford Shaneice K. , Zhao Luming , Hankey-Giblin Pamela A. , Prabhu K. Sandeep , Xiong Na , Paulson Robert F.
Published: January 2023

Bone marrow steady state erythropoiesis maintains erythrocyte homeostasis. However, during inflammation it is unable to generate sufficient erythrocytes as pro-inflammatory cytokines inhibit medullary output. At these times extra-medullary stress erythropoiesis (SE) predominates. SE utilizes progenitors and signals that are distinct from the steady state. Using a model of sterile inflammation, we have recently shown that the inflammatory signals that suppress steady state erythropoiesis activate SE. While we have demonstrated an important role for SE in maintaining erythroid homeostasis during inflammation, the potential for SE to directly influence inflammation remains largely unexplored. We report the first study to demonstrate the importance of SE in the context of a bona fide infection using the model pathogen Citrobacter rodentium. We observed that infection rapidly induces SE in the spleen, however mice mutant for either of the two key signals (BMP4 and GDF15) that regulate SE exhibited compromised recovery following infection. Inflammation was significantly increased while FoxP3+ regulatory T-cells (Tregs) were decreased in these mice. In vitro, induction of Tregs was defective when naïve CD4+ T cells from mutant mice were used. The addition of BMP4 or GDF15 into these cultures rescued Treg induction. These data suggest a new model that includes a more extensive interplay between erythroid homeostasis and the immune response. In this model Treg induction, like the activation of SE, depends on the action of both BMP4 and GDF15. Thus, BMP4 and GDF15 may function together during the critical period immediately following inflammation to synchronously maintain both erythroid homeostasis and regulate inflammation in vivo.
Tregs orchestrate antigen specific suppression via stripping cognate peptide-MHCII from the DC surface
Authors: Akkaya Billur , Akkaya Munir , Oya Yoshihiro , Al Souz Jafar A. , Holstein Amanda H. , Kamenyeva Olena , Kabat Juraj , Dorward Dave , Glass Deborah D. , Shevach Ethan M.
Published: January 2023

Tregs are professional suppressors of the immune response, yet their mechanism of action in vivo remains unclear. We compared the morphology and stoichiometry of the interactions of activated 5CC7 T cells and 5CC7 iTregs with MCC88–103 pulsed splenic DCs by electron and confocal microscopy. Image analyses revealed that Tregs interacting with DC displayed a distinct morphology with finger like membrane projections at the DC binding site and uropods at the rear end within three hours of in vitro culture. In contrast, activated T cells maintained their round morphology. Tregs occupied a greater extent of the DC surface than activated T cells consistent with a higher binding avidity. Dynamic intravital two-photon microscopy of adoptively transferred OTII iTregs and activated OTII cells demonstrated that Treg displayed a greater volume and duration of contact with OVA323–339 pulsed DC compared to that of activated OTII cells. Subsequent to their high avidity interactions with DC, 5CC7 iTregs captured MCC88–103-I-Ek complexes from DC surface reducing the amount of MCC presented on DC. Reduced antigen presentation was not due to global suppression of the DC’s ability to present antigen as 5CC7 and 3A9 iTregs only captured their cognate peptide-MHCII from MCC/HEL double pulsed DC, reducing the DC presentation in an antigen specific manner. When double pulsed DCs were cultured with iTregs specific for one peptide, separated from the iTregs, the DC failed to prime T cells specific for the antigen seen by the iTreg, but primed T cells specific for the second antigen. Altogether, we propose antigen specific depletion of peptide-MHCII complexes as a new mechanism for Treg-mediated suppression.
Subset-specific neurotransmitter receptor expression tunes T cell activation
Authors: Rosenberg Kenneth M. , Singh Nevil J.
Published: January 2023

T cells continually patrol and invade other tissues and are exposed to varying tissue-specific cues. Different tissues are typically innervated by neurons using characteristic neurotransmitters. Therefore, encounter with particular neurotransmitters has the potential to influence the tissue-specific behavior of T cells. Although neurons utilize a complex array of over 180 neurotransmitter receptor (NR) genes, we find that murine T cells in total express only a limited set (26 detected) of them. Furthermore, the expression is T cell subset-specific suggesting distinct functional roles. Several receptors, including Adrb2, Gabrr2, and Chrnb2, are most highly expressed in naïve CD4 T cells while CD8 T cells specifically express the glutamate receptor Gria3 and have high expression of the cannabinoid receptor Cnr2. Within the CD4 population, memory T cells upregulate Hrh4 and P2ry1 while the VIP receptor Vipr1 is uniquely absent from regulatory T cells. In order to understand how these distinct patterns affect immune responses, we are analyzing the functional impact of signaling T cells through them. Importantly, NR signaling pathways considerably overlap with T cell receptor (TCR) signaling pathways. Accordingly, preliminary data shows that a competing signal from NR receptors such as the β2 adrenergic, histamine H2, and VPAC1 (VIP) receptors dampens direct T cell activation through the CD3 complex. Determining how T cells integrate the complex contextual information they encounter in vivo guides our understanding of T cell decision making and will allow for the development of more targeted therapeutics.
CD11bhigh CD11clow Ialow DCs regulate T-cells activation in the experimental autoimmune uveitis
Authors: Lin Wei , Wang Xin , Fan Zhichao
Published: January 2023

Uveitis is a serious inflammatory disease that can cause visual disability and blindness. Uveitogenic antigen-specific CD4+ T cells which secrete IFN-γ or IL-17, are believed to be crucial effector cells to infiltrate in the sites of inflammatory eyes to drive inflammation and tissue damage. Recently, dendritic cells (DCs) act as unique antigen presenting cells, are reported to involve into the pathogenic process of uveitis. Regulating the status of DCs could prevent the generation of Ag-specific Th1 and Th17 cells to attenuated EAU. Herein, in a mouse model of experimental autoimmune uveitis (EAU), we found CD11bhighCD11chighIahigh DCs increased at the inflammation stage of EAU, while CD11bhighCD11clowIalowDCs, a regulatory DCs subset, increased at the recovery stage. CD11bhighCD11chighIahigh DCs expressed high level of CD80, CD86 and CD54 to promote T cells secreting IFN-γ, While CD11bhighCD11clowIalowDCs promote T cells to express high level of CTLA-4 and to be CD4+CD25+FoxP3+ T cells. Furthermore, CD11bhighCD11clowIalowDCs were found to form a regulatory immunological synapse with T cells, and CTLA-4 localized at the contact of DC-T to prevent the localization of TCR signaling molecules and the localization of calcium microdomains at the contact of DC-T to inhibit T-cell activation. However, CD11bhighCD11chighIahigh DCs promote the accumulation of TCR signaling molecules in the IS and calcium releasing in T cells. Transferring CD11bhighCD11clowIalowDCs into EAU could relive the symptom of EAU and decrease Ag-specific T cells in EAU. Overall, CD11bhighCD11clowIalowDCs could suppress T-cells activation by forming a regulatory synapse to decrease calcium releasing in T cells to prevent the development of EAU.