bluuebunny/crossref_metadata_2025 huggingface dataset:
PAM3 supports the generation of M2-like macrophages from lupus patient monocytes and improves disease outcome in murine lupus
Authors: Horuluoglu Begum Han , Bayik Defne , Kayraklioglu Neslihan , Goguet Emilie , Blanco Luz P , Kaplan Mariana J , Klinman Dennis M
Published: January 2023

Systematic Lupus Erythematosus (SLE) is an autoimmune syndrome of unclear etiology. While T and B cell abnormalities contribute to disease pathogenesis, recent work suggests that inflammatory M1-like macrophages also play a role. Previous work showed that the TLR2/1 agonist PAM3CSK4 (PAM3) could stimulate normal human monocytes to preferentially differentiate into immunosuppressive M2-like rather than inflammatory M1-like macrophages. This raised the possibility of PAM3 being used to normalize the M1:M2 ratio in SLE. Consistent with that possibility, monocytes from lupus patients differentiated into M2-like macrophages when treated with PAM3 in vitro. Furthermore, lupus-prone NZB x NZW F1 mice responded similarly to weekly PAM3 treatment. Normalization of the M2 macrophage frequency was associated with delayed disease progression, decreased autoantibody and inflammatory cytokine synthesis, reduced proteinuria and prolonged survival in NZB x NZW F1 mice. The ability of PAM3 to bias monocyte differentiation in favor of immunosuppressive macrophages may represent a novel approach to the therapy of SLE.
Mitochondrial changes synergize with long chain fatty acid derivatives to support Th17 inflammation in diabetes
Authors: Agrawal Madhur , Nicholas Dequina A , Proctor Elizabeth A , Belkina Anna C , Jones Albert , Panneerseelan-Bharath Leena , Raval Forum , Ip Blanche , Zhu Min , Cacicedo Jose , Habib Chloe , Sainz-Rueda Nestor , Persky Leah , Sullivan Patrick G , Corkey Barbara E , Apovian Caroline M , Kern Philip A , Lauffenburger Douglas A , Nikolajczyk Barbara S
Published: January 2023

Obesity-associated Type 2 diabetes (T2D) is driven by chronic inflammation. A combinatorial Th17 cytokine profile characterizes and mathematically predicts T2D in people, but the mechanisms that generate the Th17 profile are not clear. We tested the possibility that anaerobic glycolysis, which fuels inflammatory cytokine production from multiple immune cell types, drives the T2D-associated Th17 profile. We showed that activated PBMCs and purified CD4+T cells from T2D subjects prefer anaerobic glucose metabolism to produce ATP regardless of fuel availability. Unexpectedly, glucose starvation did not abrogate the T2D-predictive Th17 profile. Gene expression array suggested that mitochondrial fatty acid uptake catalyzing protein CPT1a differentiated PBMCs from T2D and ND subjects. CPT1a inhibition by etomoxir strongly down-regulated the Th17 profile, surprisingly independent of CPT1a-mediated fatty acid oxidation (OXPHOS). These data suggest that OXPHOS-independent mitochondrial changes that are also glucose-independent support the Th17 profile in T2D. Analyses from T2D vs ND PBMCs showed a lower CACT:CPT1a protein ratio in T2D, indicating defects in lipid uptake and thus defects in lipid flux. We knocked down CACT protein and overloaded PBMCs from lean subjects with fatty acylcarnitines to mimic lipid uptake defects in T2D. 16C-fatty acylcarnitine, but not 6C- or 10C-derivatives, increased frequency of CD4+IL-17+T cells, and phenocopied the Th17 profile only in CACT knockdown cells. 16C-fatty acylcarnitine alone had no effect. We conclude that excessive long chain fatty acylcarnitine combines with dysfunctional mitochondria to support a T2D-associated Th17 profile largely independent of glycolysis.
Newfound role for noncanonical NF-κB signaling in Inflammatory Bowel Disease: Gene expression levels serve as a proxy for predicting anti-TNF therapy responsiveness
Authors: Morrison Holly , Eden Kristin , Nguyen Vu Q , Sorrentino Dario R , Brock Rebecca M , Allen Irving C
Published: January 2023

The canonical arm of NF-κB signaling is well studied in Inflammatory Bowel Disease (IBD) while its noncanonical counterpart has yet to be fully defined. The activation of the noncanonical pathway initiates the nuclear transcription of chemokines associated with chronic inflammation and other diverse immunological processes. Infliximab is an effective anti-TNF treatment that reduces inflammation in IBD, yet a significant percentage of patients are unresponsive or lose responsiveness. Here, we show that noncanonical NF-κB signaling has more relevant implications on IBD and its expression levels correlate to infliximab responsiveness. Our study evaluated noncanonical NF-κB signaling in 27 IBD patient and 9 non-IBD control patient tissue biopsies. Data from these studies were analyzed using bioinformatics to predict its influence on cellular pathways. Genes related to the noncanonical NF-κB pathway were significantly upregulated in IBD lesions compared to healthy tissue. IBD patients that positively respond to infliximab had significantly decreased expression levels compared to nonresponsive patients. These results suggest infliximab, when effective, decreases noncanonical signaling to lessen inflammation. Through targeting in part, the noncanonical NF-κB pathway has prominent emerging roles in IBD pathobiology and is an indicator for improving drug efficacy.
Enhanced adenosinergic signaling and immune cell exhaustion after trauma
Authors: Tiwari-Heckler Silpa , Longhi Maria Serena , Harbison James , Otterbein Leo E. , Hauser Carl J. , Robson Simon C.
Published: January 2023

Trauma is currently the leading cause of death in young adults, in some cases due to infection as a consequence of acquired immune suppression. The pathogenesis for this is poorly understood. Extracellular ATP released from cells during injury serves as a danger molecule, but is rapidly converted to AMP by the ectonucleotidase CD39; AMP is then hydrolyzed to immunosuppressive adenosine by CD73 ecto-5’-nucleotidase.

Here, we studied the impact of purinergic signaling in trauma patients over time (day 0/1 after trauma (D1) vs day 2 after trauma (D2) vs controls undergoing elective surgery). We noted higher expression of Adenosine A2A- and A2B- receptor mRNA levels in blood PBMCs on D2 and later, when compared to D1 trauma patients and control samples. Analyses of BAL-derived mononuclear cells revealed decreases in CD39 activity in D1 trauma patients and marked decreases in CD73 activity in D2 trauma patients, when compared to controls. Notably, there was increased CD39 and CD73 expression in PBMC-derived CD8 T-cells of trauma patients, suggesting these cells as the prominent subset involved in the purinergic signaling during trauma. Importantly, CD39+CD8+cells from trauma patients displayed features of “immune exhaustion” phenotype (i.e. high PD-1 and Tim-3 levels) and exhibited less cytotoxicity.

We conclude that the early phase after trauma is initially characterized by heightened systemic inflammation with boosted ATP-mediated signaling, linked with loss of CD39 activity. This is followed by an immunosuppressive phase characterized by enhanced adenosinergic signaling and by increase in exhausted CD39+CD8+cells, which might impair the innate immune responses and lead to increased susceptibility to infections in trauma patients.
Dysregulated monocyte compartment in human obesity
Authors: Friedrich Kathleen , Sommer Miriam , Thrum Stephan , Blüher Matthias , Wagner Ulf , Rossol Manuela
Published: January 2023

Background

Monocytes can be divided in classical monocytes (CM), intermediate monocytes (IM) and non-classical monocytes (NCM). CD56+ monocytes are part of the classical monocytes. Aim of the study was to assess the occurrence of the different monocyte subpopulations in human obesity.

Methods

110 age-matched subjects were included in the study and classified according to the definition of the World Health Organization into lean (n=27), overweight (n=23) and obese (n=60). Monocyte subpopulations were analyzed by flow cytometry (antibody staining of CD14, CD16, CD56).

Results

Obese subject showed a higher BMI, fat mass, and waist circumference, deterioration of metabolic parameters, and the absolute amount of leukocytes and monocytes were increased. The calculated absolute numbers of CM, IM and CD56+ monocytes were significantly increased in obese subjects compared to lean and overweight. In contrast, the absolute numbers of NCM were equal between lean, overweight and obese subjects. Strong associations between the absolute numbers of CM, IM and CD56+ monocytes with the BMI, fat mass, and waist circumference were detectable in the whole cohort. Triglyceride, HDL-c values and HbA1c were also strongly correlated with numbers of CM, IM and CD56+ monocytes. Obese subjects with impaired glucose tolerance had increased numbers of CD56+ monocytes in comparison to obese subsects with normal glucose tolerance. Stratification of obese subjects in subjects with and without type 2 diabetes mellitus showed no difference in absolute numbers of CM but significant increased numbers of IM and NCM.

Conclusion

The amount of CM, IM and CD56+ monocytes are expanded in the blood of obesity subjects and contribute to monocytosis in obesity.
Mathematica as a Tool for Interactive Display of Flow Cytometry and Clinical Data
Authors: Loewendorf Andrea I , Collier Ai-ris Y , Kahn Daniel A , Nevell Julian , Nguyen Tina A
Published: January 2023

In a prior study, we used flow cytometry to characterize lymphocytes from mother-infant pairs from healthy and preeclamptic pregnancies. Despite a relatively low number of study participants, we acquired a large, multidimensional dataset and felt limited in our capacity to fully explore it. Here we describe the use of Mathematica to create an interactive display tool that allows dynamic data exploration. The end result is a HIPAA compliant data library and code that can be exported, published, and explored by readers with the freely available MathReader application.

Our database included flow cytometry data from 8 staining panels with up to 15 markers each, clinical, and obstetric data in MS Excel. By combining Mathematica’s Chart, Manipulate, and Tooltip functions, we incorporated the clinical variables including neonatal sex, birth weight, and prior obstetric history into each graph, while the Tooltip function displays the participant study ID.

We have coded an interactive tool that allows the rapid display of data from multiple complex datasets in a single, interactive graph allowing the scientist to compare immunologic parameters between specific clinical conditions. This makes it possible to highlight, for example: activated NK-, NKT-, γδT -, naïve CD4 T, CD8 T-, memory CD4 T, and memory CD8 T cells from mothers with PE who gave birth to female neonates who were small for gestational age, and easily change all those graphs to highlight those patients who gave birth normal size fetuses. Interactive tools are ideal to display complex, multidimensional data such as human immune phenotyping. Ideally, they should also be publishable as interactive tools for the reader to explore on their own. Mathematica allows for this.
Construction of humanized mice, kinetics of human immune system development and induction of matured and specific T-dependent and T-independent antibody responses
Authors: Chupp Daniel P , Acosta Ometeotl , Zan Hong , Casali Paolo
Published: January 2023

Inbred mice, the most widely used surrogates for human biology, recapitulate many features of the human immune system and immune response. They, however, diverge from humans in important immune elements and functions, such as differential TLR expression, certain antibody and T cell responses, species-specific viral and/or bacterial infections and drug interactions, thereby underscoring the need for a human model that mimics the in vivo human immune response. Humanized mice have recently provided some data on human infections, autoimmunity and cancer, with limited results.

We have undertaken a systematic approach to the generation of humanized mice by grafting NOD.Cg-KitW-41J Prkdcscid Il2rgtm1Wjl/WaskJ (NSGW41) mice within 48 hours of birth, devoid of prior irradiation, with human hematopoietic (CD34+) stem cells. Human umbilical cord hematopoietic stem cells were collected within 30 minutes post-partum and injected intracardially, consistently yielding up to 95% human cell peripheral reconstitution. Human leukocyte development, differentiation and long-term persistence in our CD34+ cell-grafted NSGW41 mice revealed B and T cell maturation as part of a full immune system development, which led to emergence of IgM, IgG, IgA and IgE antibody titers comparable to wild type mice. CD34+ cell-grafted NSGW41 mice supported specific T-dependent and T-independent antibody responses that include human B cell class switch DNA recombination, plasma cell and memory B cell differentiation. They also supported a hydrocarbon-induced autoantibody response leading to symptoms mimicking systemic lupus. Thus, we have devised a robust in vivo platform allowing for generation and maturation of human antibody and autoantibody responses.
The effect of bee venom phospholipase A2 (bvPLA2) on high-fat-diet-induced obesity in mice
Authors: JEONG hYUNJU , Lee Hyunji , Bae Hyunsu
Published: January 2023

Obesity is a medical condition which shows excess body fat accumulation and is a complex metabolic disorder accompanied with cardiovascular disease, high blood pressure, type 2 diabetes and high cholesterol. Further, it is characterized by the accumulation of inflammatory immune cells into white adipose tissue (WAT) in obesity as a feature of the dysfunction of AT. Bee venom is a complex mixture of proteins and compounds and has been reported to have anti-tumor, anti-inflammatory and anti-obesity function. However, the specific compound of bee venom which is responsible for its anti-obesity effect in obesity is still unknown. In this study, the therapeutic effect of PLA2 on high-fat-diet (HFD)-induced obesity was confirmed. As a result, we confirmed that bvPLA2 markedly decreased body weight and WAT weight and significantly lowered the liver and kidney weight in HFD-fed mice. The fat accumulation in liver was also decreased by bvPLA2 in HFD-fed mice. bvPLA2 also decreased adipocyte size. We further confirmed that bvPLA2 decreased the inflammatory cell accumulation in HFD-fed mice. Thus, bvPLA2 can be used as a therapeutic agent for obesity by regulating immune homeostasis.
Keratinocyte-derived CCL20 orchestrates epidermal localization of IL-17-producing γδ T cells and ILCs to mediate psoriasis-like skin inflammation
Authors: Singh Tej Pratap , Lu Xinping , Singh Satya P , Zhang Hongwei , Doucet Michele , Kominsky Scott L , Farber Joshua M
Published: January 2023

CCR6 is important for imiquimod (IMQ)-induced psoriatic dermatitis in mice, which depends on motile CCR6+ γδ T cells that produce IL-17 and IL-22. CCR6 ligands include CCL20 and β-defensins. We used new strains of Ccl20 tdTomato knock-in mice, Ccl20fl/flmice, and Ccr6fl/fl GFP knock-in mice to determine sites of production and action of CCL20 and its role in the localization of γδ T cells and ILCs during imiquimod (IMQ)-induced inflammation. IMQ treatment of Ccl20+/tdT mice led to expression of Ccl20 in keratinocytes (KCs), including in the infundibulum and isthmus of the hair follicles. Ccl20−/− mice showed reduced inflammation and expression of Il17 and Il22 in the treated skin. Deletion of Ccl20 specifically in the KCs by treating Ccl20fl/flK14-CreERT mice with tamoxifen before applying IMQ significantly decreased the accumulation of IL-17-producing gd T cells and ILCs in the epidermis. Depleting KC-derived CCL20 at day 4 when inflammation was well established also reduced the number of epidermal γδ T cells and ILCs. In both cases the percent of IL-17-producing γδ T cells and ILCs did not change, demonstrating that migration to the epidermis and the ability to produce IL-17 are regulated independently. Multi-photon microscopy of IMQ-treated Ccr6+/gfp and Ccr6gfp/gfp(CCR6-deficient) micerevealed a CCR6-independent step whereby dermal CCR6+ cells accumulate at the hair follicles, followed by a CCR6-dependent step whereby the cells move into the superficial epidermis.
Engagement of CD45 differentially alters early signaling events in human T cells costimulated through TCR+CD28 compared with TCR+ICAM-1
Authors: Bhatta Anuja , Chan Marcia , Benedict Stephen H
Published: January 2023

Our lab has been developing the concept that co-stimulation of human naïve CD4+ T cells through different costimulatory molecules can tune differentiation to different phenotypes. Much of our work has compared costimulation through TCR/CD3+CD28 with costimulation through TCR/CD3+ICAM-1. In the absence of exogenously added cytokines, we observed differences in phenotypic outcomes among Th1, Th2 and Treg cells. An open question is where in the signaling pathways induced by these two costimulation regimens do differences occur that contribute to outcome of differentiation. The cell surface glycoprotein, CD45, exerts intracellular tyrosine phosphatase activity immediately after recognition of cognate Ag by the TCR. CD45 plays a crucial role to initiate T cell signaling by dephosphorylating a negatively regulatory tyrosine residue in the C-terminal region of Src family kinases such as Lck. Here, we investigate the early signaling process by comparing events that follow engagement of CD45 alone or in association with the two co-stimulatory molecules: ICAM-1 or CD28. Using protein blotting, we have confirmed that engagement of CD45 alone induces changes in protein phosphorylation. We have observed that CD45 engagement affected differently the composition of signaling complexes formed during costimulation through CD3+CD28 compared with CD3+ICAM-1. We hypothesize that during TCR recognition of cognate antigen, engagement of CD45 differentially guides signaling events in T cells depending on the mode of costimulation. A goal is to learn whether different outcomes of naïve T cell differentiation can begin at the earliest steps of T cell activation and ultimately how these signals progress during the activation process.
Molecular background of Orai1 accumulation in the immunological synapse
Authors: Vörös Orsolya , Panyi György , Hajdu Péter
Published: January 2023

CRAC channels have a vital role in the activation of T lymphocytes, through their calcium influx. CRAC is composed of the ER-membrane resident STIM1 (stromal interaction molecule 1) and the plasma membrane Orai1 pore-forming subunit. Both are accumulated at the interface of the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). CRAC channelopathies due to mutations in either Orai1 or STIM1 are characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia. The down-regulation of actin-binding protein expression (e.g. HS1), which may be responsible for the immobilization of ion channels, hampers Ca2+-signaling and IS formation of T lymphocytes. We hypothesized that actin-binding proteins modulate the trafficking of the CRAC channels to the IS, which is crucial for the Ca2+-signaling. By means of GST-affinity assay we could show that HS1, which is an actin-regulatory adaptor protein, binds to the N-terminus of the Orai1. N-terminal truncated Orai1 channels (ΔN1: 1–74, ΔN2: 1–88) were created and expressed in Jurkat cells. Then Orai1 channel expressing Jurkat cells were conjugated to CD3/CD28 beads as an APC and Orai1 polarization was determined: the Orai1-ΔN1 mutant similar to the wild-type Orai1 redistributed to the IS but the Orai1-ΔN2 did not. Furthermore, the HS1 protein did not show IS-accumulation in cells expressing the Orai1-ΔN2 subunits unlike for those transfected with the Orai1-ΔN1 or the wild-type constructs. Overall, these data indicate that Orai1-HS1 interaction could contribute to the anchoring of the Orai1 in the IS and can affect the Ca2+-signaling upon T cell activation.
Role of PAFR-MHC-II-CD38 interactions in infections
Authors: Khan Aslam A
Published: January 2023

Infectious diseases are the most prevalent cause of deaths around the world. Chronic contagious ailments will account for seven out of ten deaths in the world by 2020. The most significant problem in the treatment of the infectious disease is the ever-increasing rate of resistance to drugs mounted by pathogens. Our preliminary results suggest that during antigen presentation the major histocompatibility complex II (MHC-II) engages two associate protein molecules CD38 and Platelet Activating Factor Receptor (PAFR). CD38 and PAFR elicit two counteractive signaling pathways in APCs resulting in the release of either proinflammatory cytokine such as IL-12 or anti-inflammatory cytokines such as IL-10 respectively. Pathogens show remarkable adaptability to the drugs by either metabolizing the drug molecules or by changing the surface profile to neutralize binding of drugs to the pathogens. Therefore, there is a requirement to find new treatment modalities that do not target the pathogens but aid the immune system to eliminate the pathogens. Our preliminary data further suggest that the use of associate molecule during antigen presentation decides the fate of pathogens such as Salmonella Typhimurium or Staphylococcus aureus in macrophages. For example, if MHC-II engages CD38 to signal inside the macrophages leading to reduced pathogen survival in macrophages and mice, while MHC-II-signaling via PAFR aid the pathogen survival in macrophages and mice. Our studies also suggest that combinatorial use of PAFR inhibitor (WEB2086) and antibiotics ampicillin reduced the growth of S. Typhimurium or S. aureus in macrophages and mice.
microRNA-21 controls inflammatory and metabolic program of macrophage and neutrophils during sepsis
Authors: Melo Paulo Henrique , Alvarez Annie Rocio Piñeiros , Serezani Carlos Henrique
Published: January 2023

Sepsis is associated with a hyper inflammatory state and switch of metabolic profile of immune cells leading to organ damage and lethality. MiR-21 is one of the most abundant microRNAs in phagocytes and plays important roles in both increasing and dampening inflammation. Here, we aim to investigate the role of miR-21 in inflammatory and metabolic state of phagocytes during acute sepsis. Sepsis induced by CLP (Cecal ligation and puncture) enhanced miR-21 expression in neutrophils and macrophages from peritoneal cavity and lung. MiR-21 deficient myeloid cells mice (miR21Δmyel) show increased sepsis survival, which was correlated with increased bacterial clearance and decreased production of IL6 and IL1β, and reduced of heart and liver damage markers (CK-MB and AST, respectively). Next, we sought to test whether decreased inflammation in miR21Δmyel is dependent on the aerobic glycolysis. Both peritoneal and BALF cells from miR21Δmyel septic mice showed decreased expression of genes encoding glycolytic molecules. Also, decreased HK1 was associated with lower lactate release than septic miR21fl/fl mice. Corroborating the in vivo data, LPS-challenged BMDM and peritoneal neutrophils from miR21Δmyel also exhibited lower expression of glycolytic genes and inflammatory cytokines than miR21fl/fl cells. Both, macrophages and neutrophils were submitted to glycolytic stress assay after LPS stimulation in order to confirm the functional impact of miR-21 over glycolysis. Similarity in both cells the lack of miR-21 leads to decrease of ECAR (extracellular acidification rate). In conclusion, higher expression of miR-21 after sepsis and LPS stimulation leads to assembly of inflammatory and glycolytic metabolic program in myeloid cells.
CD73-dependent adenosine dampens IL-1β-induced CXCL8 production in gingival fibroblasts via HO-1 and pAMPK
Authors: Ramos Erivan Schnaider , Pedram Michael , Lee Renee E. , Exstrom Drake , Yilmaz Ozlem , Coutinho-Silva Robson , Ojcius David M. , Morandini Ana Carolina
Published: January 2023

During inflammation, stressed or infected cells can release adenosine tri-phosphate (ATP), which can be hydrolyzed to adenosine by ectonucleotidases such as CD39 and CD73. The role of CD73 in the modulation of cytokine release by human gingival fibroblasts (HGF) remains underexplored. Here, we investigated whether CD73-mediated hydrolysis of extracellular ATP (eATP) could affect IL-1β-induced CXCL8 secretion. The mRNA expression of adenosine receptors, CD39, and CD73 of periodontitis samples was retrieved from a public database. HGF mRNA levels were measured after 3, 6 or 24 h of IL-1β stimulation. IL-1β-induced CXCL8 protein levels were measured after pretreatment with 100 μM eATP in the presence of absence of CD73 inhibitor. The effect of eATP degradation to adenosine on CXCL8 levels was investigated using agonist and antagonist of adenosine receptors. The CD39, CD73 and adenosine receptor mRNA were differentially modulated by IL-1β. ATP pretreatment impaired IL-1β-induced CXCL8 secretion and required activation of HO-1 and pAMPK. The inhibition of CD73 or adenosine receptors abrogated the ATP effect on CXCL8 secretion. CD73-generated adenosine dampens IL-1β-induced CXCL8 in HGF via HO-1 and pAMPK. These results imply that CD73 is a negative regulator of the inflammatory microenvironment, suggesting this ectoenzyme should be considered as a target for therapeutic strategies to control periodontal disease.
Resistin-like molecule alpha (RELMα) dampens lung inflammation and promotes wound healing in helminth infection and a 3D lung repair model
Authors: Kim Sang Yong , Li Jiang , Burr Abigail C. , Batugedara Hashini , Nordgren Tara , Zang Xingxing , Nair Meera G
Published: January 2023

RELMα is a small secreted and immunoregulatory protein, also known as hypoxia-induced mitogenic factor (HIMF) and found in inflammatory zone (FIZZ). RELMα, produced by macrophages and epithelial cells in the lung and intestine, has recently been shown by our lab and others to induce wound healing during Nippostrongylus brasiliensis (Nb) infection. However, the mechanisms by which RELMα activates wound healing pathways and what cell-types are activated by RELMα are unclear. We generated constitutive RELMα−/−/TdTomato-red (TdT) reporter mice to delete the RELMα gene and track RELMα promoter activity. We found that following Nb infection, RELMα−/− macrophages exhibited reduced expression of genes associated with wound healing such as Arg1, Mmp19 and Pdgfra. To complement the RELMα−/− mouse in vivo studies, a new endotoxin-free RELMα-human Fc fusion protein was constructed and purified. A RELMα capture assay with the fusion protein demonstrated that RELMα binds to macrophage cell line RAW 264.7. Moreover, 3D lung scaffold and wound healing assays showed that RELMα-Fc promoted tissue repair by lung epithelial cells and mesenchymal stem cells. Lastly, RELMα function in vivo was characterized by RELMα-Fc fusion protein treatment of Nb-infected RELMα−/−/TdT mice, which downregulated immune cell recruitment in the lung compared to control Fc. Ongoing studies include identification of the RELMα receptor using the RELMα fusion proteins and testing whether the wound healing properties of RELMα are effective in the intestine following Heligosomoides polygyrus infection.
Plexin B1 expression on human Treg cells is required for regulation of their phenotypic stability by semaphorin 4A
Authors: Chapoval Svetlana P , Hritzo Molly , Qi Xiulan , Tamagnone Luca , Golding Amit , Keegan Achsah Dorsey
Published: January 2023

We previously reported that neuroimmune semaphorin (Sema) 4A regulates the severity of experimental allergic asthma and increases regulatory T-cell (Treg) numbers in vivo, however the mechanisms of Sema4A action remain unknown. It was also reported that Sema4A controls murine Treg cell function and survival acting through Neuropilin 1 (NRP-1) receptor. To clarify Sema4A action on human T cells, we employed T-cell lines, HuT78 and HuT102, as well as human PBMCs and CD4+ T cells in phenotypic and functional assays. We found that HuT78 demonstrated a T-effector-like phenotype (CD4+CD25lowFoxp3-), while HuT102 expressed a Treg-like phenotype (CD4+CD25hi Foxp3+). Neither cell line expressed NRP-1. However, HuT102 cells expressed high levels of the Sema4A-counter receptor, Plexin B1 whereas HuT78 cells demonstrated high cell-surface expression of Sema4A. All human peripheral blood CD4+ T cells, including Treg cells, expressed PlexinB1 and lacked both, NRP-1 and −2. Culture of HuT cells with soluble Sema4A led to an upregulation of CD25 and Foxp3 markers on HuT102 cells. Addition of soluble Sema4A increased the relative numbers of CD4+CD25+Foxp3+ cells in PBMC and CD4+ T cells which did not express NRP-1 but were PlexinB1+, suggesting the role of this receptor in Treg cell stability. The inclusion of anti-PlexinB1 blocking Ab in cultures before rSema4A addition significantly decreased Treg cell numbers as compared to cultures with rSema4A alone. Sema4A was as effective as TGF-b in iTreg induction from CD4+CD25depleted cells but did not affect Treg cell suppressive activity in vitro. These results suggest strategies for the development of new Sema4A-based therapeutic measures to combat allergic inflammatory diseases.
Identification of distinct highly pathogenic allergen-specific T cells in common allergy and asthma
Authors: Seumois Gregory , Suastegui Ciro Ramirez , Shmiedel Benjamin J , Liang Shu , Shulten Veronique , Panwar Bharat , Madrigal Ariel , Sette Alessandro , Peters Bjoern , Vijayanand Pandurangan
Published: January 2023

Despite the proven critical role of the aero-allergen house dust mite (HDM) in the pathogenesis of airway allergic diseases and asthma, the molecular mechanisms leading to allergen-specific T cell activation, as well as the molecular differences driving to disease between different type of HDM-sensitized patients are unknown. The recent development of robust allergen activation assays combined with single-cell RNA-Seq technologies made us able to perform the first large single-cell RNA-Seq study for around 50,000 cells. We collected blood from 12 asthmatic patients with or without HDM sensitization, 6 patients with Allergic Rhinitis HDM-sensitized and 6 healthy subjects. We stimulated PBMCs with a pool HDM peptides for a period of 6 hours and sorted activated cells based on the expression of 2 surface markers: CD154+ (effector T cells) and CD137high/CD154low (regulatory T cells). We performed standard bulk RNA-Seq and single-cell RNA-Seq. Differential gene expression analysis showed important changes in gene expression upon TCR engagement in both populations. Comparison of disease groups identified several known but also new genes with relevant functional significance in allergy and asthma. Clustering analysis of Effector T cells single-cell RNA-Seq revealed an unexpected level of heterogeneity of gene expression among T cells. Major changes were observed between disease groups, revealing important pathological consequences to the understanding of immunity in allergy. Our study was the first attempt to generate a large-scale of single-cell RNA-Seq datasets from activated HDM-specific T cell. We showed presence of new T-cell subsets with unknown function as well as new molecules of therapeutic interest in allergy and asthma.
Alcohol effects on immune responses of classical pro-inflammatory macrophages
Authors: Hu Yawen , Yoshimura Masami
Published: January 2023

Alcohol consumption modulates immune responses. However, the mechanisms of ethanol’s action on the immune system are not fully understood. Previous studies demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform-specific manner, that type 7 AC (AC7) is most enhanced by ethanol, and that AC7 is highly expressed in immune cells. Therefore, we hypothesized that AC7 is a central player in regulating alcohol’s effects on immune responses. To examine this hypothesis, we generated AC7 knockout (KO) cell lines from a mouse microglia cell line, BV-2, and isolated primary bone marrow-derived macrophages (BMDM) from myeloid lineage-specific AC7 KO mice. Cells were activated by lipopolysaccharides (LPS) and interferon gamma (IFN-γ) to classical pro-inflammatory macrophages. The effects of ethanol on nitric oxide (NO) production of macrophages were examined via Griess reagent. LPS and IFN-γ synergistically increased nitric oxide (NO) production in wild type (WT) BV-2 cells in a dose-dependent manner. Ethanol suppressed LPS and IFN-γ co-activated NO production. AC7 KO BV-2 cells did not respond to LPS or IFN-γ activation. LPS increased NO production of both WT and AC7 KO BMDM. NO production of KO BMDM was significantly lower than that of WT BMDM. IFN-γ increased LPS-induced NO production of both WT and AC7 KO BMDM. Ethanol suppressed LPS-induced NO production in a dose-dependent manner in WT BMDM, while this effect was reduced in AC7 KO BMDM. The results suggest that AC7 is a key factor in the regulation of NO production and that the effect of ethanol on NO production depends on AC7. In the future, we will investigate the iNOS expression signaling cascade of classical pro-inflammatory macrophages in the presence of ethanol.
Myeloid CFTR Loss-of-function Causes Persistent Neutrophilic Inflammation in Cystic Fibrosis
Authors: Wang Guoshun , Ng Hang Pong
Published: January 2023

Persistent neutrophilic inflammation is a hallmark manifestation of cystic fibrosis (CF). However, the mechanism underlying this phenomenal clinical symptom remains incompletely understood. Here we report a pivotal role of CFTR in myeloid immune cells in control of neutrophilic inflammation. Myeloid CFTR-Knockout (Mye-Cftr−/−) mice and Wild-type (WT) mice were challenged peritoneally with zymosan at different doses. The lethal-dose challenge resulted in significantly higher mortality in Mye-Cftr−/− mice, indicating an intrinsic defect in host protection against inflammation in CF. The sub-lethal-dose challenge demonstrated an impaired resolution of inflammation in Mye-Cftr−/− mice, reflected by persistent neutrophilic inflammation, and hyper-inflammation with significantly higher levels of pro-inflammatory cytokines, including the neutrophil-recruiting chemokines MIP-2 and KC, which led to excessive neutrophil recruitment in vivo. Pulmonary challenge with zymosan confirmed the peritoneal finding. To determine the major types of cells responsible for the over-recruitment of neutrophils, zymosan-elicited peritoneal neutrophils and macrophages from Mye-Cftr−/− and WT mice were FACS-sorted and cultured ex vivo. The CF neutrophils produced significantly more neutrophil chemokine MIP-2. Moreover, peripheral blood neutrophils and monocytes from Mye-Cftr−/− and WT mice were cultured and stimulated with zymosan in vitro. Similarly, the CF neutrophils produced significantly more MIP-2. These data altogether suggest that CFTR dysfunction in myeloid immune cells leads to excessive neutrophil recruitment, thus serving as a mechanism for the long-observed neutrophilic inflammation in CF.
A role for allosteric disulfides in regulating the function of human killer cell receptor KIR2DL4
Authors: Rajagopalan Sumati , Mastorakos George , Long Eric O
Published: January 2023

KIR2DL4 is an unusual member of the killer cell Ig-like receptor (KIR) family expressed in NK cells. It resides mostly in endosomes and activates NK cells in response to soluble agonists, such as soluble HLA-G and a monoclonal antibody. KIR2DL4 signals from endosomes for a unique proinflammatory and proangiogenic response, which has relevance to placental development due to the restricted expression of its ligand, HLA-G, by fetal trophoblast cells during early pregnancy. To understand the nature and regulation of KIR2DL4–HLA-G interactions, we used an unbiased random mutagenesis approach and screened mutants of KIR2DL4 in a cellular assay for their ability to load soluble HLA-G into KIR2DL4+ endosomes. With this approach, amino acid residues in KIR2DL4 that are important for endosomal localization and HLA-G recognition were identified. Specifically, we have identified a putative allosteric cysteine in the extracellular domain of this receptor, which may be involved in a disulfide switching mechanism to control receptor internalization and ligand binding. Protein disulfide isomerase (PDI) acting at the cell surface may be responsible for this switch as inhibition of PDI interfered with internalization of KIR2DL4 and uptake of HLA-G into endosomes. The configuration of the relevant disulfide bond is -RHstaple, which is a hallmark of many allosteric disulfides. Thus, KIR2DL4 localization and function is regulated by allosteric disulfide bonds at the cell surface. Studies are underway to explore the dynamic regulation of receptor structure by this allosteric disulfide switch to control ligand binding, localization and signaling by this receptor.
Protein Kinase CK2 Drives Plasma Cell Differentiation
Authors: Wei Hairong , Yang Wei , Yan Zhaoqi , Qin Hongwei , Benveniste Etty
Published: January 2023

Casein kinase 2 (CK2) is a serine/threonine protein kinase composed of three subunits: two catalytic subunits, CK2α and CK2α’, and the regulatory subunit CK2β. It is overexpressed and overactive in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphomas, leading to inappropriate activation of NF-κB, JAK/STAT and PI3K/AKT/mTOR signaling, but little is known about how CK2 functions in normal B-cell differentiation. Here, we demonstrate that CK2 mRNA and protein expression is induced upon B-cell activation. Using the small molecule inhibitor CX-4945, which targets the catalytic activity of CK2, a significant inhibition of plasma cell differentiation was observed in vitro under T-cell-independent (TI) or T-cell-dependent (TD) conditions via stimulation with lipopolysaccharide (LPS) or CD40L and interleukin 4 (IL-4) +IL-5, respectively. At the cellular level, we found that inhibition of CK2 kinase activity in B-cells resulted in defective mTORC1 signaling upon stimulation with LPS. These results were confirmed in purified mature B-cells from mice with tamoxifen-induced global deletion of CK2α. These data demonstrate that CK2α is an important regulator of B-cell differentiation. Ongoing experiments with B-cell specific deletion of CK2α will further examine the impact of CK2α on B-cell differentiation in vivo.
H3K27me3-specific demethylases modulate B cell development and differentiation
Authors: Kania Anna , Scharer Christopher D. , Price Madeline J , Haines Robert Richard , Alexander-George Lou-Ella , Boss Jeremy
Published: January 2023

B cell terminal differentiation into antibody secreting plasma cells (ASC) must be properly regulated to ensure robust humoral immune responses against foreign but not self-antigens. While the role of transcription factors in this process is well-established, there is also a growing appreciation of the epigenetic regulation of B cell differentiation. The histone modification H3 lysine 27 trimethylation (H3K27me3) is associated with a repressive chromatin state and gene silencing and is highly dynamic during B cell differentiation. EZH2, the enzyme that deposits H3K27me3, is required for proper B cell development, as well as germinal center and ASC formation. However, the role of active demethylation of H3K27me3 by the two demethylases UTX and JMJD3 in B cells is still to be elucidated. To determine the requirements for these enzymes in B lymphocytes, we crossed Utxfl/flJmjd3fl/flmice onto Cd19Cre/+background (dKO mice). In dKO mice, we observed a significant increase in T2 transitional and marginal zone B cells in the spleen and a reduction in circulating mature B cells in the bone marrow. To determine the role of UTX and JMJD3 in T cell dependent B cell responses, we infected CreCtrl and dKO mice with PR8 influenza virus. Fourteen days post infection, we observed a modest increase in GC B cells and skewing in the GC polarity in dKO mice. RNA-seq of CreCtrl and dKO GC B cells revealed that majority of differentially expressed genes (DEGs) were downregulated in the absence of UTX and JMJD3. Furthermore, a subset of down-DEGs were enriched for H3K27me3 in dKO supporting the role of UTX and JMJD3 in promoting gene. expression. Taken together, these data place H3K27me3 demethylases as critical enzymes that regulate the epigenetic dynamics of B cell fate.
Elucidating the nature of long-lived plasma cell precursors
Authors: Vijay Godhev Kumar Manakkat , Chawla Amanpreet Singh , Singh Harinder
Published: January 2023

Plasma cells (PCs) are terminally differentiated B cells that secrete protective antibodies and serve as a source of long-term humoral memory. PC precursors appear to be generated late in the germinal center B cell response in the spleen and migrate to the bone marrow (BM) to differentiate into PCs. However, the developmental and molecular mechanisms that control the generation, migration and differentiation of precursors of long-lived PCs (LLPCs) remain to be elucidated. In order to identify and characterize the precursors of LLPCs we established a novel adoptive transfer system. C57Bl/6J mice were immunized with NP-KLH along with LPS and alum. Post-immunization, splenic CD138+ cells were isolated on days 21, 28, 35 and 42 and then adoptively transferred into the B cell deficient μMT mouse strain. Sustained NP-specific IgG1 responses were observed in the μMT recipients, with a peak following adoptive transfer of day 35, CD138+ cells. The adoptively transferred cells were demonstrated to contain LLPC precursors as they generated NP-specific IgG1+ antibody secreting cells (ASCs) in the BM that were sustained for over 2 months following transfer. These results directly demonstrate that precursors of LLPCs arise between days 28 to 42 post-immunization. Genomic analyses of LLPC precursors using scRNA-seq and Ig profiling is being used to uncover molecular mechanisms that regulate their generation and differentiation.
A Single Administration of Fc-Fused Recombinant Human IL-7 Induces Expansions of T Lymphocytes in Healthy Adult Volunteers
Authors: Kim Sojeong , Choi Dong-Hoon , Heo MinKyu , Lee Byung Ha , Yang Se-Hwan , Sung Young Chul , Lee Howard , Shin Eui-Chul , Park Su-Hyung
Published: January 2023

Interleukin-7 (IL-7) is a cytokine that plays important roles in the development and homeostasis of T lymphocytes, which supports its application to reconstruct T cell populations in patients with a low lymphocyte count caused by various reasons such as hematopoietic stem cell transplantation or intensive chemotherapy. We investigated the effects of Fc-fused recombinant human IL-7 (rhIL-7-Fc) on T cell immunity in 24 healthy volunteers, who received a single subcutaneous (20 and 60 mg/kg) or intramuscular (60 mg/kg) dose of rhIL-7-Fc. We observed a significant increase in the number of CD8 and CD4 T cells after rhIL-7-Fc administration, the peak of which was seen on days 14–21 post dose. Importantly, single administration of rhIL-7-Fc increased the number of all subsets (naïve, EM, CM) of both CD8 and CD4 T cells, coupled with an enhanced expression of Ki-67, a marker of T cell proliferation. Furthermore, the numbers of other immune cells such as NK cells, NKT cells and γδ T cells were also increased after rhIL-7-Fc administration, whereas the number of B cells was transiently decreased on 7–14 days after rhIL-7-Fc administration and gradually recovered to the pre-treatment value. In conclusion, our results indicated that rhIL7-Fc’s potential immunomodulatory role can be further tested in humans to develop it as a beneficial agent to treat patients with a low lymphocyte count.
Themis integrates T cell receptor and cytokine signals in CD8+ T cells
Authors: Gascoigne Nicholas R J , Brzostek Joanna , Zhao Xiang , Fu Guo , Chen Elijah W. , Mehta Monika
Published: January 2023

T cell receptor (TCR) signaling controls T cell development in the thymus and T cell functions in the periphery. Themis is a T-cell lineage-specific protein with a critical role in T cell development, where it regulates TCR signal strength though modulation of Shp1 phosphatase activity. The role of Themis in mature peripheral T cells is unknown. To address this, we generated Themis conditional knockout (cKO) mice, with post-selection Themis deletion mediated by distal Lck-Cre. The cKO mice have reduced CD8+ T cell numbers. Themis-deficient CD8+ T cells on the OT-I TCR transgenic background show normal functional responses to agonistic pMHC ligands of different affinities. However, Themis-deficient OT-I CD8+ T cells have reduced cell surface expression of CD5 and increased cell surface expression of CD8, suggesting decreased signal from self pMHC. Moreover, cKO OTI CD8+ T cells display severe reduction in lymphopenia-induced (homeostatic) proliferation in vivo, as well as reduction in proliferative responses to low affinity pMHC and pro-inflammatory cytokines in vitro. Signals from low affinity pMHC and cytokines synergistically induce phosphorylation of Akt, metabolic changes, and induction of c-Myc in CD8 T cells. This does not occur in the absence of Themis. Themis performs these functions through the phosphatase Shp1, as double deletion of Themis and Shp1 in peripheral T cells rescues the CD8+ T cell maintenance. This work shows a novel role for cytokines and very low affinity pMHC in altering cellular metabolism to drive CD8+ T cell proliferation. It also demonstrates that Themis and Shp1 act in concert to integrate T cell receptor and cytokine signals.
STAT4 programs CD103− tissue-resident memory cells during infection
Authors: Bergsbaken Tessa , Fung Helen , Wilson Nicolette , Teryek Matthew
Published: January 2023

Tissue-resident memory (Trm) CD8+ T cells represent a distinct population of memory T cells that are maintained independently of the circulation and are positioned to respond rapidly to reinfection of the tissue. Using the bacterial pathogen Yersinia pseudotuberculosis (Yptb), we identified two distinct intestinal CD8+ Trm populations that are differentiated by their expression of the integrin CD103. Proximity of T cells to areas of inflammation within the intestinal tissue regulated Trm differentiation, with IL-12 leading to increased numbers of CD103− Trm cells. Therefore, we examined the role of the transcription factor STAT4 in the programming of this Trm subset. During infection, both wild-type and Stat4−/− T cells expanded and entered the intestinal tissue at similar rates. Stat4−/− T cells localized to areas of inflammation along with wild-type cells, but failed to express CD103− Trm signature genes. After infection, Stat4−/− CD103+ intestinal Trm cells formed a stable memory population; however, the number of Stat4−/− CD103− Trm cells was significantly reduced relative to wild-type Trm cells. We also observed impaired persistence of Stat4−/− CD103− Trm cells in other tissues with prominent CD103− Trm populations, including the liver, colon, and mesenteric adipose. RNA-seq analysis of wild-type and Stat4−/− CD103− Trm populations was used to identify genes required for the maintenance of this Trm subset. This work has identified STAT4 as a regulator of CD103− Trm differentiation and maintenance across multiple tissues, and these results will help identify strategies to maximize the number and persistence of Trm cells during vaccination and allow us to address the role of Trm heterogeneity in tissue-specific immunity.