bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetic modulation of immune response in salmonid cells infected with the infectious pancreatic necrosis virus (VIR2P.1173)
Authors: Carcamo Juan , Manriquez Rene , Vera Tamara , Avendaño-Herrera Ruben
Published: January 2023

The great growing of salmon farming has brought a noteworthy increase in infectious diseases, generating important economic losses in the aquaculture industry. The infectious pancreatic necrosis virus (IPNV) is an agent of high mortalities in fish farmed. The aim of this study is elucidate whether the antiviral response caused by IPNV is through epigenetic mechanisms. Studies of methylation in CpG islands of the promoters of cytokines in infected salmonids cells with IPNV and treatments with DNA methyltransferase (DNMT) inhibitors were performed to evaluate the effect on the expression of cytokines. We observed that expression of cytokines, in salmonids cells infected with IPNV was associated to the promoter methylation status. Moreover, DNA methyltransferase inhibitors were able to modulate the expression of these cytokines. Our work suggested that epigenetic mechanisms participate in the regulation of host genes expression in IPNV infection.
The intracellular pathway for MR1 presentation of vitamin B-related antigens
Authors: Villadangos Jose A
Published: January 2023

Compounds related to Vitamin B metabolism (“VitB Ags”) produced by pathogens were recently discovered as a novel family of Ags that are presented by the MHC-I like molecule, MR1, to Mucosal-Associated Invariant T (MAIT) cells. In stark contrast with the mechanisms involved in presentation pathways for peptide and lipid Ags by MHC and CD1 molecules, respectively, the MR1 presentation pathway for VitB Ags remains largely uncharacterized.

We show that MR1 does not constitutively present self-ligands in the steady-state, but accumulates in a ligand-receptive conformation within the endoplasmic reticulum (ER). Exogenous VitB Ags reach the ER, where they form a Schiff base with a lysine residue in the MR1 Ag-binding cleft. Formation of this covalent bond serves as a “molecular switch” that allows complete folding and egress of MR1-VitB Ag complexes out of the ER. The MR1-Ag complexes follow the secretory pathway, whereupon their half-life on the plasma membrane is independent of the affinity of the bound ligand. They undergo endocytosis and although some MR1 molecules acquire new ligands during passage through endosomes and recycle back to the surface, most are degraded intracellularly. Presentation of VitB Ags on the plasma membrane of MR1 antigen presenting cells thus decreases rapidly after the cells are no longer exposed to the ligand.

In summary, the MR1 Ag presentation pathway possesses two main features that set it apart from the MHC and CD1 presentation pathways: (i) it presents exogenous VitB Ags but binding of these antigens to MR1 occurs within the ER rather than in the endocytic route; (ii) It is characterized by a rapid “off-on-off” mechanism strictly dependent on Ag availability.
An animal component-free, serum-free culture method for generation of human dendritic cells
Authors: Tabatabaei-Zavareh Nooshin , Le Fevre Timothy A. , Wognum Albertus W. , Szilvassy Stephen J. , Thomas Terry E. , Eaves Allen C.
Published: January 2023

Dendritic cells (DCs) are central regulators of adaptive immune responses that potentially have major applications in the immunotherapy of cancer and autoimmune disorders. DCs are rare in peripheral blood (PB) and are therefore often generated in vitro from PB monocytes. We developed an animal component-free (ACF) and serum-free medium, ImmunoCult-ACF DC, that supports the generation of DCs from monocytes in culture. CD14+ monocytes were isolated using EasySep immunomagnetic separation and cultured for 5 days in ImmunoCult-ACF DC medium and cytokines (GM-CSF and IL-4) to promote their differentiation into CD14−CD83− immature DCs. The immature DCs were then stimulated for 2 days with the ImmunoCult-DC Maturation Supplement, a combination of cytokines and pro-inflammatory mediators, to promote their maturation to CD14− CD83+ DCs (93 ± 5% CD83+, n=23). The yield of mature DCs was 42 ± 23% (n=23), similar to the yield in a control serum-free medium (49 ± 24%, n=16). Mature DCs produced high levels of IL-12, on average 371 pg/mL (range: 27–1756, n=9 in cultures initiated with 106 monocytes). Mature DCs loaded with CMV, EBV and Flu virus peptides efficiently stimulated the proliferation of autologous CD8+ T cells in 7 day co-cultures. An 18-fold increase in T cell numbers (range: 5–31, n=4) was observed when compared to control culture conditions containing only T cells without mature DCs. In conclusion, functional DCs can be efficiently generated by differentiation of monocytes in a completely animal (including human) component-free and serum-free medium. This medium and culture method will enable further research into the development and application of DCs for cellular therapy.
TAPBPR, a Peptide Editor – interactions with MHC complexes and SAXS structural studies
Authors: Natarajan Kannan , Morozov Giora , Jiang Jiansheng , Boyd Lisa F , Mage Michael G , Margulies David H
Published: January 2023

TAPBPR (TAP Binding Protein-Related), a tapasin homolog, functions as a peptide editor independent of the classical peptide loading complex (PLC) that consists of tapasin, transporter associated with antigen processing (TAP), ERp57, and calreticulin. Using insect-cell expressed recombinant luminal human TAPBPR, we previously reported interaction with HLA-A*02:01 molecules freed of peptide following photolysis of HLA-A2/beta2m complexes prepared with photolabile peptides. Here, we extend our analysis of the TABPBPR/MHC interaction by examining the binding of a set of MHC/peptide complexes prepared with different MHC-I molecules refolded with a variety of peptides. Using surface plasmon resonance, we show that a variety of refolded complexes, with or without photolysis of the bound peptide, bind to TAPBPR, raising the possibility that TAPBPR may interact either with a small proportion of peptide free molecules found in some MHC/peptide preparations, or with molecular conformations containing peptide in dynamic equilibrium with the lowest energy state. We extend our analysis of TAPBPR by determination of the low resolution small angle X-ray scattering structures of both TAPBPR and tapasin luminal domains, revealing expected structural similarities.
The eotaxin-CCR3 pathway is required for eosinophil trafficking to the heart in eosinophilic myocarditis
Authors: Diny Nicola , Hou Xuezhou , Barin Jobert G , Talor Monica V , Rose Noel R , Cihakova Daniela
Published: January 2023

Cardiac manifestations are a major cause of morbidity and mortality in patients with eosinophil-associated diseases. Eosinophils have been proposed to play a pathologic role in the heart. Nevertheless, the pathways that recruit eosinophils to the heart have not been described. In IFNγ−/− IL-17A−/− mice, induction of experimental autoimmune myocarditis results in a Th-2-driven, eosinophilic inflammation. IFNγ−/− IL-17A−/− mice had much higher expression of the eosinophil-attracting chemokines eotaxin-1 (Ccl11) and eotaxin-2 (Ccl24) in the heart than wildtype mice. Genetic ablation of the eotaxin receptor CCR3 resulted in a dramatic decrease in heart infiltrating eosinophils. Adoptive transfer experiments with CCR3+/+ or CCR3−/− eosinophils into eosinophil-deficient ΔdblGATA1 or IFNγ−/− IL-17A−/− ΔdblGATA1 recipients showed that two conditions have to be met for efficient eosinophil trafficking to the inflamed heart: high eotaxin expression in the heart and expression of CCR3 by eosinophils. We identified the source of cardiac eotaxins by RT-PCR of FACS sorted heart cells and by immunohistochemistry. Eotaxin-1 was mainly produced by cardiac fibroblasts with interstitial localization in the heart. In vitro culture of cardiac fibroblasts with IL-4 and IL-13 induced eotaxin-1 expression. In contrast, eotaxin-2 was expressed by multiple inflammatory cell types, located at inflammatory foci, and substantial expression was only found in mice lacking IFNγ and IL-17A during myocarditis. In conclusion, eosinophil trafficking to the heart is dependent on the eotaxin-CCR3 pathway in mice with eosinophilic myocarditis. Blockade of this pathway may be a useful therapeutic approach to prevent eosinophil-mediated heart damage.
Syndecan-1 controls the role of iNKT cells in regulating body fat mass and glucose homeostasis in mice
Authors: Jaiswal Anil Kumar , Dai Hong , Hamad Abdel Rahim A
Published: January 2023

Obesity is a major risk factor for metabolic disease, with white adipose tissue inflammation emerging as a key underlying pathology that coupled with insulin resistance, type 2 diabetes and cardiovascular diseases. Immune cells play a central role in maintaining immune homeostasis of these adipose tissues. Formerly, significant efforts have been made in understanding the roles of conventional and regulatory T cells. Natural killer T17 cells (NKT17 cells) are lipid-sensing innate T cells, restricted by the antigen-presenting molecule CD1d and express the transcription factor RORyt. Though, the role of invariant natural killer T (iNKT) cells is still remains unclear. Recently, we have discovered that syndecan-1 (sdc1) highly express on the surface of NKT17 cells. After thymus, sdc1-expressing NKT17 cells specifically localize the adipose tissues.

Encouraged with previous results, iNKT cells were characterized in both wild type (wt) and sdc1 knock out (sdc1ko) BALB/c mice. The iNKT cells with sdc-1ko mice have shown higher level of proliferation compared to wt. The proliferation assay has shown significantly high number of iNKT cells in S-phase of cell cycle dominating with double positive (CD4+CD8+T cells) T cells in sdc1ko mice. Further, sdc-1ko mice have significantly reduced body fat and improved glucose tolerance. Future study with siRNA, targeting sdc1 in iNKT cells could be a contributing factor in regulating obesity and glucose homeostasis and to understand the physiological significance of sdc1 expression by NKT17 cells. Thus, developing the strategies that maintain immune homeostasis in obese adipose tissue could lead to novel therapeutics that improve and/or prevent the metabolic and cardiovascular risks of obesity.
TLR8 activation shifts neutrophils from phagocytosis to NETosis through furin-dependent shedding of FcgRIIA
Authors: Lood Christian , Arve Sabine , Ledbetter Jeffrey A , Elkon Keith B
Published: January 2023

Background

Immune complex (IC)-mediated formation of neutrophil extracellular traps (NETs) has emerged as a mechanism to increase the autoantigenic burden as well as promote the prominent type I interferon (IFN) signature seen in patients with the autoimmune disease systemic lupus erythematosus (SLE). The aim of the current study was to explore properties of ICs regulating their ability to induce phagocytosis and NETosis.

Results

Whereas immobilized IgG, mimicking tissue-deposited ICs, induced NETs in a TLR-independent manner, both FcgR- and TLR-engagement were required for induction of NETosis by soluble RNA-ICs, as demonstrated by FcgR blocking antibodies as well as RNase-treatment. Despite that degradation of RNA inhibited NETosis, removal of the TLR ligand by RNase markedly increased the phagocytosis of RNA-ICs by neutrophils suggesting that TLR activation suppressed phagocytosis. Consistent with this hypothesis, addition of a TLR8 agonist (R848) inhibited phagocytosis of ICs, but not beads, in neutrophils. Mechanistically, we observed that TLR8 induced ROS-dependent activation of the serine protease furin, participating in shedding of cell surface FcgRIIA reducing the phagocytic capacity but promoting progression into NETosis.

Conclusions

i) In the fluid phase, both FcgRIIA and TLR8 ligation are necessary for IC-mediated NETosis; ii) TLR8 activation induces furin-mediated shedding of FcgRIIA, thereby inhibiting further phagocytosis of ICs but enabling NETosis. Therapeutic approaches aimed at degrading the TLR8 ligand, such as RNases, would be predicted to increase uptake of circulating ICs, while disarming their inflammatory potential and ability to induce NETs.
T-bet expression in NK cells is critical in the pathogenicity of Th17-induced neuroinflammation
Authors: Kwong Brandon , Flickinger John , Lazarevic Vanja
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is a murine model for multiple sclerosis (MS) in which self-reactive CD4+ T cells induce autoimmune inflammation in the central nervous system (CNS). Although mice deficient in the transcription factor T-bet are resistant to EAE disease, the mechanism by which T-bet deficiency confers protection against EAE is poorly understood. Here, we demonstrate that EAE induction via the adoptive transfer of 2D2 TCR-transgenic (CNS Ag-specific) Th17 cells requires T-bet expression in both the donor T cells and a host immune cell subset(s) within the CNS. While T-bet-expressing 2D2 Th17 cells induced severe neuropathology upon transfer into wild-type recipient mice, T-bet-deficient recipients were highly resistant to EAE induction by 2D2 Th17 adoptive transfer. Lineage-specific deletions of T-bet in either the endogenous T cells or dendritic cells of host mice had no effect on EAE induction. Strikingly, the deletion of T-bet specifically in natural killer (NK) cells significantly abrogated EAE disease, indicating a critical pathogenic role for T-bet expression in CNS-infiltrating NK cells. Resistance to EAE was similarly observed following the systemic depletion of NK cells in recipient mice. Although T-bet-deficient NK cells infiltrating the CNS did not show intrinsic defects in classical pro-inflammatory effector functions, preliminary data suggests that T-bet regulates the trafficking of NK cells into the CNS as well as their activation state within the CNS. Elucidating the mechanisms by which T-bet expression in NK cells and CD4+ T cells regulates EAE disease pathogenicity could ultimately lead to novel and improved therapies against clinical MS disease.
Deletion of the exon 2 of mouse Foxp3 results in systemic lupus erythematosus-like disease
Authors: Wang Qun , Du Jianguang , Wang Lina , Zhou Baohua
Published: January 2023

Regulatory CD4 T cells (Tregs) are indispensable in maintaining self-tolerance and deficiency in Treg development and function leads to autoimmunity. Foxp3 is the master transcription factor determining Treg differentiation and function in both humans and mice. Unlike the mouse Foxp3 gene which only expresses a single product, human FOXP3 gene encodes at least two major isoforms (a full length and a short isoform lacking exon 2) through alternative splicing. The region of human FOXP3 encoded by exon2 binds to RORα and RORγt in vitro and regulates their transcription activity. However, how Foxp3 exon 2 regulates Treg development and function in vivo remains largely unknown. In order to understand the role of exon 2 in vivo, we deleted the exon 2 of mouse Foxp3 gene. Exon 2 knockout (dE2) mice showed splenomegaly/lymphadenopathy and produced higher ratio of Tfh and Tfr when compared with wildtype mice. Despite increased Treg compartment, dE2 mice generated autoreactive IgG against dsDNA and nuclear antigens. Histological examination of kidney revealed immune-complex deposits and glomerulonephritis. The dE2 mice also exhibited skin lesions on face, neck and back of the body starting 6 month after birth. Our data suggest that exon 2 of Foxp3 is critical in maintaining proper Treg function and self-tolerance.
Micromanging lupus nephritis: miR17-92 modulates regulatory T cell activity by targeting foxp3 co-regulators
Authors: Yang Huang-Yu , Wu Chao-Yi , Pan Fan , Pardoll Drew , Huang Jing-Long , Hung Cheng-Chieh , Yang Chih-Wei
Published: January 2023

Regulatory T (Treg) cells play a critical role in maintaining self-tolerance and controlling the magnitude of physiologic immune response. The Treg transcription factor forkhead box P3 (Foxp3) works in concert with other co-regulator molecules including Eos to determine suppressive phenotype of Treg. A strong correlation between systemic lupus erythematous and deficiencies of Treg has been reported. We induce pristane nephropathy on T cell specific miR-17-92 knockout mice. Mir17-92 T cell specific deficiency mitigates pristane induced-lupus nephropathy in mice. The mice showed less Th17 cells and lower autoantibody formation. Consistent with the reduction in autoantibody production, histological analysis revealed a lower mean renal histopathology score and less compliment deposition. We showed for the first time that one microRNA of the miR17-92 cluster, miR-17, regulates the suppression function of Tregs. We identify a gene target of miR-17, Eos, which regulates Tregs through Foxp3-mediated gene suppression. Ectopic expression of miR-17 downmodulates the suppression functions of Tregs and provides Tregs with partial effector activity via de-repression of cytokine genes. Our studies suggest that miR17-92 modulates Treg cell function by targeting Eos, unveiling the future therapeutic potential of microRNA manipulation in lupus nephritis.
The development of autoimmunity in NZB/NZW embryonic chimeras.
Authors: Hankins Julia , Li Xiaohong , Beutler Bruce
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease resulting from autoantibodies against nuclear antigens. These autoantibodies can form complexes that sediment in joints, skin, kidneys, and vasculature to cause tissue damage, but can also cross-react with other self-antigens to induce injury. It is unclear how the immune system breaks tolerance, and in most cases, what genes or environmental factors initiate the disease. A traditional mouse model of SLE, the NZB/NZW F1, has many of the features of human SLE, including autoantibodies, nephritis, a female sex bias, as well as the complex genetic basis of the disease. Although this mouse model has proven to be useful in understanding SLE progression, it is still unknown why the F1 between two strains without SLE develop disease. To clarify the role of the two parental strains, NZB and NZW, in the development of autoimmunity we have generated NZB/NZW embryonic chimeras. These chimeras can develop autoantibodies 6 months earlier than the NZB/NZW F1 and at titers that are 10 fold greater. Furthermore, these mice also develop nephritis, the end-organ disease of SLE. Embryonic chimeras permit the study of intercellular interactions and the independent contributions of the two parental strains. Analysis has shown that most B cells are produced by NZW, and that these make autoantibodies. In addition, most T cells, and almost all activated T cells, are derived from NZB. Moreover, disease severity is associated with higher quantities of NZB T cells. Thus, understanding the pathogenesis of SLE in the NZB/NZW embryonic chimeras may elucidate possible causes of autoimmunity by identifying the intercellular transactions between these two strains that do not have SLE in isolation of one another.
Posttranslational modification of islet autoantigens in type 1 diabetes
Authors: Yang Mei-Ling , Wen Li , Herold Kevan C , Mamula Mark J
Published: January 2023

Autoimmune diseases are typically initiated by self-proteins, either native proteins or those with posttranslational modifications (PTMs), that break immune tolerance and lead to tissue pathology. Inflammation and oxidative stress amplifies various PTMs such as glutathionylation, nitrosylation and carbonylation, although these pathways are not clearly understood in the genesis of type 1 diabetes (T1D). By various proteomic analyses, including two-dimensional gel electrophoresis and mass spectroscopy, we identified more than a dozen carbonyl modified islet proteins bound by autoantibodies in non-obese diabetic (NOD) mice and in human T1D. In particular, we identified the beta subunit of prolyl-4-hydroxylase (P4Hb) as a carbonylated autoantigen in both human and murine T1D. P4Hb is a conserved protein catalyst known to be critical for proinsulin folding and trafficking in insulin biosynthesis. Of interest, we detected circulating autoantibodies against P4Hb in prediabetic NOD mice, before the onset of hyperglycemia and anti-insulin autoimmunity. Similarly, autoantibodies arise in early onset human T1D prior to anti-insulin autoimmunity. Finally, we demonstrate that carbonyl modified P4Hb is elevated in human pancreatic islets stressed with inflammatory cytokines (INF-γ, IL-1β and TNF-α). These studies provide mechanistic insight into the pathways of creating autoantigens and define a linked set of autoimmune responses (P4Hb and insulin) in the development of T1D.
G-alpha-s-coupled receptor signaling controls circadian rhythm in integrin-mediated cell adhesion of virus-specific human CD8+ T cells
Authors: Dimitrov Stoyan I , Besedovsky Luciana , Jensen Anja Tatiana Ramstedt , Born Jan , Gouttefangeas Cecile , Rammensee Hans-Georg , Lange Tanja
Published: January 2023

Beta2-integrin activation in response to inside-out signaling by chemokines plays a major role in T-cell adhesion to the endothelium and the following extravasation into the inflamed tissue. We and others have previously shown that daytime increase in G-alpha-s-coupled receptor (GαsPCR) signaling, such as via beta2-adrenergic receptors counteracts integrin activation and promotes mobilization of effector CD8+ T cells from the marginal pool into the recirculation. In T cells, integrin activation is also rapidly induced by stimulation of antigen-specific T-cell receptors (TCR), resulting in the formation of stable immunological synapses. Therefore, we asked whether daytime increase in GαsPCR signaling inhibits integrin activation induced by TCR stimulation of human virus-specific T cells. Using ligand-complex-based adhesion assay to measure beta2-integrin activation, we show that affinity of integrins, which were activated by soluble cytomegalovirus virus peptide-major histocompatibility complex class I HLA-multimers is minimal during daytime. Additional in vitro experiments with GαsPCR agonists showed that epinephrine, norepinephrine, histamine, adenosine, and prostaglandins E2/D2 down-modulate the beta2-integrin activation; however, only epinephrine did so in physiological daytime concentrations, suggesting its mayor role in circadian regulation of T-cell adhesion. This active down-modulation of integrin activation through GαsPCR might be an essential mechanism of circadian immune defense regulation.
Label-free, Live Cell Imaging of Transendothelial Migration
Authors: OClair Belinda , Li Guangnan , Appledorn Daniel
Published: January 2023

The recruitment of leukocytes to sites of infection and their migration through the endothelium are critical to immune responses. Transendothelial migration is essential for leukocytes to respond to foreign microorganisms, but if uncontrolled, can cause autoimmune diseases such as inflammatory bowel disease and rheumatoid arthritis. In order to evaluate the transmigration of leukocytes, we have developed a kinetic, label-free in vitro assay to automatically acquire and analyze transendothelial migration, with the added ability to monitor monolayer integrity.

Using primary T cells and Human Umbilical Vein Endothelial cells (HUVEC), we evaluated the ability of this novel assay to quantify leukocyte transmigration in the absence of cell labeling. Briefly, endothelial cells were grown to confluence on a physiological surface. Leukocytes were added to the monolayer, and the upper chamber was placed into a reservoir plate containing chemoattractant. Live cell images were captured at regular intervals, and directed transendothelial migration was quantified using image analysis algorithms. Significant diapedesis was observed in response to increasing concentrations of SDF-1α, and completely inhibited by AMD3100, a CXCR4 antagonist. We show that migration across the endothelial layer was partially dependent on ICAM-1, as neutralizing ICAM-1 antibodies, and treatment with an allosteric inhibitor of LFA-1, BIRT 377, significantly inhibited SDF-1α mediated diapedesis of T cells. This label-free transendothelial assay is suitable for testing multiple cell type pairings, evaluation of immunomodulation with blocking antibodies or inhibitors with the associated ability to assess monolayer integrity throughout the assay.
The role of ICAM-1 isoforms in neutrophil recruitment to liver
Authors: Yuki Koichi
Published: January 2023

Intercellular adhesion molecule-1 (ICAM-1) is one of important molecules involved in neutrophil recruitment and works as a ligand for integrin leukocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1) on neutrophils. Full-length ICAM-1 consists of five domains (D1–D2–D3–D4–D5) and D1 is a ligand binding domain for LFA-1 and D3 is for Mac-1. In addition, several alternative splicing variants exist as shown in transgenic mice. ICAM-1 Exon 4 deletion mice express D1D4D5, D1D2D5 and D1D5, while exon 5 deletion mice express D1D2D3D5, D1D2D5 and D1D5. The previous report of sepsis model showed exon 4 deletion mice showed significant reduction in neutrophil recruitment to liver with no mortality, while almost all of exon 5 deletion mice died with a large neutrophil recruitment. The mortality of WT mice was better than that of exon 5 deletion mice and worse than that of exon 4 deletion mice. These results suggest that ICAM-1 isoforms have functional roles in neutrophil recruitment. Our sepsis model demonstrated that neutrophil recruitment to liver was not affected in LFA-1 knockout mice and was reduced significantly in Mac-1 knockout mice. We speculated that ICAM-1 isoforms have functional roles via the interaction with Mac-1. Our PCR experiments demonstrated that liver highly expressed full length ICAM-1, D1D3D4D5 and D1D2D3D5 with full length ICAM-1 at the most abundant amount. Both D1D3D4D5 and D1D2D3D5 contain D3 and potentially bind to Mac-1. We expressed D1D3D4D5 and D1D2D3D5 along with full-length ICAM-1 in 293T cells, and examined the binding to Mac-1. Our preliminary data suggest that D1D2D3D5 bound more strongly to Mac-1 than full-length, supporting our hypothesis.
Defective monocyte motility disrupts alveolar macrophage development in mice deficient for L-plastin
Authors: Morley Sharon Celeste , Todd Elizabeth M , Zhou Julie Y. , Deady Lauren E. , D’Angelo June Ann , Szasz Taylor
Published: January 2023

Alveolar macrophages, sentinel cells essential to pulmonary immunity, mature during perinatal development. Fetal monocytes arrive in the lungs during late embryonic development and mature into alveolar macrophages through a pre-alveolar macrophage intermediate. Here we identify a requirement for the actin-bundling protein L-plastin (LPL) in a key transition during neonatal alveolar macrophage development. LPL−/− mice demonstrated a two-fold reduction in alveolar macrophages. The requirement for LPL was cell-intrinsic, as mice expressing a CD11c-specific deletion of LPL (CD11c.Cre-LPLfl/fl mice) also exhibited a significant (> 75%) reduction in alveolar macrophages. Impaired alveolar macrophage development in LPL−/− mice resulted from a block in the transition of the intermediate pre-alveolar macrophages to mature alveolar macrophages. Differentiation of monocytes into alveolar macrophages requires transit to the alveolar space, which exposes precursor cells to the essential growth factor GM-CSF, which in turn upregulates the transcription factor PPAR-γ. LPL is required for monocyte migration into the alveoli, providing a mechanism by which LPL deficiency impairs alveolar macrophage maturation. As predicted by this model, pre-alveolar macrophages in LPL−/− mice did not upregulate PPAR-γ, indicating that exposure to GM-CSF is reduced. Finally, LPL−/−, CD11c.Cre-LPLfl/fl and CD11c.Cre-LPLfl/+ mice failed to efficiently clear pulmonary pneumococcal infection, revealing a physiological consequence of impaired alveolar macrophage development. In summary, we show that genetic disruption of monocyte motility can impair differentiation of a key cell type, subsequently impairing host immunity.
Peroxisome Proliferator-Activated Receptor Delta (PPARδ) activity is required for normal T cell development
Authors: Zhao Fei , Ahn Jeeyoon Jennifer , Drohomyrecky Paulina , Zuniga-Pflucker Juan Carlos , Dunn Shannon
Published: January 2023

PPARδ is a nuclear receptor that functions as a transcriptional regulator of various processes including metabolic homeostasis, cell proliferation and differentiation; however, whether PPARδ regulates T cell development is not known. We observed that PPARδ-deficient (PPARδ−/−) mice have smaller spleens and thymi compared to wild-type (WT) mice. This reduction in thymus cellularity was apparent across all stages of thymocyte development. To further narrow in on the cellular basis of this effect, radiation bone marrow chimeras were generated where WT or PPARδ−/− mice were reconstituted with WT or PPARδ−/− bone marrow. These experiments clearly revealed that the defects in thymus and spleen mapped to the radio-resistant compartment. This was furthered confirmed by the finding that mice with T cell-restricted deficiency of PPARδ did not exhibit defective thymocyte homeostasis. Given that thymic epithelial cells (TECs) are the major radio-resistant cell population in the thymus, we next generated mice with a TEC-restricted deficiency of PPARδ (PPARδflox/floxFoxN1-Cre+). We observed that PPARδflox/floxFoxN1-Cre+ mice exhibit a trend for reduced thymus and spleen cellularity compared to PPARδflox/flox mice; however, this phenotype was not as striking as seen in PPARδ−/− mice. These results suggest that, in addition to acting within TECs, PPARδ may regulate the production of a circulating factor that in turn regulates thymocyte development. Large-scale serum profiling studies are underway to identify this factor. Altogether, this work highlights a role for PPARδ in T cell development.
Fusion of Early B Cell Factor 1 with Platelet-Derived Growth Factor Receptor Beta in B-ALL disrupts functions necessary for normal B lymphopoiesis
Authors: Hagman James R , Welsh Seth J
Published: January 2023

Human B cell Acute Lymphoblastic Leukemia (B-ALL) cells often lack functional genes encoding Early B cell Factor 1 (EBF1). EBF1 is essential for B lineage specification, where it regulates >500 genes necessary for normal B cell development. Here, we describe molecular mechanisms that contribute to EBF1-associated B-ALL. Intra-chromosomal deletions result in fusion of the nearly complete EBF1 gene with the 3′ half of the Platelet-Derived Growth Factor Receptor Beta gene (EBF1:PDGFRB) in a subset of high risk pediatric B-ALL. The fusion protein, EBF1:PDGFRβ, unifies loss of EBF1 function with a proliferative advantage due to unregulated Receptor Tyrosine Kinase (RTK) activity of PDGFRβ. Our laboratory has utilized biochemical, cell-based, and fluorescence microscopy approaches to study DNA binding, transcriptional activation and repression, oligomerization, and localization of EBF1:PDGFRβ in B cells. EBF1:PDGFRβ transforms B cell progenitors by eliminating their dependence on IL-7. Although the fusion protein includes nearly intact EBF1 and its nuclear localization signal, EBF1:PDGFRβ localizes predominately in the cytosol. Localization of EBF1:PDGFRβ is dependent upon the transmembrane (TM) domain of PDGFRβ; however, rather than docking EBF1:PDGFRβ in the plasma membrane, the TM functions as a nuclear export signal. Treatment with the tyrosine kinase inhibitor (TKI) imatinib reduces RTK activity and partially restores transactivation of EBF1 target genes by EBF1:PDGFRβ. This suggests a potentially undesirable effect of TKI chemotherapy, which could allow evasion of leukemic cells and disease relapse. Our data suggests that the TM domain of PDGFRβ could serve as a therapeutic target to supplement TKI chemotherapy.
BCAP (B-cell adaptor for phosphoinositide 3-kinase) plays a role in development of Marginal Zone B cells in mice
Authors: He Xiao , Dai Hu , Wang Xiaomin , Skidmore David , Kurosaki Tomohiro , Jensen Peter E
Published: January 2023

B-cell adaptor for phosphoinositide 3-kinase (BCAP) connects B cell receptor (BCR) signaling to the phosphoinositide 3-kinase (PI3K)-Akt pathway. Upon BCR engagement, tyrosine kinases Syk and Btk phosphorylate BCAP and phosphorylated BCAP provides the binding site for the regulatory subunit (p85) of PI3K, leading to relocation of PI3K to cytoplasmic membrane and PI3K activation. In response to CD19 engagement, BCAP phosphorylation is also required for p85 binding of BCAP prior to PI3K and Akt activation. It has been shown that disruption of the BCAP gene in the chicken DT40 B cell line inhibits BCR-mediated phosphatidylinositol 3,4,5-trisphosphate generation, leading to an impaired Akt response through a significant reduction in the recruitment of PI3K to glycolipid-enriched microdomains (lipid rafts). Furthermore, using BCAP knock out (KO) mice, BCAP has been shown to be essential for normal Follicular B and B1 B cell development and function. Here we report that the development of another peripheral B cell subpopulation, Marginal Zone B (MZB) cells, is impaired in BCAP KO mice as well. The numbers of MZB (B220+CD23−CD24midCD21high) cells and their surface expression level of CD80 are significantly reduced, compared to the littermate or age-matched B6 controls. The serum IgG3 level of the BCAP KO mice is also significantly lower than that of control mice. This result is further supported by an in vitro co-culture system, using bone marrow (BM) cells with the OP9-DL1 cells, in which the development of MZB cells from BCAP KO BM is also reduced, compared to B6 BM. Moreover, addition of exogenous BAFF could not fully restore the development of MZB cells from BCAP KO BM. Our data ascribes a role of BCAP to MZB cell development in mice.
Anti-FcγRIIB mAb suppresses IgG-dependent anaphylaxis by Fc domain targeting of FcγRIII
Authors: Clay Corey D , Strait Rick T , Mahler Ashley , Khodoun Marat , Finkelman Fred D
Published: January 2023

The inhibitory Fcγ receptor (FcγRIIB) is expressed on human and murine bone marrow-derived cells. FcγRIIB-mediated inhibition of stimulatory Fcγ receptor signaling limits inflammation in autoimmune, infectious and atopic diseases. We evaluated the ability of the anti-mouse FcγRIIB mAb, k9.361, to influence IgG-mediated anaphylaxis.

Methods

Wildtype and Fcγ receptor (FcγR)-deficient mice were used to study IgG-mediated anaphylaxis, which was induced by i.v. injection of 2.4G2 (a mAb that binds both FcγRIIB and the stimulatory receptor, FcγRIII), or by actively immunizing IgE-deficient mice, then challenging them with the immunizing antigen. Pretreatment with k9.361 was assessed for ability to influence anaphylaxis in these models.

Results

Unexpectedly, a single k9.361 injection induced mild anaphylaxis (assessed as hypothermia), which was both FcγRIIB- and FcγRIII-dependent. Injection with k9.361 also prevented the ability of a subsequent 2.4G2 injection to induce anaphylaxis and suppressed myeloid cell expression of stimulatory FcγRs, particularly FcγRIII. Treatment with k9.361 F(ab′)2 did not cause anaphylaxis, deplete FcγRIII, or suppress 2.4G2-induced anaphylaxis, but prevented the ability of intact k9.361 to induce anaphylaxis, without preventing the ability of intact k9.361 to suppress IgG-mediated anaphylaxis.

Conclusion

Anti-FcγRIIB mAb induces and then suppresses IgG-mediated anaphylaxis by targeting myeloid cell FcγRIII through its Fc domain. Sequential treatment with anti-FcγRIIB F(ab′)2, intact anti-FcγRIIB, and intact anti-FcγRIIB/III mAbs may provide a safe way to prevent IgG-mediated anaphylaxis.
Accumulation of CD23+ CD21-high Bin-like cells in human reactive and rheumatoid arthritis lymph nodes
Authors: Bottaro Andrea , Kuzin Igor , Kates Stephen , Wu Yawen , Rahimi Homaira , Wojciechowski Wojciech , Bernstein Steven , Burack Richard , Schwarz Edward
Published: January 2023

We have previously described a unique population of CD23+ CD21-high B cells in inflamed nodes (Bin) which accumulate in lymph nodes (LN) draining inflamed joints of TNF-transgenic (TNF-tg) mice, and contribute to arthritis flare by distorting node architecture and hampering lymphatic flow. The existence of Bin-like cells in human inflamed LN is yet unknown. Here we report the characterization of resident B cell populations in fresh popliteal lymph nodes (PLN) harvested from discarded tissues from rheumatoid arthritis (RA) patients and patients with other severe lower limb diseases (non-RA), undergoing total knee arthroplasty or lower limb amputation. We also analyzed banked, cryopreserved reactive and normal human LN single cell suspension samples. Bin-phenotype cells were shown to be significantly increased in reactive LN, and strikingly elevated (>30% of total) in RA samples. Histopathology and immunofluorescence analyses were consistent with Bin expansion and histological alterations in RA vs. non-RA PLN. This is the first description of Bin-like cells in human inflamed LN. Consistent with published mouse data, this population appears to be associated with inflammatory arthritis and distortion of LN architecture. Further analysis of this population will be important to assess its role in RA pathogenesis and arthritic flare.
A potential link between immune response to a periodontal bacterium, Aggregatibacter actinomycetemcomintans and Systemic Lupus Erythematosus
Authors: Bagavant Harini , Wolska Nina , Rybakowska Paulina , Kamp Stan , Guthridge Joel , James Judith A , Merrill Joan , Deshmukh Umesh
Published: January 2023

Infections are a significant cause of morbidity in Systemic Lupus Erythematosus (SLE). Although oral health is considerably compromised in SLE patients, the role of oral pathogens in influencing lupus pathogenesis is not known. In the present study, antibody responses to a periodontogenic, facultative anaerobic bacterium, Aggregatibacter actinomycetemcomitans (Aa), (which serve as an indicator of past or ongoing infection with Aa), were measured in SLE patients (n=587) and healthy controls (n=75). The associations between anti-Aa antibody titers and clinical measures of SLE were evaluated. The effect of Aa infection on development of lupus was investigated in the NZM2328 mice. The ability of Aa to induce neutrophil extracellular traps (NETs) was studied in vitro.

Both SLE patients and controls showed the presence of anti-Aa. In SLE patients, the anti-Aa titers correlated strongly with anti-dsDNA, anti-chromatin, anti-RNP, and anti-Sm; but not with anti-Ro52, anti-Ro60, and anti-La antibodies. NZM2328 mice developed a robust immune response to Aa, and the Aa infection caused an accelerated onset of autoantibody and renal disease. In addition, Aa readily induced NETosis in NZM2328 neutrophils.

Our data suggest for the first time that SLE patients are exposed to oral pathogen Aa, and this exposure significantly influences immune responses against a subset of lupus-associated autoantigens, possibly through NETosis. Furthermore, our studies suggest that management of oral health in SLE patients might prove to be beneficial for controlling lupus.
Identification of human effector B lymphocytes that express ARID3a and secrete interferon alpha
Authors: Webb Carol F , Ward Julie M. , Ratliff Michelle L. , Dozmorov Mikhail G. , Wiley Graham , Guthridge Joel M. , Gaffney Patrick M. , James Judith A.
Published: January 2023

Previously, we observed increased numbers of B lymphocytes expressing the transcription factor ARID3a in patients with systemic lupus erythematosus (SLE).* Although numbers of ARID3a+ B cells in individual patients varied over time, enhanced disease activity was associated with increased numbers of circulating ARID3a+ B cells. Because increased disease activity has also been correlated with interferon alpha (IFNa) responses in many SLE patients, we hypothesized that ARID3a expression might be associated with IFNa in SLE. Our new data show increased levels of IFNa in plasma from patients with increased numbers of circulating ARID3a+ B cells. Surprisingly, we found that ARID3a+ SLE B cells secrete IFNa. In vitro stimulation of healthy control B cells with the TLR 9 agonist, CpG, resulted in expression of both ARID3a and IFNa. Furthermore, co-cultures of healthy ARID3a+ B cells with autologous plasmacytoid dendritic cells increased IFNa production in the dendritic cells by up to 9-fold. These data identify ARID3a+ B cells as a novel type of effector B cell, and link ARID3a expression in B lymphocytes to IFN-associated inflammatory responses in SLE.
Choroid Plexus Infiltrates in MRL/lpr Lupus-Prone Mice Represent Tertiary Lymphoid Structures
Authors: Stock Ariel D , Laban Sivan , Ben-Zvi Ayal , Putterman Chaim
Published: January 2023

In addition to an overt neuropsychiatric phenotype (depression and cognitive dysfunction), MRL/lpr mice display robust infiltration of lymphocytes through the brain choroid plexus (CP). However, the functional features of these cells, or their potential contribution to neuropsychiatric disease, have not been explored in detail.

Localized tertiary lymphoid structures have been described in the kidney and peritoneum in spontaneous and inducible lupus models. Given the immunoprivileged nature of the brain and recent studies describing the critical role of the CP in immune surveillance, we evaluated 3D reconstructions of B-cell and T-cell infiltrates. We hypothesized that these CP infiltrates consist of tertiary lymphoid structures (ectopic germinal centers), induced in response to local and/or systemic inflammation.

We found dramatically increased lymphotoxin-β and CXCL13, key molecules in lymphoid follicle organization, in the CP of MRL/lpr mice. Lymphotoxin-β and CXCL13 were upregulated by 8 weeks of age, well before any cellular infiltration was present. Organizational and phenotypic features of lymphoid follicles, including clusters of proliferating (PCNA+) T and B cells, organized according to local chemokine gradients, were present. Furthermore, we identified phenotypically distinct groups of B-cells (e.g. IgM+, GL7+, BCL6+) and T-cells (e.g. FoxP3+, BCL6+, GL7+), as well as follicular dendritic cells and extracellular reticular networks, consistent with lymphoid follicles. We therefore conclude that the CP of MRL/lpr mice contain germinal-center like structures. The contribution of these structures to the pathogenesis, including local production of neuropathic autoantibodies, remains to be determined.
The α2,3 sialyltransferase ST3Gal1 regulates galectin-3 binding to human B cells
Authors: Giovannone Nicholas , Sweeney Jenna Geddes , Liang Jennifer , Antonopoulos Aristotelis , Pochebit Stephen M , Bhattacharyya Neil , Barthel Steven R. , Haslam Stuart M. , Dimitroff Charles J
Published: January 2023

Activated leukocytes commonly remodel cell surface carbohydrates (glycans) in order to modulate lectin-glycoprotein interactions involved in trafficking, adhesion, antigen receptor signaling, and cell fate. Because germinal center (GC) B cells upregulate glycan ligands for peanut lectin (PNA), we hypothesized that B cells remodel cell surface glycans upon GC differentiation in order to alter interactions with endogenous lectins. To test this, we studied cell surface glycosylation of ex vivo tonsillar naive, GC, memory, and plasmablast (PB) B cells using a panel of lectin probes. We found that in addition to PNA ligands, polylactosamine chains (polyLNs) are >4-fold enriched on GC B cells compared to naive, memory, and PB subsets, and that polyLNs are predominantly present as O-glycans on CD45. Moreover, qRT-PCR analysis and overexpression studies in tonsil and Ramos B cells, respectively, revealed polyLNs are inversely correlated with expression of ST3Gal1, an α2,3 sialyltransferase that competes for polyLN precursors. Given that polyLNs are preferred ligands of galectin-3 (Gal-3), a known regulator of apoptosis enriched in GCs, we assessed whether ST3Gal1 regulates Gal-3 binding to B cells. Unexpectedly, B cells with the lowest ST3Gal1 expression and most polyLNs (GC, ST3Gal1loRamos B cells) showed poor Gal3 binding, whereas B cells with natively high or enforced ST3Gal1 expression (Naive, Memory, ST3Gal1hi Ramos B cells) exhibited robust Gal-3 binding. Thus, our data implicate ST3Gal1 as a novel regulator of Gal-3 binding in B cells by a non-canonical mechanism. We propose GC B cells reduce Gal-3 ligand expression by repressing ST3Gal1 transcription, and as a result tune CD45-related signaling and susceptibility to apoptosis.
Complement mannan-binding lectin associated serine proteases 1 and 3 generated by allo-liver transplantation cleave in vivo pro-Factor D generated by the adipose tissue into active Factor D
Authors: Banda Nirmal K , Acharya Sumitra , Scheinman Robert I. , Mehta Gaurav , Takahashi Minoru , Thiel Steffen , Sekine Hideharu , Fujita Teizo , Holers V Michael
Published: January 2023

The alternative pathway (AP) of the complement system plays an important role in human diseases such as rheumatoid arthritis. MBL-associated serine proteases 1 & 3 (MASP-1/3)of the lectin pathway (LP) cleave pro-Factor D (proDf) (inactive) into Df(active), a necessary step to allow the AP to be activated. However, the site(s) of this cleaving activity are unknown; MASP-1/3 are predominantly liver-derived and proDf is produced by adipocytes. We hypothesized that the cleavage event can take place in the circulation, and may be facilitated by a complement activating surface. Liver from fD−/− but MASP-1/3sufficient (donor) was transplanted under the kidney capsule of MASP-1/3−/− (recipient)mice. H & E staining showed the presence of healthy hepatocytes under the kidney capsule of recipient mice. After five weeks of transplantation, the transplanted liver was found to generate MASP-1/3 proteases in the circulation, and concurrently proDf was cleaved into Df. Following liver transplantation, adherent anti-collagen antibodies in vitro also now activated C3 and generated C5a in the serum from transplanted MASP-1/3−/− mouse through the AP. This result illustrates one point of control of the AP by the LP. Furthermore, in vitro mixing and incubation of the sera from mice lacking MASP-1/3 or fD in the absence of anti-collagen antibodies did not cleave proDf. These results show that MASP-1/3 cleaves circulating proDf in a process facilitated by a complement activating target surface. Because the AP of complement is necessary for the development of collagen antibody-induced arthritis (CAIA), MASP-1 and/or MASP-3 inhibition, either at the mRNA or protein activity level, may be used as a new therapeutic approach for CAIA and its deleterious effects.
Role of retinoic acid in diet induced obesity
Authors: Gundra Uma Mahesh , Poles Jordan , Loke Png
Published: January 2023

Obesity is a complex disease associated with chronic, low-grade inflammation of the adipose tissue, which promotes insulin resistance and type-2 diabetes. Adipose tissue macrophages (ATMs) with an inflammatory M1 profile are involved in the development and progression of obesity associated metabolic diseases. In contrast, alternatively activated M2 macrophages (AAMs) may promote anti-inflammatory responses and polarization of CD4+Foxp3+ regulatory T cells (Tregs), attenuating adipose tissue inflammation. We now show that AAMs in the adipose tissue promote the polarization and maintenance of Tregs, through expression of the enzyme retinal dehydrogenase 2 (Raldh2). Diet induced obesity reduces expression of Raldh2 by adipose tissue AAMs and disrupts the polarization of Tregs. Treatment of lean mice with a pan-retinoic acid receptor inverse agonist BMS493 disrupts polarization of Tregs in the adipose tissue. In contrast, retinoic acid (RA) treatment of obese mice reverses defects in Tregs. Induction of AAMs and Tregs in adipose tissue occurs through a Stat6 dependent mechanism. Conditional deletion of Raldh2 with LysM-Cre or CD11c-Cre resulted in the disruption of Treg polarization. These findings suggest that RA may be important for maintaining adipose tissue Tregs by AAMs under steady state non-obese conditions and could perhaps be used as a novel therapeutic agent for diet induced metabolic diseases.