bluuebunny/crossref_metadata_2025 huggingface dataset:
Utilization of Two Seven-Transmembrane, G Protein-Coupled Receptors, Formyl Peptide Receptor-Like 1 and Formyl Peptide Receptor, by the Synthetic Hexapeptide WKYMVm for Human Phagocyte Activation
Authors: Le Yingying , Gong Wanghua , Li Baoqun , Dunlop Nancy M. , Shen Weiping , Su Shao Bo , Ye Richard D. , Wang Ji Ming
Published: December 2022

Trp-Lys-Tyr-Val-d-Met (WKYMVm) is a synthetic leukocyte-activating peptide postulated to use seven-transmembrane, G protein-coupled receptor(s). In the study to characterize the receptor(s) for WKYMVm, we found that this peptide induced marked chemotaxis and calcium flux in human phagocytes. The signaling induced by WKYMVm in phagocytes was attenuated by high concentrations of the bacterial chemotactic peptide fMLP, suggesting that WKYMVm might use receptor(s) for fMLP. This hypothesis was tested by using cells over expressing genes encoding two seven-transmembrane receptors, formyl peptide receptor (FPR) and formyl peptide receptor-like 1 (FPRL1), which are with high and low affinity for fMLP, respectively. Both FPR- and FPRL1-expressing cells mobilized calcium in response to picomolar concentrations of WKYMVm. While FPRL1-expressing cells migrated to picomolar concentrations of WKYMVm, nanomolar concentrations of the peptide were required to induce migration of FPR-expressing cells. In contrast, fMLP elicited both calcium flux and chemotaxis only in FPR-expressing cells with an efficacy comparable with WKYMVm. Thus, WKYMVm uses both FPR and FPRL1 to stimulate phagocytes with a markedly higher efficacy for FPRL1. Our study suggests that FPR and FPRL1 in phagocytes react to a broad spectrum of agonists and WKYMVm as a remarkably potent agonist provides a valuable tool for studying leukocyte signaling via these receptors.
Blockade of T Cell Costimulation by CTLA4-Ig Inhibits Lung Inflammation in Murine Hypersensitivity Pneumonitis
Authors: Israël-Assayag Evelyne , Fournier Marcien , Cormier Yvon
Published: December 2022

Hypersensitivity pneumonitis (HP) is characterized by an influx of activated T cells in the lungs. The CD28/B7 system provides costimulatory signals essential for complete T cell activation and differentiation. We have previously demonstrated that alveolar macrophages from patients with HP have an up-regulated expression of B7 molecules. In the present study, we investigated the effect of i.p. administration of CTLA4-Ig, a CD28/B7 antagonist, on the lung inflammation of mice inoculated with Saccharoplyspora rectivirgula (SR), a major causative agent of HP. Five groups of C57BL/6 mice were intranasally instilled with SR or saline for 3 consecutive days per wk during 3 wk. CTLA4-Ig was administered starting either after 1 wk of SR challenge or 6 h before the first antigenic exposure and continued during the whole period of sensitization. A control-IgG was given similarly during the 3 wk of SR exposure. The groups included: 1, saline; 2, SR; 3, SR + control-Ig; 4, SR + CTLA4-Ig for the last 2 wk; and 5, SR + CTLA4-Ig for 3 wk. CTLA4-Ig treatment markedly decreased lung inflammation as shown by significantly fewer inflammatory cells in the bronchoalveolar lavage and in lung tissue and reduced SR-specific serum and bronchoalveolar lavage Ig levels. Production of IL-4, IL-10, and IFN-γ by IL-2-stimulated pulmonary T cells was also decreased by CTLA4-Ig. Administration of CTLA4-Ig did not affect the SR-induced up-regulation of B7-2 expression. These results show that blockade of CD28/B7 interactions by CTLA4-Ig inhibits SR-induced lung inflammation and immune response to SR Ag in mice and may provide a novel approach in the treatment of HP.
Determination of the Contribution of Cysteinyl Leukotrienes and Leukotriene B4 in Acute Inflammatory Responses Using 5-Lipoxygenase- and Leukotriene A4 Hydrolase-Deficient Mice
Authors: Byrum Robert S. , Goulet Jennifer L. , Snouwaert John N. , Griffiths Richard J. , Koller Beverly H.
Published: December 2022

Arachidonic acid metabolism by 5-lipoxygenase leads to production of the potent inflammatory mediators, leukotriene (LT) B4 and the cysteinyl LT. Relative synthesis of these subclasses of LT, each with different proinflammatory properties, depends on the expression and subsequent activity of LTA4 hydrolase and LTC4 synthase, respectively. LTA4 hydrolase differs from other proteins required for LT synthesis because it is expressed ubiquitously. Also, in vitro studies indicate that it possesses an aminopeptidase activity. Introduction of cysteinyl LT and LTB4 into animals has shown LTB4 is a potent chemoattractant, while the cysteinyl LT alter vascular permeability and smooth muscle tone. It has been impossible to determine the relative contributions of these two classes of LT to inflammatory responses in vivo or to define possible synergy resulting from the synthesis of both classes of mediators. To address this question, we have generated LTA4 hydrolase-deficient mice. These mice develop normally and are healthy. Using these animals, we show that LTA4 hydrolase is required for the production of LTB4 in an in vivo inflammatory response. We show that LTB4 is responsible for the characteristic influx of neutrophils accompanying topical arachidonic acid and that it contributes to the vascular changes seen in this model. In contrast, LTB4 influences only the cellular component of zymosan A-induced peritonitis. Furthermore, LTA4 hydrolase-deficient mice are resistant to platelet-activating factor, identifying LTB4 as one mediator of the physiological changes seen in systemic shock. We do not identify an in vivo role for the aminopeptidase activity of LTA4 hydrolase.
Functional Analysis of Antigen-Specific T Lymphocytes by Serial Measurement of Gene Expression in Peripheral Blood Mononuclear Cells and Tumor Specimens
Authors: Kammula Udai S. , Lee Kang-Hun , Riker Adam I , Wang Ena , Ohnmacht Galen A. , Rosenberg Steven A. , Marincola Francesco M.
Published: December 2022

The cloning of cancer Ags recognized by T cells has provided potentially new tools to enhance immunity against metastatic cancer. The biological monitoring of effective immunization has, however, remained a dilemma. We describe here a sensitive molecular quantitation methodology that allows analysis of in vivo immune response to vaccination. Metastatic melanoma patients were immunized with a synthetically modified peptide epitope (209-2M) from the melanoma self-Ag gp100. Using serial gene expression analysis, we report functional evidence of vaccine-induced CTL reactivity in fresh cells obtained directly from the peripheral blood of postimmunized patients. Further, we demonstrate in vivo localization of vaccine-induced immune response within the tumor microenvironment. The results of these molecular assays provide direct evidence that peptide immunization in humans can result in tumor-specific CTL that localize to metastatic sites.
Activation of Eotaxin Gene Transcription by NF-κB and STAT6 in Human Airway Epithelial Cells
Authors: Matsukura Satoshi , Stellato Cristiana , Plitt James R. , Bickel Carol , Miura Katsushi , Georas Steve N. , Casolaro Vincenzo , Schleimer Robert P.
Published: December 2022

The C-C chemokine eotaxin is a potent chemoattractant for eosinophils and probably plays an important role in the pathogenesis of asthma, although the mechanisms of its regulation are not well known. Airway epithelial cells express eotaxin mRNA and protein after stimulation with a variety of cytokines. We focused on the molecular mechanisms of eotaxin gene regulation by TNF-α and IL-4 in the airway epithelial cell line, BEAS-2B. Cells were transfected with luciferase reporter plasmids, which contained up to 1363 bp of the eotaxin promoter. Eotaxin promoter activity was increased by TNF-α (2.5-fold) and IL-4 (1.5-fold), respectively. The combination of TNF-α and IL-4 produced 3.6-fold activation of the eotaxin promoter. The eotaxin promoter contains overlapping consensus binding sites for transcription factors, NF-κB and STAT6, which are known to mediate responses to TNF-α and IL-4, respectively. Electrophoretic mobility shift assays revealed NF-κB binding after TNF-α stimulation and STAT6 binding after IL-4 stimulation using a DNA probe derived from the eotaxin promoter. Mutant plasmids were generated to define the roles of these transcription factors in eotaxin promoter activity. TNF-α stimulation, but not IL-4 stimulation, was lost in plasmids mutated at the NF-κB binding site, whereas IL-4 stimulation, but not TNF-α stimulation, was lost in plasmids mutated at the STAT6 binding site. When both sites were mutated, all transcriptional activation was lost. These results imply that TNF-α and IL-4 stimulate expression of the eotaxin gene by activating NF-κB and STAT6.
Antibody Repertoires of Four- and Five-Feature Translocus Mice Carrying Human Immunoglobulin Heavy Chain and κ and λ Light Chain Yeast Artificial Chromosomes
Authors: Nicholson Ian C. , Zou Xiangang , Popov Andrei V. , Cook Graham P. , Corps Elaine M. , Humphries Sally , Ayling Christine , Goyenechea Beatriz , Xian Jian , Taussig Michael J. , Neuberger Michael S. , Brüggemann Marianne
Published: December 2022

We have produced mice that carry the human Ig heavy (IgH) and both κ and λ light chain transloci in a background in which the endogenous IgH and κ loci have been inactivated. The B lymphocyte population in these translocus mice is restored to about one-third of normal levels, with preferential (3:1) expression of human λ over human κ. Human IgM is found in the serum at levels between 50 and 400 μg/ml and is elevated following immunization. This primary human Ab repertoire is sufficient to yield diverse Ag-specific responses as judged by analysis of mAbs. The use of DH and J segments is similar to that seen in human B cells, with an analogous pattern of N nucleotide insertion. Maturation of the response is accompanied by somatic hypermutation, which is particularly effective in the light chain transloci. These mice therefore allow the production of Ag-specific repertoires of both IgM,κ and IgM,λ Abs and should prove useful for the production of human mAbs for clinical use.
Production of Soluble Granulocyte Colony-Stimulating Factor Receptors from Myelomonocytic Cells
Authors: Iwasaki Hiromi , Shimoda Kazuya , Okamura Seiichi , Otsuka Teruhisa , Nagafuji Koji , Harada Naoki , Ohno Yuju , Miyamoto Toshihiro , Akashi Koichi , Harada Mine , Niho Yoshiyuki
Published: December 2022

It has been speculated that a soluble form of G-CSFR might be physiologically present in humans, since G-CSFR mRNA that lacks a transmembrane domain has been identified from a human myelomonocytic cell line. Here, we demonstrate human soluble G-CSFR (sG-CSFR) of two different molecular sizes (80 and 85 kDa) on an immunoblot analysis using Abs generated against the amino-terminal, extracellular domain of the full-length G-CSFR. Both isoforms of sG-CSFR were able to bind recombinant human G-CSF (rhG-CSF). RT-PCR analysis with primers targeted outside of the transmenbrane region revealed that membrane-anchored G-CSFR is expressed at all maturation stages of purified myeloid cells, including CD34+CD13+ cells (blasts), CD11b−CD15+ cells (promyelocytes or myelocytes), CD11b+CD15+ cells (metamyelocytes and mature neutrophils), and CD14+ cells (monocytes). On the other hand, sG-CSFR mRNA was detectable in CD11b−CD15+, CD11b+CD15+, and CD14+ cells, but not in the CD34+CD13+ blast population. The serum concentration of both isoforms of sG-CSFR appeared to be correlated with the numbers of neutrophils/monocytes before and after rhG-CSF treatment in normal individuals. Thus, two isoforms of sG-CSFR are physiologically secreted from relatively mature myeloid cells and might play an important role in myelopoiesis through their binding to serum G-CSF.
Monomeric Complement-Activating IgG Paraproteins
Authors: Trendelenburg Marten , Hess Christoph , Kondo-Oestreicher Mitsuko , Tissot Jean D. , Späth Peter , Schifferli Jürg A.
Published: December 2022

Three patients presented a unique syndrome of recurrent panniculitis with an IgGκ paraprotein and depletion of the early components of the classical pathway of complement. The IgGκ paraproteins were monomers with a normal structure, and with no evidence for aggregation, as assessed by electron microscopy and ultracentrifugation. Both heavy and light chains were of normal molecular size (SDS-PAGE), and the paraproteins were not heavily glycosylated. However, the paraproteins from all three patients had unusual features that included abnormal behavior on gel filtration chromatography and a heavy chain of high pI. When analyzed by fast protein liquid chromatography (Superdex 200), elution of the paraproteins was retarded, particularly when the ionic strength was increased. This retardation was partially reversed in 20% alcohol, and fully reversed in 6 M guanidine-HCl. Neither anti-C1 inhibitor nor anti-C1q autoantibodies were found in any of the patients’ sera. However, the paraproteins bound to the globular heads of C1q at normal ionic strength. They activated C4 in normal human serum, but not in C1q-deficient serum. Activation led to the formation of C1s-C1 inhibitor complexes. Taken together, the data suggest that the unusual paraproteins have the capacity to bind C1q, which then leads to activation of C1. The ability of these paraproteins to activate C1, in spite of their being soluble monomers, is likely to be related to their unique physicochemical features.
The Sensitiveness of Tuberculous Guinea Pigs One Month Old to the Toxicity of Tuberculin
Authors: Freund Jules
Published: December 2022

In a previous paper (1) it was demonstrated that young experimentally infected guinea pigs show slight if any reaction to the injection into the dermis of a watery extract of tubercle bacilli despite the presence of gross lesions of tuberculosis in the regional lymph nodes and in the spleen. The control adult guinea pigs infected under the same conditions reacted to the same extract with marked redness, edema and necrosis at the site of injection.

This observation was confirmed subsequently by Valtis (2), who employed old tuberculin instead of the watery extract used in our work.

The purpose of the experiments reported here is to establish whether young experimentally infected guinea pigs are sensitive to the toxic action of old tuberculin. By young guinea pigs is meant, in this paper, guinea pigs not more than one month old; by adults, those weighing from 400 to 500 grams.
Utilization of distinct antigen-presenting cells for induction of CD4+ T cell-dependent humoral immunity in response to an intact bacterium versus soluble conjugate vaccine (APP3P.105)
Authors: KAR SWAGATA , Snapper Clifford
Published: December 2022

We hypothesized that CD4+ T cell-dependent polysaccharide (PS)- and protein-specific IgG responses to intact bacteria versus soluble PS-protein conjugates involve utilization of distinct APC. Confocal microscopy demonstrated that intact Streptococcus pneumoniae (Pn) co-localized within splenic mouse monocytic CD11b+CD11c- cells following i.p. immunization, whereas a pneumococcal conjugate vaccine co-localized within splenic CD11c+ cells. To explore the functional implications of this dichotomy we used clodronate liposomes (CL), to selectively deplete phagocytic cells, including monocytic CD11b+ cells, marginal zone macrophages and marginal metallophilic macrophages but not CD11c+ cells. CL injection prior to i.p. immunization with OVA peptide-expressing Pn resulted in marked inhibition of OVA-specific CD4+ T cell proliferation, whereas it had no effect on OVA-specific CD4+ T cell proliferation in response to an OVA-polysaccharide conjugate or OVA protein alone. Of interest, CL markedly reduced primary and secondary protein-specific IgG responses to Pn, as well as conjugate vaccine or protein alone. CL only modestly reduced PS-specific IgG responses to intact Pn and conjugate vaccine. These data suggest that CL-sensitive cells critically mediate early CD4+ T cell proliferation in response to an intact bacterium, but that they are also required for downstream events in response to a soluble antigen.
Macrophages-releasing antigen-carrying exosomes enhance dendritic cells-mediated CD4+ T cell response (APP3P.107)
Authors: Xu Yingping , Liu Yi , Yang Chunqing , Zhu Guangming , Wang Meixiang , Hu Jingxia , Song Wengang , Tang Hua
Published: December 2022

The mutual interplay of immune cells plays a critical role in the optimal adaptive immune response. Macrophages (Mφs) and dendritic cells (DCs) are major professional antigen presenting cells (APCs) and are jointly involved in clearance of invading pathogens and maintenance of homeostasis. However, the underlying interaction mechanism remains poorly understood. In this study, we demonstrated that Mφs, DCs, and neutrophils were predominant phagocytes that uptook apoptotic cell-associated antigens (Ags) in the spleen. Moreover, by utilizing deletion approaches with clodronate liposome (CLL) to delete Mφ, we found that Mφs were required for fully initiating a CD4+ T response to apoptotic cell-associated Ags. Although Mφs themselves were inefficient to present Ags to CD4+ T cell, it was capable of transferring Ags to DCs to induce a perfect CD4+ T cell proliferation. We further discovered that the process of Ags transfer to activate CD4+ T cell was related to cell-migration and soluble factor released by Mφs. More importantly, an exosome secretion inhibitor GW4869 could inhibit CD4+ T cell proliferation to cell-associate Ags in vitro and in vivo, which suggesting exosomes released by Mφs acted as one of vectors to bridge DCs and Mφs. Thus, we illustrated a new crosstalk mechanism between Mφs and DCs, contributing to regulate the optimal adaptive immunity and maintain immune homeostasis.
Evidence of a functional CD1d lipid antigen presentation pathway in murine alveolar epithelial cells (APP3P.108)
Authors: Rizvi Zaigham , Puri Niti , Saxena Rajiv
Published: December 2022

Lipid antigen presentation by CD1 molecule is an evolutionary conserved pathway that elicits rapid yet efficient cytotoxic response against fast mutating pathogens. CD1d molecule, the only isoform of CD1 family in mouse, has been shown to recognise important component of the Mycobacterium tuberculosis (Mtb) cell wall like phosphatidylinositol mannosides. We have recently shown that alveolar epithelial cells (AECs) present Mycobacterium bovis - BCG (an avirulent model for Mtb infection) antigens to sensitized Th cells in vitro, however, it remains unexplored whether AECs can also participate in the presentation of lipid antigens through CD1d pathway; that is, so far, know to operate only in professional antigen presenting cells like dendritic cells (DCs) and B cells. We started by establishing the expression of CD1d on LA4 (mouse airway epithelial) cell line and murine AECs by flow cytometry and immuno-histochemistry. qPCR showed significant up-regulation of mRNA expression of some of the crucial molecules involved in CD1d pathway in AECs upon BCG infection. The protein CD1d expression was also found to be boosted upon BCG infection. Finally, a functional CD1d antigen presentation by AECs (in vitro) was shown by using α-GalactosylCeramide (α-GalCer) antigen as model. Our findings suggest that AECs are capable of lipid antigen presentation through the CD1d pathway and this ability of AECs may contribute to the immune defences against pathogens like Mtb.
Early overexpression of FAS in beta cells protects NOD mice from autoimmune diabetes (BA6P.119)
Authors: Mora-Giral Concepcion , Wong Florence , Panosa Anaïs , Green E , Flavell Richard
Published: December 2022

Fas receptor signaling is essential for the induction of pancreatic β cell death in autoimmune diabetes, upon being engaged by its ligand FasL (CD95L). We found that early overexpression of this proapoptotic receptor on pancreatic β cells delayed diabetes onset and reduced diabetes incidence in the NOD mice, and its late overexpression in β cells does not have any protective effect on the disease onset. We have further explored whether early Fas expression on β cells promotes the immunodeviation of the autorreactive CD4+T repertoire to the T regulatory phenotype, at the expense of the proinflammatory, diabetes-causing Th1-Th17 phenotypes. We did not find an increased frequency of CD4+Treg cells in the peripheral lymphoid organs or increased FoxP3 expression in islets from NOD mice overexpressing Fas early in life. Diabetes protection was improved by neutralizing the Th17 response in transgenic mice, which suggests that protection mediated by Fas overerexpression is not due to the abrogation of the Th17 response, at least not exclusively.
Bacterial amyloids promote type I interferon production and accelerate autoimmunity (BA6P.124)
Authors: Gallucci Stefania , Gallo Paul , Rapsinski Glenn , Oppong Gertrude , Sriram Uma , Wilson Ronald , Buttaro Bettina , Tukel Cagla
Published: December 2022

Infections can be pathogenic in systemic lupus erythematosus (SLE). Curli is a bacterial amyloid part of enteric bacterial biofilms, like those of E. coli and Salmonella. We have recently found that curli fibers form highly immunogenic composites with DNA within the biofilm. Here we studied the immune response to curli-DNA complexes in the context of murine SLE. For in vitro studies, we stimulated bone marrow-derived dendritic cells (cDCs) from WT and lupus-prone mice with curli amyloid from S. Typhimurium biofilms. A dose titration of curli amyloid induced robust proinflammatory cytokine and type I-IFN responsive gene expression in cDCs. Using synthetic curli peptide, we found that curli and nucleic acids synergize to activate cDCs. We injected in vivo pre-diseased lupus-prone NZB/W-F1 mice i.p. with curli-DNA complexes and found that they accelerate disease, inducing high titers of anti-dsDNA and anti-chromatin autoantibodies. Curli-DNA complexes induced autoantibodies also in non-autoimmune C57BL/6 mice. Finally, we infected NZB/W-F1 mice i.p. with curli-expressing or curli mutant E. coli or Salmonella. Curli-competent bacteria triggered far higher autoantibody titers than curli mutant strains. In conclusion, biofilm-derived curli-DNA complexes are potent activators of DCs and can rapidly break tolerance and accelerate autoimmunity in mice, suggesting that bacterial amyloids and biofilm-based bacterial infections are important environmental triggers in lupus.
Drak2 contributes to type 1 diabetes by altering regulatory T cell development. (BA6P.128)
Authors: Harris Tarsha , Creasy Blaine , Wehenkel Marie , Lukens John , McGargill Maureen Ann
Published: December 2022

Mice deficient in the kinase, Drak2, are resistant to disease in mouse models of type 1 diabetes (T1D) and multiple sclerosis (MS) due to diminished survival of autoreactive T cells. Yet, Drak2-/- mice effectively eliminate infectious pathogens and retain the ability to combat tumors similar to wildtype mice. Thus, Drak2 is an ideal protein to target to treat autoimmune disorders without compromising immunity to infections or tumors. However, the molecular mechanisms and functions of Drak2 are unclear. It is important to discern how Drak2 contributes to T1D to further understand the etiology of this disease, as well as to validate Drak2 as a therapeutic target. We show here that the resistance to T1D in NOD.Drak2-/- mice was due to the absence of Drak2 in T cells rather than the pancreatic b-cells. Furthermore, the resistance to disease required regulatory T cells (Tregs). Interestingly, the development of thymic Tregs was increased in Drak2-/- mice due to augmented IL-2 signaling, which led to an increase in the proportion of Foxp3-expressing cells. These studies provide important insight into the etiology of T1D by showing that Drak2 contributes to autoimmune disease by reducing the proportion of Tregs through reduction of IL-2 signaling in T cells.
A novel B7 family member, B7-H3 is preferentially expressed on the cell surface of synovial monocytes in rheumatoid arthritis (RA) (BA7P.153)
Authors: Yoon Bo Ruem , Chung Yeon-Ho , Kim Dong Hyun , Kim Bonah , Yoo Su-Jin , Chung Doo Hyun , Kang Seong Wook , Lee Won-Woo
Published: December 2022

B7-H3, a newly identified B7 family member, has functional duality as a T-cell costimulator and coinhibitor that fine-tunes T-cell mediated immune responses. Given that B7-H3 expression on human monocytes and dendritic cells is enhanced by inflammatory cytokines, its potential inmmunoregulatory role at sites of inflammation has been suggested. Monocytes also play crucial roles in the pathophysiology of various autoimmune diseases. However, the immunological role of B7-H3 in RA has not been defined. We aimed to investigate the possible roles of monocyte B7-H3 in the pathogenesis of RA. Synovial monocytes, but not peripheral monocytes, in RA patients predominantly express surface B7-H3. The 4Ig isoform of B7-H3 is exclusively induced on the cell surface, whereas the 2Ig B7-H3 isoform is constitutively expressed in the intracytoplasmic region of both peripheral and synovial monocytes. B7-H3 knockdown experiments provide evidence that surface B7-H3 has an inhibitory effect on IFN-γ production in CD4 memory cells. Moreover, the expression level of 4Ig B7-H3 on synovial monocytes inversely correlates with clinical parameters such as CRP levels, ESR, and DAS28. Our findings demonstrate that activation-induced B7-H3 expression on synovial monocytes has the potential to inhibit Th1-mediated immune responses and immunomodulatory roles thereby affecting the pathogenesis of RA.
Eosinophils and IL-4 are essential drivers of inflammatory dilated cardiomyopathy (BA7P.160)
Authors: Diny Nicola , Barin Jobert , Talor Monica , Ong SuFey , Wu Lei , Rose Noel , Cihakova Daniela
Published: December 2022

Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in children and young adults, and patients often require a heart transplant. DCMi develops in up to 30% of myocarditis patients, but the mechanisms involved in disease progression are poorly understood. In wildtype (WT) BALB/cJ mice, experimental autoimmune myocarditis (EAM) progresses to DCMi with heart failure. Surprisingly, eosinophil-deficient ΔGATA1 mice developed myocarditis similar to WT mice, but were completely protected from DCMi. This indicates that eosinophils are crucial for progression of myocarditis to DCMi. Moreover, the level of eosinophilia correlated with DCMi severity. We used two models of eosinophilic myocarditis: mice expressing IL-5 under the CD3 promoter and IFNγ-IL-17A-double knockout mice. Both strains developed massive eosinophil infiltration in the heart and even more severe DCMi than WT mice. Recently, eosinophils were shown to affect several physiological processes through their production of IL-4. We therefore assessed IL-4 producing cell types in myocarditis using IL-4 reporter mice. Indeed, eosinophils were the major IL-4 expressing cell type in EAM. Additionally, IL-4-deficient mice developed myocarditis but were completely protected from DCMi, matching the phenotype of ΔGATA1 mice. In conclusion, eosinophils are required for progression of myocarditis to DCMi, drive severe DCMi when present in large numbers, and likely mediate this process through IL-4.
Macrophage depletion attenuates skin and kidney disease in lupus mice (BA7P.143)
Authors: Chalmers Samantha , Doerner Jessica , Wen Jing , Putterman Chaim
Published: December 2022

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of anti-nuclear autoantibodies and inflammatory damage in target organs, commonly the kidney and skin. Macrophages are thought to be important in the pathogenesis of lupus end organ disease, although studies to date have not been sufficiently conclusive. In this study, we investigated the role of macrophages in a spontaneous mouse model of SLE, the MRL/lpr mouse. PLX3397 (Plexxikon), a small molecule inhibitor of CSF1R, c-Kit, and FMS-like tyrosine kinase 3a, was administered in chow form to MRL/lpr mice starting at 9 weeks of age. Compared to matched control treated mice, PLX3397-treated MRL/lpr mice exhibited significantly less proteinuria and improved renal function. Furthermore, anti-dsDNA antibodies were significantly reduced at several time points. IBA-1 staining confirmed macrophage depletion within the kidneys of the PLX3397 treated mice. Interestingly, PLX3397-treated mice were also protected from the development of spontaneous skin lesions common to MRL/lpr mice, with control treated mice displaying significantly worse cutaneous disease macroscopically at 16 and 22 weeks of age. The improvement in skin lesions in PLX3397-treated mice was confirmed histologically in skin obtained at sacrifice. Our results indicate an important role for macrophages in the pathogenesis of skin and kidney disease in MRL/lpr mice, and implicate macrophages as a potential therapeutic target in SLE.
Batf3-dependent dendritic cells are essential for development of diabetes in the NOD strain of mouse (BA7P.145)
Authors: Ferris Stephen , Carrero Javier , Mohan James , Calderon Boris , Murphy Kenneth , Unanue Emil
Published: December 2022

Little is known about the early events that instigate autoimmune diabetogenesis. To understand these initiating events, we examined the early antigen presenting cells (APCs) within the islets of Langerhans in the NOD mouse. Islets contain a set of resident macrophages that are long lived and positioned next to blood vessels. We found, concurrent with T cell entry, a small subset of CD103+ dendritic cells (DCs) belonging to the Batf3-dependent lineage of DCs entering the islets during the early stages of diabetes. These DCs increase in the islets over the course of the disease paralleling the increase of T cells. In order to further investigate the function of these DCs during diabetes, we backcrossed the Batf3 deficient mouse to the NOD strain. These Batf3 deficient NOD mice displayed no diabetes when followed for over a year. No lymphocytic infiltration into the islets, innate or adaptive, was observed during their lifetime. Further examination showed that antigens presented to CD8+ T cells were absent, in conjunction with a reduction in antigen presentation to CD4+ T cells, in the draining lymph nodes. Finally, NOD.Rag1-/- bone marrow reconstitution rescued the CD103+ DCs and T cell infiltration to the islets. The reconstitution illustrates that Batf3 deficient T cells were intrinsically capable of being diabetic and confirms the necessity of the CD103+ DC lineage in diabetogensis. In sum, the CD103+DC controls the initiation of autoimmune diabetes.
IL-33 drives alum-induced cytokine production and provides a robust secondary antibody response independently of alum (CCR4P.211)
Authors: Rose William , Okragly Angela , Patel Chetan , Benschop Robert
Published: December 2022

For over 80 years, human vaccines have used aluminium-based adjuvants (alum) despite incomplete understanding of how alum enhances the immune response. One proposed mechanism is that alum induces cellular necrosis and release of endogenous danger signals. These signals induce inflammation-associated cytokines that lead to humoral immunity. IL-33 is proposed to be released from necrotic cells and can elicit TH2-biased cytokines. Therefore, we investigated a role for IL-33 in the adjuvant activity of alum. We show that alum induces cellular necrosis and elevates IL-33 levels shortly after intraperitoneal administration. Alum or IL-33 injection induces similar significant increases in IL-5, IL-6, KC, MCP-1 and MIP-1β. We establish that many of the alum-induced cytokines are dependent on IL-33 using IL-33 knockout mice or an IL-33-neutralizing recombinant ST2 receptor. Interestingly, IL-33 itself can function as an adjuvant. While IL-33 only induces a marginal primary response, it facilitates a secondary response comparable to alum and correlates with delayed splenic germinal center formation. Alum-induced antibody responses in IL-33 knockout mice are similar to those in wild-type mice, demonstrating that IL-33 is not absolutely required for the alum-induced humoral response. Our results provide novel insights into the mechanism of action behind alum-induced cytokine responses and show that IL-33 is sufficient to provide a robust secondary antibody response independently of alum.
Neuroprotective effects of IL-22 during CNS inflammation (CCR4P.203)
Authors: Caspi Rachel , Mattapallil Mary , Rigden Rachael , Zarate-Blades Carlos , Silver Phyllis , Luger Dror , Chan Chi Chao
Published: December 2022

IL-22 has opposing effects in different tissues, from pro-inflammatory (skin, joints) to protective (liver, intestine) but little is known about its effects on neuronal tissue. We examined this using the induced models of experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE). During EAU, IL-22 was produced by CD4+ eye-infiltrating cells, most of which co-produced IL-17. IL-22-/- mice immunized for EAU or for EAE had exacerbated disease, as did wild type mice treated with anti-IL-22 systemically or intraocularly during the expression phase of EAU. Retinal glial Müller cells in uveitic eyes had upregulated IL-22rα1 expression. Furthermore, IL-22 upregulated IL-22rα1 on Müller cells in culture and enhanced their ability to suppress effector T cells. Finally, IL-22 injected concurrently with IL-1β into the eye inhibited IL-1-induced expression of multiple proinflammatory and proapoptotic genes in retinal tissue. Our results suggest that IL-22 can function locally within the CNS to ameliorate inflammatory damage and provide neuroprotection by affecting multiple molecular and cellular pathways.
Astrocytes express novel members of the IL-10 cytokine family in response to bacterial challenge (CCR4P.204)
Authors: Burmeister Amanda , Marriott Ian
Published: December 2022

We have previously demonstrated that resident cells of the central nervous system (CNS) express the anti-inflammatory cytokine IL-10 in a delayed manner following bacterial challenge. More recently, we have shown the ability of astrocytes to express the recently identified IL-10 family member IL-19 in a similar manner and demonstrated the ability of this cytokine to limit inflammatory mediator production by this cell type. In the present study we have further investigated the ability of astrocytes to express novel IL-10 family members. We show that primary murine astrocytes can express IL-24 in response to bacterial LPS as determined by mRNA expression and protein production. Interestingly, and in contrast to the delayed production of IL-10 and IL-19 by this cell type, IL-24 production by astrocytes is rapid and transient. Such kinetics is consistent with those previously reported in peripheral cell types, its reportedly pro-inflammatory effects, and its concomitant production with other inflammatory cytokines by astrocytes. Finally, we have determined that the production of these IL-10 family members can be modulated by soluble neuronal factors such as the neuropeptide substance P (SP). We show that SP significantly augments the production of IL-19 by astrocytes in response to LPS. These data suggest that microbe challenged astrocytes are a significant source of these novel immune mediators, and that their production is influenced by the CNS environment.
Identification of a human clonogenic monocyte/macrophage progenitor in fetal and adult hematopoiesis (HEM5P.232)
Authors: Seet Christopher , Miao Runfeng , Chin Chee Jia , He Chong Bin , Zhu Yuhua , Chan Rebecca , Crooks Gay
Published: December 2022

During steady-state hematopoiesis, the monocytic lineage, which includes macrophages, osteoclasts, monocytes, and monocyte-derived dendritic cells (moDCs), arises from a progenitor hierarchy in bone marrow. In humans, multipotent myeloid progenitors such as the granulocyte-monocyte progenitor (GMP) are proximal in this hierarchy, however the distal stages of monocytopoieis are unclear. We report here identification of a human clonogenic, monocytic-committed progenitor with a lineage negative, CD34+ CD38+ CD45RA+ CD123int CD115+ phenotype, which was developmentally conserved across bone marrow, cord blood, and fetal liver. Ex vivo analysis demonstrated a high cloning efficiency, and monocytic but not granulocytic CFU potential. Differentiation in the presence of M-CSF resulted in exclusive production of functional M2-like macrophages, whereas cultures modified with RANKL and GM-CSF/IL-4 resulted in highly enriched cultures of functional osteoclasts and moDCs, respectively. Transplantation into immunodeficient mice reconstituted bone marrow, splenic, pulmonary, and hepatic macrophage subsets, as well as circulating CD14+ monocytic cells. Of note, GMP depleted of CD115+ cells could regenerate the monocytic progenitor phenotype in vitro and in vivo, consistent with a step-wise program of lineage restriction during monocytopoiesis. Identification of the steady-state human monocytic progenitor offers opportunities for therapeutic expansion or modification of the monocytic lineage.
Rupture-type platelet biogenesis from bone marrow megakaryocytes is regulated by IL-1alpha in response to acute inflammatory or thrombocytopenic conditions (HEM5P.236)
Authors: Nishimura Satoshi
Published: December 2022

Acute inflammatory conditions were frequently associated with increased platelet counts. However, their regulation and cellular mechanisms of acute thrombopoiesis from bone marrow megakaryocytes (MKs) were unclear. Herein, we determined the novel rupture type thrombopoiesis regulated by inflammatory cytokines, IL-1alpha, in response to acute platelet demands. The hematopoietic cytokine thrombopoietin (TPO) is believed to maintain thrombopoiesis via formation of proplatelets. However, proplatelet formation (PPF) is not the only path to platelet biogenesis. We elucidate that the IL-1alpha is a rapidly acting platelet releasing factor that induces an alternative MK rupture-type thrombopoietic pathway, characterized by cytoplasmic fragmentation with acute particle releases. During recovery after platelet depletion or under inflammatory conditions, serum IL-1alpha levels are acutely increased (within 24 h), whereas TPO is elevated only at later times (day 3 or later). In vivo, IL-1alpha administration rapidly induces rupture-type thrombopoiesis in MKs, mediating an increase in platelet counts. IL-1alpha/IL-1R1 signaling activates pERK, pAKT and caspase-3/-9, which reduces intracellular K+ concentrations, plasma membrane potential and membrane stability. Thus the balance between IL-1alpha and TPO dynamically determines MK fate, with IL-1alpha controlling rupture-type platelet release under inflammatory or acutely thrombocytopenic conditions.
Mir-34a promotes the development of dendritic cells and inhibits the function of DC on activating the CD4+ T cells by inducing high expression of IL-17 (HEM5P.237)
Authors: Zhang Jinping
Published: December 2022

Many evidence suggests that miRNAs are involved in the hematopoietic lineage commitment. Dendritic cells (DCs) play pivotal role in immune system. But the role of miRNAs in DC development is unclear. In this study, we first established mir-34a chimeric and transgenic mice. FACS analysis showed that both cDCs and pDCs were increased in miR-34a chimeric and TG mice. Brdu and annexin V staining experiments showed that miR-34a-induced increasing of cDCs and pDCs were not due to the alteration of proliferation and apoptosis of DCs. GMP\CMP\ProDC and PreDC were comparable between WT and mir-34a TG mice, but mir-34a TG PreDC could produced more DC in vitro compared to WT preDC. Luciferase report assay and Western blot demonstrated that WNT1 is the target of miR-34a. Overexpressing WNT1 could preserve the development of cDC and pDC in miR-34a TG mice. all above evidence suggests that WNT1 is the functional target of miR-34a in DC. To our surprise, cDCs from miR-34a TG mice produced large amount of IL-17a and these cDC could inhibit CD4+ T cell activation, but did not affect antigen-uptaking and maturation of cDC. Addition of IL-17a neutralizing antibody made this inhibition disappeared. Further experiments showed that mir-34a regulated Wnt1-TCF1-RORγT-IL-17a pathway. Taken together, miR-34a promotes preDC development into cDCs and pDCs by targeting WNT1, and inhibits the function of cDCs on the activation of CD4+ T cells by up-regulating IL-17a production in cDCs.
Agelasine D suppresses RANKL-induced osteoclastogenesis via down-regulation of c-Fos, NFATc1 and NF-κB (HEM5P.240)
Authors: Kang Jong Soon , Kang Moo Rim , Jo Sun Ah , Han Sang-Bae , Kim Youngsoo
Published: December 2022

Osteoclasts are considered as innate immune cells of a bone and involved in the regulation of hematopoiesis as well. In the present study, we investigated the effect of agelasine D (AD) on osteoclastogenesis. Treatment of bone marrow macrophages (BMMs) with receptor activator of nuclear factor κB ligand (RANKL) resulted in a differentiation of BMMs into osteoclasts. However, RANKL-induced osteoclastogenesis was significantly suppressed by AD treatment. We also confirmed the increased mRNA and protein expression of osteoclastic markers during RANKL-induced osteoclast differentiation and this was down-regulated by AD treatment. Moreover, AD treatment significantly suppressed RANKL-induced mRNA expression of DC-STAMP and OC-STAMP and cell fusion of TRAP-positive mononuclear osteoclast precursors. In addition, AD suppressed RANKL-induced expression of transcription factors, c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important transcription factors involved in differentiation of BMMs into osteoclasts. Furthermore, RANKL-induced phosphorylation of extracellular signal-related kinase (ERK) and activation of NF-κB were also inhibited by AD treatment. Collectively, these results suggest that AD inhibits RANKL-induced osteoclastogenesis by down-regulation of multiple signaling pathways involving c-Fos, NFATc1, NF-κB and ERK. Our results also suggest that AD might be a potential therapeutic agent for prevention and treatment of osteoporosis.
Immunological Evidence on the Role of the Lymphocyte in Antibody Formation
Authors: Habel Karl , Endicott Kenneth M. , Bell J. Frederick , Spear Frances
Published: December 2022

In summary, it seems from the evidence here presented that the relatively high antibody content of lymph may be accounted for, at least in part, by physical transfer from blood. The ratio of lymph antibody to serum antibody increases when the node has been stimulated by an irritant such as a heterologous antigen. There is very little significant evidence in these experiments to indicate that the lymphocytes or lymph node cells per se carry large amounts of antibody. Until more critical immunological technics and better controlled experiments are employed, the immunological evidence for lymphocyte production of antibodies must be considered incomplete.
Flagellar Antigens of Escherichia Coli
Authors: Stuart C. A. , Carpenter Philip L.
Published: December 2022

Kauffmann (1947) has reviewed the extensive work of himself and several colleagues on the serology of the Escherichia coli group. According to this report, cultures from pathologic and normal sources could be typed serologically by a system patterned after that used with the genus Salmonella. Three kinds of antigens were used: the usual H and O antigens, and K antigens, which were either thermolabile (L and B) or thermostable (A) envelope or capsular substances.

This work is important in that it helps to clarify the serological picture of a puzzling group of organisms. It will be especially valuable from the phylogenetic point of view when it has been extended to show the interrelationships of Escherichia coli with other genera and species of the Enterobacteriaceae.

Kauffmann experienced great difficulty in determining the H antigens of the coli-group because of inability to obtain actively motile cultures. Only by use of U-tubes with 0.1 per cent agar were suitable antigens secured.
Blood Groups and Subgroups of the Newborn
Authors: Witebsky Ernest , Engasser Lillian M.
Published: December 2022

Adult blood cells of subgroups A1 and A2 as well as cord blood cells of group A are agglutinated by native immune anti-A sera of human origin almost to the same extent, provided saline solution is used as the diluent. If saline solution is replaced as a diluent by normal adult serum of group A or AB, immune anti-A sera of human origin allow the differentiation of adult A1 and adult A2 cells. Under these conditions, cord blood cells of group A frequently resemble adult cells of subgroup A2 more than adult cells of subgroup A1. The addition of a solution of purified A substance to human immune anti-A sera results in the neutralization of the antibody content against all subgroups of A when saline solution is used as the diluent. Neutralized anti-A sera frequently agglutinate A1 cells up to the original titer, without agglutinating A2 cells at all, if saline solution is replaced as a diluent by normal adult serum. Cord blood cells of 34 babies belonging to the blood group A revealed wide variations of agglutinability when tested with this type of reagent, exhibiting differences corresponding to the subgroups of adult A bloods, though on a lower level of agglutinability. About half of the babies' cells reacted like adult A2 cells or even weaker. The other half could be placed between adult A2 and adult A1 cells, rarely reaching the agglutinability of adult A1 cells. Hirszfeld's theory of “pleiades” is discussed in its relation to the experimental results described in this paper.
Antigen-Antibody Reactions in Agar
Authors: Becker Elmer L. , Neff J. C.
Published: December 2022

In simple diffusion in tubes (technique of Oudin) plotting x, the distance moved by the leading edge of the precipitate band against t½, the square root of the time, yields a straight line with slope equal to k (1). Oudin showed empircally that at sufficiently low antigen (Ag) and high antibody (Ab) concentrations the following equation held where m is a constant and Agi and Abi are the antigen and antibody concentrations (1) respectively at zero movement of the band (1). In the preceding paper of this series we attempted to give theoretic significance to this equation by equating m with an apparent diffusion coefficient of the antigen. This was based upon a relationship (equation 2) derived previously (2), where R' is the ratio of antigen to antibody in the leading edge of the precipitate band which we have postulated to be equal to the ratio of antigen to antibody at equivalence as determined in liquid media by the usual quantitative precipitin technique.
Immunologic Studies of Japanese Encephalitis Virus in Japan
Authors: Scherer William F. , Moyer Jack T. , Izumi Toshiaki
Published: December 2022

Discussion and Summary

Antibodies to JE virus of maternal origin were found in pigs 2–7 months of age near Tokyo, Japan. These passively acquired antibodies were detected most frequently by the neutralization test, less often by hemagglutination-inhibition and never by complement fixation. Detectable maternal antibodies disappeared in the majority of pigs by 4–6 months of age, at which time swine became susceptible to virus. No information was sought or obtained in these studies concerning the mechanism of antibody transfer from sows to shoats although transfer via the colostrum and placenta were two recognized possibilities.

HI and neutralizing antibodies developed regularly within 1–4 weeks of natural or experimental swine infection by JE virus, but CF antibodies formed in only 18 per cent of 61 pigs infected naturally at farms and in only two of four pigs after subcutaneous inoculation of virus at the laboratory. Although the neutralization test was most sensitive for detection of maternal antibody, the HI test, because of the ease and and inexpense of performance, and the promptness with which the test became positive after infection, proved best for evaluating recent swine infection near Tokyo, where no other Group B arbor virus was endemic. The lack of consistent quantitative relationships or temporal concurence in development of the different antibodies following infection supported the conclusion that HI, CF and neutralization tests measured three different antibodies to JE virus in pigs.

Viremia was demonstrated following natural infection of pigs in mosquito traps. After subcutaneous inoculation of JE virus at the laboratory, virus was found in blood for 4 days and to titers of 2.5–2.6 log LD50/0.03 ml of blood.