bluuebunny/crossref_metadata_2025 huggingface dataset:
The Disappearance of Agglutinin from the Blood of Anaphylactic and Normal Animals
Authors: Hempl Hilda
Published: January 2023

In order to throw light on the question of the origin of “anaphylatoxin,” Doerr and Pick (Centralbl., 62, Orig., p 146) and Römer and Viereck (Zeitschrift f. Immunitätsforsch., Orig., Bd. xxi, p. 32) measured the disappeaance of antibodies in heterologous sera in anaphylactic animals.

Doerr and Pick injected a rabbit sensitized to horse serum and a normal rabbit intravenously with cholera agglutinating horse serum, and they found that the amount of agglutinin and of the precipitable horse albumen remained the same in both for about a day and then disappeared faster from the anaphylactic animal than from the other. They injected guinea pigs sensitized to horse serum intraperitoneally with 1 to 2 cc. of cholera agglutinin from the horse and found that the agglutinin and the precipitable horse albumen appeared in the circulation of the sensitized animals more quickly than in that of the others and, up to six hours after injection, they were present in greater quantity.
The Bacteriolytic Power of Normal and Immune Rabbit Serum for Typhoid Bacilli and the Influence of the Intravenous Injection of Vaccine Upon the Same
Authors: Teague Oscar , McWilliams Helen I.
Published: January 2023

The work of Buxton and others showing that normal rabbit serum is capable of killing large numbers of typhoid and paratyphoid bacilli and that the sera of rabbits highly immunized against typhoid and paratyphoid bacilli do not kill these organisms is confirmed; it is emphasized, therefore, that the titer of an immune serum as determined by the usual dilution method with the addition of complement is not an indication of the bactericidal power of the blood plasma in vivo.

The bacteriolytic power of normal and highly immune antityphoid rabbit serum for typhoid bacilli is neither increased nor decreased by the intravenous injection of a large dose of typhoid bacilli; the serum of a rabbit that has received only one or two immunizing injections shows a distinct, but not a great, increase in its bactericidal power at five and twenty-four hour intervals after such an injection.
ST8Sia6 attenuates diabetes progression and severity
Authors: Belmonte Paul , Chung Ji Young , Arocha Sinibaldo Romero , Schwab Aaron , Shapiro Michael J. , Fife Brian T. , Shapiro Virginia
Published: January 2023

Nearly 1.25 million Americans suffer from type 1 diabetes, an autoimmune disease which destroys insulin-producing beta cells of the pancreatic islets. Although insulin therapy is a lifesaving treatment, it is not a cure and secondary complications remain a significant concern. Experimental islet transplantation as a curative measure has yet to overcome the barrier of immune-mediated destruction, as patients eventually revert back to insulin-dependency. Immunotherapies to date have focused on targeting the adaptive immune response, largely ignoring the innate immune system, and have had limited success. Siglec-E is an inhibitory receptor expressed on innate immune cells such as macrophages and dendritic cells; it is known to dampen inflammation through several mechanisms. Here, we demonstrate that the sialic acid transferase ST8Sia6, which catalyzes addition of a2,8-linked disialic acids to glycoproteins, produces cell surface ligands for Siglec-E. We selectively expressed ST8Sia6 in pancreatic beta cells, hypothesizing this would have a beneficial effect in delaying disease in the murine multiple low-dose streptozocin model of diabetes. We observe that when diabetes is induced, mice that express ST8Sia6 in beta cells have delayed onset of diabetes and less severe hyperglycemia as compared to littermate controls. Notably, expression of ST8Sia6 in beta cells does not interfere with normal glucose response. Therefore, ST8Sia6 expression in pancreatic beta cells is safe and attenuates diabetes progression and severity. Because cell surface glycans play a major role in recognition of self, this work has significance in islet-targeted gene therapy, transplantation, and graft survival.
The transcription factor NFAT5 inhibits experimental autoimmune encephalomyelitis (EAE) in mice
Authors: Packialakshmi Balamurugan , Hira Sharanpreet , Halterman Julia , Lees Jason R. , Zhou Xiaoming
Published: January 2023

Th1 and Th17 cells contribute to the pathogenesis of multiple sclerosis (MS) and EAE, an animal model of MS. Th1 cells are characterized by the abundant expression of IFN-γ, whereas Th17 cells express high levels of IL-17. T cell activation leads to the nuclear translocation of nuclear factors and activators of T-cells (NFATs), which contribute to the expression of IFN-γ and IL-17. NFAT5 is the newest member of the NFAT family, however, its role in the pathogenesis of EAE is unknown. We are therefore studying MOG35–55-induced EAE in mice with a global heterozygous knockdown of NFAT5 (NFAT5+/−, NFAT5−/−is lethal) with NFAT5+/+ mice as a control. Interestingly, we found that the knockdown of NFAT5 actually increased EAE induction rate from 65% to 74% and delayed the recovery of EAE, indicating that NFAT5 is protective against the disease. Naïve CD4 cell differentiation reveals that the NFAT5 knockdown increased IFN-γ expression without significantly affecting IL-10 expression in Th1 cells. In vitro differentiation of Th17 cells from NFAT5+/− mice was not successful. IL-6 and protein tyrosine phosphatase SHP-1 have been demonstrated to inhibit IFN-γ expression in Th1 cells. To study the mechanisms by which NFAT5 knockdown increases IFN-γ expression in Th1 cells, we have examined the effects of NFAT5 knockdown on IL-6 and SHP-1. NFAT5 knockdown had no significant effect on IL-6 mRNA level. Instead, our ongoing experiments show that knockdown of NFAT5 reduces SHP-1 mRNA level by 38% (n=3, p=0.11) with a concomitant 44% increase in phosphorylation of Y705 of STAT3, one of its targets (n=3, p=0.2), and has no significant effect on SHP-2 mRNA level. In conclusion: NFAT5 inhibits EAE possibly by suppressing the expression of IFN-γ in Th1 cells.
Structural characteristics of the serum endonuclease Dnase1L3
Authors: McCord Jon Joseph , Harsini Faraz , Sutton Roger Bryan , Keyel Peter A.
Published: January 2023

Systemic Lupus Erythematosus (SLE) is a debilitating autoimmune disease with poly- and mono-genic etiologies. One cause of monogenic SLE is the disruption of Dnase1L3. Dnase1L3 is a serum endonuclease that degrades chromatin in apoptotic microparticles. Dnase1L3 could represent a novel therapy for some cases of SLE. However, the structure of Dnase1L3 is predicted by the 42% identical protein Dnase1. To better understand the structure-function of Dnase1L3, we set out to solve the crystal structure. To solve the crystal structure, we first generated a recombinant His6-Maltose Binding Protein-Dnase1L3 fusion protein. We developed a protocol for purifying Dnase1L3. We isolated Dnase1L3 from the cell lysate using Nickel-NTA beads in maltose buffer, removed purification tags on Dnase1L3 with Tobacco Etch Virus protease, and purified Dnase1L3 on a negatively charged ion exchange column. We then purified Dnase1L3 by size exclusion chromatography to 96.7% final purity. We verified purity at each step by Coomassie staining and, confirmed identity of the final product by Western blot. Recombinant Dnase1L3 robustly degraded plasmid DNA, with a specific activity of 1.83 x 104 U/mg. We found Dnase1L3 had a propensity for small mosaic crystals in a Tris and MgCl2 crystallization buffer. However, larger crystals were grown at 7 °C, compared to 22 °C. Overall, we have developed a new purification method for generating active Dnase1L3 useful for crystallography and other high-purity applications. These applications will further our understanding of Dnase1L3 structure and its function in SLE.
Inflammation targets cell-cycle actors at the onset of autoimmune diabetes (T1D) in pancreatic endocrine cells from the NOD mouse model
Authors: Mora Conchi , Saavedra-Ávila Noemí Alejandra , Sala Ester , Sengupta Upasana , Verdaguer Joan , Stratmann Thomas , Sicinski Peter , Lahti Jill
Published: January 2023

Prior to the massive beta cell demise that leads to autoimmune diabetes (Type 1 Diabetes or T1D), profound profiling changes are imprinted onto the beta cell by an aggressive pro-inflammatory environment that interferes negatively with cell functionality and viability. Ourgoal is to determine key signaling pathways altered in beta cells by the autoimmune assault that are responsible for T1D progression. To this end we have used the T1D-prone NOD (Non Obese Diabetic) mouse model. We have identified several candidate genes the expression of which is altered in the NOD isletendocrine cells prior to the diabetes onset. Interestingly, several of these genes are related to cell cycle progression and are downregulated due to the insulitic attack to the islet. One of these genes is cyclin D3, a D-type cyclin that complexes with either Cdk4 or Cdk6 topromote G1/S cycle progression. The cyclin D3 promoter has binding sequences to NF-kB. However, we have reported that cyclin D3 protects beta cells against cytokine-induced apoptosis and is required for proper beta cell function in a cell-cycle independent fashion. Cdk11 is also affected by inflammation in islet cells and is a cyclin-dependent kinase which is involved in transcription, mitosis and apoptosis. The natural partners of Cdk11 are L-type cyclins, but cyclin D3 has been reported to interact with Cdk11 too. We have addressed whether cdk11 downregulationis responsible for beta cell death.
Thymic development of regulatory T cells by dendritic cells presenting modified peripheral beta cell antigens.
Authors: Lin Yong , Lee Thomas , Zurawski Gerard , Bettini Maria , Bettini Matthew L.
Published: January 2023

Type I Diabetes (T1D) is a T cell-mediated autoimmune disease that destroys insulin producing β cells found within the pancreatic islets of Langerhans. Insulin and Chromogranin A (ChgA) are the only currently known β cell antigens (βAg) necessary to initiate autoimmune diabetes in the non-obese diabetic (NOD) mice; however, expression of ChgA within medullary thymic epithelial cells (mTECs) has not yet been detected. Therefore, tolerance to ChgA may be due in part to peripheral dendritic cells (DCs) presenting ChgA to developing thymocytes and naïve T cells. For example, post-translational modification (PTM) of ChgA by fusion with an insulin peptide increases epitope immunogenicity by forming the neo-antigen, insulin-ChgA fusion peptide (2.5HIP). Pathogenic T cells that are specific for 2.5HIP, which is uniquely expressed in the pancreas, may escape thymic selection due to immunologic ignorance. The relative contribution of thymic and peripheral derived Ag in the selection of βAg specific CD4+ T cells and regulatory T cells (Tregs) remains unclear. To test the role of peripheral Ag exposure on thymic development of βAg specific Tregs, we generated a 50:50 mixed bone marrow chimera using NOD WT (CD45.2 × CD45.1) and BDC2.5 TCR Tg (CD45.1) bone marrow to study the role of peripheral DCs (CD45.2) on developing ChgA T cells. Next, we targeted thymic Langerin+ DCs with anti-Langerin linked to ChgA peptides in BDC2.5 TCR Tg mice. In both experiments, we found robust increase in the ratio and number of ChgA specific Tregs in the presence of the 2.5HIP, in adult and neonatal mice. The results suggest thymic exposure to PTM peptides by DCs enhances β cell specific Treg development and may alter T1D pathogenesis.
Foxp3 domain-swap interface is required to suppress T helper type 2 transcriptional program in Regulatory T cells.
Authors: Van Gool Frederic , Nguyen Michelle L. , Mumbach Maxwell R. , Satpathy Ansuman T. , Anderson Mark S. , Marson Alexander , Chang Howard Y. , Bluestone Jeffrey A.
Published: January 2023

Regulatory T cells (Treg) play a fundamental role in maintaining immune tolerance. These cells are defined and characterized by expression of the master regulator Foxp3, a transcription factor that is crucial for Treg function and homeostasis. Natural occurring mutations in Foxp3 gene have been shown to be responsible for the immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, which is characterized by widespread autoimmune diseases. We recently identified a Foxp3 mutation in a boy that manifested a severe IPEX syndrome. This mutation, resulting in the substitution of methionine for isoleucine at amino acid 370 was determined to be part of a class of Foxp3 mutations that disrupt the dimerization motif in the domain swap interface of the protein. We have shown that, although dimerization mutants retain DNA binding, allows Treg development and certain Treg characteristics, it inevitably led to an IPEX syndrome in humans and mice. Genomic analysis of M370I mutant Treg by RNAseq, ChIPseq and HiChIP revealed a de-repression of the T helper type 2 (Th2) transcriptional program leading to the generation of Th2-like Treg. We have shown that, dimerization mutant proteins bind to multiple locations in the Th2 locus leading to increased intrachromosomal interactions and Th2 cytokines production. As expected, M370I Tregs were unable to suppress Th2 immune responses. Transgenic mice expressing M370I Foxp3 developed autoimmune diseases characterized by systemic Th2 cytokines production and skin and lung inflammation. In conclusion, disruption of the domain swap interface of Foxp3 by M370I mutation led to the development of Th2-like Treg which are functionally unable to suppress the development/function of Th2 cells.
Protein Kinase 2 (CK2) is Required for CD4+ T Cell Function in the Pathogenesis of Colitis
Authors: Yang Wei , Gibson Sara , Yan Zhaoqi , Qin Hongwei , Benveniste Etty
Published: January 2023

Crohn’s disease (CD) is one of the major forms of inflammatory bowel disease (IBD), characterized by chronic inflammation of the gastrointestinal tract. CD is associated with aberrant Th1 and Th17 responses accompanied by high levels of IFN-γ and IL-17, respectively. Protein kinase CK2 is a highly conserved serine-threonine kinase that is involved in several signal transduction pathways, including the JAK/STAT, NF-κB, PI3K/Akt/mTOR and Wnt pathways. Inhibition of CK2 kinase activity by small molecule inhibitor (CX-4945) or genetic deletion of CK2α in CD4+ T cells shifts the balance between Th17 and Tregs. However, the function of CK2 in the pathogenesis of colitis is unknown. Here, we demonstrate that CD4+ T cells from CK2αfl/fl dLck-Cre mice failed to induce wasting disease and intestinal inflammation, accompanied by lower levels of inflammatory cytokines and chemokines in the serum, and decreased IL-17A+, IFN-γ+ and IL-17A+/IFN-γ+ CD4+ T cells in the spleen and colon. Furthermore, CK2α controls CD4+ T cell accumulation in the spleen and colon through a cell-intrinsic mechanism. Thus, our data suggest that CK2α contributes to the pathogenesis of colitis, and that inhibition of CK2 kinase activity may be a novel therapeutic treatment for patients with CD.
Regulation of beta1 Integrin Recycling, Cell Migration and Focal Adhesion Turnover by Cell Adhesion molecule- CD13
Authors: Ghosh Mallika , Lo Robin , Devarakonda Charan , Aguilera Brian , Ivic Ivan , Shapiro Linda H.
Published: January 2023

CD13 is a multifunctional cell adhesion molecule that is expressed on a variety of cells where we have shown that it modulates receptor mediated endocytosis and ligand internalization to control downstream signaling pathways. In fibroblasts and epithelial cells, CD13 is localized with β1- integrin at focal adhesions (FAs), the sites of communication between the extracellular matrix and the actin cytoskeleton, prompting our exploration of potential contributions of CD13 in focal adhesion turnover, integrin endocytosis and trafficking. Phenotypically, FAs in CD13KO fibroblasts are elongated and irregular with displaced FA accessory proteins, markedly reduced actin stress fibers and fewer microtubule extensions, consistent with a link between CD13 and the control of FA dynamics. In wild type cells, CD13 and β1-integrin co internalize, traffic to Rab5+ early endosomes and recycle to the plasma membrane via Rab11a+ recycling endosomes. Pulse-chase assays with wild type and CD13 phospho tyrosine mutant confirmed that tyrosine phosphorylated CD13 must associate with the active ARF6 for β1-integrin recycling to the membrane to occur. Conversely, the absence of CD13 results in trafficking of internalized β1-integrin from early endosomes to Rab7+ late endosomes/lysosomes and a failure to return to the cell surface. Functionally, in migrating cells CD13 accumulates at the leading edge and co-localizes with IQGAP1 to regulate cell migration and adhesion. In conclusion, CD13 interacts with FAK-Src to control proper localization of focal adhesion proteins regulating focal adhesion turnover and associates with active ARF6-IQGAP1 to promote b1 integrin recycling and cell motility.
Epigallocatechin-3-Gallate suppresses neutrophil accumulation and the IL-1 beta pathway in a transgenic zebrafish model of inflammation
Authors: Nguyen Thao , Payan Brittany A. , Bondesson Maria , Mohan Chandra
Published: January 2023

Background

The zebrafish represents the simplest and most convenient vertebrate model for undertaking large screens because of its small size, rapid reproduction time and transparency of the body. Here, we adopt a well-established model of inflammation in zebrafish to assess the impact of Epigallocatechin-3-Gallate (EGCG), the main catechin in green tea.

Method

Tg (MPX:GFP) or Tg (MPX:mCHERRY) transgenic zebrafish were used, bearing GFP- or mCherry-tagged neutrophils, respectively. 4dpf embryos were anesthetized, and inflammation was induced by sterile tailfin-resection. The inflamed groups were separated into those that were non-treated or exposed to EGCG from 0 – 600 uM for 6 hours. 10 fish per group were then mounted alive in methyl cellulose for wide-field fluorescence imaging and analysis was done using ImageJ. Animation of neutrophil migration or accumulation was recorded using an inverted fluorescence confocal microscope till 6-hours after resection. To examine the mechanistic basis of EGCG’s effect, 37 zebrafish inflammatory genes were studied using qPCR.

Results

The static accumulation of neutrophils at the resection site was highly elevated after wounding (n=32, p<0.0001), and this accumulation was reduced significantly by 31% and 43%, respectively, after exposure to EGCG 300 uM (p<0.05) and 600 uM (p<0.01). Dynamic neutrophil tracking studies are in progress. qPCR studies revealed 6 signature genes in the IL-1 beta pathway to be significantly down-regulated at least 2-fold after EGCG treatment.

Conclusion

EGCG suppresses inflammation in zebrafish through similar biochemical pathways as in other species. This novel animal model allows facile screening of inflammatory modulators in real-time.
Engineering antigen-specific T cells from human pluripotent stem cells
Authors: Vo Linda Thuy , Nguyen David H. , Roth Theodore , Marson Alexander , Daley George Q. , Bluestone Jeffrey A.
Published: January 2023

Immunotherapy has rapidly emerged as a promising treatment for many different types of cancer. Ongoing clinical trials continue to validate the efficacy of T cell-based cancer immunotherapy, such as chimeric antigen receptor (CAR) T cells, as a valid therapeutic approach. As such, new sources of both autologous and “off the shelf” T cells available in large qualities would be invaluable to widespread application of these treatments. Human induced pluripotent stem cells (iPSCs) represent a potentially inexhaustible source of clinically useful cell types. Previous studies have demonstrated the feasibility of generating T cells from iPSCs, but yields have been low due to the inefficiency of lymphoid differentiation. Here, we report a cell engineering platform capable of generating large numbers of T cells from iPSCs using a combination of transcription factors and chromatin modifiers. We initially performed a loss-of-function screen to identify regulators that enhance lymphoid potential in iPSC-derived hematopoietic progenitors. We found that repression of the Polycomb group protein EZH1 uniquely enhanced multilineage hematopoietic output from human iPSCs in vitro and in vivo. EZH1 directly modulates the expression of HSC and lymphoid genes in iPSC-derived hematopoietic progenitors and its knockdown enhances chromatin accessibility of these loci. Coupled with CAR and genome editing technologies, iPSC-derived CAR T cells release effector cytokines and mediate antigen-specific anti-tumor responses. Future work is aimed at improving antigen-specificity of iPSC-derived T cells. Together, this work highlights the utility of chromatin modifiers as cell engineering targets to enhance blood differentiation from human iPSCs.
Discrete roles and bifurcation of PTEN signaling and mTORC1-mediated anabolic metabolism underlie IL-7-driven B lymphopoiesis
Authors: Zeng Hu , Su Wei , Shi Hao , Chi Hongbo
Published: January 2023

IL-7 drives early B lymphopoiesis, but the underlying molecular circuits remain poorly understood, especially how Stat5-dependent and independent pathways contribute to this process. Combining transcriptome and proteome analyses and mouse genetic models, we show that IL-7 promotes anabolic metabolism and biosynthetic programs in pro-B cells. IL-7-mediated mTORC1 activation supported cell proliferation and metabolism in a Stat5-independent, Myc-dependent manner, but was largely dispensable for cell survival or Rag1 and Rag2 gene expression. mTORC1 was also required for Myc-driven lymphomagenesis. Importantly, PI3K and mTORC1 had discrete effects on Stat5 signaling and independently controlled B cell development. In fact, PI3K was actively suppressed by PTEN in pro-B cells to ensure proper IL-7R expression, Stat5 activation, heavy chain rearrangement and survival, suggesting the unexpected bifurcation of the classical PI3K-mTOR signaling. Altogether, our integrative analyses establish IL-7R-mTORC1-Myc and PTEN-mediated PI3K suppression as discrete signaling axes driving B cell development, with differential effects on IL-7R-Stat5 signaling.
Transgenic expression of human IL15 in NOD-scid IL2rg
null (NSG) mice enhances the development and survival of functional human NK cells
Authors: Brehm Michael A. , Aryee Ken-Edwin , Bruzenksi Lisa , Greiner Dale L. , Shultz Leonard D. , Keck James
Published: January 2023

Human NK cells have critical roles in tumor surveillance and in immunoregulation within the tumor microenvironment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function. Humanized mice are a powerful alternative to study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However human NK cell development and survival in humanized mice are severely limited. Based on previous studies showing the importance of human IL15 for NK cell development, we established NSG mice that constitutively expresses human IL15 (7.1 ± 0.3 pg/ml). To evaluate human NK cell development, 8 to 12-week-old NSG and NSG-Tg(Hu-IL15) mice were irradiated and injected intravenously with CD34+ HSC derived from umbilical cord blood. Levels of circulating human CD45+ cells, T cells and B cells were similar between the HSC-engrafted NSG-Tg(Hu-IL15) and NSG mice. Significantly higher levels of human CD56+ NK cells were found in NSG-Tg(Hu-IL15) mice as compared to NSG mice at all time points and in all tissues tested. A higher proportion of human CD56+ NK cells recovered from the blood and spleen of NSG-Tg(Hu-IL15) mice expressed granzyme A, granzyme B and perforin as compared to NK cells from NSG mice, suggesting that the NK cells were functional. Moreover, human NK cells enriched from the NSG-Tg(Hu-IL15) mice lysed K562 cells in an in vitro cytotoxicity assay. These data demonstrate that HSC-engrafted NSG mice expressing human IL15 support enhanced development of functional human NK cells and suggest that HSC-engrafted NSG-Tg(Hu-IL15) mice are a powerful model to study human NK cells.
Delineating the ILC Development Pathway
Authors: Kasal Darshan , Bendelac Albert S.
Published: January 2023

We investigated the hierarchical relationship of previously identified innate lymphoid cell (ILC) precursor populations in the murine bone marrow (BM) and fetal liver (FL) through the use of novel transcription factor reporter combinations and single cell potential analysis. We found that the common helper ILC progenitor (CHILP) comprises a heterogeneous population of ILC Precursors (ILCPs) and LTi precursors (LTiP), but contains no common progenitor to either as originally proposed. Remarkably, the Early ILC Progenitor (EILP), a population that is marked by the transient loss of IL-7R expression and arises prior to the ILCP and LTiP, is heterogeneous, containing both ILCP-like and LTiP-like cells with limited potential. Taken together, these results suggest that the point of helper ILC and LTi bifurcation is situated earlier than previously supposed, potentially from the EILP.
MHCII+ Macrophages in Peritoneal and Pleural Cavities Require IRF4 for Development from Circulating Monocytes
Authors: Kim Kiwook , Williams Jesse W. , Wang Ya-Ting , Ivanov Stoyan , Gilfillan Susan , Colonna Marco , Virgin Herbert W. , Gautier Emmanuel L. , Randolph Gwendalyn J.
Published: January 2023

Resident macrophages in peritoenal and pleural cavities exist as two distinct populations: F4/80+ICAM2+macrophages and MHCII+ macrophages. F4/80+ ICAM2+macrophages are derived from CX3CR1+embryonic precursors, and maitained by the transcription factor Gata6. However, the origin and regulating factors that maintain MHCII+ macrophages remain unknown. Here, we show that the MHCII+ macrophages arise postnatally from monocytes, which continuously replenish this subset through adulthood. Gene expression analysis identified distinct surface markers such as CD226 and revealed that the transcription factor IRF4 was selectively expressed in these macrophages relative to other organs. Monocytes first enter peritoneal or pleural cavities to become MHC II+ cells that up-regulate CD226 later as they continue to mature. In the absence of IRF4 or after administration of oral antibiotics, MHCII+CD226−CD11c− monocyte-derived cells accumulated in peritoneal and pleural cavities, but CD11c+ CD226+ macrophages were lost. Thus, MHC II+ resident peritoneal and pleural macrophages are continuously replenished by blood monocytes recruited to the peritoneal and pleural cavities constitutively, starting after birth, where they require IRF4 and signals likely derived from the microbiome to fully differentiate.
Differentiation of hematopoietic stem and progenitor cells to NK cells in a stroma-free, serum-free culture system
Authors: Tabatabaei-Zavareh Nooshin , Fawcett Kent A. , Le Fevre Tim A. , Szilvassy Stephen J. , Thomas Terry E. , Eaves Allen C. , Wognum Albertus W.
Published: January 2023

Natural Killer (NK) cells play an important role in innate immunity by secreting proinflammatory cytokines and killing tumor cells and virus-infected cells. Human NK cells can be generated by culturing CD34+ hematopoietic stem and progenitor cells (HSPCs) with stromal cells and cytokines. The use of stroma (and serum) is, however, not desirable in clinical applications. We developed a culture method for generating large numbers of NK cells from purified CD34+ cord blood (CB) HSPCs in the absence of serum and stromal cells. CD34+ CB cells were isolated by EasySep magnetic separation, seeded into culture plates coated with a Notch ligand and cultured for two weeks in StemSpan Serum-Free Expansion Medium (SFEM II) supplemented with SCF, TPO, Flt3L and IL-7. These conditions promote expansion of HSPCs and their differentiation into CD7+CD5+ lymphoid progenitors. Cells were then transferred to non-coated plates and cultured for two more weeks in StemSpan SFEM II supplemented with SCF, Flt3L, IL-7, IL-15 and a small molecule, UM729, to promote differentiation into NK cells. After culture, on average 76% (range: 54–90%, n=12) of cells expressed the NK cell marker CD56 and 58% (range: 25–80%) coexpressed CD56 and NKp46 (a NK cell activating receptor). A more mature CD56+CD16+ NK cell subset also emerged in these cultures (5%, range: 1–11%). The yield of CD56+ NK cells was ~13,000 (600–41,000) per initial CD34+ cell. The NK cells were functional and able to kill K562 target cells. These results show that HSPCs can expand and differentiate into NK cells under stroma- and serum-free culture conditions. This novel culture system will be useful for studies directed toward the development of cancer immunotherapies where large numbers of NK cells are needed.
Lymphoid biased hematopoietic stem cells acquire a myeloid pattern of gene expression with age.
Authors: Kong Ying , Pioli Peter D. , Montecino-Rodriguez Encarnacion , Casero David , Dorshkind Kenneth
Published: January 2023

Lymphocyte development declines with age, which in turn contributes to the reduced replenishment of naïve lymphocytes in secondary lymphoid organs. An impaired ability of old hematopoietic stem cells (HSCs) to generate lymphoid progeny is thought to contribute to this attenuation of lymphopoiesis. It is now recognized that the HSC compartment is heterogeneous and includes lymphoid biased (Ly-HSCs) and myeloid biased (My-HSCs) stem cells. Previous studies have demonstrated that the frequency of Ly-HSCs is reduced with age, and this observation has resulted in the formulation of a model of stem cell aging which proposes that the age-related decline in lymphocyte development is due to a reduction in the number of these lymphoid biased precursors. We now show that when total HSC number is taken into account, Ly-HSCs do not decline in number with age and that compartment size is maintained in old mice. In order to obtain insights into why lymphocyte development is reduced with age, we compared the transcriptomes of young and old Ly-HSCs. We found that 371 genes were differentially expressed between young and old Ly-HSCs, and Gene Ontology analysis revealed that multiple processes that included regulation of apoptosis, chromatin remodeling, cytoskeleton organization, and cell cycle progression were altered by aging. We also transcriptionally profiled young and old My-HSCs and analysis of the data unexpectedly revealed that the transcriptomes of old Ly-HSCs resembled that of old My-HSCs. Based on these results, we propose a revised model of HSC aging in which altered patterns of gene expression that occur in aging Ly-HSCs, rather than a reduction in their number, contribute to declines in lymphocyte production in old individuals.
Paneth cells regulate allergic sensitization in the gastrointestinal tract
Authors: Kim Eunsoo , Burnett Drew J. , Rudinsky Adam J. , Mori-Akiyama Yuko , Cormet-Boyaka Estelle , Boyaka Prosper N.
Published: January 2023

The gut immune homeostasis plays a major role in controlling sensitization to food allergens and the development of allergic responses in the gastrointestinal tract, but also to skin or lung. Epithelial cells sensing of commensal microbes is crucial for the maturation of the gut immune system and antibiotic treatment during early life is believed to have contributed to the increased incidence of food allergy. Paneth cells secrete antimicrobial products in the crypts of the small intestine, but their role in food allergic remains elusive. We found that SOX9ΔIEC mice, which lack of Paneth cells, display dysbiosis with increased microbial diversity and the presence of a large number of unclassified bacterial species. Upon oral sensitization with OVA as a model of food antigen in the presence of cholera toxin, SOX9ΔIEC mice developed similar levels of allergen-specific serum IgA and IgG responses to control wild-type mice with the exception of IgG3 responses, which were elevated in SOX9ΔIEC mice. Interestingly, SOX9ΔIEC mice exhibited significantly increased allergen-specific serum IgE responses, and exacerbated signs of allergic responses (i.e., swelling, hypothermia, impairment of lung functions) after nasal allergen challenge. Transplantation of fecal materials from SOX9ΔIEC mice was able to transfer the hyper-allergenic phenotype to control wild-type mice. Ongoing studies are assessing the metabolites present in the intestinal lumen of SOX9ΔIEC mice and their effect on intestinal epithelial cells and myeloid cells to elucidate the mechanisms by which impaired or defective anti-microbial functions of Paneth cells regulate allergic sensitization of the GI tract.
Development and validation of a novel method to evaluate differences in allergenicity among wheat genotypes
Authors: Gangur Venugopal , Gao Haoran , Jin Yining , Ng Perry KW. , Olson Eric
Published: January 2023

Wheat allergy is one of the major food allergies that is growing at an alarming rate for reasons that are not completely understood. Variation in allergenicity among various wheat genotypes is not well studied. Here we sought to develop a novel method to evaluate the relative allergenicity among wheat genotypes. Groups of Balb/c mice weaned onto a plant-free diet received four IP injections with AABB or AABBDD wheat salt-soluble protein extract and alum. After confirming IgE responses and clinical sensitization for anaphylaxis, mice were boosted repeatedly over a 6-month period. Bi-weekly blood samples were collected and pooled to create AABB and AABBDD plasma banks. Using this plasma, IgE inhibition ELISA systems were optimized and used to determine the relative allergenicity. In the AABB system, AABBDD abolished IgE binding by 81+/−5%, and DD SSWP abolished it by 69+/−4%. In the AABBDD system, AABB abolished IgE binding by 76+/−4% and DD abolished it by 80+/−8%. In the AABB system, other genotypes were less allergenic by 7% (AABBDD) and 21% (DD). In the AABBDD system, other genotypes were less allergenic by 12% (DD) and 17% (AABB).
Oral electrophilic nitro-fatty acids inhibit cutaneous immune responses
Authors: Wang Peng , Killeen Meaghan , Mathers Alicia R.
Published: January 2023

Allergic contact dermatitis (ACD) is a common inflammatory skin disease affecting as many as 15–20% of the population. ACD results from a delayed-type hypersensitivity reaction occurring in the skin upon re-exposure to the offending hapten and mediated by the activation of hapten-specific T cells. An increasing body of literature demonstrates that electrophilic nitro-fatty acids (NO2-FAs) exert potent anti-inflammatory actions by modulating metabolic and inflammatory pathways. Therefore, we evaluated the potential regulatory effects of NO2-FAs on ACD by a well-established model of contact hypersensitivity in mice utilizing 2,4-dinitrofluorobenzene (DNFB) as the hapten. Contrary to our hypothesis, topical pretreatment of nitro-oleic acid (OA-NO2) prior to DNFB application potentiated ACD. Given that ACD is mediated by adaptive immunity, topical administration of OA-NO2 may have an adverse effect on keratinocytes thereby limiting OA-NO2’s potential to systemically suppress the adaptive immune response. Therefore, we next compared the effect of oral OA-NO2 on ACD. In contrast to topical administration, oral OA-NO2 dose-dependently suppressed ACD. Moreover, oral OA-NO2 downregulated the production of cutaneous inflammatory cytokines. Interestingly, in vitro experiments demonstrate that OA-NO2 leads to a dose-dependent inhibition of keratinocyte proliferation and expression of IL-1β, TNF-α, and IL-6. Overall, these data emphasize the complexity of the cutaneous microenvironment. A better understanding of the immunoregulatory effects of NO2-FAs in the skin microenvironment will contribute to therapeutics and to our understanding of the pathophysiology of inflammatory skin diseases, such as ACD and psoriasis.
The effect of the common food additive tBHQ in OVA-elicited food allergy
Authors: Jin Yining , Bursley Jenna , Dover Heather , Gangur Venugopal , Rockwell Cheryl Elizabeth
Published: January 2023

Immune-mediated adverse reactions to food allergens are rising at an alarming rate on a global scale for reasons that are not completely understood. Although many factors such as microbiota, diet, obesity, and environmental chemical exposure have been proposed to contribute to this marked increase, the identification of specific causative factors has remained elusive. Here we tested the hypothesis that a common food additive, tert-butylhydroquinone (tBHQ), at concentrations relevant to human exposure, exacerbates allergic response in ovalbumin (OVA) sensitized mice. Female Balb/c mice (4 weeks old) were maintained on control diet or diet with 0.001% tBHQ for the duration of these studies. The mice were exposed to OVA once per week for 4 weeks during the sensitization phase. Sensitization to OVA was assessed by the rise in OVA-specific IgE and IgG1. Upon oral challenge, mice were monitored for hypothermia shock response (HSR) and mast cell protease (mMCP)-1 response. Although sensitization with OVA elicited a robust OVA-specific IgE antibody response in both the control and tBHQ diet groups, IgE levels were markedly higher in the mice on the tBHQ diet as compared to control diet. Likewise, in response to OVA challenge, a more marked decrease in body temperature was observed in the mice on the tBHQ diet. Furthermore, the mMCP-1 response to OVA challenge was 3-fold greater in the mice on the tBHQ diet as compared to control diet. Taken together, we report low concentrations of the food additive tBHQ promote OVA sensitization and exacerbate anaphylactic response to OVA challenge in a mouse model of food allergy.
Development of a novel mouse model to study tick-borne onset of red meat allergy
Authors: Chandrasekhar Jessica , Cox Kelly , Loo William , Tung Ken , Erickson Loren
Published: January 2023

Bites from Amblyomma americanum, also known as the lone star tick, cause a life-threatening food allergy that induces IgE-mediated allergic reactions in affected individuals after eating dietary ‘red’ meat such as beef, pork, and lamb. Currently, there is no treatment to prevent or cure red meat allergy. Thus, establishing how lone star ticks cause red meat allergy is important for protecting individuals against this allergy. Our lab has successfully developed a novel mouse model of hypersensitivity to tick exposure to better understand how bites from the lone star tick sensitize the host to produce and maintain allergic IgE. Using this model, we identified increased T follicular helper cell and germinal center B cell responses and elevated serum titers of tick-specific IgE and IgG1 in mice exposed to lone star ticks subcutaneously. Furthermore, these mice generate a hypersensitivity response after oral challenge with red meat, as measured by basophil activation. Finally, we found that both the formation of inflammatory skin lesions at the site of tick exposure and the production of IgE were dependent on CD4+ helper T cells. These findings suggest that contained within the tick are factors that markedly influence priming of CD4+ T cells leading to the production of allergic IgE. Based on these data, we propose that manipulation of the factors within lone star ticks that drive CD4+ helper T cell activity could be used locally in the skin at the tick bite site to prevent the onset of meat allergy and systemically to stop an allergic reaction from progressing to a state of severe hypersensitivity.
Arid5a orchestrates IL-17-mediated inflammation through post-transcriptional control of mRNA
Authors: Amatya Nilesh , Cruz J. Agustin , Aggor Felix , Garg Abhishek , Berman Andrea J. , Atasoy Ulus , Gaffen Sarah L.
Published: January 2023

Interleukin-17A (IL-17) is a proinflammatory cytokine essential for clearance of microbial pathogens, but conversely drives immunopathology in autoimmune settings. Surprisingly, very little is known about IL-17 receptor-mediated signaling pathway. Although IL-17 is consistently found to be a modest activator of gene transcription in experimental settings, its biological impact in vivo is profound. In addition to de novo transcription, IL-17 also upregulates inflammatory gene expression by stabilizing their transcripts. mRNA stabilization is a central, but still poorly understood facet of IL-17 signaling. Here, we report an RNA binding protein Arid5a (AT-rich interactive domain-containing protein 5A) as a novel mediator of IL-17-induced stabilization of inflammatory mRNAs. IL-17 stimulation upregulated Arid5a expression, whereupon Arid5a was rapidly recruited to TRAF2. Arid5a stabilized multiple IL-17-induced cytokine mRNA transcripts, including Il6, Cxcl1 and Cxcl5, by binding to their 3′ untranslated regions (UTR). In some cases, Arid5a counteracted mRNA degradation mediated by the endoribonuclease MCPIP1 (Regnase-1). Additionally, Arid5a enhanced IL-17-induced expression of the transcription factors IκBz (Nfkbiz) and C/EBPβ (Cebpb), which could then transactivate IL-17-dependent promoters. Arid5a bound to Nfkbiz or Cebpb transcripts, but surprisingly did not detectably affect total Nfkbiz or Cebpb mRNA levels. Rather, Arid5a facilitated translation of IkBz and C/EBPb. Thus, Arid5a orchestrates a feed-forward amplification loop within the IL-17 pathway, revealing a new function for Arid5a in controlling translation and demonstrating a role in controlling downstream IL-17 signaling.
Type II Cytokines Guide Early Thymic Progenitor Lineage Choice and Influence Negative Selection of Myelin-Reactive T cells
Authors: Cattin-Roy Alexis N. , Barik Subhasis , Miller Mindy M. , Ukah Tobechukwu Kenneth , Zaghouani Habib
Published: January 2023

IL-4 and IL-13 are considered anti-inflammatory cytokines and have been effective in sustaining peripheral T cell tolerance and modulation of experimental allergic encephalomyelitis (EAE), an animal model for human multiple sclerosis. Herein, evidence is presented suggesting that both IL-4 and IL-13 contribute to central tolerance of myelin-reactive T cells and restrain the development of EAE. Indeed, IL-4 and IL-13 signaling through their IL-4Rα/IL-13Rα1 heteroreceptor (HR) activates STAT6 transcription factor to inhibit the potential of early thymic progenitors (ETPs) to mature to T cells. In parallel, the cytokines trigger phosphorylation of STAT1 which promote ETPs to give rise to myeloid cells most of which belong to the CD11c+CD8α+ dendritic cell (DC) subset which reside in the thymus. These ETP-derived thymic resident DCs are able to function as antigen presenting cells (APCs) and are proven effective in supporting central tolerance of myelin-reactive T cells in vivo leading to resistance against the development of EAE. Since IL-4 and IL-13 are usually induced in response to parasitic infection or exposure to allergen, the findings are interpreted to mean that these cytokines provide a link between environmental triggers and central tolerance, a concept that bodes well with theories of the hygiene hypothesis and the discrepancies in autoimmune diseases incidence among undeveloped and western countries
MIR205HG is a Long Noncoding RNA with Distinct Functions in the Thymus versus the Anterior Pituitary
Authors: van Oers Nicolai S. , Du Qiumei , Dozmorov Igor , Raj Prithvi , Molina Erika , de la Morena M. Teresa , Mendell Joshua T. , Cleaver Ondine
Published: January 2023

MicroRNAs (miRNAs) are processed from primary RNA transcripts (pri-miRNAs) encoded in host genes. Several miRNAs including miR-205 are present within long noncoding RNAs (lncRNAs). MiR-205 is an epithelial-specific miRNA that supports thymopoiesis by positively regulating Forkhead Box N1 (Foxn1) expression. We assessed whether the host gene for miR-205, MIR205HG, has independent functions as a lncRNA. The more severe stress-induced thymic atrophy reported in miR-205-deficient mice is also evident in MIR205HG knockout lines. However, MIR205HG knockouts have a small stature phenotype. A new set of miR-205KI mice did not have this phenotype. The smaller mouse size of the MIR205HG null animals is partly a consequence of reduced levels of endocrine hormones produced by the anterior pituitary. In addition, the MIR205HG null animals had abnormal development of the lacrimal and harderian glands that produce eye secretions. Transcriptome analyses revealed that MIR205HG regulates gene expression in the anterior pituitary unlike miR-205. In contrast, transcripts regulated by miR-205 and MIR205HG in the epithelial cells of the skin and thymus overlap significantly. These data indicate that MIR205HG has a specific function as a lncRNA in the anterior pituitary in addition to its primary role as the host gene for miR-205 in thymic epithelial cells.
The role CTCF and transcription in sculpting the TCRβ repertoire
Authors: Nganga Vincent K. , Majumder kinjal , Mishra Bikash , Kyle Katherine E. , Oltz Eugene M.
Published: January 2023

Primary Tcrb diversification is preceded by distinct changes in chromatin conformation and transcription during the early stages of T-cell development. However, the mechanisms by which these changes occur and how they shape the repertoire remain unclear. We have shown that distal and proximal Trbv segments are distinctly regulated. For instance, a CTCF binding site, known as 5′ PC, mediates long-range interactions that bring distal Trbvs into spatial proximity with the DβJβ segments. The 5′PC and several other CTCF sites in the 3′ end of the locus are oriented in a manner that we predict to facilitate specific contact, and thus recombination, between groups of Trbv segments the DβJβ segments. To specifically test this, we individually deleted key CTCF sites from the mouse Tcrb locus and sequenced Trbv gene usage in DN thymocytes. We find that loss of the 5′ PC results in a repertoire skewed against distal Trbv segments but favors proximal Trbv usage. Deletion of another CTCF site at the 3′ end of the locus, near the DβJβ clusters, does not significantly alter Trbv repertoire, presumably due to functional redundancy with another set of DβJβ-flanking CTCF sites. To test how transcriptional state affects the Trbv repertoire, we deleted an area spanning the Trbv12-2 and Trbv13-2 segments, which displays the highest levels of transcriptional and chromatin activation. We find that Trbv12/13 deletion enhances the usage of neighboring Trbv genes in the primary repertoire. Collectively, these data show that specific CTCF sites and regional transcription, at least in part, shape Tcrb repertoire.