bluuebunny/crossref_metadata_2025 huggingface dataset:
Treg-specific ablation of HuR results in systemic autoimmunity associated with decreased TLR4 activation and IL-1β production
Authors: Atasoy Ulus , Fattahi Fatemeh , Ellis Jason S , Socha Sarah , Bahleda Kristin
Published: January 2023

The RNA-binding protein HuR (Elavl1) plays a critical role in T cell activation and function by regulating the stability and transport of its mRNA targets. To study the role of HuR in regulatory T cell (Treg) development and function, we generated the Foxp3YFP-Cre HuRfl/fl mouse in which HuR is selectively deleted in CD4+ Foxp3+ cells, a lineage with crucial roles in self-tolerance and immune homeostasis. Foxp3+ Treg-specific HuR deletion results in abnormal phenotypic features (scurfy-like mice, hair loss, stippled/banded tails, significant failure to thrive, splenomegaly and multi-organ inflammation, including lung and lymphoid organs), likely the result of reduced Treg function, a phenotype similar to human IPEX syndrome (an autoimmune disorder). Unexpectedly, our RNAseq data showed reduced levels of TLR4, IL-1β, IL-18 and NLRP3 mRNA expression in HuR deficient Tregs (Foxp3YFP-Cre HuRfl/fl). In control WT mice, in vitro LPS treatment of Tregs induced increased expression of TLR4 and IL-1β production, associated with increased nucleocytoplasmic shuttling of HuR. In contrast, the expression of TLR4 and IL-1β was significantly decreased in Tregs from Foxp3YFP-Cre HuRfl/fl mice. Homozygous Foxp3YFP-Cre HuRfl/fl mice had statistically significant reductions in Treg numbers and increased frequency of activated effector T cells. We were also able to detect IL-1β secretion from WT Tregs in vitro following LPS treatment, which was amplified by the addition of DAMP agonists (e.g., ATP or Nigericin). In contrast, HuR KO Tregs had decreases in IL-1β secretion. Together, our data show that HuR is critical for normal CD4+ Foxp3+ regulatory T cell function.

Supported by grants from NIH (R01 AI080870 and R21 AI079341).
Distinct super-enhancer elements differentially control Il2ra gene expression in a cell-type specific fashion
Authors: Spolski Rosanne , Li Peng , Chandra Vivek , Shin Boyoung , Liu Chengyu , Oh Janksuk , West Erin , Vijayanand Pandurangan , Rothenberg Ellen , Leonard Warren
Published: January 2023

Expression of the IL-2 receptor a-chain (IL-2Ra, also known as CD25) is tightly regulated during T cell development and in mature lymphoid populations. IL-2Ra is constitutively expressed on double negative DN2/DN3 thymocytes and regulatory T cells, but its expression is inducible on mature T and natural killer (NK) cells. The Il2ra gene is regulated in part by a super-enhancer that is extensively bound by STAT5, but whether the super-enhancer elements are differentially used to control the cell-specific expression of Il2ra in distinct cell populations is unknown. Here, we generated a comprehensive series of mutant mice harboring deletion of individual or multiple elements within this super-enhancer. Strikingly, deletions in the upstream super-enhancer region dominantly affected CD25 expression on DN2/DN3 thymocytes and Treg cells, whereas deleting an intronic region primarily decreased IL-2-induced CD25 expression on peripheral CD4+ and CD8+ T and NK cells. Thus, distinct elements control constitutive versus inducible expression of the Il2ra gene in a cell-type-specific manner rather than behaving as simple enhancers whose activity is additive/cooperative, as has been observed for the a-globin and Wap super-enhancers. Moreover, the intronic super-enhancer region was critical for chromatin looping, and deletion of this region altered super-enhancer structure throughout the locus. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating ways to manipulate CD25 expression in a cell-type specific fashion, an area of importance in autoimmune and other disease states.
TRAF3 enhances type I interferon (IFN I) receptor signaling in T cells through modulation of the phosphatase PTPN22
Authors: Hornick Emma , Wallis Alicia Michelle , Bishop Gail A
Published: January 2023

Type I interferons (IFN I) are among the most powerful tools host cells deploy against intracellular pathogens. Their effectiveness is due to both the rapid, directly anti-viral effects of IFN-stimulated gene products, and to the effects of IFN I on responding immune cells. IFN I signaling through its receptor, IFNAR, is tightly regulated at multiple steps in the signaling cascade. Despite the distinct phenotypic changes induced by IFN I in different immune cell types, few studies have addressed the possibility of cell type-specific differences in regulation. In this study, we show that TNFR-associated factor 3 (TRAF3) promotes robust IFNAR signaling specifically in CD4+ T cells by preventing recruitment of the negative regulatory phosphatase PTPN22 to the IFNAR complex. The balance between signals through IFNAR and other differentiating cytokine receptors strongly influences CD4+ T cell differentiation and function during infections. Our work introduces TRAF3 and PTPN22 as newly appreciated regulators key to influencing CD4+ T cell activation by IFN I.

Supported by R01 AI123107, T32 AI007260, T32 HL007344, Department of Veterans Affairs
Interferon kappa (IFNk) in keratinocytes is critical for normal wound repair and is decreased in diabetic wounds
Authors: Wolf Sonya , Audu Christopher O , Joshi Amrita D , denDekker Aaron , Melvin William J , Xing Xianying , Wasikowski Rachael , Tsoi Lam C , Gudjonsson Johann E , Kahlenberg J Michelle , Gallagher Katherine A
Published: January 2023

Wound repair following acute injury requires a coordinated inflammatory response. Type I interferon (IFN) signaling is important for regulating the inflammatory response post- skin injury. IFN kappa (IFNk), a type I IFN, has recently been found to drive skin inflammation in lupus and psoriasis; however, the role of IFNk in the context of normal or dysregulated wound healing is unclear. Thus, this project explores the role of IFNk in wound repair. Here, we found that IFNk expression is upregulated in keratinocytes early post-injury and is essential for normal tissue repair. Under diabetic conditions, IFNk was decreased in wound keratinocytes, and early inflammation was impaired. Further, we found that the histone methyltransferase mixed lineage leukemia protein-1(MLL1) regulates IFNk expression in diabetic wound keratinocytes via an H3K4me3 mediated mechanism. Using a series of in vivo studies with a genetically engineered mouse model(Mll1fl/fl K14cre−) and human wound tissues from patients with T2D, we demonstrate that MLL1 controls wound keratinocyte-mediated IFNk and MLL1 is decreased in T2D keratinocytes. Importantly, we find the administration of IFNk early following injury improves diabetic tissue repair. These findings have significant implications for understanding the complex role type I interferons play in keratinocytes in normal and diabetic wound healing. Additionally, they suggest IFNk may be a viable therapeutic target to improve diabetic wound repair.

Supported by MNORC P30-DK089503
Loss of Ten-Eleven Translocation 2 (TET2) reduces CCR6 expression and increases B1 B cell number in the peritoneal cavity
Authors: Dennis Emily A , Bontha Sai Vineela , Marshall Melissa A , Srikakulapu Prasad , Garmey James , Blackburn Cassidy M R , McNamara Coleen A
Published: January 2023

TET2 is an evolutionarily conserved dioxygenase that catalyzes the conversion of 5-methyl-cytosine to 5- hydroxymethyl-cytosine, promotes DNA demethylation and regulates transcription. TET2 has been reported to regulate germinal center formation and class switch recombination in B2 B cells, yet its role in B1 B cell subsets remains largely unexplored. To investigate the role of TET2 in B1 subsets, flow cytometry of cells from the peritoneal cavity (PEC), spleen and bone marrow of TET2−/− mice and wildtype littermate controls (n = 14/group) was performed. In addition, sort purified PEC B1a, B1b and B2 cells were analyzed for methylation status (n = 24) and RNA expression (n = 24). Results demonstrated increased B1a (p = 0.0162) and B1b (p = 0.0056) cells in TET2−/− mice in the PEC, their primary niche, but not in the spleen, while in the bone marrow only TET2−/− B1a cells were increased (p = 0.0021). RNAseq analysis of PEC B1a, B1b, and B2 cells from TET2−/− and wildtype mice revealed reduced chemokine receptor CCR6 expression in B1a (−4.879068141 fold change, 1. 8.03E-08 padj.) and B1b cells (−2.17044776 fold change, 0.34771255 padj.) from the TET2−/− mice. Further, methylation analysis showed significant hypermethylation of CCR6 in B1a and B1b TET2−/− cells compared to wildtype B1a and B1b cells. This was corroborated by gene pathway analysis of the RNAseq data which showed significant downregulation of chemotaxis and migration pathways in B1a and B1b TET2−/− cells. We conclude that loss of TET2 increases hypermethylation of CCR6 and suppresses CCR6 RNA expression in B1 cells which may impair trafficking out of the PEC to the spleen, but not necessarily the bone marrow, leading to PEC accumulation of B1 subtypes.

Supported by grants from the NIH: 1R01HL 136098-01 (McNamara, C PI), R01 HL141123 (McNamara, C PI), T32 HL007284 (Dennis, E)
The Effect of Various Stimulants on Cytokine Secretion Profiles in Freshly Isolated Peripheral Blood Mononuclear Cells (PBMC)
Authors: Lefavor Rebecca , Clarke Emer
Published: January 2023

Cytokines play critical roles in in cancer, in regulating autoimmune diseases and in chemically induced tissue damage repair. In response to antibody treatment or as a result of corona virus disease (COVID), a cytokine storm may occur where high levels of inflammatory cytokines are produced leading to organ damage. To understand how to regulate cytokine levels, primary cell in vitro assays may assess the effects of small molecule compounds or antibodies. We evaluated OKT3, Polyinosinic-polycytidylic acid (Poly I:C), Resiquimod, CpG oligodeoxynucleotides (CpG ODN), DynaBeads with aCD3/aCD28, Phytohemagglutinin (PHA)-M and lipoplolysaccharide (LPS) on cytokine secretion from 6 normal donors. PBMCs isolated from fresh blood using a density gradient medium were plated at 1 × 105 cells per well in the presence and absence of the test compounds for 48 hours. Supernatants were assessed for interleukin 2 (IL-2), tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ). The remaining cells were assessed for viability to evaluate cytotoxicity. Flow cytometric profiles of T cell subsets were determined. The data generated from the 6 normal donors varied greatly, though the effects of the stimulants followed the same trends. The Dynabeads (CD3/28) generated the highest values of IL-2, IFNγ and TNFα in all donors. OKT3 and PHA-M, Resiquimod and LPS generated lesser amounts of the cytokines whereas Poly I:C and CpG ODN did not induce these cytokines over background levels. Only PHA-M caused a decrease in cell viability. Additionally, the release of cytokines was not associated with activation of T cells. Cytokine release assays provide a robust readout with certain stimuli and may facilitate evaluation of novel test compounds for functional activity.
Sex differences in IL-3, IL-4, and IL-7 correlation with cholesterol and triglyceride levels in African Americans with high HbA1c
Authors: Kimbro K Sean , Wamble Ezekiel , Williams Ariel
Published: January 2023

Background

Nearly 30 million Americans have diabetes; majority are diagnosed with type 2 diabetes (T2D). Low grade inflammation is associated with T2D which influences a network of serum cytokines. IL-3, IL-4, and IL-7 play coordinated pathogenic roles in many inflammatory diseases including T2D and non-fatty liver disease. It has been previously reported high circulating glucose influences serum cytokine levels and cholesterol metabolism. However, further studies are needed to delineate the relationship between cytokine serum levels, cholesterol, and triglycerides metabolism.

Methods

A total of 232 serum samples were collected from African Americans: 161 women and 71 men. This group consists of 95 normal HbA1c and 106 high HbA1c participants. Most participants had a BMI > 30 (n=171). Cytokines were measured using multiplexing assays from Luminex X-MAP® technology.

Results

In the presence of high HbA1c, there is a trend increase in serum levels of IL-4 in both men and women. In women participants, IL-3 (R=−0.41, P=0.02) is inversely correlated to BMI, but not in men. IL-3 is positively correlated to VLDL cholesterol in women with normal HbA1c. HDL is positively correlated with IL-3 (R=0.20, P=0.04). VLDL is positively correlated with IL-4 (R=0.79, P=0.007), in men with normal HbA1c. Triglycerides were positively correlated to IL-4 (R=0.80, P=0.006) and IL-3 (R=0.56, P=0.05) in men with normal HbA1c.

Conclusion

The involvement of IL-3, IL-4, IL-7 are linked to cholesterol metabolism but are possibly regulated differently between sexes. The evidence of these correlations provides a basis of understanding that could be utilized to provide intervention to reduce the complications of T2D.

Supported by NIH NIMHD U54MD012392
IL-11 induces monocyte inflammasome activation and migration to the CNS in IL-11R+ cells in relapsing remitting multiple sclerosis (RRMS)
Authors: Seyedsadr Maryamsadat , Wang Yan , Alzoheiry Manal , Kasimoglu Ezgi , Hwang Daniel , Garifallou James , Miskin Dhanashri , Markovic-Plese Silva
Published: January 2023

Objective

To examine the effect of IL-11 on the transcriptome changes in the blood and cerebrospinal fluid (CSF)-derived cells from RRMS patients.

Methods

IL-11+ expressing cells in PBMC or CSF were characterized by flow cytometry (n=14 and 9 respectively). Single cell RNA sequencing (scRNAseq) was performed for IL-11R+ sorted peripheral blood-derived cells following IL-11 stimulation (1 hour) (n=3 RRMS patients) or CSF and blood samples (n=2 RRMS patients). The animal model of RRMS (RREAE) was used to test the therapeutic effect of anti-IL11 mAb.

Results

RRMS patients have an increased frequency of IL-11+ monocytes, IL-11+ and IL-11R+ CD4+ lymphocytes and IL-11R+ neutrophils in comparison to matched HCs. IL-11 expressing monocytes, CD4+ lymphocytes and neutrophils accumulate in the CSF of RRMS patients in comparison to paired blood samples. scRNAseq for IL-11R+ sorted cells following IL-11 stimulation, revealed the highest number of differentially expressed genes (DEGs) in classical monocytes with upregulation of NFKB1, NLRP3 and IL1B genes involved in the inflammasome activation. All CD4+ cell subsets had an increased expression of S100A8/9 genes involved in the inflammasome activation. ScRNAseq for IL-11R+-sorted cells from the CSF revealed that classical and intermediate monocytes upregulate multiple complement, inflammasome-induced (IL-18), and migratory genes. aIL-11 mAb in RREAE decreased clinical scores, CNS inflammatory infiltrates, demyelination, IL-11+ CD4+ cells and monocytes, and numbers of NFkBp65+, NLRP3+ and IL-1b+ monocytes within the CNS.

Conclusion

IL-11/IL-11R signaling in monocytes represents a therapeutic target in early RRMS.

The study was supported by NIH 1R01AI131238-01A1 and PA Cure SAP4100083100 grant.
The roles of IL-22 and its receptor in the regulation of inflammatory responses in the brain
Authors: Lee DaHae , Jo HyeJung , Go Cheolhyeon , Kim Yejin , Kang Jae Seung
Published: January 2023

Interleukin (IL)-22 is a potent mediator of inflammatory responses. The IL-22 receptor consists of the IL-22Rα and IL-10Rβ subunits. Previous studies have shown that IL-22Rα expression is restricted to non-hematopoietic cells in the skin, pancreas, intestine, liver, lung, and kidney. Although IL-22 is involved in the development of inflammatory responses, there have been no re-ports of its role in brain inflammation. Here, we used RT-PCR, western blotting, flow cytometry, immunohistochemical, and microarray analyses to examine the role of IL-22 and expression of IL-22Rα in the brain, using the microglial cell line, hippocampal neuronal cell line, and inflamed mouse brain tissue. Treatment of BV2 and HT22 cells with recombinant IL-22 increased the ex-pression levels of the pro-inflammatory cytokines IL-6 and TNF-α, as well as cyclooxygenase (COX)-2 and prostaglandin E2. We also found that the JNK and STAT3 signaling pathways play an important role in IL-22-mediated increases in inflammatory mediators. Microarray analyses revealed up-regulated expression of inflammation-related genes in IL-22-treated HT22 cells. Finally, we found that IL-22Rα is spontaneously expressed in the brain and is upregulated in in-flamed mouse brain. Overall, our results demonstrate that interaction of IL-22 with IL-22Rα plays a role in the development of inflammatory responses in the brain.

This study was funded by the NRF of Korea (No.: 2017R1A2B2010948, 2017R1A6A3A11032576, 2020R1C1C1009334)
Pancreatic Interleukin-22 Receptor Signaling is Critical in Maintaining Beta-Cell Insulin Production and is Hepatoprotective
Authors: Sajiir Haressh , Wong Kuan Yau , Mueller Alexandra , Keshvari Sahar , Wang Ran , Wiid Percival , Macdonald Graeme , Prins John , McGuckin Michael A , Hasnain Sumaira Z
Published: January 2023

We discovered that the cytokine interleukin-22 (IL-22) is an efficient natural inhibitor of cellular stress and exogenous IL-22 improved insulin quality in pancreatic β-cells in preclinical models of Type 2 diabetes. Importantly, IL-22 completely restored glucose tolerance, suppressed fasting hyperinsulinemia/hyperproinsulinemia, and restored insulin sensitivity in obese animals. Moreover, metabolic improvements were accompanied by significant improvements in circulating triglycerides, liver function (AST:ALT ratio) and a reduction in hepatic lipid accumulation. Whilst the IL-22 receptor, IL-22RA1 is highly expressed in the pancreas and liver, its endogenous role in these tissues remains elusive. To explore this, we generated tissue specific IL-22ra1 knockout mice lacking the receptor in pancreatic β-cells (Il-22ra1fl/fl x Ins2Cre: IL-22ra1β-cell−/−) and hepatocytes (Il-22ra1fl/fl x AlbCre: IL-22ra1Hep−/−). We assessed their metabolic phenotype including glycemic control, hepatic lipid accumulation, and hepatic markers of cellular stress, lipid, and glucose metabolism with age or with obesity. We discovered that IL-22ra1β-cell−/− animals developed hyperglycemia with age and had defective insulin secretion, which was exacerbated when on a high fat diet. Interestingly, they also had a significant increase in markers of hepatic inflammation and cellular stress. In conclusion we demonstrate, for the first time, that local endogenous IL-22 secretion in pancreatic β-cells maintains insulin biosynthesis. Moreover, our work highlights the importance of the pancreatic-β-cell-liver axis as the absence of pancreatic IL-22ra1 signaling leads to a marked increase in liver inflammation and cellular stress.
Perturbations of Marrow Stromal Cell function during Acute Inflammation
Authors: Matkins Victoria , Camacho Virginia , Hoang Ashley , Patel Sweta , Welner Robert S
Published: January 2023

Mesenchymal stromal cells (MSCs) reside in a complex network of blood cells and can differentiate into chondrocytes, osteoblasts, and adipocytes. MSCs differentiation process is tightly regulated through intrinsic and extrinsic signals within the marrow microenvironment, leading to transcriptional programming of balanced lineage output. Inflammatory insults to this system can occur through the upregulation of activating cytokines. Pro-inflammatory cytokines’ impact on the hematopoietic lineage is well-defined, but similar impacts to stromal cells was largely undefined. To delineate these changes, we studied two well-characterized forms of inflammation: polyinosinic-polycytidylic acid (Poly(I:C)), that activates IFNα, and Lipopolysaccharide (LPS), that promotes IFNγ. Prior studies of inflammatory conditions showed bone loss; therefore, we hypothesized that there would be an increase in the MSC pool to compensate for loss of bone maturation. Using flow cytometry and lineage-tracing models of stroma, we characterize the non-hematopoietic cells’ changes following inflammatory perturbation. Our data show an increase in MSCs with decreases in osteoblasts and adipocytes following stimulation. Furthermore, we used reverse-phase protein and cytokine arrays to map the signaling mechanisms and activated pathways. We found an increase in the NFκB pathway with an accompanying increase IL-1β. Targeting of the IL-1 pathway did not phenotypically reverse the stromal perturbations but corrected the MSCs functionally. Understanding these alterations within the bone marrow microenvironment during inflammation will allow for therapeutic interventions to mediate hematopoietic diseases such as sepsis or leukemia.

Supported by NIH R01 HL150078
Collaboration of therapeutic, systemic IL12p40 with locally released IL12p35 focuses immunity to the tumor microenvironment
Authors: Hill Elizabeth M , Gerber Allison N , Singh Nevil J
Published: January 2023

The cytokine IL-12 was an early candidate for cancer immunotherapy due to its role in stimulating the release of IFN-γ from T and NK cells to promote tumor clearance. Administering IL-12 therapeutically was challenging because systemic treatments led to excessive cytokine storm and inflammation. Developing more targeted ways to focus IL-12 activity to the tumor microenvironment is a significant priority. IL-12 is a heterodimer formed by the subunits, IL-12p35 (p35) and IL-12p40 (p40) which are canonically thought to be made together in the same cell. Although p40 can be released as a monomer, this is inert. We have shown that these p40 monomers however can combine extracellularly with p35 from stromal cells to form functional IL-12 and help control infection. Based on this newly described pathway, we hypothesized that treatment with recombinant p40 monomers would allow it to combine with tumor-released p35 and focus IL-12 formation to the local microenvironment. This would facilitate targeted tumor clearance while limiting systemic immunopathology. To test this hypothesis, we evaluated treatment with p40 monomers in mice challenged with a subcutaneous tumor (MB49). We found that monomeric 40 itself was inert – neither capable of inducing IFN-γ or inhibiting the activity of heterodimeric IL-12. Nevertheless, recombinant p40 monomers helped control tumor growth in p40-deficient mice and increased the amount of CD4 T cells infiltrating the tumor. This suggests that p40 can combine with p35 released in the tumor microenvironment to form functional IL-12. Our data provide evidence for an alternate pathway of IL-12 formation in vivo and suggest that this can be exploited therapeutically to avoid systemic inflammation.
Loss of ADAM17 from macrophages induces ST2+ T regulatory cells that limit obesity-induced metabolic inflammation in mice through changes in both membrane and soluble TNF
Authors: Martin Rebecca , Lownik Joseph Cornelius , Farrar Jared , Way Grayson , Zellner Matthew , Ni Bin , Celi Francesco , Conrad Daniel H
Published: January 2023

Obesity is known to contribute to changes in immune cells, cytokines and metabolism. Secreted TNF (sTNF) levels are directly correlated in obesity with weight gain and morbidity, as well as metabolic dysregulation. ADAM17 cleaves transmembrane TNF (mTNF) into its soluble form. This is well defined, but the mechanism behind the regulation of TNF, the source of TNF, as well as the downstream action of TNF is less clear. Our data showed that mice with ADAM17 deleted from the myeloid lineage (ADAM17LysM) had a significant metabolic advantage as illustrated by an increase in glucose tolerance and decrease in body weight. Immunophenotyping of the fatpad of these mice using high dimensional flow cytometry after high fat diet (HFD) feeding showed an increase in activated ST2+ T regulatory cells (Tregs). Based on these findings, we hypothesized that modulation of TNF on macrophages through loss of ADAM17, helps maintain a healthy metabolic state through recruitment and activation of ST2+ Treg cells that limit metabolic dysregulation. Next, we showed that the accumulation and activation of these Tregs occurred through two separate mechanistic actions of TNF. First, the loss of sTNF production from macrophages decreased soluble ST2 (sST2) production by adipocytes. This loss of sST2 augments ST2+ Treg recruitment and expansion in the fatpad due to increased bioavailable IL-33. These experiments were controlled using a myeloid-specific knockout of TNF. Second, utilizing TNFR blocking antibodies, we demonstrated that ST2+ Treg activation was dependent on macrophage mTNF acting through TNFR2.

Supported by grants from NIH (R56 AI139658 and R01 AI018697)
Characterization of a CLCF1 conditional knock-out mice model
Authors: Laplante Veronique , Rousseau Marine , Fajardo Ernesto , Pasquin Sarah , Lesage Sylvie , Gauchat Jean-François
Published: January 2023

The Cardiotrophin-like Cytokine Factor 1 (CLCF1) is a cytokine of the IL6 family with important pro-neurotrophic and immuno-modulating functions. However, the mechanisms behind CLCF1 activities and CLCF1 properties in pathological models remain poorly understood. This is partly due to a lack of tools for the study of CLCF1 functions in vivo. Indeed, the complete knock-out of CLCF1 in mice is lethal at P1: underdeveloped motor neurons of the face and jaw prevent the pups from suckling. To overcome this obstacle, we obtained a CLCF1 conditional knock-out mice model generated using the CRISPR-Cas9 technology. We then bred these mice with Vav-Cre mice to induce CLCF1 deletion in hematopoietic cells. We are now conducting immuno-phenotyping experiments of the main immune populations in those animals. Considering the activities of CLCF1 that were previously shown in vitro or in vivo using overexpression models, we hypothesize that the knock-out of CLCF1 in immune cells will lead to decreased numbers of B and myeloid cells. This new CLCF1 conditional knock-out mice model will be a potent tool to confirm and further study the activities, mechanisms, and pathological roles of CLCF1 in vivo.

Supported by grants from CIHR (Canadian Institutes of Health Research)
Notch signaling is required for generation of conventional type 1 dendritic cells from human induced pluripotent stem cells
Authors: Ito Fumito , Long Mark D , Kajihara Ryutaro , Matsueda Satoko , Oba Takaaki , Kanehira Kazunori , Liu Song , Makino Kenichi
Published: January 2023

Compelling evidence shows a critical role of conventional type 1 dendritic cells (cDC1) in T-cell mediated antitumor immunity; however, obtaining large numbers of cDC1 is difficult. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSC) that could provide an unlimited source of autologous cDC1. However, generation of cDC1 from human iPSC remains elusive. Here, we hypothesized that Notch signaling would facilitate generation of cDC1 from human iPSC. We found that human iPSC gave rise to CD141+ XCR1+ CLEC9A+ HLA-DR+ cells on OP9 feeder cells expressing the Notch ligand delta-like 1 (OP9-DL1) while the majority of iPSC-derived cells differentiated on OP9 cells were monocytic cells. Single-cell RNA sequencing analyses confirmed the presence of cDC1 as well as identified the heterogeneity of iPSC-derived CD1c+ cells; cDC2A, cDC2B, CD5+ cDC2, and DC3. Inhibition of Notch signaling during co-culture of iPSC-derived CD34+ hematopoietic progenitor cells with OP9-DL1 cells abrogated generation of cDC1, and made expression of CD5, CLEC4A, CLEC10A and CD163 in CD1c+ DC similar to those differentiated on OP9 cells. Notch-activated human iPSC-derived XCR1+ CLEC9A+ HLA-DR+ CD11c+ cells exhibited similar gene expression profile with peripheral blood cDC1, and phagocytotic, T-cell proliferative, and cytokine producing functions. Our study demonstrates that Notch signaling is required for the differentiation of cDC1-like cells, and facilitates generation of various cDC subsets from human iPSC. These findings provide insights into the future development of personalized treatment with unlimited numbers of autologous cDC1 from human iPSC.

This work was supported by National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park’s Flow and Image Cytometry, Bioinformatics, and Genomic Shared Resources. This work was supported by the Melanoma Research Alliance and the Sarcoma Foundation of America (F. Ito), Uehara Memorial Foundation (T. Oba), and National Cancer Institute (NCI) grant, U24CA232979 (M. Long and S. Liu), K08CA197966 and R01CA255240-01A1 (F. Ito).
Stress Induced Eosinophilpoiesis Contributes to Mortality after Pulmonary Resection
Authors: Mei Zhongcheng , Guo Yizhan , Li Dongge , Khalil May , Banerjee Anirban , Kreisel Daniel , Gelman Andrew , Krupnick Alexander
Published: January 2023

Factors affecting mortality after lung surgery remain poorly explored. We performed pulmonary lobar resections(removing □ 1/5 of the total pulmonary volume) from C57BL/6 mice and evaluated quantitative changes in various leucocytes in the remaining lung, blood and spleen. A nearly four-fold increase in eosinophils was evident by day-7 post resection with a gradual return to baseline by day 30. Evaluation of the bone marrow demonstrated no change in common myeloid, granulocyte-monocyte or eosinophil progenitors. However the number of early stage and mature eosinophils doubled after lobar resection suggesting that pulmonary surgery augmented maturation of eosinophils. Neutralization of IL-5 decreased overall eosinophil numbers but the relative differences between resting and post-resection mice remained. Further evaluation of bone marrow demonstrated an increase in IL-25, IL-6, IL-9, KC and MCP-1 after pulmonary resection. We next performed right pneumonectomies in saline or diphtheria toxin-treated iPHIL mice, where diphtheria toxin receptor expression is limited to eosinophils, and noted a 70% mortality in eosinophil sufficient vs. 20% mortality in eosinophil depleted mice (p=.005). Severe hypoxia was noted in eosinophils sufficient vs. deficient mice (pO2 of 54.3+/−5.7 vs. 83.6+/−11.63; p=.04) four hours prior to resection. Taken together out data demonstrate that pulmonary surgery leads to bone marrow-specific maturation of eosinophils and systemic eosinophilia which directly contributes to perioperative mortality. Thus defining factors which may disrupt this process offers the opportunity to improve outcomes after lung surgery.

Supported by PO1 AI116501, RO1 AI145108-01
Maternal immune activation (MIA) impairs neonatal lung ILC2 establishment, function, and airway hyperresponsiveness
Authors: Lopez Diego A , Griffin Aleah , Beaudin Anna E
Published: January 2023

We have recently shown that prenatal inflammation induced by maternal immune activation (MIA) imparts lasting changes to innate immunity by driving the inappropriate expansion and persistence of lymphoid-biased progenitors during fetal life. To test how perturbation during this critical window drives immune dysfunction, we examined underlying changes to lung type-2 innate lymphoid cells (ILC2s) and susceptibility to airway hyperresponsiveness following MIA. MIA induction via a single-low dose injection of poly (I:C) at mid-gestation altered the establishment of a fetal liver (FL) ILC-committed progenitor, increasing cell number and frequency both one- and three-days post MIA. MIA also enhanced lung ILC2 proliferation postnatally, resulting in a robustly expanded ILC2 compartment at postnatal day (P)14 that persisted into adulthood. Concomitant with a greater inflammatory profile, MIA-treated ILC2s were hyperactivated, producing more IL5 and IL13 in-vitro. Lung cytokine levels remained relatively static across early lung development in MIA or saline conditions, suggesting changes to ILC2 proliferation and hyperactivation are cell-intrinsic. Furthermore, hyperactivated ILC2s led to remodeling of the lung immune landscape as observed by expansion of B-, T-, NK-, NKT-cells, alveolar macrophages and eosinophils, but ILC2s were the first immune population to expand in cell number. Altogether, our data suggest MIA imparts lasting changes to lung immunity by perturbing the establishment of committed ILC progenitors in the FL, altering the production of neonatal lung ILC2s poised to hyperactively respond to secondary immune activation, potentially contributing to greater allergic airway hyperresponsiveness.

Supported by grants from NIH/NICHD (T32HD007491
Differential Response of Dendritic Cells and Macrophages to Signals from Airway Epithelial Cells in Humans: Changes with Age
Authors: Agrawal Anshu , Agrawal Sudhanshu , Monteiro Clarice
Published: January 2023

Advancing age significantly enhances the risk of respiratory diseases such as COPD, fibrosis etc. Immune response to respiratory viral infections is also impaired often leading to hospitalization. The underlying mechanisms are not well understood. Airway epithelial cells (AECs) and dendritic cells (DCs) and alveolar macrophages are the primary cells involved in respiratory immunity and tolerance. Since DCs and macrophages exist in close proximity to AECs, the function of both these cells is influenced by AECs. Using a 2D model to study the interaction of human primary bronchial epithelial cells (representative of AECs) with DCs we have recently demonstrated that AECs enhance the immune surveillance capacity of myeloid DCs (mDCs) from the circulation at homeostasis. Culture of mDCs with AECs resulted in the upregulation of pathogen recognition receptors (PRRs) and downstream signaling molecules. In contrast to circulatory mDCs, culture of macrophages with AECs led to a significant inhibition of LPS induced pro-inflammatory responses. More importantly, AECs induced the secretion of TGF-β and IL-10 from macrophages even in the absence of LPS stimulation. Thus, it seems AECs induce tolerance in macrophages but enhance the immunogenicity of mDCs suggesting a division of labor between DCs and macrophages in the respiratory mucosa. Investigation into the possible mechanisms for this differential response indicates that metabolites and growth factors from AECs may be responsible for inducing tolerance in macrophages and priming DCs. We also find that the crosstalk between AECs and macrophages and DCs is altered with age which may contribute to the increased susceptibility of elderly to respiratory diseases and infections.

This study was supported by grant from the NIH AG045216 (to AA), and from the National Center for Research Resources and the National Center for Advancing Translational Sciences # UL1 TR000153.
Tobacco smoke activated fibrogenic MARCKS/AXL complex promotes macrophage reprogramming and pulmonary fibrosis
Authors: Yang David Cheng , Zhang Jun , Li Ji-Min , Chu Chih-Wei , Hsu Ssu-Wei , Chen Ching-Hsien
Published: January 2023

Macrophages and tobacco smoke (TS) exposure have been demonstrated to play significant roles in modulating pulmonary fibrosis (PF). However, the mechanisms of how TS exposure modulates pro-fibrotic macrophage polarization and drives lung fibrosis is unclear. In our study, we investigated how TS modulates macrophage polarization and the functional consequences of this polarization. Multicolor flow cytometric data indicated that markers of M2 macrophage polarization were elevated in both human and mouse macrophage cells and tissues upon TS exposure. In addition, multiple primary lung fibroblast cells demonstrated elevated pro-fibrotic markers and aggressive phenotypes upon interacting with TS-exposed macrophage cells in a co-culture system. Elucidation of the signaling pathways activated by TS exposure through a receptor tyrosine kinase array screen revealed AXL receptor as a novel smoke-responsive molecule in macrophage cells. We noted elevated secretion of AXL ligand, Gas6, and AXL activity in TS exposed cells and tissues. Prior work had demonstrated an interaction between MARCKS, a smoke-responsive protein, and AXL in promoting a pro-fibrotic phenotype in lung fibroblasts. Similarly, we observed AXL activity positively correlated with MARCKS phosphorylation in macrophage cells. Pharmacologic and genetic targeting of the MARCKS/AXL signaling complex reduced M2 markers and profibrotic cytokine production in macrophages and reduced fibrotic changes in the co-culture model and in an animal model of smoke-mediated lung fibrosis. In all, our work suggests that the MARCKS/AXL fibrogenic complex is a potential target in attenuating macrophage activity in TS-mediated fibrosis.

Supported by grants from NIH/NHLBI (R01HL146802), DOD DCMRP/PRMRP (PR202411), and UCOP TRDRP (28IR-0061 and T31DT1849).
The role of CD45 in the regulation of B cell subset functions in Atherosclerosis
Authors: Ma Shelby , Moriarty Alina K , Keeter William Coles , Waseem Tayab , Zyskin Alexander , Gauronskas Philip , Mussbacher Marion , Galkina Elena
Published: January 2023

Atherosclerosis is an inflammatory disease of the large and medium size arteries characterized by deposition of oxidized lipids within the vessel. While specific self-antigens are not well characterized, data show that the immune response is involved in atherogenesis. The role of B cells in atherosclerosis is subset specific. FO B cells are proatherogenic, while B1 and MZ B cells serve as protective. B cell receptor signaling is important in B cell activation, differentiation, and functions, while important costimulatory molecules, like CD45, assist in initiating signal propagation. To date, it is unknown whether B cell subsets respond differently to BCR signaling or if CD45-dependent regulation of BCR signaling is subset specific in homeostatic or atherosclerotic conditions. Taking advantage of transgenic mice that express low levels of CD45(CD45L/L), we examined activation of CD45L/L B cells through Ca2+ flux. We show that BCR-induced B cell activation differs between subsets and CD45-dependent modulation of BCR signaling is subset specific. MZ and B1 have high levels of Ca2+ flux, with MZ B cell activation the most affected by CD45 expression. To test the effects of CD45L/L B in atherosclerosis, we performed adoptive transfer of CD45L/L or WT B cells into B cell-deficient atherosclerotic prone mice. After 23–38 wks of western diet feeding, CD45L/L B cell recipients had increased lesion formation despite an increase of protective MZ B cells. This increase in atherogenesis could be due to MZ CD45L/L B cells having reduced activation, disrupting their protective ability. This suggests that the subset-specific modulation of BCR signaling through CD45 plays a role in the regulation of B cell functions in atherosclerosis.

This work was supported by American Heart Association AIREA grant 8AIREA33960546.
Multispectral imaging to detect immune phenotypes associated with the tumor microenvironment in a multi-tissue study: A full automated 7-color mIF immuno-oncology workflow
Authors: Mehra Navi , Patel Bhavika , Allen Stephanie , Boldt Brittney , Ramirez Noah , Mammadova Najiba , Haggerty Agnes
Published: January 2023

Immunotherapy and precision medicine are rapidly developing approaches to cancer therapy. Biomarkers that detect the tumor and tumor microenvironment allow for the development of strategies that accelerate the advancement of treatments to enhance a patient’s immune system. Akoya’s MOTiF™ PD-1/PD-L1 Panel is a validated, multiplex immunoassay enabling detection of the 6 most clinically relevant immuno-oncology and spatial biomarkers: PD-1, PD-L1, FoxP3, CD8, CD68, and PanCK. The MOTiF™ PD-1/PD-L1 Panel was used to analyze the tumor microenvironment and specifically assess immune phenotypes of different types of cancers: non-small cell lung cancer (NSCLC), colon adenocarcinoma, head and neck squamous cell carcinoma (HNSCC), and breast cancer. We demonstrate the utility of Akoya’s MOTiF™ PD-1/PD-L1 panel kit in studying the cellular diversity of various cancers while retaining spatial context. Stained slides were analyzed using the InForm® and PhenoptrReports image analysis platforms to identify and better understand spatial relationships between a variety of simple and complex cell phenotypes. The MOTiF™ PD-1/PD-L1 panel kit provides reproducibility across different tissue types. These data provide insight into the innate and adaptive immune environment for targeted design of new immunotherapies and have implications for improving the treatment paradigm across many tumor types.
Synthetically glycosylated antigens as an inverse vaccine platform to prevent and treat food allergies
Authors: Cao Shijie , Maulloo Chitavi D , Sabados Matthew , Raczy Michal M , Wilson D Scott , Nagler Cathryn R , Hubbell Jeffrey A
Published: January 2023

The only FDA-approved oral immunotherapy for a food allergy provides protection against accidental exposure to peanuts. However, this therapy often causes discomfort or side effects and requires long-term commitment. Better preventive and therapeutic solutions are urgently needed. We have developed a technology for inverse vaccination using glyco-polymerized antigens to induce antigen-specific non-responsiveness. We have demonstrated that following subcutaneous (SC) administration, glyco-polymer conjugates traffic to draining lymph nodes (LNs) and are preferentially internalized by antigen presenting cells, educating the immune system to respond to these antigens in an innocuous way. Here, we tested SC administration of glyco-polymerized β-lactoglobulin (BLG) in a murine model of cow’s milk allergy. Two doses of glyco-polymerized BLG given one week apart (prior to sensitization) prevented an allergic response to BLG upon intragastric challenge as measured by reductions in BLG-specific IgG, IgG1, and IgE production as well as BLG-specific type 2 T helper cell (Th2) responses in vitro. We further explored the therapeutic potential of glyco-polymerized BLG. Unlike unmodified BLG, two SC injections of glyco-polymerized BLG after sensitization did not cause anaphylactic reactions in allergic mice and inhibited cellular Th2 cytokines upon BLG restimulation. Since pre-existing humoral immunity inhibited the therapeutic effect of our glyco-polymerized BLG, we introduced a co-therapy (anti-CD20) to blunt the humoral response and observed ameliorated anaphylactic responses following glyco-polymerized BLG therapy. This platform may provide a potential T cell-modulating strategy to prevent and treat food allergies.

This work was supported in part by seed funding from the Chicago Immunoengineering Innovation Center at the University of Chicago as well as the Food Allergy Fund.
Defining a functionally distinct, lineage dependent B1 cell population in Nippostrongylus brasiliensis infection
Authors: Zellner Matthew , Lownik Joseph Cornelius , Urban Joseph F , Martin Rebecca
Published: January 2023

Nippostrongylus brasiliensis (Nb) is the strongest natural inducer of IgE. Despite this fact, asthma rates have an inverse correlation in countries endemic with parasitic infection. Our lab as well as others have sought to explain this phenomenon with little success. The mechanism behind this relationship is complicated by the fact that two different lineages of B cells, that are difficult to distinguish, are capable of making IgE. These B cell populations, B1 cells and B2 cells, have been classically separated using cell surface markers, but our data shows that these markers do not truly define these populations. Our lab has previously shown that IgE, using classical separation of these B cells, have functional differences in a Nb response. Current work shows that these populations have increased plasticity in a location dependent manner. High dimensional flow cytometry analysis of B cell populations, before and after Nb infection, illustrate this phenomenon. Using a lineage specific mouse model, reconstituted with fetal derived B cells that secrete FLAG-tagged IgE, we demonstrate that fetal derived B1 cells are not responsible for IgE production in Nb infection. However, hematopoietic cells expressing B1 markers are a significant source for this IgE. These data support a functionally distinct B cell population that is hematopoietic derived and has been previously classified as B1 cells.

Supported by grants from the NIH-NIAID (R56 AI139658-01AI)
P2X3 is a Female-Dominant Activator of Mast Cells
Authors: Kee Sydney Ann , Haque Tamara , Dailey Jordan , Ryan John J
Published: January 2023

Asthma is characterized by increased mucus production, airway hyperresponsiveness and airway inflammation, all promoted by mast cell activation. There is a clear bias for greater incidence and acuity of asthma among females, but the reasons for this are not clear. Women are 60% more likely to be diagnosed with asthma and three times more likely to be hospitalized for it. In a search for possible new and upcoming drugs that might be repurposed to suppress mast cell function, we discovered that the ATP receptor P2X3, most often studied in neuropathic pain, has a role in mast cell activation. The P2X3 antagonist BLU-5937 suppressed IL-33 mediated-mast cell cytokine secretion in vitro and reduced neutrophilic peritonitis in vivo in female mice. In contrast, male mice and mast cells cultured from them showed no suppression when treated with BLU-5937. In addition, female mast cell responses were much stronger than males when activated with IL-33 in vitro. We also found that IL-33 elicits rapid ATP secretion by mast cells, possibly forming an autocrine loop via P2X3. These data coincided with little detectable P2X3 expression on male mast cells. We hypothesize that IL-33-mediated mast cell activation is more potent in females due to P2X3 receptor signaling. Our data support the theory that P2X3 is a female-dominant mast cell activator that can be targeted to reduce allergic inflammation. This research is novel because it could explain the sexual dimorphism in allergic disease and elucidate the role of P2X3 in the immune system.

Supported by 1RO1 AI164710
Identification of redundancy between human FcɛRIβ and MS4A6A proteins points toward additional mechanisms for FcɛRI trafficking and signaling
Authors: Bitting Katie Jane , Hedgespeth Barry , Ehrhardt-Humbert Lauren C , Arthur Greer , Bradding Peter , Tilley Stephen L , Cruse Glenn
Published: January 2023

Allergic diseases are triggered by signaling through the high affinity IgE receptor, FcɛRI. In mast cells (MCs) and basophils, FcɛRI is a tetrameric receptor complex comprising a ligand binding α subunit (FcɛRIα); a tetraspan β subunit (FcɛRIβ) responsible for trafficking the complex and amplifying signaling; and a signal transducing dimer of single transmembrane γ subunits (FcɛRIγ). However, FcɛRI also exist as presumed trimeric complexes that lack FcɛRIβ and are expressed on several cell types outside of the MC and basophil compartments. There are known differences in the trimeric FcɛRI complex between humans and mice and questions remain as to how this trimeric FcɛRI complex traffics and whether it signals in the absence of FcɛRIβ. We have previously reported that targeting FcɛRIβ with exon skipping oligonucleotides diminishes IgE-mediated degranulation in mouse MCs, but equivalent targeting in human MCs was not effective at reducing degranulation. Here, we report that an FcɛRIβ-like protein exists in humans and we identify that the Membrane Spanning 4A (MS4A) gene cluster member, MS4A6A compensates for FcɛRIβ in FcɛRI trafficking and signaling. The function of MS4A6A is currently unknown, but MS4A6A and FcɛRIβ are encoded by highly related genes within the same family (MS4A gene family) that cluster within the human gene loci 11q12–q13, a region linked to allergy and asthma susceptibility. Human MS4A6A promotes surface expression of FcɛRI complexes and promotes degranulation. Indeed, our data suggest the presence of either FcɛRIβ or MS4A6A is sufficient for degranulation. This study suggests that MS4A6A could be an elusive FcɛRIβ-like protein in human MCs that performs compensatory functions to FcɛRIβ.

Research was supported by the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID), Award Number R01AI143985, Department of Molecular Biomedical Sciences, College of Veterinary Medicine, start-up funds, Institutional National Research Service Award from the Office of the Director of National Institutes of Health, NIH T32OD011130.