bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin 6 (IL6) is an important upstream contributor towards the progression of SLE-like disease in BXSB.Yaa mice (BA11P.134)
Authors: Jain Shweta , Adkins Elisabeth , Christianson Gregory , Sproule Thomas , Roopenian Derry , Morse Herbert
Published: January 2023

Systemic lupus erythematosus (SLE) is a multigenic autoimmune disorder that results in the production of autoantibodies (autoAbs) and deposition of immune complexes leading to tissue inflammation and organ damage. Typical disease characteristics include glomerulonephritis, splenomegaly and monocytosis. To understand disease mechanism, we studied spontaneous autoimmune BXSB.Yaa strain in which male mice develops SLE-like features with intricacy paralleling human SLE. It was shown that an extended pseudo-autosomal region on Y chromosome carrying a duplication of Tlr7 gene is responsible for the disease. TLR7 binds with ssRNA that consequently leads to production of inflammatory cytokines. We previously reported that IL21, produced by CD4 T follicular helper cells (TFH), plays a critical role in the disease of BXSB.Yaa mice. Since TFH cells require IL6 for their differentiation and maintenance from naïve T cells and dysregulated IL6 production has pathological consequences in SLE patients, we sought to determine if IL6 plays important upstream role in disease progression. We found that increased serum levels of IL6 in BXSB.Yaa mice correlated with the production of autoAbs, Ig secretion and hyperactive B cells. Moreover, abrogating IL6 signaling not only improved survival of BXSB.Yaa mice but they also had reduced numbers of TFH and lesser B cell activation. Thus, we conclude that IL6 signaling is an important determinant in the progression of SLE-like disease in BXSB.Yaa mice.
Selective deletion of estrogen receptor alpha in FoxP3+ CD4+ T cells influences peripheral T cell homeostasis (BA11P.139)
Authors: McKarns Susan
Published: January 2023

Being female confers a greater risk of developing an autoimmune disease than any other known genetic or environmental risk factor. Given that a loss of regulatory T cell (Treg) function contributes to autoimmunity and estrogen receptor alpha (ERα) promotes Tregs and suppresses experimental autoimmune encephalomyelitis (EAE), ERα-induced Tregs offers a new strategy to treat autoimmune disorders. The objective of these studies was to determine whether selective ablation of ERα in FoxP3+ Tregs dysregulates peripheral T cell homeostasis. To the end, mice harboring a floxed Esr1 gene (ERαf/f) were bred to mice expressing a YFP-Cre fusion protein driven by an IRES following foxp3 to obtain FoxP3+ Treg restricted deletion of Ers1. Thymic and peripheral CD4+ and CD8+ T cell frequency appeared unaffected with Esr1 ablation except for a sex-biased reduction in the total number of thymic and peripheral FoxP3+ Tregs and an increase in total number of splenocytes in ERαf/f FoxP3Cre mice compared to ERα+/+ FoxP3Cre mice. Spotaneous chronic inflammation and autoimmunity was undetected in ERαf/f FoxP3Cre mice. In a CD4+ T cell transfer model of experiment colitis, selective ablation of the ERα in FoxP3+ Tregs resulted in exacerbated inflammation compared to mice harboring ERα-sufficient Treg cells. Overall, these preliminary results suggest that FoxP3+ Treg-specific ERα is essential for the function of peripheral Tregs and immune homeostasis in a sex-biased manner.
Tolerance induction in recent thymic emigrants (BA11P.121)
Authors: Friesen Travis , Ji Qingyong , Fink Pamela
Published: January 2023

Recent thymic emigrants (RTEs) comprise the subset of peripheral T cells that have most recently undergone thymic maturation and egress to the lymphoid periphery. RTEs are an important lymphocyte population to study because they represent the majority of T cells in neonates and contribute to T cell diversity throughout life. Using RAG2p-GFP transgenic (Tg) mice to identify RTEs in the periphery, our lab has discovered that T cell development continues for several weeks after cells have left the thymus and entered the lymphoid periphery. We hypothesize that post-thymic maturation represents a period of heightened susceptibility to tolerance induction. We tested this notion using T cell receptor (TCR) Tg ovalbumin (OVA)-specific CD4 (OT-II) and CD8 (OT-I) RTE and MN T cells transferred into mice expressing a membrane-bound form of the OVA protein in the pancreatic islets and kidney proximal tubular cells (RIP-mOVA Tg). Following co-transfer of congenically marked OT-II and OT-I RTE and MN T cells to RIP-mOVA Tg mice, we found that, compared to mature T cells, both CD4 and CD8 RTEs proliferate, accumulate, and secrete less IL-2 and IFNg, but express elevated levels anergy-associated genes. Finally, both OT-II and OT-I RTEs were less diabetogenic than their mature T cell counterparts when transferred into RIP-mOVA adoptive hosts. These results further support our hypothesis that post-thymic maturation represents a period of heightened susceptibility to tolerance induction.
Mechanisms underlying cytokine receptor suppression of type 1 diabetes (BA11P.127)
Authors: Ukah Tobechukwu , Chen Weirong , Miller Mindy , Barik Subhasis , Ellis Jason , Zaghouani Habib
Published: January 2023

Type 1 or pediatric diabetes (T1D) manifests when the insulin-producing pancreatic beta-cells are destroyed as a consequence of an inflammatory process initiated by lymphocytes of the immune system. IL-4 and IL-13 cytokines which utilize a shared heteroreceptor (HR) comprising IL-4Rα and IL-13Rα1 are viewed as anti-inflammatory. The mechanism by which these cytokines drive suppression of T1D remains undefined. In this study, we used non-obese diabetic (NOD) mice, which develop the disease spontaneously to determine how the cytokines and the shared HR drive modulatory functions. The findings indicate that NOD mice in which the shared HR is made non-functional by ablation of IL-13Rα1 gene resist the disease relative to wild type mice with intact heteroreceptor. In addition, their lymphocytes could not transfer the disease into NOD.Scid mice effectively. This was due to an increase in the frequency of membrane-bound TGF-β T cells expressing intermediate levels of FoxP3 marker (mTGFβ+FoxP3int). Also, there was relocation to the pancreas of anti-inflammatory macrophage (MΦ) expressing the mannose receptor, a marker of M2 phenotype. Thus, the HR modulates both arms of adaptive and innate immune suppressors including Tregs and MΦ. These insights on the role of HR in modulation of T1D will likely inform on the development of therapeutic approaches against the disease.
HIV-1 Infection increases sphingosine-1-phosphate receptor 1 expression in the human thymus (CAM4P.162)
Authors: Resop Rachel , Craft Joshua , Uittenbogaart Christel , Vatakis Dimitrios , Blom Bianca
Published: January 2023

Sphingosine-1-phosphate receptor 1 (S1P-R1) is essential in mice for homing of lymphoid cells as well as egress of mature thymocytes from the thymus to the periphery. We have recently demonstrated that in the human thymus, response of S1P-R1 to its ligand, Sphingosine-1-phosphate (S1P), is required for egress of mature human thymocytes to the periphery. Herein we demonstrate that immune dysregulation accompanying Human Immunodeficiency Virus (HIV) infection alters the expression of S1P-R1 on human thymocytes, increasing expression of the receptor at both the mRNA and protein levels. Moreover, the function of the receptor is maintained, as assayed by flow cytometry based detection of phosphorylated Akt (pAkt). Finally, we found that several cytokines including Tumor Necrosis Factor Alpha and Interferon Alpha are perturbed during infection of the thymus, and exogenous treatment of human thymocytes with these cytokines increases or attenuates S1P-R1 expression. Therefore we have identified a contributing mechanism to alteration of S1P-R1 expression during HIV infection of the human thymus, which may have important implications for therapeutics for HIV infected individuals.
Role of dendritic cell-derived amphiregulin in pulmonary fibrosis (CCR5P.203)
Authors: Liu Tianju , Ding Lin , Wu Zhe , Gonzalez De Los Santos Francina , Phan Sem
Published: January 2023

Recruitment of dendritic cells (DCs) has been implicated in lung fibrosis. Accumulation of BM-derived DC (BMDC) was shown to play an important role in bleomycin (BLM)-induced fibrotic lungs. A microarray analysis of lung DCs derived from control and BLM-treated mice revealed amphiregulin (AREG), an epidermal growth factor receptor ligand was highly upregulated in cells from fibrotic lung. Moreover BLM-induced fibrosis and AREG induction were attenuated when BMDCs were depleted. Thus AREG induced primarily in BMDCs appear to play a significant role in this model of fibrosis. To further confirm the in vivo importance of AREG fibrosis was induced in wild type or AREG knockout (KO) mice by injecting BLM endotracheally, and the fibrotic response was analyzed biochemically and histologically. The results showed that BLM induced significant induction of HYP in WT mice was significantly decreased in AREG KO mice. The fibrotic markers collagen I and α-smooth muscle actin expressions were also significantly reduced compared to the WT mice, along with impaired proinflammatory cytokine/chemokine expression. These fibrotic responses were also reflected by tissue histopathology. Finally, unlike BMDCs isolated from wild type mice, those from AREG KO mice lacked the ability to induce myofibroblast differentiation when co-cultured with lung fibroblasts. Thus BMDC-derived AREG played an important role in pulmonary fibrosis probably by promoting fibroblast proliferation and survival.
Developing soluble mimetics of the chemokine receptor CCR5 for the study of HIV Env/receptor interactions (CCR6P.216)
Authors: Hajduczki Agnes , Bundoc Virgilio , Berger Edward
Published: January 2023

During HIV entry, the surface-exposed gp120 subunit of the envelope glycoprotein (Env) undergoes a series of stepwise conformational changes in response to interactions with the primary receptor CD4, and co-receptors, CCR5 or CXCR4. Obtaining structural information on the various intermediates during viral entry is a key focus of antiviral and vaccine research and could open the doors for more effective treatment and prevention. This project aims to develop recombinant soluble co-receptor mimetics featuring critical determinants of the chemokine receptor, CCR5, using two approaches: fusing the CCR5 moieties to soluble CD4 by flexible polypeptide linkers and using a globular, stable scaffold protein to orient the co-receptor determinants in an optimal conformation to recreate the binding interaction with gp120. We have successfully overexpressed and purified the recombinant proteins from mammalian cells. Characterization of the gp120-binding properties of the variants is underway using a vaccinia-based cell fusion assay where soluble CD4 has been shown to induce membrane fusion between Env-expressing effector cells and target cells bearing CCR5, but no CD4. The CCR5 moieties of the sCD4-fused constructs compete with cell-surface CCR5 for binding to gp120, leading to impaired activation of membrane fusion compared with sCD4. The soluble variants of CCR5 will be used in experiments to elucidate the conformational changes in gp120 that immediately precede membrane fusion.
The role of interleukin-12 in breast cancer (CCR6P.220)
Authors: Nunez Angel , Arroyo Nelly , Godoy Lenin , Dutil Julie
Published: January 2023

Interleukin-12 (IL-12) is a dimeric cytokine which has demonstrated anti-cancer effects in vitro and in vivo. However, the role of this cytokine in breast cancer (BC) has not been studied thoroughly. In this pilot study, the levels of the IL-12 p70 and IL-12 p40 secreted from PBMCs of de novo-BC patients, BC patients in remission, and non-BC controls were measured and compared. Peripheral blood mononuclear cells (PBMCs) from participants (n=8) were stimulated with endotoxin at specific time points to induce IL-12 secretion. We expect to obtain differences on the levels of IL-12 p40 and IL-12 p70 among the study groups. This pilot study will give an initial comparative standpoint to see if there are differences in the levels of IL-12 among the groups. Further studies can be conducted to analyze the mRNA expression and protein levels of other downstream IL-12 signaling pathway components.
Loss of ΔNp63 impairs thymic epithelial progenitor survival and differentiation (HEM7P.234)
Authors: Bain Virginia , Reeh Kaitlin , Carter Carla , Flores Elsa , Richie Ellen
Published: January 2023

The thymic stroma consists primarily of thymic epithelial cells (TECs) that originate from endodermal progenitors in the 3rd pharyngeal pouch (pp). A common epithelial progenitor in fetal thymus differentiates into networks of cortical (cTEC) and medullary (mTEC) cells, which provide distinct maturation signals that support T cell generation. The transcription factor p63 is expressed in cTECs and mTECs and global deletion results in a severely hypoplastic thymus. Alternative promoters generate two p63 isoforms, TAp63 containing a transactivation (TA) domain and ΔNp63, lacking a TA domain. The roles of TAp63 and ΔNp63 in thymus organogenesis have not been determined. We find that TAp63 is undetectable in fetal or steady state adult thymus and deletion of TAp63 in fetal TECs does not affect thymus size or TEC development. In contrast, ΔNp63, is highly expressed by E9.5 and persists in cTECs and mTECs in the postnatal thymus. Using Foxn1Cre to delete ΔNp63fl/fl in TEC progenitors of the 3rd pp results in a severely hypoplastic thymic phenotype by E14.5. Patterning of 3rd pp endoderm into thymus- and parathyroid-fated domains is not disrupted. Plet1+ progenitor TECs are found at a normal frequency at E12.5; however, Plet1- TECs lacking ΔNp63 undergo apoptosis at a higher frequency than wildtype. Furthermore, surviving ΔNp63 null TECs fail to differentiate beyond an early progenitor state. We conclude that ΔNp63 is necessary for survival and differentiation of developing TECs.
MicroRNA-205 regulates thymopoiesis under steady state and stress conditions (HEM7P.222)
Authors: Hoover Ashley , Khan Shaheen , Cleaver Ondine , de la Morena Maite , van Oers Nicolai
Published: January 2023

The thymus comprises an interconnected meshwork of epithelial cells (TECs) and thymocytes. This organ is exceedingly sensitive to stress, undergoing rapid involution in response to infection, trauma, radiation exposure, and/or glucocorticoid treatments. The nature of the stress predicates whether the epithelial cells, developing thymocytes, and/or both are affected. MicroRNAs are small noncoding RNAs that regulate cellular responses to stress and during regeneration. We discovered that miR-205 is a stress responsive, epithelial-specific miR that modulates thymopoiesis. Mice lacking miR-205 selectively in TECs exhibited a significant decline in thymopoiesis beginning at 8 weeks of age. When stressed with a dsRNA mimic (polyI:C), these mice exhibited a much more severe thymic atrophy compared to littermate controls. Quantification of the TEC subsets revealed a defect in the proliferative recovery of the cortical TECs lacking miR-205. This contributed to a significant reduction in the number of single positive CD4 and CD8 T cells, indicating that the capacity of the TECs to support selection processes at the CD4+CD8+ stage of thymopoiesis is compromised. Current experiments to determine how miR-205 regulates TEC function and proliferation are ongoing. The findings reveal an important role for miR-205 in thymopoiesis, which may be of therapeutic importance.
Wnt signaling during thymic involution and reconstitution. (HEM7P.226)
Authors: Singh Varan , Schwartz Carla , Jamil Nawshin , Hoque Mohammed , Osada Masako , Pezzano Mark
Published: January 2023

Despite its critical role in T-cell development, the thymus undergoes profound age-related involution. Inhibition of Wnt signaling in thymic epithelium (TECs) leads to rapid involution. Lack of a Wnt reporter that was functional in TECs hindered characterization of Wnt signaling during TEC ontogeny. Using the TCF/LEF-H2BGFP Wnt reporter mouse, TECs undergoing active Wnt signaling were characterized during different stages of thymic aging and recovery. In the postnatal thymus, H2BGFP expression was restricted to TECs in the medulla and CMJ including 60% of the Aire-expressing mTECs, suggesting a role for Wnt signaling in the development of this critical TEC subset. Comparison of the TECs undergoing Wnt signaling in old versus young thymus revealed an increase in the frequency of TECs undergoing Wnt signaling even though overall TEC numbers decreased significantly with age. The decrease in both total TECs and TECs undergoing Wnt signaling was most apparent in the UEA1hi mTEC subset and suggests that the age related decline in AIRE expression is correlated with the decline in mTECs undergoing Wnt signaling. SCID/TCF/LEF1-H2BGFP mice were used to observe Wnt signaling during thymic reconstitution induced by either bone marrow transplant (BMT) (full thymic development) or injection of mature T cells (mTEC development). During thymic reconstitution induced by both BMT and mature T-cells, Wnt signaling was primarily associated with Aire expressing mTECs, critical for self-tolerance.
a conversed gene regulatory pathway underwrites the development of innate lymphoid cells and adaptive T cells (HEM8P.239)
Authors: Yang Qi , Xue Hai-Hui Howard , Bhandoola Avinash
Published: January 2023

Innate lymphoid cells (ILC) are a specialized family of lymphocytes that functionally mirror subsets of effector T cells but lack adaptive antigen receptors. ILC express many T-lineage characteristic genes, and are closer to T cells than to other leukocytes at the transcriptome level. Here we show that the transcription factor TCF-1 drives a shared T/ILC lineage gene pathway that underwrites the development of both ILC and T lineages. TCF-1-/- bone marrow progenitors failed to give rise to all known adult ILC and T lineage subsets as well as their precursors. Ectopic expression of TCF-1 elicited the expression of many key genes shared by ILC and T lineages. Like in early T cell development, Notch signaling promoted pulmonary ILC2 development in part through TCF-1, and TCF-1 acted through both GATA-3 dependent and independent mechanisms. Our data suggested that a shared gene regulatory circuit underwrites the development of innate lymphoid cells and T cells.
Atg7 plays a critical role in the development of iNKT cells with altered PLZF expression. (HEM8P.242)
Authors: Jeong Dongjin , Jin Young-Hee , Chung Doo Hyun
Published: January 2023

Invariant Natural Killer T (iNKT) cell is a distinct subset of T cells that plays a regulatory role in various immune diseases. Although many studies have reported that various factors regulate development of iNKT cells, the role of autophagy in the development is still unknown. Here, we report that autophagy-related protein 7(Atg7) is required for development of iNKT cells in the thymus. iNKT cells of the thymus and liver expressed Atg7 and their development is completely attenuated in the thymus of CD4-specific Atg7 knock-out (CD4-Cre/Atg7f/f) mice compared with WT mice. The levels of PLZF expression were altered in thymocytes from CD4-Cre/Atg7f/f mice as compared with WT mice. CD4-Cre/Atg7f/f mice showed a few iNKT cells in peripheral organs, which led to functional deficient in iNKT cell-specific activation in vitro and in vivo. Accordingly, autoantibody-induced arthritis was attenuated in CD4-Cre/Atg7f/f mice. Therefore, our findings suggest that Atg7 regulates iNKT cell development with altered PLZF expression.
Pregnancy does not attenuate the antibody or plasmablast response to inactivated influenza vaccine (HUM6P.247)
Authors: Bayless Nicholas , Kay Alexander , Fukuayama Julia , Aziz Natali , Dekker Cornelia , Mackey Sally , Swan Gary , Davis Mark , Holmes Susan , Blish Catherine
Published: January 2023

Background. Inactivated influenza vaccine (IIV) is recommended during pregnancy to prevent influenza infection and its complications in pregnant women and their infants. However, the extent to which pregnancy modifies the antibody response to vaccination remains unclear, and prior studies have focused primarily on hemagglutinin inhibition (HI) titers. A more comprehensive understanding of how pregnancy modifies the humoral immune response to influenza vaccination will aid in maximizing vaccine efficacy. Methods. Healthy pregnant women and control women were studied prior to, 7 days after and 28 days after vaccination with IIV. HI titers, microneutralization (MN) titers and the frequency of circulating plasmablasts were evaluated in pregnant vs. control women. Results. Pregnant women and control women mount similarly robust serologic immune responses to IIV, with no significant differences for any influenza strain in post-vaccination geometric mean HI or MN titers. HI and MN titers correlate, though MN titers demonstrate more robust changes pre- vs. post-vaccination. The induction of circulating plasmablasts is increased in pregnant women vs. controls (median fold-change 2.60 vs. 1.49 [Interquartile Range 0.94-7.53 vs. 0.63-2.67]; p = 0.03). Conclusion. Pregnant women do not have impaired humoral immune responses to IIV, and may have increased circulating plasmablast production compared to control women.
Early exposure to phosphorylcholine-bearing microbes dampens the development of house dust mite allergy during adult life (HYP7P.263)
Authors: Patel Preeyam
Published: January 2023

A suggested mechanism to explain the dramatic rise in the incidence of allergic disease among individuals living in developed countries has been an imbalance in the TH1/TH2 subsets; however, a majority of neonatal pathogens evoke potent B cell responses. These B cell clones, with shared specificity for allergens, can be protective against the development of allergic disease. Streptococcus pneumoniae and house dust mite (HDM) bear similar phosphorylcholine epitopes. Therefore, we hypothesized that B cell clones generated as a result of early pneumococcal exposure would dampen the development of HDM allergy during adult life. To test our hypothesis, we immunized neonatal mice with PC-deficient or PC-bearing pneumococcus and then challenged these mice with HDM as adults. Mice receiving the PC-bearing pneumococcus had a large frequency of PC-specific B cells that resided in their spleen and continually secreted antibodies. Upon pulmonary exposure to PC-bearing HDM, these B cells migrated to the lung and secreted antibody locally into the pulmonary space. As a result, mice immunized with PC-bearing pneumococcus, presented with dampened production of serum IgE and TH2-associated cytokines, development of airway hyperreactivity, and infiltration of allergic mediators into the lung as compared to mice exposed to PC-devoid pneumococcus as neonates. We further demonstrated that neonatal exposure to bacteria was not sufficient enough to disrupt the balance in the TH1/TH2 subsets.
Increased survival and exocytosis of toxic primary granules by neutrophils in an in vitro model of Cystic Fibrosis airway disease (INC6P.310)
Authors: Forrest Osric , Ingersoll Sarah , Preininger Marcela , Laval Julie , Brown Milton , Tirouvanziam Rabindra
Published: January 2023

Cystic Fibrosis (CF) lung disease is characterized by the massive recruitment of neutrophils (PMNs) into the bronchiolar lumen. Extracellular neutrophil elastase (NE) that is released from PMN primary granules, is a strong predictor of lung function and survival in CF patients. However, the process by which CF airway PMNs exocytose HNE is not well understood and not reproduced in animal models of CF airway disease. Methods: We used a surface collagen-coated 3D porous polystyrene scaffold (Alvetex) to mimic the airway lamina propria and grew human bronchiolar H441 cells at air-liquid interface on top. In the transepithelial migration assay, blood PMNs were placed on the basal side and allowed to migrate towards to CF airway fluid placed apically, after which PMNs were analyzed by flow cytometry for rate of migration, survival, and degranulation. Results: Using this model we show that (i) CF airway fluid induces rapid transepithelial migration and survival of neutrophils, (ii) live PMNs actively release of primary granules upon exposure to CF airway fluid, and (iii) migration towards CF airway fluid induces PMN reprogramming allowing for the activation of the inflammasome and pinocytosis. Conclusions: These results suggest a primary role for the CF microenviroment in inducing increased survival and phenotypic reprogramming of PMNs and establish our model as a robust platform for deconstructing CF airway PMN dysfunction and developing new therapies for CF inflammation.
Alternatively activated macrophages attenuate influenza-induced acute lung injury in mice heterozygous for the F508del mutation in CFTR. (INC6P.314)
Authors: Woods Parker , Davis Ian
Published: January 2023

Infection with influenza A virus can result in acute lung injury (ALI), characterized by pulmonary edema and hypoxemia. We found that influenza-induced ALI in C57BL/6 (WT) mice was associated with increased bronchoalveolar epithelial cell Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Influenza-induced ALI was highly attenuated in C57BL/6-congenic mice that are heterozygous for the F508del CFTR mutation (HETs), which exhibit a 50% reduction in CFTR expression and CFTR-mediated Cl- transport. This beneficial phenotype was alveolar macrophage (AM)-dependent and was not due to differences in viral replication between mouse strains. The aim of this study was to evaluate the role of HET AMs in the attenuation of influenza-induced ALI. We hypothesized that ALI attenuation in influenza-infected HET mice results from “alternative” activation (M2 polarization) of HET AMs in response to infection. Compared to WT AMs, Arginase 1 (Arg1) protein expression was higher while inducible nitric oxide synthase (iNOS) protein was lower in HET AMs following infection. Arg1 enzymatic activity was also higher in HET AM lysates. BALF from HET mice contained higher levels of urea and lower levels of nitrate/nitrite (end-products of Arg1 and iNOS, respectively) than WT. These findings indicate that reduced CFTR expression/activity promotes an anti-inflammatory, M2 AM polarization response to influenza infection, which contributes to ALI attenuation.
Human CCL1 antisense oligodeoxynucleotide (ODN) screened as a potent inhibitor of M2b monocytes (INC6P.322)
Authors: Ito Ichiaki , Bhopale Kamlesh , Nishiguchi Tomoki , Loucas Bradford , Suzuki Sumihiro , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

In a murine model of γ-irradiation, bacterial translocation and subsequent sepsis have been effectively controlled by CCL1 antisense ODN, a M2b macrophage reverter. In the present study, we tried to obtain human CCL1 antisense ODN which is inhibitory on human M2b macrophages. Twenty human CCL1 antisense ODNs were synthesized based on the prediction of CCL1 mRNA secondary structure, calculation of GC content, and binding energy of ODNs. To evaluate the activity of these ODNs, an amount of CCL1 in culture fluids of M2b monocytes cultured with the ODNs was determined by ELISA. M2b monocytes were generated in vitro from healthy donor peripheral blood monocytes after stimulation with IL-1β and IgG in combination. In the results, 2 ODNs (HCA-15 ; CAACATCTGGAGAAGGGT : HCA-17 ; GGGCTGTGGTGATGATC), at doses of 20 and 100 µg/ml, suppressed 70-80% or more of CCL1 production by M2b monocytes. Also, decreased expression of IL-10 and CD163 was shown in M2b monocytes cultured with HCA-15 and HCA-17. The other 18 ODNs were not significantly active on the M2b macrophage reversion. These results suggest that HCA-15 and HCA-17 may be beneficial in controlling bacterial translocation and subsequent sepsis in patients with γ-irradiation.
Rapid, simple, column-free isolation of CD11b+ cells from various mouse organs (INC6P.323)
Authors: Valdez Yanet , Tabatabaei-Zavareh Nooshin , de Jong Susan , Morin Peter , Karas Evan , Kollar Anita , Fairhurst Maureen , Eaves Allen , Thomas Terry
Published: January 2023

CD11b+ cells are distributed throughout the body and act as sentinels for the immune system. The frequency of CD11b+ myeloid cells varies in different organs, ranging from approximately 48 % of the brain myeloid (microglia) cells, 30% of bone marrow and 6% of total nucleated cells in gut and spleen. The integrin CD11b heterodimerizes with CD18 to form the mature complex Mac-1. Mac-1 facilitates a variety of immune cell responses including phagocytosis, adhesion, migration and chemotaxis, all functions that are crucial for normal tissue homeostasis and inflammatory responses. CD11b+ cells are difficult and time consuming to isolate, due to their variable frequency using traditional methods. Here, we describe EasySepTM, a simple, column-free immunomagnetic method for the positive selection of highly purified CD11b+ cells from brain, bone marrow, spleen and gut. Labelled CD11b+ cells are retained in a tube in an EasySepTM magnet while unwanted cells are poured off. Selection can be fully automated using RoboSepTM. Starting with a frequency of 7.6 ± 1.3 % CD11b+ cells in spleen, purities of 93.9 ± 4.5 % (n=41) were achieved. Fully competent cells, as shown by functional assays (phagocytosis, ROS), were isolated from the above tissues. This EasySepTM technology provides a fast and simple method to access highly purified functional CD11b+ cells from a variety of tissues, facilitating their study ex-vivo.
Intoxication reduces the number of alveolar macrophages and increases apoptosis after burn and pulmonary infection (INC6P.307)
Authors: Ippolito Jill , Ramirez Luis
Published: January 2023

Alcohol intoxication is involved in 50% of all burn injuries and results in an increased risk of lung infection and mortality, relative to non-drinking patients. Experimental models of intoxication, burn and Pseudomonas aeruginosa (PA) intratracheal (i.t.) infection have revealed amplified lung inflammation, relative to controls. Since lung inflammation is mediated in part by alveolar macrophages (AMs), we chose to examine AMs in this combined injury model. Mice were subjected to ethanol 30 min prior to a 15% total body surface area scald injury, followed by an i.t. PA infection (3,000 CFUs). At 24 h, bronchoalveolar lavage cells were analyzed by flow cytometry for absolute numbers of AMs. Relative to shams, burn and infected mice had 60% less AMs (p<0.05), while combined injured, intoxicated, burn and infected mice had 80% less AMs (p<0.05). Between burn groups, combined injury had 60% less AMs, relative to burn and infection alone. Lungs from shams revealed 3 apoptotic cells per low powered field. This was increased 2-fold after burn and infection and by 8-fold with combined injury (p<0.05). Combined injured mice also had 5-fold more apoptotic cells than burn and infection alone (p<0.05). These data suggest a decrease in AM numbers may be due to an increase in AM apoptosis. Overall, the loss of AMs may contribute to the exacerbated lung inflammation observed with combined injury. [R01AA012034 (EJK), T32 AA013527 (EJK), F31 AA022566 (JAI), and the Falk Foundation (EJK)].
TIGIT facilitates liver regeneration via limiting natural killer cell over-activation (INM6P.341)
Authors: Sun Rui , Bi Jiacheng , Zheng Xiaodong , Tian Zhigang
Published: January 2023

Innate immune cells, particularly natural killer (NK) cells, are known to negatively regulate liver regeneration, and inhibition of NK cell overactivation could facilitate liver regeneration. However, the molecular mechanisms that limit NK cell overactivation during liver regeneration are still elusive. In our study, we show that T cell Ig and ITIM domain (TIGIT), a coinhibitory receptor, is selectively up-regulated on NK cells, along with high expression of its ligand, poliovirus receptor (PVR/CD155), on hepatocytes during liver regeneration. Moreover, TIGIT absence impairs liver regeneration in vivo, along with NK cell overactivation and higher IFN-γ production by NK cells. We also show that NK cell-derived higher IFN-γ production is the major account of impaired liver regeneration caused by the absence of TIGIT. Adoptive transfer of Tigit-/- NK cells into NK-deficient Nfil3-/- mice sufficiently led to impairment of liver regeneration. On the other hand, silencing PVR in hepatocytes rescued impaired liver regeneration caused by TIGIT deficiency in vivo, while blockade of TIGIT in NK-hepatocyte coculture increased IFN-γ production by NK cells in vitro. Therefore, these results reveal that TIGIT is a safeguard molecule to facilitate liver regeneration through negatively regulating NK-hepatocyte crosstalk.
IL-1β secretion is dependent upon a calcium-mediated interaction with calmodulin (INM6P.329)
Authors: Ainscough joseph
Published: January 2023

IL-1β secretion is a central event in the inflammatory process. Unlike most other cytokines, which are secreted via the classical pathway, 2 independent signals are required for IL-1β release. The first induces the intracellular up-regulation of pro-IL-1β, and the second drives secretion of the bioactive molecule. The release of pro-IL-1β is a complex process involving proteolytic cleavage by caspase-1. However, the exact mechanism of secretion is poorly understood. Here, we sought to identify novel proteins involved in IL-1β secretion and intracellular processing, in order to gain further insight into the mechanism of release. A human proteome microarray containing 19,951 unique proteins was used to identify globally proteins that bind Human recombinant pro-IL-1β. Probes with a signal to noise ratio (SNR) of >3 were defined as relevant biologically. In these analyses, calmodulin was identified with a mean SNR of ~11; one of the strongest interactions recorded. Using an ELISA-based protein binding assay, the interaction between recombinant calmodulin and pro-IL-1β, but not mature IL-1β, was confirmed and shown to be calcium dependent. Finally, using small molecule inhibitors it was demonstrated that both calcium and calmodulin were required for IL-1β secretion by the human monocytic cell line THP1. These data suggest that following calcium influx into the cell, pro-IL-1β interacts with calmodulin and that this interaction is important for the secretion of IL-1β.
IL-33-mediated immune responses contribute to renal pathology and endothelial damage in Orientia tsutsugamushi-infected mice. (INM6P.332)
Authors: Shelite Thomas , Liang Yuejin , Wang Hui , Xu Guang , Sun Jiaren , Gong Bin , Walker David , Soong Lynn
Published: January 2023

Endothelial cells (EC) are the main target for Orientia tsutsugamushi infection, and EC dysfunction is a hallmark of severe scrub typhus in patients. However, very little is known regarding the molecular basis of EC dysfunction and its impact on infection outcomes. To address these issues, we recently developed a mouse model of severe scrub typhus and examined gene expression kinetics for IL-33, a DAMP molecule preferentially expressed in EC. Following the infection, mice showed a tissue-specific expression profile for IL-33 and its ST2 receptor, and both genes were highly expressed in the kidneys at the onset of disease (day 6) and prior to host death (day 10). The attenuated disease in IL-33-/- mice implied a role of IL-33/ST2 signaling in disease pathogenesis. We found that WT mice displayed exacerbated EC activation and apoptosis, as judged by the altered expression of angiopoietin (elevated Ang2/Ang1 ratio), increased endothelin-1 (ET-1) expression, and increased vascular damage. In IL-33-/- mice, however, EC stress and apoptosis were markedly attenuated, partially via the expression of molecules known to promote endothelial integrity (Ang1, eNOS, BCL2). The effect of Ang1-based treatment on disease outcome is being examined. These data indicate that IL-33-mediated immune responses contribute to renal pathology and endothelial damage in severe scrub typhus. This study also points to potential therapeutic targets in the treatment of Orientia-induced acute kidney injury.
The role of SARM1 in Toll-like receptor and viral-induced neuronal apoptosis (INM7P.347)
Authors: Khan Burhan , Mukherjee Piyali , Taylor Katherine , Woods Tyson , Winkler Clayton , Peterson Karin
Published: January 2023

Neuronal apoptosis is a key aspect of several neurological diseases but the mechanisms leading to apoptosis are not well understood. Recent studies have suggested that activation of Pattern Recognition Receptors (PRRs) of neurons leads to neuronal apoptosis, even in the absence of pathogen insult. For example, combined stimulation of neurons through toll-like receptors (TLRs) TLR7 and TLR9 results in significant apoptosis. Using neurons from transgenic mice, we found that TLR-induced apoptosis required endosomal localization of TLRs but was independent of MyD88 signaling. Instead, apoptosis required sterile alpha armadillo motif (SARM1), which localized to the mitochondria following TLR activation and was associated with mitochondrial accumulation in neurites. Deficiency in SARM1 inhibited both mitochondrial accumulation and TLR-induced apoptosis. These studies indicate a previously unknown pathway for TLR-mediated neuronal apoptosis and provide a mechanism for how innate immune responses in the CNS directly induce neuronal damage. SARM1 also induces neuronal apoptosis in a Bunyavirus infection model through another PRR pathway, suggesting that SARM1 may be a common mechanism for innate-immune driven neuronal apoptosis. Our current studies are focused on understanding the interaction of SARM1 with other cellular proteins to identify: 1) the mechanism that regulates SARM1 localization to mitochondria, and 2) the mechanism by which SARM1 influences mitochondrial trafficking.
Macrophage-specific deletion of PPARγ impairs lung function and immune responses in an mouse model of Pseudomonas aeruginosa infection (INM8P.363)
Authors: Curtis Brenda , Palmer Jessica , Hoo Melissa , Thomassen Mary , Kovacs Elizabeth
Published: January 2023

Alveolar macrophages (AM) play a pivotal role in response to infection by first releasing pro-inflammatory molecules and phagocytosing pathogens and then resolving inflammation and clearing apoptotic neutrophils. This is accomplished largely based on their ability to switch from a pro-inflammatory to an anti-inflammatory phenotype. PPARγ is a transcription factor important for promoting resolution. To study the role of AM PPARγ in regulating respiratory function and immune responses in the context of bacterial infection, we utilized macrophage-specific PPARγ knockout mice (Mac PPARγ KO). Wild-type (C57Bl/6) or Mac PPARγ KO mice were given intratracheal Pseudomonas aeruginosa (40,000 CFU) or PBS control. After 24 hours, plethysmography was performed, followed by euthanasia. Compared to other groups, infected Mac PPARγ KO mice had impaired respiratory function, with a 5-fold increase in PenH (p<0.05) and a 50% reduction in minute volume and breath rate (p<0.05). Lungs of infected Mac PPARγ KO mice had dramatically increased cellularity, alveolar collapse, and interstitial thickening. Bacterial dissemination to the spleen was observed. Lastly, interleukin-6 levels were 40-fold higher in infected Mac PPARγ KO, compared to infected controls. Together, these findings suggest AM-specific PPARγ loss prolongs inflammation and weakens the ability to clear P. aeruginosa infection.
Lipid metabolism regulator, Acetyl-coA carboxylase: a possible therapeutic target for dendritic cell-based immunotherapy (IRC10P.414)
Authors: Nguyen Thuy , Kim Jung-Sik , Kang Seong-Jun , Park Chung-Gyu
Published: January 2023

Dendritic cells (DCs) are proficient antigen-presenting cells that regulate the activation and suppression of the innate and adaptive immunity. There has been a growing interest in the regulation of DC function by tailoring metabolic requirements in DC-based immunotherapy of cancer. Here we report that targeting acetyl-CoA carboxylase, a key regulator of de novo fatty acid synthesis and lipid oxidation, facilitates the down-modulation of DC functions during the maturation process. DCs treated with ACC blockade, CP-640186, dramatically down-regulated the expression of co-stimulatory molecules including CD80, CD86 and CD40 together with MHC II(major histocompatibility complex II) during LPS-mediated maturation by a dose-dependent manner. Coincidently, the secretion of pro-inflammatory cytokines (IFN-r, IL-6, IL-12) was decreased as well. Blocking fatty acid synthesis by targeting ACC down-regulated the expression of CCR7, a homing receptor of DCs, but up-regulated the expression of CCR5, a chemokine receptor largely expressed in immature DCs. Our data elucidate the role of fatty-acid synthesis in DC maturation and have implications for the development of metabolism based-approaches in DC-mediated immunotherapies.
Dietary vitamin D suppresses delayed type hypersensitivity without effecting bacterial burden during experimental tuberculosis infection (IRC10P.410)
Authors: Reeme Allison , Robinson Richard
Published: January 2023

Mycobacterium tuberculosis (Mtb), the causative agent of the disease tuberculosis (TB), is a significant and extremely successful intracellular pathogen. Protective immunity against Mtb is initiated by phagocytic cells in the lung that activate CD4+ T cells to produce cytokines essential for combating the infection (e.g. IFNγ). However, a negative consequence of this robust T cell response is damaging immunopathology in the host lungs. Recently there is a renewed interest in using vitamin D (VitD) as a host-directed therapy to help treat TB. We sought to understand the effects of VitD on the immune response using an in vivo mouse model of Mtb infection. When C3HeB/FeJ mice were fed a VitD high (VitDHI) or VitD deficient (VitDLO) diet and aerosol infected with the Mtb strain H37Rv, we did not see a significant difference in bacterial burden in the lung or spleen. However, VitDHI mice had significantly less recruitment of lymphocytes to the lung granuloma and decreased production of IFNγ, indicating that VitD is inducing an anti-inflammatory immune environment. Currently, we are using VitD receptor knockout mice to further understand the specific cell types VitD is exerting its effects on to modulate the immune response. Overall the results from these experiments will help define VitD’s effects on the immune response during Mtb infection and help determine if VitD is a reasonable therapeutic option to reduce TB-associated immunopathology.
In vivo activation of the aryl hydrocarbon receptor during viral infection up-regulates immunoregulatory factors in dendritic cells (IRC10P.411)
Authors: Franchini Anthony , Jin Guang-Bi , Boule Lisbeth , Lawrence B.
Published: January 2023

Activation of the aryl hydrocarbon receptor (AHR) substantially dampens host resistance to infection with influenza A viruses. We previously reported that AHR activation negatively regulates the expansion and differentiation of CD8+ cytotoxic T lymphocytes (CTL), and reduces the ability of dendritic cells (DC) to prime naïve CD8 T cells. However, the mechanism by which AHR activation dampens DC function in vivo is unknown. Given that AHR is a ligand inducible transcription factor, we used unbiased gene expression profiling to identify DC specific pathways modulated by in vivo AHR activation. Lung DCs from mice that were exposed to the prototype AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or control were sorted and used for unbias transcriptome analysis with RNA-seq. This approach revealed multiple gene targets of AHR in DCs, which may play a role in modulating DC function. For example, indoleamine-2,3-dioxygenase (Ido1) was highly induced. Accompanying increased Ido1 expression were significant increases in the proportion of plasmacytoid DCs (pDCs) and regulatory T cells. Expanding our findings into an in vitro human system, monocyte-derived DCs from healthy human donors exposed to TCDD and influenza A virus presented a similar suite of altered genes, including elevated Ido1. This is further evidence that AHR activation in DCs intrinsically changes gene expression, inducing an immunoregulatory phenotype that exerts strong influence on the antiviral immune response.
Distinct glucocorticoid sensitivity of Th17 cytokines in murine T hybridomas and primary cells (IRC11P.428)
Authors: Banuelos Jesus , Shin Soon , Lu Nick
Published: January 2023

Cytokine suppression contributes to the anti-inflammatory actions of glucocorticoids (GCs). However, IL-17 has been reported to be resistant to GC regulation in asthma, nasal polyps, and Crohn’s disease. In contrast, a negative GC-response element has been identified in the promoter region of the IL-17 gene and IL-17 was sensitive to GC suppression in a murine asthma model. To clarify whether IL-17 and other Th17 cytokines are sensitive to GC regulation, we generated Th17 hybridoma cells by fusing in vitro differentiated murine (bl/6) Th17 cells with BW5147 thymoma. Two clones selectively expressing IL-17A, IL-17F, IL-22 and GM-CSF were stable for over 30 generations. Additional clones selectively expressing IFN-γ or IL-4, but not Th17 cytokines and BW5147 cells that produce little or no cytokines were used as controls. Dexamethasone, a potent GC, significantly inhibited anti-CD3 and anti-CD28-stimulated IFN-γ, IL-4, as well as IL-17A, IL-17F, and IL-22 in various clones. Interestingly, IL-17A and IL-17F, but not IL-22, were resistant to GC suppression in in vitro differentiated Th17 cells. Furthermore, hybridomas, but not in vitro differentiated Th17 cells underwent GC-induced apoptosis. IL-6 partially rescues hybridomas from GC-induced apoptosis, however it did not modulate IL-17 suppression by GCs. Our findings support that IL-17A and IL-17F are capable of being downregulated by GCs and Th17 cell microenvironment and cellular milleu engender GC resistance.
Progressive differentiation of follicular B helper T cells regulates the germinal center response (IRC11P.430)
Authors: Herman Edward , Weinstein Jason , Lainez Begoña , Licona-Limón Paula , Esplugues Enric , Flavell Richard , Craft Joseph
Published: January 2023

Initiation of a thymus-dependent humoral immune response requires interaction of activated B cells and follicular B helper T (Tfh) cells, a specialized subset of CD4 T cells. Differentiation of Tfh cells results in their localization to the B-cell follicle and germinal center (GC) of secondary lymphoid organs and expression of the transcription factor Bcl6. Here we show Tfh cells differentiate further within the follicle and GC, with development of discrete subsets that regulate distinct aspects of B-cell maturation. Using mice expressing reporter genes for IL-21 and IL-4, we found that Tfh cells initially produce IL-21 mRNA and protein and support selection of high-affinity B cell mutations. Gradually, Tfh cells began to produce IL-4 and while ceasing production of IL-21, and demonstrated the ability to induce class-switch recombination and development of antibody-secreting cells. These changes in phenotype and function occurred as these Tfh21- and Tfh4-subsets exhibited differences in global gene expression as well as localization within the GC. These data support a model in which ongoing Tfh-cell differentiation orchestrates the formation of an effective antibody response.