bluuebunny/crossref_metadata_2025 huggingface dataset:
The variation of CD3+CD4-CD44HighCD62L- T cells in the development of asthma in mice (HYP7P.302)
Authors: Li Yanchun , Hua Shucheng , He Xiaoyan , Li Yun , Zhu Yaping
Published: January 2023

Objective CD44HighCD62L- is the marker for effector memory T cell. It is well-known that CD4+ T memory cells (Tms) play important role in asthma, but the role of CD8+ T memory cells (Tms) in asthma remains unknown. Here, we analyzed the variation of CD4-Tms in asthmatic mice so to have a general understanding of the role of CD8+Tms in asthma indirectly. Method To construct asthma mice model, BALB/c mice were sensitized and challenged with OVA for continuous 7 days. We detected the ratio of CD3+CD4-CD44HighCD62L- T cells in CD3+CD4-T cells from 1 day before challenge to 1 month after challenge by using a flow cytometry. Results In lungs, the ratio of CD4-Tms increased gradually after 1st challenge from 23.56% to 30.245% and reached the peak value with 61.97% at 2nd day after the last challenge. Interestingly and notably, it decreased rapidly to the level before challenge after that. This is quite different from CD4+Tms in which CD4+Tms maintained high level for much longer time. In spleens, the ratio of CD4-Tms maintained low level with about 20% during challenge. It increased to 35.86% after the last challenge and maintained at a stable level of 30% for 2 week then returned to the level before challenge. This variation is also different from CD4+Tms in spleens which is almost synchronize with CD4+Tms in lungs. Conclusion These results suggest that CD8+Tm may play a role in asthma with different mechanism from CD4+Tm, the detailed mechanisms warrant further exploration.
Signaling pathways in eosinophils that regulate allergic asthma and inflammation (HYP7P.304)
Authors: Stokes Kindra , LaMarche Nelson , August Avery
Published: January 2023

Allergic asthma and inflammation is a disease of the airways. The prevalence of allergic responses and asthma are increasing worldwide, but it is still unclear why some predisposed individuals develop disease. Eosinophils and T helper 2 cells (TH2) and eosinophils are critical cellular mediators in asthma. The signal transducer and activator of transcription (STAT)-6 is a crucial mediator of TH2 cytokine responses, acting downstream of interleukin -4 and -13 (IL-4 and IL-13). We have previously shown that eosinophil derived IL-13 plays an important role in the recruitment of T cells to the lung, and subsequent development of allergic asthma. However whether eosinophils respond to TH2 signals to control allergic airway inflammation is unclear. The objective of this study is to understand the role of STAT6 in eosinophil-dependent regulation of allergic asthma and inflammation. Using ΔdblGATA mice lacking eosinophils and transfer of STAT6-/- eosinophils, we found that compared to ΔdblGATA reconstituted with WT eosinophils, eosinophils lacking STAT6 were unable to rescue recruitment of CD4+ T cells and eosinophils into the lungs compared to WT eosinophils. These data indicates that STAT6 is important in the recruitment of both T cells and eosinophils to the lung during the development of allergic airway inflammation. Ultimately, deciphering the mechanisms of intracellular and cytokine signals will afford us improved insights on developing therapeutic strategies in asthma.
Fungal aeroallergen challenge results in increased granulocytic inflammation in Jh-/- mice in a murine model of allergic airway disease (HYP7P.280)
Authors: Ghosh Sumit , Asbach Scott , Hoselton Scott , Dorsam Glenn , Schuh Jane
Published: January 2023

B lymphocytes are well recognized in the development of IgE responses, which exacerbates asthma symptoms, but may also be important as a protective response against inhaled fungi. Further, these lymphocytes may play a direct role in the maintenance of airway wall fibrosis. Jh-/- mice have been used extensively to study the role of B lymphocytes in immune responses. These mice lack the Jh gene, which codes for the antibody heavy chain and is thought to be critical for B cell survival and function. In this study, we examined the role of B lymphocytes using a murine fungal aeroallergen model to mimic human fungal allergic airway disease. We compared disease progression in the BALB/c and Jh-/- mice and found no differences in airway hyperresponsiveness, collagen deposition, or overall pulmonary inflammation. However, the Jh-/- mice had a significantly higher number of neutrophils and eosinophils when compared to the BALB/c mice. IL-17A and IL-6 cytokines, which play a role in driving eosinophilia and neutrophilia, were also significantly higher in the Jh-/- mice after fungal challenge. We plan to characterize temporal activation and the relative contribution of these B lymphocytes in the maintenance of Tregs and Th17 lymphocytes within the pulmonary compartment. These studies will help expand our knowledge of how B lymphocytes regulate T lymphocyte responses in allergic asthma.
The Nrf2 activator, tBHQ, exacerbates immediate hypersensitivity response to food allergen (HYP7P.315)
Authors: Rockwell Cheryl , Gangur Venugopal , Pestka James , Para Radhakrishna , Turley Alexandra , Zagorski Joseph , Bursley Jenna , Dover Heather
Published: January 2023

Nuclear erythroid 2 related factor 2 (Nrf2) is a transcription factor activated by cell stress, including oxidative and electrophilic stimuli, resulting in the upregulation of cytoprotective genes. Nrf2 can be activated experimentally by a number of different pharmacological agents, including tert-butylhydroquinone (tBHQ), which is also used commercially as a food additive. We recently showed that activation of Nrf2 by tBHQ promotes Th2 differentiation and inhibits Th1 differentiation in isolated CD4+ T cells. Given that tBHQ is a food additive and promotes Th2 differentiation, the purpose of the present studies was to determine the effect of tBHQ in a mouse model of food allergy. Mice were administered a diet either with or without tBHQ (0.001%) for two weeks prior to sensitization with ovalbumin. Notably, this dose of tBHQ is lower than that normally present in standard rodent chow (0.0016%). The mice were sensitized weekly transdermally for 4 weeks. Sensitization to ovalbumin caused an increase in ovalbumin-specific IgE in plasma, which was greater in animals exposed to tBHQ. After sensitization, the mice were orally challenged with ovalbumin, causing mild/moderate immediate hypersensitivity, which was exacerbated by tBHQ. Overall, these studies suggest that low doses of the food additive, tBHQ, increase IgE response to food allergen and exacerbate clinical signs of immediate hypersensitivity. (This work was funded by NIH grant: ES018885.)
IgE and FcεRIα do not promote allergic airway disease in mice inoculated intratracheally with house dust mite extract (HYP7P.316)
Authors: McKnight Christopher , Perkins Charles , Finkelman Fred
Published: January 2023

The role of IgE in asthma pathogenesis is controversial. Early studies showed IgE contributed to allergic airway disease (AAD) when mice were sensitized passively by anti-ovalbumin (OVA) IgE or sensitized actively by OVA without adjuvant. Other groups found that IgE did not promote AAD that was provoked by fungal extracts or by OVA with adjuvant. We have used IgE-deficient mice and FcεRIα-deficient mice and their respective FVBN and BALB/c wild-type controls to study the IgE- and FcεRIα-dependence of AAD induced by a clinically relevant allergen and route of administration. Mice were inoculated intratracheally with house dust mite extract without adjuvant every 2 days for 7 doses. We measured airway hyperresponsiveness by both unrestrained plethysmography (Buxco) and forced oscillation (flexiVent), analyzed pulmonary inflammation by differential cell count on BALF, and sectioned lungs to quantitate goblet cell metaplasia by microscopic examination. We found no difference in any of these disease parameters between wild-type mice and gene-deficient mice, regardless of whether minimal, moderate, or more severe AAD was induced. These results complement the clinical observation that anti-IgE therapy fails to improve the underlying lung function of humans with long standing asthma, although this treatment does reduce acute exacerbations. We are currently studying the acute changes in lung function that occur upon inhalation of allergen in AAD and their dependence on the IgE axis.
Dermal exposure to the commonly used antimicrobial chemical triclosan promotes allergic responses in skin and lung (HYP7P.317)
Authors: Marshall Nikki , Long Carrie , Anderson Katie , Lukomska Ewa , Anderson Stacey
Published: January 2023

There has been a dramatic increase in the incidence of allergies and asthma in the U.S. thought to be influenced by environmental exposures. The antimicrobial chemical triclosan is found in countless products such as soap, lotion, toothpaste and kitchen utensils (0.1-1%). Triclosan is detected in the urine of the majority of Americans and recent epidemiological studies have found a positive association between urinary triclosan levels and allergic disease. Interestingly, exposure to triclosan, a non-sensitizer, enhanced allergic responses in a mouse ovalbumin (OVA) asthma model. To help elucidate the mechanisms behind the augmented allergic responses, triclosan (0-3%) was applied to the dorsal surface of the ears of BALB/c mice sensitized to OVA. Analysis of skin revealed infiltration of leukocytes and a dramatic increase in thymic stromal lymphopoietin (TSLP) expression even after a single dose. Analysis of cells in the draining LN of triclosan-exposed mice revealed a peak cell response at 7 days, including increased numbers and frequencies of GATA-3+ IL-4+ CD4 T cells, and CD11b+ CD80+ CD86+ MHC class II+ dendritic cells. Mice exposed to triclosan and challenged with OVA via airway aspiration also showed increased Th2 responses and OVA+ APC in their BAL fluid. These results suggest that triclosan augments allergic responses in skin and lung in part through mechanisms that involve TSLP and enhanced Th2 responses.
Reduced C epsilon transcription by stabilization of CD23 in B cells (HYP7P.320)
Authors: Fellmann Marc , Buschor Patrick , Röthlisberger Silvan , Stadler Beda , Vogel Monique
Published: January 2023

The low affinity receptor for IgE (CD23, FcepsilonRII) is part of the broad regulatory network controlling IgE synthesis. It occurs as membrane and as soluble receptor generated in vivo by specific proteolysis. Targeting membrane CD23 with either IgE or anti-CD23 antibodies is believed to inhibit IgE synthesis. However, the mechanism behind this negative feedback remains unclear. This mechanism was investigated by examining the effect of anti-CD23 molecules in form of designed-ankyrin-repeat-proteins (DARPins) specific for CD23. Previous experiments using anti-CD23 antibodies were difficult to interpret as their Fc portion also mediate feedback mechanisms. The DARPins were produced as monovalent and bivalent molecules that were able to compete with the binding of IgE to CD23. The effects of anti-CD23 DARPins were assessed in an in vitro system using anti-CD40/IL-4 stimulated B cells. Treatment of B cells with anti-CD23 DARPins resulted in stabilization of membrane CD23. The released amount of soluble CD23 was reduced while CD23 mRNA expression was unchanged suggesting that the anti-CD23 DARPins stabilize CD23 and prevent its proteolysis. IgE secretion was significantly reduced by anti-CD23 DARPins. In addition, analysis of the epsilon transcript confirmed the reduction of IgE synthesis whereas IgG secretion was not influenced by the anti-CD23 DARPins. Further experiments will be performed to assess signalling pathways triggered by CD23 cross-linking.
The association of complement protein C1q with apoptotic cells ingested by human dendritic cells modulates dendritic cell-mediated T cell activation (INC5P.332)
Authors: Clarke Elizabeth , Tenner Andrea
Published: January 2023

Humans deficient in complement protein C1q develop systemic lupus erythematosus (SLE) with nearly 100% penetrance, but the molecular mechanisms underlying this association have not yet been uncovered. Our previous studies have shown that human monocyte-derived immature dendritic cells (DC) ingesting C1q-bound autologous apoptotic cells (AC) (C1q-polarized DC) increase secretion of anti-inflammatory cytokines such as IL-10 and reduce pro-inflammatory cytokines such as TNFα. In this study, we demonstrate that C1q-polarized DC express significantly more surface PD-L2 and CD39 and significantly less CD86 relative to DC ingesting AC alone. To elucidate whether C1q-polarized DC functionally modulate the adaptive immune responses, human autologous T cells were incubated with DC or C1q-polarized DC and the proliferation and induction of CD4+ and CD8+ T cell subset was assessed. We discovered that relative to DC ingesting AC in the absence of C1q, C1q-polarized DC decrease autologous Th17 proliferation and induction and Th1 proliferation when the T cells were maintained in IL-7 prior to the MLR. These data imply that C1q-polarized DC can switch T cell function to reduce T effector cell activation and/or suppress the induction of autoimmunity, identifying new potential pathways for therapeutic intervention in SLE and other inflammatory diseases.
Deficiency in transient receptor potential melastatin 2 impairs alveolar macrophage activation and protects mice against endotoxin-induced lung inflammation and injury (INC5P.333)
Authors: Botta Davide , Mudunuru Uma , Southworth Kristen , Simpler Thomas , Bradley John , Randall Troy , Stevenson Christopher , Lund Frances
Published: January 2023

Cellular responses to reactive oxygen species (ROS) are induced by many receptors including the non-selective cation channel TRPM2 that promotes cation influx and membrane depolarization following ROS exposure, and potentiates inflammatory cytokine production and cell death. Conflicting reports on the harmful vs. protective roles of TRPM2 in ROS-mediated disease models led us to test whether TRPM2 is required for protection against lung endotoxin exposure, one of the most common etiologies of Acute Lung Injury (ALI). We found that Trpm2-/- mice exposed to intranasal lipopolysaccharide (LPS) were protected from lung inflammation and injury and that protection was due to loss of TRPM2 in radiation-resistant lung cells. Next, we examined the role of TRPM2 in radiation-resistant alveolar macrophages (AMs) as these cells are known contributors of ROS in ALI. Using congenic mice harboring the CD45.1/CD45.2 allelic variants we generated bone marrow chimeras in which radiation-resistant host and donor-derived AMs could be distinguished. We observed increased resistance to cell death in host Trpm2-/- AMs. Furthermore, LPS-activated Trpm2-/- AMs showed decreased expression of iNOS, inflammatory cytokines and proteases. Macrophage depletion experiments are underway to investigate the protective role of Trpm2-/- AMs in vivo. Our data support a harmful role of TRPM2 in ALI, and attribute the protective phenotype of Trpm2-/- mice to impaired macrophage-mediated lung inflammatory responses.
Changes in zebrafish phagocytic activity induced by acute ethanol exposure vary by time of day and leukocyte type (INC5P.327)
Authors: Macklis Paul , Curran Kaitlin , Newton-Acquah Antoinette , Lopez Juvenal , Hannum Lynn
Published: January 2023

Chronic alcohol abuse and acute alcohol consumption (binge drinking) have been associated with suppression of immune function. Reduced phagocytic activity in macrophages and altered levels of inflammatory mediators have been reported in mammalian studies of ethanol exposure. To explore whether the zebrafish model could be valuable in understanding the effects of ethanol on innate immune activity, we assessed the activity of zebrafish phagocytes after acute exposure to ethanol in tank water. Experiments were conducted at different times of day to also determine whether effects of ethanol vary in a diurnal pattern. Treating wild-type AB strain fish with ethanol in the morning reduced the percentage of total phagocytic cells that ingested bacteria. The opposite results were observed in the evening, when a higher proportion of cells participated in phagocytosis compared to controls. To distinguish between the activity of macrophages and neutrophils, mpx:GFP zebrafish were also used. Exposure to ethanol in tank water resulted in a reduction in the amount of bacteria engulfed by neutrophils, whereas changes in macrophage phagocytic activity were not as evident. These initial results indicate that ethanol affects subsets of phagocytic cells differently, and that the effects may be time-of-day dependent.
Activation-specific metabolic regulation of NK cell IFN-gamma production (INC6P.334)
Authors: Cooper Megan , Keppel Molly , Topcagic Nermina
Published: January 2023

Many factors that stimulate NK cell functional responses have been described; however metabolic fuels that drive NK cell function are unknown. Metabolism is significantly altered in disease states and metabolic regulation of T cell function is well-described. We hypothesize that NK cells require distinct metabolic fuels for survival, proliferation, and functional responses. NK cell metabolic phenotype was determined with an extracellular flux analyzer to determine basal oxidative phosphorylation (oxphos) and glycolysis of resting and activated NK cells. NK cells were then cultured under a variety of conditions using inhibitors and nutrient deprivation to determine cellular requirements for glucose, fatty acids, and oxphos for NK cell survival, proliferation, and function. NK cells primarily utilize oxphos at baseline and, in contrast to T cells, upon proliferation. However, there is significant plasticity in the metabolic requirements for NK cell IFN-gamma production. NK cell IFN-gamma production in response to cytokine stimulation is not dependent on either glycolysis or oxphos. By contrast, NK cell IFN-gamma production in response to receptor stimulation requires oxphos and is partially dependent on glycolysis. The metabolic requirements for NK cell IFN-gamma production are not altered by priming in vitro or in vivo, but do change with proliferation. These findings suggest that the metabolic microenvironment in vivo may significantly alter NK cell function.
Increased IRAK-M in peritoneal macrophages of NOD mice suggests in vivo exposure to bacterial endotoxins from the gut (INC6P.343)
Authors: Emani Rohini , Alam Catharina , Hanninen Arno
Published: January 2023

Macrophages play an important role in innate immune reactions, and peritoneal macrophages guard the sterility of this compartment mainly against microbial threat from the gut. Type-1 Diabetes (T1D) is an autoimmune disease in which gut microbiota and gut immune system appear to play a role. We have observed increased permeability and free radical production of gut epithelium in nonobese diabetic (NOD) mice. Impaired barrier function could lead to bacterial endotoxin leakage to peritoneal cavity. To seek for extraintestinal consequences of impaired barrier function, we characterized peritoneal lavage cells from young NOD mice after weaning. We detected a rapid increase in the number of macrophages in 1-2 weeks from weaning in NOD mice compared to C57BL/6 and BALB/c mice, and to NOD mice fed an antidiabetogenic diet (ProSobee), which improves gut barrier function. Upon LPS stimulation ex vivo, macrophages produced low amounts of TNF-α and showed reduced expression of CD40. Expression of TLR signalling pathway negative regulator, Interleukin-1 Associated Kinase -M (IRAK-M) was increased, indicating prior in vivo exposure to TLR-4 ligand(s). Upon further phenotyping, increased levels of ARG1 were observed suggesting in vivo differentiation towards M2-like phenotype. Bacterial endotoxins in peritoneal cavity may contribute to autoreactive T-cell activation by increased production of cytokines and activation of antigen-presenting cells in pancreatic lymph nodes.
NKp46 modulates NK immune synapse function (INC6P.346)
Authors: Hadad Uzi , Thauland Timothy , Butte Manish , Porgador Angel , Krams Sheri
Published: January 2023

Natural killer cells play an important role in the first-line defense against tumor and virus-infected cells. The activity of NK cells is tightly regulated by a repertoire of cell-surface expressed inhibitory and activating receptors. NKp46 is a major NK cell activating receptor that is involved in the elimination of viral infected cells. NKp46 also regulates interactions with other immune cells including T cells and dendritic cells. Recent studies revealed that complex integration of NK receptor signaling controls the cytoskeleton rearrangement and other immune synapse related events. However the distinct nature by which NKp46 participates in NK immunological synapse formation and function remains unknown. In this study we found that NKp46 forms microcluster structures at the immune synapse between NK cells and target cells. Labeling of F-actin showed that higher expression of NKp46 is correlated with increased accumulation of actin mesh at the immune synapse. Concordantly, knock-down of NKp46 in primary NK cells abridged recruitment of F-actin to the synapse. Live cell imaging experiments showed a linear correlation between NKp46 expression and lytic granule polarization to the immune synapse. Taken together, our data suggests that NKp46 signaling directly effects both the early formation and late function of the NK lytic immune synapse. Moreover, this is a continuous rather than threshold effect of activation vs non-activation.
Inhibition of PI3K contributes to nanoparticle-mediated exaggeration of endotoxin-induced leukocyte procoagulant activity (INC6P.340)
Authors: Ilinskaya Anna , Man Sonny , Patri Anil , Clogston Jeffrey , McNeil Scott , Dobrovolskaia Marina
Published: January 2023

Nanoparticles are increasingly used in biomedical applications due to their ability to change biodistribution, increase safety and improve efficacy of drugs. However, application of engineered nanomaterials in medicine is often halted by safety concerns. Endotoxin contamination is very common in nanomaterials and there is an increasing number of studies reporting that certain nanoparticles can exaggerate endotoxin-mediated inflammation. We studied the relationship between physicochemical properties of polyamidoamine (PAMAM) dendrimers and their ability to exaggerate LPS-induced procoagulant activity (PCA) of human leukocytes in vitro. The PCA is phospholipid-protein complex composed of tissue factor (TF) and phosphotidylserine (PS). We found that cationic but not anionic or neutral PAMAM dendrimers, increase LPS-induced leukocyte PCA. Cationic PAMAM dendrimers enhanced both LPS-induced TF expression and PS exposure on the cell surface. The mechanism of LPS-induced PCA exaggeration by PAMAM dendrimers is not mediated by the electrostatic interaction between cationic dendrimers and anionic LPS, calcium influx and secondary messengers released by dendrimer activated platelets. We hypothesize that dendrimers exaggerate LPS-mediated PCA through the inhibition of negative regulation of inflammation. Specifically, we demonstrate that cationic PAMAM dendrimers inhibit phosphoinositol-3-kinase (PI3K), a negative regulator of TF expression in response to endotoxin.
Proteasome defects in candle patients’ cells lead to IFN production in both hemopoietic and non-hemopoietic cells (INM6P.416)
Authors: Liu Yin , Biancotto Angelique , Huang Yan , Gadina Massimo , McCoy John Philip , Goldbach-Mansky Rapheala
Published: January 2023

CANDLE (Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated Temperature) syndrome is an autoinflammatory disease caused by autosomal recessive mutations in PSMB8 and other subunits of the proteasome. Many IFN regulated genes in CANDLE patients’ whole blood were strongly upregulated and patient monocytes showed STAT1 hyperactivation after IFN-γ stimulation. We hypothesized that cellular stress in CANDLE patients leads to IFN production and asked whether it is restricted to hematopoietic cells. We studied IFN production in PBMCs and fibroblasts from CANDLE patients or in healthy cells treated with proteasome inhibitor epoxomicin. We found increased production of IFN-α and IFN-γ in NK, T and dendritic cells from the two CANDLE patients tested compared with healthy controls. Increased IFN production was reproduced in PBMCs of healthy individuals treated with epoxomicin. We also observed increased IFN-α message in CANDLE patients’ fibroblasts compared to healthy controls. Similarly, epoxomicin treated healthy fibroblasts increased IFN-α message. Interestingly message for other inflammatory cytokines, including TNF-α, IL-1 and IL-6, were either not change or even suppressed. Microarray study showed that IFN regulated genes were upregulated in sorted T, B, NK cells, monocytes and neutrophils. We conclude that proteasome defect causes IFN production in both hemopoietic and non-hemopoietic cells in CANDLE patients that can lead to increased IFN signaling.
Distinct actions of oligomers and fibrils of islet amyloid polypeptide on Toll-like receptor 2 and NLRP3 inflammasome activation (INM7P.423)
Authors: Westwell-Roper Clara , Ehses Jan , Verchere Bruce
Published: January 2023

Aggregation of islet amyloid polypeptide (IAPP) to form amyloid contributes to beta cell dysfunction in type 2 diabetes. Human but not non-amyloidogenic rodent IAPP activates TLR2 and NLRP3 to induce IL-1β secretion by islet macrophages. It is unclear which form of IAPP aggregate induces IL-1β and whether this pathway is relevant to disease pathogenesis. We therefore evaluated the contributions of soluble and fibrillar IAPP to IL-1β secretion by bone marrow-derived macrophages (BMDMs) and assessed the effects of IL-1 receptor antagonist (IL-1Ra) on glucose homeostasis in transgenic mice with beta cell expression of human IAPP. BMDMs treated with soluble but not fibrillar IAPP provided a TLR2-dependent priming stimulus for ATP-induced IL-1β secretion, suggesting that pre-fibrillar species interact with TLR2. Fibrillar but not soluble IAPP aggregates were required for IL-1β secretion in LPS-primed BMDMs and are therefore likely the principal stimulus for NLRP3. Moreover, IL-1Ra (50 mg/kg/d for 8 weeks) improved glucose tolerance in obese human IAPP transgenic mice (AUC=1360±110 vs. 880±70; p<0.05) with no effect in wild-type littermates. These data provide the first in vivo evidence that IAPP-induced islet dysfunction in type 2 diabetes is mediated by IL-1, and suggest that both soluble and fibrillar IAPP aggregates participate in this process. Islet inflammation and amyloid formation are therefore important therapeutic targets to improve beta cell function in type 2 diabetes.
Silencing airway epithelial cell-derived hepcidin exacerbates acute lung injury by regulating the intracellular iron status in alveolar macrophages (INM7P.434)
Authors: Chen QiXing
Published: January 2023

Objective: Hepcidin is a β-defensin-like antimicrobial peptide and acts as a principal iron regulatory hormone. Hepcidin is mostly produced by hepatocytes, but is also expressed by other cells, such as airway epithelial cells. However, nothing is known about its function in lung infectious and inflammatory disease such as acute lung injury. Methods: Acute lung injury was induced by polymicrobial sepsis via cecal ligation and puncture surgery. Adenovirus-mediated short hairpin RNA specific for the mouse hepcidin gene hepc1 and control adenovirus were intratracheally injected into mice twice. The adenovirus-mediated knockdown of hepcidin in airway epithelial cells was evaluated in vivo. Lung injury and the 7-day survival rate were assessed. The local and systemic iron content was assayed. Results: The hepcidin level in airway epithelial cells was upregulated during polymicrobial sepsis. The knockdown of airway epithelial cell-derived hepcidin aggravated the polymicrobial sepsis-induced lung injury and pulmonary bacterial infection and increased the mortality. Moreover, the knockdown of hepcidin in airway epithelial cells led to reduced ferroportin degradation and a low intracellular iron content in alveolar macrophages, but did not affect the STAT3/SOCS3 axis. Conclusions: Airway epithelial cell-derived hepcidin plays an important role in protection against acute lung injury via regulating the intracellular iron status in alveolar macrophages.
Blockade of mTOR in innate immune system represents an effective strategy to boost Toll-like receptor-induced Th1 response for vaccine (INM7P.428)
Authors: Xiao Hui , Zang Aiping , Ma Dapeng , Yuan Chuangping , Du Min , Huang Zhong , Xiao Jianhua , Lan Ke , Li Xiaoxia
Published: January 2023

Development of better adjuvant is urgently needed for vaccines whose efficacy depends on potent Th1 response. While TLR ligands represent the most promising adjuvants for Th1 response, induction of only suboptimal IL-12 but evident IL-10 in DCs may limit their efficacy. In this study, we attempted to test the notion that maximizing TLR-induced IL-12 versus IL-10 ratio in innate immune system can be a viable approach to elicit robust Th1 response for vaccine. Systemic screening identified a dozen of small molecule compounds capable of elevating LPS-induced IL-12p70 production in DCs. Interestingly, blocking mTOR directly by Rapamycin, or indirectly by compounds targeting mTOR’s upstream regulator PI3K or ERK, respectively, was able to consistently increase IL-12 but decrease IL-10 induction in LPS-stimulated DCs and macrophages. Our study further demonstrated that blocking mTOR in the innate immune system by a variety of compounds can effectively improve MPL-induced Th1 and antibody responses for HBV vaccine. Moreover, we identified a subset of transcriptional factors whose expression in innate immune cells depends on mTOR activation, whereby revealing novel mechanisms by which mTOR regulates TLR-induced IL-12 and IL-10 expression. In summary, our results suggest that maximizing IL-12 vs IL-10 ratio in innate immune system by blocking mTOR can be a novel approach to boost TLR-induced T cell response for vaccine.
Allele-specific induction of interleukin-1beta expression by CEBP-beta and PU.1 contributes to increased tuberculosis susceptibility (INM8P.451)
Authors: Chen Xinchun , Zhang Guoliang , Zhou Boping , Li Shaoyuan , Wen Yuxin , Yang Hui , Zhan Senlin , Zhang Mingxia , Zeng Gucheng , Feng Carl , Sassetti Christopher
Published: January 2023

Mycobacterium tuberculosis infection is associated with a spectrum of clinical outcomes, from long-term latent infection to different manifestations of progressive disease. Pro-inflammatory pathways, such as those controlled by IL-1beta, have the contrasting potential both to prevent disease by restricting bacterial replication and to promote disease by inflicting tissue damage. Thus, the ultimate contribution of individual inflammatory pathways to the outcome of M. tuberculosis infection remains ambiguous. In this study, we identified a naturally-occurring polymorphism in the human IL1B promoter region, which alters the association of the CEBPbeta and PU.1 transcription factors and controls the expression of IL-1β. The high-IL-1β expressing genotype was associated with the development of active tuberculosis, the severity of pulmonary disease and poor treatment outcome in TB patients. Higher IL-1β expression did not suppress the activity of IFN-γ-producing T cells, but instead correlated with neutrophil accumulation in the lung. These observations support a specific role for IL-1β and granulocytic inflammation as a driver of TB disease progression in humans, and suggest novel strategies for the prevention and treatment of tuberculosis.
Actelated dextran microparticle delivery as a means to generate regulatory myeloid cells (IRC5P.470)
Authors: Papenfuss Tracey , VanGundy Zachary , Strange Heather , Bachelder Eric
Published: January 2023

Myeloid cells (MC) have potent regulatory abilities but factors influencing the development of such regulatory myeloid cells (MC-regs) remain poorly understood. Retinoic acid (RA) is a metabolite of vitamin A that has regulatory abilities and impacts myeloid cell differentiation. We have previously shown that RA produces MC-regs that expressed increased IL-10, regulatory T (Treg) cells, and were able to suppress the proliferation and cytotoxicity of CD4+ and CD8+ T cells, respectively. We hypothesize that encapsulation of RA within acetalated dextran (AC-DEX) microparticles (MPs) can be used for targeted generation of MC-regs in vivo. We used MPs containing encapsulated FITC-labeled BSA to determine the relative uptake of Ac-DEX MPs by myeloid cell populations in vitro and in vivo. We found that labeled MPs were readily taken up by macrophages, DCs and myeloid progenitors in vitro. Following in vivo injection, increased percentage of FITC+ cells was seen in both CD11c+CD11b- and CD11c-CD11b+ cells in draining lymph nodes (DLN) over controls of free FITC or empty MP administration. When RA was encapsulated into MPs, CD11b+ cells within the DLNs had a increased PD-L1 expression and proliferation in DLNs was decreased. These results suggest that in vivo targeted delivery of regulatory compounds by Ac-DEX MPs can induce MC-regs which may be useful to treat inlammatory and immune-mediated diseases.
In vivo role of HMGB1 in endotoxemia and bacterial infection (IRC5P.454)
Authors: Matsuda Atsushi , Yanai Hideyuki , Taniguchi Tadatsugu
Published: January 2023

High-mobility group box protein 1 is nuclear protein conserved among almost all eukaryotic cells. HMGB1 is thought to be a danger signal mediator when it is released both actively from activated immune cells and passively from dead cells into extracellular milieu upon pathogen infections or sterile inflammations. Released HMGB1 act as an inflammatory cytokine and promote inflammatory disorders. However, in vivo function of HMGB1 is still elusive since Hmgb1 gene-deficient mice are lethal. In this study, by using Hmgb1 gene conditional knockout mice which we have established recently, we examined the role of HMGB1 in innate immune responses and inflammation in vivo. We successfully generated Hmgb1fl/flLysM-Cre+ mice because it has been reported that HMGB1 is released from myeloid cells. They developed without significant anomaly and HMGB1 is deleted from macrophages. Interestingly, we observed that, in LPS induced endotoxin shock model, Hmgb1fl/flLysM-Cre+ mice became more sensitive while plasma HMGB1 level of Hmgb1fl/flLysM-Cre+ mice is comparable with wild-type control. These mice also became more susceptible to bacterial infection. In this poster session, we would like to discuss these and recent data further.
Suppressive monocytes: homeostatic mechanism to restrain chronic intestinal inflammation. (IRC5P.455)
Authors: Ostanin Dmitry , Kurmaeva Elvira , Bhattacharya Dhruva
Published: January 2023

Chronic intestinal inflammation in patients with inflammatory bowel diseases (IBD) is fueled by complex and poorly understood interactions between T cells and myeloid cells that are constantly recruited from blood into the affected regions of the gut. We undertook this study to characterize the interplay between colitis-induced myeloid cells and CD4 T cells in CD45RBhigh T cell-induced colitis model. Within massively expanded CD11b+Gr-1+ splenocytes in colitic mice, only Ly6Chigh inflammatory monocytes suppressed proliferation and production of cytokines by CD4 T cells. Suppression was mediated by cell-contact, nitric oxide (NO), and partially by IFN-γ and prostaglandins. Ly6Chigh cells isolated from colitic colons showed upregulation of inducible NO synthase (iNOS) and arginase-1 and were more potent suppressors than those from spleen. Morphological and phenotypical analysis revealed that colon Ly6Chigh cells were macrophage-like (CD115-CD64+) but did not express MHC-II, CD11c, or F4/80. They secreted IL-10 and IL-1β/IL-6 and, upon co-culture with naïve T cells, promoted their differentiation into foxp3+ T cells and Th17 cells, respectively. Taken together, recruitment of inflammatory monocytes into inflamed intestine enhances their suppressive properties and may also arrest their further differentiation. Thus, monocyte recruitment to areas of chronic inflammation may represent an important homeostatic mechanism aimed at restraining and/or reshaping T cell responses.
Characterization of human CS1 gene promoter in NK and B cells (IRM7P.485)
Authors: Mathew Stephen , Kim Jongrok , Mathew Porunelloor
Published: January 2023

CS1 (CRACC, SLAMF7 or CD319) is a member of SLAM (Signaling Lymphocyte Activation Molecule) family receptors and is expressed on NK cells, CD8+ T lymphocytes, mature dendritic cells and activated B cells. Unlike other SLAM members, CS1 shows SAP (SLAM-associated protein) independent but EAT-2 (Ewing’s Sarcoma associated transcript) dependent signaling in human NK cells. CS1 has been shown to be overexpressed in multiple myeloma (MM) and a humanized monoclonal antibody against CS1 is in phase 2 clinical trial for MM. In this study, we analyzed the transcriptional regulation of human CS1 gene. In contrast to the multiple transcription initiation sites in human and mouse 2B4 genes that are driven by TATA-less promoters, we found that proximal region of CS1 promoter contains CAAT box and atypical TATA-box that might result in common transcription initiation at -29 nucleotides upstream of the ATG translation start codon. Relative luciferase activities of successive deletion mutants of CS1 promoter were different between Blimp-1-positive YT cells and Blimp-1-negative DB cells. Our electrophoretic mobility shift assay indicates that Blimp-1 binds to putative Blimp-1 binding site at -750 to -746 region of CS1 promoter. This suggests a possible activating role of Blimp-1 transcription factor in certain circumstances. Understanding CS1 gene expression and regulation will help in developing immune based therapy for B cell-mediated diseases, such as SLE and MM.
FCRL5 promotes T cell-independent innate-like B cell responses and counter-regulates tolerance through the Lyn/SHP-1 circuit (IRM8P.717)
Authors: Becker Eugene , Zhu Zilu , Won Woong-Jai , Dalla-Favera Riccardo , Davis Randall
Published: January 2023

The earliest phases of humoral defense are orchestrated by splenic marginal zone (MZ) and body cavity-derived B1 B cells. These frontline responders have been termed “innate-like” given their broadly-reactive natural antibody repertoires and capacity for rapid responses to T cell-independent (TI) antigens. Despite their key contributions to primary responses, why these cells possess such prompt responsivity to innate-stimulation is poorly understood. The discovery of a family of Fc receptor-like (FCRL) molecules with homology to the classical FCRs for IgG and IgE, complex tyrosine-based function, and preferential B cell expression has introduced a new level of humoral regulation. Although their in vivo functions have not yet been investigated, human FCRL (hFCRL) members are becoming increasingly appreciated for their associations with immune-related diseases. In particular, hFCRL3 has emerged as a generalized risk factor in autoimmunity. To better understand the role of hFCRL3 in B cells, we investigated its closest relative mouse FCRL5 (mFCRL5). This molecule shares homologous extracellular domains, binary tyrosine-based signaling, and innate-like B cell expression. Here we demonstrate that Fcrl5 deficiency impairs humoral responses to TI antigens and differentially modulates autoimmunity in lupus-prone SHP-1 and Lyn mutant mice. These data indicate a critical role for mFCRL5 in regulating innate-like B cell function at the interface of host defense and tolerance.
AID-initiated DNA lesions are differentially processed in distinct B cell populations (IRM8P.701)
Authors: Wang Jing , Chen Zhangguo , Ranganath Sheila , Viboolsittiseri Sawanee , Eder Maxwell , Elos Mihret , Chen Xiaomi , Hansen Erica
Published: January 2023

Activation-induced deaminase (AID) initiates U:G mismatches, causing point mutations or DNA double-stranded breaks at immunoglobulin (Ig) loci. How AID-initiated lesions are prevented from inducing genome-wide damage is not completely understood. Prior studies proposed a differential DNA repair mechanism that protects certain non-Ig loci such as c-myc from AID attack. However, determinants that regulate such protective mechanisms remain largely unknown. To test whether target DNA sequences might modulate protective mechanisms via modifying the processing manner of AID-initiated lesions, we established a knock-in model by inserting an Sγ2b region, a bona fide AID target, into the first intron of c-myc. Unexpectedly, we found that the inserted S region did not mutate or enhance genomic instability of c-myc, due to error-free repair of AID-initiated lesions, in antigen-stimulated germinal center B cells. In contrast, cytokine-activated B cells display a much higher level of c-myc genomic instability in an AID- and S region-dependent manner. Our findings reveal a complex interplay between target sequence, DNA repair mechanism, and cellular context associated with differential transcription, which coordinately regulate genome stability. Our studies could provide mechanistic insight into differential routes to AID-mediated genetic alterations in distinct subtypes of common B cell lymphomas.
Novel role of the E26 transformation-specific transcription factor Spi-C for B cell development and function (IRM8P.703)
Authors: Li Stephen , DeKoter Rodney
Published: January 2023

Transcription factors of the E26 transformation-specific family are critically important for regulating B cell development and function. Spi-B and Spi-C are highly related E26 transformation-specific transcription factors that can bind to identical DNA sequences in the genome. Spib-/- mice have reduced follicular and increased marginal zone B cell frequencies compared to WT mice. Spib-/- B cells are defective in BCR signaling, and cannot generate T-dependent antibody responses. Spi-C is expressed in developing B cells, but its function in B cell development and function is not clear. To determine if Spi-B and Spi-C are functionally redundant, we generated mice that are germline knockout for Spib and heterozygous for Spic (Spib-/-Spic+/-). Spib-/-Spic-/- mice were not born, potentially due to embryonic lethality. Interestingly, Spib-/-Spic+/- mice had restored number of B cells in the spleen, including a rescue of follicular B cells, and had restored proliferative responses compared to Spib-/- mice. Transcripts of genes important for BCR signaling were rescued in sorted follicular Spib-/-Spic+/- B cells compared to Spib-/- B cells. These results suggest a novel role for Spi-C in opposing Spi-B in B cell development and function. These findings have important implications for understanding antibody forming responses, such as those seen in vaccinations.
Receptor interacting protein-2 plays a critical role in human lung epithelial cells survival in response to Fas-induced cell-death (IRM9P.731)
Authors: Rahman Mohd. , Sundaram Kruthika , Srabani Das , Gavrilin Mikhail , Wewers Mark
Published: January 2023

Lung epithelial cell death is critical to acute lung injury. It is known that FasL plays a prominent role in lung cell death and may work in part through activation of the receptor interacting protein-2 (RIP2). RIP2 is serine/threonine kinase with a C-terminal caspase activation and recruitment domain (CARD). This CARD contains a highly conserved, predicted tyrosine phosphorylation site. Thus, involvement of tyrosine phosphorylation in the CARD domain of RIP2 may play a critical role in Fas-mediated apoptosis in the human lung. To test this hypothesis, human lung epithelial cells (BEAS-2B) were induced to undergo cell death in response to the Fas agonist antibody CH11 with and without manipulation of endogenous RIP2 concentrations. We show that CH11 increases lung epithelial cell death in a dose-dependent manner as determined by LDH release and nuclear condensation. Fas-induced LDH release was inhibited by RIP2 knock-down. Reduced levels of RIP2 in BEAS-2B cells after treatment with RIP2 siRNA were confirmed by immunoblot. Overexpression of RIP2 in BEAS-2B cells synergized with Fas ligand-induced LDH release in a dose-dependent manner. Finally, mutation of the tyrosine phosphorylation site in CARD of RIP2 protected BEAS-2B cells from Fas ligand induced cell death. Thus RIP2’s CARD tyrosine phosphorylation may represent a new therapeutic target to promote the survival of human lung epithelial cells in disorders that lead to acute lung injury and ARDS.
Structural motifs suggestive of hydrolytic activity are common among three-dimensional antibody structures (IRM9P.722)
Authors: Bowen Anthony , Casadevall Arturo
Published: January 2023

Naturally occurring catalytic antibodies are associated with both positive and negative outcomes in autoimmune disease and infection, but their function and prevalence remain unclear. Two monoclonal antibodies to Cryptococcus neoformans were shown to hydrolyze several peptide antigen mimetics. These two antibodies had distinct peptide hydrolysis patterns, which were altered, but not blocked, by alanine substitution of peptide residues, indicating a non-sequence-specific mechanism. A putative serine-protease-like active site was identified in the light chain variable region of both molecules. A computational algorithm was developed to detect similar sites present in other antibody structures. A three-dimensional reference template corresponding to the putative active site was optimally superposed with all possible templates in the other antibody structures. Hits were defined as those superpositions with an RMSD ≤ 1Å. The putative site was found in 24 of 80 known catalytic antibody structures and was found in 126 of 1367 studied antibodies with no annotation of catalytic activity. The ability of many antibodies to cleave antigen, albeit slowly, would provide a new function to immunoglobulin molecules in host defense. Confirmation of this active site is being pursued through site-directed mutagenesis. We anticipate that these studies will provide insights into the design of anti-cryptococcal therapies and the role of catalysis as a function of humoral immunity.
PIK3IP1- A novel negative regulator of PI3K (IRM10P.743)
Authors: Uche Uzodinma , Kane Lawrence
Published: January 2023

Phosphatidylinositide-3-Kinases (PI3Ks) are a family of lipid kinases that play important intracellular signaling roles in cellular functions such as cell proliferation, motility, growth, intracellular trafficking, differentiation and survival. PI3K produces PIP3 which further facilitates the activation of downstream effectors such as Akt and PDK1. These effectors facilitate the cellular processes associated with PI3K activity. Conversely, because of the nature of PI3Ks roles, dysregulation of PI3K is implicated in many cancers. PTEN, SHIP and INPP4B are negative regulators of PI3K activity that function downstream of PI3K and have been shown to act as tumor suppressors. Recently, PI3K Interacting Protein 1 (PIK3IP1), a novel negative regulator that functions upstream and proximal to PI3K, has been identified. PIK3IP1, is a transmembrane protein that down-regulates PI3K activity, leading to reduced Akt phosphorylation. Our lab recently showed that PIK3IP1 can inhibit TCR signaling and TCR activation. To better understand the role of PIK3IP1 in T cells, we have begun to carefully define its expression pattern in various T cell subsets. In addition, we have developed a PIK3IP1 ecto-domain Fc fusion protein, which we are using to define possible ligands for PIK3IP1. Finally, we have obtained knockout mice lacking expression of PIK3IP1, to better define the requirements for PIK3IP1 in primary T cells.