bluuebunny/crossref_metadata_2025 huggingface dataset:
Using social media platforms Twitter and Pinterest to engage undergraduate students in vaccinology and immunology.
Authors: Gorski Stacey A
Published: January 2023

Recent studies have shown that biomedical scientists and healthcare professionals are increasingly more likely to use social networking sites such as Twitter for professional correspondence and to remain informed of advancements in their fields. For this reason, pre-professional health students in vaccinology and immunology courses engaged in out-of-class social media assignments using Twitter and Pinterest, respectively. The objectives of these assignments were to demonstrate appropriate social media etiquette, enhance scientific communication skills, and provide a channel for students to relate in-class material to current events. Students in the vaccinology course were tasked with maintaining an active presence on Twitter to share accurate and personal experiences regarding vaccines and immunizations. At the end of the course, 20/20 students (100%) reported that participating on Twitter “enhanced their familiarity with current events in vaccinology,” while 90% reported that it also enhanced scientific communication skills and social media skills. Additionally, >95% of students felt that the assignment was worthwhile for teaching others, perhaps increasing their feelings of contributions to the field. Students in the immunology course used Pinterest to collect information regarding immunology-related study tools, prominent immunologists, current events in immunology, and immunotherapies. Students reported that the Pinterest assignment helped them learn in-class material (40%) and felt that it helped them relate in-class material to ‘real-world’ issues (60%). The majority of students in both classes reported that they were willing to continue to use the respective social media platform for professional use.
Determinants of phagocytic receptor mobility: cytoskeletal picket fences and the pericellular glycocalyx coat.
Authors: Freeman Spencer A , Riedl Magdalena , Vega Anthony , Johnson Pauline , Lidke Diane , Mayor Satyajit , Tammi Markku , Jaqaman Khuloud , Grinstein Sergio
Published: January 2023

Phagocytosis is initiated by clustering of receptors upon exposure to target particles bearing multiple ligands. Clustering depends on the ability of receptors to move laterally in the plane of the membrane. Intrinsic proteins were originally thought to diffuse freely along the fluid mosaic of the membrane, but more recent observations indicate that mobility can be severely restricted by the existence of a cytoskeletal fence anchored to the plasmalemma via transmembrane “pickets”. However, the molecular nature of these putative pickets, the manner whereby they associate with the cytoskeleton, and their role in phagocytosis have not been investigated. Based on its abundance and structural features, we surmised that CD44 may serve as a picket in macrophages. We used single-molecule tracking to study its behavior and how it affects the mobility of phagocytic receptors. In addition, because its extracellular domain can bind hyaluronic acid, we found that CD44 serves as a transmembrane connector between the pericellular (glycocalyx) coat and the cytoskeleton. The pericellular coat curtails access of particles to the comparatively short phagocytic receptors. The implications of this transmembrane framework to phagocytosis were investigated and will be discussed.
A systems approach to the characterization of Toll-like receptor response to different ligand stimulation in macrophages.
Authors: Nita-Lazar Aleksandra , Sjoelund Virginie Helle , Koppenol-Raab Marijke , Manes Nathan P. , Fraser Iain D , Benet Zachary , Dutta Bhaskar , Khan Mohd M.
Published: January 2023

One of the major questions in innate immunity is: How do the multiple TLRs stimulated during pathogen challenge act in concert to promote an appropriate host response? To begin to develop a comprehensive proteomic interrogation of TLR signaling under varying conditions, we used quantitative mass spectrometry to analyze the phosphoproteome, proteome and secretome changes in mouse macrophages. The cells were activated with each of three selective TLR ligands (LPS, P3C and R848) and with intact pathogens. We have identified and quantified on average more than 500 phosphosites per ligand after TLR stimulation at five time points within a time frame of 30 minutes. We have characterized the differences and similarities in the phosphoproteome dynamics between the three TLR stimulations, and we identified key differences between the phosphorylation profiles induced by the three TLR stimulations. The pathways most affected by the treatments were cytoskeletal signaling, endocytosis and phagocytosis. Furthermore, we compared the intracellular (proteome) and extracellular (secretome) responses of macrophages to TLR ligands. Comparison to the transcriptome yielded a significant number of proteins regulated at the post-transcriptional level, emphasizing the importance of systematic and global studies that reach beyond gene expression profiling. We have also generated preliminary data of the absolute quantification of the number of molecules per cell for the core TLR signaling pathway using targeted mass spectrometry. This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.
Identification of novel and potent synthetic TLR agonists
Authors: WANG YING , Su Lijing , Morin Matthew , Jones Brian , Whitby Landon , Surakattula Murali , Huang Hua , Shi Hexin , Choi Jin Huk , Wang Kuan-wen , Moresco Eva Marie Y , Berger Michael , Zhan Xiaoming , Zhan Hong , Boger Dale , Beutler Bruce
Published: January 2023

Agonists and antagonists of Toll-like receptors (TLRs) may be useful as vaccine adjuvants or suppressors of inflammation, respectively. In an effort to identify compounds capable of activating macrophages via TLRs or other sensors, a synthetic compound library was screened using mouse peritoneal macrophages and human THP-1 cells. Through extensive SAR studies of initial hits, we developed two strong synthetic agonists: Neoseptin-3 and Diprovocim. Genetic studies established that neoseptin-3 is a mouse TLR4/MD-2 agonist with no structural similarity to LPS. It activates mTLR4/MD-2 independently of CD14 and triggers canonical MyD88- and TRIF-dependent signaling. Diprovocim was found by a combination of genetic and antibody blockade analyses to be a TLR1/2 agonist, active on both mouse and human receptors. Its EC50 in human THP-1 cells is 110 pM. Diprovocim showed TLR1/2 dependent adjuvant activity when co-administered with ovalbumin (OVA). It not only promoted antigen-specific humoral responses but also activated cytotoxic T lymphocyte responses in a TLR1/2 dependent manner. Since neither Neoseptin-3 nor Diprovocim resemble the natural ligands for TLR4/MD-2 nor TLR1/2, respectively, we surmise that other “unconventional” ligands for these TLRs may exist in nature. However, the exquisite SAR of both compounds makes it clear that TLRs are not highly promiscuous receptors; on the contrary, they are activated only by compounds that fulfill strict structural rules. In finding these agonists, we also identified antagonists that bind the TLR complexes but do not activate them. The parent compounds Neoseptin-3 and Diprovocim will be used to develop new agonists and antagonists optimized for clinical application.
2′-5′ Oligoadenylate like (OASL) inhibits interferon induction by inhibiting cGAS activity during DNA virus infection.
Authors: Ghosh Arundhati , Shao Lulu , Sampath Padmavati , Patel Nidhi , Zhu Jianzhong , Hornung Viet , Thorne Stephen , Sarkar Saumendra N
Published: January 2023

Oligoadenylate Synthetases-like (OASL) is a type I interferon (IFN)-stimulated gene (ISG). During RNA virus infection OASL promotes IFN induction by augmenting RIG-I activation and inhibits virus replication. Here we report a completely new role of OASL in the context of DNA virus infection. Unlike RNA viruses, human OASL or mouse Oasl2 knockout cells showed reduced replication when infected with DNA viruses such as Vaccinia (VV) and Herpes simplex virus (HSV). Consequently, Oasl2 knockout mice showed delayed and reduced VV spread and mortality as opposed to enhanced virus multiplication and spread with RNA virus such as Vesicular Stomatitis virus. Mechanistically, OASL negatively regulates DNA-sensor-mediated IFN induction in vitro and in vivo. Stimulation of both mouse and human OASL-deficient cells with intracellular DNA showed increased induction of IFN and ISG compared to the Wt cells. However, OASL-deficient cells did not exhibit any difference in IFN induction compared to Wt cells when stimulated with the STING ligand cGAMP, indicating the upstream effect of OASL on cGAS and not on STING stimulation. In vitro immunoprecipitation experiments showed OASL specifically interacted with cGAS, and this interaction is mediated by the OAS domain of OASL protein. Furthermore, this OASL-cGAS interaction led to the inhibition of cGAS enzyme activity and reduced cGAMP production. Together these results establish the unique ability of OASL to differentially modulate RNA and DNA virus replication and IFN induction.
The advancing age affects the frequency, functions and antibody repertoire of human B-1 cells, which secrete anti-tumor antibodies
Authors: Hernandez Ana Maria , Rodriguez-Zhurbenko Nely , Quach Tam D. , Hopkins Thomas J. , Rothstein Thomas L.
Published: January 2023

N-glycolylated (NeuGc) gangliosides are not naturally expressed in human tissues but are overexpressed in several tumors and have immunosuppressive capacity. The present work shows the existence of natural cytotoxic antibodies against NeuGcGM3, whose levels decrease with age. Furthermore, this reactivity was not detected in the sera of non-small cell lung cancer patients that matched in sex and age the assessed healthy donors. Aging decreases the efficiency of immune functions, which could be associated with an increased incidence of cancer. In order to identify and study the effect of aging on the B cell population involved in the response against NeuGcGM3 naïve, memory, plasma and B-1 cells were purified from healthy human donors PBMC and stimulated in vitro to produce immunoglobulins. Only B-1 cell supernatants recognized tumor cells expressing this ganglioside. Interestingly, B-1 cells from NSCLC patients had a reduced activation capacity. The percentage of B-1 cells and their capacity to spontaneously secrete IgM decreased with age in healthy donors. In fact, expression levels of Xbp1 and Blimp1 were significantly lower, while those of Pax5 were significantly higher, in elderly donors. Elderly donors showed a B-1 antibody repertoire with higher number of repeated clones and therefore a reduced variability in comparison with young individuals. It was interesting to observe in aged individuals an increased frequency of VH3-23 and VH4-34 usage, genes which have been linked to autoimmune diseases and hematopoietic malignancies, while there is a significant reduction of clones expressing the sequence VH3-33, frequently found on antibodies against PC, oxidized LDL, apoptotic cells and induced after vaccination with Pneumavax23.
Splenic microenvironment is important in the survival and growth of Chronic Lymphocytic Leukemia in mice
Authors: McKenna Mary Kathryn , Noothi Sunil K , Alhakeem Sara Samir , Byrd John C , Muthusamy Natarajan , Rangnekar Vivek , Bondada Subbarao
Published: January 2023

Chronic Lymphocytic Leukemia (CLL), the most common adult leukemia in the western world, is characterized by accumulation of clonally expanded CD5+CD19+ B lymphocytes in the peripheral blood and secondary lymphoid organs. CLL cell survival is dependent on B cell receptor signaling and cross-talk within the leukemia microenvironment. CLL presents with enlargement of the spleen, and lymph nodes due to accumulation of long-lived lymphocytes with impaired apoptotic mechanisms. The Eμ-Tcl1 mice, with a B cell specific overexpression of the oncogene, T cell Leukemia 1 (Tcl1), serve as an excellent model to study CLL as they develop a CLL like disease by 9–13 months of age. They exhibit splenomegaly, enlarged lymph nodes and a high peripheral blood white count. Adoptive transfer of primary CD5+CD19+ CLL cells from the Eμ-Tcl1 mice into syngeneic recipients leads to rapid disease development. Ultrasonography of recipient mice showed a dramatic enlargement of the spleen, which preceded detection of CLL cells in the blood. Unlike various genetic modifications, which can only delay the onset of CLL, splenectomy in these models, cures or significantly delays disease development from 30–35 days to 250–270 days. Interestingly, splenectomy did not delay CLL development as significantly in animals deficient for prostate apoptotic response-4 (Par-4) compared to C57BL/6 wild type mice. Par-4 is a known tumor suppressor protein that can be secreted and induce apoptosis selectively in cancer cells but not in normal cells. Par-4 appears to regulate a specific microenvironment required for CLL growth. Presently we are investigating the role of Par-4 in the microenvironment and the cell types that are critical for CLL growth within the splenic niche.
Modulation of signaling preference of NK cells by different forms of NKG2D ligands and its implications
Authors: Dhar Payal , Basher Fahmin , Zhang Jinyu , Wu Jennifer
Published: January 2023

The significance of ligand-induced activation of NK cell activating receptor NKG2D has been well established in controlling tumor growth in various experimental animal models. Amongst different types of NKG2D ligands, MHC I chain related molecule MICA/B is expressed most prevalently in various human cancers. In particular, proteolytically shed form of MICA/B (sMIC) has been shown to suppress the immune system and found to correlate with advanced disease stages in cancer patients. Previous studies in our lab using two humanized bi-transgenic mice, one expressing native ligand (TRAMP/MICB) and the other expressing the shedding-resistant mutant (TRAMP/MICB.A2) revealed opposite roles of these ligands in regulating tumor immunity. MICB expressing mice exhibited increased tumor progression, whereas MICB.A2 expressing mice had tumor free survival. These findings raised the intriguing question of why do these structurally similar forms of MIC have vastly different effects on tumor immunity. To address this question, we investigated the signaling events and functional outcomes in NK cells upon stimulation by the two forms of ligands. In vitro co-culture studies of NK cells with tumor cell lines expressing sMICB and MICB.A2 revealed elevated pro-inflammatory and immunosuppressive cytokine production by NK cells upon stimulation with sMICB. In contrast, NK cells stimulated with MICB.A2 displayed enhanced expression of cytotoxicity mediators and signaling molecules of cytotoxicity pathway. This suggests that signaling through sMICB may polarize the NKG2D signaling pathways with preferential activation of inflammatory cytokine pathways. Our data has uncovered a new potential mechanism whereby sMIC promotes tumor progression.
The role of TFEB in tumor-associated macrophages
Authors: Hodge Johnie O’Neil , Fang Liang , Fan Daping
Published: January 2023

Alternatively activated (M2-like) tumor-associated macrophages (TAMs) have been shown to promote immune suppression, invasion, angiogenesis, the establishment of distant metastases, and maintain the breast cancer stem cell population. However, the mechanisms governing TAM polarization are not fully understood. Transcription factor EB (TFEB) has been recently investigated due to its role as the master regulator of autophagy, but little is known regarding its role in governing macrophage activation or other immune response pathways. We show here that TFEB transcription and activation are reduced in TAMs, and TGF-β-induced activation of ERK signaling is the driving force behind this phenomenon. siRNA knockdown of TFEB in macrophages significantly enhances their alternative activation. Co-inoculation of these macrophages with breast tumor cells results in an increase of both tumor growth and infiltration of M2-like TAMs. Further mechanistic studies reveal that TFEB knockdown regulates M2 polarization by decreasing activation of SOCS3, which acts as an inhibitor of STAT3. These findings led us to further investigate whether increasing TFEB activation in TAMs could decrease their alternative activation and their tumor promoting properties. We found that activation of macrophage TFEB suppresses alternative activation, and introduction of these macrophages at the time of tumor initiation decreases tumor growth in multiple murine models. Therefore, our data demonstrate that the downregulation of TFEB is an integral part of immune editing in the tumor microenvironment, and provides a rationale for a new cancer treatment strategy involving the modulation of TAM polarization through activating TFEB.
Analysis of granulocytes and macrophages infiltrating a primary tumor site within the first 72 hours
Authors: Kurt Robert A , Zhang Chenyu , Newsom-Stewart Catherine M , Ptasinski Anna M , Greis William H
Published: January 2023

While a great deal of work has been reported on innate and adaptive white blood cells infiltrating solid tumors, as well as the pre-metastatic niche, there is a paucity of information concerning the early innate immune response to primary tumors. This is particularly evident in the very early stages; before a solid tumor is palpable. Here we used a gelatin sponge model to investigate white blood cells infiltrating a tumor site 12–72 hours following injection. Initial studies focused on identifying the total number of infiltrating cells as well as the number of lymphocytes, macrophages and granulocytes. While 12 hours after injection there was no difference in the total number of cells infiltrating the tumor site versus a control site injected with saline, at 24 and 48 hours there was a small, yet significant increase in the number of cells at the tumor site. The majority of the cells to arrive at the tumor and saline injected sites within the first 12 hours were Gr1+F4/80+ (60%), and this population of cells decreased to 36% by 72 hours at both sites. These data contrasted with the F4/80+Gr1− cells which increased over time from about 10% at 12 hours to 25% at 72 hours. Moreover, two significant differences were noted between the tumor and saline sites. At 24 hours there were significantly more F4/80+Gr1-cells at the tumor site, and the number of Gr1+F4/80− cells remained higher at the tumor site over time. These data indicate early macrophage recruitment and neutrophil retention at the primary tumor site. Gene expression analysis by quantitative RT-PCR is underway to investigate additional differences in these cell populations.
Control of gut bacteria-associated sepsis by an inhibitor of group 2 innate lymphoid cells in severely burned mice
Authors: Ito Ichiaki , Kobayashi Makiko , Finnerty Celeste C , Herndon David N , Suzuki Fujio
Published: January 2023

The effect of SR3335 on infectious complications caused by enterococcal translocation was studied in severely burned mice. SR3335 is an RORα selective inverse agonist for group 2 innate lymphoid cells (ILC2). Sepsis caused by Enterococcus faecalis oral infection (106 CFU/mouse) was developed in decontaminated and proton pump inhibitor treated mice subjected to burn injuries (25%TBSA burn), as previously described. These mice were treated i.p. with 100 μg/mouse of SR3335 once daily for 3 days started 1 h after burn injury. SR3335 inhibits the maturation of ILC2 through the suppression of the RORα transcriptional activity. ILC2 were isolated form the intestines and analyzed by flow cytometry. The severity of infection was evaluated by mortality and bacterial growth in organs of these mice, as previously described. In the results, all severely burned mice died within 6 days of E. faecalis oral infection, while 100% of the same mice treated with SR3335 survived more than 10 days after the infection. E. faecalis grew seriously in the organs of burned mice after the infection, but the pathogen did not grow in burned mice treated with SR3335. The increased number of ILC2 and their cytokine products (IL-5 and IL-13) were detected in the lamina propria of mice 1 to 7 days after severe burn injury. However, the number of ILC2 was not increased in the lamina propria of burned mice treated with SR3335. SCIDbg mice resisted E. faecalis oral infection, while the same mice adoptively transferred with ILC2 were shown to be susceptible to the infection. These results indicate that ILC2 play a role in the severity of gut bacteria-associated sepsis, and the ILC2 inhibitor (SR3335) is suggested to be protective against these infections in severely burned mice.
Pulmonary innate type 2 inflammatory responses protect against Staphylococcus aureus-induced sepsis
Authors: Krishack Paulette , Louviere Tyler J , Sperling Anne I , Verhoef Philip A
Published: January 2023

Sepsis is defined as life-threatening organ dysfunction caused by the body’s response to infection. Sepsis is associated with overwhelming type 1 or type 17 immunologic inflammation, but the role of type 2 immune responses in sepsis remains unknown. To test the role of type 2 immune responses in sepsis, we treated mice with intratracheal IL-33 to induce an innate pulmonary type 2 response, followed by intravenous infection with S. aureus to induce sepsis. Surprisingly, type 2 responses were beneficial during acute infection, as IL-33 treatment protected mice from S. aureus-induced death. An increase in the ratio of lung eosinophils to neutrophils was revealed in IL-33 treated, S. aureus infected mice, suggesting that type 2 responses suppress lung neutrophilia. Further, no difference was found in splenic granulocytes or lymphocytes, indicating that activation of pulmonary immune responses alone was sufficient for protection. Infection of PLZF null mice, which have defective NKT and ILC2 cells normally required for type 2 innate responses after IL-33 treatment, resulted in accelerated mortality compared with wild type littermate controls. Moreover, IL-33 did not rescue PLZF null mice from death. However, infection of CD1d null mice, which lack only NKT cells but have intact ILC2s, demonstrated no difference in mortality. Thus, pulmonary type 2 innate immunity provided by ILC2s is critical for protection against S. aureus-mediated death.
Glutathione reductase facilates immune defense against fungal infections
Authors: Liu Yusen , Kim Victoria Y. , Jin Yi , Meng Xiaomei , Zhang Jian , Rogers Lynette K. , Nelin Leif D.
Published: January 2023

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in Redox regulation. We expressed Gsr as a GST-fusion protein and produced a rabbit polyclonal antibody against Gsr. Using this antibody we examined Gsr expression levels in various mouse tissues. We found that Gsr was highly expressed in a variety of tissues, including lung, kidney, eyes, spleen, thymus, and bone marrow, while Gsr expression was very low in muscle and heart. Importantly, Gsr was not detected in any tissues of the Gsr hypomorphic mice. Because Gsr is expressed in lymphoid tissues and is implicated in phagocytic functions, we assessed the effect of Gsr deficiency on immune defense against a fungal pathogen, Candida albicans. We report in this study that Gsr-deficient mice exhibited substantially enhanced susceptibility to C. albicans challenge. Upon C. albicans infection, Gsr null mice exhibited dramatically increased fungal burdens in the kidneys, cytokine and chemokine storm, striking neutrophil infiltration and histological abnormalities in both the kidneys and hearts, and substantially elevated mortality. Large fungal foci surrounded by massive numbers of neutrophils were detected outside of glomerulus in the kidneys of Gsr null mice but not in wildtype mice. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed normal phagocytosis and yet attenuated oxidative burst activity. Thus, Gsr-mediated redox regulation is crucial for fungal clearance during host defense against fungal challenge.
Immature myeloid cells (IMCs) increased in the peripheral blood of patients with alcohol use disorder (AUD) suppress the bactericidal activity of monocytes
Authors: Asai Akira , Yasuoka Hidetaka , Ohama Hideko , Tsuchimoto Yusuke , Fukunishi Shinya , Kobayashi Makiko , Higuchi Kazuhide , Suzuki Fujio
Published: January 2023

AUD patients have an increased risk of serious opportunistic infections. IMCs have been described as suppressor cells for host antibacterial defenses. In this study, IMCs isolated from AUD patients were examined for their inhibitory functions on the bactericidal activity of healthy donor (HD) monocytes. IMCs and monocytes were isolated from the peripheral blood of 10 AUD patients admitted to Osaka Medical Collage and 5 HD. HD monocytes (1 × 105 cells/ml) were stimulated with heat-killed Enterococcus faecalis, then cultured in media supplemented with the conditioned media of patient IMCs (24 hr-culture fluids, 10% v/v, 1 × 106 cells/ml). Twenty-four hrs after the cultivation, monocytes were harvested and tested for their bactericidal activities against E. faecalis. In some cases, patient IMCs, cultured with 5 μg/ml of retinoic acid (RA) for 24 hrs, were utilized for the experiments. Also, both groups of IMCs were analyzed for RA receptor (RAR) expression by flow cytometry. In the results, as compared to HD peripheral blood IMCs, the numbers of IMCs increased in AUD patients. HD monocytes were shown to be bactericidal against E. faecalis. However, the bactericidal activity of HD monocytes was not demonstrated when they were cultured with media supplemented with the conditioned media of patient IMCs. HD IMCs differentiated to monocytes or dendritic cells after cultivation with RA. However, AUD patient IMCs were shown to be not susceptible to RA treatment (patient IMCs, RAR−; HD IMCs, RAR+). These results suggest that, through the production of some soluble factors active against killing functions of monocytes, patient IMCs increase the susceptibility of AUD patients to opportunistic infections.
Protective role of ILC2 in sepsis-induced acute lung injury
Authors: Shu Qiang , Lai Dengming , Fan Jie
Published: January 2023

Objective

Group 2 innate lymphoid cells (ILC2), a recently identified population of innate immune cells, were suggested to be protective in infection. However, the underlying mechanism remains unknown. The current study aimed to determine the protective role of ILC2 in sepsis-induced acute lung inflammation and the underlying mechanism.

Methods

WT and IL-33 knockout mice were subjected to cecal ligation and puncture (CLP) to induce sepsis. The number of ILC2 and the concentration of cytokines including IL-4, IL-9 and IL-13 in the lung tissue were measured by flow cytometry and ELISA, respectively, at different time after CLP. In the in vitro studies, lung-derived ILC2 were co-cultured with mouse lung endothelial cells (LEC) and macrophages (MΦ), respectively, and challenged with LPS and/or TNF-α for up to 48 h. The LEC apoptosis and the ability of MΦ phagocytosis of bacteria were then detected by confocal fluorescence microscopy.

Results

Sepsis induced IL-33-dependent recruitment of ILC2 in the lung after CLP. The levels of ILC2-derived IL-9 and IL-13 significantly increased in the lung tissue of WT septic mice and in the supernatant of cultured ILC2 after LPS stimulation. IL-9 protects LPS/TNFα-induced LEC apoptosis, whereas, IL-13 decreased MΦ apoptosis in response to LPS/TNF-α. In addition, both IL-9 and IL-13 promote MΦ phagocytosis of bacteria.

Conclusions

IL-33-mediated ILC2 recruitment in the lung following sepsis protects lung injury through reducing apoptosis of LEC and MΦ and promotes bacteria clearance by MΦ. Increasing ILC2 and/or ILC2-derived cytokines in the lung may present a new therapeutic strategy for acute lung injury in sepsis.
GM-CSF controls the balance between effector and regulatory T cell lineage in experimental autoimmune encephalomyelitis (EAE)
Authors: Ghosh Debjani , Curtis Alan Dale , Wilkinson Daniel S , Mannie Mark D
Published: January 2023

Previous studies showed that GM-CSF-deficient (Csf2-deficient, Csf2−/−) mice exhibited profound resistance to experimental autoimmune encephalomyelitis (EAE). The present study focused on the myelin oligodendrocyte glycoprotein (MOG) 35–55-specific model of murine EAE in Csf2-deficient and wildtype C57BL/6 mice. The study addressed whether disease resistance of Csf2-deficient mice reflected a role for GM-CSF in controlling the functional balance between effector and regulatory T cells. The main finding was that treatment with the anti-CD25 mAb PC61 rendered Csf2-deficient mice fully susceptible to severe EAE, similar to that of C57BL/6 mice. When both donors and recipients were treated with PC61 in a passive model of EAE, adoptive transfer of myelin-specific Csf2−/− T cells into Csf2−/− recipients resulted in chronic severe EAE. Csf2-deficient T cell repertoire was characterized by elevated CD3+ T cell frequencies that reflected accumulations of naïve CD44null-low CD4+ and CD8+ T cells but normal frequencies of CD4+ CD25+ FOXP3+ T cells. Overall, this finding suggested that Csf2−/− mice had secondary deficiencies in T cell sensitization to environmental antigens. In accordance, MOG35-55/ Complete Freund’s Adjuvant -sensitized Csf2−/− mice exhibited deficient peripheral sensitization to MOG, whereas pretreatment of sensitized Csf2−/− mice with PC61 enabled the robust induction of MOG-specific T cell responses in the draining lymphatics. In conclusion, the EAE resistance of Csf2−/− mice, at least in part, reflects deficient induction of effector T cell function that cannot surmount normal Treg barriers. EAE effector responses in Csf2-deficient mice however are unleashed upon depletion of regulatory CD25+ T cells.
The Crystal Structure of the IL-33 Signaling Complex Reveals the Molecular Basis for Receptor Sharing in the IL-1 Family.
Authors: Guenther Sebastian , Bowers Amanda L. , Deredge Daniel , Bonsor Daniel A. , Beadenkopf Robert , Wintrode Patrick L. , Sundberg Eric J.
Published: January 2023

Cytokines of the IL-1 family are potent stimulators of the immune system. As seen in other cytokine families, cytokine redundancy due to receptor sharing is characteristic for the IL-1 family. Today five primary receptors have been described that each bind a subset of the 11 known cytokines within this family. Once an agonistic cytokine-receptor pair has formed, it can engage one of the two co-receptors and initiate signaling through MyD88. While IL-18Rβ is the unique co-receptor for the IL-18/IL-18Rα pair, IL-1RAcP is shared between the other four primary receptors, IL-1RI, IL-1RII, ST2 and IL-36R. Crystal structures of IL-1β bound to IL-1RI or IL-1RII in complex with IL-1RAcP previously revealed the general architecture of signaling complexes in the IL-1 family. IL-33 is the latest cytokine to be discovered in the IL-1 family. Since its first description in 2005 it has been recognized as a central driver of type 2 immunity. Here we present the crystal structure of IL-33 in complex with its receptors ST2 and IL-1RAcP. The structure reveals a remarkable conservation of the ternary architecture of signaling complexes in the IL-1 family. We observe common principles of co-receptor binding, also seen in the other two ternary structures containing IL-1RAcP. However, hydrogen/deuterium mass exchange spectroscopy and extensive alanine-scanning analysis combined with direct binding analysis of the IL-1β and IL-33 signaling complexes demonstrate that the binding energy is distributed quite differently within the two. Co-receptor binding of ST2/IL-33 is much less dependent on the cytokine than on the receptor, while for IL-1β/IL-1RI the cytokine plays a much more important role.
CCL22 impedes T cell activation capacities of dendritic cells by reducing membrane expression of MHC molecules and CD80
Authors: Kühnemuth Benjamin , Piseddu Ignazio , Knott Maximilian , Vetter Viola , Layritz Patrick , Endres Stefan , Anz David
Published: January 2023

The chemokine CCL22 is a well known modulator of immune responses. By binding to its receptor CCR4, which is highly expressed on several T cell subtypes as well as dendritic cells (DC), CCL22 mediates chemotaxis of responsive cells, thus influencing the distribution of these cells within tissues. For instance, CCL22 is known to be expressed in human and murine tumors contributing to the recruitment of regulatory T cells. Given that many chemokines show effects exceeding pure chemotaxis we hypothesized that CCL22 mediates modulation of immune responses by further activities.

In order to identify additional immune modulating properties of CCL22 on DCs and T cells we stimulated murine splenocytes isolated from healthy donor mice with recombinant CCL22 in vitro. We did not find any significant alteration in expression of cell surface activation markers like CTLA-4, PD-1 or CD62L on CD4+, CD8+ and FoxP3+ T cells. However, splenic CD11c+ DCs showed a significant reduction in MHC class I molecules on cell surface upon stimulation with CCL22. Notably, this reduction was most prominent in CD11c+ CD8α+ DCs which also showed decreased levels of MHC class II molecules as well as the costimulatory receptor CD80 after encountering CCL22.

In summary, our data indicate an alternative mode of action for CCL22 on Dendritic cells, especially on CD11c+ CD8α+ DCs. The CCL22-mediated modification of DC’s capabilities to present antigens and to activate T cells might represent a fine-tuning mechanism of adaptive immune responses.
Kinetics modeling of myeloid cells in rhesus macaques with pulse-chase BrdU labeling
Authors: He Ziyuan , Sugimoto Chie , Allers Carolina , Fujioka Hideki , Didier Elizabeth , Kuroda Marcelo J
Published: January 2023

Myeloid cells are continuously produced via bone marrow hematopoiesis and then cleared from the circulation to maintain homeostasis. To better understand the kinetics of such cell movement, we applied in vivo BrdU pulse-chase labeling and flow cytometry analysis to follow the kinetics of cell division during homeostasis of each subset of myeloid cells in peripheral blood in rhesus macaques. Results were applied to a mathematical model to estimate the half-life and daily production of each subset as follows; neutrophils (1.63±0.16 days, 1.42×109cells/L/day), basophils (1.78±0.30 days, 5.89×106 cells/L/day), classical monocytes (1.01±0.15 days, 3.09×108 cells/L/day), intermediate monocytes (1.23±0.14 days, 2.90×107 cells/L/day), non-classical monocytes (3.45±0.93 days, 1.58×107 cells/L/day), CD1c+ myeloid dendritic cells (DCs) (1.11±0.16 days, 1.65×107 cells/L/day), CD123+ plasmacytoid DCs (2.69±0.38 days, 9.67×105 cells/L/day). Unlike monocytes which were released into the blood circulation as soon as 1 day after dividing, neutrophils required ~4–5 days in bone marrow before being released. Interestingly, even within the granulocyte population, neutrophils and basophils exhibited distinct kinetics in peripheral blood and different bone marrow maturation times. We also applied our model to healthy rhesus macaques ranging from 3 to 19 years of age and observed that while the half-life of neutrophils did not change with increasing age, the older animals produced fewer neutrophils per day. In addition to neutrophils and monocytes, the non-classical (inflammatory) monocytes, pDCs and mDCs, as well as basophils, were relatively shorter-lived cells that may contribute to protection and homeostasis at steady state.
Sclerostin expression in distinct osteolineage cell types differentially regulates hematopoietic stem cell and B lymphocyte development
Authors: Manilay Jennifer , Murugesh Deepa , Yee Cristal , Economides Aris , Loots Gabriela
Published: January 2023

Investigation of the reciprocal effects of hematopoietic cell types and osteolineage cells on each other’s cell fates have clinical applications to improve bone fracture healing, prevent age-related bone loss and immune deficiencies. A significant knowledge gap exists as to the contribution of different osteolineage cells to the differentiation, proliferation, and long term survival of B cells in the BM. In global sclerostin knockout (SostKO) mice, we previously uncovered a cell-extrinsic requirement for sclerostin on B lymphocyte development. It is widely accepted that sclerostin is transcribed primarily in osteocytes (OCYs), but whether is it expressed and/or active in other bone progenitors has been controversial. In order to identify the Sost-expressing osteolineage cell type that is responsible for B cell support, we analyzed hematopoietic differentiation in mice lacking Sost in mesenchymal stem cells (MSCs) [Prx1-Cre], osteoblasts (OBs) [Col1a-Cre] and OCYs [Dmp1-Cre] through the use of conditional Sost-KO (cKO) mice. Loss of Sost expression in MSCs resulted in reduced BM cellularity, whereas this was unaffected by Sost-deficiency in OB and OCYs. Furthermore, MSC-specific depletion of Sost caused an accumulation of early B cell progenitors. In contrast, loss of Sost in OBs and OCYs resulted in a reduction of mature “recirculating” B cells in the BM. Remarkably, the percentage of long-term CD150+ CD48− HSCs was significantly reduced in MSC-cKO mice, but was not affected in OB- and OCY-cKOs. The data described herein suggest that Sost deficiency in MSCs, OBs, and OCYs differentially regulates hematopoietic stem cell retention, survival, maintenance and B cell differentiation in the bone microenvironment.
ERM family proteins regulate T cell signal initiation by limiting Lck activity and T cell receptor clustering.
Authors: Robertson Tanner Ford , Blumenthal Daniel , Chandra Vidhi , Burkhardt Janis K
Published: January 2023

Ezrin, radixin, and moesin (ERM) proteins create specialized membrane subdomains by linking phosphatidyl inositol lipids and protein binding partners in the plasma membrane to the underlying actin cortex. These proteins are widely expressed and have well-characterized roles in cell polarity, development, and cytokinesis. In recent years, it has become clear that these proteins are also intimately involved in signal transduction. In humans, mutations in ERM proteins cause severe immunodeficiency characterized by lymphopenia and poor T cell proliferation. To investigate the role of ERM proteins in T cell activation, we generated mice with T cells deficient in ezrin and moesin, the two ERM family members expressed by T cells. Preliminary analysis shows that ERM-deficient CD4+ T cells proliferate poorly in vitro. Interestingly, however, these T cells signal more robustly in response to TCR ligation and produce high amounts of IFNγ. At rest, ERM-deficient T cells exhibit enhanced phosphorylation of Lck and larger pre-clustering of the T cell receptor. These data are consistent with a model in which ERM proteins both maintain cortical actin fences to limit TCR clustering and recruit negative regulators of proximal signaling events to the plasma membrane, thereby governing early events in the TCR signaling cascade.
Human T-cell lymphoma and leukemia cell lines display Treg cell phenotypes and respond to external semaphorin 4A
Authors: Chapoval Svetlana P , Hritzo Molly , Qi Xiulan , Tamagnone Luca , Golding Amit , Keegan Achsah Dorsey
Published: January 2023

We have previously shown that neuroimmune semaphorin Sema4A regulates experimental allergic asthma severity and Treg cell number in vivo (Mucosal Immunol., 2012, 5:409). Later, a study from the Vignali laboratory (Nature, 2013, 501:252) demonstrated that Sema4 controls Treg cell function and survival acting through its Neuropilin 1 receptor. In our current study, we employed the human cell lines HuT78 and HuT102 to further investigate the contribution of Sema4A to Treg phenotypes. These cell lines were selected for phenotypic and functional assays as they variably express Treg cell markers including CD25 and Foxp3. Moreover, they both express high levels of Sema4A. Although we did not detect Neuropilin 1 on these cell lines, they both were found to express another Sema4A counter receptor, Plexin B1. Culture of these cell lines with soluble Sema4A led to downregulation of Plexin B1 and upregulation of Sema4A and CD25 expression with slightly increased yield of CD4+CD25+ and CD25+Foxp3+ cells. Our future research is aimed at defining the role of Sema4A-PlexinB1 signaling pathways in Treg cell stability and function. We will extend this study to Treg cells obtained from normal PBMC and from patients with autoimmune and cancerous conditions. This will help us to develop new Sema4A-based therapeutic measures to combat these diseases.
NEUTROPHILS PLASTICITY DURING Mycobacterium tuberculosis INFECTION IS RELATED TO THE DISEASE PROGRESSION
Authors: Silva Priscilla Mariane Cardoso , Zambuzi Fabiana Albani , Lima Leonardo , Brauer Vêronica , Soares Luana , Fontanari Carolina , Bollela Valdes , Frantz Fabiani
Published: January 2023

Tuberculosis is an infectious disease that affects one third of world population. The bacilli are spread by the illness bearer and can cause a serious pulmonary infection. Together with resident macrophages, neutrophils appear to be an important cell in the control of Mycobacterium tuberculosis infection. Recently, some studies are breaking paradigms about neutrophils, revealing that these cells can polarize to N1 and N2 profiles. These findings opened a different perspective in the study of Mtb infection and progress to active disease. Thus, the aim of this study is to determine how the polarization of neutrophils in N1/N2 profiles could influence the control of Mtb infection. For this purpose, neutrophils were isolated from peripheral blood of ten healthy volunteers. These cells were stimulated for one hour with GM-CSF and INF-γ for differentiation in the N1 profile or with IL-13 and TGF-β for N2. The polarized neutrophils were characterized by their morphology and production of reactive oxygen species. After the differentiation neutrophils were infected with Mtb and were evaluated the formation of NET and cytokine production. As results, the caracterization of polarized neutrophils revealed a different nuclear morphology and metabolic activity. When neutrophils are challenged with Mtb the N1 group shows an increase of IL-8, IL-1-β, INF-γ and the formation of NETs. By contrast N2 profile reveals a decline of these inflammatory cytokines and an increase of IL-4 and TGF-β. Further, in this condition is no longer possible to observe the formation of NETs. This result supports the idea of neutrophils plasticity and helps to understand better how the neutrophils may have different actions against Mtb.
A natural product inhibitor of the NLRP3 inflammasome derived from Glycyrrhiza uralensis for the treatment of inflammatory diseases
Authors: Yang Gabsik , Lee Hye Eun , Han Sin-Hee , An Tae-Jin , Jang Jae-Ki , Lee Joo Young
Published: January 2023

The NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome is a pattern recognition receptor complex recognizing invading pathogens and activating caspase-1 to induce maturation of interleukin (IL)-1β, thereby playing a critical role in the regulation of immune responses. Its aberrant activation is pathogenic to inherited issues for example, cryopyrin-associated occasional syndrome, multiple sclerosis, type 2 diabetes, Alzheimer’s disease and atherosclerosis. The root of Glycyrrhiza uralensis (GU) has been used as herbal medicine worldwide for over 4000 years. GU is a well-recognized, natural sweetener and used as a traditional medicine to treat several diseases and symptoms, including diabetes, lung diseases, and coughs. Although the effects of GU on immunity have been reported, its effects on inflammasomes, have not been fully studied yet. In this study, we elucidated the effect of GU on inflammasome activation using primary mouse macrophages. GU inhibited the production of caspase-1(p10) and mature IL-1β induced by NLRP3 inflammasome activation in primary mouse macrophages. Furthermore, GU markedly inhibited diet-induced adipose tissue inflammation and IL-1β and caspase-1 production in white adipose tissue. The results demonstrate that GU inhibits NLRP3 inflammasome activation in macrophages, suggesting that GU is a potential drug candidate for the treatment of NLRP3 inflammasome-associated inflammatory diseases.

Acknowledgement

This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01199002)” Rural Development Administration, Republic of Korea.
Four-dimensional correlation between frequency of degranulation and Natural Killer (NK) cell cytotoxicity
Authors: Gwalani Lavesh A , Orange Jordan S
Published: January 2023

NK cells are cytotoxic lymphocytes required for early control of viral infections and tumor immunosurveillance. NK cytotoxicity occurs through directed secretion of lytic granules (LG) containing effector molecules such as perforin and granzymes. Cytoskeletal reorganization during the formation of an NK-cell immunological synapse (IS) enables delivery of LG to the IS. Fusion of LG with the NK cell membrane results in exocytosis of its contents into the IS leading to target cell death. While the requirements for LG transport and exocytosis are largely known, the minimum number of degranulation events sufficient for an NK cell to kill its target is unknown. In this study we performed four-dimensional quantification of degranulation events using a degranulation indicator (LAMP1-pHluorin) in concert with a target cell death sensor. Confocal microscopy of live single NK-target cell conjugates allowed visualization and measurement of the entire NK-cell kill. Degranulation events were quantified in LAMP1-pHluorin transduced human NK cell lines against their known targets. Killing exhibited two distinct patterns, fast and slow. In one NK cell line the minimal effective degranulation events for fast killing were almost half of that for slow killing and were similar to the minimal effective degranulation events in the other NK cell line. LD50 for vital dye extinction was calculated and was mediated by a surprisingly small number of degranulation events. Importantly, a strong temporal correlation existed between the appearance of degranulation events and target cell killing. With this study we have quantified NK cell degranulation at a single cell level and defined parameters of degranulation that correlate with NK-cell cytotoxicity.