bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thermodynamic effects of mutating hot-spot tyrosine residues in the NKG2D binding site (134.19)
Authors: Maddox Michael K. , McFarland Benjamin J. 
Published: January 2023

Abstract
Immune responses are largely governed by tight, highly specific protein-protein interactions. This study focused on one such interaction between the homodimeric immunoreceptor NKG2D, found on natural killer cells, and its MHC-like ligands, which are expressed on stressed cells. Previous research has shown that the NKG2D-ligand interface structure is dominated by 4 central tyrosine residues at positions 152 and 199. We mutated these residues to smaller and larger amino acids in order to investigate the role of the tyrosines in NKG2D-ligand complexes. Eight tyrosine mutants were tested for binding to MICA, MICB and a high-affinity MICA mutant using surface plasmon resonance. For example, mutating Y199 to phenylalanine had a larger detrimental effect on binding than did Y152F, implying that the former tyrosine is more sensitive to the removal of the hydroxyl. Alanine mutations were most detrimental to complex affinity but we were still able to detect binding. We found the vant Hoff enthalpies of each mutant NKG2D-ligand interaction. We correlated observed binding energies with those predicted by a fixed-backbone RosettaDesign mutation simulation. Even simple fixed-backbone RosettaDesign provided qualitative agreement with results for 14 of 15 mutant NKG2D-ligand interactions, and quantitative agreement for 11 of those. To complete this study we plan to investigate the hydrophobic effect in the center of the site by making an additional 3 mutations (I,L, and V) at each residue.
Identification of a cell surface ligand for NKp30 (134.20)
Authors: Levin Steven D , Brandt Cameron S , Yi Eugene C , Kennedy Jacob , Gao Zeren , Fox Brian , Haldeman Betty , Ostrander Craig , West Robert , Xu Wenfeng 
Published: January 2023

Abstract
We have identified a common gene structure found in all known members of the CD28 family and have used this conserved structure to include the Natural Cytotoxicity Receptor NKp30 as a CD28 family member. Soluble forms of NKp30 specifically bound to the chronic myelogenous leukemia cell-line K562 and inhibited cytolysis by the human NK line NK-92. This suggested the presence of a functional counter-structure for NKp30 on the surface of K562 and we employed a proteomic approach to identify this molecule. Our analysis identified an unannontated gene specifically from K562 lysates, which when transfected into P815 cells conveyed NKp30-specific binding activity and sensitivity to NKp30-mediated cytolysis by NK cells. The predicted protein and gene structure of this molecule suggested it was a member of the B7 family and we therefore designated it B7h6. Expression analysis suggested limited expression on normal tissues but expression was apparent on a subset of tumor lines and expression correlated well with sensitivity to NKp30-mediated cytolysis further supporting the notion that this molecule serves as a functional counter-structure for NKp30.
Acute inflammation with IL-18/IL-12 or α-GalCer treatment induces liver iNKT cell apoptosis and repopulation from peripheral tissues, whereas chronic inflammation ablates systemic iNKT cells with thymic-dependent repopulation (134.24)
Authors: Subleski Jeff J , Hall Veronica L , Scarzello Anthony J , Wolfe Thomas B , Ortaldo John R , Hodge Deborah L , Weiss Jon M , Chan Tim , Wiltrout Robert H 
Published: January 2023

Abstract
We previously showed that IL-18/IL-12 treatment of tumor bearing mice elicited significant anti-tumor activity concurrent with a decrease in the detectable number of invariant NKT (iNKT) cells. We now demonstrate that acute treatment with IL-18/IL-12 initially decreased the number of NK1.1(+) iNKT cells while α-GalCer decreased both NK1.1(+) and NK1.1(-) iNKT cells in the liver. Genomic qPCR analysis of iNKT cell's restricted Vα14Jα18 TCR confirmed the loss of cells at 24hrs in the liver rather than receptor down modulation. A small or no decrease in the genomic levels of Vα14Jα18 from other lymphoid tissues indicates the loss of iNKT cells from IL-18/IL-12 or α-GalCer treatment of mice was regulated by the liver microenvironment. Furthermore, an increase in AnnexinV was detected on liver iNKT cells following treatment with IL-18/IL-12 and an increase in cells that were tunnel positive was observed in the liver following α-GalCer treatment of mice. Although iNKT cells initially (24hrs) decrease in the liver following acute administration of α-GalCer or IL-18/IL-12, these cells subsequently (72hrs) reappear and expand in the liver. To study the expansion of iNKT cells in the liver, we administered BrdU to mice 24hrs after acute treatment with IL-18/IL-12 or α-GalCer and found a high percentage of proliferating liver iNKT cells were recent emigrating NK1.1(-) iNKT cells.
A requirement for early neonatal exposure to vitamin D for iNKT cells (134.26)
Authors: Yu Sanhong , Cantorna Margherita 
Published: January 2023

Abstract
CD1d-reactive invariant natural killer T (iNKT) cells are a unique subset of lymphocytes, which play important roles in immune regulation. We have shown that the vitamin D receptor (VDR) is required for the development of iNKT cells. Here we determined the effect of the vitamin D ligand (calcitriol) on iNKT cells. Like the VDR KO mice fewer iNKT cells were identified in the calcitriol deficient mice. However, ligand deficiency did not affect the function of the remaining iNKT cells. In addition, calcitirol deficiency did not affect the development of iNKT cells in the thymus, while VDR KO iNKT cells were blocked at the CD44+/NK1.1- stage of development. The 1alpha-hydroxylase (encoded by cyp27B1) is an enzyme required for production of calcitriol. Cyp27B1 KO mice are calcitriol deficient because they cannot convert vitamin D to the active form. Cyp27B1 ko/+ breeders were fed vitamin D deficient diets. At three weeks of age WT and Cyp27B1 KO littermates were weaned and fed diets that contain vitamin D for the next 5 weeks. Analysis of iNKT cells from these mice showed that both the WT and Cyp27B1 KO littermates had very few iNKT cells. In fact the percentage of iNKT cells in the liver of vitamin D deficient mice littermates were very low 1.2-1.8% and lower than in the VDR KO mice (6%). Early exposure to calcitriol in utero is required for iNKT cell development and iNKT cells failed to recover in either WT or Cyp27B1 KO littermates that were calcitriol sufficient from the age of 5-8 wks. The data show that there are differences in the role of the VDR and the vitamin D ligand on iNKT cells. In addition, the data suggest a critical role for calcitriol early either during neonatal development or the first 3 weeks post-birth in inducing iNKT cells.
Detection of fungal particles by Dectin-1 triggers reorganization of macrophage membrane proteins to form a “phagocytic synapse” (134.35)
Authors: Goodridge Helen S , Wawrowsky Kolja A , Reyes Christopher N , Ma Jun , Wolf Andrea J , Becker Courtney A , Vasilakos John P , Underhill David M 
Published: January 2023

Abstract
The β-glucan receptor Dectin-1 is a central player in the anti-fungal responses of myeloid phagocytes. Detection of β-glucan-containing particles by Dectin-1, which signals via an ITAM-like motif in its cytoplasmic tail, triggers phagocytosis, the induction of an oxidative burst, and production of a variety of inflammatory mediators by macrophages and dendritic cells. In contrast, soluble β-glucans, which are too small to trigger phagocytosis, fail to activate Dectin-1 signaling. However, soluble β-glucans immobilized on latex beads are capable of inducing Dectin-1-mediated responses. We used immunofluorescence microscopy to study the rearrangement of macrophage surface proteins upon binding of β-glucan particles to Dectin-1, and observed the formation of a synapse analogous to the immunological synapse that forms between a T cell and an antigen presenting cell. Our data indicate that the formation of this "phagocytic synapse" is required to initiate Dectin-1 signaling, and that the inability of soluble β-glucans to trigger such reorganization accounts for their failure to activate Dectin-1 signaling.
This work is supported by grants to DMU from the NIH (AI071116, GM085796) and AHA (0640100N); HSG is a CCFA Research Fellow
Exploring the development of endotoxin tolerance and anti-endotoxin activity of LL-37 in mammalian cells (134.55)
Authors: Pena (Serrato) Olga M , Pistolic Jelena , Falsafi Reza , Gardy Jennifer , Hancock Robert E. W. 
Published: January 2023

Abstract
The innate immune response is the first line of defense against microorganisms. However, its over-stimulation can result in excessive inflammation that can lead to deadly syndromes like sepsis. Initially, septic patients exhibit a high immune response, which later shifts towards an immunosuppressive state. This state may be related with a phenomenon called endotoxin tolerance, which is a reduced capacity to respond to antigens. Recent studies have demonstrated that cationic host defense peptides, such as LL-37, have broad immunomodulatory activities like diminishing inflammatory responses. Therefore, the purpose of these two phenomena may be to reduce the immune response and control excessive inflammation. Although much studied, mechanisms that lead to their development are not clear. In this work, time course analysis by RT-qPCR, western blot, and microarrays on human peripheral blood mononuclear cells and monocytic cell line, have shown similar patterns in the up- and down-regulation of important immune players like pro/anti-inflammatory mediators, and negative regulators during the development of endotoxin tolerance and anti-endotoxin activity of LL-37. Results from this work may not only help clarify how each of these phenomena occur, but also may find a link between them.
This work has been supported by Genome British Columbia and Genome Prairie for the Pathogenomics of Innate Immunity Research Program, and by the Foundation for the National Institutes of Health and Canadian Institutes for Health Research through the Grand Challenges in Global Health Initiative.
Complement components modulate activation of mouse NK cells (134.67)
Authors: Qing Xiaoping , Koo Gloria C , Tomlinson Stephen , Salmon Jane E 
Published: January 2023

Abstract
Activation of TLR pathway and complement cascade are two critical defense mechanisms of the innate immune system recently found to interact and influence effector cell function. Natural killer (NK) cells, essential participants in anti-viral and anti-tumor responses, are activated by TLR-3, -4, -7, and -9 pathways via priming by dendritic cells. It is not known whether products of complement activation influence NK cell responses. To examine this possibility, we analyzed NK cell activation in mice lacking complement components or their receptors. Splenocytes from C3-/- mice had significantly more IFN-ã positive NK cells than C3+/+ mice (2-4 fold higher, P&lt;0.01-0.05) when stimulated with PMA and ionomycin, or with TLR agonists: poly I:C (TLR3), Imiquinoid (TLR7), and LPS (TLR4). Increased NK cell activation by Poly IC was also evident in mice lacking C3a and C5a receptors (C3aR-/- 33%, C5aR-/- 45% vs. BALB/c WT 23% p&lt;0.05). Furthermore, intravenous injection of CR2-Crry, an inhibitor of C3 convertase targeted to the sites of complement activation, markedly enhanced IFN-ã production in NK cells in response to poly I:C (CR2-Crry 35% vs PBS 17%, p&lt;0.01). Our results indicate that NK cell activation was negatively regulated by complement components. Blockade of complement activation is a potential therapy to amplify NK cell responses and promote enhanced host defense against viral infections and tumors.
This study is supported by NIH grant R01AI055007 awarded to Dr Jane Salmon.
Mechanism of M-CSF pathway-mediated innate resistance to tuberculosis (134.76)
Authors: Yang Chao-Tsung , Swaim Laura E , Ramakrishnan Lalita 
Published: January 2023

Abstract
The outcome of mycobacterial infections is determined by interactions between host and bacterial factors yet only very few host factors that affect the susceptibility to the infections are known. In particular, there has been an increasing appreciation that both innate immunity and hematopoietic growth factors such as M-CSF play important roles in modulating tuberculosis.
The Ramakrishnan laboratory has established a model for mycobacterial pathogenesis using Mycobacterium marinum (Mm) in the optically transparent and genetically tractable zebrafish. This model allows us to dissect the host cellular and genetic mechanisms that regulate macrophage function during infection. We have found that zebrafish with mutations in the M-CSF-receptor, fms, are hypersusceptible to Mm. M-CSF has pleitropic effects on macrophage development and function. To determine its mechanism of resistance in infection, we performed a step-wise comparison of infection in wildtype and fms-mutant fish. We found that initial macrophage production, as well as macrophage migration to infection sites, phagocytosis and microbicidal capacity were all normal in the fms mutants. Hypersusceptibility became apparent only after granulomas had formed. We are currently testing the hypothesis that the M-CSF pathway is required for macrophage replenishment during infection to maintain granuloma integrity and retain the bacteria within a more restrictive intracellular niche. This study may shed light on the genetics of macrophage homeostasis during mycobacterial infection.
Generation of C4 Binding Protein Deficient Mice: Evaluation of Putative Biological Functions (134.92)
Authors: Soimo Kipruto , Morales John E , Mueller-Ortiz Stacey , Nonaka Maumi , Nonaka Masura , Blom Ana M , Wetsel Rick A 
Published: January 2023

Abstract
The complement C4 binding protein (C4BP) has been suggested to modulate the antibody response to T dependent antigens by binding CD40 on B cells and to confer serum resistance when bound to certain bacteria, including M. catarrhalis. To date, all functions attributed to C4BP have been delineated in vitro. To evaluate functions of C4BP in vivo, we have generated C4bBP deficient mice by targeted disruption of mouse C4BP alpha gene. As expected, the C4bBP-/- serum exhibited significant increased hemolytic activity, and C3 deposition on M. catarrhalis was significantly increased. C4BP-/- mice and C4BP+/+ mice were injected i.p. with M catarrhalis to determine if elevated C3 deposition in C4BP-/- sera would correspond to increased killing of M. catarrhalis in vivo. Contrary to our expectation, the absence of C4BP did not impact the clearance (CFUs) of M.catarrhalis. To investigate if the absence of C4BP would impair the immune response to T-dependent Ags, mice were immunized with DNP-OVA. In contrast to the prediction of Brodeur et al (Immunity 2003), C4bBP-/- mice produced DNP specific IgG1 and IgG2a antibodies at similar levels as did C4bBP+/+ mice. Collectively, these studies provide evidence for the importance of C4BP in regulating complement activation and that C3 deposition on M. catarrhalis is impaired by binding C4BP. However, the absence of C4BP did not cause increased killing of M. catarrhalis in vivo, suggesting that other mechanisms of bacterial clearance compensate for the absence of C4BP. Moreover, C4BP as a major modulator of the antibody response was not supported in these studies, suggesting species dependent differences in C4BP biological functions.
TLR2 and TLR4 differentially regulate DC to stimulate Th17-associated cytokines in response to mycoplasma superantigen (135.30)
Authors: Mu Hong-Hua , Hasebe Akira , Cole Barry 
Published: January 2023

Abstract
Mycoplasma arthritidis induces toxic shock, arthritis and skin necrosis in mice. M. arthritidis mitogen (MAM), a potent superantigen (SAg) secreted by M. arthritidis, contributes to some of these symptoms in certain mouse strains. We previously demonstrated that C3H mouse strains differing in expression of TLR4, differ in immune reactivity to MAM due to differential engagement of TLR2 and TLR4. Herein, we demonstrated that in TLR4-defect C3H mice, following injection of MAM SAg, the splenic DCs were activated to express significantly higher CD8α surface markers than expressed in wild-type mice, and produced remarkably elevated IL-12p40, TNFα and IL-23. In vivo injection of anti-IL-23 antibody, or anti-CD8α antibody greatly suppressed IL-17 in splenic CD4+ cell cultures in comparison with untreated groups. In contrast, in wild-type C3H mice the interaction of MAM with both TLR4 and TLR2 led to reduction of the CD8α+ DC subset; as a consequence the IL-23 is down-regulated, but IL-10 are greatly up-regulated. The results suggest that TLRs control Th17 cell-associated cytokine development in response to MAM, and the CD8α+ DC subset is a major cell type responsible for IL-23 production. DC function that is regulated by discrete TLRs and CD8α molecules in response to MAM SAg might largely influence the outcome of subsequent adaptive immune responses in murine inflammatory diseases caused by M. arthritidis.
Calcium hydroxide attenuates inflammatory potential of lipoteichoic acid from Enterococcus faecalis (135.33)
Authors: Baik Jung Eun , Ryu Young Hee , Han Ji Young , Im Jintaek , Yang Jae Seung , Yun Cheol-Heui , Han Seung Hyun 
Published: January 2023

Abstract
Enterococcus faecalis is a pathogenic gram-positive bacterium closely related with refractory apical periodontitis. Since lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we prepared highly pure LTA from E. faecalis and investigated its inflammatory responses in a murine macrophage-like cell line, RAW 264.7. Upon exposure to E. faecalis LTA, RAW 264.7 cells substantially produced tumor necrosis factor-α (TNF-α) in concentration- and time-dependent manner. Notably, the LTA was able to stimulate Toll-like receptor 2 (TLR2), but not TLR4. Concomitantly, LTA enhanced the DNA-binding activity of a transcription factor, NF-κB, which plays an important role in the transcriptional activation of genes encoding inflammatory mediators. However, induction of TNF-α production was not observed in the cells exposed to LTA pre-treated with calcium hydroxide, which is widely used endodontic medicament for eliminating viable bacteria and inactivating virulence factors. Furthermore, calcium hydroxide-treated LTA was not able to stimulate TLR2. These results suggest that E. faecalis LTA could stimulate inflammatory responses through TLR2 and calcium hydroxide could detoxify LTA, resulting in attenuation of the inflammatory responses. Supported by the Korea Research Foundation Grant (KRF-2007-314-E00186).
The immune adaptor MPYS/STING/MITA signals via multiple differentially regulated pathways (135.60)
Authors: jin lei , xu liang-guo , cambier john c 
Published: January 2023

Abstract
MPYS, a.k.a. STING and MITA, is a newly described signaling adaptor that functions in both innate and adaptive immunity. MPYS transduces apoptotic death signals when aggregated on the B cell surface, but also plays an obligate role in the intracellular sensing of B-DNA and infectious agents. The basis of MPYS signal transduction is unclear. We report that MPYS signals by two distinct pathways. It associates via its juxtamembrane Box 1 and phosphotyrosine motifs with Tyk2 and STAT1, and utilizes these effectors in ISRE activation. Activation of IFNbeta occurs via MPYS association with and activation of TBK1 and IRF3, and is independent of VISA. MPYS (but not VISA) activation of IFNbeta expression is sensitive to LPS-induced TLR4-independent inhibition, but is positively cooperative with TLR signals. Thus MPYS activates multiple, uniquely regulated signaling pathways to mediate resistance to infection and regulation of adaptive immunity.
TLT2 Ligation Potentiates Neutrophil Migration and the Respiratory Burst Response (135.62)
Authors: Halpert Matthew M , King R. Glenn , Justement Louis B. 
Published: January 2023

Abstract
TREM-like Transcript 2 (TLT2) is a transmembrane receptor expressed on B cells, macrophages and neutrophils in the mouse and human. As of yet, an endogenous ligand has not been identified and the function of TLT2 remains to be elucidated. Because neutrophils are a primary mediator of the host innate immune response to bacterial infection, the analysis of factors that control neutrophil function is of great interest. Preliminary evidence suggests that TLT2 potentiates multiple neutrophil functions, including the respiratory burst and chemotaxis. Using a chemiluminescence system, it was shown that TLT2 ligation increases the primary phase of the respiratory burst by 20-25% in response to the bacterial peptide FMLP, although there is a slight inhibitory delay during the initial phase of the response. Transwell assays have been used to determine the effect that α-TLT2 mAb has on murine neutrophil chemotaxis. In vitro data demonstrate that TLT2 ligation increases migration 1.5-2.5 times in response to different chemotactic stimuli, such as FMLP, IL-8, and MIP-2. Importantly, α-TLT2 mAb treatment potentiates chemotaxis in response to agonists that signal via G-protein coupled receptors, but not in response to agonists that signal via tyrosine kinase-dependent pathways (e.g. G-CSF). These data suggest that TLT2 ligation effectively potentiates multiple neutrophil functional responses.
C-Cbl Regulates Neutrophil Integrin-dependent Respiratory Burst (135.67)
Authors: Peterson Erik J , Zou Liangxing , Ellingson Sonja 
Published: January 2023

Abstract
Signaling through integrin receptors is required for optimal reactive oxygen species (ROS) production by polymorphonuclear neutrophils (PMN). The E3 ubiquitin ligase and adapter protein C-Cbl has been implicated in integrin-mediated adhesion, and is a substrate for integrin-stimulated protein tyrosine kinases. We hypothesized that c-Cbl regulates integrin-driven PMN functions. We observed tyrosine phosphorylation of c-Cbl after beta-2 integrin cross-linking in the NB4 myeloid cell line. To address the requirement for c-Cbl in primary PMN signaling, we utilized c-Cbl deficient mice. We found that while c-Cbl is dispensable for PMN maturation, signaling through both immunoreceptors and integrins on PMNs is modulated by c-Cbl. Upon stimulation of c-Cbl-deficient bone marrow-derived PMNs with beta-2 integrin ligands (poly-RGD or fibrinogen plus TNF-alpha), we observed an 80% decrease in ROS generation compared to stimulated control cells. Phorbol ester-driven ROS generation is unaffected by c-Cbl deficiency. We next addressed the biochemical mechanism of c-Cbl action, investigating MAPK, PI3K, and Ca2+dependent pathways downstream of integrins. We observed no defect in either ERK or Akt phosphorylation after integrin engagement in c-Cbl deficient PMNs; furthermore, we observed that poly-RGD stimulated ROS production is unimpaired in PMNs lacking the PI3K p110gamma subunit. However, c-Cbl-null PMNs displayed decreased phospholipase C phosphorylation. Together, these data strongly suggest that c-Cbl selectively regulates Ca2+driven signaling downstream of beta2 integrins and upstream of ROS generation in PMNs.
Supported by the Minnesota Medical Foundation