bluuebunny/crossref_metadata_2025 huggingface dataset:
Receptor-independent, lipid dependent mechanism of Syk kinase activation in dendritic cells (134.71)
Authors: Ng Gilbert , Sharma Karan , Ward Sandra M , Desrosiers Melanie D , Stephens Leslie A , Schoel Michael , Li Tonglei , Lowell Clifford A , Ling Chang-Chun , Amrein Matthias W , Shi Yan
Published: January 2023

The binding of antigens to professional antigen-presenting cells (APCs), particularly dendritic cells (DCs), is essential for immune activation. Previously, it was shown that uric acid crystals, the causative agent of gout, were released from dying and injured cells as a "danger signal" to initiate a robust adaptive immune response. But how uric acid crystals lead to APC activation at a molecular level is unknown. Using atomic force microscopy (AFM) to analyze single cell activation in real time, we demonstrate that uric acid crystals directly bind cellular membranes, particularly cholesterol components, with very high affinity. Binding is dependent on intracellular Syk-kinase and PI3-kinase signaling and is independent of extracellular protein receptors. These findings suggest a mechanism that activates the immune system without specific cell surface protein receptors and presents a testable hypothesis for inflammation to materials without evolutionarily sensible receptors such as materials produced post-industrially. This research has applications in vaccine development, biomaterials, inflammation, and crystal-associated arthropathies.
The Mechanism of Invariant NKT Cell Responses to Viral Danger Signals (134.8)
Authors: Tyznik Aaron J , Tupin Emmanuel , Nagarajan Niranjana A , Her Min J , Benedict Chris A , Kronenberg Mitchell
Published: January 2023

Invariant (i)NKT cells influence the response to viral infections, although the mechanisms are poorly defined. Here we show that these innate-like lymphocytes secrete IFN-γ upon culture with CpG oligodeoxynucleotide-stimulated dendritic cells (DCs) from mouse bone marrow. This requires TLR9 signaling and IL-12 secretion by the activated DCs, but does not require CD1d expression. iNKT cells also produce IFN-γ in response to mouse CMV (MCMV) infection. Their mechanism of MCMV detection is quite similar to that of CpG, requiring bothTLR9 signaling and IL-12 secretion, while the need for CD1d expression is relatively minor. Consequently, iNKT cells have the ability to respond to a variety of microbes, including viruses, in an antigen-independent manner, suggesting they may play a broad role in anti-pathogen defenses despite their limited TCR repertoire.
Drosophila glycoprotein 93 is an ortholog of mammalian heat shock protein glycoprotein 96 and retains chaperone function for both Toll-like receptors and integrins (134.82)
Authors: Morales Crystal , Wu Shuang , Yang Yi , Hao Bing , Li Zihai
Published: January 2023

Mammalian heat shock protein (HSP) glycoprotein 96 (gp96) is an obligate chaperone for multiple integrins and Toll-like receptors (TLR). A database search revealed glycoprotein 93 (gp93) in Drosophila with high sequence homology to gp96, with unknown function. To determine if gp93 and gp96 are functionally conserved, we expressed gp93 in gp96 deficient mouse cells. We found that gp93 is able to chaperone multiple gp96 clients including α4, αL, β2 integrins and TLR2. Whereas gp96 undergoes intermolecular disulfide bond formation, gp93 is unable to do so due to absence of a critical cysteine residue at the N-terminus. However, site-directed mutagenesis of both gp96 and gp93 demonstrated that intermolecular disulfide bond formation is not critical for chaperone function. We conclude that gp93 is the Drosophila ortholog of gp96 and that its chaperone function is conserved. Further study of gp96 from an evolutionary angle should be informative to uncover the detailed mechanism of this understudied HSP90 paralogue in its chaperone function.

Z.L. is supported by NIH grant R01AI070603. C.M. is supported by a NIH predoctoral training grant 08-TA1007080F.
The use of ENU-mutagenesis mouse model to study the role of IL-15 alternative splice variant in the control of herpes simplex virus-1 infection (134.85)
Authors: Ku Chia-Chi , Yang Fu-Ju , Chang Pin-Fang , Wang Jy-Hsiu , Yeh Chao-Wei , Chang Mei-Ling , Kung John
Published: January 2023

Cytokine splice variants generated from alternative splicing of pre-mRNA have been broadly described and are implicated to influence cytokine signaling. Nonetheless, their importance in regulating the immune response remains unclear. The ENU-mutagenized pedigree 191 (P191) generated by the Mouse Mutagenesis Program Core Facility funded by the National Science Council was characterized by its depressed expression of CD44 on CD8 T cells. The mutant gene was linked to chromosome 8 and was mapped to IL-15 mRNA. Infection of P191 mutant mice with HSV-1 via flank skin revealed insufficient control of zosteriform as results of severe and delayed pathological manifestations, increased viral yields and lacking of Gr1+ cell recruitment to the lesional site. Whereas antigen specific CD8 T cells were generated in P191 mutant mice after HSV-1 infection detected with MHC class I tetramer in complex with HSV-1 gB498-505 epitope by flow cytometric analysis, the number was declined later time as opposed to be accumulated as generated in the wild type mice. These results have suggested that expression of IL-15 alternative splice variant could not only have effect ongenerating effective innate immune response in skin but also affect the function and maintenance of CD8 memory cells against HSV-1 infection. However, how the IL-15 splice variant modulates these immune mechanism remains to be clarified.
How caspase-7 controls intracellular infection in macrophages (134.86)
Authors: Amer Amal , Kanneganti Thirumala-Devi , Tridandapani Susheela , Wewers Mark
Published: January 2023

Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Ipaf-/- and caspase-1-/- derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that upon infection of murine macrophages, caspase-7 was activated downstream of the Ipaf inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin. Remarkably, mice lacking caspase-7 and their macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7-/- macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Ipaf inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium.
The effects of HSP72 expression on adhesion receptor activation and phagolysosome function in human PMNs (134.93)
Authors: Osterburg Andrew R. , Schwemberger Sandy , Babcock George F
Published: January 2023

Heat shock protein 72 (HSP72) expression is a hallmark of functionally suppressed neutrophils from pediatric burn patients. CD11b and CD15 are critical receptors that mediate neutrophil (PMN) adhesion. In this study, we examined the effects of HSP72 on these receptors as well as phagolysosome function. Expression of CD11b, CD15 and pHrodo were measured by flow cytometry. HSP72 expression in PMNs, induced by 60-90 min incubation at 42C, suppressed the upregulation of high affinity CD11b conformation induced by fMLP (~90% reduction vs controls). Additionally, CD15 expression is reduced in HSP72 positive PMNs. GM-CSF treatment (5-25 ng/ml) appears to have a modest ability to preserve PMN function in stressed PMNs. HSP72 positive PMNs also have a reduced capacity phagocytose. Opsonized pHrodo labelled Staphylococcus aureus were incubated with HSP72 positive or negative PMNs. Control samples had significantly more pHrodo positive bacteria (~2-fold increase, controls vs stressed PMNs, P<0.01) than the thermally stressed cells. However, the HSP72 positive PMNs appeared to acidify phagolysosomes to the same extent as control PMNs. Taken together, these data suggest that HSP72 expression reduces adhesion as well as the extent of phagocytosis. These deficits may contribute to the immune suppression found in thermally injured patients. Supported by Shriners of North America.
Induction of MyD88-Dependent Pro-inflammatory Cytokines Contributes to Lethal Toxicity of Staphylococcal Enterotoxins in Mice (135.13)
Authors: Saikh Kamal U , Kissner Teri L , Cisney Emily D , Ruthel Gordon , Ulrich Robert G
Published: January 2023

An elevated pro-inflammatory cytokine response is a primary cause of death by toxic shock after exposure to staphylococcal enterotoxin (SE). Identifying all host targets that control signaling for toxic shock is vital for developing a therapeutic strategy. We examined the role of the signaling adaptor MyD88 in cell culture and a mouse model of toxic shock. Our results indicated that elevated TNF-a, IFN-g, IL-1, and IL-6 production from mouse spleen cells treated with SEA alone, or in combination with lipopolysaccharide (LPS), was regulated by MyD88. Consistent with these results, MyD88-/- mice had impaired pro-inflammatory cytokine production in response to SEA and were resistant to a lethal SEA challenge. MyD88- dependent SEA- or SEB- induced lethality in mice was further confirmed in LPS- independent, D-galactosamine-sensitized mice. Our results demonstrate that MyD88-dependent pro-inflammatory signaling is responsible for SE toxicity. These findings offer important insight upon SE exposure and subsequent therapy targeting MyD88.
Downregulation of the TLR2-mediated signal by the DC-SIGN ligand Man-LAM (135.14)
Authors: Ohtani Makoto , Hasebe Akira , Iyori Mitsuhiro , Mohammad Haque Shamsul , Totsuka Yasunori , Shibata Ken-ichiro
Published: January 2023

C-type lectins have been shown to function both as adhesion and as pathogen recognition receptors. We have demonstrated that the Toll-like receptor 2 (TLR2) ligand FSL-1 enhanced phagocytosis of bacteria by enhancing expression level of scavenger receptors and C-type lectins such as DC-SIGN (dendritic cell-specific ICAM3 grabbing nonintegrin). This study demonstrated that the DC-SIGN ligand Man-LAM inhibits the TLR2-mediated signaling pathway and DC-SIGN forms a complex with TLR2 on the cell surface.
The role of IRF-1 on T cell subsets shift during pathogen infection (135.15)
Authors: Maruyama Saho , Kanoh Makoto , Matsumoto Akira , Asano Yoshihiro
Published: January 2023

[Purpose] Pathogen infection induces a shift to Th1 and inhibits a shift to Th2. The transcription factor IRF-1 is known to be essential for inducing IL-12 production and a shift to Th1. However, IRF-1 deficient cells express IL-12 p40 gene and induce Th1 cells during Listeria monocytogenes (Lm) infection. On the other hand, the effect of IRF-1 on Th2 is unknown. Since there is no inhibition of IL-4 production in the absence of IRF-1, we investigated the role of IRF-1 on IL-4 transcription during Lm infection.

[Results] (1) IL-4 production of cloned Th2 (MS-SB) cells was not inhibited by Lm infection of the clone. (2) When the mixture of MS-SB and T-depleted wild type or IRF-1 KO splenic cells were infected with Lm, Il-4 production was inhibited. Therefore, IRF-1 functions to inhibit IL-4 production in Th2 cells. (3) We transfected MS-SB cells with Luciferase reporter plasmid containing IL-4 promoter and IRF-1 expression plasmid. When MS-SB cells were stimulated with PMA plus Ionomycin, IL-4 production was inhibited without affecting Luciferase activity. Therefore, IRF-1 does not inhibit IL-4 promoter activity.

[Conclusion] There are two silencer regions in down stream of IL-4 coding region, and these silencers contain IRF-1 binding motif. Our results suggest that IRF-1 functions as a negative regulator for IL-4 gene transcription via silencer regions in Th2 cells during Lm infection.
TLR9 activation is an important mechanism of antigen-induced chronic lung inflammation (135.19)
Authors: Ito Toshihiro , Schaller Matthew , Hogaboam Cory M. , Lukacs Nicholas W. , Chensue Stephen W. , Kunkel Steven L.
Published: January 2023

It is likely that the continuous host response to a persistent challenge polarizes the cytokine environment toward a Th2 cytokine phenotype. We investigated the role of TLR9 in a Th2-driven pulmonary granulomatous response, initiated via the embolization of Schistosoma mansoni eggs to the lungs of mice. TLR9-/- mice showed an increase in pulmonary granuloma size, augmented collagen deposition, an increase in the Th2 cytokine phenotype, impaired accumulation of dendritic cells (DCs), and an increase in numbers of lung macrophages compared with WT mice. BM-derived DCs, but not macrophages, promoted the production of Th2 cytokines from CD4+ T cells during the S. mansoni egg antigen-induced immune response. In addition, DCs from TLR9-/- mice induced impaired Th1 cytokine (IFN-γ) and enhanced Th2 cytokine (IL-4, IL-5, and IL-13) compared with DCs from WT mice. Macrophages from TLR9-/- mice expressed significantly higher alternatively-activated (M2) phenotype characterized by increased "found in inflammatory zone-1" (FIZZ-1) expression. Passive immunization with neutralizing antibodies specific for FIZZ-1 during granuloma formation resulted in a decrease in granuloma size and collagen deposition. Together, these results suggest TLR9 plays an important role in the maintenance stage of specific chronic lung disorders.

This work was supported by NIH grants P01-HL031963, HL092845, HL074024, and HL031237.
Control of Interleukin-27 p28 Gene Transcription by Interferon Regulatory Factors (135.21)
Authors: Liu Jianguo
Published: January 2023

Interferon Regulatory Factor (IRF) family members, especially interferon regulatory factor-1 (IRF-1) and interferon regulatory factor-8 (IRF-8 or ICSBP), play important roles in interferon signaling in a wide range of host responses to infection and malignant growth. Interleukin-27 (IL-27), as a member of IL-12 family cytokines, not only acts like a pro-inflammatory cytokine that plays an important role in the regulation of differentiation of naive T helper cells but also possesses anti-inflammatory properties. IL-27 is consisted of Epstein-Barr virus (EBV)-induced gene 3 (EBI3), an IL-12p40-related protein, and p28, an IL-12p35-related polypeptide. Our recent work has revealed an intimate relationship between IRF-1 and IL-27 p28 in that IRF-1 regulates IL-27 p28 gene transcription by specifically binding to the IRF-1 response elements in the p28 promoter. In this study, we find that IRF-8 deficient macrophages are highly defective in the production of IL-27 at both mRNA and protein levels. Transcriptional analyses reveal that IRF-8 is a potent activator of the IL-27 p28 gene. It acts independent of the existing IRF-1 binding site located at -57 to -48 in the IL-27 p28 promoter. Consistent with this observation, over-expression of IRF-8 induces the IL-27 p28 promoter activity in a dose-dependent manner. This study provides further novel mechanistic information regarding how signals initiated during innate and adaptive immune responses synergize to yield greater IL-27 production and sustained cellular immunity.
Reprogramming of TLR4 pathways in endotoxin-tolerant cells (135.26)
Authors: Medvedev Andrei E , Chen Haiyan , Song Chang , Wahl Larry M , Fitzgerald Katherine A , Li Liwu , Piao Wenji
Published: January 2023

Endotoxin tolerance is important in the pathogenesis of the anti-inflammatory response syndrome and secondary infections in septic shock survivors. We elucidated whether reprogramming of TLR4 responses in endotoxin tolerance is mediated by altered signalosome assembly, kinase activation and up-regulation of negative regulators of TLR4 signaling. LPS stimulation of medium-pretreated human monocytes induced TAK1 activation, IRAK1-IKKγ association and TRAF6/IKKγ ubiquitination, while LPS tolerization diminished these responses. LPS-tolerant cells showed increased expression of Tollip, SOCS1, IRAKM, and SHIP1 that correlated with reduced expression of TNF-α, IL-6 and IL-8, while exhibiting similar levels of TNFRII, formyl peptide receptor I, PgE synthase and bactericidal/ permeability-increasing protein-like 2. Endotoxin tolerization suppressed LPS-mediated TLR4-TRIF-TBK1 associations, activation of TBK1 and IRF3, expression of RANTES and IFN-β, but increased levels of SIKE and SARM. Expression of Tollip in 293/TLR4/MD-2 cells blunted activation of NF-κB and RANTES in response to LPS. Thus, endotoxin tolerance interferes with LPS-inducible TAK1 activation, TLR4-TRIF-TBK1 and IRAK1-IKKγ assembly, TRAF6/IKKγ ubiquitination, TBK1/IRF3 activation and increases expression of negative regulators of TLR signaling (supported by AI067497 (KAF.), AI64414 (LL) and AI059524 (AEM)).
Muramyl dipeptide potentiates lipoteichoic acid-induced nitric oxide production in macrophage (135.32)
Authors: Jeon Jun-Ho , Yang Jihyun , Im Jintaek , Han Ji Young , Kim Sun Kyung , Baik Jung Eun , Yun Cheol-Heui , Han Seung Hyun
Published: January 2023

Pathogen-associated molecular patterns, such as lipoteichoic acid (LTA) and peptidoglycan (PGN) of gram-positive bacteria, often cooperate together to induce immune responses in the host. In this study, we investigated nitric oxide (NO) production by macrophages in response to LTA and muramyl dipeptide (MDP), a minimal structural unit of PGN. MDP alone could not induce NO production in both murine macrophages and a murine macrophage-like cell line, RAW 264.7, whereas LTA induced NO production. However, MDP potentiated LTA-induced NO production under the same condition. This was due to enhanced activation of Toll-like receptor 2 (TLR2) recognizing LTA since MDP increased TLR2 expression on the surface of macrophages and the potentiation effect was abolished in the macrophages derived from TLR2-deficient mice. Furthermore, MyD88 and MAP kinases such as ERK and p38 kinase also appear to be essentially required for such potentiation, which was determined by using MyD88-deficient macrophages and their specific inhibitors, PD98059 and SB203580, respectively. Subsequently, MDP enhanced the LTA-induced activation of NF-κB in RAW 264.7. These findings suggest that MDP potentiates the ability of LTA to stimulate macrophages via upregulating TLR2 activity. Supported by a grant from 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science and Technology (11-2008-15-005-01).
The Pseudosubstrate domain of Protein Kinase C-epsilon directs translocation to phagosomes via binding to phosphomonoinositides (135.46)
Authors: Lennartz Michelle R. , Cheeseman Keylon , Hanes Cheryl M , Kashiwagi Kaori , Shirai Yasuhito , Saito Naoaki , Loegering Daniel J.
Published: January 2023

Protein kinase C-epsilon (PKC-ε) translocation is required for efficient IgG-mediated phagocytosis. We have published that the C1B region, binding to DAG generated by PLCγ1, is necessary for translocation (Cheeseman et al, MCB, 2006). However, C1B is not sufficient for translocation. Deletion and mutational analysis, as well as the use of PKC-ε/PKC-δ chimeras, indicate that the pseudosubstrate domain (εPS) is necessary for PKC-ε translocation. A PKC-ε fragment containing only the C1 and PS regions, as well as a chimera of PKC-δ containing εPS and εC1B, accumulated, demonstrating that these two regions are necessary and sufficient for targeting PKC-ε to phagosomes. Protein-lipid overlays of εPS-GST indicate that εPS preferentially associates with phosphomonoinositides (PI5P, PI3P>PI4P>>>polyphospho-inositides). Of the PI3 Kinase inhibitors, 100 nM wortmannin, but not 50 µM LY294002 inhibits PKC-ε translocation to phagosomes. That both drugs block the formation of PI3P as indicated by 2XFYVE staining argues against PI3P as the docking lipid for PKC-ε. Similar to PKC-ε, wortmannin, but not LY294002, blocked accumulation of PLCδPH (a PI4,5P2 reporter) at forming phagosomes. Together, these data suggest that εPS binding to PI4P and/or PI5P is necessary for PKC-ε translocation to phagosomes. Efforts are underway to identify the specific lipid binding partner for PKC-ε. Funded by NIH R01 AI05821 (MRL).
Chicken scavenger receptors and their ligand-induced cellular immune responses (135.57)
Authors: He Haiqi , MacKinnon Kathryn M. , Genovese Kenneth J. , Nerren Jessica R. , Swaggerty Christi L. , Kogut Michael H.
Published: January 2023

Scavenger receptors (SR) play an important role in the innate immunity. To evaluate the role of SR in the chicken immune system, we investigated immune responses, including oxidative burst, degranulation, phagocytosis, nitric oxide (NO) production, and cytokine gene expression of chicken heterophils, peripheral blood mononuclear cells, and a chicken macrophage cell line (HD11) to stimulations with various SR class A and B ligands. Our results show that ligands for SR-A, fucoidan, poly (I) and poly (G), but not ligands for SR-B, phosphatidylserine (PdS) and native LDL, stimulated a dose-dependent NO production in HD11 cells. However, these SR-A ligands failed to induce NO in monocytes isolated from peripheral blood. Quantitative RT-PCR indicated that SR ligands differentially affected the gene expression of several cytokines and chemokine in HD11 cells. SR-A ligands induced a strong up-regulation of proinflammatory cytokine IL-1β and to a lesser extent, IL-6, IL-12 and Mip-1β, with down-regulation of IL-4. However, SR-B ligands did not alter expression of these genes. Heterophil degranulation was not induced by any SR ligands. Stimulation of heterophils and monocytes with SR ligands also did not increase oxidative burst, with the exception of native LDL. However, LDL induced oxidative burst is probably mediated by the LDL receptor, not by SR.
MOLECULAR LINK BETWEEN TOLL LIKE RECEPTORS, TUMOROGENESIS AND OBESITY (135.61)
Authors: Kumaraguru Uday , Nandakumar Subhadra
Published: January 2023

Hygiene hypothesis states that "Lack of early childhood exposure to infectious agents and symbiotic microbes increases the susceptibility to allergic diseases by modulating the immune system". We predicted that neonate's exposure to microbes or its components will have long term protective effect against infectious diseases and cancer. To this end, we simulated dirty environment (exposure to microbial components) for a group of neonatal mice and compared them to mice kept in sterile conditions. The mice were allowed to grow up to 5-6 weeks after which they were analyzed for their ability to resist infection and tumor challenge. The mice that were in a simulated dirty environment were better able to resists HSV disease manifestation and tumor challenge with adeno carcinoma cells. Interestingly, the mice kept in sterile conditions gained significant body weight. Taken together, mice in cleaner environment became obese and were prone to infection and cancer indicating a similarity with human epidemiological data. We found that microbial exposure resulted in the 2.5 times more bone morphogenetic protein -7 in microbes exposed mice compared to mice kept in cleaner environment. BMP-7 is involved in the generation of brown fat that is known to be efficient in energy utilization, metabolism and weight reduction. Hence we conclude that bmp-7 induced by exposure to microbial components may be the molecular link between the microbial exposure pattern, obesity development and immune-protection. Increasing the levels of BMP-7 may well be employed as a therapeutic tool.

Acknowledgements:

ETSU-College of Medicine-Start up funds
Pathogen-induced activation of inflammasomes requires intracellular redistribution of the apoptosis associated speck-like protein containing a caspase recruitment domain (ASC) (135.70)
Authors: Bryan Nicole B , Dorfleutner Andrea , Rojanasakul Yon , Stehlik Christian
Published: January 2023

Activation of caspase-1, which is necessary for the maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18, occurs upon assembly of multi-protein complexes known as inflammasomes. Apoptosis associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein, which is essential for the recruitment of caspase-1 into inflammasomes. However, distribution of endogenous ASC has not been thoroughly examined. In the current study, we used laser scanning confocal microscopy and subcellular fractionation to demonstrate that endogenous ASC localized primarily to the nucleus in resting human macrophages. Upon pathogenic stimulation, ASC rapidly redistributed to the cytosol, and subsequently formed a characteristic perinuclear aggregate. These aggregates were found to incorporate other key inflammasome proteins including NLRP3 and caspase-1. Furthermore, sequestering ASC to the nucleus impaired formation of the inflammasome and abolished caspase-1 activation, as measured by IL-1β secretion. Our results indicate that the redistribution of ASC from the nucleus to the cytosol represents a novel and essential mechanism that regulates inflammasome assembly and activation.
Nod1 and Nod2 regulation of pulmonary immunity to Legionella pneumophila (135.77)
Authors: Berrington William Richard , Smith Kelley D , Wells Rick D , Jansson-Hutson Malinka , Iyer Ravi , Skerrett Shawn J , Hawn Thomas R
Published: January 2023

The role of Nod1 and Nod2 in pulmonary innate immune responses is poorly understood. We hypothesized that Nod1 and Nod2 regulate non-redundant and specific immune responses during murine in vivo infection with Legionella pneumophila (Lp). We first examined whether Nod1 and Nod2 regulate detection of Lp in HEK293 cells transfected with luciferase reporter constructs and either Nod1 or Nod2. Heat-killed Lp stimulated NF-κB and interferon-β-dependent promoter activity in cells transfected with either Nod1 or Nod2. To examine the role of Nod1 and Nod2 during in vivo infection, we exposed Nod1-/- and Nod2-/- and C57Bl/6 control mice to aerosolized Lp. Bronchoalveolar lavage fluid from Nod1-/- animals had decreased neutrophils compared to wild type animals at 4 and 24 hours post-infection. Furthermore, monocyte recruitment was also impaired at 24 hours. In addition Nod1-/- mice had increased lung bacterial loads at 72 hours in comparison to wild type and Nod2-/- mice. In contrast, there was no reproducible difference in cytokines in Nod1-/- lung homogenates at 4 hours and 24 hours. Conversely, Nod2-/- mice had impaired neutrophil cell recruitment at 24 hours, and only a trend to increased organism burden at 72 hours (p=0.07). Nod2-/- animals, however, showed reproducible impairment in cytokine production from lung homogenates. Lung histologic sections at later time points (3-7 days) had no detectable differences in inflammation in Nod1-/- or Nod2-/- mice in comparison to wild type mice. Overall these data indicate that Nod1 regulates early inflammatory cell recruitment of both neutrophils and monocytes to the alveolar space without detectable differences in inflammatory cytokine productions or changes in lung histology at later time points.
Catecholamine mediated S49 T lymphoma apoptosis is regulated by p38 MAPK (136.15)
Authors: LaJevic Melissa D , Suleiman Samia , Cohen Rhonna L , Chambers Donald A.
Published: January 2023

p38 MAPK is an important intracellular signaling mediator for a variety of cellular and environmental stressors and plays a role in cell survival, apoptosis, and inflammation. Previously, our lab has shown that norepinephrine (NE) treatment of S49 T lymphoma cells leads to increased activity of p38 MAPK by the classical βAR/AC/cAMP/PKA pathway. Exogenous cAMP or βAR stimulation of S49 cells leads to apoptosis. To study the relationship between p38 MAPK activity and apoptosis, S49 cells were incubated in the presence and absence of a cAMP analogue (8-bromo) with or without the p38 MAPK inhibitor SB203580. After 72 hours of cAMP treatment, S49 cell viability decreased by 50%, yet in the presence of SB203580 cell viability decreased by 35%. The steady state levels of FasL mRNA (an early biochemical marker of apoptosis) were analyzed after two hours of S49 cell treatment with 8-bromo cAMP in the absence or presence of SB203580. FasL mRNA levels increased 9.8 fold with 8-bromo cAMP treatment for 2 hours and was reversed 50% by p38 MAPK inhibition. These results suggest that activation of p38 MAPK by NE or cAMP is associated with T lymphoma cell apoptosis. Supported by NIH
Deletion of miRNAs in bone morrow disrupts iNKT cell development and function (136.19)
Authors: Zhou Li , Seo Kook-Heon , He Hong-Zhi , Pacholczyk Rafal , Meng Dong-Mei , Dong Zhen , Mi Qing-Sheng
Published: January 2023

MicroRNAs (miRNAs), a class of non-coding small RNAs, function as endogenous translation repressors of protein-coding genes. Accumulated evidences indicate that miRNAs are differentially regulated in developmental hematopoietic organs during hematopoietic lineage differentiation. Invariant Natural Killer T (iNKT) cells are potent regulators of diverse immune responses. The role of miRNAs in iNKT cell development is unknown. Here we test if lack of miRNAs following the deletion of Dicer, the miRNAs-processing enzyme, will affect iNKT cell development and function. We generated a mouse strain with tissue-specific disruption of Dicer in the hematopoietic progenitors, which is designated as Dicerfl/flTie2Cre+ (Dicer KO). Significantly decreased iNKT cell ratio and munber were found in the thymus, spleen, and liver of Dicer KO mice compared to littermate control (Dicerfl/flTie2cre-). The frequencies of mature CD44hiNK-1.1+ iNKT cells were significantly decreased in Dicer KO mice, suggesting that miRNAs may regulate iNKT cell maturation at the CD44low to CD44hi and NK1.1- to NK1.1+ checkpoints. Furthermore, miRNA-deficient peripheral iNKT cells display profound defects in alpha-GalCer-induced activation and cytokine production. Bone marrow (BM) from Dicer KO mice poorly reconstituted iNKT cells compared to BM from WT control mice, while thymic iNKT cells from WT mice poorly migrate to liver and spleen in irradiated Dicer KO mice compared to that in WT control mice. In summary, miRNAs are potent regulators of iNKT cell development, migration, and function. (Supported by JDRF).
MicroRNA-30c is down-regulated during NKL cell activation and cytotoxicity induced by CD226 crosslinking (136.20)
Authors: Chen Lihua , Gong Jiuyu , Wang Tao , Xu Zhuwei , Zhuang Ran , Zhang Yun , Yang Angang , Jin Boquan
Published: January 2023

MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. There have been little report on NK cell associated miRs until now. CD226 is a kind of activation receptors located on NK cells and involved in the activation and cytotoxixity of NK cells. Previous study showed that CD226 cross-linking increased the CD107a expression on NKL cells. In the present study, we identified miRs induced in NKL cells after exposure to CD226 crosslinking using microarray and quantitive RT-PCR technology. It has been found that miR-30c level was down-regulated in NKL cells after CD226 crosslinking. Furthermore transfection of miR-30c into NKL cells decreased the CD107a expression on NKL cells induced by CD226 crosslinking, suggesting that miR-30c plays a role in NKL activation and cytotoxicity induced by CD226 crosslinking. Meanwhile miR-30c was predicted to base-pair with sequences in the 3' UTRs of the HMBOX gene. Altogether, our studies propose that miR-30c may play a role in control of CD226 signalling in NKL cells through a negative feedback regulation loop involving in down-regulation of HMBOX expression.
C8orf13/BLK association with systemic lupus erythematosus across different ethnicities (136.28)
Authors: Lu Rufei , Dominguez N , Lessard C , Deshmukh H , Vidal G , Macwana S , Kelly J , Kim X , Cobb B , Kaufman K , Bae S-C , Tsao B , Shen N , Langefeld C , Niewold T , Gilkeson G , Merrill J , Moser K , Harley J , Nath S , Gaffney P , James J , Guthridge J
Published: January 2023

SLE is a complex autoimmune disease with immunological abnormalities that targets B cell functions and development. Previous genome-wide association studies identified C8orf13/BLK as a novel SLE risk factor. BLK (B lymphoid tyrosine kinase) has been shown to have potential effects on B cell develoment. The main objectives of this study are to: 1.) assess this association in independent populations and 2.) determine if BLK risk alleles correlate with alteration in peripheral blood B cell development and protein expression. We genotyped 52 SNPs within the C8orf13/BLK region in European-derived, Asian and African American populations. rs13277113 is associated with SLE in all three populations. A few novel upstream SNPs within the intergenic region demonstrate a stronger association in Asians. Flow cytometry data suggests there is a significant difference in BLK expression and T2 cells B cell numbers between high-risk and non-risk individuals based on the risk haplotype. Our results suggest that the BLK genetic polymorphisms alter immune system development and function to increase the risk for SLE development. Supported by NIH RR020143, RR015577, NIAID-DAIT-BAA-05-11, AI031584, AR053483, AR48940, AI063274, AR052125, AR043247 and OCAST # HR08-037.
Determining the copy number variation (CNV) of human Complement Factor H Related Genes: from Southern blots to Real-time PCRs (136.30)
Authors: Kitzmiller Kathryn J , Yang Yan , Wu Yee Ling , Zhou Bi , Chung Erwin K , Yu C. Yung
Published: January 2023

Current applications for detecting the CNV of Complement Factor H-Related Genes 3 and 1, CFHR3 and CFHR1, such as Multiplex Ligation-Dependent Probe Amplification, MLPA, rely on the presence of single nucleotide polymorphisms that associate with but do not necessarily reflect the actual physical variation of gene copy numbers. A 76-kb "deletion" of genomic region for CFHR3-CFHR1was detectable using long range-mapping of PmeI-digested DNA resolved by Pulsed-Field Electrophoresis using a CFHR1 cDNA probe. The deletion was further characterized by Southern blots of genomic DNA digested by TaqI using CFHR2 cDNA probe, or PshAI-PvuII using CFH exons 21-22 probe. Thus, we developed quantitative Real-time PCR (RT-PCR) assays to detect the deleted region using 36 samples of genomic DNA characterized by Southern blot for CNV of CFHR3-CFHR1. Three RT-PCR assays were designed to measure a common region between CFHR3 and CFHR4, and CFHR1 and CFHR2, as well as intergenic region between CFHR3 and CFHR1using the standard curve method and obtaining the ratio between the target gene(s) and endogenous control. The 3 RT-PCR assays were able to correctly identify the CNV of CFHR3-R1 in 35/36 samples, for 97.5% accuracy and 100% sensitivity. This method will enable further investigation of CFHR3-R1CNVs in Age-Related Macular Degeneration, atypical Hemolytic Anemia and Systemic Lupus Erythematosus.
Epigenetic regulation of BCL6 and other genes important for B-cell maturation (136.32)
Authors: Ramachandrareddy Himabindu , Shen Yulei , Chan Wing C , McKeithan Timothy W
Published: January 2023

BCL6, a proto-oncogene frequently mutated and translocated in B-cell lymphoma, encodes a transcriptional repressor essential for the germinal center (GC) reaction. BCL6 mRNA and protein have higher expression in GC-B cells than in pre- and post-GC-B cells despite strong autorepression. Hypothesizing that histone modifications epigenetically regulate BCL6 during B-cell differentiation, we mapped a number of histone modifications associated with active transcription far upstream to the promoters of BCL6 and ~100 other genes in pre-GC-B, GC-B and plasma cell lines at nucleosomal resolution using the ChIP-chip assay. We identified numerous regions of histone H3 acetylation and H3 lysine-4 mono- and trimethylation (H3K4me1 and -3) within and upstream of BCL6; most striking was a vast region of H3K4me1 extending 140-260 kb upstream of BCL6 seen in a GC-B-cell line, but not in pre- or post-GC-B-cells. We also observed high expression of an intergenic transcript within this far upstream region in GC-B-cell lines but not in numerous other lines, suggesting distant regulatory regions, active only in GC-B-cells, account for high BCL6 expression. Many other genes also showed evidence for stage-specific distal regulatory regions. Defining a histone code for B cell master regulators will elucidate transcriptional networks central to both the immune response and cancer. The work is supported by UNMC Eppley Cancer Institute.
Identification of corticosteroid regulated genes in human Th2 cells (136.37)
Authors: Bowen Holly , Lee Tak H , Cousins David J , Lavender Paul
Published: January 2023

Glucocorticoids (GCs) are the mainstay treatment for many inflammatory diseases including asthma, which in the majority of patients can be controlled by inhaled GC. The molecular mechanisms of this regulation are however still not fully understood, especially in the small percentage of corticosteroid insensitive patients, who fail to respond well to even high doses of GCs.

In order to gain a deeper understanding of the molecular mechanisms of GC mediated gene regulation we have performed microarrays on in vitro differentiated Th1 & Th2 cells treated with the synthetic GC Dexamethasone (Dex). Naïve human CD4+ T cells were cultured for 28 days under either Th1 or Th2 skewing conditions. At day 28 these highly polarised cells (>95% Th1 cells are IFNγ positive, and >50% Th2 cells are interleukin (IL)-5 positive) were activated in the presence or absence of Dex at 10-7M. The isolated total RNA was used to probe the U133Plus2 gene chip (n=2), and results were confirmed via quantitative real-time PCR (n=4).

Our results have identified 64 genes consistently altered > 2-fold by Dex treatment in differentiated human T cells. These genes include documented Dex targets such as GILZ and FKBP51, as well as some novel targets. We are currently identifying the pathways that these novel targets fall into, as well as investigating potential differences between to corticosteroid sensitive and insensitive asthmatic patients.

This work is funded by the MRC and Asthma UK.
Antisense transcripts negatively regulate transcription of multiple variegated killer immunoglobulin-like receptor (KIR) genes (136.38)
Authors: Cichocki Frank , Lenvik Todd , Anderson Stephen K , Miller Jeffrey S
Published: January 2023

The objective of this study is to understand the epigenetic mechanisms that regulate Killer Immunoglobulin-Like Receptor (KIR) gene regulation in human natural killer (NK) cells. We have previously identified both bidirectional transcription within the previously characterized KIR promoter and a novel promoter element upstream of the previously characterized promoter. Based on these findings, we propose that antisense transcripts homologous to the KIR promoter region participate in the establishment of the epigenetic modifications that silence individual KIR genes during human NK cell development. To test this prediction, we overexpressed KIR3DL1 antisense transcripts in developing NK cells and found that mature cells from these cultures exhibited a four-fold decrease in KIR expression relative to GFP-expressing controls. We have also identified 324 base pair and 28 base pair double-stranded RNA molecules homologous to the KIR3DL1 promoter region, suggesting that a siRNA mechanism may be responsible for establishment of the promoter methylation patterns within the KIR locus. Understanding the mechanisms of KIR expression, a requisite for NK cell education/licensing, may allow us to understand the acquisition of effector function and manipulate it for therapeutic benefit.

R01 HL 55417
Genetic potential of macrophage TNF production regulates immune mediated diseases (136.5)
Authors: Gillett Alan M , Marta Monica , Olsson Tomas , Harris Robert , Jagodic Maja
Published: January 2023

Tumor necrosis factor (TNF) is produced by several immune cell types in response to injury or infection. A region on rat chromosome 4 regulates several inflammatory diseases including experimental autoimmune encephalomyelitis (EAE), experimental arthritis and experimental autoimmune neuritis. We hypothesized that central inflammatory processes may be controlled by genetic determinants in this region and therefore mapped TNF production using linkage analysis in an advanced intercross line between DA and PVG.AV1 rats. We identified a single TNF regulating quantitative trait locus with a LOD score of 6.16 and confirmed the biological effect in a congenic rat strain. Congenic strain macrophages produced less TNF upon lipopolysaccharide administration and subsequently had a reduced pro-inflammatory phenotype. The reduced TNF potential associated with reduced disease. Congenic rats had reduced EAE compared to control DA rats. We have identified genetically determined regulation of TNF, a central pro-inflammatory cytokine in many diseases. Determination of genes and mechanisms involved in this regulation will be valuable in pathological understanding and treatment development.
Differential regulation of maturation genes in human dendritic cells stimulated with respiratory syncytial virus and TLR agonists (136.8)
Authors: Kallal Lara E , Lukacs Nicholas W
Published: January 2023

Dendritic cells (DCs) undergo rapid phenotypic change upon stimulation of surface and intracellular Toll Like Receptors (TLRs), including upregulation of the lymph node homing receptor, CCR7, and production of cytokines such as IL-10, IL-12 and Type I interferon (IFN). Pathogens are known to interfere with these signatures of DC maturation, and respiratory syncytial virus (RSV) in particular has been shown to antagonize TLR-mediated IFNβ production in dendritic cells. We thus sought to investigate the regulation of CCR7 and IFNβ in human DCs after activation by RSV and TLR agonists. We found that DCs stimulated with polyI:C and LPS, TLR3 and TLR4 agonists, respectively, significantly upregulated CCR7 after 6 and 24 hours. PolyI:C was the most potent upregulator of IFNβ at both 6 and 24 hours, with LPS upregulating this cytokine only at 6 hrs. Though other RSV-induced genes were activated, DCs stimulated with RSV failed to upregulate CCR7 or IFNβ. Analysis of activating (H3K4) and silencing (H3K27) histone modifications at the promoters of CCR7 and IFNβ showed, compared to unstimulated DCs, high H3K4 methylation of CCR7 and IFNβ upon PolyI:C stimulation, whereas LPS showed high H3K4 at CCR7, but high H3K27 at IFNβ. Methylation marks after RSV stimulation did not differ markedly from unstimulated DCs, suggesting that RSV may interfere with cellular machinery that controls gene expression, potentially delaying DC maturation.

This work is supported by NIH R01 AI073876
Non-lymphoid expression of B7-H1 regulates severity of proteoglycan-induced arthritis (137.24)
Authors: Hamel Keith M , Doodes Paul , Cao Yanxia , Wang Yumei , Rodeghero Rachel L , Chen Lieping , Finnegan Alison
Published: January 2023

The immune system has several mechanisms to prevent autoreative T cell activation in rheumatoid arthritis (RA) including the co-inhibitory signal delivered to T-cells by PD-1 ligation of B7-H1/PD-L1 on antigen presenting cells (APCs) and on non-lymphoid tissue. B-cells express B7-H1 and are an important APC population as highlighted by the efficacy of B cell depletion therapy in RA patients. Here we tested B7-H1 expression on B cells in a murine model of RA, proteoglycan-induced arthritis (PGIA). Wildtype (WT) and B7-H1-/- female BALB/c (≥3 months of age) were immunized intraperitoneally with 150 μg human PG in adjuvant [dimethy dioctadecy ammonium bromide (DDA)] 3 times at 3-week intervals. Exacerbation of PGIA was observed in B7-H1-/- mice along with increased titers of autoantibodies and CD4+ T cell proliferation. SCID mice reconstituted with B7-H1-/- B-cells or both B7-H1-/- B and T cells developed a similar intensity of PGIA as mice reconstituted with WT cells. Our results indicate that B7-H1 regulates the onset and severity of PGIA and that non-lymphoid B7-H1 expression is sufficient to control the more aggressive arthritis observed in the complete B7-H1-/- mice. Funding was provided by NIAMS NIH grant AR47657.
Constitutive expression of Foxp3 in conventional T cells reduces cellular proliferation and alleviates autoimmune arthritis (137.25)
Authors: Guo Linjie , Zheng Biao , Han Shuhua
Published: January 2023

The forkhead family transicrption factor Foxp3 is critically important for the development and function of regulatory T cells (Tregs). Lack of Foxp3 leads to the development of fatal autoimmune lymphoproliferative diseases. Previously studies have shown that ectopic Foxp3 expression can phenotypically convert effector T cells to regulatory T cells. However, we have recently generated Foxp3 transgenic mice under the control of lck distal promoter. The transgene (Tg) Foxp3 is expressed in the majority of CD4+ and CD8+ T cells but at modulate levels. The ectopic expression of Foxp3 renders T cells hyporesponsive but conferred neither Treg phenotype nor suppressive function. In addition, we found that Foxp3 transgenic mice exhibit reduced susceptibility to collagen-induced arthritis (CIA). Foxp3 Tg mice had delayed disease onset and developed milder arthritis compare to wild type controls. The serum autoantibody levels were reduced in Foxp3 Tg mice. Both cytokine production and antigen-specific proliferation were decreased in T cells from draining lymph nodes of Foxp3 Tg mice. It has been shown that in human Foxp3 expression is not only confined to CD4+CD25+ Tregs but also in a high percentage of activated T cells that do not have suppressive capabilities. Thus, our results indicate that Foxp3 may play an important role in regulating immune responses and inflammation by attenuating proliferative responses and cytokine production of antigen-specific T cells.
Mouse Models of Autoimmunity at The Jackson Laboratory (137.40)
Authors: Merriam Jen J , Donahue Leah R. , Lutz Cathleen M , Rockwood Stephen F , Sasner Michael
Published: January 2023

The Jackson Laboratory Repository was established to serve as a centralized resource for the archiving and distribution of genetically engineered mice of high health status. Nearly 400 strains are added to the collection each year. New strains for Systemic Lupus Erythematosus work include NOD Faslgld mice (8223) which develop autoimmunity at ~8 weeks of age. BcN/LmoJ inbreds (7228) develop fatal autoimmune glomerulonephritis. Nba2 congenics (7716) enable genetic study of an NZB-derived QTL. For Type I Diabetes researchers: NOD Itgax-DTR/EGFP (Cd11c-DTR) transgenics (8549) are transiently depleted of dendritic cells upon DT treatment. NOD Foxp3-EGFP/cre mice (8694) enable GFP expression in FoxP3+ cells when crossed with a floxed allele strain (useful for studies of regulatory T cell involvement). Of general interest, we have undertaken a project to comprehensively characterize Cre transgenic strains. A public searchable database presenting embryonic and adult expression patterns and images using terms from the Mouse Anatomical Dictionary is now under construction. Researchers wishing to have strains considered for inclusion in the Repository may submit their strains at: http://www.jax.org/grc/.