bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of IL-17 in the mucosal adjuvant activity of cholera toxin for epicutaneous vaccines (134.53)
Authors: Boyaka Prosper N , Iwakura Yoichiro , Chaplin David D
Published: January 2023

It is now established that the mucosal adjuvant activity of cholera toxin (CT) involves the induction of IL-1 and other proinflammatory cytokines. Interleukin-17 (IL-17) is a cytokine implicated in the regulation of inflammation. In addition, Th-17 associated cytokines TGF-beta and IL-6 act as Ab mu to alpha class switch factor and promote the terminal differentiation of B cells into plasma cells, respectively. We examined the role played by IL-17 in the mucosal adjuvant activity of CT for epicutaneous vaccines. Groups of IL-17 KO and control C57BL/6 mice were immunized by epicutaneous application of 50 micrograms of CT with 1 mg ovalbumin (OVA). Control mice developed high levels of OVA- and CT-specific IgA Abs in the serum, and in fecal extracts. The adjuvant activity of CT in control mice also resulted in high antigen-specific IgG responses in the serum with a profile of IgG subclasses characterized by a IgG1>IgG2a. The IL-17 KO mice given the same epicutaneous vaccine exhibited enhanced serum and fecal IgA responses. On the other hand, serum IgG responses were of the same magnitude and profile than control littermates, suggesting that IL-17 signaling affects CT-induced IgA but not IgG Abs.
Anaphylatoxin Signaling in Murine Glial Cells (134.58)
Authors: Ingersoll Sarah A , Martin Carol B , Martin Brian K
Published: January 2023

Autoimmune destruction of myelin in diseases of the CNS is known to involve several immune effector mechanisms, including complement. The brain is considered an immunoprivileged site and is protected by the blood brain barrier; however, it has been shown that complement proteins are produced locally by glial cells. It has been found that glia express receptors for anaphylatoxins, C3a and C5a. Currently, our lab is investigating the effect of anaphylatoxins on glial cell activation as well as their overall role in demyelination and remyelination pathology. To study the role of anaphylatoxins in vitro, our lab created novel adenoviral vectors to express murine C3a or C5a. To study the role of anaphylatoxin signaling in glia we treated murine astrocyte and microglial cells with C3a, C5a or both anaphylatoxins, then examined various downstream effects. Using Western blots, we found that MAPK pathway proteins, ERK1/2, were highly phosphorylated when microglia were stimulated with C5a, but not C3a. In contrast, ERK1/2 were not activated in astrocytes treated with either anaphylatoxin. Finally, C5a was able to induce chemtoaxis in J774 murine macrophages; however we did not see significant migration of primary astrocytes or BV-2 microglia. These studies suggest that anaphylatoxin signaling and/or the functional consequences of that signaling is differentially regulated in a cell-specific manner.
Complement C5 deficiency in inbred mouse strains (134.61)
Authors: Lake Jeffrey
Published: January 2023

The Jackson Laboratory repository (JAX) serves as a centralized facility to import, develop, preserve, and distribute mouse models to the scientific community. Approximately 300 strains are added annually to one of the largest collection of characterized mouse strains available. JAX currently has 194 inbred mouse strains; 103 strains are maintained in living colonies and 91 strains are cryopreserved. Selecting appropriate inbred strains for study may be difficult requiring careful selection. For example, at least 14 inbred strains and many of the BXD recombinant inbred strains have a deficiency of the fifth complement component, C5. Mice deficient in C5 are more susceptible to a variety of infectious diseases. C5 also plays an important role in autoimmune diseases, allergic airway disease, and in various other biological processes. Other factors for model selection of inbred strain selection include strain background including breeding performance, MHC haplotype, other known mutations, and other associated phenotypes. Supported by NCRR (RR09781, RR11083, RR16049), NIA, HHMI, and donations from several private foundations
Structure-function analysis of gC1qR/p33 using deletion mutants show additional binding sites for C1q. (134.69)
Authors: Xu Sulan , Menzies Catherine A , Vinayagasundaram Rama , Mathew Sally , Vinayagasundaram Uma , Peerschke Ellinor I.B , Jesty Jolyon
Published: January 2023

Surface expressed or soluble gC1qR has been shown to activate complement and to play a role in various inflammatory processes. By peptide mapping and antibody inhibition studies, we had previously identified a binding site for C1q in a gC1qR domain contained within residues 76-93. However, although mAbs 60.11, which recognizes residues 76-93, can inhibit C1q binding to gC1qR, the inhibition is not complete. Similarly, binding of C1q to a truncated form of gC1qR lacking residues 76-93 is reduced by only 40-50% when compared to the full-length gC1qR. These findings therefore prompted us to look for additional binding sites elsewhere in the molecule. To this end, we generated several recombinant gC1qR proteins by deletion of domains of very high negative charge— and which, based on their location in the crystal structure—were predicted to interact with solution-phase ligands. The capabilities of these mutants to bind to C1q were therefore examined by comparing to the binding of the full-length gC1qR. Analyses of the data reveal the presence of two additional binding sites for C1q—one contained within residues 190-192 and another within residues 144-162. These results not only demonstrate well the value of crystal-based structure-function predictions, but also allow the identification of potential targets for drug design to alleviate complement-mediated inflammation. [Supported by NIH-NIAID Grant R01 AI-060866]
Plasma lipoproteins are important components of the host defense system? (134.74)
Authors: Han Runlin
Published: January 2023

Plasma lipoproteins [VLDL, LDL, and HDL] function primarily in lipid transport among tissues and organs. However, the cumulative evidence may support that as part of innate immunity, lipoproteins may also broadly prevent or inhibit bacterial, viral and parasitic infections. Lipoproteins can detoxify lipopolysaccharide and lipoteichoic acid. Lipoproteins neutralize a vast of enveloped and non-enveloped DNA and RNA viruses. Staphylococcus aureus α-toxin is bound and inactivated by purified LDL. LDL also directly binds to some bacteria, but the pathophysiological significance of these LDL-pathogen interactions remains to be elucidated. The oxidized LDL (ox-LDL), which can be induced by infections, has several anti-infective activities. It protects against endotoxin-induced tissue damage. The antibody against phosphorylcholine (PC) elicited by ox-LDL may provide a broad protection against pathogens since PC has been detected on a number of prokaryotes and many nematodes; Ox-LDL-activated macrophages enhance phagocytosis of pathogens. Thus, the oxidation of LDL may be an important response of host against infections. Importantly, Lipoprotein(a) might inhibit the binding of a number of pathogens to plasminogen, subsequently protect against the invasion of pathogens. Taken together, it might be reasonable to introduce the anti-infective role of plasma lipoproteins into immunology textbooks.
Crystal structure of a MIC-reactive gamma-delta T cell receptor - insights into gamma-delta TCR antigen recognition (134.79)
Authors: Xu Bin , Pizarro Juan C. , Holmes Meg , Strong Roland K.
Published: January 2023

Gamma-delta T cells play essential roles in the immune surveillance and elimination of tumor cells and various types of infection. However, little is known about the recognition mechanisms of human gamma-delta TCRs. MHC class I-related chain (MIC) is one of only two validated protein ligands for human gamma-delta TCRs. To investigate the recognition mechanism of gamma-delta TCR - MIC interaction, we have determined crystal structure of a MIC-reactive Vdelta1 gamma-delta TCR at 3.0 angstrom resolution. The structure reveals significantly altered loop conformations, particularly in CDR3 loops in both gamma and delta chains comparing to three other gamma-delta TCR structures that are available so far. Our structure suggests novel MIC antigen recognition mode. We further identified conserved surface patches in both Vdelta1 receptor and MIC antigen that may be important for recognition. To our knowledge, this is the first structure of a human gamma-delta TCR that recognizes human self-antigen. Supported by NIH grant AI48675.
Effector proteins of FcγRI: inside-out and outside-in regulation (134.81)
Authors: van der Poel Cees E , Beekman Jeffrey M , van der Linden Joke A , van de Winkel Jan GJ , Leusen Jeanette HW
Published: January 2023

FcγRI is the only IgG receptor that is capable of binding monomeric IgG. It depends on the FcR γ-chain for its function. However, some functions such as antigen presentation, are mediated by the ligand binding α-chain. Here we describe two binding partners of FcγRI: periplakin (PPL) and protein 4.1G.

Yeast-two hybrid screenings identified PPL and 4.1G as binding partners of the cytosolic tail of FcγRI. Alanine scans and truncations of the tail were made to study the binding site of the proteins to the receptor. Plate immobilized IgG adhesion assays were used to measure affinity of FcγRI stabily expressed on IIa1.6 cell lines with and without c-terminal PPL.

PPL and 4.1G were found to bind to the proximal region of the receptor. PPL affected the binding of FcγRI to opsonised RBCs and adhesion to IgG coated plates. Experiments with tat-peptides blocking PPL- FcγRI interaction suggested that endogenous PPL lowers FcγRI affinity.

Among the Fcγ receptors, FcγRI is unique in its capability to bind monomeric IgG and in its cytosolic tail sequence. We propose a role for PPL in modulating the affinity of FcγRI, leading to accessibility of the receptor for immunecomplexes.

Funding

NWO #07764/ALW
The local inflammatory response to Leishmania chagasi infantum infection (134.89)
Authors: Thalhofer Colin J , Wilson Mary E
Published: January 2023

Leishmania chagasi infantum (Lci) causes human visceral leishmaniasis. To study the nature of the early inflammatory response, we introduced metacyclic wild type and mCherry-transgenic parasites intradermally in ears of BALB/c mice. Inflammatory cells were collected from the dermal tissue and draining lymph nodes (dLN) at various times post-infection. FACS analysis revealed a rapid and abundant influx of neutrophils in the infected ears, which peaked between 6 and 24 hrs, but diminished by 48 hrs after infection. The neutrophil influx was dose dependent and specific for the local infection site. NK cells peaked at 24-48 hrs in the ear and 48-72 hrs in the dLN. Total cellularity of the dLN increased significantly after infection, primarily due to an expansion in the B cell population. mCherry parasites were observed infecting nearly 50% of neutrophils in the ear lesions during the earliest times (6-24 hrs), whereas parasites predominantly infected macrophages by 48 hrs. These data suggest intradermal Lci infection induces a rapid innate immune response in the local site and support the hypothesis that infected neutrophils provide one of the first major points of Leishmania entry into the infected host.
Plasticity in licensing of mature natural killer cells (134.9)
Authors: Elliott Julie M , Yang Liping , Yokoyama Wayne M
Published: January 2023

Natural killer (NK) cells are innate immune lymphocytes with potent effector functions against infected and tumor cells. NK cell activity relies on integration of signals from both activating and inhibitory effector receptors. Additional control of NK cell effector function includes a tolerance mechanism, termed licensing, whereby only NK cells with inhibitory receptors for self-MHC class I acquire the capacity to be triggered by their activation receptors. In an environment devoid of ligands to engage an inhibitory Ly49 receptor, such as in a β2-microglobulin (β2m)-deficient host, NK cells are hypo-responsive. Currently, it is assumed that the complete NK developmental pathway, including licensing, occurs in the bone marrow, fixing the phenotype of peripheral NK cells as either competent or hypo-responsive. In adoptive transfer studies, we find that mature splenic NK cells possess the capacity to change their phenotype based upon the environment to which they are transferred. In particular, when recovered after residence in a wild type host, β2m-deficient NK cells acquire the ability to produce interferon-gamma at levels comparable to WT NK after stimulation through activation receptor crosslinking. These results indicate that the process of NK cell licensing occurs independently of development per se.
Monocyte antimicrobial activities are differentially triggered by TLR2/1 and TLR2/6 activation (134.91)
Authors: Zhang Xiaoran , Liu Philip T , Stenger Steffen , Modlin Robert L
Published: January 2023

Human innate immune responses against microbial pathogens are mediated in part by Toll-like receptors (TLR). We have previously demonstrated that the 19kD lipoprotein, a TLR2/1 ligand, induces a vitamin D-dependent killing of Mycobacterium tuberculosis in infected monocytes; however the ability of the other TLR family members to induce this pathway is unclear. Given that TLRs instruct the innate immune response to different types of pathogens, we hypothesize the TLR family members will differentially regulate antimicrobial activity. While 19kD is able to induce killing in infected macrophages, macrophage activating lipopeptide 2 (MALP-2), a TLR2/6 ligand, did not. We investigated the differences in gene expression profiles between 19kD and MALP-2 treated monocytes, which yielded differentially expressed gene programs. Interleukin-10 family members were found to be upregulated in 19kD treated monocytes as compared to monocytes treated with MALP-2. In MALP-2 treated monocytes, leukocyte immunoglobulin-like receptors (LIR) were found to be upregulated. Investigating these differentially regulated genes can yield information that could results therapeutic targets against intracellular pathogens.
Role of IFNG in Granuloma Formation in a Zebafish Model of Francisella Infection (134.94)
Authors: Hansen John  D , Badil Samantha , Vojtech Lucia N
Published: January 2023

Members of the bacterial genus Francisella are highly virulent, gram-negative pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. Using a fish-specific species of Francisella, we recently demonstrated that acutely infected zebrafish undergo a disease process and mount an innate immune response that shares many features with tularemia. In heavily colonized zebrafish immune tissues, bacterial antigens begin to localize to focal regions by five days post-infection as assessed by immunohistochemistry. Currently, we are exploring how Francisella establishes chronic infections in zebrafish. The early focal regions appear to correspond to areas of initial granuloma formation. We hypothesize that the establishment of Francisella-mediated granulomas in zebrafish tissues may represent a host-protective mechanism, confining bacteria and thereby allowing for a strong innate immune response to acute infection to abate. In particular, we are interested in the role that IFNG produced by helper T-cells plays in the formation and maintenance of granulomas during chronic Francisella infections. To do so, we are assessing the formation of granulomas over time in wild type and RAG1 knockout strains of zebrafish via histopathology and immunohistochemistry. In addition, the induction of granuloma formation will also be correlated with the transcriptional response of IFNG and other immune response genes by qRT-PCR in wild type and RAG1 knockout fish. This study will illustrate specific aspects of granuloma formation and host immune responses that are attributable to adaptive immunity.
The differential roles of Act-1 and NF-κB in the regulation of various IL-17A-induced genes in A549 cells (134.95)
Authors: Huang Fei , Hung Li-Yin , Wu Reen
Published: January 2023

It has been shown that IL-17A is a potent inducer of human beta-defensin (HBD)-2 in airway epithelia through the Act1-dependent NF-kB transcriptional regulation. In our recent findings, the induction of HBD-2 is very robust in human primary airway epithelia and human bronchial epithelial-1 (HBE-1). In contrast, A549, an alveolar epithelial cell line, has a very weak IL-17A-stimulated HBD-2 gene expression. We found A549 cell has an impaired NF-kB activation specific to IL-17A cytokine, since other pro-inflammatory cytokines could stimulate significant NF-kB and HBD-2 gene expression. In addition, over-expression of Act-1 protein in HBE-1 can significantly induce HBD-2 promoter activity in the absence of IL-17A, while in A549, no HBD-2 induction was observed regardless of the presence of IL-17A. Interestingly, the IL-17A-stimulated genes that have been discovered so far are mostly dependent on Act-1 and NF-kB activation. However, we found the extent of the regulation by Act-1 and NF-kB varies between different genes. By use of siRNA knockdown, we found some IL-17A-induced genes are strictly regulated by Act1 but only partially by NF-kB. Therefore, IL-17A can stimulate certain gene expressions even under a deficient NF-kB activation in A549 cells. These findings suggested there might be some molecules in addition to NF-kB regulated by Act1 to stimulate IL-17A gene expression.
Different innate immune profiles by astrocytes and microglia following TLR7 and/or TLR9 agonist stimulation (135.12)
Authors: Butchi Niranjan B , Du Min , karin Peterson
Published: January 2023

Astrocytes and microglia are phagocytic cell types in the central nervous system (CNS) that are often activated following virus infection in the brain and may play an important role in initiating the innate immune response to virus infection. However, there is lack of information on the responses by astrocytes and microglia following activation by virus-sensing Toll like-receptors such as TLR7, which recognizes viral ssRNA, and TLR9, which recognizes unmethylated CpG DNA. In the present study, we examined the cytokine and chemokine profiles, as well as the signaling molecules induced in astrocytes and microglia following stimulation of TLR7 and/or TLR9. Although both astrocytes and microglia produced proinflammatory cytokines and chemokines as a response to TLR7/9 stimulation, microglia also produced anti-inflammatory and neuroprotective cytokines while astrocytes did not. In general, TLR7 stimulation induced higher levels of proinflammatory cytokines and chemokines than TLR9 stimulation in both cell types. However, a high concentration of TLR7 agonist suppressed both TLR7 and TLR9-induced proinflammatory responses, suggesting a negative feedback mechanism in astrocytes and microglia in response to high levels of TLR7 ligands.
Toll-like receptor expression is altered by stress of intense physical conditioning (135.28)
Authors: Hallam Justus , Sanderson Cole , Sim Young Je , Senchina David , Sultana Ishrat , Kohut Marian
Published: January 2023

Periods of intense physical training are associated with an increased incidence of infection. We hypothesize that the TLR pathways involved in the initial response to multiple pathogens are compromised during intense physical exercise stress. In order to test this hypothesis, eight male collegiate wrestlers participated in one month of intense physical conditioning stress. Blood was collected at rest both before and after a 1 month period of intense exercise, and after completing a single 90 minute session of exhaustive exercise. TLR receptor pathway expression was assessed by microarray. The results showed that 1 month of intense exercise conditioning decreased expression of genes that may be important in antiviral defense (IFN-alpha, TLR3, PKRKA, SARM1), and inflammatory response (TNFSRF, PTGS2, REL). Ninety minutes of intense exercise decreased IFN-gamma and IRF1, but expression of TLR pathways associated with anti-bacterial defense increased (TLR5, TIRAP, PGLYRP2, LY86). These findings are consistent with epidemiological data suggesting an increased incidence of respiratory viral infections during periods of intense physical exercise stress. The results also implicate several genes important in inflammation, and further research is needed to determine whether these genes play a significant role in the recently reported anti-inflammatory effects of repeated exercise.
Anthrax lethal toxin enhances I kappa B kinase (IKK) activation and differentially regulates pro-inflammatory genes in human endothelium (135.3)
Authors: Warfel Jason M , D'Agnillo Felice
Published: January 2023

Anthrax lethal toxin (LT), a key virulence factor of Bacillus anthracis, was previously shown to enhance transcriptional activity of NF-κB in primary human endothelial cells. Here, we report that LT enhancement of NF-κB activation in tumor necrosis factor-α (TNF)-activated endothelium is driven by increased IκB kinase (IKK) phosphorylation which in turn correlates with enhanced phosphorylation of the IKK substrates, IκBα and p65. This effect was not associated with a general inhibition of cellular phosphatase activity. Moreover, a specific IKK inhibitor prevented the previously reported LT enhancement of IκBα degradation. Consistent with these data showing augmentation of the IKK-NF-κB pathway, LT increased the transcription of the pro-inflammatory genes CD40 and HLA-C. Conversely, LT inhibited transcription of another NF-κB regulated gene, CCL2. This inhibition was linked to the LT-mediated suppression of another CCL2-regulating transcription factor, activator protein-1 (AP-1) as measured by a DNA-binding ELISA. These data suggest that LT-mediated enhancement of NF-κB is IKK-dependent but, importantly, the net effect of LT on the transcription of pro-inflammatory genes is driven by the cumulative effect of LT on the particular set of transcription factors that regulate a given promoter. Together, these findings provide new mechanistic insight on how LT may disrupt the host response to anthrax.
Flagellin-induced IL-8 expression requires formation of lipid rafts and activation of PKC and MAP kinases (135.31)
Authors: Im Jintaek , Cho Min Kyung , Woo Sang Su , Kang Seok-Seong , Jeon Jun-Ho , Yun Cheol-Heui , Han Seung Hyun
Published: January 2023

At infections in the lung, alveolar type II epithelial cells produce chemokines upon recognizing pathogen-associated molecular patterns. Here, we investigated the induction mechanism of a chemokine, IL-8 in the human alveolar type II epithelial cell-line, A549 by bacterial flagellins from Salmonella typhimurium. The flagellins induced the expression of IL-8 in both mRNA and protein levels, mediated through lipid rafts but not clathrin-coated pits. Interestingly, Toll-like receptor 5 recognizing flagellins was observed at the cytosol of A549 but rarely on the cell surface. IL-8 expression was attenuated by inhibitors of protein kinase C (PKC) and mitogen activated protein (MAP) kinases, whereas no such effect was observed with inhibitors for reactive oxygen species, phosphatidylinositol 3-kinase or protein kinase A. Moreover, flagellins enhanced DNA binding activity of transcription factors, NF-κB, AP-1, and NF-IL6, all of which are known to be involved in the transcription of the IL-8 gene. Taken together, these results suggest that flagellin-induced IL-8 expression requires formation of lipid rafts and activation of PKC and MAP kinases leading to the activation of the transcription factors, NF-κB, AP-1, and NF-IL6 in human alveolar type II epithelial cells. Supported by Agricultural R&D Promotion Center (107092-03-1-HD120).
Activation of TLR2 by fibrillar albumin (135.34)
Authors: Liang Shu-Mei , Chen Yen-Po , Cheng Yung-Chih , Huang Chun-Yung , Chu Chiao-Li , Liang Chi-Ming
Published: January 2023

Toll-like receptors (TLRs) are pattern recognition receptors and play important roles in initiating innate and adaptive immunity. Among TLRs, TLR2 has the widest range of known agonists which have been applied as vaccines and cancer adjuvant. We have recently found a process that converted globular bovine serum albumin (G-BSA) into fibrillar BSA (F-BSA). Here, we studied the immunological effects of F-BSA and found that F-BSA, but not G-BSA, activated NF£eB in exogenous TLR2 expressing HEK293T cells. Immunofluorescence microscopy, flow cytometry and immunoprecipitation results revealed that F-BSA bound to TLR-2 directly. F-BSA induced macrophages of normal mice but not macrophages of TLR2-/- mice to produce cytokines. The TLR2 activating ability of F-BSA was blocked by TLR2 antibody and was not due to contamination of TLR2 agonist £]-glucan and peptidoglycan from microbes. The activation of TLR2 by F-BSA was associated with TLR1 and CD14 as indicated by using co-transfection of expressing plasmids and antibody blocking. These results suggest that F-BSA is a new agonist of TLR2 and might be used as vaccine adjuvant.

This study was supported by the grant NSC 96-2313-B-001-005-MY3 from the National Science Council of Taiwan
NFκB-p65 up regulates P2Y2 receptor expression following induction of infla mmationin intestinal epithelial cells (135.35)
Authors: Degagné Emilie , Grbic Djordje M , Gendron Fernand-Pierre
Published: January 2023

There is an up-regulation in P2Y2 receptor (P2Y2R) expression in tissues of patient suffering from inflammatory bowel diseases. Our study investigates the molecular mechanisms responsible for the transcriptional regulation of the P2Y2R during inflammation. Hypothesis: Under inflammatory conditions, P2Y2R gene expression is regulated at the transcriptionnal level by transcription factor NFκB/p65. P2Y2R gene expression was determined by Q-PCR in Caco-2 cells treated or not with 0.5% DSS. Proximal region of the human P2Y2R promoter was cloned into pGL4.10-Luc vector (pGL4/P2Y2). Caco-2 cells were co-transfected with the pGL4/P2Y2 construction or with deletion mutants and with the transcription factor p65. Inflammation was then induced to Caco-2 cells by a 6h treatment with 0.5% DSS. The interaction between NFκB-p65 and P2Y2R promoter region was determined by luciferase assays and identification of NFκB/p65 potential binding sites determined by EMSA and ChIP assays. We demonstrated that P2Y2R mRNA expression was up regulated in cells treated with 0.5% DSS. We established that p65 can bind to the promoter region of P2Y2 and we demonstrated in vivo, by ChIP assays, that p65 can bind to the P2Y2R proximal promoter. We ultimately identified a binding site for NFκB-p65 in the P2Y2R proximal promoter. These results showed that inflammation up regulates the expression of P2Y2R and that the expression of this gene is regulated at the transcriptional level by p65.
Histone deacetylase regulation of MAP kinase phosphatase-1 acetylation and inflammation (135.36)
Authors: Jeong Youngtae , Cao Wangsen , Lowenstein Charles
Published: January 2023

The mitogen activated protein kinase (MAPK) pathway mediates Toll-like receptor signaling during innate immune responses. Histone deacetylases (HDACs) modulate inflammation by deacetylating histone and non-histone proteins. Previous studies in our lab identified MAP Kinase Phosphatase-1 (MKP-1) as a potential acetylation target. Thus, we hypothesize that HDACs regulate LPS-induced inflammation by deacetylating MKP-1. RAW264.7 mouse macrophages preferentially expressed HDAC1, 2, and 3 (class I HDAC isoforms). HDAC1, 2, and 3 interacted with MKP-1. To support our hypothesis, we adopted both genetic and pharmacologic approaches, using HDAC1, 2, and 3 siRNAs and MS-275, a HDAC1, 2, 3 specific inhibitor. Both silencing and pharmacologic inhibition of HDAC1, 2, and 3 increased MKP-1 acetylation in cells. HDAC1 decreased MKP-1 acetylation in vitro. Furthermore, both silencing and pharmacologic inhibition of HDAC1, 2, and 3 decreased LPS-induced p38 MAPK activation and iNOS expression. Finally, pharmacologic inhibition of HDAC1, 2, and 3 decreased LPS-induced NO and IL-6 production. Taken together, our results show that class I HDACs deacetylate MKP-1, and that this modification can significantly affect MAPK signaling and inflammation. Furthermore, our results suggest that MKP-1 is a potential therapeutic target in inflammatory diseases.

Supported by the American Heart Association grant (0815093E).
TLR7 tolerance in B cells: Prevention and rescue by antigen receptor signals (135.40)
Authors: Poovassery Jayakumar , Vanden Bush Tony , Bishop Gail
Published: January 2023

Toll-like receptors (TLR) play crucial roles in detecting microbial pathogens and subsequent development of immune responses. However, chronic or sustained stimulation through TLRs can render immune cells hyporesponsive to subsequent stimulation with the same or different TLR ligands; a phenomenon known as TLR tolerance. Although TLR tolerance has been well characterized in myeloid cells, it has not been studied in detail in B lymphocytes. In this study, we examined the effects of primary stimulation with a synthetic TLR7 ligand, R848, on subsequent restimulation with the same ligand in B lymphocytes. We found that mouse splenic or human peripheral blood B cells that received primary stimulation with R848 produced markedly reduced amounts of IL-6 in response to subsequent R848 restimulation. The hyporesponsive state was associated with reduced NF-kB and MAP kinase activation. However, B cells that received primary stimulation via both B-cell receptor (BCR) and TLR7 produced large amounts of IL-6 in response to TLR7 restimulation, indicating that combined BCR and TLR stimulation can prevent or rescue B cells from TLR tolerance.
Surfactant protein-A interacts with TLR4 and affects the phagocytic function of dendritic cells (135.41)
Authors: Awasthi Shanjana , Wolf Roman , White Gary , Awasthi Vibhudutta
Published: January 2023

The surfactant protein (SP)-A and Toll-like receptor (TLR4) proteins are known as secretory and cellular pathogen pattern recognition receptors, respectively, and play an important role in the recognition of pathogens and host defense. In lung, the TLR4 is expressed by immune cells, and SP-A is synthesized by type II epithelial cells and secreted in alveoli. In this study, we investigated the interaction between purified baboon lung SP-A and recombinant TLR4 (extracellular domain)-MD2 proteins and effects of these proteins on phagocytic function of the human myelomonocytic cells (KG1) derived- and primary baboon lung-dendritic cells. The interaction between the two proteins was studied in vitro by a microwell based method. For measuring the changes in phagocytic ability of the dendritic cells, the cells were incubated with pHrodo-labeled, heat-killed Escherichia coli bioparticles in presence of native SP-A and/or recombinant TLR4-MD proteins and fluorescence released from ingested E. coli bioparticles inside the acidic phagosome was read on a fluorometer. We found that the SP-A protein binds to TLR4-MD2 protein. The exogenous addition of SP-A and TLR4-MD2 proteins was found to significantly increase the phagocytosis of E. coli bioparticles by dendritic cells. However, when the SP-A and TLR4-MD2 proteins were added together, the phagocytic ability of dendritic cells either remained unaltered or decreased from those treated with SP-A or TLR4-MD2 alone. These results suggest that the interaction between SP-A and TLR4 affects the bacterial phagocytosis by dendritic cells.
Expression analysis of chicken adaptors MyD88, TRIF, TIRAP, SARM and TRAM used in toll-like receptor signaling (135.45)
Authors: MacKinnon Kathryn M , He Haiqi , Kogut Michael H
Published: January 2023

Activated toll-like receptors (TLR) trigger innate immunity through signaling adaptors including MyD88, TRIF/TICAM-1, TIRAP/MAL, SARM and TRAM/TICAM-2. With limited information available on these adaptors in chickens, we chose to examine the distribution and expression of these adaptors in multiple chicken tissues and cells. All adaptor molecules were detected in ileum, ceca, cecal tonsils, large intestine, bursa, heart, kidney, lung, muscle, skin and spleen tissues using quantitative real-time RT-PCR. Within each tissue, expression of TRAM was greatest and SARM was lowest. Across tissues, spleen and lung tissues had the highest level of expression and muscle had the lowest. Heterophils and peripheral-blood mononuclear cells isolated from 2 to 3-day-old chickens, in addition to the avian macrophage cell line (HD-11), expressed mRNA from all adaptor molecules. However stimulation of HD-11 cells for up to 2 hours with TLR agonists did not induce changes in mRNA expression. Comparison of Salmonella-infected and control birds showed infected birds had a 1.5-fold increase (P < 0.02) in expression of MyD88 in the ileum and a 1.65-fold decrease (P < 0.02) of TIRAP in the cecal tonsils. Overall, these TLR-adaptor molecules appear to be ubiquitously expressed in chicken cells and tissues and expression levels may play a role in immune regulation during infection.
PI3K and potentially autophagy are involved in HSV, Flu, SeV, and CpG A -stimulated IFN-α production by human plasmacytoid dendritic cells (135.51)
Authors: Deng Jing , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia
Published: January 2023

IFN-α production plays a critical role in the immune system and as "professional" type I interferon (IFN) producing cells, plasmacytoid dendritic cells (pDC) can produce much more IFN-α in response to viruses than other cell types in response to TLR-7 and -9 stimulation. Phosphatidylinositol-3 kinase (PI3K) has been shown to be activated by TLR stimulation in many cell types and is essential in the regulation of many processes, including autophagy. A role for PI3K and autophagy in pDC has been recently described. The role of autophagy in induction of IFN-α in human pDC, however, has not been described. We investigated the effects of wortmannin, a PI3K inhibitor, and 3-methyladenine (3MA), a specific inhibitor of autophagy, but also an inhibitor of PI3K, on IFN-α?production by HSV, Flu, SeV, and CpG A -stimulated human pDC. pDC were pretreated with the inhibitors then stimulated with virus for 6hr for detection of intracellular IFN-α by intracellular flow cytometry or overnight for stimulation and release of IFN-α, which was detected by ELISA. Inhibition of PI3K with wortmannin, inhibited IFN-α production by pDC. Moreover, 3MA also inhibited IFN-α production by HSV, Flu, SeV, and CpG A-stimulated pDC. In addition, 3MA inhibited HSV-induced LC3B aggregation in pDC, a marker for autophagy. These results support key roles for the PI3K pathway and perhaps, autophagy, in mediating IFN-α secretion and virus detection by human pDC. Supported by AI26806.
Characterization, function and targeting of dendritic cell associated c-type lectin-2 (DCAL-2) (135.54)
Authors: Kasahara Shinji , Hayden-Ledbetter Martha , Chen Chang-Hung , Clark Edward A
Published: January 2023

Dendritic Cell (DC) Associated C-type Lectin Receptors (CLRs) play important roles in immunity through their abilities in antigen capturing and presentation, modulation of TLR signaling, co-stimulation and adhesion. Murine DCAL-2 (mDCAL-2/ mMICL/ mKLRL-1) is a type-II CLR with a cytosolic immunoreceptor tyrosine inhibitory motif (ITIM). Using a mAb we developed, we found that mDCAL-2 is expressed at high levels on plasmacytoid DCs (pDCs), CD8α+ DCs and a subset of CD8α- DCs, at moderate levels on immature CD4-CD8- T cells and low levels on a subset of B-cells, but not on mature T cells and NK cells. DCAL-2 expression subdivides CD8α- DCs into mDCAL-2High33D1- and mDCAL-2Low33D1+ populations. These two subsets of DCs express different levels of TLRs. In addition, these subsets have significantly distinct cytokine profiles in response to TLR ligands. Whereas mDCAL-2High33D1- produced high levels of TNFα, IL-6, IL-10 and IL-12, mDCAL-2Low33D1+ DCs produced extremely low levels of these cytokines. Thus, we hypothesize that mDCAL-2High DC subsets induce T-cell responses that are distinct from that of mDCAL-2Low DCs. To investigate what type of T-cell responses are induced through mDCAL-2 in an Ag specific manner, we developed an anti-mDCAL-2 single chain fragment variable (scFv) fused with antigens (OVA or HEL). We found that anti-mDCAL-2 scFv successfully targets mDCAL-2+ DC subsets in vivo. What type of T cell responses are induced by OVA-anti-mDCAL-2 scFv injection in OT-II transgenic mice will be presented.

This study is supported by NIH grants AI52203, DE16381, and RR00166
mRNA Expression of the Avian-Specific Toll-Like Receptor 15 is Induced by Multiple Species of Bacteria (135.58)
Authors: Nerren Jessica R , He Haiqi , Genovese Kenneth J , Kogut Michael H
Published: January 2023

Pattern recognition receptors (PRRs) are essential for recognition of conserved molecular constituents found on infectious microbes, and Toll-like receptors (TLRs) are a critical component of the PRR repertoire. mRNA expression of the avian-specific TLR 15 has recently been shown to be increased in response to multiple Salmonella serovars, suggesting that the ligand for TLR 15 is some component of Salmonella. The objective of this study was to determine whether mRNA expression of TLR 15 was Salmonella-specific or could be induced by exposure to other species of bacteria commonly found in the chicken. We also wanted to determine whether TLR 15 mRNA expression could be induced by exposure to known TLR ligands that are components of Salmonella. To test this hypothesis, we stimulated chicken heterophils with live Salmonella enterica serovar Enteritidis (SE), heat-killed or formalin-killed SE, an avian isolate of Escherichia coli, and an avian isolate of Enterococcus gallinarum. TLR ligands used as stimuli included LPS, flagellin, and CpG. Gene expression was measured using real-time quantitative RT-PCR. We found that mRNA expression of TLR 15 was significantly increased (P < 0.05) in response to stimulation with all of the bacteria tested. There were no significant differences in stimulation between live and dead bacteria nor between species of bacteria. Across bacteria tested, the average increase in TLR 15 expression was 8-fold greater (range, 6.5 to 9.1) than in unstimulated heterophils. In contrast, stimulation of heterophils with the TLR ligands LPS, flagellin, and CpG induced no significant increase in expression of TLR 15 mRNA. These data demonstrate that TLR 15 recognizes some component of both Gram-positive and Gram-negative bacteria, but that LPS, flagellin, and CpG are likely not the components of these bacteria that are recognized.
Regulation of Notch ligands by the TLR and IFN-γ pathways in macrophages (135.63)
Authors: Foldi Julia , Hu Xiaoyu , Chung Allen Y. , Ivashkiv Lionel B.
Published: January 2023

Jagged and Delta proteins are transmembrane ligands for the Notch family of receptors. The role for Notch and its ligands in innate immunity remains largely unknown. Our lab recently identified a subset of Toll like receptor (TLR)-inducible genes that were activated synergistically, by TLR-ligands and the Notch pathway. Some of these genes were also regulated by interferon (IFN)-γ, a potent macrophage activator. To further elucidate the role of the Notch pathway in innate immunity, we would like to know how Notch ligands are regulated in macrophages. We found that Jagged-1 and Delta-like (Dll)-1 were induced by TLR ligation and were regulated by IFN-γ in CD14+ primary human monocytes and in mouse bone-marrow-derived macrophages. TLR-induced upregulation of Jagged-1 was independent of de novo protein synthesis and the mitogen activated protein kinase (MAPK) pathway, while the nuclear factor (NF)-κB and Notch pathways played a role in this process. IFN-γ pre-treatment of cells super-induced TLR-stimulated Jagged-1, while it inhibited Dll-1 upregulation. In conclusion, Jagged and Delta proteins are synergistically regulated by the TLR, Notch and IFN-γ pathways in macrophages. This work is supported by the Cancer Research Institute (CRI) Predoctoral Fellowship.
Functional Characterization of a Novel Interferon-inducible Protein, Mouse ISG12(b2) (135.66)
Authors: Lu Mei-Yi , Liao Fang
Published: January 2023

Interferons (IFNs) play crucial roles in the host defense against virus. Many interferon-stimulated genes (ISGs) induced by viral infection exert antiviral effects. ISG12(b2) was dramatically induced in liver from dengue-infected mice as well as in Hepa 1-6 (mouse hepatoma cell line) following dengue virus infection. We have biochemically and functionally characterized mouse ISG12(b2). ISG12(b2) is mainly localized to the mitochondria. Overexpression of ISG12(b2) in Hepa 1-6 resulted in the changes in mitochondrial morphology, the mitochondrial membrane potential, the accumulation of Sub-G1 population, the activation of caspase-8, caspase-9, and caspase-3, and the increase of the PARP cleavage. Knockdown of ISG12(b2) by siRNA followed by dengue virus infection in Hepa 1-6 revealed the attenuation of caspase activation and PARP cleavage. Taken together, our results suggest that ISG12(b2) is involved in the activation of the mitochondrial apoptotic pathway, which may subsequently eliminate virus-infected cells.
The immune response of macrophages to Mycobacterium leprae infection via Naip5, a member of Nod-like receptor protein (135.75)
Authors: Kang Tae Jin , Kim Se-Kon , Chae Gue-Tae
Published: January 2023

Naip5 is a member of the NOD-like receptors (NLRs) involved in the cytosolic recognition of pathogen-associated molecular patterns. Recently, A/J mice have been found to have amino acid differences in the Naip5, one of the adaptor proteins for caspase-1 activation, and this defect is associated with a susceptibility to Legionella infection suggesting an important role of Naip5 in the immune response to intracellular pathogens, such as M. tuberculosis and M. leprae. In this study, the immune responses of macrophages from A/J mice against M. leprae infection were studied and compared to those of macrophages from C57BL/6 (B6) mice. A/J and B6 macrophages induced high levels of NF-¥ê B activation when infected with M.leprae. Caspase-1 activation and IL-1beta secretion were also induced in both macrophages. However, macrophages from A/J mice have impaired caspase-1 activation, IL-1beta expression, and nitric oxide (NO) production, partially, compared to macrophages from B6 mice. These results suggest that NOD family proteins may have a role in the innate immune response to M. leprae.
Inflammasome-activating biodegradable nanoparticulates as vaccine delivery systems (135.80)
Authors: Fahmy Tarek M , DEMENTO Stacey , Eisenbarth Stephanie C , Caplan Michael J , Saltzman W Mark , Mellman Ira , Ledizet Michel , Fikrig Erol , Flavell Richard
Published: January 2023

Inflammasome activation requires signaling of both the Toll-like receptor (TLR) and NLRP3 in antigen-presenting cells. We present a class of nanomaterials endowed with these two signals for rapid optimization of vaccine design. We constructed this system using an approach that incorporates lipopolysaccharides (LPS) onto the surface of nanoparticles constructed from a biocompatible polyester loaded with antigen. We demonstrate that LPS-modified particles, elicits potent humoral and cellular immunity against a model antigen, ovalbumin. Wild type macrophages pulsed with LPS-modified nanoparticles resulted in production of the proinflammatory cytokine IL-1β consistent with inflammasome activation. In comparison, NLRP3-deficient and caspase-1-deficient macrophages showed negligible production of IL-1β. Furthermore, when endocytosis and lysosomal destabilization were inhibited, inflammasome activity was diminished, supporting the notion that nanoparticles rupture lysosomal compartments and behave as 'danger signals'. The generality of this vaccination approach is tested by encapsulation of a recombinant West Nile envelope protein and demonstrated by protection against a murine model of West Nile encephalitis. The design of such an antigen delivery mechanism with the ability to stimulate two potent innate immune pathways represents a new approach to simultaneous antigen and adjuvant delivery.
Chlamydial-induced TLR2 signaling leads to increased neutrophil production of proinflammatory molecules and delayed spontaneous apoptosis (135.9)
Authors: Frazer Lauren , O'Connell Catherine , Darville Toni
Published: January 2023

Chlamydial-induced TLR2 signaling is involved in development of genital tract pathology. We hypothesized that chlamydial-induced activation of PMN TLR2 induces the release of cytokines and matrix metalloproteinases (MMPs), and prolongs PMN longevity. Human PMNs were incubated with wild-type C.trachomatis strain D/UW-3/Cx, TLR2 signaling deficient strain CTD153, TLR2 agonist Pam3Cys, or media for 4 or 20 hrs. Supernatants were analyzed for cytokine and MMP9 production via bead array. TNFa, IL-6, IL-8, G-CSF, and IL-1β were increased in PMNs incubated with D/UW-3/Cx or Pam3Cys vs. CTD153 for 20 hrs (p< 0.05). MMP9 production was increased in PMNs incubated with D/UW-3/Cx or Pam3Cys vs. CTD153 for 4 hrs. or 20 hrs.. At 20 hrs, MMP9 = 455 ± 61 ng/ml, D/UW-3/Cx; 435 ± 64 ng/ml, Pam3Cys; 322 ± 144 ng/ml, CTD153; 132 ± 84 ng/ml, media; p<0.02 for D vs. CTD153; p < 0.001 for D or Pam3Cys vs. UI. Flow cytometric enumeration of TUNEL and microscopic analysis of Giemsa staining revealed incubation with D/UW/3Cx for 20 hrs but not 4 hrs. induced significant protection from spontaneous apoptosis when compared to CTD153 (Giemsa: % apoptotic = 37 ± 2%, D/UW/3Cx; 78±2%, CTD153; 76±3%, media; (p<0.01 for D vs. CTD153 and media). PMNs have increased longevity, cytokine and MMP9 production when incubated with chlamydiae capable of activating TLR2. Chlamydial-induced activation of PMN TLR2 may contribute to development of tissue pathology.

NIH A1054624; Pennsylvania Department of Health; Howard Hughes Institute Research Training Fellowship
mRNA stability influences the temporal order of inflammatory gene induction (136.1)
Authors: Hao Shengli , Baltimore David
Published: January 2023

The inflammatory response is regulated by a great number of genes. It is still unknown whether and how these genes are organized to function as groups. We have performed a global analysis of about 200 genes induced in cultured cells by TNF, a proinflammatory cytokine. We found that genes can be divided roughly into three groups with different induction kinetics. Group I genes are activated quickly but then quickly repressed. Group II genes are also activated rapidly but reach maximal activity later and remain at the induced level longer. The Group III genes are activated in a delayed, slow but steady manner. The activation pattern of a gene remains largely the same in different cell types and species and is not specified by the extracellular signals. In addition, some genes within the same group have similar related functions. This suggests that these genes are temporally organized to function as groups. While transcriptional regulation is important, we found that a key parameter characterizing the grouping of genes is the stability of their mRNAs. The early mRNAs are very unstable, while the late mRNAs are highly stable. The instability of mRNAs correlates with the number of AU-rich elements in their 3'-untranslated regions (3'UTR). Changing mRNA stability either through extracellular LPS treatment or by switching the 3'UTRs but not the promoter/enhancer regions that control expression of GFP transgenes resulted in corresponding changes of the gene activation kinetics. These show that the inflammatory response is pre-set at the genome level and that the 3'UTR of a gene is a key element for organizing genes into functional groups.