bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyriboinosinic Polyribocytidylic Acid (Poly(I:C)) Induces Stable Maturation of Functionally Active Human Dendritic Cells
Authors: Verdijk Rob M. , Mutis Tuna , Esendam Ben , Kamp Janine , Melief Cees J. M. , Brand Anneke , Goulmy Els
Published: January 2023

For vaccination strategies and adoptive immunotherapy purposes, immature dendritic cells (DC) can be generated from adherent monocytes using GM-CSF and IL-4. Presently, the only clinically applicable method to induce stable maturation of DC is the use of supernatants of activated monocytes (monocyte-conditioned medium (MCM)). MCM contains an undefined mixture of cytokines and is difficult to standardize. Here we report that stable maturation of DC can be simply induced by the addition of polyriboinosinic polyribocytidylic acid (poly(I:C)), a synthetic dsRNA clinically applied as an immunomodulator. Poly(I:C)-treated DC show a mature phenotype with high expression levels of HLA-DR, CD86, and the DC maturation marker CD83. This mature phenotype is retained for 48 h after cytokine withdrawal. In contrast to untreated DC, poly(I:C)-treated DC down-regulate pinocytosis, produce high levels of IL-12 and low levels of IL-10, induce strong T cell proliferation in a primary allo MLR, and effectively present peptide Ags to HLA class I-restricted CTL. In conclusion, we present a simple methodology for the preparation of clinically applicable mature DC.
Specialized Ability of Thymic Epithelial Cells to Mediate Positive Selection Does Not Require Expression of the Steroidogenic Enzyme P450scc
Authors: Jenkinson Eric J. , Parnell Sonia , Shuttleworth John , Owen John J. T. , Anderson Graham
Published: January 2023

Thymic epithelial cells are uniquely efficient in mediating positive selection, suggesting that in addition to providing peptide/MHC complexes for TCR ligation, they may also provide additional support for this process. Recent studies have shown that although engagement of either the TCR or glucocorticoid (GC) receptors can individually induce apoptosis in thymocytes, together these signals are mutually antagonistic. This had led to the suggestion that local GC production by thymic epithelial cells, by opposing TCR signaling for apoptosis, provides the basis of the ability of these cells to mediate thymocyte positive selection. In this paper we have examined this possibility directly and shown that highly purified cortical epithelial cells, which have the functional ability to mediate positive selection in reaggregate cultures, do not express mRNA for the key steroidogenic enzyme P405scc. Thus we conclude that the ability of thymic epithelial cells to support positive selection does not rely on their ability to produce GC. However, we find that P450scc mRNA is up-regulated in thymocytes on the initiation of positive selection, raising the possibility that any local protective effect of steroid production is mediated at the level of thymocytes themselves.
Generation of an Immunodominant CTL Epitope Is Affected by Proteasome Subunit Composition and Stability of the Antigenic Protein
Authors: Gileadi Uzi , Moins-Teisserenc Hélène T. , Correa Isabel , Booth Bruce L. , Dunbar P. Rod , Sewell Andrew K. , Trowsdale John , Phillips Rodney E. , Cerundolo Vincenzo
Published: January 2023

Generation of the HLA-A0201 (A2) influenza Matrix 58–66 epitope contained within the full-length Matrix protein is impaired in cells lacking the proteasome subunits low molecular protein 2 (LMP2) and LMP7. This Ag presentation block can be relieved by transfecting the wild-type LMP7 cDNA into LMP7-deficient cells. A mutated form of LMP7, lacking the two threonines at the catalytic active site, was equally capable of relieving the block in presentation of the influenza Matrix A2 epitope. These observations were extended by analyzing whether modification of the influenza Matrix protein could overcome the block in presentation of the A2 Matrix epitope. Expression of either a rapidly degraded form of the full-length Matrix protein or shorter Matrix fragments led to an efficient presentation of the A2 influenza Matrix epitope by LMP7-negative cells. These findings demonstrate two main points: 1) LMP7 incorporation into the proteasome is of greater importance for the generation of the influenza A2 Matrix epitope than the presence of the LMP7’s catalytic site; and 2) the interplay between cytosolic proteases and stability of target proteins is of importance in optimization of Ag presentation. These observations may have relevance to the immunodominance of tumor and viral epitopes and raise the possibility that generation of shorter protein fragments could be a mechanism to ensure optimal Ag presentation by cells expressing low levels of LMP7.
Peptide Presentation to an Alloreactive CTL Clone Is Modulated Through Multiple Mechanisms Involving Polymorphic and Conserved Residues in HLA-B27
Authors: García-Peydró Marina , Paradela Alberto , Lamas José R. , López de Castro José A.
Published: January 2023

This study addressed the mechanisms by which HLA class I polymorphism modulates allorecognition. CTL 27S69 is an alloreactive clone raised against HLA-B*2705, with a known peptide epitope. This CTL cross-reacts with B*2702, which differs from B*2705 in the D77N, T80I, and L81A changes, but not with B*2701, which has D74Y, D77N, and L81A changes. To explain this differential recognition, B*2705 mutants mimicking subtype changes were used. The A81 mutant was not recognized, despite binding the natural epitope in vivo, suggesting that, when bound to this mutant, this peptide adopts an inappropriate conformation. The N77 and I80 mutations restored recognition in the N77A81 or I80A81 mutants. These compensatory effects explain the cross-reaction with B*2702. The Y74 and the Y74N77 mutants were weakly recognized or not recognized by CTL 27S69. This correlated with the absence or marginal presence of the peptide epitope in the Y74N77-bound pool. As with B*2701, exogenous addition of the peptide epitope sensitized Y74 and Y74N77 targets for lysis, indicating that failure to cross-react with B*2701 or these mutants was due to poor binding of the peptide in vivo and not to inappropriate presentation. The abrogating effect of Y74 was critically dependent upon the K70 residue, conserved among subtypes, as demonstrated with mutants at this position. Thus, HLA polymorphism affects allorecognition by modulating peptide binding or the conformation of bound peptides. Compensatory mutations and indirect effects of a polymorphic residue on residues conserved play a critical role.
CXC Chemokine Receptor-2 Ligands Are Necessary Components of Neutrophil-Mediated Host Defense in Invasive Pulmonary Aspergillosis
Authors: Mehrad Borna , Strieter Robert M. , Moore Thomas A. , Tsai Wan C. , Lira Sergio A. , Standiford Theodore J.
Published: January 2023

Invasive pulmonary aspergillosis is a devastating complication of immunosuppression, which occurs in association with neutrophil dysfunction or deficiency. ELR+ CXC chemokines are a subfamily of chemokines that play a critical role in neutrophil chemotaxis and activation both in vitro and in vivo. We hypothesized that interaction of these ligands with CXC chemokine receptor-2 (CXCR2), their sole murine receptor, is a major component of neutrophil-dependent pulmonary host defense against Aspergillus fumigatus. In immunocompetent animals, neutrophils were recruited to the lung in response to intratracheally administered A. fumigatus conidia. In a model of transient in vivo depletion of neutrophils, animals developed invasive pulmonary aspergillosis, associated with delayed influx of neutrophils into the lung. In both normal and neutrophil-depleted animals, the ELR+ CXC chemokines MIP-2 and KC were induced in response to intratracheal administration of conidia. Ab-mediated neutralization of the common ELR+ CXC chemokine receptor, CXCR2, resulted in development of invasive disease indistinguishable from the disease in neutrophil-depleted animals, while control animals were highly resistant to the development of infection. CXCR2 neutralization was associated with reduced lung neutrophil influx and resulted in a marked increase in mortality compared with controls. In contrast, animals with constitutive lung-specific transgenic expression of KC were resistant to the organism, with reduced mortality and lower lung burden of fungus. We conclude that CXCR2 ligands are essential mediators of host defense against A. fumigatus, and may be important targets in devising future therapeutic strategies in this disease.
Prostaglandin E2 Selectively Inhibits Human CD4+ T Cells Secreting Low Amounts of Both IL-2 and IL-4
Authors: He Xiaowen , Stuart John M.
Published: January 2023

PGE2 is a potent inflammatory mediator with profound immune regulatory actions. The present study examined the effects of PGE2 on the activation/proliferation of CD4+ T cells using 37 cloned CD4+ T cell lines. Ten T cell clones sensitive to PGE2 and 10 T cell clones resistant to PGE2, as measured by proliferation in response to anti-CD3 Ab, were selected for comparison. It was found that the PGE2-sensitive T cells were characterized by low production (<200 pg/ml) of both IL-2 and IL-4, while PGE2-resistant T cells secreted high levels (>1000 pg/ml) of IL-2, IL-4, or both. The roles of IL-2 and IL-4 were confirmed by the finding that addition of exogenous lymphokines could restore PGE2-inhibited proliferation, and PGE2-resistant Th1-, Th2-, and Th0-like clones became PGE2 sensitive when IL-2, IL-4, or both were removed using Abs specific for the respective lymphokines. In addition, we showed that the CD45RA expression in PGE2-sensitive T cells was significantly lower than that in PGE2-resistant cells (mean intensity, 1.2 ± 0.6 vs 7.8 ± 5.7; p = 0.001). In contrast, CD45RO expression in PGE2-sensitive T cells was significantly higher that that in PGE2-resistant cells (mean intensity, 55.7 ± 15.1 vs 33.4 ± 12.9; p = 0.02). In summary, PGE2 predominantly suppressed CD45RA−RO+ CD4+ T cells with low secretion of both IL-2 and IL-4.
Inactivation of NF-κB by EBV BZLF-1-Encoded ZEBRA Protein in Human T Cells
Authors: Dreyfus David H. , Nagasawa Masayuki , Pratt Joanne C. , Kelleher Colm A. , Gelfand Erwin W.
Published: January 2023

We have previously shown that the EBV ZEBRA protein (also denoted EB1, Z, or Zta) encoded by the BZLF open reading frame is expressed in primary human thymocytes and in human T lymphoblastoid cell lines infected by EBV. Expression of EBV-encoded gene products in T lymphocytes could contribute to viral pathogenesis during acute EBV infection as well as in individuals coinfected with EBV and HIV. HPB-ALL and Jurkat T lymphoblastoid cell lines transiently and stably expressing ZEBRA were characterized in this work. Expression of ZEBRA protein in human T lymphoblastoid cells was associated with decreased expression of an NF-κB reporter gene, altered expression of the NF-κB p50 protein subunit, and decreased DNA binding by components of NF-κB. These observations suggest that inactivation of NF-κB transcription by ZEBRA in EBV-infected T cells may be a novel mechanism of viral pathogenesis analogous in part to over-expression of the endogenous cytoplasmic inhibitor of NF-κB, IκBα.
VSVΔG MARV GP and VSVΔG ZEBOV GP induce strong and rapid anti-viral state in mouse peritoneal macrophages through type I interferon stimulation (128.11)
Authors: Silaghi Alex , Fernando Lisa , Alimonti Judie , Stroeher Ute , Feldmann Heinz , Jones Steven M
Published: January 2023

Replacement of Vesicular Stomatitis Virus (VSV) G protein with the Marburg (MARV) and Ebola (ZEBOV) viruses' glycoproteins (GP) led to the most effective experimental post-exposure therapeutics available against filoviruses.

We hypothesize that the therapeutic effect is partly dependent on activation of innate immune mechanisms, as animals die before the adaptive immune response is activated. Macrophages are primary target cells of MARV, ZEBOV and the recombinant VSVs. Thus, we investigated the innate anti-viral mechanisms that are activated in J774A.1 murine macrophages by VSVΔG MARV GP and VSVΔG ZEBOV GP.

The infections result in rapid induction of anti-viral state, as measured by resistance to challenge with a high MOI of a VSV expressing GFP. There is a significant induction of anti-viral state by 6 hours post-infection and by 24 hours post-infection the cells are almost completely resistant to subsequent infections, which correlates with phosphorylation of IRF-3 and IFN-α and IFN-β production. Blockage of the IFN-α/β Receptor, neutralizes 70%-90% of the anti-viral effects suggesting that the anti-viral state induced after VSVΔG MARV GP or VSVΔG ZEBOV GP infections are type I interferon-dependent. In conclusion, the post-exposure protection may be dependent on type I interferon induction and subsequent activation of anti-viral pathways, which may inhibit filovirus replication in target cells.
A novel mechanism for Galectin-1 inhibition of endothelial cell fusion in Nipah virus infection; a critical component of vascular mediated innate immunity and host defense (128.3)
Authors: Garner Omai B , Aguilar Hector , Fulcher Jennifer , Levroney Ernest , Lee Benhur , Baum Linda
Published: January 2023

The vascular endothelium plays a critical role in the pathogenesis of many viral infections, either via direct infection of endothelial cells, or via damage to endothelia by the immune system. Nipah virus (NiV) is an emerging viral pathogen, of the genus Paramyxoviridae, but little is known about NiV biology or the immune response to NiV. The virus targets endothelial cells, with endothelial syncytia formation being the pathogenic hallmark of infection. Endothelial cell fusion requires expression of NiV-F and NiV-G envelope glycoproteins, and specific N-glycans on NiV-F regulate the extent of endothelial cell fusion.

We have found that galectin-1, an endogenous lectin expressed by activated endothelia, inhibits NiV mediated endothelial cell fusion and syncytia formation by a direct effect on NiV-F. Specifically, galectin-1 regulates endothelial cell fusion triggered by NiV-F by binding glycans on NiV-F and thus retaining immature NiV-F on the cell surface, restricting endocytosis necessary for NiV-F maturation, and inhibiting the mature NiV-F protein from fusing endothelial cell membranes. This suggests that endogenous galectin-1 may regulate the extent of endothelial cell damage in Nipah virus infection. These findings may be broadly applicable to other vascular diseases in which endothelial damage and syncytia formation occur.
Reduced antigen-specific CD8+ T cell responses as a result of increased bacterial translocation in chronic ethanol consuming mice (129.17)
Authors: Gurung Prajwal , Young Betty M , Turner Lucas E , Coleman Ruth A , Wiechert Susan , Waldschmidt Thomas J , Legge Kevin L , Cook Robert T
Published: January 2023

Chronic ethanol consumption leads to increased translocation of bacterial products such as LPS and PGN. Previously, we have shown that chronic ethanol consumption results in reduced antigen-specific CD8+ T cell responses following attenuated Listeria monocytogenes (actA-LM) inoculation. We hypothesized that reduced antigen-specific CD8+ T cell response in chronic ethanol mice can be modeled by treatment with bacterial products. Control water mice were pretreated with either LPS or TLR-2 agonist to mimic systemic bacterial product translocation and their response to actA-LM inoculation was evaluated. Antigen-specific CD8+ T cell responses to actA- LM were reduced in Pam3CSK4 or LPS treated mice, similar to ethanol mice. Further analyses showed that the reduction of antigen-specific CD8+ T cells in TLR agonist treated mice was due to an indirect effect on antigen presenting cells, as well as a direct effect on CD8+ T cells. None of the TLR agonist effects on CD8+ T cells was reversed by prior depletion of natural Treg cells, or by the depletion of all CD4+ T cells, nor was the reduction of IL2Rb reversed by several days in culture. Reduction of antigen-specific CD8+ T cells was also independent of IL-10. The data as a whole clearly indicate that TLR agonists resulting from bacterial translocation represent one potent mechanism for alteration of antigen-specific CD8+ T cell responses.
Flow-cytometric evaluation of T cell responses elicited by an alphavirus replicon particle vaccine for cytomegalovirus (CMV) in healthy adults (132.14)
Authors: Reap Elizabeth A , Katen Kevin S , Watson Aubrey D , Smith Kaitlin M , Norberg Pamela K , Chulay Jeffrey D , Olmsted Robert A
Published: January 2023

We recently completed a Phase I evaluation of a bivalent, virus-like replicon particle (VRP) vaccine (AVX601) expressing three CMV proteins in CMV seronegative adults. One VRP component expressed the pp65 and IE1 genes as a polyprotein and the second expressed gB. AVX601 was well-tolerated, with no significant safety issues identified. Analysis of the immune responses by IFN-γ ELISPOT and CMV neutralization showed the vaccine to be highly immunogenic. In this study, multiparameter flow cytometry was used to further characterize the AVX601-induced T cell responses. Overall, the CD8 T cell responses to all 3 antigens were of higher magnitude than the CD4 T cell responses. The highest levels of CD8 and CD4 antigen-specific responses detected were 0.97% and 0.17% for pp65, 0.12% and 0.10% for IE1, and 0.18% and 0.08% for gB. Antigen-specific T cells were analyzed for their ability to produce IFNγ, TNFα and/or IL-2 after CMV peptide stimulation. Polyfunctional subsets of both CD4 and CD8 lineages specific for all 3 antigens were detected at all time points tested. The maximum % of CD8 T cells positive for 2 or 3 cytokines at any time point studied was 0.58% for pp65, 0.03% for IE1 and 0.07% for gB, and for CD4 T cells was 0.12% for pp65, 0.06% for IE1 and 0.05% for gB. These data demonstrate that this novel alphavirus replicon particle vaccine for CMV elicited polyfunctional CD4 and CD8 T cells to all 3 CMV immunogens.
IgG1, but not IgG2a/c, is pathogenic in Leishmania mexicana infection (133.18)
Authors: Buxbaum Laurence U. , Thomas Bolaji N.
Published: January 2023

FcγRs and IL-10 are both required for chronic disease caused by L. mexicana infection of C57BL/6 mice. We recently showed that IgG1 and IgG2a/c can both induce IL-10 from macrophages and dendritic cells when bound to Leishmania amastigotes, but through different FcγR types. IgG1 induces IL-10 through FcγRIII, whereas IgG2a/c induces IL-10 primarily through FcγRI. Given that FcγRIII KO mice are resistant to L. mexicana infection and because IgG1 responses occur much earlier than IgG2a/c responses, we examined whether IgG1 is specifically required for chronic L. mexicana disease. In fact, mice lacking IgG1 are more resistant to L. mexicana infection. Furthermore, we found that IgG1 KO mice had an earlier and stronger IgG2a/c response to L. mexicana infection. This demonstrates that IgG1 is pathogenic and that IgG2a/c is not, and may in fact be protective. Thus IgG2a/c does not appear to induce a suppressive IL-10 response in vivo. This opens up the question of whether macrophages are the source of suppressive IL-10 responsible for chronic disease in vivo, or whether another cell type is responsible. In addition it has important implications for vaccines for leishmaniasis and potentially diseases caused by other pathogens that share this FcγR/IL-10 mechanism, as vaccines must be engineered to minimize IgG1 responses to amastigote surface epitopes.

Funded by VA Merit and the University of Pennsylvania.
TL1A, a TNF-like molecule, regulates the differentiation of human TH17 cells (133.21)
Authors: Michelsen Kathrin , Yu Qi , Ko Brian , Thomas Lisa , Takedatsu Hidetoshi , Landers Carol , Targan Stephan
Published: January 2023

TL1A is a member of the TNF superfamily that mediates a strong co-stimulation of TH1 cells by enhancing IFN-γ production by CD4+ T cells. The expression of TL1A is increased in inflamed mucosa of Crohn's Disease patients and in murine models of ileitis. We have shown that TL1A is induced in human antigen-presenting cells (APCs) by Fc γ Receptor but not TLR signaling. Furthermore, neutralizing TL1A antibodies attenuate colitis by attenuating TH1 and TH17 responses in a chronic model of colitis. However, it remains to be elucidated if TL1A enhances de novo differentiation of TH17 cells. Here we demonstrate that FcγR signaling in APCs leads to the concomitant induction of TL1A, IL-6, TGF-β, and IL-23. TL1A, in combination with TGF-β and IL-6, promotes the differentiation of human TH17 cells from naive CD4+ T cells and leads to a unique cell population of IL-17/IFN-γ producing TH17 cells. Furthermore, TL1A enhances the IL-17 production by committed CD45RO+CCR6+ TH17 cells. TL1A also enhances the induction of RORA mRNA but not RORC mRNA. However, the enhanced RORA mRNA levels corresponded to an increase in IL-17 mRNA under the same conditions suggesting that enhancement of RORA mRNA by TL1A is sufficient to enhance production of IL-17. Our findings establish an important role of TL1A in promoting human TH17 cell differentiation and function and may provide a potential target for therapeutic intervention in TH17 driven diseases.
The major Th2 polarizing component in schistosome eggs is a T2 ribonuclease (omega-1) that inhibits dendritic-T cell interaction (133.23)
Authors: Jankovic Dragana , Andersen John , Steinfelder Svenja , Cannons Jennifer L. , Schwartzberg Pamela L. , Sher Alan
Published: January 2023

Although helminth parasites are the most potent infectious stimuli for the induction of Th2 cells, the identity of their Th2-inducing components has remained elusive. Schistosoma mansoni eggs contain factors that trigger potent Th2 responses in vivo and condition murine dendritic cells (DC) to promote Th2 lymphocyte differentiation. We have performed a classical biochemical purification of the DC-dependent Th2-polarizing molecule(s) in both secreted egg products and in a water-soluble egg extract (SEA) and identified the active Th2-inducing component as a 31 kDa molecule identical to the previously described S. mansoni ribonuclease T2, omega-1. The Th2-promoting activity of this glycoprotein is sensitive to ribonuclease inhibition and does not require MyD88 or TRIF signaling in DC. In common with unfractioned SEA the purified native protein suppresses LPS-induced DC activation, but unlike SEA fails to trigger IL-4 production from basophils. We further demonstrate that SEA as well as purified omega-1 directly affect both DC morphology and the ability of these APC to interact physically with CD4 T lymphocytes. Based on this evidence, we propose that SEA and its omega-1 bioactive component promote Th2 polarization by lowering the strength of the activation signal delivered by DC.

This work was supported by the Intramural Research Program of the NIAID.
Immune Responses during Respiratory Syncytial Virus Pathogenesis in a Neonatal Ovine Model (133.46)
Authors: Sow Fatoumata Ba , Gallup Jack M , Olivier Alicia , Krishnan Subramaniam , Suzich JoAnn , Ackermann Mark R
Published: January 2023

Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory tract infection in infants and young children worldwide, causing severe pneumonia and bronchiolitis. The newborn ovine lung is well suited for comparison to the human perinatal lung, due to similarities in alveolar development, immune responses, and RSV susceptibility. To understand the role of innate and adaptive immunity in RSV pathogenesis, we inoculated lambs intratracheally at 3 days of age with human RSV (hRSV), and lung tissue was collected from the right cranial lobe at 3, 6, and 14 days post-inoculation for histopathology, viral load, cellular infiltration and cytokine production. hRSV antigen was present at low levels at day 3 of infection, increased 29.8-fold at day 6, and decreased to baseline levels at day 14. The presence of hRSV in the lung correlated positively with an increased accumulation of CD11b+ cells. Infection was associated with a decreased expression of IFN-β, TNF-α, TGF-β, IL-10, RANTES and PD-1, but with an increased expression of IL-8, MCP-1, MIP-1α, PD-L1, and IFN-γ. Laser capture microdissection analysis of macrophage-enriched populations from lung sections determined that MCP-1 was highly up-regulated in those cells, but that alveolar macrophages were not the source of increased IL-8 mRNA. The modulation of host immune responses by RSV may contribute to the enhanced susceptibility and severity of disease.

This project is funded by MedImmune LLC and by NIH grant # RAI062787A
Urushiol regulates the expression of TLR2, HBD and TSLP via NF-κB pathway in Staphylococcus aureus-treated HaCaT cells (134.50)
Authors: PARK JU YOUN , Chang Jae-Ho , Kim Myong-Jo , Kim Soo-Ki
Published: January 2023

TLR2, HBDs(beta-defensins) and TSLP(thymic stromal lymphopoietin) expressed from keratinocytes play key roles in controlling the progression of atopic dermatitis. These molecules might account for the accelerated colonization with S. aureus into eczematous skin barrier. In the course of screening natural substance in regulating TLR2, HBD and TSLP expression by using the inflamed keratinocytes, we found urushiol, major active ingredient of Rhus verniciflua Stokes which has been used as barrier protective in traditional medicine. The aim of this study was to clarify how urushiol regulates innate immune molecules, focused on TLR2, HBD and TSLP, in our in vitro infectious keratinocyte model, S. aureus-treated HaCaT cells. To the end, the expression of TLRs, cytokines and chemokines were assessed by by siRNA, RT-PCR, ELISA and Western blots. We found that urushiol treatment on HaCaT cells induced the expression of TLR2 and HBD2 but not that of TNF-α and TSLP. In S. aureus-treated HaCaT cells, urushiol pretreatment enhanced the expression of TLR2 and HBD2, whereas TNF-α and TSLP expression were in decrement. Further to explore the mechanism, inhibition of NF-κB decreased the expression of TLR2, HBD2 and TSLP, albeit increased TNF-α release. Cumulatively, our data indicate that urushiol regulates the expression of TLR2, HBD and TSLP via NF-κB pathway in S. aureus-treated HaCaT cells.
Demonstration of a severe complement defect in FcRγ-deficient mice (134.63)
Authors: Strait Richard T , Khodoun Marat , Mahler Ashley , Potter Crystal , Köhl Jörg , Finkelman Fred D
Published: January 2023

Several studies have examined the relative roles of complement vs. Fc receptors (FcRs) in host protection and inflammatory disease. Many of these studies have concluded that the more important role is played by stimulatory Fc receptors. These conclusions were based, however, on the assumption that complement activation occurs normally in the absence of stimulatory Fc receptors. We have used FcRγ-deficient mice, which lack all stimulatory FcRs, to examine this assumption. FcRγ-sufficient and -deficient mice were found to have similar levels of C3 in blood. Also, as expected, similar amounts of IgG2a became associated with blood leukocytes from both mouse strains after intravenous injection of a complement fixing IgG2a mAb to MHC class I Ag. Surprisingly, however, >10-fold more C3 became associated with the blood leukocytes from FcRγ-sufficient mice than those from FcRγ-deficient mice. Additionally, >6-fold more C5a was generated in blood from FcRγ-sufficient than deficient mice. These results demonstrate that mice that lack all stimulatory FcRs have a severe abnormality in complement activation. This abnormality may well contribute to the decreased protective immunity and increased resistance to inflammatory disease characteristic of FcRγ-deficient mice that have previously been ascribed solely to direct effects of the absence of stimulatory FcRs.
A Meta Analysis of Relationship between C - Reactive Protein and Respiratory Tract Infection in Children (134.64)
Authors: Shen Chao-Bin , Gu Jun , Xia You-Jia
Published: January 2023

BACKGROUND: Although several studies have evaluated serum concentrations of C-reactive protein (CRP) as a predictor of bacterial respiratory tract infections or pneumonia in this patient population, the utility of this test remains unclear. OBJECTIVE: The purpose of this meta-analysis was to quantitatively define the utility of serum CRP as a predictor of bacterial respiratory tract infections or pneumonia in acutely ill children. METHODS: Multiple databases were searched, bibliographies reviewed,and 13 authorities in the field were queried.The quality of each included study was determined have 6 metrics: diagnostic criteria; study author; CRP agent; patient age; CRP assay of method; and CRP results before patients who received antibiotics. Data was extracted from each article; the primary outcome measure was the means of patients with bacterial or not bacterial infection and serum CRP concentrations exceeding 8 mg/L. RESULTS: Thirteen studies fulfilled inclusion criteria. Combining all of the studies demonstrated a pooled study population of 2760 patients.The level of serum CRP in the children with bacterial infection were higher 3.13 times than viral infection(Z=7.61,p£1/40.00001,95% confidence interval=2.33-3.94),and were higher 3.02 times than control group(Z=10.26,p£1/40.00001,95% confidence interval =2.44-3.60).The level of serum CRP in children with viral infection were higher 0.68 times than control group(Z=4.74,p£¼ 0.00001,95% confidence interval=0.40-0.96). Children with bacterial infection were significantly serum CRP concentrations than children with nonbacterial infections.CONCLUSIONS:The serum CRP level has value for early recognition of bacterial infections could guide treatments,reduce misuse of antibiotics.
Complement Factor H Regulation in the Central Nervous System (134.70)
Authors: Fraczek Laura , Martin Carol , Martin Brian
Published: January 2023

The complement system is an important component of innate immunity that requires proper regulation to avoid inappropriate activation. This regulation is provided by many proteins including complement Factor H (CFH), a secreted protein of the alternative pathway. Despite its regulatory function, gene and promoter polymorphisms of CFH have been implicated in disease of many patients with age-related macular degeneration and atypical hemolytic uremic syndrome, indicating that a better understanding of this gene's regulation is valuable. Presently, little work has been done to address the role of CFH in the central nervous system (CNS). We have cloned the murine CFH promoter and have made truncation constructs to examine CFH gene regulation in cells of the CNS. Our studies show that CFH mRNA is present in CNS cell types, and that specific regions throughout the promoter contain possible enhancers and repressors. Database mining of these regions indicates that there are potential transcription factor binding sites conserved between many different species, which led us to investigate specific transcription factor binding interactions in these regions. Through these studies we hope to elucidate the transcriptional regulation of CFH in the CNS.

This work is supported by the National Multiple Sclerosis Society.
In Vivo Inhibition of Type I Interferon Transcription in Genital HSV-2 Lesions (134.72)
Authors: Peng Tao , Zhu Jia , Klock Alexis , Koelle David , Wald Anna , Corey Larry
Published: January 2023

We performed transcriptional analysis and immunocytochemistry (ICC) of sequential biopsies of lesional tissue of immunocompetent persons with recurrent HSV-2 infection. Histologic analysis of these biopsies indicated a massive infiltration of monocytes/macrophages along with a large amount of myeloid and a small number of plasmacytoid dendritic cells in the dermis of these lesional biopsies. IFNB1 and IFN-α were weakly expressed and IFNG potently induced during time periods in which there was abundant detection of HSV-2 antigens and gene expression. Transcriptional arrays of the same anatomic area over time (newly healed, 2 and 4 weeks post healing) in which no HSV DNA/RNA or antigen was detected continued to show that IFNB1 and IFN-α were barely detectable. IFNG persisted in lesional tissue, albeit at lower levels as compared with active lesions. Interferon-stimulated genes (ISGs) were also markedly up-regulated with expression patterns more clearly resembling those in primary human fibroblasts treated by IFNG than by IFNB1. The presence of a very large number of innate cells capable of sensing HSV-2 infection and synthesizing type I IFN by both histologic and transcriptional analyses associated with extremely low levels of IFN-α and IFNB1 even in the earliest lesional biopsies suggests a potent alteration in host defense during HSV-2 infection in vivo. This block of type I IFN by HSV-2 may be a major factor in allowing the virus to breakthrough host mucosal defenses.
Toll-Like Receptor 3 Signaling on Macrophages is Required for Survival Following Coxsackievirus B4 Infection (134.77)
Authors: Richer Martin Joseph , Lavallée Danielle J , Shanina Iryna , Horwitz Marc S.
Published: January 2023

Toll-like receptor 3 (TLR3) has been proposed to play a critical role in the innate detection of viruses. However, as several reports have demonstrated that TLR3 signaling is either dispensable or even harmful following certain viral infections, the role of TLR3 in anti-viral immunity remains controversial. Here, we asked whether TLR3 is required for the immune response to coxsackievirus B4 (CB4), a common human pathogen associated with pancreatitis, myocarditis and diabetes. We demonstrate that TLR3 signaling is required for survival following CB4 infection. TLR3 deficient mice produce lower levels pro-inflammatory mediators and are unable to control viral replication resulting in severe cardiac damage and increased mortality. We further demonstrate that the MyD88-dependent signaling pathways are dispensable in the response to this RNA virus and are unable to compensate for the loss of TLR3. Finally, we demonstrate that adoptive transfer of WT macrophages is sufficient to reestablish protection in a TLR3 deficient host by reducing cardiac pathology suggesting that TLR3 signaling on macrophages is critical for survival following CB4 infection. Our results demonstrate that TLR3 is not simply part of a redundant system of viral recognition, but rather TLR3 plays an essential role in recognizing the molecular signatures associated with specific viruses including CB4.

This work was supported by a CIHR grant to MSH.
Poly-T oligonucleotides enhance TLR7/8 agonist induced responses in glial cells through a TLR7-dependent mechanism (135.11)
Authors: Du Min , Butchi Niranjian , Peterson Karin E
Published: January 2023

The brain does not contain a resident dendritic cell population so resident cells such as microglia and astrocytes play an important role in initiating innate immune responses in the central nervous system (CNS) following virus infection. However, the activation of these cells following stimulation of virus-recognizing toll-like receptors (TLRs) such as TLR7 and TLR8 remains unclear. Poly T oligonucleotides (pT-ODNs) were recently shown to enhance signaling through TLR8, including murine TLR8 which was previously thought to be non-functional. In the current study, we analyzed the ability of pT-ODNs to enhance TLR7/8 activation of murine astrocytes and microglia. We found that the TLR7/8 agonist CL075 (3M002) induced the expression of glial cell activation markers and induced the production of multiple proinflammatory cytokines and chemokines. Addition of pT-ODNs enhanced the expression of proinflammatory cytokines and chemokines as well as the microglia/macrophage activation markers F4/80 and Iba1. Surprisingly, pT-ODNs enhancement of CL075-induced glial activation was abolished in TLR7 deficient mice, with the exception of Gfap (glial fibrillary acidic protein) mRNA upregulation. Thus in glial cells, pT-ODNs enhanced TLR7/8-induced proinflammatory responses through a TLR7-dependent pathway.

This research was supported by the Intramural Research Program of the NIH.
Desialylation of TLR4/MD2/CD14 enhances the response to LPS (135.16)
Authors: Feng Chiguang , Whitford Melissa , Zhang Lei , Rallabhandi Prasad , Vogel Stefanie N. , Stamatos Nicholas M.
Published: January 2023

Objective: Since bacterial neuraminidase (NANase) treatment of intact human PBMCs led to a synergistic increase in cytokine expression, we hypothesized that components of the TLR4 receptor complex were sialylated, and that removal of sialyl residues enhanced LPS-initiated signaling.

Methods: HEK293T cells were transfected with TLR4, MD2, CD14 and ELAM-Luc expression vectors. They were treated with NANase, stimulated with LPS, and assayed for NF-kB-driven luciferase expression. Sialylation was assayed by lectin blot after immunoprecipitation from lysed transfected cells.

Results: LPS stimulated NF-kB activation and IL-8 expression only in cells transfected with TLR4/MD2/CD14 or in cells transfected with TLR4/CD14 supplemented with supernatants (SNT) of MD2-transfected cells. NANase treatment of TLR4/CD14/MD2 cells led to enhanced NF-kB activation. While NANase treatment of either TLR4/CD14 cells or MD2 SNTs each enhanced the LPS response in the reconstituted culture, optimal responses occurred when both were treated. Lectin blots revealed MD2 to be sialylated with a α2,6 linkage since SNA but not Maackia bound to MD2.

Conclusions: We conclude that removal of sialyl residues from MD2 and perhaps from TLR4/CD14 enhances the response to LPS. Thus mobilization of endogenous sialidase in PBMCs could modulate the activity of the TLR4/CD14/MD2 complex and serve as a therapeutic target.

Support: NIH grant no. HL086933-01A1
Regulation of pro-inflammatory cytokines by interferon-gamma during endotoxin tolerance (135.38)
Authors: Chen Janice , Ivashkiv Lionel B.
Published: January 2023

Endotoxin tolerance is a phenomenon whereby previous exposure of macrophages to microbial products, such as LPS, induces a transient period of hyporesponsiveness upon subsequent LPS challenge and is characterized by diminished release of pro-inflammatory cytokines. Previous reports have shown that interferon-gamma (IFN-γ) can prevent and reverse endotoxin tolerance, and our study aims to define the mechanisms by which this occurs. We found that exposure of CD14+ human monocytes to 100 pg/mL LPS for 24 h was sufficient to blunt their ability to produce IL-6 and TNFα upon secondary LPS challenge. However, IFN-γ pretreatment abrogated tolerization of IL-6, and to a lesser extent, TNFα, induced by LPS as well as cross-tolerization induced by Pam3Cys and IL-1β. IFN-γ did not reverse the defects in LPS signaling induced by tolerization. Tolerized monocytes exhibited greatly reduced LPS-induced IL-6 gene expression, while IFN-γ-activated monocytes retained the capacity to upregulate IL-6 mRNA despite tolerizing signals. IFN-γ-mediated regulation of tolerance occurred at the level of transcription, as revealed by IL-6 primary transcript measurements and RNA polymerase II binding to the IL-6 promoter. Current efforts are directed at identifying key factors that might mediate the ability of IFN-γ to abrogate endotoxin tolerance. This work is supported by the CRI Predoctoral Fellowship and NIH T32 AIO7621.
The role of PI3-Kinase signalling in immunomodulation by a protective innate defence regulator peptide (135.6)
Authors: Madera Laurence , Nijnik Anastasia , Waldbrook Matt , Mookherjee Neeloffer , Gardy Jennifer , Falsafi Reza , Elliott Melissa , Hancock Robert EW
Published: January 2023

The rise of antibiotic resistance has lead to the search for new therapeutics against bacterial infections. Innate defence regulators (IDRs), synthetic versions of natural host defence peptides, are being developed as anti-infective agents due to their immunomodulatory properties. HDPs have been shown to suppress infections by boosting leukocyte recruitment, while limiting harmful inflammation. To optimize the IDR regulatory function, we developed peptide 1002, a bactenecin derivative, by screening a peptide library for enhanced leukocyte chemoattractants induction in human blood mononuclear cells (hBMC). 1002 enhanced protection in a Staphylococcus aureus murine infection model, correlating with increased chemokine production and leukocyte recruitment. 1002 also suppressed inflammatory cytokine production by lipopolysaccharide-stimulated hBMC. IDRs are being deciphered to assist in their development as therapeutics. We found that 1002 regulation is dependent on the PI3-Kinase pathway, a signal cascade that regulates immune and inflammatory responses. PI3-K inhibition abrogated 1002-mediated chemokine induction and reduced its anti-inflammatory properties. 1002 influence on PI3-K signalling was confirmed by examining responses of pathway elements and by systems biology microarray analysis of the immune network by InnateDB. Understanding the mechanisms of IDR immune regulation via PI3-K modulation will aid in their development as novel anti-infective agents. This work was supported by Genome BC and Genome Prairie for the Pathogenomics of Innate Immunity Research Program, and by FNIH and CIHR through the Grand Challenges in Global Health Initiative.
The inflammasome as a therapeutic target to improve outcomes after injury to the central nervous system (135.73)
Authors: de Rivero Vaccari Juan Pablo , Lotocki George , Dietrich W. Dalton , Keane Robert W.
Published: January 2023

The inflammasome constitutes one of the host's defenses against pathogens and deleterious stimuli by activating caspase-1. Inflammation with production of cytokines processed upon inflammasome activation is a crucial part of the secondary cascade of cell death induced after central nervous system (CNS) injury. The molecular mechanisms underlying trauma-induced inflammation in the CNS are poorly defined. In this study, the cellular distribution and protein association of inflammasome components were investigated in spinal cords and brains of rats subjected to moderate cervical injury and fluid-percussion brain injury, respectively. Coimmunoprecipitation experiments revealed that in the CNS, Apoptosis-Associated Speck-like protein containing a CARD (ASC) associates with caspase-1, caspase-11, NLR family, Pyrin domain containing 1 (NLRP1) and the inhibitor of apoptosis XIAP, forming the NLRP1 inflammasome. Injury to the CNS affects the composition of the inflammasome resulting in increased association of caspases-1 and -11 and NLRP1 with ASC and cleavage of XIAP. Immunohistochemistry revealed that motor neurons and cortical neurons express the NLRP1 inflammasome proteins. Therapeutic neutralization of ASC after injury inhibited inflammasome activation as shown by decreased caspase-1 activation and XIAP cleavage, resulting in reduced lesion volume and improved functional outcomes after spinal cord injury. Thus the inflammasome forms part of the innate immune response in the

CNS and its inhibition may offer a novel therapeutic strategy to improve outcomes after injury and CNS inflammatory diseases.

This research was supported by the NIH/NINDS and the US Army.
Catecholamine and PKA-dependent regulation of Foxo1 mRNA and protein in S49 lymphosarcoma cells (136.12)
Authors: Davis Daniel H. , Cohen Rhonna , Chambers Donald A.
Published: January 2023

We previously linked psychological stress to lymphocyte physiology by demonstrating the presence of a norepinephrine (NE)/beta-2-adrenergic receptor/cAMP/ PKA-dependent mRNA decay system in T lymphocytes. Foxo1 and the PKA pathway can participate in the promotion of cell cycle arrest and apoptosis of T lymphocytes, so we explored whether a PKA-selective cAMP analog (8CPT-cAMP) or NE could regulate BALB/c murine thymomas, S49wt and S49kin- (PKA-deficient), by affecting the genetic expression of Foxo1. Real-time PCR studies showed a 2-3 fold upregulation of Foxo1 mRNA in S49wt, but not S49kin- cells with NE or 8CPT-cAMP 2hr after treatment. Foxo1 total protein, determined by immunoblotting, was increased 6hr after treatments. Actinomycin D treatment of S49wt cells incubated with NE or 8CPT-cAMP revealed that the increase in Foxo1 was not associated with altered mRNA stability. Transfection of S49wt and S49kin- cells with a Foxo1 promoter luciferase reporter suggested Foxo1 mRNA increases due to enhanced Foxo1 message transcription. These results associate NE/cAMP/PKA in modulating increased Foxo1 mRNA and protein expression, and links Foxo1 to PKA-mediated gene regulation of lymphocyte physiology.
Catecholamine/cAMP regulation of Thy1 mRNA stability in S49 cells involves a 3’UTR cis-acting element (136.13)
Authors: Koduvayur Sujatha P , LaJevic Melissa D , Cohen Rhonna L , Chambers Donald A.
Published: January 2023

Under conditions of psychological stress, release of the catecholamine, norepinephrine (NE), by sympathetic nerves has been reported. We described Thy1 mRNA regulation via a NE-ß2 adrenergic receptor-cAMP-PKA dependent pathway (NE pathway) in murine thymoma (S49) cells. To understand the molecular mechanism(s) underlying this regulation we studied the involvement of a 116nt region containing a putative ARE in the 3'UTR of Thy1 mRNA. The Thy1 ARE region was inserted downstream of the GFP coding region and the resultant plasmid was transfected into S49 cells. The presence of this region reduced the expression of exogenous GFP mRNA upon stimulation of the NE pathway. This ARE region also binds to known ARE binding proteins (bps) including AUF1, HUR, TIAR and Hsp70 and binding of ARE bps to known AREs like TNFα mRNA is competed away by the Thy1 ARE region. Additionally, silencing the ARE bp HUR, a known enhancer of mRNA stability, increased Thy-1 mRNA decay upon stimulation of the NE pathway in S49 cells. These results suggest that Thy1 mRNA UTR can bind RNA bps and contains a novel cis-acting, 116 nt. ARE region that participates in the mechanism of NE pathway regulated mRNA decay. Supported by NIH.
PRDI-BF1/Blimp-1 Associates with Cytokine-Mediated Activation of Human Natural Killer Cells (136.25)
Authors: Smith Matthew A , Wei Sheng , Djeu Julie , Caligiuri Michael , Wright Kenneth L
Published: January 2023

PRDI-BF1 (Positive Regulatory Domain I - Binding Factor 1,) is a zinc finger transcription factor, originally identified as a suppressor of interferon-beta in virally-infected human fibroblasts. It is most widely recognized as a key regulator of B-cell terminal differentiation into antibody-producing plasma cells. More recently, several reports have described functions in T-cell homeostasis. Here, we demonstrate that PRDI-BF1 is expressed in human natural killer (NK) cells and is involved in the attenuation of activation-induced cytokine gene transcription. Ex vivo stimulation of negatively-selected human CD56+ lymphocytes with activating cytokines increases levels of PRDI-BF1 mRNA and protein. This induction occurs primarily in the CD56dim population. The combination of IL-2, IL-12 and IL-18 leads to synergistic increases in PRDI-BF1 and IFN? as part of a global transcriptional profile that we have characterized via microarray analysis. Blockade of PRDI-BF1 induction via siRNA leads to enhanced TNFα and IFNγ transcription. Chromatin immunoprecipitation analysis of both NK cell lines and primary CD56+ lymphocytes reveals occupancy of several promoters, including IFNγ. Collectively, these data provide evidence for novel contributions of PRDI-BF1 to natural killer cell function and innate immunity.