bluuebunny/crossref_metadata_2025 huggingface dataset:
Toll-like Receptor 4-Dependent activation of Dendritic cells by heme binding protein from Ascaris suum (90.9)
Authors: MADALA SATISH KUMAR , Bundoc Virgilio Garcia , Hodges Marcus Gomez , Trivedi Shweta , Urban Joseph F. , Keane-Myers Andrea
Published: January 2023

Toll-like receptors (TLRs) recognize specific molecular patterns on invading pathogens leading to the development of host immunity. A number of pathogens including helminths have used pattern recognition by TLRs to modulate host immunity and inflammation to establish a chronic infection. To understand the molecular basis for the TLR-induced tolerance induction observed in individuals infected with helminth parasites, we have established a mouse model combining allergic inflammation and Ascaris suum infection. These in vivo studies demonstrated that concomitant infection with chronic A. suum can inhibit allergic inflammation in response to a non-parasite allergen, ragweed (RW). In the current study, we have purified heme-binding protein (HBP) from pseudocoelomic fluid of A.suum and studied its antigenic and immunomodulatory properties using bone marrow derived dendritic cells. Recognition of HBP by antiserum from pigs infected with Ascaris larvae indicate that HBP is one of the antigenic proteins present in PCF. Furthermore, HBP exposure increases activation marker surface expression on DC including CD40, CD86 and MHC II. This increase in activation marker expression is abrogated in TLR4, but not TLR2, knockout mice implicating that HBP is a TLR4 ligand. Pretreatment of dendritic cells with HBP prior to LPS stimulation results in increased production of IL-10 but not IL-12 compared with DC exposed to LPS alone. These results provide the first evidence for a novel TLR4-mediated response to Ascaris suum HBP resulting in altered APC activation and cytokine production.
The role of LacCer inhibitor in modulation of established Pneumocystis pneumonia (91.1)
Authors: Vuk-Pavlovic Zvezdana , Standing Joseph E , Limper Andrew H
Published: January 2023

Pneumocystis pneumonia is caused, at least partly, by inflammatory action of β-glucan, the major component of Pneumocystis cell wall. Glucan induces release of pro-inflammatory cytokines in alveolar macrophages and lung epithelial cells. While macrophages recognize glucan mainly by dectin receptors, epithelial cells utilize membrane glycosphingolipid (GSL) lactosyl ceramide (LacCer) as glucan-binding molecule. The use of GSL inhibitors in vitro causes reduced NF-κB activation and inhibited IL-8 release in epithelial cells. To assess the impact of LacCer in lung inflammation, we treated Pneumocystis-infected mice with GSL inhibitor PDMP. The animals were immunosuppressed by GK 1.5 antibody, infected with Pneumocystis by intratracheal injection and, starting at 8 weeks post infection, treated with either 150 mg PDMP/kg of body weight - dissolved in saline, or equal volume of saline alone (daily i.p. injections for 10 days). The analysis of sacrificed animals showed that PDMP-treated mice had fewer leukocytes in the bronchoalveolar lavage fluid (BALF) than saline-treated controls with the most marked difference in the number of neutrophils. Interestingly, BALF concentrations of MIP-2 and TNF-α (determined by ELISA) did not differ between these two groups. The analysis of Pneumocystis burden in the lung, as determined by Q-PCR, showed a trend of decreased Pneumocystis-specific DNA copies in PDMP-treated animals. These results suggest that PDMP might have an impact on inflammatory response to Pneumocystis. Other GSL inhibitors are yet to be tested in similar in vivo experiments. This might lead to new options in treatment of Pneumocystis pneumonia.

Supported by NIH RO1 HL62150 and Mayo
Target APC polarization at immunological synapses (IS) in vivo: infected astrocytes polarize towards CTL cells during the brain antiviral immune response. (91.13)
Authors: Lowenstein Pedro R , Barcia Carlos , Liu Chunyan , Wawrowsky Kolja , Kroeger Kurt M , Castro Maria G
Published: January 2023

IS mediate intercellular communication between T cells and APCs. T cells’ structure and membrane proteins are polarized at IS. T cell polarization at the IS consists of a peripheral supramolecular activation cluster (pSMAC), which is high in LFA-1, and low in TCR, and a central SMAC, high in TCR, and low in LFA-1. The MTOC, Golgi and secretory vesicles are also polarized towards the IS. Here we demonstrate that during an anti-viral immune response in the brain, APCs are also polarized. Brain astrocytes were infected with an adenoviral (Ad) vector expressing Thymidine Kinase (TK), a marker of infected cells. 30 days later we immunized systemically against Ads, and 14 days later animals were sacrificed and brains prepared for microscopical analysis. Brain sections were immunostained for TCR, LFA-1, TK, EAAT2, γ-Tubulin, α-Tubulin and GM130. Confocal analysis revealed that target infected astrocytes, normally a non-polarized cell, became completely repolarized. Target astrocytes retract all processes and form a single large protrusion towards the T cell. The MTOC and Golgi are then targeted towards and into the protrusion, and membrane proteins become polarized. These results demonstrate that CTL signaling, potentially mediated through IS, induces profound morphological changes in target APCs.

This work was funded by grants from NINDS/NIH.
Antigen receptor-dependent and -independent regulation of NKT patrolling and activation in liver sinusoids (91.7)
Authors: Velazquez Peter , Cameron Thomas O , Kinjo Yuki , Nagarajan Niranjana , Kronenberg Mitchell , Dustin Michael L
Published: January 2023

Natural killer T (NKT) cells are most abundant in liver and spleen and, recognize lipid antigen presented by CD1d. Utilizing intravital fluorescence microscopy, we previously demonstrated NKT patrolling in the liver sinusoids, meaning cell migration both with and against the blood flow in surveillance of antigen. Here, we report the relationship between patrolling and activation. First, both a Sphingomonas bacteria derived weak agonist, GalA-GSL at 2 μg/g, and a synthetic strong agonist, aGalCer at 10 ng/g, induced serum IL-4, CD69 upregualtion and acute NKT arrest. Surprisingly, a low dose of aGalCer (2 ng/g) did not significantly affect NKT patrolling, although a comparable biological response was elicited. Second, IL-12 and IL-18 synergized to activate NKT cells in the absence of exogenous antigen. These cytokines activated liver NKT cells and induced serum IFN- ? but not IL-4. NKT patrolling was not significantly affected. Therefore, glycolipid antigens can induce NKT cell activation and arrest, although the requirement for arrest may be restricted to weak agonists. Importantly, IL-12 and IL-18 activate NKT cells without affecting NKT patrolling thereby allowing these primed cells to continue to survey tissue for antigen. Current studies are aimed at understanding the hierarchy of antigen receptor versus cytokine driven activation and further defining their relationship to NKT cell patrolling.
CD45RB expression refines delineation of memory CD4 T cells and aids in understanding their development (91.8)
Authors: Caucheteux Stephan M , Younes Souheil A , Paul William E
Published: January 2023

Upon immunization, CD4 T cell stimulation leads to the development of effectors cells and of 2 major subsets of memory cells: central and effector memory cells, the latter often defined by their differential expression of CD62L and CD44. It is still unclear whether central memory cells derive from effectors or are directly induced. Our results have shown that CD44hi cells can be divided into 2 different subtypes depending on their expression level of CD45RB, providing a precise distinction among central and effector memory CD4 T cells. Using these markers, we show that while central memory CD4 T cells are absent in young mice, they are enriched in old and thymectomized animals To understand the properties of the subsets defined by CD45RB and the establishment of the central and effector memory pools, we followed the fate of transferred naïve 5C.C7 T cell receptor transgenic cells, specific for pigeon cytochrome. During the first days following immunization, virtually all the transferred cells adopt the phenotype associated with effector or effector memory cells. After a month, essentially all 5C.C7 cells in lymphoid organs express phenotypes associated with central memory cells, while in the lung effector T cells are still found. Analysis of the mean “life” of these memory cells and their cycling within the lymphoid and the non-lymphoid organs is under investigation.

Taken together, these observations suggest that CD45RB, together with CD44 and CD62L, provides a powerful tool to identify and quantitate naïve, effector and memory cells in lymphoid and non-lymphoid organs.

This work was supported by the Intramural Research Program of the NIAID.
Identification of human B cells with immunoregulatory properties (92.12)
Authors: Tretter Theresa , Venigalla Ram KC , Eckstein Volker , Lorenz Hanns-Martin
Published: January 2023

Regulation of immune responses and tolerance is mediated by a variety of mechanisms and cells. So far the role of B cells in this process is not well known, yet.

Methods: Highly purified CD19+B and CD4+T cells were separated from human PBMC by MACS. B cells were prestimulated with SAC, αIgM/IgG or αCD40, washed and set up in cocultures with freshly isolated autologous CD4+T cells under addition of αCD3+IL-2 or αCD28. CD4+ T cell proliferation was determined after 3–6d by 3H Thymidine incorporation and PKH-26; apoptosis by AnnexinV.

Results: under optimal stimulatory conditions T cell proliferation was inhibited more than 50% in presence of SAC-activated B cells and to a lesser extent by αIgM/IgG stimulated B cells. Enrichment of SAC-activated large CD25+ B cells by FACSsorting enhanced suppression further up to 68%, while the small CD25− population had no significant effect. Separation of B and T cells by cell culture inserts abolished inhibition, suggesting a requirement for direct cell-contact. In addition to growth inhibition, T cell specific cytokine production (IFNγ, IL-10) was downregulated, and a significant proportion of T cells (37% vs 17%) went into apoptosis.

Conclusions: B cells are able to inhibit T cell mediated responses depending on their mode of activation. Further experiments are dealing with the mechanisms of B cell mediated suppression and their pathophysiological impact also in autoimmune diseases.
The Proinflammatory Cytokine Interleukin-32 is expressed in Keratinocytes and Dendritic Cells Obtained from Patients with Chronic Plaque Psoriasis (CPPs) (92.15)
Authors: Hu Ling-Jia , Li Li , Fitzpatrick James E. , Francis Shayla O. , Fujita Mayumi , Takashi Miyatake K. , Shroyer Kenneth R. , Azam Tania K. , Kim Soo-Hyun K. , Dinarello Charles A. , Norris David A. , Edwards Carl K
Published: January 2023

CPPs is characterized by hyperproliferation of keratinocytes (KC) and T-cell (Tc)/DCs activation. IL-32, which induces TNF and activates NFkB and p38 MAPK, is involved in early inflammation. We investigated the role of IL-32 in the pathogenesis of CPPs by IHC, ECL and qRT-PCR analysis. Biopsy tissues were obtained from CPPs patients with active disease (N=20) and healthy human controls (N=15). A mouse Mab against human IL-32 and mouse IgG1 isotype antibody were used. IHC staining of IL-32 was performed on deparaffinized sections of formalin-fixed tissues by an indirect imunoperoxidase method (EnVision, Dako). IL-32 was expressed in basal KC with cytoplasmic granular staining. IL-32 was also expressed in DCs with strong nuclear and cytoplasmic staining in dermis. IL-32 was weakly expressed in the nucleus of KC in healthy human skin. Induction of IL-32 was detected in human KC lysates after stimulation with TNF and IL-1 or cell contact with activated Tc, but not in KC supernatants, indicating IL-32 is not secreted from KC in vitro. ECL/qRT-PCR results showed IL-32 and other CPPs-relevant cytokines (IL-18, IL-20, 22 and 23) were significantly (P<0.05) increased. Our data support the hypothesis that IL-32 is a novel proinflammatory cytokine which is involved in the pathogenesis of CPPs, especially in epidermal regions of the skin where innate immune mechanisms are central to host defense.
Induction of a Th1 Response from Th2 Polarized T Cells by Activated Dendritic Cells: Dependence on TCR:pMHC Interaction, ICAM-1, IL-12 (92.7)
Authors: Radhakrishnan Suresh , Wiehagen Karla R , Pulko Vesna , Knutson Keith , WIlliam Faubion , Pease Larry R
Published: January 2023

Dendritic cells (DC) are important regulators of T cell immunity. The degree of stimulation, the pattern of co-stimulatory molecules expressed, and the cytokines secreted by DC dictate the nature of the effector and memory cells generated, particularly with respect to their Th1 or Th2 phenotypes. In this report we demonstrate that addition of activated DC to spleen cultures containing established Th2 polarized CD4+ T cells was sufficient to suppress Th2 and to induce Th1 cytokines in a recall response, a phenomenon referred to as phenotype reversal. The ability of activated DC to induce phenotype reversal displayed exquisite antigen specificity. The DC activator B7-DC XAb was more than 10,000-fold more efficient at inducing phenotype reversal than the TLR agonists, CpG-ODN and Gardiquimod. Characterization of the mechanisms governing phenotype reversal revealed the requirement for cognate interaction between the TCR: pMHC complex, the expression of the co-stimulation/adhesion molecule ICAM-1, and secretion of IL-12 and IFNƒ×ƒnby the activated DC. Requirement for the co-stimulation/adhesion molecule SLAM was found to be quantitative. Thus, activation of DC, particularly by cross linking B7-DC, can modulate well established Th2 T cell responses in an antigen specific manner.
Similarities and differences in tapasin interaction with disparate murine MHC class I allotypes (93.11)
Authors: Groening Laura Christina , Wang Xiaojian , Tuli Amit , Ormsbee Briana D. , McIlhaney Mary M. , Solheim Joyce C.
Published: January 2023

Tapasin is a key molecule in the major histocompatibility complex (MHC) class I peptide-loading complex. In this study, we first assessed the influence of the tapasin C-terminus on H2-Kb, -Kd, and -Ld in mouse cells. Truncated mouse tapasin (lacking the transmembrane and C-terminal region) was unable to associate with TAP or any of three murine MHC class I allotypes, and did not assist murine MHC class I folding and surface expression. A tapasin mutant with a single substitution in the C-terminus (K408W) increased the amount of Kd (but not Ld) in the open, peptide-free form, relative to wild type tapasin. We investigated whether Kd was also particularly responsive to other tapasin mutations, and found that a C95S substitution in tapasin also caused an elevation in open Kd (but not open Ld or Kb). Folded Kd was found to be amply present in association with wild type tapasin, and even more so with tapasin K408W and C95S. In summary, our results suggest that the C-terminus of mouse tapasin plays a vital role in the surface expression of murine MHC class I molecules, and that mouse MHC allotypes vary in their sensitivity to specific alterations in tapasin sequence and in their release from tapasin after folding. Overall, our results reveal new complexity in tapasin’s interactions with polymorphic MHC class I molecules. (NIH Grant GM57428)
Tapasin functions to assemble MHC class I subunits but does not affect their lifetime on the cell surface (93.12)
Authors: Everett Maya W , Edidin Michael
Published: January 2023

Proper assembly of MHC class I (MHCI) molecules is dependent on the dedicated ER-resident chaperone tapasin (tpn). Tpn binds both MHCI and TAP through different domains. We created three fluorescently tagged constructs: wild type tpn, soluble tpn, which no longer interacts with TAP, and N300 tpn, which no longer interacts with MHCI. Despite previous biochemical data, using fluorescence microscopy we find that soluble tpn is mostly located within the ER, similarly to wild type tpn. FRAP analysis shows that soluble tpn is more mobile than both wild type and N300, possibly because the latter constructs interact with TAP. Transfecting tpn negative cells with soluble tpn does not increase MHCI surface expression to the same level as wild type. When wild type is transfected in combination with N300, MHCI surface levels are reduced to those found with soluble, suggesting that N300 blocks wild type tpn access to TAP. Although resulting MHCI surface levels differ between wild type, soluble, and N300 tpn constructs, MHCI surface lifetimes are the same. Our data suggests that tpn functions to bring together the MHCI subunits, a process which is aided by proximity to TAP, but does not act to increase stability of the resulting MHCI complex.
The double mutation of DRBW61A/N82A changed the MHC II-peptide complex stability and DM catalytic activity (93.24)
Authors: Zhou Zemin , He Xiao , Chen Xinjian , Jensen Peter E.
Published: January 2023

The MHC class II protein HLA-DM (DM) is required for efficient antigen presentation; however the molecular basis of how DM facilitates this process remains incompletely understood. We have generated the HLA-DR1 β chain double mutant, replacing W61 and N82 with alanine (DRβ61/82), and a single mutant, DRβH81A (DRβ81), to investigate the role of conserved hydrogen bonds in the DM mechanism. Previous in vitro experiment showed that DRβ61/82 is relatively DM independent in peptide binding or dissociation. Expression of the DRβ mutants with DRα in MHC class II deficient T2 cells showed that there is no detectable CLIP on the surface of DRβ61/82 T2 cells, while CLIP is present on the surface of DR-WT or DRβ81 T2 cells. When DM was transduced into the DR-WT, DRβ81 or DRβ61/82 T2 cell line, the amount of CLIP on the surface of DM-DR-WT or DM-DRβ81 T2 cell line was decreased substantially, indicating functional interaction with DM. Our results suggest that the two sites, W61 and N82, but not H81, play an important role in DR/CLIP complex stability and DM-catalyzed peptide exchange

(Supported by NIH grant AI030554).
The TCP-1 Ring Complex (TRiC) binds antigenic peptides and facilitates their cross-presentation by APCs (93.5)
Authors: Vatner Ralph Eric , Srivastava Pramod K
Published: January 2023

The multimeric chaperonin TCP-1 Ring Complex (TRiC), an hsp60 homologue, was recently found to associate with the precursors of the ovalbumin-derived SIINFEKL epitope during processing and loading onto MHC I. The TRiC-peptide association is essential for loading of MHC I. Here, we have sought to characterize the peptide-binding properties of TRiC in vitro, and study the behavior of TRiC-peptide complexes in cross-presentation assays. TRiC is shown to bind a variety of peptides in vitro in much the same manner as previously described peptide chaperones of the hsp90 family. TRiC-peptide complexes are efficiently taken up by APCs and the peptides are processed and represented on MHC class I for recognition by specific CD8+ T cells. These results as well as the immunological properties of TRiC-peptide complexes formed in vivo shall be reported.
Developing a novel approach to follow the processing of viral antigens for MHC class I presentation. (93.7)
Authors: CARDINAUD Sylvain J , SHASTRI Nilabh
Published: January 2023

Cytotoxic T cells recognize their targets by the presence of unique peptides displayed by MHC class I molecules on the surface of antigen presenting cells. The peptides are generated by the antigen processing pathway and some proteins are known to inhibit this processing to escape the immune response. This is the case of EBNA1 (EBV nuclear antigen), a viral antigen detected in EBV-associated tumors. It is now established that the glycine-alanine repeat sequence present in EBNA1 is responsible for the absence of cell surface antigen presentation. Nevertheless how the antigen processing is modified remains unknown. Indeed, using conventional T cell assays, only the final peptide can be efficiently detected on the cell surface. We developed a novel assay, based on HPLC fractionation, allowing the detection of antigenic precursors in vivo. So far, we have successfully used this system to isolate, quantify and characterize antigenic intermediates derived from EBNA1 proteolysis. Using inhibitors as well as RNA interference, we can determine for the first time how EBNA1 interacts intracellularly with key steps of its antigen processing: degradation of newly synthezed proteins (DRiPs), proteasome proteolysis, TAP transport, and the role of chaperonin molecules.
Role of IL-18 on phosphorylation of STAT-6 in monocyte/macrophages (94.11)
Authors: Tamma Seetha M , Bhavsar Alap , Patel Tejal , Costa Kevin
Published: January 2023

Cytokine interactions in health & disease are very complex. There are conflicting reports on the cytokine IL-18 and its role in Th1 versus Th2 shift. The hypothesis that STAT-6 is expressed in monocyte/macrophages and that cytokine IL-18 has an effect on the phosphorylation of STAT-6 is addressed. Monocyte/macrophages were isolated by negative selection. The data indicates that the optimum concentration of IL-18 required for stimulation is 10μg/1x106 cells and 72 hour culture period is the optimum time for the induction of maximum levels of STAT-6 phosphorylation. We also observed total tyrosine phosphorylation of several intracellular proteins. Based on these studies, we propose that macrophages respond to IL-18 via STAT-6 phosphorylation and that IL-18 induces mitotic activity in monocyte/macrophages. Our data suggest that IL-18 actsin a more subtle manner as a costimulatory factor in the activation of STAT-6 in monocytes and provides evidence for a new immunoregulatoryeffect of the proinflammatory cytokine IL-18 on monocytes.

Grant from C.W.Post Campus Research Committee of Long Island University, New York supported this work.
Role of IL-18 on Th-2 shift and on phosphorylation of STAT-6 (94.12)
Authors: Tamma Seetha M , Bhavsar Alap
Published: January 2023

STAT-6 is a central mediator of IL-4-induced gene responses. It has been shown that STAT-6 is expressed in B cells and T cells upon stimulation with IL-4. Interleukin-12 (IL-12) and IL-18 are the key factors for the induction of Th1 Cells, early signals being involved in Th2 differentiation if any by IL-18 are less well characterized. We have investigatedthe mechanisms employed by IL-18 and IL-4 to control expression of STATs. Since we have shown in earlier studies that Th2 cytokines protect cells from apoptosis, we explored the possible role of the controversial cytokine IL-18 on Th2 shift and analyzed the effects of IL-18 on CD4+ T cells. Cells were cultured either with IL-18, IL-4, IL-18 + CD28 for different time points. The optimum time required for the phosphorylation of STAT-6 when stimulated with IL-18 was 72 hours. This effect of STAT-6 phosphorylation was blocked by addition of anti-IL-18 antibody in the culture. However, phosphorylation of STAT-1 and STAT-4 was seen by 24 hours. Furthermore, when cells were stimulated with anti-CD28 antibody plus IL-18, there is accelerated and enhanced phosphorylation of STAT-6. These studies may suggest that IL-18 serves as a negative feed back mechanism on inflammatory responses.

Grant from C.W.Post Campus Research Committee of Long Island University, New York supported this work.
Exploring the cross-talk between p38 MAPK and canonical Wnt signaling pathways (94.15)
Authors: Flick Lisa M , Florczyk Stephen J
Published: January 2023

The Wnt signaling pathway is active in many stages of embryonic development and tissue maintenance in adult organisms including bone homeostasis. Further understanding of the Wnt pathway could allow for improvements in therapeutics for metabolic bone disease and biomaterials used for bone replacement. To explore the potential crosstalk between the canonical Wnt and p38 mitogen-activated protein kinase (MAPK) pathways, MC3T3-E1 pre-osteoblasts were cultured and stimulated with LiCl in the presence or absence of the p38 MAPK inhibitor SB239063. Activation of the canonical Wnt signaling pathway was assessed by quantifying the lymphoid enhancer binding factor 1 (LEF1) binding activity in a non-radioactive colorimetric assay. The p38 MAPK pathway is typically considered independent of the Wnt signaling pathway and therefore, the SB239063 was hypothesized to have no effect on the LEF1 binding activity. Interestingly, cell extracts from cultures stimulated with LiCl and SB239063 showed a 37% increase in LEF1 binding activity compared to LiCl treated controls. The data suggests that p38 or a molecule downstream from p38 acts as an inhibitor on the canonical Wnt pathway. One candidate repressor protein is the transcriptional repressor high-mobility group box protein 1 (HBP-1) which has been shown to be a target of p38 MAPK and can also suppress LEF1 activity resulting from canonical Wnt signaling.
Cyclosporin A and FK506 Inhibit IL-12p40 Production through the Calmodulin/Calmodulin-dependent Protein Kinase-activated PI3K in LPS-Stimulated Human Monocytic Cells (94.19)
Authors: Ma Wei , Mishra Sasmita , Gee Katrina , Mishra Jyoti Prasad , Angel Jonathan , Kumar Ashok
Published: January 2023

Cyclosporine-A (CyA) and FK506 are immunosuppressive agents that are widely used to treat inflammatory and autoimmune disorders because of their ability to suppress the production of Th1 cytokines including IL-12. Both CyA and FK506 are potent inhibitors of calcineurin in the calcium signalling pathway. However, the role of calcium signalling in the regulation of human IL-12p40 production remains unknown. Interestingly, calcium and phosphoinositide 3-kinase (PI3K) signalling pathways have been shown to negatively regulate LPS-induced murine IL-12p40 production. Contrary to these observations, we show here that LPS-induced IL-12p40 production in human monocytic cells is positively regulated by calcium activated calmodulin- (CaM) and CaM-dependent protein kinase-II (CaMK-II)-activated PI3K. Furthermore, LPS induced the production of IL-12p40 by NFκB and AP-1 transcription factors through the activation of upstream CaM/CaMK-II. LPS-induced IL-12p40 production is known to be regulated by the c-Jun-N-terminal kinase (JNK) pathway. Importantly, both CyA and FK506 downregulated LPS-induced IL-12p40 transcription by inhibiting CaM/CaMK-II-activated PI3K and their downstream transcription factors NFκB and AP-1 independent of the JNK pathway.
C3a desArg/Acylation stimulating protein (ASP) and C5L2 correlation with expression of inflammatory factors and metabolic factors in human adipose tissue (94.25)
Authors: Cianflone Katherine , Lu HuiLing , MacLaren Robin , Smith Jessica
Published: January 2023

C5L2 is a recently identified receptor for C5a and C3a/C3adesArg (ASP). C5a/C5adesArg binds C5L2 with high affinity but no functional activation. By contrast, C5L2 is a functional receptor for ASP and in adipocytes plays a role in lipid metabolism. In this study, we used adipose tissue microarray analysis to explore the overlap between metabolism and immunology. Subcutaneous (SC AT) and omental (OM AT) adipose tissue were collected from 14 subjects (normal + obese) and divided into two groups based on plasma ASP level (assayed by ELISA): high ASP group (↑ASP) (ASP>40nM) and low ASP group (↓ASP ) (ASP<40 nM).

C5L2 (p=0.06), C5aR (p=0.03), IL-6 (p=0.01), and IL-6R (p=0.03) expression were increased significantly in ↑ ASP vs. ↓ASP in OM AT, with no change in SC AT. Plasma ASP correlated with lipid metabolic factors GPAT-1, FABP1, FABP5, FABP7 and adipophilin, and inflammatory factors CRP, C5aR and IL-6R in OM AT. ASP was mainly correlated with lipid metabolic factors, such as LPL, CD36, FABP3, and FABP6 in SC AT.

ASP-C5L2 may play a role in mediating metabolic- immune interactions in adipose tissue.

Funding: CIHR
Profiling Cytokine Levels Using a Multiplex Human Cytokine ELISA Array (94.26)
Authors: You Min , Lo Jing-Yi , Wang Yanqiu , Yu Xiang
Published: January 2023

The induction of multiple cytokines is a common phenomenon in many immunological responses; therefore, a method to quickly and simultaneously screen in vitro or in vivo samples for multiple cytokine levels is needed. We developed a “multiplex” sandwich ELISA Array on a 96-well plate allowing researchers to screen several human serum, plasma or cell culture supernatant samples for multiple cytokines in one experiment. The ELISA Array is easy to perform and only requires a standard ELISA plate reader. It can be also used in conjunction with corresponding single analyte ELISA kits to perform validation and absolute quantification. As an illustrative example, we monitored the time-dependent (0, 6, 24, and 48 hours) patterns of Th1/Th2 cytokine induction by human peripheral blood mononuclear cells (PBMC) in response to PMA (50 ng/ml) and ionomycin (1 μg/ml). Large amounts of IL-2, IFN-gamma and TNF-alpha are induced after the 6-h stimulation when only more moderate amounts of IL-4, IL-5, IL-10 and IL-13 are secreted. IFN-gamma levels are the highest among the eight cytokines tested after 6 hours, while IL-2 has the highest level after 48 hours. The amount of IL-12 p70 is not detectable even after 48 hours. The ELISA Array thus provides a powerful, easy-to-use solution for profiling the relative levels of several cytokines across multiple experimental conditions at the same time.
Sensitivity of TLR4- and 7-induced NFkappaB1 p105-ERK pathway to TNF-receptor-associated factor 6 (TRAF6) revealed by RNA interference in mouse macrophages (94.6)
Authors: Parameswaran Narayanan , Patial Sonika , Loniewski Katie J
Published: January 2023

TLR4- and -7-induced ERK activation in macrophages (MAC) involves the IκB kinase complex (IKK)-mediated phosphorylation of NFκB1 p105, a member of the IκB family. Major goal of this study was to examine the sensitivity of TLR4- and -7-induced IKK-p105-ERK pathway to TRAF6 in MAC. Using an RNAi approach we reduced the cellular levels of TRAF6 by ~80% in MAC. Although TLR4-stimulated ERK pathway, including p105 phosphorylation by IKK, and ERK phosphorylation, were not affected with TRAF6 knockdown (KD), TLR7-stimulated ERK pathway was completely blocked. In contrast, both TLR4- and 7- induced IKK-mediated IκBα phosphorylation was completely blocked by TRAF6 KD. These results demonstrate that TLR4 activates a TRAF6-sensitive and -insensitive IKK pathways in MAC. Surprisingly, compared to the role of TRAF6 in ERK activation, TLR4- and 7-induced JNK phosphorylation were completely blocked by TRAF6 KD, suggesting differential regulation of MAPK pathways by TRAF6. TLR4-induced TNFα mRNA in MAC is mediated in part via the ERK pathway. Consistent with signaling data, TLR4-induced TNFα mRNA was only partially blocked with TRAF6 KD. Taken together, these results indicate the differential roles of TRAF6 not only between TLRs but also demonstrate differential utilization of TRAF6 in IKK and ERK/JNK activation with in TLR4 signaling. This has important implications for drug development against TRAF6 in inflammatory diseases.
Type I Inteferons Differentially Stimulate Interferon-Gamma Secretion in Human Natural Killer Cells. (95.10)
Authors: Lavoie Thomas B. , Hung Finn , Crisafulli Sara , Reynolds Della , Scarzello Anthony , Moolchan Karlene , Skawinski Michael , Koltchev Doranelly , Pestka Sidney , Young Howard A , Clarke William A
Published: January 2023

The human lymphoma cell line NK-92 has many characteristics of human natural killer cells. We have examined NK-92 activation by a full panel of human alpha Interferons (IFNs) by measuring IFN gamma secretion. We confirmed that NK-92 cells secrete IFN gamma in response to IFN alpha 2 in a dose-dependent manner. The IFN gamma response is enhanced by addition of IL-2, IL-18 or PMA, as has been seen with human donor NK cells. The EC50 for IFN alpha 2 ranges between 700–3000 pg/ml. Inclusion of IL-18 exhibited little effect on IFN potency but significant enhancement of IFN efficacy. EC50 values differ by >1000 fold between the IFN subtypes. Alpha 1 demonstrated the weakest activity while alpha 10 and beta 1a were the most potent. The relative potency of the IFNs correlates well with ability to phosphorylate STAT1 and 2. When NK-92 potency is compared with antiviral activity on A549 cells and antiproliferative activity on OVCAR3 cells, there are subtle but significant differences in the rank orders of potency and potencies relative to IFN alpha 2. These studies will be extended to examination of donor NK cells, additional signal transduction pathways, and the secretion of other cyto/chemokines
Rapid Generation of a Functional NK Cell Compartment (95.12)
Authors: Prlic Martin , Bevan Michael J
Published: January 2023

Anti-IL-2 antibodies have been used with the premise that they block IL-2 signaling in vitro and in vivo, but recent findings demonstrate that anti-IL-2 antibodies form a complex with IL-2 in vivo that actually enhances IL-2 activity. NK cells are one cell type affected by these IL-2 complexes. We asked at what stage IL-2 complexes act on NK cells and whether they could be utilized to generate functional NK cells with potential therapeutic value.

We found that the administration of IL-2 complexes resulted in the rapid generation of NK cells in IL-15−/− animals. IL-15 is required for the development and survival of NK cells. Mature, transferred NK cells survived in IL-15−/− animals treated with IL-2 complexes, but proliferated only modestly. These data suggest that the IL-2 complex preferentially expands an immature subset of NK cells. To determine whether this expansion results in the generation of functional NK cells, we treated lethally irradiated, bone marrow reconstituted mice with IL-2 complexes and assessed the activity of NK cells by an in vivo killing assay. Bone marrow chimeras that were treated with IL-2 complexes displayed greatly enhanced NK lytic activity compared to control chimeras 4 days after initiation of treatment. We conclude that IL-2 complex has the potential to rapidly generate functional NK cells that may reduce the risk of viral infections and influence GVHD processes.
Characterization of foam cell formation in human macrophage subsets (95.17)
Authors: Montoya Dennis , Cruz Daniel , Modlin Robert
Published: January 2023

The hallmark of the human atherosclerotic plaque is the presence of lipid-laden macrophages, or foam cells. However, many macrophage subsets are found within atherosclerotic lesions and it is not well understood how monocytes differentiate into these subsets. We focused on characterizing macrophages derived in vitro from human peripheral blood monocytes treated with IL-15, IL-4 or IL-10. We show these macrophages to have differing phenotypes: CD209+CD64+, CD209+CD23+, or CD209+CD163+ for macrophages derived from IL-15, IL-4, or IL-10 respectively. To characterize the macrophage subsets ability to become foam cells we measured their uptake of fluorescently-labeled oxidized LDL (oxLDL). IL-10 derived macrophages had the greatest amount of oxLDL uptake. We then investigated the mechanism of uptake and found that fucoidan, a class-A scavenger receptor competitor, significantly inhibited uptake of oxLDL in IL-10 cells. On the other hand a blocking antibody against the class B scavenger receptor, CD36, did not inhibit uptake. Furthermore, we found IL-10 macrophages to express higher mRNA levels of the class A scavenger receptors macrophage receptor with collagenous structure (MARCO) and macrophage scavenger receptor (MSR) expression. These data suggests that IL-10 may promote foam cell formation and is dependent on class-A, but not class B scavenger receptors.
A developmental maturation of a dendritic cell subset controls the Th2 bias of neonatal immunity through IL-12 production and downregulation of IL-13Rα1 (95.2)
Authors: Lee Hyun-Hee , Hoeman Christine , Hardaway J.C. , Jain Renu , Divekar Rohit , Ellis Jason E. , Nast Danielle , Haymaker Cara L. , Guloglu F.Betul , Zaghouani Habib
Published: January 2023

Neonates are susceptible to microbial infections and allergic reactions due to lack of Th1 immunity and skewing of Th2 response. Analysis of primary response in neonates showed that Th1 and Th2 cells develop upon priming with antigen on the day of birth. However, Th1 cells display up-regulated expression of IL-13Rα1 that was found to associate with IL-4Rα and provide a receptor that can be used by both IL-4 and IL-13. Upon rechallenge with antigen, IL-4 produced by Th2 cells utilizes IL-13Rα1/ IL-4Rα receptor to drive apoptosis of Th1 cells hence bias of neonatal immunity towards Th2. Here, we demonstrate that up-regulation of IL-13Rα1 is due to inability of neonatal antigen presenting cells to produce IL-12. In fact, supply of exogenous IL-12 during priming with antigen minimizes IL-13Rα1 expression, leading to inhibition of apoptosis and restoration of Th1 secondary immunity. Interestingly, lack of IL-12 is due to delayed maturation of CD8α+CD4− subset of dendritic cells. However, by day 6 after birth CD8α+CD4− DCs accumulate and produce significant amounts of IL-12 that diminishes IL-13Rα1 expression, leading to inhibition of Th1 apoptosis and restoration of an adult-like Th1 immunity. Thus, developmental maturation of DC subsets is responsible for bias of neonatal immunity towards Th2 providing susceptibility to microbial infections and allergic reactions.

Research supported by grant R01 AI 48541 from NIH.
Cloning and functional analysis of an IL-13 splice variant lacking exon 2 which has inflammatory activity (95.22)
Authors: Yu Jiayi , Tse Harley , Lu Hong , Li Jinzhu , Shaw Michael
Published: January 2023

Interleukin 13 is a Th2 type (anti-inflammatory) cytokine produced by activated T cells. We have cloned a naturally occurring mRNA splice variant of IL-13 in which the second exon is omitted (termed δE2). We have developed RT-PCR assays for quantitation and discrimination of δE2/IL-13 gene expression, and cloned each of the molecules as HIS-tagged proteins for use in in vivo and in vitro studies.

We find that unlike IL-13, δE2 is expressed predominantly under Th1 conditions: δE2 is expressed by spleen cells stimulated with polyclonal activators and in the lymph nodes of mice immunized with CFA as adjuvant. Addition of exogenous δE2 to cultured antigen presenting cells induces phenotypic changes characteristic of an inflammatory (Th1) response, such as increased inflammatory cytokine secretion (TNF-α and IL-12). Addition of exogenous δE2 to antigen specific T cell cultures results in an increase in TNF-α producing cells as determined by ELIspot. Treatment of mice with exogenous δE2 at the time of immunization with CFA and antigen results in significant increases in the expression of inflammatory genes, including IL-12, CD28, and CD5. Lastly, we find that this splice variant is produced primarily by activated (CD19+, CD86+) B cells

Given the expression pattern of δE2 and its ability to modulate the expression of inflammatory cytokines, we hypothesis that δE2 is a unique cytokine involved in inflammatory immune responses
A critical role for IL-6 secretion by dendritic cells promoting Th2 and limiting Th1 response (95.24)
Authors: Krishnamoorthy Nandini , oriss Timothy , Paglia Melissa , Ray Anuradha , Ray Prabir
Published: January 2023

The mechanisms by which dendritic cells (DCs) induce differentiation of naïve CD4+ T cells along the Th2 lineage is not well understood given that DCs themselves do not produce IL-4. In the present study we undertook a microarray approach to identify genes involved in the induction of Th2 differentiation by DCs treated with a Th2-skewing adjuvant cholera toxin (CT). In the microarray analysis, murine bone marrow derived immature DCs were treated with CT. The analysis showed that CT upregulated IL-6 but not IL-12p70 both at the RNA and protein levels. DCs stimulated with CT and antigen preferentially upregulated GATA-3 expression in CD4+ T cells. This was associated with production of high levels of the Th2 cytokines IL-4 and IL-13. This suggested that low IL-12 and high IL-6 cytokine profile in the CT-treated DC cultures had a Th2-stimulatory capacity. To further investigate the role of IL-6 in Th2 response in vitro, IL-6 was neutralized with anti-IL-6 in DC –T cell co-cultures. This resulted in a decrease in Th2 and an increase in Th1 response as evident from the cytokine profile of IL-13 and IFN-. We also tested the ability of house dust mite (HDM) extract, an allergen known to induce asthma in humans, to induce IL-6 secretion from DCs. BMDC-stimulated with HDM also secreted high levels of IL-6, but low levels of IL-12p70. IL-6-deficient mice when administered HDM intranasally showed diminished Th2 response as evidenced by decreased IL-13 production. Our ongoing studies are focused on elucidating the role of specific differentially regulated genes in CT-induced IL-6 gene expression. Taken together, these studies will help identify pathways in DCs that participate in Th2 induction via IL-6 upregulation.

(Supported by NIH grants RO1 HL60207 to P.R. and RO1 HL77430 to A.R.)
Constitutive expression of IL-27 in neuro-retina negatively regulates THIL-17 cell development (95.25)
Authors: Yu Cheng-Rong , Amadi-Obi Ahjoko , Mahdi Rashid M. , Liu Xuebin , Lee Yunsang , Gery Igal , Egwuagu Charles
Published: January 2023

Ocular immune privilege is maintained by constitutive expression of inhibitory molecules such as TGF-b, FAS and FAS-ligand. Here, we describe a model of immune privilege for the eye based on IL-27-induced suppression of Th17 responses. We show that Th17 cells are elevated in retina of mice during experimental autoimmune uveoretinitis (EAU) and treatment with anti-IL17 Abs reduces EAU severity. It is of note that the increase and subsequent trafficking of Th17 cells into the retina during EAU is accompanied by corresponding increases in IL-27 and IFN? in the retina. We further show that IL-27 is constitutively expressed in human or mouse retina and that IFN? upregulates its expression in retinal cells. Of particular interest is inhibition of the proliferation of uveitogenic Th17 cells by IL-27, suggesting that IFN? may mitigate uveitis by antagonizing Th17 expansion through induction of IL-27 in the target tissue. In a co-culture system the expansion of Th17 cells (propagated under Th17 polarizing conditions) is inhibited by primary retina cells. However, addition of anti-IL-27 antibody into the culture overrides the inhibitory effects of the retinal cells on the expansion of Th17 cells. The data presented suggests that inhibitory effects on inflammatory cells by the retina are mediated in part by endogenously produced IL-27 and establishes mechanistic links between IL-27 expression and anti- Th17 effects in the eye.
Therapeutic efficacy of immunomodulatory polysaccharides may be explained by synergism with interferon-γ (95.29)
Authors: Sun-A Im , Young-Ran Lee , Young-Hee Lee , Kyungjae Kim , Chong-Kil Lee
Published: January 2023

Immunomodulatory polysaccharides have been implicated for the therapeutic efficacy of many plants or mushrooms. The absorption efficacy of immunomodulatory polysaccharides has been a matter of concern due to the high molecular weight nature. The present study shows that low in vivo concentration of polysaccharides may be enough to exert strong immunomodulatory activity in a microenvironment where a small amount of IFN-γ is present. Immunomodulatory polysaccharides, termed SHP, were isolated from Salicornia herbacea and examined their macrophage-activating activity alone and in combination with interferon (IFN)-γ. The combination of SHP and IFN-γ synergistically inhibited the growth of the mouse monocytic cell line, RAW 264.7, inducing further differentiation to strongly adherent macrophages. The differentiation-inducing activity of SHP alone and in combination with IFN-γ was confirmed by changes in the expression of differentiation antigens such as CD11b, CD18 and CD24. In addition, the combination of SHP and IFN-γ synergistically activated RAW cells to produce cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and nitric oxide (NO). The synergistic activity of SHP was more prominent when SHP concentration was low. Increased production of TNF-α, IL-1β and NO was correlated with an increased level of their respective transcripts. These results confirm that immunomodulatory polysaccharides exert synergistic monocyte-activating activity with small doses of IFN-γ.
IL-1β, not IL-18, is the primary IFNγ inducing factor released by activated monocytes (95.30)
Authors: Raices Raquel Marie , Sarkar Anasuya , Wewers Mark
Published: January 2023

IFNγ and caspase-1 have a reciprocal relationship. INFγ induces caspase-1 expression but caspase-1 is critical to the activation of the INFγ inducing factor IL-18. To investigate the INFγ inducing function of caspase-1 we characterized the INFγ inducing activity produced in response to caspase-1 activation (LPS + ATP) of human monocytes. Preliminary experiments demonstrate that IL-18 is not alone as an INFγ inducing product of caspase-1. Purified monocytes were stimulated with LPS/ATP and supernatants analyzed for IL-1β and IL-18 release, as well as for their ability to induce IFN-γ release by KG-1 cells. Monocytes released IL-1β and IL-18, as well as an IFN-γ inducing factor. The IFN-γ inducing activity was enhanced with ATP added to LPS and suppressed by a caspase-1 inhibitor, consistent with a caspase-1 regulated factor. However, the IFN-γ inducing activity was neutralized by IL-1β, not IL-18, blocking agents. In contrast, KG-1 cells released IFN-γ in response to rIL-18, not rIL-1β. When supernatants were fractionated by gel filtration, IL-18 eluted at a high molecular weight peak, indicating that it may be complexed to an inhibitory protein. IL-1β eluted at a single molecular weight peak corresponding to its mature form. However, the IL-1β and IL-18 peaks had minimal IFN-γ inducing activity. Taken together, these findings suggest that IL-1β is required, but not sufficient for the induction of IFNγ production.

Support: NIH R01 HL 746767 and HL 40871, F31 AI 061828-02
Epstein-Barr virus-induced gene 3 is a negative regulator of IL-17 and protective immunity against Listeria monocytogenes (95.4)
Authors: Yang Jianfei , Yang Min , Htut Tin Min , Hanidu Adedayo , Li Xiang , Sellati Sosemarie , Jiang Huiping , Zhang Shu , Li Hongxing , Ting Adrian T , Mayer Lloyd , Unkeless Jay C , Labadia Mark E , Hodge Martin , Li Jun , Xiong Huabao
Published: January 2023

The product of the Epstein-Barr virus-induced gene 3 (EBI3) associates with p28, an IL-12p35-related protein, to form IL-27. EBI3 also associates with IL-12p35 to form a distinct heterodimer. The function of EBI3 in innate as well as adaptive immunity to intracellular bacteria has not been established. Here we demonstrate that EBI3 deficient mice exhibit a reduced bacterial load following an acute challenge with Listeria monocytogenes or a re-challenge of animals previously immunized with L. monocytogenes, suggesting EBI3 plays a negative role in both innate and adaptive immunity. The enhanced protective immunity in EBI3-deficient mice is not due to modulation of the Th1 immune response since the production of IFNγ and TNFα was not significantly altered. Importantly, spleen cells from EBI3-deficient mice immunized with L. monocytogenes produced significantly higher levels of IL-17 than wild type cells. Furthermore splenocytes as well as Th17 cells from EBI3-deficient mice secreted increased levels of IL-17. Elevated numbers of IL-17-producing cells were observed when EBI3 deficient CD4+ T cells were cultured under conditions to induce Th17 cell differentiation. Taken together, these data provide direct evidence that EBI3 is an important negative regulator of protective immunity against L. monocytogenes as well as IL-17 expression.