bluuebunny/crossref_metadata_2025 huggingface dataset:
FCGR genetics in HIV disease (88.21)
Authors: Poonia Bhawna , Pauza David
Published: January 2023

Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism for HIV-specific antibodies to target infected cells for destruction by cytotoxic effectors. The Fc portion of IgG antibodies binds cytotoxic effector cells through a family of Fc receptors that include FCGR3A (CD16). Antibody binding to Fc receptor also delivers an activating signal to the effector cells. Thus there is a balance between effector cell stimulation and target cell killing during ADCC. Single nucleotide polymorphisms (SNP) in the Fc receptor gene FCGR3A alter the amino acid sequence and encode high or low functioning proteins. We predicted that HIV elite controller groups would have increased frequencies of the high functioning allele compared to HIV progressors or uninfected controls. Genomic DNA was analyzed from individuals with no HIV infection, Elite controllers and HIV progressors on HAART for polymorphism rs396991, that results in a T (F amino acid) or a G (V amino acid) allele at the SNP position. PCR was used to amplify a region of FCGR3A and an analysis of rs396991 was done using the ABISnapshot multiplex kit. An allele frequency of T/G 0.82/0.18 for normal donors, T/G 0.75/0.25 for elite controllers and T/G 0.58/0.42 for HIV progressors was found. These results of higher frequencies of high functioning alleles in HIV progressors suggest that this allele may be responsible for chronic lymphocyte activation that is a feature of progressing HIV disease.
Developmental Regulation of Immunoglobulin CDR-H3 Content through Control of DH and JH Usage in Mice (88.4)
Authors: Szymanska Ewa , Schelonka Robert , Vale Andre , Zhuang Yingxin , Gartland G. , Schroeder, Jr. Harry
Published: January 2023

The ability to generate antibodies to specific antigens develops in a controlled, programmed fashion during ontogeny. This process occurs in parallel with the regulated development of antibody repertoire diversity. Compared to adult, the human fetal antibody repertoire is enriched for use of most DH-proximal VH gene segment. The CDR-H3 repertoire is depleted of N nucleotides and enriched for DQ52, the most JH-proximal DH; and for DH-proximal JH2 and 3. The murine fetal antibody repertoire is also enriched for the use of the most DH proximal VH gene segment and depleted of N nucleotides. However, the extent to which DH and JH usage is controlled is unclear. We used surface expression of CD43, BP-1, CD19, IgM and IgD to sort B lineage cells from BALB/cJ perinatal liver, then used RT-PCR to amplify VH7183-containing VDJCμ transcripts. We observed increased use of JH-proximal, DHQ52 and DH-proximal JH2 at all stages of development. Patterns of VH7183-DH-JH usage and CDR-H3 content during B cell development in 18 day fetal liver and neonatal liver were compared to those of adult bone marrow. When evaluated by length and hydrophobicity, evidence of CDR-H3 repertoire selection was most evident in the transition from Hardy Fraction C to D. Our results indicate that regulation of the antibody repertoire ontogeny in mouse shares more similarities with human than previously appreciated. Supported by NIH AI48115.
Neutrophil gelatinase-associated lipocalin (NGAL): Endogenous activators of the immune system (89.15)
Authors: Tewary Poonam , Redmond Chris , Clifton Mathew , Yang De , Oppenheim Joost
Published: January 2023

Neutrophil gelatinase-associated lipocalin (NGAL), also known as Lipocalin 2 (Lcn2), is a member of the extremely diverse family of molecules called lipocalins. Recent studies have shown that NGAL is secreted by multiple cell types including neutrophils, and has a wide variety of functions from anti-apoptotic signaling to kidney development. We have been investigating the properties of four different forms of NGAL to determine whether they exhibit the characteristics of an alarmin. Alarmins are endogenous antimicrobial peptides or proteins which are released in response to tissue injury or infection and have the additional capacity to recruit and activate antigen presenting cells (APCs) and enhance antigen specific immune responses in vivo. Our results show that different variants of NGAL are potent stimulants of antigen presenting cells. Treatment of monocyte-derived iDCs with NGAL induced their maturation as evidenced by upregulated surface markers (CD80, CD83, CD86, and MHC class II), enhanced production of cytokines (IL-6, CXCL8, IL-12p70, and TNF alpha), and acquisition of the capacity to stimulate allogeneic T cell proliferation. Thus, NGAL can rapidly mobilize and activate pro-inflammatory innate immune responses and by generating mature DCs can also induce adaptive immune responses. If these NGAL molecules are shown to induce a significant immune response, they may prove to be useful as non-toxic vaccine adjuvants for treatment of various types of tumors.
Recognition of bacterial formyl peptides by FPR2 (89.21)
Authors: Southgate Erica , Nanamori Masakatsu , Ye Richard
Published: January 2023

Formyl peptide receptor 2 (FPR2) is a homologue to FPR1, which is known for its high-affinity binding to the E. coli-derived chemotactic peptide fMet-Leu-Phe (fMLF). Unlike FPR1, FPR2 is a low affinity receptor for fMLF, raising questions for its role in host defense. We found that FPR2 is able to respond to a variety of formylated peptides with activation of cellular functions. A pentapeptide fMet-Ile-Val-Ile-Leu from L. monocytogenes and a tetrapeptide fMet-Ile-Phe-Leu from S. aureus were ~ 100 fold more potent than fMLF in activating FPR2 suggesting that bacterial formyl peptides can be high affinity ligands for FPR2. The presence of the formyl group at the N-terminus and C-terminal interaction are both important for full activation of FPR2. In comparison, FPR1 activation through formyl peptides only requires interaction with the N-terminal formyl group. We also investigated the role of charged residues in ligand interactions of both FPR1 and FPR2 receptors. Mutation of residues 84, 85 and 284 in each receptor were not competent to effect formyl peptide activation for each receptor, however; the specificity by FPR2 agonists was directly affected in the mutational study, indicating involvement of these residues in structure conformation and downstream signaling. These data demonstrate a divergent role for FPR2 in recognizing bacterial and synthetic formyl peptides, suggesting a potential function of this receptor in host defense as well as homeostatic regulation.
Effect of lipoteichoic acid on B cells in female C57BL/6 mice (89.25)
Authors: Dasgupta Subhajit , Molano Ivan , Eudaly Jackie , Baker DeAnna , Cunningham Mellissa , Gilkeson Gary
Published: January 2023

Activation of B cells is hall mark of protective immune responses against pathogens. A wide range of antigen(s) and differences in mode of recognition of these antigens confer diversified adaptive immune responses in infection, autoimmune disease like lupus or in allergy. Our initial observations via flow cytometry indicated a shift in CD19+ B cells in spleens of female C57BL/6 mice injected TLR2 ligand LTA via the intraperitoneal route as compared to control mice received sterile PBS. The spleen cells of LTA injected mice demonstrated a population of CD19+ B cells which also found to express TLR2 (0.2% CD19+TLR2+) as compared with control (0.1% CD19+ TLR2+ cells). We also detected a small population of CD19+CD25+ cells in spleens of LTA injected mice as compared with control. There was considerable alteration in CD19+CD40+ cell population in LTA injected mice as compared with control. Our observations demonstrated a shift in CD19+CD86+ activated B cell populations in LTA injected mice. Selective estrogen receptor alpha antagonist MPP was found to alter the B cell activation markers in LTA mice indicating possible role of estrogen receptor alpha in B cell activation. The observations indicate salient role of TLR2 ligands on B cell activation signals that may in turn, responsible for regulation of adaptive immune responses.
CD34+CD31+ immature myeloid cells (IMC) as an inhibitor cell on the production of antimicrobial peptide: II. Glycyrrhizin inhibits IMC suppressor cell activities (89.27)
Authors: Yoshida Shohei , Asai Akira , Inatsu Akihito , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

In the accompanying paper I, CD34+CD31+ IMC are shown to be inhibitory on HBD-1 production by normal human epidermal keratinocytes (NHEK). CCL2 and IL-10 released from CD34+CD31+ IMC are identified as effectors in this suppressor cell activity. In this study, the suppressor cell activity of CD34+CD31+ IMC was shown to be controlled by glycyrrhizin (GL), an active component of licorice root. CD34+CD31+ IMC were isolated from peripheral blood of exercised donors, as described in the accompanying paper I. These IMC were transwell-cultured with NHEK for 48 hrs in the presence or absence of GL (Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan), and cells in the bottom chamber (NHEK) were assayed for HBD-1 mRNA expression by RT-PCR. Also, the production of CCL2 and IL-10 by CD34+CD31+ IMC (1 x 106 cells/ml) treated with GL was measured by ELISA. In the results, NHEK transwell-cultured with CD34+CD31+ IMC did not express HBD-1 mRNA, while these transwell-cultures were performed with GL (10-100 μg/ml), HBD-1 mRNA was expressed by NHEK. The production of CCL2 and IL-10 was greatly reduced in CD34+CD31+ IMC cultures added with GL. These results indicate that the suppressor cell activity of CD34+CD31+ IMC on HBD-1 production by NHEK is controlled by GL. This study was supported by SHC #8610.
Human L-ficolin can bind and defence against multiple pathogens (89.29)
Authors: Zhang Xiao-Lian , A.M. Ali Mohammed , Luo Fengling , Wang Qilong , He Ruifen , Shen Xihui , Yang Rongge
Published: January 2023

Human L-ficolin is a recently identified lectin complement pathway activator present in normal plasma and inplicated to be associated with infectious diseases, but little is known about the roles of ficolins in the infections of human important pathogens. In this study, we firstly demonstrate that human L-ficolin can bind and reduce infectivity of Human Immunodeficiency Virus (HIV), Hepatitis C virus (HCV), Mycobacteria tuberculosis (M.tb) and Salmonella typhimurium C5 in vitro or in vivo. Further investigations demonstrated C-terminal of L-ficolin specifically bound to these microbial carbohydrates, thereby activate lectin complement pathway and complement mediated cytolytic activity. Our findings support the development of L-ficolin as a novel innate immune molecule immunotherapy against the infections of these important pathogens.
CD34+CD31+ immature myeloid cells (IMC) as an inhibitor cell on the production of antimicrobial peptide: I. Inhibitory activities of CD34+CD31+ IMC on the antimicrobial peptide production by keratinocytes (89.31)
Authors: Jones Vickie , Yoshida Shohei , Kobayashi Makiko , Herndon David , Suzuki Fujio
Published: January 2023

CD34+CD31+ IMC, isolated from peripheral blood of exercised donors, are shown to be inhibitor cells on HBD-1 production by normal human epidermal keratinocytes (NHEK). Lineage cell-depleted PBMC were isolated from peripheral blood of healthy donors 24 hrs after 1-hr of treadmill exercise using a lineage cell depletion kit, then CD34+CD31+ IMC were sorted from these cells by flow cytometry. IMC were transwell-cultured with NHEK for 48 hrs, and NHEK harvested were analyzed for HBD-1 mRNA expression by RT-PCR. Also, conditioned media harvested 48 hrs after the cultivation of CD34+CD31+ IMC (IMC-culture media) were assayed for their inhibitory activities on HBD-1 production by NHEK. In the results, HBD-1 mRNA expression by NHEK was completely inhibited after transwell-cultivation with CD34+CD31+ IMC. HBD-1 was not detected in NHEK cultures supplemented with 20 to 40% of IMC-culture media. IL-10 and CCL2 were detected in the IMC-culture media. When NHEK were cultured with the IMC-culture media previously treated with a mixture of anti-CCL2 and anti-IL-10 mAbs, HBD-1 was produced in their culture fluids. These results indicate that CD34+CD31+ IMC transiently appearing in peripheral blood of exercised donors are inhibitory on HBD-1 production by NHEK. CCL2 and IL-10 produced by these cells are required on the CD34+CD31+ IMC-mediated inhibition of HBD-1 production by NHEK. This study was supported by SHC #8610.
Natural polysaccharides and nucleic acid based TLR ligand nanocomplexes improves immune response (89.41)
Authors: Tincer Gizem , Yerlikaya Seda , Gürsel Ihsan
Published: January 2023

Toll-like receptors (TLR) induce NF-κB dependent or independent cytokine/chemokine secretion and contribute to the establishment of innate and adaptive immune response. When nucleic acid ligands were given in vivo they were attacked by endonucleases and have low uptake efficiency into immune cells due to premature adsorption to serum proteins. Polysaccharides (PS) from edible mushrooms were characterized as immunostimulatory amphiphilic macromolecules. Due to their β-glucan moieties, we have established that these PS`s are recognized and induce TLR2 (but not via NOD-1 or NOD2) mediated activation (as evidenced by NO, TNFα IL6 responses) and can be naturally targeted through this extracellular receptor. Next, we complexed PS with pI:C, R848 and CpG ODN, a TLR3,7/8, and 9 ligands. Different ratio of these nanocomplexes displayed a strong synergism as evidenced by a Th1-biased cytokine secretion and NO production compared to their alone counterparts. This study showed that natural PS is an immunostimulatory compound by itself, and furthermore, it could be used to effectively complex and deliver nucleic acid based TLR ligands to relevant immune cells. The targeted delivery of the generated nanocomplexes led to a more pronounced immune activation that is not achievable either of the stimulants when given alone.
Identification of novel ligand(s) for the NK cell receptor, KIR2DL4 (89.44)
Authors: Brusilovsky Michael , Porgador Angel , Campbell Kerry
Published: January 2023

The ligands for many NK cell activating receptors remain unknown, and identification of these ligands has become a major goal for understanding the molecular basis of NK cell activation. The killer Ig-like receptor (KIR) 2DL4 is the only NK cell receptor that triggers cytokine production (IFNγ, chemokines, pro-angiogenic factors), but not cytotoxicity in resting NK cells. Several early studies reported that 2DL4 recognizes the non-classical MHC-I molecule - HLA-G. We have recently found that a soluble form of 2DL4 binds to various tumor cell lines of epithelial or fibroblast origin that lack HLA-G expression, but not to transformed hematopoietic or HLA-G positive lines. Our results demonstrate that soluble forms of HLA-G and HLA-A are both recognized by the soluble 2DL4, but binding of the 2DL4 to cell surface forms of HLA-G or HLA-A was not detected. Our studies further suggest that soluble 2DL4 or MHC-I molecules may exhibit interactions that are not characteristic of cell surface proteins and that 2DL4 recognition of the tumor cell lines is independent of surface expression of MHC-I or MHC-I like molecules.
Distinct Roles of Rab27a in Lytic Granule Movement at the Plasma Membrane and in the Cytosol (89.47)
Authors: Liu Dongfang , Meckel Tobias , Long Eric
Published: January 2023

The small GTPase Rab27a is essential for degranulation by cytotoxic T cells and natural killer (NK) cells. To test its role in granule movement we visualized lytic granules in live, unstimulated NK cells by high-speed 3-dimensional (3D) spinning disc confocal and 2-dimensional (2D) total internal reflection fluorescence (TIRF) microscopy. Movement was tracked and quantified by automated image analysis of thousands of granules. Pharmacological inhibitors showed that long-range, directed 3D movement required microtubules (MT) but not actin, whereas 2D movement under the plasma membrane required actin dynamics. The role of Rab27a was examined by shRNA-mediated knockdown in a human NK cell line and by using NK cells from Rab27a-mutant Ashen mice. Fewer lytic granules reached the plasma membrane in the absence of Rab27a. Rab27a promoted actin-dependent, directed movement under the plasma membrane. In contrast, Rab27a reduced tracking along MT and diffusion of lytic granules in the cytosol. Therefore, Rab27a promotes delivery of lytic granules to, and movement at the plasma membrane, and has a distinct role in the cytosol, where it serves as tether to constrain lytic granule movement.
Vitamin C attenuates ERK signaling induced by LL-37 in human dermal fibroblasts (89.5)
Authors: Kim Hee Jung , Choi Jung Min , Cho Baik Kee , Cho Dae Ho , Park Hyun Jeong
Published: January 2023

Vitamin C (ascorbic acid), a water-soluble micronutrient present in fruits and vegetables, is important for multiple biological functions. Vitamin C is used as an anti-aging agent because of its collagen enhancing effects. The precise cellular signaling mechanism of vitamin C is not well known. Here we investigate the profibrotic mechanism of vitamin C against LL-37. Antimicrobial peptide LL-37 decreases collagen expression at mRNA and protein levels in human dermal fibroblasts (HDFs). The ability of LL-37 to inhibit collagen expression is dependent on phosphorylation of extracellular signal-regulated kinase (ERK). HDFs were treated with vitamin C before 2 hours of LL-37 treatment. Vitamin C, at 0.5mM concentration, enhances collagen mRNA expression and total soluble collagen production inhibited by LL-37. H2O2 decreased the expression of COL1A1, COL1A2, and COL3A1 mRNA, as was collagen production. We found that collagen expression and production reduced by LL-37 were increased by N-acetyl-L-cysteine (NAC). These results indicate that ROI levels are involved in collagen expression. Furthermore, vitamin C turned off phosphorylation of ERK that was induced by LL-37. Ets-1 transcriptional factor, which is involved in the regulation of collagen expression by LL-37, was also inhibited by vitamin C. This study shows that vitamin C enhances collagen production by inhibiting the ERK pathway induced by LL-37.
MHC class I polymorphism defines NK cell-mediated resistance to murine cytomegalovirus (89.50)
Authors: Brown Michael , Xie Xuefang , Stadnisky Michael , Bullock Timothy , Coats Ebony
Published: January 2023

NK cells are vital to innate antiviral defenses; NK deficiency corresponds to susceptibility to infection, inflammation, tissue damage, and mortality. In MA/My mice, NK-mediated resistance to murine (M)CMV infection requires H-2k, but a critical gene has not been identified. We mapped the resistance effect to the MHC class I (MHC-I) D locus. A cloned Dk transgene was microinjected into MCMV-susceptible (MA/My.L-H2b x C57L)F1 embryos. MHC-I Dk founders were backcrossed to MA/My.L-H2b or C57L mice. We found that transgenic MHC-I Dk expression conferred MCMV resistance. Ly49G+ NK cells were required since their removal before infection abrogated resistance. Using bone marrow chimeras, a requirement for MHC-I Dk expression in hematopoietic or non-hematopoietic cells was found essential to full MCMV resistance. MHC-I Dk resistance through Ly49G+ NK cells further corresponded to splenic CD11c+ dendritic cell stability and acquisition of virus-specific CD8+ T cells after infection. We conclude that MHC-I Dk is essential to MCMV resistance and define a role for MHC-I Dk on both hematopoietic and non-hematopoietic cells in enhancing NK virus control and virus-specific immunity.
Enhancement of antiviral immunity by a peptide antagonist of SOCS (89.9)
Authors: Ahmed Chulbul , Dabelic Rea , Johnson Howard
Published: January 2023

Suppressors of cytokine signaling (SOCS) are negative regulators of both innate and adaptive immunity via inhibition of signaling by cytokines such as interferons (IFNs). We have developed a small peptide antagonist of SOCS-1 that corresponds to the activation loop of the Janus kinase JAK2. SOCS-1 inhibits both type I and type II IFN activities by binding to the kinase activation loop via the kinase inhibitory region (KIR) of the SOCS. The antagonist, pJAK2(1001-1013), inhibited the replication of vaccinia virus and EMC virus in cell culture, suggesting that it possesses broad antiviral activity. In addition, pJAK2(1001-1013) protected mice against lethal vaccinia and EMC virus infection. pJAK2(1001-1013) increased the intracellular level of the constitutive IFNβ, which may play a role in the antagonist antiviral effect at the cellular level. pJAK2(1001-1013) also synergizes with IFNs as per IFN gamma mimetic to exert a multiplicative antiviral effect at the level of transcription, the cell, and protection of mice against lethal viral infection. pJAK2(1001-1013) binds to the KIR of both SOCS-1 and SOCS-3 and blocks their inhibitory effects on the GAS promoter. In addition to a direct antiviral effect and synergism with IFN, the SOCS antagonist also exhibits adjuvant effects on the humoral and cellular arms as well as an enhancement of polyI:C activation of TLR3. The SOCS antagonist thus presents a novel and effective approach to enhancement of host defense against viruses.
Differentiation of Peyer’s patch M cells does not require signals from B cells: evidence from a mouse model of acute antibody-mediated depletion of B cells (90.1)
Authors: Knoop Kathryn , Butler Betsy , Kumar Nachiket , Williams Ifor
Published: January 2023

Microfold (M) cells are antigen-sampling cells in the epithelium covering Peyer’s patches (PP) and other mucosal lymphoid tissues. The previously demonstrated reduction in the number of PP M cells in B cell deficient mice (e.g. JH null and muMT models) led to a model proposing that B cells provide critical signals supporting M cell differentiation, but the specific B cell-derived factors involved in this process remain to be identified. We used an alternate model of acute antibody-mediated B cell depletion in human CD20 transgenic mice to reassess the role of B cells in PP M cell differentiation. Transgenic mice and nontransgenic littermates were treated with 1 mg of rituximab (anti-human CD20) every 4 days for a total of 4 doses. The status of PP M cells was analyzed 16 days after the start of treatment. Rituximab treatment led to loss of ≥94% of B cells from the spleen and PP of human CD20 transgenic mice. Whole mount staining of PP with the UEA-I lectin revealed that the number and pattern of UEA-I+ M cells per follicle was essentially unchanged in mice depleted of B cells (86 M cells/follicle in CD20 transgenic mice vs. 88 M cells/follicle in nontransgenic mice). We conclude that the acute depletion of B cells from PP of adult mice does not impair the normal differentiation of PP M cells. The modest decrease in the density of PP M cells observed in B cell deficient mice may be a consequence of abnormal embryonic PP development rather than lack of a B cell-derived factor.
Roquin-mediated regulation of intestinal inflammation (90.13)
Authors: Schaefer Jeremy , Montufar-Solis Dina , Klein John
Published: January 2023

Peripheral CD4+ (Th17) and CD8+ (Tc17) T cells that secrete the interleukin 17 proinflammatory cytokine play a central role in the host immune response during acute infection; however, aberrant expression of IL-17 has been implicated in a number of autoimmune and chronic inflammatory conditions. The RING finger E3 ligase, Roquin, promotes the degradation of gene transcripts in a microRNA-mediated decay process. Roquin-deficient mice develop chronic inflammation due to excessive ICOS expression. However, the role of Roquin in controlling other T cell activation signals and proinflammatory molecules has yet to be examined. Colonic intraepithelial lymphocytes (cIELs) from IL-10-/- mice, a model of human inflammatory bowel disease, and the EL4 T cell lymphoma line, were used to evaluate the contribution of Roquin in curtailing IL-17-mediated inflammation. In vitro-cultured CD4+ and CD8+ cIELs from IL-10-/- mice with severe inflammation were high producers of IL-17; conversely, Roquin expression was decreased. In IL-10-/- cIELs cultured with recombinant mouse IL-10, IL-17 expression was significantly reduced and Roquin expression was increased. Thus, during IL-10-induced immune regulation, Roquin and IL-17 are differentially modulated with Roquin expression being amplified while IL-17 is inhibited. These findings suggest a link between IL-10 and Roquin in the regulation of IL-17, and they may have significance for developing immunotherapies to control intestinal inflammation.
Dissecting the effects of microflora and antibiotics on immune and metabolic compartments in the gut (90.7)
Authors: Shulzhenko Natalia , Morgun Andrey , Dzutsev Amiran , Matzinger Polly
Published: January 2023

Because germ-free animals are difficult to generate and maintain, antibiotic-treated animals are often substituted in studies designed to determine the role of the normal microflora in health and disease. To determine the extent to which antibiotic (AB) treatment mimics the germ-free condition, and to ascertain the direct effects of AB on the host, we orally treated conventional and germ-free (GF) mice with a combination of four antibiotics for one month and performed microarray analysis of terminal ileum. We observed a decrease in the expression of T lymphocyte genes in both AB-treated and GF mice whereas B-lymphocyte transcripts had much higher expression in AB-treated and conventional than in GF mice. This result was confirmed at the cell number level in the tissue. Among the direct effects of antibiotics independent on flora, we found a decrease in the expression of genes related to mitochondrial functions and cell division. Concordantly, we detected lower amounts of mitochondria and a higher rate of cell death in the gut epithelial cells of AB-treated than of control mice. In conclusion, we found that antibiotics treatment only partially recapitulates the GF condition for mucosal immune system being able to mimic the germ-free status of mucosal T lymphocytes but not of B lymphocytes. In addition, antibiotics have some major direct effects decreasing energy metabolism in the host tissue independently on flora elimination.
The role of MyD88 in the synergy between FcϵR1, TLR4 and ST2 in mast cells. (91.5)
Authors: Collington Sarah , Zaidi Asifa , LaRosa David , Ali Hydar
Published: January 2023

Mast cells are well established as important effector cells in allergic reactions. Cross-linking of the high affinity IgE receptor (FcϵRI) on mast cells by allergen causes degranulation leading to the release and generation of a variety of inflammatory mediators. IL-33 and lipopolysaccharide (LPS) are members of the IL-1 family that use MyD88 as an adaptor molecule for signalling. Alone, IL-33 and LPS are unable to cause mast cell degranulation but they can induce cytokine generation. In addition, IL-33 and LPS synergise with antigen/IgE for cytokine production in mast cells. IL-33 and LPS-induced cytokine production was abolished in MyD88-deficient (MyD88-/-) bone marrow-derived mast cells (BMMCs). Surprisingly, antigen/IgE-mediated cytokine production was also substantially reduced in MyD88-/- BMMCs. However, MyD88 had no effect on FcϵRI expression, calcium mobilisation or degranulation. The synergy between IL-33 and antigen/IgE in IL-13 production by BMMCs was shown to be MyD88-dependent. Similarly, MyD88 was required for cytokine production in LPS and antigen/IgE stimulated BMMCs. The synergy between LPS and antigen/IgE for cytokine generation also required p38 MAP kinase activation. These findings suggest that MyD88 signalling in mast cells could provide a novel target to modulate allergic diseases.
Plasmacytoid dendritic cells kill virus-specific CD8 T cells during lethal dose influenza virus infections (92.1)
Authors: Legge Kevin , Langlois Ryan
Published: January 2023

Our previous studies have shown that the reduction in CD8 T cell immunity observed during high-dose influenza virus infection (IAV) is mediated via lymph node (LN) dendritic cells (DC) that express FasL and drive FasL:Fas (DC:T) induced apoptosis. However, the specific DC subset(s) within the LN and the additional factors required for DC mediated elimination of IAV-specific CD8 T cells remain unknown. Here, we demonstrate that plasmacytoid DC (pDC) accumulate to greater numbers within the LN of lethal, compared sublethal, dose IAV infected mice. Further our findings show that lethal, but not sublethal dose, pDC drive elimination Fas+ CD8 T cells and that this elimination occurs in the absence of protective cognate IAV-peptide-MHCI:TCR interactions. Together our results suggest that pDC play a heretofore unknown role in limiting the CD8 T cell response therein increasing mortality during high dose IAV infections.
Botanical medicines and their effects on the replication of influenza A virus and the influenza A virus-induced production of inflammatory mediators (92.14)
Authors: Cecil Chad , Oyegunwa Akinbolade , Kandhi Vamsikrishna , Eads Dawn , Hansen Rebecca , Sikes Michael , Scholle Frank , Petty Ian , Davis Jeanine , Cech Nadja , Laster Scott
Published: January 2023

Experiments are underway to evaluate plant extracts, and their constituents, for the ability to inhibit influenza A virus replication and the influenza A virus-dependent production of cytokines, chemokines and inflammatory lipids. Terameprocol (TMP), or tetra-O-methyl-nordihydroguaiaretic acid, produced by the creosote bush (Larrea tridentata), did not inhibit the growth of several strains of influenza A virus. In contrast, TMP strongly inhibited production of several inflammatory mediators including TNF-α, MCP-1, and PGE2 from infected cells. Our experiments show that TMP’s effects on cytokine and chemokine production stem from effects on the ability of NF-κB to bind DNA while effects on prostaglandin production stem from a direct inhibitory effect on the activity of COX-2. We found that extracts of Echinacea purpurea are also able to block production of TNF-α and PGE2 from infected RAW 264.7 cells, although wide variation in effectiveness is seen among plants from different locations. These effects were also seen with purified alkylamides found in Echinacea extracts, although different alkylamides displayed different activities. As with TMP, Echinacea extracts did not display an inhibitory effect on the growth of influenza A virus. Finally, extracts of Goldenseal (Hydrastis canadensis), and its primary alkaloid berberine, inhibited both influenza A virus replication and production of inflammatory mediators. Studies on the mechanisms underlying these effects are underway.
CD4 T-cells mediate enhanced immunopathology during influenza infection (92.22)
Authors: Verhoeven David , Farber Donna
Published: January 2023

Influenza infection generates robust CD4 T-cell memory that cross-reacts conserved epitopes between viral strains. We previously determined that influenza specific-memory CD4 T-cells have protective properties but enhance earlier morbidity. In this study, we found that Rag2-/- mice with memory CD4 T-cell specific for influenza A/PR/34/8 (PR8) and challenged with PR8 exhibited increased weight loss, had lower pO2 levels associated with greater lung epithelial damage, and died sooner than mice without flu memory or with naïve CD4 T-cells. To further define the immunopathologic nature of memory CD4 T-cell, we utilized adoptive transfers of PR8-specific memory into congenic hosts and challenged these mice with the heterosubtypic strain of influenza HK-X31. We determined that mice with influenza specific-memory CD4 T-cells had greater initial influenza-associated morbidity with enhanced epithelial damage in lungs despite lower viral titers as compared to primary infection. CD4 T-cell memory was associated with increased proliferation and recruitment of X31-specific effector CD4 T-cells during heterosubtypic challenge and enhanced flu specific interferon-γ and IL-17 responses. Memory CD4 T-cells tended to cluster together in the lungs during a secondary challenge possibly concentrating antiviral cytokines and further damaging the epithelium. This study clearly establishes the mechanisms driving a dual protective/immunopathologic role for CD4+ T-cell memory during infection.
Development and Maintenance of Antiviral Antibody Secreting Cells in Response to Influenza Virus Infection (92.3)
Authors: Wolf Amaya , Mozdzanowska Krystyna , Metzgar Michele , Quinn William , Bram Richard , Cancro Michael , Erikson Jan
Published: January 2023

Respiratory infection with influenza virus (flu) induces long-lasting protective humoral immunity. Although flu-specific antibody secreting cells (ASCs) increase post infection (p.i.) and are present in various organs, including the lung, it is unclear what signals promote their maintenance and/or survival. We determined that flu-specific ASCs were maintained in the lung up to half a year p.i.. These cells were not proliferating as they failed to incorporate BrDU. With the hypothesis that BLyS-family members may be important for ASC maintenance, we investigated the B cell response to flu in TACI-/- mice. TACI has been implicated in the T-independent type II response, but its role in anti-viral immunity is unknown. WT and TACI-/- mice exhibited a similar anti-flu B cell response at day 8 p.i., however, by day 33p.i., TACI-/- mice had significantly lower anti-flu antibody levels in the serum and locally in the lung. TACI deficiency did not affect flu-specific ASC numbers in spleen and lung-draining mediastinal lymph nodes. In contrast, it resulted in drastically reduced ASCs in lung and bone marrow. The results of our study will be discussed in the context of protection and possible implications for vaccine development.
Inhibiting the Inflammatory Response Following Influenza Virus Infection utilizing Sphingosine-1-phosphate receptor 1 signaling (92.4)
Authors: Teijaro John , Walsh Kevin , Rosen Hugh , Oldstone Michael
Published: January 2023

Emerging pathogenic strains of influenza A virus have caused significant morbidity and mortality in humans. During influenza virus infection, severe damage of lung tissue directly correlates with innate and adaptive cellular immune responses in addition to elevated pro-inflammatory cytokine responses. We reported previously that intra-tracheal treatment with a sphingosine analog, AAL-R, inhibits the production of pro-inflammatory cytokines and T cell accumulation within lung tissue without altering B cell responses or viral clearance (Marsolais, et al., 2008). Now we report that intra-tracheal treatment with AAL-R has profound S1P1-specific effects on innate immune responses. AAL-R inhibits neutrophil, NK and macrophage activation and recruitment into the lung via S1P1 signaling. In addition, AAL-R modulation of pro-inflammatory cytokines was also specific to S1P1 receptor signaling, as treatment with an S1P1 specific agonist significantly reduces early pro-inflammatory cytokine production after influenza virus challenge. Finally, we demonstrate that inhibition of T cell accumulation within the infected lung was independent of S1P1 receptor signaling. Here we provide mechanistic evidence for previously unrecognized inhibition of influenza virus innate and pro-inflammatory immune responses via S1P1 signaling. These findings have profound implications for designing therapeutic strategies to mitigate harmful immune responses during pathogenic influenza virus infection.
Uveitis-associated epitopes of retinal antigens are pathogenic in the humanized mouse model of EAU and identify autoaggressive T cells (93.1)
Authors: Mattapallil Mary , Silver Phyllis , Mattapallil Joseph , Horai Reiko , Karabekian Zaruhi , McDowell Hugh , Chan Chi-Chao , James Eddie , Kwok William , David Chella , Caspi Rachel
Published: January 2023

Autoimmune uveitis affects 150,000 Americans annually and is a leading cause of blindness. Pathogenesis is thought to involve T cell mediated responses to retinal proteins, such as retinal arrestin (S-Ag). Susceptibility to autoimmune uveitis is strongly associated with HLA class I and class II, but relatively little is known about the nature of HLA class II alleles and about the epitopes that might be pathogenic in the context of different HLA class II molecules. Using a ‘humanized’ mouse model for experimental autoimmune uveitis (EAU) in HLA transgenic mice, we have identified permissive and non-permissive alleles of the HLA-DR and -DQ genes and characterized allele-specific uveitogenic epitopes. The sequences of these epitopes overlap with some of the previously identified peptides of S-Ag (M and N), which elicit memory responses in lymphocytes of uveitis patients. HLA-DR restricted S-Ag specific CD4+ T cells could be detected in blood and draining lymph nodes of immunized mice using HLA class II tetramers, demonstrating the potential use of this reagent to detect and isolate antigen specific T cells in uveitis patients. Our data provide new insights into the pathogenesis of human uveitis, and may help in the development of antigen specific therapies.
An epitope from Acanthamoeba castellanii induces clinical signs of autoimmune encephalomyelitis in SJL mice by molecular mimicry. (93.25)
Authors: Massilamany Chandirasegaran , Thulasingam Siva , Steffen David , Reddy Jay
Published: January 2023

Autoimmune responses to myelin antigens have been implicated in the pathogenesis of multiple sclerosis (MS), but the mechanisms by which the disease is initiated are unknown. Exposure to environmental microbes has been suspected to initiate the disease by generating autoreactive cells as a result of the release of new antigens, bystander activation and molecular mimicry. We have identified an epitope (aa, 83-95) from Acanthamoeba castellanii (ACA) and it induces clinical signs of experimental autoimmune encephalomyelitis (EAE) in SJL/J mice reminiscent of the disease induced with myelin proteolipid protein (PLP) 139-151 regardless of gender. By using major histocompatibility complex II / IAs tetramers, we demonstrate that both ACA 83-95 and PLP 139-151 generate antigen-specific cross-reactive CD4 T cells and the T cells secrete identical patterns of T helper (Th) 1 and Th 17 cytokines. However, by contrasting the differences in TCR Vβ usage, it was revealed that the T cells from mice immunized with ACA 83-95 showed a higher proportion of Vβ2+ T cells with a corresponding decrease in Vβ17a+ cells and the reverse pattern was observed in mice immunized with PLP 139-151. These results may provide insights into the pathogenesis of multiple sclerosis and ACA-induced granulomatous encephalitis.
T cells, but not TLRs, control Act1-driven autoimmunity (93.28)
Authors: Johnson Angela , Li Xiaoxia , Jorgensen Trine
Published: January 2023

Act1 is a negative regulator of B lymphocyte-activating factor (BAFF) and CD40 signaling and is a key modulator of systemic autoimmunity, since Act1-deficient mice develop symptoms of severe autoimmune disease. As both T cell help and TLR signaling are known to contribute to autoimmune disease, mice deficient in either functional T cells (TCRb/d knockout) or TLR signaling (IRAK4 knockout) were crossed onto the Act1 autoimmune-prone background. Importantly, IRAK4-deficient animals are devoid in all TLR signaling, except TLR3. Splenomegaly, lymphademopathy, hypergammaglobulinemia, antigen-specific autoantibodies and immune complex deposition are all reduced in Act1 / TCRb/d triple deficient mice but not in Act1 / IRAK4 double deficient mice, indicating that T cells but not TLRs are required for Act1-driven autoimmunity. Additionally, as it is known that the numbers of transitional B cells are increased in the absence of functional Act1, preliminary cell population analysis demonstrates that in the absence of T cells, transitional B cells are still increased in number. Taken together, this indicates that early B cell development is not dependent upon the presence of T cells; whereas, the activation of autoreactive B cells is dependent upon T cells. Furthermore, both of these events appear to be independent of IRAK4 and potentially TLR signaling.
Dexamethasone synergistically increases secretion of the anti-inflammatory cytokine interleukin(IL)-10 in murine dendritic cells (DCs) upon viral infection: Potential implication to stress-mediated modification of susceptibility to viral infection. (94.4)
Authors: Ng Sinnie , Li Andrew , Ozato Keiko , Kino Tomoshige
Published: January 2023

Glucocorticoids (GC) are end-products of the HPA axis, which play central roles in stress response, including pathogen infection, and are widely used as therapeutic agents for inflammatory diseases. It is known that stressed condition altered the susceptibility to viral infection. Since it is not known how GC influence the function of DCs upon viral infection, we infected murine DC with Newcastle disease virus (NDV) in the presence of glucocorticoid receptor (GR) ligand dexamethasone (Dex), and examined mRNA expression of 84 genes, which play various roles in the TLR signaling cascade. We found that NDV infection potently induced mRNA expression of 24 genes. Among them, 7 genes (interferon (IFN)-γ, interleukin (IL)-6, IL-10, lymphotoxin A, C-type lectin domain family 4, nuclear factor of κ light chain gene enhancer in B cell inhibitor β and prostaglandin-endoperoxidase synthase 2) were further up- or down-regulated by Dex pre-treatment. Particularly on IL-10, Dex synergistically up-regulated NDV-induced mRNA by 7-fold. The Dex effect was mediated by GR, as inhibitor RU 486 abolished the effect. In contrast to IFN-γ and IL-6, which are Th1 cytokines and response at early time point (6 h), kinetics of IL-10 mRNA showed the highest synergistic effect of NDV and Dex at a later time point (24 h). ELISA results showed consistent results with mRNA expression. Taken together, our study indicates that GC modulate host immunity, altering TLR-related gene expression in DCs.
Improved tumor elimination by anti-tumor T cells that are resistant to adenosine-mediated immunosuppression in tumor microenvironment (95.1)
Authors: Ohta Akio , Kjaergaard Jorgen , Hatfield Stephen , Madasu Manasa , Subramanian Meenakshi , Kini Radhika , Ohta Akiko , Sitkovsky Michail
Published: January 2023

Tumors protect themselves from the anti-tumor immune response. One of the self-protective mechanisms is extracellular adenosine in tumor microenvironment, which inactivates anti-tumor T cells via A2A adenosine receptor (A2AR). Anti-tumor T cells that are resistant to adenosine-mediated immunosuppression should be of significant benefit to improve adoptive tumor immunotherapy. Our previous study demonstrated that addition of an adenosine receptor agonist NECA in CTL culture made the CTL resistant to adenosine-mediated immunosuppression. In this study, we evaluated the efficacy of NECA-treated anti-tumor T cells in MCA205 lung metastasis model. Culture-activated tumor-draining lymph node cells (TDLN) were used as anti-MCA205 effector cells. It was confirmed that NECA treatment of TDLN are less sensitive to A2AR signaling. We transferred either TDLN or NECA-treated effector cells (NECA-TDLN) into tumor-bearing mice 10 days after tumor inoculation. NECA pretreatment improved tumor rejection as indicated by the decreased number of tumor nodules from 118 ± 51 (TDLN) to 21 ± 33 (NECA-TDLN). T cell proliferation in vivo was analyzed after the adoptive transfer of CFSE-labeled effector cells. NECA-TDLN proliferated better than control TDLN in the lung (tumor site) suggesting that adenosine-resistant NECA-TDLN could activate well in tumor microenvironment. Induction of adenosine-resistance by NECA pretreatment might be a novel approach to overcome immunosuppression in tumors.
Nuclear expression of the regulatory T cell marker Foxp3 by non-small cell lung cancer (NSCLC) tumors is positively correlated with histologic grade (95.3)
Authors: Tucker Jo , Horbinski Craig , Yannelli John
Published: January 2023

Recently, expression of the regulatory T cell associated transcription factor, Foxp3, has been shown in a variety of tumors and tumor cell lines. The function of Foxp3 in tumor cells is currently unknown, but has implications for immune evasion and tumor progression. We have detected the presence of Foxp3 in several tumor cell lines, including 8 NSCLC lines, as well as in fresh tumor and normal adjacent tissue (NAT) samples by flow cytometry and western blot. To examine expression patterns in vivo, immunohistochemistry was performed on paraffin sections obtained from 25 NSCLC adenocarcinomas and 2 NAT. There was no evidence of Foxp3 expression in the normal lung tissues, however, all NSCLC samples included Foxp3+ tumor cells displaying either nuclear and/or cytoplasmic expression. Samples were graded 0-3, indicating no, weak, moderate or strong expression in each compartment. Strong nuclear expression was seen in 8 samples, moderate in 4 and weak in 8. Coefficient correlation analysis revealed a significant correlation (p=0.02) of nuclear expression strength with increasing histologic grade which was regardless of TNM staging. The analysis also suggests a relationship between nuclear Foxp3 expression in tumor cells and in infiltrating lymphocytes. As histologic grade has been reported as an independent prognostic factor for survival in NSCLC, we propose that Foxp3 may be an important element in promoting disease progression by inducing an immunosuppressive microenvironment.
Interleukin-2 treatment promoting T regulatory cells can inhibit the development of experimental autoimmune encephalomyelitis. (96.11)
Authors: Rouse Michael , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Multiple Sclerosis (MS) is a chronic and disabling autoimmune disease that affects ~400,000 people in the US. There is a pressing need for alternate and more effective treatment strategies that target the components of inflammatory cells. In recent years, regulatory T-cells (T-regs) have been found to play an important role in governing the immune system, particularly in preventing the development of autoimmunity. In our studies, we used experimental autoimmune encephalomyelitis (EAE), a murine model of MS, to investigate the potential therapeutic role of the cytokine interleukin-2 (IL-2) and its effects on FoxP3+ T-regs and IL-17 producing T-cells (Th17 cells). In our studies, clinical scores of paralysis were significantly reduced in EAE mice pre-treated with a moderate dose of IL-2. Immune cell phenotypes showed significant cellular infiltration in the brains of untreated EAE mice, which was reduced with IL-2 pre-treatment. Tissue histopathology of the central nervous system also confirmed the effectiveness of IL-2 pre-treatment by decreasing cellular infiltration in the spinal cord and preserving tissue integrity, whereas untreated EAE mice possessed numerous infiltrating cells and marked demyelination. Together, these studies demonstrate that IL-2 treatment prior to MOG administration may suppress the development of EAE by activation of T-regs and down-regulation of Th17 cells.