bluuebunny/crossref_metadata_2025 huggingface dataset:
Adjuvant-free nanogel-based PspA nasal vaccine for the induction of protective immunity against Pneumococcus (166.7)
Authors: Yuki Yoshikazu , Kong Il , Sato Ayuko , Nochi Tomonori , Mejima Mio , Kurokawa Shiho , Hiroiwa Tomoko , Fukuyama Yoshiko , Sawada Shinichi , Takahashi Haruko , Akiyoshi Kazunari , Kiyono Hiroshi
Published: January 2023

To establish strategically effective and attractive vaccine against pneumococcal respiratory infections, combining the current knowledge and technology for common antigen throughout pneumococcal strains and the new delivery system is essential. Here, we introduce a new pneumococcal nasal vaccine using the advantages of Pneumococcal surface protein A (PspA) antigen and a new adjuvant-free intranasal vaccine-delivery system with a nanometer-sized hydrogel (nanogel) consisting of a cationic type of cholesteryl group-bearing pullulan (cCHP). Nanogel-based PspA nasal vaccination induced high levels of antigen-specific serum IgG, and nasal and bronchial secretory IgA (SIgA) antibodies. The levels of PspA-specific antibodies were as high as those in mice nasally immunized with PspA and mucosal adjuvant, cholera toxin. The nanogel induced PspA-specific immune responses provided protective immunity against the lethal challenge with Streptococcus pneumoniae Xen10. Nanogel-PspA vaccinated group thus had less numbers of pneumococcus on the surface of the bronchial mucosa, and perfectly protected from the pneumococcal invasion of the lung parenchyma. These results demonstrate the effectiveness of the nanogel-based PspA nasal vaccine system as an adjuvant free mucosal vaccine against the respiratory infection of pneumococcus.
Anti-Group B streptococci Ib antibodies protect against invasive aspergillosis. (166.9)
Authors: Wharton Rebekah , Dizon Brian , Kearney John
Published: January 2023

Aspergillus fumigatus (A.f.) the cause of invasive Aspergillosis (IA) has become a leading cause of death in immunosuppressed populations, due to the lack of effective therapies or vaccines. Studies using anti-A.f. monoclonal antibodies indicate that they can protect against IA. However, the lack of detailed information on the antigenic targets of protective anti-A.f. antibodies has hampered the development of an effective anti-A.f. vaccine. Sialylated epitopes found on a variety of pathogens including fungi and Group B Streptococci (GBS) are thought to be major virulence factors and are involved in attachment to host cells, uptake by phagocytes, and complement activation. Sialylated epitopes on A.f. may therefore provide a potential target for new therapies. Our preliminary data show that a purified, calcium-dependent GBSIb type-specific antibody, SMB19 (IgM,κ) also binds to A.f. and prolongs survival in a murine model of IA. Similarly, immunization of wild type and SMB19 VH bearing immunoglobulin transgenic mice with a GBSIb vaccine also provides protection. These results support our hypothesis that induction of GBSIb specific antibodies, perhaps supplemented with antifungal drugs, will lead to new vaccines or passive antibody prophylaxis to prevent and treat IA.
Insights into MHC evolution provided by interesting genes on Gallus gallus chromosome 16 (167.10)
Authors: Miller Marcia , Zhang Lei , Goto Ronald , Abernathy Jason , Zhou Huaijun , Delany Mary
Published: January 2023

The MHC is viewed as ancient and polymorphic as result of enduring selection by diverse pathogens. But even now little is known about MHC polymorphism outside of a few mammalian species. There is little evidence as to how this region contributions in resistance to infectious disease. The MHC of the domesticated chicken (Gallus gallus) has long been observed to influence disease resistance. With a significant portion of the G. gallus MHC sequenced it is now possible to begin to examine individual loci in detail. Our studies have provided evidence for three diverse features contributing to G. gallus MHC diversity and polymorphism: 1) It has become clear that chicken natural killer cells distinguish among different MHC class I isoforms. Natural killing of target cells can be stimulated or inhibited by the expression of different MHC class I alleles. So it seems that innate immunity likely contributes to the long-observed association between chicken MHC haplotype and disease. 2) Expression of BG1, a polymorphic MHC inhibitory receptor contributing in MHC-linked resistance to a herpesvirus-induced lymphoma, is modulated in at least one allele by a retroviral insert targeted by endogenous miRNA. 3) By combining trisomy mapping with array Comparative Genomic Hybridization (aCGH) we have been able to assign to chicken chromosome 16 unmapped genomic sequence contigs containing additional genes long posited to be critical in maintaining pathogen selected MHC diversity.
Transcription factor E2F1 suppresses dendritic cell maturation. (167.3)
Authors: Fang Fang , Wang Yan , Li Rui , Zhao Ying , Guo Yang , Jiang Ming , Sun Jie , Ma Yang , Ren Zijia , Tian Zhigang , Yang De , Xiao Weihua
Published: January 2023

Recent studies report that transcription factor E2F1 regulates a number of genes in macrophage in response to immune stimulus, and involves in regulating T cell proliferation and differentiation. These studies indicate that E2F1 has impacts on the immune regulation additionally to its roles of regulating cell cycle and apoptosis. Our work shows that during the lipopolysaccharides (LPS) induced dendritic cell (DC) maturation, the level of E2F1 expression was transiently down regulated. The further study showed that knockdown of E2F1 induced both phenotypic and functional maturation in DCs. Conversely, DCs maintained in immature state when E2F1 was ectopically overexpressed. Moreover, E2F1 induced activation of multiple signaling pathways including PI3K, Erk1/2, NF-kappaB, which are generally thought to be involved in DC maturation. Overall, our study demonstrated that E2F1 served as a negative feedback factor to prevent overreaction in DC maturation.
DUSP11 is required for innate immunity (167.4)
Authors: Nallaparaju Kalyan , Zhang Yongliang , Liu Xikui , Reynolds Joseph , Nurieva Roza , Dong Chen
Published: January 2023

Dual specificity phosphatases (DUSPs) have been shown to play a critical role in regulation of various cellular processes. DUSP11 was previously demonstrated to bind to RNA-RNP complexes and RNA splicing factors and is a p53 target gene. Our in vitro studies showed that DUSP11 expression is induced in dendritic cells (DCs) upon stimulation by TLR ligands and in T cells upon CD3/CD28 activation suggesting the prominence of DUSP11 in immune responses. To examine the function of DUSP11 in regulating immune responses, we generated and analyzed DUSP11 deficient mice. We found that DUSP11 deficient antigen presenting cells produce decreased levels of proinflammatory cytokines during innate immune responses and CD4 T cells showed increased proliferation after activation but no apparent defect in in vitro T cell differentiation. However, our in vivo experiments demonstrated that DUSP11 deficient mice are defective in antigen-specific T cell responses and that they are more susceptible to Listeria monocytogenes infection. Using DC and T cell co-culture experiments we established that the DUSP11 signalling in DCs eventually regulates CD4 T cell activation. Together, these results demonstrate the critical role of DUSP11 in innate immunity.
A mechanistic view of the effects of manzamine A (MZA) on rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) (167.8)
Authors: Klosowska Karolina , Hoppe Michael , Volin Michael , Woods James
Published: January 2023

Previous studies suggested that MZA, an alkaloid extracted from a marine sponge, has anti-inflammatory properties which may act via mitogen-activated protein (MAP) kinases. We hypothesized that MZA would decrease phosphorylation of MAP kinases in RA FLS and reduce secretion of proinflammatory cytokines from RA synovial tissue (ST) explants. RA FLS were treated with 10 μM MZA or DMSO (vehicle) for 1 hour prior to stimulation with various dilutions of RA synovial fluid (SF) for 5 minutes and Western blotting was performed on cell lysates. Western blotting with phospho-specific antibodies demonstrated that RA SF increases the phosphorylation of the MAP kinases ERK 1/2 and JNK in RA FLS at dilutions of 1:10 and 1:25. However, pretreatment with MZA did not consistently alter RA SF-stimulated RA FLS phosphorylation of ERK 1/2 or JNK. RA ST explants were incubated with either DMSO or MZA (10, 1.0 or 0.1 µM) and conditioned media (CM) was collected after 24 hours for analysis by ELISA. CM collected 24 hrs after treatment of RA ST explants with MZA or vehicle revealed no significant differences in secretion of RANTES (CCL5) or interleukin (IL)-10. We conclude that RA SF dilutions can be used as a biologically relevant stimulant of RA FLS MAP kinase phosphorylation. We have also shown that MZA has no consistent effect on the phosphorylation of the MAP kinases ERK 1/2 or JNK. We also note that MZA may not alter secretion of IL-10 or RANTES using a RA ST explant model.
Phenotypic and epigenetic approach evaluating human M1 and M2 macrophages (167.9)
Authors: Kittan Nicolai , Allen Ronald , Hogaboam Cory , Kunkel Steven
Published: January 2023

Macrophages can be divided into a classically (M1) or alternatively activated (M2) phenotype depending on the cytokine environment. Commonly, IFNg±LPS and IL-4±IL-13 drive the M1 and M2 phenotype, respectively. In humans however, understanding of the M1/M2 paradigm is still evolving. Recent findings also suggest that the growth factors GM-CSF and M-CSF influence human macrophage development towards a M1- or M2-like phenotype. We analyzed macrophages grown from human blood monocytes under GM-CSF or M-CSF culture conditions that were treated with either IFNg ± LPS or IL-4/IL-13. Cells were analyzed for the expression of genes associated with the M1 and M2 phenotype. To further explore mechanisms defining the M1/M2 cells, superarray was performed to evaluate mRNA differences in the expression of chromatin modifying enzymes. In both GM-CSF and M-CSF cultured cells IFNg/LPS increased the expression of genes associated with M1 macrophages, e.g. CXCL10 and CCR7; while treatment with IL-4/IL-13 lead to an increase of M2 markers, Mannose Receptor and most prominent 15-Lipoxygenase. In addition, preliminary data also point to an involvement of histone modifying enzymes including MLL and KDM6B in the M1setting, whereas there was an association of DNA methyltransferase(s) like DNMT1 with the M2 phenotype. Overall, further analysis of the epigenetic mechanisms governing the M1/M2 phenotype will hopefully provide a better understanding of this important macrophage fate decision.
The roles of combination of dextran with gp120 on human macrophage immune response in HIV infection (168.16)
Authors: Wen Jianjun , Pang Lan , Estes Don
Published: January 2023

In this study, high molecular weight dextran only or combination with gp120 was used to stimulate human monocyte-derived macrophages to study the immune response to treatments and to elucidate the preliminary possibly related pathways. Our data demonstrated that 40 µg/ml of dextran did not induce a significant alteration of important immune-related gene expressions but decreased the gene expressions of CXCR4 and CCR5 mediated with reactive oxygen stress (ROS) generated by mitochondria. Cell viability measured by flow cytometry indicated that HIV induced 84% of macrophage deaths but combination of dextran with gp120 restored 48% of the mortality rate. Interestingly, a combination of dextran with gp120 can improve the enhancement of CD244 and perforin expressions induced by HIV, indicating that HIV infection is mediated to cytotoxicity. In addition, PI13 K and NFκB pathway played a very important role in the HIV-infected cell viability and ROS generation had partially influenced the cell viability. Regarding Toll-like receptors, dextran stimulated TLR1 increase, TLR9 decrease via PI13K pathway; however the alterations were restored by HIV-1 gp 120. Our data also showed that PI13K, mitochondrial ETC and NFκB pathways play very key roles in regulating perforin, CD244 and CD260. In conclusion, combination of dextran with gp120 is a good conjugate of polysaccharides to develop an HIV vaccine.
An investigation for IL-6 responses in patients with pandemic influenza A (H1N1) infection (168.20)
Authors: Bagriacik Emin , Albayrak Nurhan , Altas Basak , Ertek Mustafa
Published: January 2023

The pandemic H1N1 or the swine flu virus infected many people in 2009, and contributed an important morbidity and mortality around the world. Unfortunately very few markers related to the prognosis during the course of the disease have been reported so far. The purpose of this study is to investigate serum levels of IL-6 for prognosis evaluation of H1N1. A total of 29 sera from 18 patients that were confirmed to be positive for H1N1 by real time PCR in The National Influenza Center in Turkey were studied for determination of IL-6 levels using ELISA. Two groups of serum specimens collected at different time points were evaluated. The first group was collected at the acute phase of the viral infection (0-7th days) while the second group was collected at the late phase (10-20th days). Only one patient with respiratory failure died. We found a dramatic difference (p<0.05) in levels of serum IL-6 between the early and the late phases of the infection. We also compared the serum IL-6 levels with the clinical symptoms obtained from patients who had a previous asthma history. The asthma patients and a patient with respiratory failure had significantly high levels (p< 0.05) of IL-6 during the course of the disease. These results indicated that serum levels of a pleiotropic cytokine IL-6 might be an important marker for prognosis of H1N1 infections.
Impaired recruitment of inflammatory cells to aged lungs following influenza infection contributes to delayed virus clearance (168.24)
Authors: Eaton Sheri , Lanthier Paula , Tighe Michael , Jelley-Gibbs Dawn , Haynes Laura
Published: January 2023

Elderly people are very susceptible to influenza virus and often exhibit complications following infection including secondary bacterial infections and exacerbation of lung diseases. Using mouse models, it has been well established that, much like humans, aged animals exhibit a delay in clearing sublethal influenza virus infection compared to young. However, the mechanisms responsible for this delay are not well understood. In this study, we have determined that following influenza infection, the levels of early IFNg production (days 3-6) are significantly lower in aged lungs. Reduced IFNg levels lead to the reduced production of important IFNg-dependent chemokines by innate lung cells, including CCL3, CCL4 and CXCL10, which are necessary for the proper recruitment of inflammatory cells such as neutrophils. This observation is important since it is now appreciated that neutrophils are critical for efficient viral clearance. Reduced lung neutrophil numbers and reduced lung immunopathology were observed in aged flu-infected animals, indicating an overall less vigorous immune response. This diminished initial response plays an important role in the delay in virus clearance. In addition, it also subsequently contributes to lingering inflammation in aged lungs, including high levels of inflammatory cytokines and chemokines, which can contribute to exacerbating pre-existing lung disease.
Guanylate binding protein (GBP) 4 negatively regulates virus induced type I interferon and antiviral response by targeting interferon regulatory factor (IRF) 7 (168.30)
Authors: Sun Bing , Wang Jie
Published: January 2023

In this study, we identify GBP4 as a virus-induced protein interacting with IRF7, which is the master regulator in virus triggered induction of type I IFNs. Overexpression of GBP4 inhibits virus triggered activation of IRF7 dependent signaling, but has no effect on NF-kB signaling, whereas the knockdown of GBP4 has opposite effects. Consistently, the supernatant from sendai virus (SeV) infected cells in which GBP4 has been silenced inhibits the replication of vesicular stomatitis virus (VSV) more efficiently. Competitive coimmunoprecipitation experiments indicate that GBP4 disrupts the interactions between TRAF6 and IRF7, resulting in the impaired virus induced IRF7 phosphorylation. Collectively, our results suggest that GBP4 is a negative regulator of virus triggered type I interferon production, and it is identified as a novel protein targeting IRF7 and inhibiting its function.
The susceptibility of mice suffered from burn and irradiation injuries to pseudomonal wound infections (169.11)
Authors: Nakamura Kiwamu , Okita Yamato , Cornforth Michael , Herndon David , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

In huge nuclear power plant accidents, victims suffered from injuries combined with burn and irradiation have been reported. In this study, the susceptibility of these victims to infection with P. aeruginosa was studied in a mouse model. One day after 3-5 Gy of whole body 137Cs γ-irradiation, mice were exposed to 5% total body surface area flame burn. These mice were subcutaneously infected with 50 CFU/mouse of P. aeruginosa surrounding the burn site. The susceptibility of mice with combined injuries to the infection was evaluated by (i) mortality rates and (ii) growth of the pathogen in various organs. In the results, all mice with combined injuries died within 3 days of infection, while all groups of control mice (normal, burned, and irradiated mice) survived. Higher numbers of bacteria were observed in organs of mice with combined injuries. The numbers of CD49b(+)/TCR(−) cells greatly increased in the spleens of mice with combined injuries, as compared with those of 3 other controls (23.7%, mice with combined injury; 2.7%, normal mice; 11.6%, burned mice; 12.5%, irradiated mice). CD49b(+)/TCR(−) cells demonstrated in mice with combined injuries inhibited the bactericidal activity of macrophages. These results suggest that CD49b(+)/TCR(−) cells may play a role on the increased susceptibility of mice to P. aeruginosa burn wound infection.
Engagement of Heterogeneous Nuclear Ribonucleoprotein M with Listeriolysin O Induces Type I Interferon Expression and Restricts Listeria Monocytogenes Growth in Host Cells (169.12)
Authors: Zheng Shijun , Luo Zheng , Li Zhonghua , Chen Kun , Li Xiaoqi , Cao Hong
Published: January 2023

Listeriolysin O (LLO) is a key virulence factor secreted by the Gram-positive, facultative intracellular pathogen Listeria monocytogenes (LM). Its role in host cell response is still not very clear. Using pull-down assay, mass spectrometry analysis and immunoprecipitation approaches, we found that LLO interacted with heterogeneous nuclear ribonucleoprotein M (hnRNP M), a member of RNA splicing complex apparatus, and the binding domain of LLO for hnRNP M was located between amino acids 26-176. Knockdown of hnRNP M inhibited LLO-induced activation of IFN-α, IFN-β and AP-1 promoters and enhanced LM growth in host cells. Thus, engagement of hnRNP M with LLO induces type I interferon expression and restricts LM growth in host cells, suggesting a critical role of hnRNP M in LLO-induced immune responses in host cells. These findings will contribute to further understandings of the molecular mechanisms underlying the host defense against LM infection.
IL-1 signaling contributes to the chronic inflammatory response to the oral pathogen, P. gingivalis (169.17)
Authors: Weinberg Ellen , Kramer Carolyn , Beaulieu Lea , Orlowski Anthony , Morgan Elise , Freedman Jane , Genco Caroline
Published: January 2023

Objective. Interleukin-1 signaling is an important mediator of the pro-inflammatory response to pathogens. We have shown that the oral pathogen P. gingivalis (Pg) induces the release of IL-1alpha (IL-1α) and IL-1beta (IL-1β) in cultured cells in vitro and is associated with chronic inflammation including platelet activation, oral bone loss and atherosclerosis. We sought to define the specificity (IL-1α vs. IL-1β) of IL-1 receptor signaling on platelet function and oral bone loss in response to Pg. Methods. Wild type (WT), IL-1β knockout (IL-1β KO), and IL-1 receptor knockout (IL1R KO) mice were challenged orally with Pg or control (Con) 3 times/week for one week. Platelet activation (flow cytometry) was determined one day (acute, N=3/group) and 6 weeks (chronic, N=5/group) after the third Pg challenge. Oral bone loss (micro-CT analysis) was determined 6 weeks after the third Pg challenge (N=5/group). Results. Challenge with Pg resulted in significant platelet activation in acute and chronic samples in WT but not in IL-1β KO or IL1R KO mice. Challenge with Pg resulted in significant oral bone loss in WT mice. Inflammatory bone loss was prevented in IL-1R KO mice but was not prevented in IL-1β KO mice. Conclusions. Platelet activation following Pg challenge is likely mediated by Pg-induced IL-1β release and signaling. In contrast, oral bone loss does not appear to be mediated by IL-1β and may involve Pg-induced IL-1α release and signaling through IL1R.
Cellular mechanisms linking pattern recognition and autophagy in Dictyostelieum discoideum (169.19)
Authors: Snyder Michelle , Pflaum Katherine , Gerdes Kimberly , Yovo Kossi
Published: January 2023

Pattern recognition machinery is used by cells of the innate immune system to detect conserved patterns on invading pathogens. Dictyostelieum discoideum is a model organism that phagocytizes bacteria for nutritional purposes, and we have shown recently that D. discoideum may use conserved pattern recognition machinery to detect and respond to their bacterial prey, since D. discoideum cells stimulated with the microbial pattern lipopolysaccharide (LPS) more efficiently clear phagocytized bacteria. Here we extend these findings, showing that LPS stimulates maturation of autophagosomes and transport of autophagosomal contents to lysosomes in D. discoideum cells exposed to Staphylococcus aureus. Induction of autophagy appears sufficient for increased bacterial killing in D. discoideum cells, as D. discoideum clears bacteria at in increased rate when cells are treated with the autophagy-inducing drug, rapamycin. Finally, we illustrate that LPS-enhanced transport of phagocytized S. aureus to the degradative lysosomes is significantly reduced in D. discoideum cells lacking the autophagy-related protein Atg1 or the toll/interleukin-1 receptor domain-containing protein TirA. Thus, for the first time, we link microbial pattern recognition and autophagy in this model organism, providing greater understanding of the highly conserved mechanisms underlying pathogen detection.
Ifitm proteins are required for appropriate localization and function of membrane proteins following type I interferon activation (169.2)
Authors: Wee Yin Shen , Roundy Kirstin , Weis Janis , Weis John
Published: January 2023

The innate immune response Interferon-inducible transmembrane (Ifitm) proteins have been characterized as influencing immune cellular proliferation, aiding signaling complexes and restricting virus infections following interferon activation although a specific functional mechanism for the Ifitm proteins has not been described. We have analyzed the function(s) of these proteins in the mouse by comparing the localization of known membrane endocytic constituents between cells obtained from wild type (WT) mice and mice lacking the five Ifitm genes (IfitmDel). Cells obtained from IfitmDel mice and treated with IFNβ demonstrated a loss of clathrin from the surface of cells, compared to WT, and a loss of clathrin-mediated endocytosis. The pH of endosomes of cells obtained from the IfitmDel, with or without interferon activation, was also altered (more basic) suggesting the localization and/or function of the vacuolar ATPase proton pumps in such cells was altered by the loss of the Ifitm proteins. Co-immunoprecipation of Ifitm proteins with the proton-transporter complex constituent Atp6v0b demonstrated a direct interaction between the Ifitm proteins and the vacuolar ATPase’s. These data, in total, suggest that the Ifitm proteins serve as protein complex organizers and function to maintain the appropriate cellular localization and function of membrane proteins following type I interferon activation.
Caspase-1 is hepatoprotective through up-regulation of autophagy after hemorrhagic shock (169.20)
Authors: Sun Qian , Billiar Timothy , Scott Melanie
Published: January 2023

Hemorrhagic shock induces tissue hypoxia, cellular oxidative stress and production of reactive oxygen species, which we have previously shown leads to activation of the inflammasome and caspase-1. Our previous data also show that caspase-1 is hepatoprotective after hemorrhagic shock/resuscitation (HS/R). Autophagy is a protective response to cellular stress and we hypothesized that caspase-1 is hepatoprotective by regulating autophagy after HS/R. Autophagic markers beclin1 were upregulated in WT liver after HS/R but to significantly lower levels in caspase-1-/- liver, and caspase-1-/- liver also had higher levels of apoptosis. Adenovirus encoding beclin1 (Adbeclin1) or control AdGFP was injected via tail vein in WT and caspase-1-/- mice. After 48h mice were subjected to HS/R. Immunoblotting of whole liver lysates confirmed beclin1 overexpression after Adbeclin1. Caspase-1-/- mice expressing Adbeclin1 had significantly less liver injury after HS/R and lower cytochrome c release (marker of apoptosis/mitochondrial damage) compared with AdGFP-caspase-1-/- mice and similar to WT levels. Similarly, in vitro primary hepatocytes isolated from caspase-1 -/- mice had decreased beclin1 expression, reduced autophagic flux and increased cells death after hypoxia/reoxygenation compared with WT cells. Together our data suggest that upregulation of beclin1 is vital for hepatoprotection after HS/R and that caspase-1 regulates beclin1 expression and autophagy through a novel pathway.
Critical role of TLR4 in human metapneumovirus mediated innate immune responses and disease pathogenesis (169.21)
Authors: Kolli Deepthi , Velayutham Thangam , Garofalo Roberto , Casola Antonella
Published: January 2023

Human metapneumovirus (hMPV), described for the first time in 2001, has become one of the main viral pathogens responsible for acute respiratory tract infections in children, elderly and immunocompromised patients. The mammalian Toll-like receptors (TLR) were identified as critical regulators of innate immunity to a variety of microbes, including bacteria and fungi. Respiratory syncytial virus (RSV) fusion protein has been shown to activate TLR4, but their role in other viral infections, such as hMPV, has been largely unexplored. In this study, we examine the in vivo innate immune response in TLR4-deficient (C57BL/10ScNJ) mice and wild-type (C57BL/10SnSnJ) mice challenged with hMPV virus to address the role of TLR4 in the innate immune response to this respiratory virus infection. Our results demonstrate that mice lacking TLR4 loose less body weight compared to wild type mice. When inflammatory mediators was measured in bronchoalveolar lavage at different days post-infection, significantly lower levels of proinflammatory cytokines (IL-6, TNF-α) immunomodulatory cytokines (IL-12 p40, IL-17) and chemokines (MCP-1, MIP-1α) were detected. Further, consistent with bodyweight loss, TLR4 deficient mice show better pulmonary function demonstrated by reduced airway obstruction and airway hyperresponsiveness. The results of this study indicate that TLR4 is important for activation of the innate immune response to hMPV infection however it can also contribute to disease pathogenesis.
Expression of non-classical HLA molecule, HLA-E during Japanese encephalitis virus (JEV) infection (170.13)
Authors: Verma Shwetank , Kim Kwang , Carbone Ennio , Manjunath Ramanathapuram
Published: January 2023

Endothelial cells play an important role in transmission of JEV across the blood brain barrier while fibroblasts are involved in peripheral spread of infection. JEV, a neurotropic flavivirus was found to replicate and establish productive infection in human endothelial cell lines leading to production of IFN-β and TNF-α. Expression of HLA-E mRNA and protein showed significant upregulation during JEV infection of the human endothelial cell line, ECV304, human brain microvascular endothelial cells (HBMECs) and a primary human foreskin fibroblast (HFF) cell line. This is the first report to show that the infection of endothelial cells by JEV can result in the release of HLA-E as soluble molecules which is mediated by matrix metalloproteinases. However, HFF cells showed only upregulation of HLA-E on cell surface upon JEV infection. Experiments using inhibitors of MAPK and NF-κB suggest that the release of soluble HLA-E upon JEV infection was dependent on the MAPK pathway but independent of NF-kB. The soluble HLA-E that was released upon JEV infection was functionally active since it inhibited the IL-2 and PMA induced phosphorylation of ERK1/2 in NK cell lines. Inhibition of ERK1/2 phosphorylation has been shown to inhibit cytotoxicity of NK cells by inhibiting mobilization of granzyme granules to immunological synapse. Thus, it is possible that release of soluble HLA-E can have far reaching consequences on activation of NK cells as well as CTLs upon JEV infection.
Expression of suppressor of cytokine signaling 1 (SOCS1) impairs immune defense against influenza infection. (170.24)
Authors: Sun Keer , Metzger Dennis
Published: January 2023

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. Of interest, Influenza infection induces high levels of SOCS1 in airway immune cells. We have now found that Socs1-/-IFN-γ-/- mice exhibited increased resistance to influenza infection as shown by improved viral clearance and survival rate compared to both IFN-γ-/- and WT mice. Nevertheless, Socs1-/-IFN-γ-/- mice had pulmonary viral burdens on day 4 post-infection that were similar to IFN-γ-/- and WT controls, suggesting little effect of SOCS1 on direct innate control of viral replication in epithelial cells. Instead, SOCS1 deficiency was associated with increased humoral and cellular antiviral immunity, suggesting a regulatory effect of SOCS1 on adaptive immune-mediated viral clearance. RAG-/- mice were reconstituted with T and B cells isolated from naïve Socs1-/- IFN-γ-/- to establish animals with a SOCS1 deficiency only in adaptive immune cells. Despite increased levels of airway CD8+ T cells, RAG-/- mice reconstituted with T and B cells from Socs1-/- IFN-γ-/- mice exhibited similar levels of viral titers, airway DCs, CD4+ T cells, and antibody production compared to RAG-/- mice that received T and B cells from IFN-γ-/- mice. We are now examining whether extended type I IFN activity in Socs1-deficient mice results in improved induction of influenza-specific adaptive immunity, which then translates to increased resistance to influenza infection.
Characterization of regulatory and conventional T cell phenotype and function in HIV exposed seronegative subjects. (170.29)
Authors: Pattacini Laura , Lund Jennifer
Published: January 2023

The role of regulatory T cells (Tregs) in preventing or facilitating HIV infection remains unclear. To address this question, we analyzed samples from 129 HIV-exposed seronegative (HESN) individuals, divided into high and low exposure groups based on partner viral load. The two groups had comparable frequencies of Tregs, but in the higher exposure group there was a trend for increased expression of the Treg suppressive markers CD39 and CTLA4. CD4+ and CD8+ T cell activation was similar in the two groups. To assess T cell function, we measured the proliferation of T cells in response to four HIV peptide pools by a CFSE-based assay. Both low and high exposure groups showed a comparable percentage of CD4+ (12.7 % versus 9.7% respectively) and CD8+T cell proliferation (3.2% versus 4.9%). To assess the Treg suppressive function, we compared the proliferation of peripheral blood mononuclear cells (PBMCs) and Treg-depleted PBMCs in response to the peptide pools. In 41% of the samples that showed proliferation in response to at least one peptide, T reg depletion induced an increased proliferation of CD4+ T cells. The suppression was similar in high and low exposure groups. In the remaining samples, Treg depletion led to a decrease in CD4+ T cell proliferation. In conclusion, our study suggests that HESN can mount an HIV-specific T cell response and further, Treg can either suppress or assist this response, though the mechanism for this difference remains under investigation.
Absence of PI3K p110d isoform reduces viral load and morbidity of mice infected with Influenza virus (170.3)
Authors: Boesteanu Alina , Gracias Donald , Turner Martin , Katsikis Peter
Published: January 2023

Experimental infection of C57Bl/6 mice with influenza virus is accompanied by significant morbidity. In contrast to C57BL/6 mice, we have found that mice that lack the phosphoinositide kinase 3 (PIK3) catalytic isoform p110 delta (p110dKO) manifest significantly reduced morbidity and mortality after influenza virus infection. Throughout the infection inflammation was reduced in p110dKO mice compared to C57Bl/6 mice as determined by the total numbers of lymphocytes, B cells, T cells, macrophages and granulocytes infiltrating the lungs. Additionally lung pro-inflammatory cytokine mRNA (TNFa, MIP-2, MCP-1, IFNg) was also reduced in p110dKO mice. This was accompanied by reduced lung viral loads in p110dKO mice. We examined whether the lack of p110d in lung epithelial cells affects influenza virus replication by performing bone-marrow chimeras between p110dKO and C57Bl/6 mice. Finally, treatment of mice with a specific pharmacological inhibitor of p110d reduced significantly the mortality of mice challenged with a lethal dose of Influenza virus. These studies suggest that PI3K p110d may be a target for therapeutic intervention against influenza virus infection.
The emergence of specialized immunoregulatory antigen presenting cells in persistent infection (170.8)
Authors: Wilson Elizabeth , Brooks David
Published: January 2023

Persistent viral infections initially elicit robust immune responses, however multiple suppressive mechanisms are also triggered that rapidly disarm antiviral T cell responses and allow for long-term persistence. The degree of T cell suppression is determined by the integration of a concert of stimulatory and inhibitory signals. However, the mechanisms by which virus-specific T cells encounter and integrate immunomodulatory signals during viral infection are unclear. IL-10 is a key factor in suppression of anti-viral T cell responses in persistent infections. We demonstrate the emergence of a unique population of IL-10 expressing immunoregulatory antigen presenting cells (iAPC) in viral persistence. iAPC are metabolically distinct and express T cell interacting molecules in combination with high levels of multiple negative regulatory factors. This clustering of inhibitory molecules on a single APC allows for potent signals to be simultaneously delivered to T cells tipping their threshold from activation to exhaustion. We have also identified a novel mechanism, induced by infection and maintained in persistence, that leads to sustained upregulation of multiple immunoregulatory molecules, thereby serving as a rheostat to gauge levels of virus replication and translate this information to immunosuppression. Ultimately, blockade of these regulating factors in combination with targeting of iAPC populations may enhance antiviral immunity to in persistent infection.
Rapid recirculation of migrating lymphocytes between blood, lung, and liver (173.26)
Authors: Ganusov Vitaly , Auerbach Jeremy
Published: January 2023

Migration patterns of naive, effector, and memory lymphocytes have important implications for activation of primary and secondary immune responses and for efficient control of pathogens in peripheral tissues. Yet, quantitative details of how long it takes for lymphocytes to migrate from one tissue to another has been largely unknown. Using experimental data and mathematical modeling we estimate the rates of migration of thoracic duct lymphocytes (TDLs) from blood to multiple lymphoid and nonlymphoid tissues of a rat and back to the blood. Our model suggests extremely rapid recirculation of TDLs between blood, lung, and liver with an average residence time of cells in these tissues being less than 1 minute. The model also predicts longer residence times of TDLs in the spleen (2 hours) and major lymph nodes and Peyer's patches (8-10 hours). The same model provides a good match of independently measured output of TDLs via thoracic duct in cannulated animals. By comparing TDL accumulation in resting and antigen-stimulated lymph nodes we predict that enlargement of lymph nodes occurs because of increase entrance of lymphocytes into the inflamed LN and not decreased output as often viewed. Thus, we illustrate how within one modeling framework we can accurately describe multiple phenomena on lymphocyte recirculation in the whole organism.
Leukocyte recruitment induced by exogenous methylglyoxal: the role of endothelial adhesion molecules (173.27)
Authors: Su Yang , Lei Xi , Wu Lingyun , Liu Lixin
Published: January 2023

Methylglyoxal (MG) is a reactive dicarbonyl metabolite formed during glucose, protein and fatty acid metabolism. In condition of hyperglycemia, excessive MG produced endogenously can lead to diabetic macro- and micro-vascular complications where inflammation plays an important part. However, the role of MG in the induction of inflammation and the underlying mechanisms are poorly understood. In this study, we applied MG to local, healthy tissue and used intravital microscopy to investigate the role of endothelial adhesion molecules in MG-induced leukocyte recruitment in mice. Acute administration of MG to the tissue dose-dependently induced leukocyte recruitment with ~90% recruited cells being neutrophils. The MG treatment upregulated the expression of endothelial adhesion molecules P-selectin, E-selectin, ICAM-1, but not VCAM-1. The role of these upregulated endothelial adhesion molecules in MG-induced leukocyte recruitment was confirmed by applying specific functional blocking antibodies to acute MG-treated live animals. Our data demonstrate that exogenous MG induces leukocyte recruitment through the upregulation of the expression of P-selectin, E-selectin, ICAM-1 but not VCAM-1. Our results provided mechanistic insights into the role of MG in the induction of inflammation response in conditions related to diabetic complications and revealed the role of endothelial adhesion molecules in this process.
Distinctive roles of neutrophil LSP1 and endothelial cell LSP1 during neutrophil recruitment (173.29)
Authors: Xu Najia
Published: January 2023

Leukocyte-specific protein 1 (LSP1) is an intracellular, F-actin- and Ca2+-binding, phosphoprotein expressed in leukocytes and endothelial cells (ECs). Using intravital microscopy and time-lapse video photography, we studied the role of neutrophil LSP1 (N-LSP1) and endothelial cell LSP1 (EC-LSP1) during neutrophil recruitment in the post-capillary venules in mice. The wild-type control, LSP1-deficient (Lsp1−/−) and their chimeric mice were used in this study. Neutrophil recruitment was induced by addition of chemokine MIP-2-containing gel to the observed cremasteric venule. Neutrophil intraluminal crawling, transendothelial migration, and chemotaxis in tissue were determined. We found that both N-LSP1 and EC-LSP1 were involved in neutrophil intraluminal crawling and neutrophil transendothelial migration. After neutrophil transmigration, only N-LSP1 was found to be important for neutrophil migration in the tissue, whereas EC-LSP1, but not N-LSP1, was essential for the directional neutrophil chemotaxis. Although N-LSP1 did not play an important role in neutrophil directional chemotaxis, both neutrophil migration and directional chemotaxis were dependent on the function of p38 mitogen-activated protein kinase, an upstream signaling molecule for N-LSP1, in the emigrated neutrophils in the tissue. Our results suggest that N-LSP1 and EC-LSP1 are both important for neutrophil recruitment but play distinctive roles in different steps of neutrophil recruitment in vivo.
Leukocyte Ig-like Receptor B4 (LILRB4) Downregulates Key Steps in the Migration of Antigen-bearing Lung Dendritic Cells to Lymph Nodes in Th2 Inflammation (173.30)
Authors: Fanning Laura , Buckley Carolyn , Xing Wei , Breslow Rebecca , Katz Howard
Published: January 2023

We previously reported that Lilrb4-/- mice have exacerbated Th2 immune responses and pulmonary inflammation compared with Lilrb4+/+ animals when sensitized with OVA and low-dose LPS followed by challenge with OVA. In addition, LILRB4 is selectively upregulated on Ag-bearing dendritic cells (DCs) in the lung and draining lymph nodes (LNs). Ag-challenged Lilrb4-/- mice exhibit increased migration of Ag-bearing DCs to LNs and accumulation of IL-4- and IL-5-producing LN lymphocytes. To determine how the absence of LILRB4 leads to more DCs in the LNs of Ag-challenged mice, animals were sensitized intranasally with PBS alone or OVA/LPS and were challenged with OVA. Four hours later, lungs were evaluated by immunohistology, and lung mononuclear cells were isolated for flow cytometry. The lung lymphatic vessels of Lilrb4-/- mice expressed significantly more CCL21, a chemokine that directs the migration of DCs to draining LNs. In addition, lung DCs of Lilrb4-/- mice expressed more CCR7, the CCL21 receptor. The lungs of Lilrb4-/- mice also contained significantly greater numbers of CD4+ cells expressing IL-4 or IL-5, consistent with the greater number of Ag-bearing DCs and Th2 cells in LNs and the attendant exacerbated Th2 lung pathology. Our data reveal that LILRB4 downregulates expression of two fundamental molecules that induce the migration of Ag-bearing DCs to LNs, thereby decreasing Th2 cell accumulation in LNs and lung and the ensuing pathologic pulmonary inflammation.
Intravital two-photon microscopy of immune cells in visceral adipose tissue (173.38)
Authors: Stutte Susanne , Lynch Lydia , von Andrian Ulrich
Published: January 2023

Obesity causes systemic low-grade inflammation predisposing to metabolic disorders including insulin resistance, diabetes, dyslipidemia and hepatosteatosis and vascular diseases. The unique repertoire of the immune population, in particular T cells and high number of macrophages within the white adipose tissue is an intriguing phenomenon. Previously, it has been shown that both cell types are implicated in adipose-derived inflammation and insulin resistance. We analyzed their cell-cell interactions, and their interactions with adipocytes to see if they result in an increased expression of leukocyte adhesion molecules, chemokines, and cytokines in adipose tissue, thereby enhancing the inflammatory response and affecting insulin signaling. For this purpose, we established the intravital two-photon microscopy on adipose tissue. Furthermore, we compare macrophages and different T cell subtypes in lean and obese mice and describe their localization and proximity to different sized adipocytes, and their migratory behavior within the tissue.
Ascorbic acid ameliorates T cell-mediated acute liver injury in gulo-/- mice (173.41)
Authors: Bae Seyeon , Kim Hyemin , Yu Yeonsil , Kim Yejin , Kim Hang-Rae , Hwang Young-il , Kang Jae Seung , Lee Wang Jae
Published: January 2023

Deficiency of ascorbic acid (vitamin C) is thought to be contributed to the pathogenesis of liver diseases. To clarify the role of vitamin C in protection from liver injury, we induced concanavalin A (Con A)-mediated hepatitis in vitamin C-insufficient gulo-/- mice, which cannot synthesize vitamin C like humans. In Con A-treated vitamin C-insufficient gulo-/- mice, the extensive production of pro-inflammatory cytokines, such as IFN-gamma and TNF-alpha was observed in plasma and liver. Moreover, extensive liver damage and apoptosis of hepatocytes were also found. Increased numbers of liver-infiltrating T cells in vitamin C-insufficient gulo-/- mice correlated with increase of hepatic levels of IFN-inducible factor (IP-10). Even though IL-22, a hepato-protective cytokine, was highly increased in vitamin C-insufficient gulo-/- mice, IL-22R alpha expression and STAT3 activation followed by IL-22 were not induced in hepatocyte. Finally, we confirmed that vitamin C-insufficient T cells has hyper-proliferating activity and extensive inflammatory cytokine production to Con A. Taken together, vitamin C-insufficiency in vivo causes uncontrolled production of pro-inflammatory cytokines by liver-infiltrating T cells and severe defects on STAT3 activation at the down-stream of IL-22R alpha on hepatocytes and hepatic inflammation deteriorated. It suggests that severe hepatic damages by autoimmune or chronic inflammation could be prevented by the sufficient vitamin C supplementation.
IL-7 induces rapid internalization of CD127 by clathrin mediated endocytosis, leading to subsequent degradation by the proteasome. (174.1)
Authors: Faller Elliott , Al Ghazawi Feras , MacPherson Paul
Published: January 2023

IL-7, a potent immunomodulatory cytokine, is currently being investigated as a potential therapy and vaccine adjuvant in the treatment of HIV infection. In order to maximize these therapeutic strategies, we must first understand how the IL-7 receptor is regulated. We show here that in pimary human CD8 T cells, IL-7 decreases surface CD127 expression on CD8 T-cells in a dose- and time-dependent manner with initial suppression as early as 20 minutes and maximal suppression at 12 hours. This decrease at the cell surface is followed by loss of total CD127 protein within the cell as demonstrated by western blot. IL-7 induces internalization of surface CD127 through endocytic vesicles. The endocytosis inhibitors Filipin, and Dynasore both prevent IL-7 induced down regulation of CD127 at the cell membrane. Consistent with this we have shown by confocal microscopy that in resting CD8 T-cells CD127 is distributed evenly throughout the cell. After treatment with IL-7, CD127 forms multiple intracellular punctae with increased co-localization with clathrin by 5 minutes followed by co-localization with the early endosomal marker EEA1 after 30 minutes. By 2 hours CD127 staining associates with the late endosome marker RAB7 and the proteasomal 20S subunit, while at the same time showing decreased colocalization with the lysosomal marker LAMP1. Interestingly, the proteasome inhibitors lactacystin and MG132 both blocked IL-7’s ability to down regulate CD127.
Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation (174.10)
Authors: Yeh Trai-Ming , Chuang Yung-Chun
Published: January 2023

Autophagy is an evolutionarily conserved catabolic process which maintains cellular homeostasis under stress conditions such as starvation and pathogen infection. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that plays important roles in inflammation and tumorigenesis. Cytokines such as IL-1β and TNF-α that are induced by MIF have been shown to be involved in the induction of autophagy. However, the actually role of MIF in autophagy is still unclear. Here, we demonstrated that incubation of human hepatoma cell line Huh-7 cells with recombinant MIF (rMIF) induced reactive oxygen species (ROS) production and autophagy formation, including LC3-II expression, LC3 punctae formation and mitochondria membrane potential loss. The autophagy induced by rMIF was inhibited in the presence of MIF inhibitor, ISO-1 as well as ROS scavenger N-acetyl-L-cysteine (NAC). In addition, serum starvation-induced MIF release and autophagy of Huh-7 cells were partly blocked in the presence of NAC. Moreover, diminished MIF expression by shRNA transfection or inhibition of MIF by ISO-1 decreased serum starvation-induced autophagy of Huh-7 cells. Taken together, these data suggest that MIF is involved in autophagy through ROS generation to prevent cell death.