bluuebunny/crossref_metadata_2025 huggingface dataset:
Fc receptor-targeting of immunogen as a strategy for enhanced antigen loading, vaccination, and protection using intranasally-administered ex vivo antigen-pulsed dendritic cells (P4292)
Authors: Pham Giang , Iglesias Bibiana , Rosa Sarah , Gosselin Edmund
Published: January 2023

Dendritic cells play a critical role in the generation of an effective adaptive immune response. This is primarily due to their ability to capture, process, and present Ag to naïve T cells. Administration of Ag-pulsed DCs has proven to be an effective strategy for enhancing immunity to both tumors and infectious disease organisms. In addition, our own studies using an F. tularensis infectious disease vaccine model have demonstrated that targeting immunogens to FcγR via intranasal administration of inactivated F. tularensis-mAb complexes enhances protection against F. tularensis challenge. Therefore, we sought to determine if ex vivo targeting of Ag to FcγR would enhance the potency of intranasally administered Ag-pulsed DCs. In this study, bone marrow-derived DCs were pulsed ex vivo with inactivated F. tularensis alone or complexed to mAb. Immunization of mice, via intranasal injection of F. tularensis-mAb-pulsed DCs, enhanced humoral and cellular immune responses, as well as protection against F. tularensis infection. These observations correlated with increased loading of Ag on the DCs as a consequence of FcR targeting. Importantly, engagement of the inhibitory receptor FcγRIIB had no impact on this enhancement. In conclusion, targeting Ag ex vivo via FcγR on DCs provides a method for enhanced Ag loading of DCs, while bypassing the inhibitory impact of FcγRIIB. Thus, this represents an important strategy for increasing the potency of ex vivo-pulsed DC vaccines.
Induction of central memory to BCG vaccine in mice through rapamycin (P4300)
Authors: Jagannath Chinnaswamy , Khan Arshad , Bakhru Pearl , Soudani Emily , Lewis Dorothy
Published: January 2023

BCG vaccine induced Th1 immunity protects against childhood tuberculosis failing to protect adults. BCG induces short lived effector T cells (SLECs) but poor memory precursor effector T cells (MPECs) in mice. Rapamycin prolongs antigen presentation by macrophages and also affects MTOR of CD8 T cells. Thus, we examined whether rapamycin co-treatment with BCG enhances MPECs. Methods: C57Bl/6 mice were immunized with BCG alone or BCG-rapamycin. Mice were challenged 4 weeks later (primary challenge) with an aerosol dose of virulent Mtb and T cell responses were evaluated using flow cytometry and correlated with protection measured as a decline in the number of challenged Mtb. T cells were typed using tetramers, intracellular IFNγ, T-bet and Eomes transcription factors. Mice were then cleared of mycobacteria through drug treatment and rested before rechallenge (secondary challenge) with virulent Mtb. Results: Following primary challenge, rapamycin enhanced the efficacy of BCG vaccine through a better expansion of both CD4 SLECs and CD8 T SLEC/MPECs. Only rapamycin-BCG treated mice showed a robust recall response of CD8 T cells in the lungs of rechallenged mice, with a 1-log10 better decline in the numbers of Mtb organisms. The lungs also contained a high number of TB10.4 tetramer-IFNγ positive CD8 T cells, and CD8 T cells expressing perforin and granzyme. Thus, enhancing the ability of BCG vaccine to induce MPECs, leads to a vaccine that can induce long term immunity.
Pertussis toxin-dependent inhibition of trafficking receptors on plasmacytoid dendritic cells and delay in the polarization of Th17 cells expressing mucosal-associated trafficking receptors during B. pertussis infection. (P3031)
Authors: Nguyen Tuan , Ferguson Ryan , Wu Victoria , Sequeira Christopher , Niknam Shahryar , Wan Judy , Taylor Marlene , Abramson Tzvia
Published: January 2023

Via murine infection models of B. pertussis (Bp) and BpΔPTX (Bp strain devoid of pertussis toxin [PTX]), we tested the hypothesis that PTX inhibits the expression of G-protein coupled receptors (GPCR) on dendritic cells (DC) and of mucosal-associated trafficking receptors (MATR) on CD4+ effector memory T cells. Lung plasmacytoid DC (pDC) increased during early infection with both strains. However, a reduced percentage of pDC from Bp infected lung expressed co-stimulatory molecules and the GPCR required for migration to secondary lymph nodes. Additionally, a modest rise of circulating Th1 cells was observed during early Bordetella infections. However, in Bp infection, a reduced number expressed MATR compared to those from BpΔPTX infection. Interestingly, at 10-25 days post infection, blood Th17 cells rose dramatically, followed by accumulation in the lung and a gradually elevated display of MATR. Lung T-regulatory cells arose in tandem with increased lung Th17 level, perhaps controlling the rise in lung inflammation. The temporal correlation between pDC and memory responses suggests that pDC responses, driven perhaps by opsonized Bordetella antigens, may interfere with the imprinting of MATR on CD4+ effector memory T cells. We conclude that the PTX-dependent defective MATR imprinting on Th1 cells and delayed polarization into Th17 cells expressing MATR during Bp infection in mice may be responsible for the prolonged disease and suggest a role of pDC in this mechanism.
Circulating plasmablasts of HIV-infected individuals dominantly produce polyreactive antibodies including anti-NMDAR antibodies contributing to HAND and HAD (P3037)
Authors: Liao Hongyan , Yu Yangsheng , Li Song , Yue Yinshi , Sandkovsky Uriel , Swindells Susan , Su Kaihong , Zhang Zhixin
Published: January 2023

In the post-HAART stage, a substantial percent of HIV patients develop HIV-Associated Neurocognitive Dysfunction and HIV-Associated Dementia. Finding early biomarkers is essential to diagnose and prevent these diseases. HIV infection causes B cell dysfunctions. To evaluate the functional antibody repertoire during HIV infection, we cloned the paired IgH and IgL genes from single circulating plasmablasts of 5 HIV patients and expressed 80 recombinant antibodies to determine their functions. We also expressed 80 antibodies from 5 donors without HIV infection as control. Analyses of antibodies from HIV-infected individuals showed that 30% of them weakly bind to gp120. Surprisingly, about 72% of the antibodies cloned from HIV-infected individuals were polyreactive, which is sharp contrast to the 16% of polyreactive antibodies from control donors. Up to 60% of them cross-reacted with the DWDYS peptide present in NMDA receptor. We found 17% of antibodies bound to SH-SY5Y or U251 cell surface antigens. Detailed analyses of one such antibody HIV201P5B2 derived from HIV patients showed that it induced NMDAR clustering and internalization and eventually caused neuronal cell apoptosis. Taken together, the dominant production of polyreactive antibodies in the plasmablasts of HIV patients revealed an abnormal alteration of B cell function during HIV infection. Anti-NDMAR antibodies generated in HIV patients may provide a new diagnostic biomarker for HAND and HAD in AIDS patient.
Aerosolized influenza infection during late gestation pregnancy results in severe clinical outcomes in the ferret model (P3004)
Authors: Cole Kelly , Gage Emily , Reed Doug , Trichel Anita , Oury Tim , Beigi Richard
Published: January 2023

In 2009, the emergence of pandemic A influenza virus (pH1N1) demonstrated greater severity in pregnant women compared to prior influenza pandemics. The Centers for Disease Control and Prevention reported a death rate of 20% in pregnant women in the United States hospitalized with influenza-like symptoms. To understand the immunological basis for the enhanced influenza-associated morbidity and mortality observed in pregnant women, pregnant and nonpregnant ferrets were challenged with aerosolized pH1N1 strain A/California/04/09. Pregnant ferrets had poorer gestational outcomes and a more fulminant disease course than nonpregnant controls. Viral load and tissue tropism were similar for both groups as determined by quantitative RT-PCR. Influenza virus was detected in the brains of both groups. Gross pathological analysis and H&E stains identified interstitial pneumonia in the lungs, with no significant pathology in any other tissues. Virus was also detected in placental tissue and kits of the pregnant ferrets suggesting transmission across the placental barrier. Pro-inflammatory cytokine analysis using semi-quantitative RT-PCR showed a disparity in cytokine profile in the pregnant versus the nonpregnant ferret tissues suggesting possible cytokine dysregulation. Both cohorts demonstrated comparable influenza-specific IgG responses by 5 dpi. This model will be useful in discerning the differences in host responses associated with greater morbidity and mortality during pregnancy.
Young and elderly patients with type 2 diabetes have optimal B cell responses to the seasonal influenza vaccine (P3007)
Authors: Blomberg Bonnie , Diaz Alain , Romero Maria , Landin Ana Marie , Frasca Daniela
Published: January 2023

We evaluated immune response to the seasonal influenza vaccine in young and elderly patients with type 2 diabetes (T2D). Immune measures included the in vivo serum response to the vaccine by hemagglutination inhibition (HAI) and ELISA in 22 patients (14 young, 8 elderly) and 65 healthy age-matched controls (37 young, 28 elderly). B cell-specific biomarkers of optimal vaccine response were measured ex vivo by switched memory B cells and plasmablasts and in vitro by activation-induced cytidine deaminase (AID) in stimulated cells. Markers of systemic and B cell-intrinsic inflammation were also measured. Results show that in vivo responses, as well as B cell-specific markers identified above, decrease with age in healthy individuals but not in T2D patients. This occurred despite high levels of B cell-intrinsic inflammation (TNF-α) in T2D patients, which was surprising as we had previously demonstrated this negatively impacts B cell function. We have preliminary data to indicate that the innate immune system of T2D patients is beneficially hyperactivated and we suggest that this may induce a direct stimulation of B cells. These results altogether suggest that valid protection against influenza can be achieved in T2D patients and proposed mechanisms are discussed.
Induction of neural guidance molecule expression in macrophages and dendritic cells by Mycobacterium tuberculosis. (P3017)
Authors: Bracho-Sanchez Edith , Ramkhelawon Bhama , Desvignes Ludovic , Moore Kathryn , Ernst Joel
Published: January 2023

Cellular immunity to Mycobacterium tuberculosis (Mtb) requires coordinated cell migration and localization to form granulomas. In addition to chemokines and adhesion proteins, we found that Mtb infection of mouse bone marrow-derived DC or MØ upregulated expression of members of a family of neuronal guidance molecules: netrins, semaphorins, slits, and ephrins, which have been implicated in inflammation and immunity. Since netrin-1 and its receptor Unc5b cause immobilization of MØ in atherosclerotic lesions, we hypothesized that up-regulation of these molecules during Mtb infection contributes to MΦ and DC sequestration in granulomas. Using RT-qPCR, we found that netrin-1 and its receptors: Unc5b, Adora 2a, and Adora2b are up-regulated in Mtb-infected cells. Immunoblotting revealed that netrin-1 is secreted by Mtb-infected MΦ, and immunofluorescence showed netrin-1 is expressed in the lungs of Mtb infected mice. Since netrin-1 is secreted, we studied the migration of peritoneal macrophages in response to CCL19 in the presence of supernatants from Mtb-infected or uninfected MΦ and DCs. Supernatants from Mtb-infected cells inhibited MΦ migration in a netrin-1-dependent fashion. Based on these results, we hypothesize that neuronal guidance molecules contribute to granuloma formation and/or maintenance in tuberculosis.
Chemerin is an antimicrobial agent in human epidermis. (P1257)
Authors: Cichy Joanna , Banas Magdalena , Zabieglo Katarzyna , Kasetty Gopinath , Kapinska-Mrowiecka Monika , Borowczyk Julia , Drukala Justyna , Murzyn Krzysztof , Zabel Brian , Butcher Eugene , Schroeder Jens , Schmidtchen Arthur
Published: January 2023

Chemerin is a chemoattractant ligand for chemokine like receptor 1 (CMKLR1). CMKLR1-positive human plasmacytoid dendritic cells and NK cells can be recruited to diseased skin in a chemerin-dependent manner. Chemerin is predicted to share similar tertiary structure with antibacterial cathelicidins, and recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin protein is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin derived synthetic peptides, we demonstrate that the highly positively-charged chemerin domain Val66-Pro85 embodies the majority of the anti-microbial activity, which is comparable in potency to other antimicrobial proteins. Finally, we demonstrate that Val66-Pro85 likely functions by direct bacterial lysis. Therefore, chemerin may operate at multiple levels in skin defense, as a chemoattractant and an antimicrobial agent.
Toll-like receptor 10 mediates the inflammatory response to Listeria monocytogenes infection (P1263)
Authors: Regan Tim , Nally Kenneth , Brint Elizabeth , MacSharry John
Published: January 2023

Intestinal epithelial cells (IECs) represent the initial point of invasion for the pathogen Listeria monocytogenes however; the role of epithelial pattern recognition receptors (PRRs) engagement and subsequent inflammatory cascades remains poorly characterised. The aim of our study was to determine which PRRs are involved in initiating the immune response to L. monocytogenes in IECs. We performed a PRR siRNA library screen of 50 PRRs in the HT-29 IEC prior to infection with L. monocytogenes and then measured immune gene expression by qRT-PCR. Silencing of TLR1, TLR2, TLR10 and NLRP2 resulted in reductions in CCL20 and IL8 induction following infection, while silencing of NLRP7 and NLRP10 increased expression levels. Silencing of TLR10 gave the most significant effect and subsequent stable lentivirus knockdown of TLR10 in HT-29s yielded similar results upon infection. Similarly, silencing of TLR10 in macrophage-like THP-1 cells resulted in markedly reduced cytokine production in response to L. monocytogenes infection. Moreover, TLR10 silencing resulted in greater viability of L. monocytogenes in both THP-1 macrophages and HT-29 epithelia. Using an NF-κB reporter HEK cell line we found that TLR10 requires TLR2 for efficient NF-κB activation following L. monocytogenes infection. These data indicate a previously unknown role for TLR10 in sensing of pathogenic infection and moreover have potentially identified L. monocytogenes as a specific ligand for the orphan receptor TLR10.
Cathelicidin LL-37 bloodstream surveillance is down regulated during septic shock (P1267)
Authors: Pinheiro da Silva Fabiano , Barbeiro Denise , Barbeiro Hermes , Zampieri Fernando , Machado Marcel , Torggler Filho Francisco , Cunha Debora , Goulart Alessandra , Velasco Irineu , Cruz Neto Luiz , Souza Heraldo
Published: January 2023

Antimicrobial peptides are ancient weapons of the innate immunity. Cathelicidin antimicrobial peptides (AMPs) support the epithelial barrier against infection and are constitutively secreted in the bloodstream by immune cells. Current knowledge claims that cathelicidins are up regulated upon infection. Directly bactericidal, cathelicidins possess many additional immunomodulatory properties. Here, we show that LL-37, the human cathelicidin, is down regulated during septic shock. Furthermore, we show that these effects are not related to vitamin D serum levels, a potent inducer of LL-37 gene expression, pointing out that the mechanisms that control antimicrobial peptides activity involve multiple pathways.
MARCH1 regulates macrophage polarization and host responses against parasite infections (P1271)
Authors: Galbas Tristan , Raymond Maxime , Bourgeois-Daigneault Marie-Claude , Moffat Jessica , Ishido Satoshi , Villadangos Jose , Stäger Simona , Thibodeau Jacques
Published: January 2023

Aim: Polarization of macrophages to M1 or M2 phenotypes will dictate the effectiveness of host responses against specific infections. Membrane-associated RING-CH (MARCH1) has been shown to be implicated in the activation of antigen presenting cells and is readily induced by the anti-inflammatory cytokine IL-10. Here, we set out to investigate the role of MARCH1 in the activation of macrophages and its implication in host responses. Results: We show that MARCH1-deficient bone-marrow-derived macrophages exhibit a bias toward M1 polarization as assessed by quantitative real-time PCR, cytometric bead array (CBA) and flow cytometry. Furthermore, a macrophage population showing intermediate surface markers (CD11bint and F4/80int) between M1 and M2 was observed in the peritoneal cavity of MARCH1-deficient mice. In addition, these mice were found to be more susceptible to LPS-induced shock and exhibited higher pro-inflammatory cytokine production in their serum as monitored by CBA. Finally, MARCH1-deficient mice showed an enhanced clearing of leishmania parasites in the spleen at 21 and 28 days post infection. Conclusion: These results show that MARCH1 contributes to the polarization and functional plasticity of macrophages. Conflict of Interest disclosure: No conflict to declare.
Osteopontin supports neutrophil survival and function in polymicrobial infection. (P1281)
Authors: Matsui Aritsune , Rittling Susan
Published: January 2023

Oral infections are among the most prevalent bacterial infections: they are polymicrobial and refractory to antibiotic treatment. Understanding the host response to these infections is critical for their ultimate control. Osteopontin (OPN), a secreted phosphorylated integrin binding protein, has been previously shown to protect against inflammation in a mouse model of endodontic infection. The objective of this work was to determine the mechanism by which OPN protects against polymicrobial infection. Here we have used a titanium chamber model to analyze the innate immune response to polymicrobial infection in mice in the presence or absence of OPN. Chambers were implanted subcutaneously in WT and OPN-deficient mice, and inoculated with a mixture of four endodontic pathogens. The fluid within the chamber was sampled at different times after infection. 80-90% of the cells appearing in the chamber fluid were Ly6G+ neutrophils. At early times, there were significantly lower numbers of cells in the absence of OPN and the viability of these cells was lower than in WT mice. In addition, there were significantly higher numbers of live bacteria in the absence of OPN. These results suggest that OPN functions to protect neutrophils from apoptosis and is required for maximal bacterial killing during bacterial infection. Our results are also consistent with a role for OPN in neutrophil function. Analysis of the integrins responsible for these effects is underway.
Evasion of innate immunity by Yersinia pseudotuberculosis: LPS biosynthesis genes play a key role in inhibiting the antimicrobial effects of chemokines and other antimicrobial peptides (P1241)
Authors: Wilson Eric , Lew Cynthia , Kartchner Brittany , Sullivan Shea , Porter Nathan , Jones Nathan , Erickson David
Published: January 2023

Host defense peptides, including antimicrobial chemokines, play an essential role in protecting a wide variety of organisms from bacterial infection. In an effort to identify bacterial genes involved in resistance to antimicrobial chemokine mediated cell death we performed random transposon mutagenesis on a population of Yersinia pseudotuberculosis. Through the use of a novel flow cytometric assay these mutant bacteria were then screened for mutations which led to increased binding to the antimicrobial chemokine to the bacterium. Using this strategy we have screened thousands of mutant bacteria and identified several genes that when mutated lead to increased antimicrobial chemokine binding to and killing of bacteria. These genes, which when interrupted lead to increased susceptibility to antimicrobial chemokine mediated cell death, fall into several functional categories including efflux pups, transcriptional regulators and LPS biosynthesis related genes. Understanding how chemokines and other host defense peptides recognize and bind bacteria and how bacteria evade the antimicrobial activity of host defense peptides, could lead to novel drug targets that enhance the innate immune system. In short, the therapeutic use of AMCs to fight infections may be significantly enhanced by simultaneously targeting processes, such as lipopolysaccharide biosynthesis, that are required for resistance to these peptides.
Microbiota metabolite regulation of pattern recognition receptor activation in dendritic cells (P1253)
Authors: Steinmeyer Shelby , Katepalli Madhu , Mueller Carrie , Maiti Shreya , Jayaraman Arul , Alaniz Robert
Published: January 2023

The microbiota is a vast community of microbial cells living in symbiosis with the host. The intestinal microbiota stands out due to its direct link with overall host health. The molecular complexity of the microbiota suggests broad activation of multiple pattern recognition receptors on intestinal epithelial cells and dendritic cells. Indeed, mutations in pattern recognition receptors are correlated with increased inflammatory bowel disease incidence. Clearly, the interaction between microbiota and host cells is integral to a healthy host, but what regulates this interaction is poorly understood. In our previous work we reported that indole, a tryptophan metabolite present in the GI tract and strictly produced by the microbiota (not host cells), downregulates NFκB activation and inflammation in intestinal epithelial cells. We hypothesize that indole limits inflammation by modulating pattern recognition receptor-mediated activation of dendritic cells, key sentinels of gut homeostasis. Here, we demonstrate that dendritic cells conditioned with indole at physiologic concentrations have decreased production of pro-inflammatory cytokines after differential pattern recognition receptor stimulation. Notably, indole decreases the severity of DSS-induced colitis. This suggests an anti-inflammatory role for indole in maintenance of homeostasis in the GI tract and reveals a new therapeutic strategy for the treatment of inflammatory bowel disease.
Type I IFN-independent RIG-I expression in response to double-stranded RNA (P1399)
Authors: Hayakari Ryo , Matsumiya Tomoh , Xing Fei , Imaizumi Tadaatsu , Yoshida Hidemi
Published: January 2023

Retinoic acid-inducible gene-I (RIG-I) is a pattern recognition receptor and is included in a RIG-I-like receptors (RLRs) family. Following invasion of RNA viruses into host cells, RIG-I senses the viral nucleic acids, leading to activation of antiviral innate immune responses. RIG-I itself is known as an IFN-inducible gene; however, little is known about how RIG-I is transcriptionally regulated. In this study, we studied molecular mechanisms of the expression of RIG-I in response to dsRNA. To investigate involvement of type I IFN-mediated pathway in the expression of RIG-I in response to dsRNA, we transfected with polyinosinic-polycytidylic acid (polyI:C), a synthetic dsRNA in STAT1-deficient U3A cells and type I IFN receptor-deficient U5A cells. Both type I and type II IFN are known to induce RIG-I, suggesting involvement of STAT1 in the RIG-I expression. However, the expression of RIG-I was observed in both U3A cells and their parental 2fTGH cells in response to polyI:C. We also found that the expression of RIG-I was induced in U5A cells with a slightly different kinetic. These results suggested that RIG-I expression is mainly mediated by the induction of type I IFN followed by STAT1 phosphorylation. In addition, to “fine-tuning” the RIG-I expression in response to viral infection, other molecules may participate in the induction of RIG-I.
Human Bocavirus VP2 up-regulates IFN-β pathway by inhibiting RNF125-mediated ubiquitination of RIG-I (P1411)
Authors: Wang Hanzhong , Luo Huanle , Zhang Zhenfeng , Zheng Zhenhua , Ke Xanliang
Published: January 2023

Precise regulation of innate immunity is crucial for maintaining optimal immune responses against infections. The positive regulators of IFN pathway are known to be the main targets of viruses to antagonize the innate immune system. Whether viruses target the negative regulators of IFN pathway remains to be fully investigated. Here we report that the structural protein VP2 of human bocavirus modulates IFN pathway by targeting the ring finger protein 125 (RNF125), a negative regulator of type I IFN signaling, which conjugates Lysine 48-linked ubiquitination to retinoic-acid-inducible gene-I (RIG-I) and subsequently leads to the proteasome-dependent degradation of RIG-I. VP2 not only up-regulated Sendai virus (SeV) induced IFNB promoter activity but also enhanced SeV induced IFN-beta production at both mRNA and protein levels. In agreement, the level of Ser-396-phosphorylated IFN regulatory factor 3 (IRF3) stimulated by SeV was enhanced in the presence of VP2. Furthermore, VP2 was demonstrated to physically interact with RNF125, resulting in the reduction of RNF125-mediated ubiquitination and proteasome-dependent degradation of RIG-I. Additional study indicated that endogenous RIG-I degradation was decreased in VP2-expressing cells. Our study delineates a unique phenomenon for aberrant activation of IRF3 pathway and may represent a new mechanism underlying viral manipulation of the host immune system.
Fibulin-7, a member of the extracellular matrix fibulin family, regulates immune cell migration and function (P5124)
Authors: Sarangi Pranita , Wahl Larry , Vega Susana , Yamada Yoshihiko
Published: January 2023

Interactions of immune cells with matrix proteins and their bioactive fragments via integrins could influence their activation and differentiation. Fibulins are a family of secreted glycoproteins that are associated with extracellular matrices. Fibulins form intermolecular bridges within the extracellular matrix and serve as mediators for cellular processes and tissue remodeling. We previously identified the latest member of the extracellular protein fibulin family, TM14/fibulin-7, and found that fibulin-7 (Fbln7) is a cell adhesion molecule that binds to dental mesenchymes and odontoblasts. Fbln7 is also expressed in cartilage, placenta, and eyes. Because of Fbln7 expression in immunotolerant tissues, we hypothesized that Fbln7 or its fragment may function as an immunomodulator. In this report, we examined the effect of the full-length Fbln7-d2 and its C-terminal fragment Fbln7-d3 on immune cell functions using human monocytes. We showed that monocytes interacted with both Fbln7-d2 and -d3 in part through integrins α5β1 and α2β1. We found that Fbln7-d3 inhibited cell spreading and stress fiber formation and blocked the production of inflammatory cytokine IL-6 and MMP-1/9, while Fbln7-d2 reduced production of IL-6 and also promoted the production of IL-10. Fbln7-d3 reduced phosphorylation of Erk1/2 in TNFα-activated monocytes in vitro. Our results suggest that both full length Fbln7-d2 and Fbln7-d3 fragment are negative regulators of inflammation with distinct effects.
A role for splenic monocytes/macrophages in acetaminophen-induced hepatotoxicity (P5140)
Authors: Mandal Mili , Dragomir Ana-Cristina , Sun Richard , Laskin Jeffrey , Laskin Debra
Published: January 2023

Activated macrophages (MP) has been implicated in hepatotoxicity induced by the analgesic acetaminophen (APAP). However, the origin of these cells is unknown. Here, we analyzed the role of splenic monocytes (Mo)/MP in APAP-induced liver inflammation and injury. Treatment of mice with APAP (300 mg/kg, i.p.) was associated with a time-related increase in CD11b+Ly6Chi pro-inflammatory MP in the liver after 24-96 h. In the spleen, but not the BM, this was correlated with a significant increase in the number and proliferative activity of CD11b+F4/80+Ly6Chi inflammatory Mo/MP. CD11b+F4/80-Ly6Chi inflammatory Mo/MP were also found to increase in the spleen, as well as in the BM 72-96 h post-APAP. Conversely, CD11b+F4/80+Ly6Clo anti-inflammatory Mo/MP decreased in the spleen after 48-96 h. To assess the role of splenic Mo/MP in APAP hepatotoxicity, we used splenectomized (spx) mice. APAP-induced hepatotoxicity was significantly reduced in spx mice, as measured by decreases in serum transaminases, and histological evidence of hepatic necrosis. This was associated with a decrease in pro-inflammatory Gal-3+ MP in the liver, and an increase in CX3CR1 and CCR2 expression, proteins important in recruiting anti-inflammatory MP into the liver following APAP-induced injury. Taken together, these results indicate that the spleen is a site of monocytopoiesis and that splenic Mo/MP contribute to inflammation and hepatotoxicity induced by APAP.
Human multipotent adult progenitor cells modulate leukocyte extravasation by transcriptional regulation of fucosyltransferase VII (P5142)
Authors: Burrows Gregory , Maziarz Richard , Raber Amy , Deans Robert , van’t Hof Wouter
Published: January 2023

The targeted recruitment of leukocytes to sites of inflammation is a crucial event in normal host defense against pathogens, and attachment to and rolling on activated endothelial cells is a prerequisite first step required for eventual leukocyte extravasation into sites of inflammation. These first key events are mediated by interactions between sialylated ligands expressed on leukocytes and selectins expressed on activated endothelium. Cell surface expression of sialylated ligands on leukocytes is regulated by the rate-limiting enzyme fucosyltransferase VII (Fut7), and in its absence extravasation of leukocytes is severely inhibited. Intravenous administration of multipotent adult progenitor cells (MAPC), an adherent cell isolated from adult bone marrow, provides functional improvement in multiple preclinical models of injury or disease, but the mechanism(s) by which these outcomes are achieved remain poorly understood. In this manuscript, in vitro data are presented that characterize the ability of MAPC to secrete factor(s) that transcriptionally attenuate expression of Fut7 in T cells, thereby blocking sialylation of leukocyte cell surface-expressed selectin ligands and reducing T cell binding to endothelial cells. This represents the first example of a distinct regulatory mechanism involving transcriptional down-regulation of Fut7 by MAPC that could modulate the trafficking behavior of T cells in vivo.
Migratory behavior of human CD14dimCD16+ monocytes on human macro- and micro-vascular endothelia: an in vitro approach (P5144)
Authors: Collison Joanna , Carlin Leo , Geissmann Frederic , Peakman Mark
Published: January 2023

In the mouse, much work has been done to characterize the function of Ly6c- “patrolling” monocytes by utilising intravital microscopy. However, this technique cannot be used outside of animal models, and therefore relatively little is known about the behaviour of their human counterpart, CD14dimCD16+ monocytes. To address this, we developed a re-circulating pump system to grow human endothelial cells under shear flow for 72h before adding human CD14dimCD16+ monocytes to the circulating media and performing live cell time-lapse imaging with a Nikon TiE fluorescence microscope. Using this approach we observed CD14dimCD16+ monocytes adhere and crawl on both macro-vascular HUVEC and micro-vascular HDMEC. The tracks of CD14dimCD16+ monocytes were twice as long when crawling on HDMEC compared with HUVEC under the same shear flow rate and 3 times fewer cells detached and re-entered the flow, indicating a preference for crawling on micro-vascular endothelium. HDMEC show higher levels of CD31, ICAM-1, E-selectin and VEGFR2, and lower levels of VCAM-1 and NRP-1 in a resting state, suggesting that the former represent ligands preferentially used for CD14dimCD16+ monocyte migration on micro-vascular cells, a role that will require further elucidation by studying migration under blocking conditions.
Mathematical modeling suggests rapid migration of naive lymphocytes via lung capillaries and liver sinusoids (P5100)
Authors: Ganusov Vitaly , Auerbach Jeremy
Published: January 2023

The kinetics of recirculation of naive lymphocytes in the body has important implications for the speed at which local infections are detected and controlled by immune responses. In this study using mathematical modeling we analyze experimental data on migration of 51Cr-labeled thoracic duct lymphocytes (TDLs) via major lymphoid and nonlymphoid tissues of rats. We show that 95% of naive lymphocytes in the blood must travel via capillaries in the lung or sinusoids of the liver before they enter secondary lymphoid tissues. In contrast with a widespread belief that lymphocytes take long time to travel via vasculature of the lung and liver, our analysis suggests that lymphocytes travel via lung capillaries and liver sinusoids at an extremely rapid rate with the average residence time in these tissues being less than 1 minute. The model also predicts a relatively short average residence time of TDLs in the spleen (2.5 hours) and a longer average residence time of TDLs in major lymph nodes and Peyer's patches (10 hours). Surprisingly, we find that average residence time of lymphocytes is similar in lymph nodes draining skin (subcutaneous LNs), gut (mesenteric LNs) or in Peyer's patches.Taken together, our study for the first time provides a comprehensive, systems view of recirculation kinetics of naive lymphocytes in the whole organism.
Enhanced effector function of CD8+ T cells through thrombin activation of protease-activated receptor 1 (P5108)
Authors: Smith Mindy , Hurley Amanda , Karpova Tatiana , Hasley Rebecca , Belkina Natalya , Shaw Stephen , Balenga Nariman , Druey Kirk , Nickel Erin , Packard Beverly , Imamichi Hiromi , Hu Zonghui , McNally James , Higgins Jeanette , Sneller Michael , Lane Henry , Catalfamo Marta
Published: January 2023

Disruption of vascular integrity by trauma and other tissue insult leads to inflammation and activation of the coagulation cascade. The serine protease thrombin links these two processes. The pro-inflammatory function of thrombin is mediated by activation of the Protease-activated receptor-1 (PAR-1). We found that peripheral blood effector memory CD4 and CD8 T lymphocytes expressed PAR-1, and that expression was increased in CD8 T cells from HIV-infected patients. Thrombin enhanced TcR-induced cytokine secretion (IFNγ) in CD8 T cells from healthy controls and HIV infected patients. In addition, thrombin had little chemotactic effect but it was able to inhibited chemotaxis in a CXCL9 gradient. Live microcroscopy revealed that thrombin induced chemokinesis, but not chemotaxis, of CD8 T cells, which led to structural changes, including cell polarization and formation of a structure rich in F-actin and cpERM (phosphorylated ezrin-radexin-moesin) proteins. These findings suggest that thrombin mediates cross-talk between the coagulation system and the adaptive immune system at sites of vascular injury through increased T cell motility and production of pro-inflammatory cytokines and may allow for the accumulation of memory T cells at the site of tissue injury—a physiological mechanism of immune surveillance, inflammation and tissue repair.
Role of sphingosine-1-phosphate receptor-1 in endogenous antigen specific CD8 T cell migration after infection. (P5110)
Authors: Bénéchet Alexandre , Maher Leigh , Khanna Kamal
Published: January 2023

The factors that regulate the migration of antigen-specific CD8 T cells after infection are not well understood. Although, it is known that sphingosine-1-phosphate receptor 1 (S1P1) controls naïve T cell trafficking, our understanding of how S1P1 regulates the migration of effector CD8 T cells after infection remains poorly defined. Here we used novel mouse models to investigate the role of S1P1 in the migration of effector CD8 T cells in draining lymph node (dLN). By using in-situ tetramer staining we mapped the endogenous antigen-specific CD8 T cell response after viral infection in the dLN. We observed that effector CD8 T cells followed a multistage pathway within the dLN that included the localization of CD8 T cells in T cell zones early after infection, followed by the migration of these cells into B cell zones before exiting out of the dLN via the subcapsular and cortical lymphatic sinuses. Furthermore, we used a granzyme B-YFP reporter mouse and two-photon intravital microscopy to analyze the dynamic behavior of endogenous effector CD8 T cells after infection. Finally, we generated S1P1 conditional YFP knock-out mice to disrupt S1P1 signals specifically and temporally in effector CD8 T cells after infection. Taken together, our studies clearly demonstrate that after infection, even in the absence of retention signals such as CCR7 and CD62L, S1P1 signaling is the overriding factor that regulates effector CD8 T cell migration in the dLN.
Tetraspanin CD151 is a negative regulator of FcϵRI-mediated mast cell activation (P6018)
Authors: Abdala-Valencia Hiam , Bryce Paul , Cook-Mills Joan , Berdnikovs Sergejs
Published: January 2023

Mast cells are critical in the pathogenesis of allergic disease due to the release of preformed and newly synthesized mediators, but the mechanisms controlling mast cell activation are not well understood. The tetraspanin family has recently emerged as novel signaling molecules and negative regulators of “outside-in” signals for mast cell activation. Specifically, antibodies against tetraspanins CD63 or CD81 can inhibit FcϵRI-mediated mast cells degranulation. CD151, a poorly understood member of this famility, is specifically induced in human mast cells upon FcϵRI aggregation, however, the functional significance of this response is not yet known. In this study, we found that CD151 deficiency significantly exacerbated the IgE-mediated late phase inflammation in a murine model of passive cutaneous anaphylaxis. Bone marrow-derived mast cells from CD151-deficient mice exhibited significantly enhanced de novo synthesis of pro-inflammatory cytokines IL-3, IL-4, IL-13 and TNF-α compared to controls. However, FcϵRI-induced mast cell degranulation from these mice was unaffected. At the molecular signaling level, CD151 appeared to selectively regulate IgE-induced activation of Syk and ERK1/2 associated with cytokine production but had no effect on the PLCγ1 pathway associated with degranulation. Collectively, our data indicate that tetraspanin CD151 exerts a selective negative regulation over IgE-induced late phase anaphylactic reaction and cytokine production in mast cells.
Mast cell deficiency in Stat6 transgenic mice exacerbates allergic skin inflammation (P6024)
Authors: Sehra Sarita , Nguyen Evelyn , Kaplan Mark
Published: January 2023

Mast cells are important effectors of innate and adaptive immunity; however their role in atopic dermatitis is not completely understood. Studies from our laboratory have shown that transgenic mice expressing active Stat6 (Stat6VT) in T cells spontaneously develop atopic dermatitis with increased infiltration of eosinophils, lymphocytes and mast cells. Mice with allergic skin inflammation (ASI) also had increased expression of the mast cell growth factor Il9 in the skin. In order to elucidate the role of mast cells in ASI in Stat6VT mice, we generated Stat6VT mice on mast cell deficient background (Stat6VTxB6-KitW-sh/W-sh). Mast cell deficiency in Stat6VT mice did not protect mice from disease and instead led to increased severity and occurrence of symptoms of ASI. To understand the mechanisms of the increased pathogenesis, we defined cytokine production by peripheral T cells and gene expression and histological changes in the skin. Peripheral T cells from mast cell-deficient Stat6VT mice demonstrated increased levels of IL-9, compared to Stat6VT transgenic mice on a wild type background. Further, mast cell deficiency in Stat6VT mice resulted in a substantial decrease in the expression of IL-10 concomitant with increased infiltration of inflammatory cells including eosinophils, lymphocytes and neutrophils. Our results suggest that mast cells provide an immunomodulatory function in Stat6VT mice with ASI.
Contact hypersensitivity to oxazolone provokes vulvar mechanical hyperalgesia in mice (P6028)
Authors: Chatterjea Devavani , Martinov Tijana , Glenn-Finer Rose , Balsells Evelyn
Published: January 2023

Vulvodynia can affect ~20% of women of child-bearing age. Activities such as contact with tight clothing or sexual intercourse provoke burning pain sensations whose etiology and underlying mechanisms are poorly understood. Epidemiological evidence suggests a link between a history of environmental allergies and the risk of developing vulvodynia. Clinicians find increased mast cell numbers, hyperinnervation and up-regulation of inflammatory cytokines in biopsies from vulvodynia patients vs. controls. An allergy-based mast cell focused mouse model of vulvodynia is needed to elucidate the mechanisms underlying this pathology. We show that challenge with contact hypersensitivity allergen oxazolone produces acute mechanical hyperalgesia in the vulvar region of previously sensitized female mice. Acute pain is localized to the challenge site, lasts up to 24 hours post-challenge and is accompanied by influx of neutrophils into the labiar tissue and up-regulation of several inflammatory cytokine genes including IL-6, Cxcl-1 and Cxcl-2. Pre-challenge administration of H1R-antagonist pyrilamine, tricyclic antidepressant amitryptiline, and sodium cromoglycate all abrogate the hyperalgesic response. Our findings provide the first evidence of the induction of measurable pain following an allergic response in the labiar/vulvar tissue in mice thus establishing a model that can be adapted to chronic challenges and long-term assessment of pain with and without ongoing inflammation.
Infection and activation of mast cells by influenza A virus enhances viral-induced pathology (P6030)
Authors: Graham Amy , Zickovich Julianne , Hilmer Kimberly , Obar Joshua
Published: January 2023

Influenza A virus (IAV) is a seasonal respiratory pathogen that can result in severe lung pathology. The role of mast cells has been under explored during severe pulmonary viral infections. We found that both A/WSN/33 (WSN) and A/PR/8/34 (PR8) influenza viruses cause significant immunopathology in C57BL/6 mice, but only WSN induced pathology was mast cell dependent. Using in vitro-derived bone marrow cultured mast cells (BMCMC), we found that WSN, but not PR8, directly activated BMCMC to produce histamine, leukotrienes, inflammatory cytokines, and anti-viral chemokines. Moreover, human H1N1, H3N2, and influenza B virus isolates could activate both murine BMCMC and the human mast cell line HMC-1 in vitro. BMCMC activation required infection of mast cells by IAV, which was dependent on the viral hemagglutinin. Cytokine and chemokine production from BMCMC occurs in a RIG-I/MAVS-dependent fashion which required the de novo production of vRNA; conversely, degranulation occurs through a RIG-I/MAVS-independent mechanism. Reconstitution of mast cell deficient mice with RIG-I-/- BMCMC generated lung pathology similar to wild type BMCMC, suggesting that mast cell degranulation, rather than production of cytokines, causes WSN induced lung pathology. Thus, we have identified a unique inflammatory cascade which could be therapeutically targeted to limit morbidity following infection with influenza virus.
Development of a humanized mouse model to study human immune cell responses in vivo (P1468)
Authors: Herndler-Brandstetter Dietmar , Strowig Till , Flavell Richard
Published: January 2023

Humanized mice hold great promise for modeling and studying human diseases in vivo, and to enable vaccine testing. Transplantation of human hematopoietic stem cells into immunocompromised newborn mice allows the development of a human immune system in vivo. NOD scid γc-/- (NSG) and Balb/c Rag2-/- γc-/- (BRG) mice are the most commonly used strains. However, some limitations have restricted the utility of humanized mice in translational research. In particular, the development and survival of human T cells and NK cells is suboptimal. We have therefore generated a human signal regulatory protein alpha (SIRPα) knock-in mouse, which prevents phagocytosis of human cells by mouse myeloid cells. We demonstrate that BRG Sirpα mice have an increased survival of human naïve CD8 and CD4 T cells, and NK cells in the periphery compared to NSG mice. The BRG Sirpα mice, together with human MHC and cytokine knock-in mice will thus be a crucial step towards a better environment for human immune cells in the mouse host to study human antigen-specific immune responses in vivo.
Selective T cell receptor dependencies provoke a revised model of innate-like T cell development (P1431)
Authors: Wencker Melanie , Cope Andrew , Hayday Adrian
Published: January 2023

γδ cells include prototypic “innate-like” T cells that cast new light on lymphocyte biology. For example, many have their functions developmentally pre-programmed in the thymus so that they can respond more rapidly to challenge than can conventional T cells. Likewise, rather than deleting them or converting them to T-regs, agonist engagement in the thymus reportedly skews γδ cells toward IFN-γ-producing effectors. Conversely, those cells that do not engage agonist default to IL-17 production. Paradoxically however, thymic progenitors of CD27(-) IL-17-producing γδ cells are TCRhiCD127hiCD44hi which is typical of pre-activated cells, whereas the phenotype of most CD27(+) IFN-γ producers is typical of naïve T cells. To explore this paradox, we examined the SKG mouse which carries a hypomorphic ZAP70 mutation, thus attenuating TCR signaling but retaining TCR expression so that γδ T cell subsets can be identified. Unexpectedly, reducing TCR-signaling largely prevented the development of IL-17-producing γδ cells, whereas IFN-γ-producing γδ cells appeared normal. Nonetheless, deeper analysis showed that IFN-γ-producing γδ cells with the phenotype of agonist selection were depleted. These data permit a revised model in which there are two pathways of γδ T cell development. One gives rise to innate-like T cells that require a developmental TCR signal; from the other, cells biased toward IFN-γ emerge as adaptive cells, without significant dependence on developmental TCR signaling.
Natural killer cell education results in prolonged survival through a cytokine receptor balance mediated mechanism (P1435)
Authors: Felices Martin , Lenvik Todd , Ankarlo Dave , Curtsinger Julie , Cooley Sarah , Tolar Jakub , Blazar Bruce , Anderson Stephen , Miller Jeffrey
Published: January 2023

Although NK cell differentiation leads to acquisition of function, little is known about how cell survival affects the NK cell repertoire. We found that Killer-cell immunoglobulin-like receptor positive (KIR+) NK cells, which exhibit enhanced cytotoxicity and cytokine production, survived better than KIR- NK cells under serum starving conditions. Accordingly, KIR+ NK cells have more anti-apoptotic proteins, while they express less Fas and FasL. As NK cell survival and homeostasis are mediated by cytokine signaling, we studied expression of IL-15 and IL-2 signaling components: the shared IL-2Rβ and the common γ chain, and the individual “private” components, IL-2Rα for IL-2 and IL-15Rα for IL-15. IL-2Rα was expressed at lower levels on KIR+ NK cells while the common γ chain was higher in these cells. Interestingly, IL-15 has been shown to induce survival signals better than IL-2. We hypothesized that IL-15Rα and IL-2Rα compete for common components to influence survival and tested this by overexpressing or knocking down IL-2Rα. IL-2Rα expression negatively influenced IL-15 mediated survival confirming our hypothesis. Since no crosslinking or IL-2 signals were present in this experiment, this role differs from that of IL-2 in AICD, supporting our competition mechanism. Finally, we found a direct correlation between NK cell education/licensing and enhanced survival. In the clinic these findings could lead to better NK cell persistence required for clinical efficacy.