bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokines and Chemokines Induced by Human Herpesvirus-8 Infection in Myeloid Dendritic Cells and B Cells (128.27)
Authors: Knowlton Emilee , Rappocciolo Giovanna , Piazza Paolo , Jenkins Frank , Jais Mariel , Rinaldo Charles
Published: January 2023

Objectives: Myeloid dendritic cells (DC) and B cells are professional antigen presenting cells (APC) susceptible to HHV-8 infection after DC-SIGN receptor binding. However, the virus elicits productive infection in B cells but not in DC. We examined if these different virus pathways were related to differential cytokine and chemokine production.

Methods: DC and B cells were loaded with purified HHV-8, UV-inactivated HHV-8 and/or endotoxin. Supernatants were examined for cytokines and chemokines from 0 to 48h post-infection by Cytometric Bead Array (BD).

Results: Peak levels of TNF-α, IL-6, IL-8, IL-10, MIP-1α and MIP-1β of 52 (IL-10) to 4.4 x 104 (MIP-1β) pg/ml were detected in B cell cultures by 24-48h of HHV-8 infection, which were 3.5 to 52.5 fold higher than in uninfected B cell cultures. In contrast, DC produced peak amounts of IP-10, MCP-1, RANTES, MIP-1α and MIP-1β of 19 (IP-10) to 1.5 x 104 (MIP-1β) pg/ml by 24-48h, which were 1.5-10 fold higher than in uninfected DC. HHV-8 and UV-inactivated HHV-8 suppressed production of IL-12p70 in endotoxin treated DC. Furthermore, UV inactivation diminished cytokine/chemokine production in B cells but not DC.

Conclusions: Elevated levels of several inflammatory cytokines and MIP chemokines were detected in HHV-8 infected B cells. In contrast, HHV-8 and UV-inactivated HHV-8 loaded DC produced MIP chemokines MCP-1 and IP-10, but not IL-12 p70. Distinct cytokine production by HHV-8 infected B cells and DC could play a role in viral replication in, and function of, these APC.

(RO1 CA82053,U01 AI35041)
Aged-associated resistance to viral disease correlates with decreased recruitment of mature NK cells to the lymph node draining the site of viral infection and not with defective adaptive immunity (128.31)
Authors: Fang Min , Sigal Luis
Published: January 2023

Ectromelia virus (ECTV) is an Orthopoxvirus that causes mousepox, the murine equivalent of human smallpox. The mice strains can be divided into resistant and susceptible stains. C57BL/6 (B6) mice are naturally resistant to mousepox due to the collaborative interaction of innate and adaptive immune responses. However, we have found that starting at 8 months of age, B6 mice gradually lost their natural resistance to mousepox and frequently die 7-10 days post infection (PI). Compared with young mice, aged mice had highly increased virus loads in central organs and severely reduced T cell responses at 7 day PI. Yet, the defect was not T cell intrinsic because when adoptively transferred into young mice, the T cells from aged mice responded to infection as the youngs. Further analysis showed that as compared to young mice, the virus loads in the central organs of aged mice were significantly increased at 3 days PI, a time when the T cell responses were undetectable. Together, these suggested that the susceptibility of aged mice was due to decreased innate rather than adaptive immunity. We have recently shown NK cells control the early spread of virus from the D-LN to central organs. Therefore, we next focused on this cell population. We found that the LNs of naïve aged mice have a reduced proportion of NK cells which is particularly accentuated for the mature (CD27-, CD11b+) population. Furthermore, following viral infection, the recruitment of mature NK cells to the draining LN of aged mice was impaired. Together our data suggests that a defect in the homing of mature NK cells to the LN result in uncontrolled early viral replication and dissemination. Our data sheds new light to our understanding for why older individuals are more susceptible to viral disease.

This work is supported by NIH grant R21AI077021 to LJS
Lack of oviduct pathology in mice infected with TLR2-signaling deficient chlamydiae is associated with earlier resolution of oviduct inflammation, and a decreased Th17 response (129.10)
Authors: Darville Toni , Frazer Lauren , Scurlock Amy M , Chandra-Kuntal Kumar , O'Connell Catherine
Published: January 2023

TLR2 KO mice infected with wild-type C. muridarum strain Nigg and immunologically normal mice infected with plasmid-deficient C. muridarum CM3.1, that fails to activate TLR2, are protected from oviduct pathology during primary and challenge infection. Genital tracts harvested from normal mice sacrificed 7, 14, 21, and 28 days post infection with Nigg or CM3.1 were examined histologically. On day 14, neutrophil infiltrates were significantly decreased in the oviducts from mice infected with CM3.1 versus oviducts from mice infected with Nigg. On days 21 and 28, neutrophils and lymphocytes were significantly decreased in oviducts of mice infected with CM3.1 versus Nigg. Quantitative culture of oviduct homogenates thru 35 days post infection revealed equivalent bacterial burden and resolution of infection in both groups by day 19. Significant amounts of TNF, IL-1α, IL-1β, IL-6, GM-CSF, G-CSF, MIP-2, KC and IFN-γ were detected by bead assay in the oviduct homogenates of Nigg-infected mice on days 9 through 15, but were low or undetectable in mice infected with CM3.1. ELISPOT for IFN-γ and IL-17 producing CD4 T cells in iliac nodes reveal significantly increased Th17 cells on days 7 and 20 post infection in Nigg-infected mice. Chlamydial-induced TLR2 signaling promotes enhanced cytokine production, prolonged oviduct inflammation, and activation of Th17 cells that may contribute to induction of pathology.
Enhanced severity of malaria infection in simian immunodeficiency virus (SIV) infected rhesus macaques (129.15)
Authors: Trott Kristin A , Chau Jennifer Y , Luckhart Shirley , Abel Kristina
Published: January 2023

Epidemic HIV and malaria co-occur in Africa and Asia. We developed a rhesus macaque model of SIV and Plasmodium fragile infection to model human co-infection. From available human data, we hypothesized that P. fragile infection during acute SIV infection enhances immune activation and thereby virus replication and, conversely, that SIV infection enhances parasitemia and parasite transmission in co-infected macaques.

Immunological, parasitological and virological parameters were assessed throughout the course of infection by multiparameter flow cytometry, blood smears, and PCR.

P. fragile-only infection resulted in increased CCR5 and Ki67 expressing CD4+ T cells, potential target cells of SIV, during peak parasitemia. Indeed, acute malaria infection was associated with increased viremia in co-infected animals. Further, SIV-induced immunosuppression was correlated with severe anemia and uncontrolled parasitemia in co-infected animals. Importantly, oocyst numbers in mosquitoes that fed on blood from co-infected animals were higher than those that fed on blood from P. fragile only infected animals. These data suggest that co-infection increases the rate of transmission of both SIV and malaria in co-infected animals.

Our model can be used to elucidate the mechanisms that underlie increased transmission and altered immunity during HIV-P. falciparum co-infection in humans and to test new therapies.

NIH IR21AI077373, T32 AMID, STAR, VSTP
Innate immunity is initially effective in killing Mycobacterium tuberculosis (129.20)
Authors: Walker Tracey A , Heard Gladys T , Liao Reiling , Gill Wendy P , Urdahl Kevin B , Sherman David R
Published: January 2023

Mycobacterium tuberculosis (MTB) is a slow growing pathogen often resulting in lifelong infection. To investigate whether slow replication is an immune evasion strategy for MTB, we developed a replication tracking tool consisting of an unstable plasmid that is lost from replicating MTB at a constant rate. Mice were infected with MTB harboring this plasmid and at various times total and plasmid bearing bacteria in lungs were enumerated. MTB replication and death rates were derived using a mathematical model. Lung bacterial numbers increase for 21 days until antigen specific T cells arrive, after which time they remain constant. Using the unstable plasmid, we observed that replication and death rates were highest early and then slowed during chronic infection. Closer analysis of the acute phase revealed the dynamics of MTB replication and death. High replication and death rates were observed in the first 5 days and then both abruptly slowed. Killing was also observed in Rag1-/- mice indicating that innate immunity is effective in killing MTB. Our studies demonstrate that standard plating methods provide an incomplete picture. Using a replication tracking tool, we found that innate immunity is initially effective in killing MTB but that this is short lived. Which innate immune response mechanisms are at work and why both MTB replication and death so rapidly decrease are topics of ongoing studies.

This work was funded by the Paul G Allen Family Foundation.
Burn injury induces a myeloid suppressor phenotype and susceptibility to Pseudomonas infection (129.24)
Authors: Neely Crystal J , Wang Ming-Jin , Fuchs Erin , Vadlamudi Sivaram , Wolfgang Matthew , Cairns Bruce A , Maile Rob
Published: January 2023

Following severe burn injury, the immune system is dramatically altered resulting in a susceptibility to bacterial infections. Using a mouse model of burn injury and subsequent Pseudomonas aeruginosa infection, we hypothesized that 1) clearance will be decreased after burn and 2) expression of TLR2, important for P. aeruginosa clearance, and/or Gr1.1, a marker of myeloid suppressive ability, is altered by burn. Female C57BL/6 mice were subjected to a 20% full thickness contact burn or sham procedure. After one day, mice were inoculated s/c at the wound with 2 x104 CFU of P. aeruginosa. CFU were calculated for various organs after two days. Splenocytes were analyzed by FACS for CD11b, Gr1.1 and TLR2. Burn mice had significant morbidity (weight loss) and mortality versus sham. Burn mice developed a systemic infection (e.g. mean±SEM 4.4±0.6 x104 CFU/lung, n=6) while no CFU were recovered from sham (n=6). Infected burn mice had a significantly higher number and percent of CD11b+ TLR2hi splenocytes (22.6±3.5%) than the infected sham (3.4±0.4%). A higher number and percent of burn CD11b+ cells were Gr1.1+ (75.5±5.7%) compared to sham mice (43.3±1.2%). Therefore, although we observed an increase in TLR2+ myeloid cells following burn injury, the increased percentage of GR1.1+ CD11b+ myeloid suppressor cells could contribute to the development of a systemic P. aeruginosa infection after burn.

Supported by NIH K08 067147.
Differences in antibody and immune responses between Balb/c and C57Bl/6 mice infected with Trypanosoma cruzi (129.5)
Authors: Guyach Siobhan Elysse , Bryan Marianne A , Norris Karen A
Published: January 2023

Methods: Susceptible Balb/c and resistant C57Bl/6 mice were infected with Y strain T. cruzi. Parasitemia was monitored; plasma was collected and analyzed for specific and total antibody. T. cruzi specific responses were measured by reactivity to recombinant T. cruzi complement regulatory protein (CRP). In separate experiments, Balb/c and C57Bl/6 mice were immunized intradermally via particle bombardment (gene-gun) with plasmid DNA containing the CRP gene.

Results: Experimentally infected mice increase total plasma immunoglobulin (IgM, IgG) prior to specific response. Development of T. cruzi specific IgG response was delayed in Balb/c mice compared to C57Bl/6 mice, with comparable parasitemia. After immunization, Balb/c mice generated a higher titer IgG response than C57Bl/6 mice.

Conclusion: Experimental infection of mice with T. cruzi leads to an early increase in total plasma immunoglobulin levels, but a delayed antigen-specific response in Balb/c compared to C57Bl/6 mice. The lack of specific response early in infection in the Balb/c model was not due to an inherent inability to produce IgG in response to T. cruzi CRP antigen as these mice were more responsive to the CRP antigen in an immunization model. This suggests that the delayed T. cruzi specific response in Balb/c mice is a result of a host/pathogen interaction involving a polyclonal humoral response to the parasite.
T cell immune response to liposomal influenza A tri-peptide vaccine (130.2)
Authors: Tai Wendy , Shan Ming , Roberts Luz , Seryshev Alexander , Gilbert Brian , Corry David B.
Published: January 2023

A bivalent influenza virus (INFV) A vaccine composed of purified HA and NA surface proteins have long been available. Disadvantages of this vaccine include annual reformulation. We have developed a novel T cell-based vaccine comprised of short and highly conserved peptides derived from M2, HA, and NP peptides formulated with TLR ligands in a lipid matrix. We hypothesized that IN immunization with short and well conserved peptides will confer protection to lethally infected mice.

The tri-peptide (TP) vaccine was given IN to mice on d-21, -14, and -7 prior to INFV A and B aerosol infection (d0). Control animals received only sham liposomes (DLPC). For memory studies, mice were infected on 60d or 90d post treatment. 100% survival was observed in TP treated vs. DLPC treated mice (<10%). PAS staining of lung sections showed reduced inflammation associated with TP treatment. Although TP treatments were not long lasting, it conferred partial protection to boosted mice (40% survival), and 17% NP+ CD8 T lungs cells were observed after booster. Lung viral burden was reduced by 2 log TP treated vs. naïve mice (d2). Divergent trends of IFN-γ and IL-6 production between TP treated and naïve animals on d6 and d8 post infection were observed.

Our findings indicate that T cells play an important role in viral clearance and mediation of flu. Future studies will aim at the understanding of the recruitment and egress of these T cell populations.
Searching H5N1 Strain-Specific Hemagglutinin Epitope Restricted by HLA DR4 (130.3)
Authors: Yang Junbao , Gebe John A. , James Eddie , Yu Betty B. , Tan Venus , Kwok William W.
Published: January 2023

CD4+ T cells play a pivotal role in the viral immunity, and as such identification of unique strain specific HLA class II restricted epitopes is essential for monitoring cellular strain specific viral immunity. Using Tetramer-Guided Epitope Mapping technique, we identified HLA-DR0401 restricted HA epitopes that are strain-specific to H5N1 virion. Two immunodominant epitopes H5HA441-460 and H5HA57-76 were identified from in vitro stimulated human PBMC. Both epitopes elicit strong cellular immune responses when HLA-DR0401 transgenic mice are immunized with H5N1 subvirion indicating in vivo naturally processed immunodominant epitopes. The H5HA57-76 epitope is unique for the H5N1 strain but conserved among all H5N1 clades recommended for vaccine development by World Health Organization. The unique H5HA57-76 response was uncommon in unexposed individuals and only observed in the naïve T cell subset. Thus, H5N1 strain-specific H5HA57-76 immunogenic epitope represents a unique marker for monitoring the efficacy of vaccination or as a candidate vaccine peptide.
Evidence that prolonged antigen-driven inflammation after influenza infection helps shape the specificity of the CD8 T cell recall response (130.5)
Authors: Suarez-Ramirez Jenny E , Khanna Kamal M , Aguila Carolina , Mohni Kareem , Cauley Linda S
Published: January 2023

Influenza viruses encode four major epitopes (NP, PA, PB1 and PB2) that are recognized by naïve CD8 T cells in C57BL/6 mice. MHC class I tetramer analysis has shown that CD8 T cells specific for the NP and PA epitopes are equally represented during the response to a primary infection with the reassortant HKx31 influenza virus. Secondary challenge with the serologically distinct A/PR/8 strain, however leads to a change in the dominance hierarchy so that NP-specific cells outnumber PA-specific cells in most tissues by approximately 5:1. We recently found that processed NP antigen persists in vivo long after infectious influenza virus has been eliminated. Large numbers of endogenous NP-specific CD8 T cells in the antigen bearing lymph nodes showed signs of a response to recent antigen stimulation. In contrast, there were much smaller numbers of PA-specific CD8 T cells in the mediastinal lymph node (MLN), with a much lower frequency of activated T cells. Since DC carry viral antigens to the MLN immediately after influenza infection, we have investigated whether the local specificities of antigen-specific memory CD8 T cells in the MLN at the time of reinfection help shape the specificity of the recall response. Since there is evidence in the literature that NP epitope is more protective than PA epitope, these studies will help us to elucidate the role of antigen and the biology of local specific memory CD8 T cell response in the shaping of the CD8 T cell response during secondary infections with influenza virus. This work was supported by NIH grants AI065895 & AI071213 (LSC),
Regulation of influenza virus-induced germinal center reactions (130.9)
Authors: Boyden Alexander , Legge Kevin , Waldschmidt Thomas
Published: January 2023

Previous work from our laboratory has demonstrated a steady state environment within primary splenic germinal center (GC) reactions as measured by the ratio of non-switched (IgM+) to switched (IgM-) GC B cells, following i.p. challenge with SRBC or protein Ags precipitated in alum. Since both of these experimental antigens induce a Th2-biased response, we sought to analyze the GC reaction after challenge with influenza virus, a natural pathogen that induces B cell activation under Th1 conditions. BALB/c mice were challenged i.p. or i.n. with the H1N1 PR8 virus and splenocytes subjected to multi-parameter flow cytometric analysis. After i.p. challenge, we again observed a steady ratio of IgM+ to switched GC B cells in the spleen. Surprisingly, an intense GC response was found in the spleen following respiratory (i.n.) challenge, with loss of a steady state environment at later time points. To evaluate the degree to which Tregs contribute to control of GC responses during influenza infection, mice were treated with anti-GITR or anti-CD25 mAb prior to and during viral challenge. Treg disruption resulted in a marked abrogation of GC homeostasis in the spleen, leading to exaggerated numbers of switched GC B cells.
Viral infection alters mRNA expression of nuclear hormone receptors (NRs) and their coregulators in mouse dendritic cells (DCs) (131.1)
Authors: Ng Sinnie Sin Man , Tailor Prafullakumar , Chang Tsung-Hsien , Ozato Keiko , Kino Tomoshige
Published: January 2023

DCs, versatile regulators of host immune response against viral infection, capture antigens and present them to T-cells and subsequently activate both innate and acquired immunity. NRs, on the other hand, are intracellular receptors which function as hormone-dependent transcription factors. Some of the NRs, such as the glucocorticoid receptor and the peroxisome proliferators-activated receptors (PPARs), suppress inflammatory reactions. Thus, we examined with real-time PCR using our custom NR PCR arrays for the mRNA expression of all mice NRs and their coregulators in bone marrow derived DCs (BMDCs) infected with viruses. At the baseline, BMDCs expressed 21 NRs. PPARγ, thyroid hormone receptor-α, retinoid X receptor-α, NGF-induced factor B and Nur related factor 1 were significantly down-regulated by virus infection (15-, 6-, 6-, 50- and 15-fold reduction, respectively), while liver X receptor-α and neuron-derived orphan receptor-1 were strongly upregulated (6- and 20-fold induction, respectively). All coregulators were expressed in BMDCs at the baseline, whereas histone deacetylase 4, 6 and 10, nuclear receptor corepressor-1 and Set-TAF-Iβ were significantly down-regulated by viruses (10-, 4-, 12-, 4- and 2-fold reduction, respectively). These results indicate that viral infection alters mRNA, and possibly, protein expression of some NRs and their coregulators. Our results suggest that NR signaling pathways are integral components of the global response of DCs to viral infection.
Th17 Cells Are Not Required for Host Defense Against Murine Disseminated Candidiasis, But Are Required for Vaccine-Mediated Protection (132.10)
Authors: Lin Lin , Ibrahim Ashraf S. , Avanesian Valentina , Xu Xin , Farber Joshua , Fu Yue , Baquir Beverly , Spellberg Brad
Published: January 2023

Background: We are developing a vaccine based on the recombinant N-terminus of the candidal adhesin, Als3p (rAls3p-N), which results in significant protection against disseminated candidiasis. Vaccine protection requires upstream T lymphocytes and downstream functional phagocytes. We therefore sought to determine the role of Th17 cells in vaccine-mediated protection. Methods: We compared Th1 (CD4+IFN- ƒ×+), Th17 (CD4+IL-17+), and Th1/17 (CD4+IFN- ƒ×+IL-17+) cell frequencies in mice vaccinated with rAls3p-N + Al(OH)3 or Al(OH)3 adjuvant alone. We also compared vaccine efficacy in IL-17 deficient, CCR6 (cell surface marker of Th17 cells) deficient, or congenic wild type control mice infected via the tail-vein with C. albicans. Results: Vaccination increased Th1, Th17, and Th1/Th17 frequencies in mice. Splenocytes from vaccinated mice stimulated with rAls3p-N ex vivo produced significantly higher levels of IFN- ƒ×, IL-17, and KC than splenocytes from control mice. IL-17 and CCR6 deficient mice were not more susceptible to disseminated candidiasis than control mice. However, vaccine efficacy was abrogated in both IL-17 and CCR6 deficient mice. Conclusion: Th17 cells are not required for host defense against disseminated candidiasis in unvaccinated mice. However, the rAls3p-N vaccine stimulates Th17 and Th1/17 cells, and Th17 cells are required for vaccine efficacy in mice.
Vaccinia-specific CD4 T-cell responses in humans are predominantly restricted by HLA DR (132.22)
Authors: Jing Lichen , Mayo McCaughey Stella , Davies D Huw , Chong Tiana M , Felgner Phillip L , Kwok William W , Wilson Christopher B , Koelle David M
Published: January 2023

In humans, three to five loci encode cell-surface HLA class II heterodimers capable of binding peptides. Anecdotally, HLA DR dominates the restriction of pathogen-reactive CD4 T-cells. It is rare that primary cells have been examined on a per-cell basis, or that a large number of clones or unique fine specificities have been studied. Direct PBMC assays with multiple cytokine readouts show that HLA DR had the strongest contribution for vaccinia-specific memory CD4 T-cells. These data were reinforced by detailed investigation of the CD4 response in vaccinated persons. Amongst large panels of vaccinia-specific clones, most were DR-restricted. Genetic and proteomic tools were used to define a large number of CD4 specificities. A minimum of 42 novel CD4 antigenic regions were defined with peptides. The majority of evaluable specificities were HLA DR-restricted. This is the highest resolution data set available concerning the relative contributions of HLA class II loci to the CD4 response to a pathogen, and implies that antigen processing and presentation to HLA DR has a selective advantage in vivo in the context of live vaccinia infection. Supported by NIH AI40069 and AI067496
Anti-inflammatory effects of PDMP in Pneumocystis pneumonia (133.1)
Authors: Vuk-Pavlovic Zvezdana , Henriques Forsythe Marshaleen , Krajicek Bryan J , Behrens Marshall D , Knutson Keith L , Limper Andrew H
Published: January 2023

Glycosphingolipid (GSL) Lactosyl Ceramide (LacCer) is thought to be involved in Pneumocystis interaction with the host. Our previous results show that treatment with glycosphingolipid synthesis inhibitor PDMP reduces the fungus lung burden as well as symptoms of inflammation in mice with Pneumocystis pneumonia (PCP). Experiments were set up to resolve whether anti-inflammatory effects were due to the specific mechanisms involved in innate immune responses to Pneumocystis, or to some general anti-inflammatory property of PDMP. Animals were infected with live organisms, or challenged with either beta-glucan (the pro-inflammatory component of Pneumocystis cell wall) or LPS (inducer of lung inflammation unrelated to Pneumocystis). At the experimental endpoint the following inflammatory parameters were assessed: leukocyte recruitment to the lungs, production of pro-inflammatory cytokines and chemokines, histopathology and lung damage. Results show that glucan-induced acute inflammation partially mimics the inflammation caused by the whole organism, and PDMP suppresses some of the inflammatory parameters affected in PCP. In contrast, although LPS induces comparable intensity of inflammation as live Pneumocystis or glucan, PDMP does not suppress any inflammatory parameters measured in this group. These results suggest that the mechanism of PDMP action is related to the features specific to Pneumocystis and host recognition of the microorganism. Further studies will attempt to determine the role of LacCer in these processes.

Supported by NIH-RO1-HL62150 and Mayo Clinic
The Mycobacterium marinum RD1 locus promotes virulence and macrophage aggregation into tuberculous granulomas by enhancing induction of host matrix metalloproteinase 9 in proximal epithelial cells (133.19)
Authors: Volkman Hannah E , Pozos Tamara , Zheng John , Rawls John F , Ramakrishnan Lalita
Published: January 2023

Granulomas are organized aggregates of differentiated macrophages and other immune cells that form in response to mycobacterial infection and other persistent stimuli. While the tuberculous granuloma has long been considered a host-protective structure, our recent work in the genetically tractable and optically transparent zebrafish embryo-Mycobacterium marinum model of tuberculosis, has suggested that the early granuloma actually benefits mycobacteria. The mycobacterial RD1 virulence locus, a specialized bacterial secretion system, enhances granuloma formation, which in turn, promotes bacterial expansion and dissemination. Here we present the molecular and cellular details of RD1-induced granuloma formation. RD1-secreted effectors induce host matrix metalloproteinase 9 (mmp9) in epithelial cells neighboring the infected macrophages. By specifically knocking down host mmp9, we have shown that its expression facilitates macrophage recruitment to form granulomas and bacterial expansion. Selective mmp9 induction in immotile epithelial cells neighboring infected macrophages may serve as a mechanism to fix and expand infection foci at optimal sites. Our results provide direct evidence that early granulomas benefit mycobacteria and suggest new tuberculosis therapeutic strategies that target granuloma-promoting host susceptibility determinants like mmp9.
Lipoteichoic acid synergizes with ATP to activate dendritic cells for a pro-inflammatory TH17-polarizing phenotype (133.22)
Authors: Paustian Christopher C , Johnson Terrence D , Czerniecki Brian J , Shu Suyu , Cohen Peter A , Koski Gary K
Published: January 2023

Stimulation of dendritic cells (DCs) by exogenous danger signals such as single Toll-like receptor (TLR) ligands results in generalized DC activation. Recently, Napolitani and co-workers showed select TLR ligand pairings induced a more specialized DC activation program including enhanced IL-12 p70 secretion and the capacity to polarize CD4 T cells into IFN-γ-secreting Th1 cells. We therefore examined the effect of pairing an exogenous TLR2 ligand (lipoteichoic acid) with an endogenous danger signal, ATP, which is found in high extracellular concentrations in damaged tissues, malignant tumors, and possibly other pathophysiological settings. ATP added to LTA modified somewhat the expression of common DC activation markers, but primarily induced a distinct pattern of cytokines with greatly enhanced levels of both IL-23 and IL-1β, while TNF, IL-10 and IL-6 remained mostly unchanged. IL-12 was not produced. These DC induced a Th17 response when stimulating allogeneic T cells in a manner partially dependent on IL-1β. These studies show that pairing of endogenous and exogenous danger signals can induce a specialized DC activation program that may have important physiological implications.
Interleukin-23 is required for protection against Listeria monocytogenes (133.24)
Authors: Meeks Karen D. , Sieve Amy N. , Kolls Jay K. , Berg Rance E.
Published: January 2023

Listeria monocytogenes (LM) is a gram-positive, intracellular bacterium that can induce spontaneous abortion, septicemia, and meningitis. Recently, it has been demonstrated that interleukin (IL)-17A is necessary for an optimal immune response against LM in the liver. As IL-23-dependent cytokines, IL-17A and IL-17F induce the mobilization of neutrophils to sites of infection. The importance of IL-23 during infection with LM has not been studied. We demonstrate here that IL-23 and the IL-17 receptor A (IL-17RA), which mediates both IL-17A and IL-17F signaling, are necessary for resistance against LM infection. During infection with LM, mice deficient in either IL-23 (IL-23p19 KO) or the IL-17RA (IL-17RA KO) have increased susceptibility to infection and increased bacterial burden in the spleen and liver. Interestingly, IL-17A, IL-17F, and IL-22 are decreased in supernatants from cells of LM infected IL-23p19 KO mice. Furthermore, neutrophils are decreased in IL-23p19 KO and IL-17RA KO mice at early time points. When IL-23p19 KO mice are rescued with the administration of exogenous IL-17A, a protective phenotype similar to that seen during infection of wild-type mice is achieved. Therefore, it is likely that IL-23 regulates the optimal production of IL-17A/F during LM infection which results in early neutrophil recruitment and bacterial clearance.
Association of Toll-like Receptor polymorphisms with variation in BCG-induced immunity (133.33)
Authors: Randhawa April Kaur , Peixoto Blas , Shey Muki , de Kock Marwou , Kaplan Gilla , Hanekom Willem , Hawn Thomas
Published: January 2023

While the development of an efficacious vaccine against TB remains a global priority, the mechanisms of protection and reasons for variability of the BCG vaccine remain poorly understood. Several studies suggest that variation in host genes can affect susceptibility to TB infection. We hypothesized that variation in innate immunity genes is associated with altered immune responses to BCG and its clinical efficacy. Infants (N=12000) were vaccinated at birth with BCG and followed prospectively in a nested case-control study to identify those with active TB and exposed individuals who did not develop disease. Blood samples were collected and re-stimulated with BCG to assess innate and T cell responses. We are examining single nucleotide polymorphisms in TLR pathway genes to determine which variants are associated with BCG-induced cytokine responses. In preliminary analyses, individuals with TLR1 deficiency (TLR1_T1805G) had decreased IL-10 production, but no alteration in IFN-γ or IL-2. Studies are underway to determine whether polymorphisms in 10 additional TLR pathway genes are associated with these responses. To our knowledge, this is the first study to examine whether variation in innate immune genes is associated with different types of BCG-induced immunity.

Supported by: Puget Sound Partners for Global Health, the Dana Foundation, and NIH Contract NO1-AI-70022.
Rhinovirus infection modulates MHC class I-related chain A and B molecules in human respiratory epithelial cells (133.37)
Authors: Stanciu Luminita A , Rushwan Nagla , Telcian Aurica G , Johnston Sebastian L
Published: January 2023

Cell surface and soluble MICA and MICB molecules induced by stress/viruses could activate, respectively impair, cytotoxic activity of NKG2D+ NK and CD8 T cells.

We hypothesised that rhinovirus (RV) infection of bronchial epithelial cells alters the balance between activatory (surface) and inhibitory (soluble) MICA and MICB molecule.

MIC molecules were investigated in BEAS-2B cells (a bronchial epithelial cell line) exposed in vitro to RV1B MOI 1 ± IL-4 10ng/ml up to 72 hours by flow cytometry (intracellular and cell surface) and ELISA (soluble molecules in supernatants).

RV1B infection of BEAS-2B cells increased the levels of surface MICA in a dose- and replication-dependent manner, peaking at 24h and gradually decreased during 72h. RV1B-induced soluble MICA molecule levels increased in a time-dependent manner, peaking at 72h. IL-4 presence during RV1B infection increased RV1B-iduced surface MICA (MFI: 72±9 versus 45±10, p<0.05) and soluble MICA levels (12±8 versus 9±7, p=0.3).

Intracellular and surface MICB molecules were decreased by RV1B infection and IL-4 further decreased intracellular levels.

To conclude, RV1B infection in BEAS-2B cells in the presence of IL-4 up-regulated the surface MICA levels but also increased soluble MICA levels which could inhibit in vivo the activation of NKG2D+ cells and by doing this delaying virus clearance and prolonging the airway inflammation in virus-induced asthma.
Calpain-dependent degradation of transcription factors and X-linked inhibitor of apoptosis (XIAP) in colon epithelial cells during cell death induced by Entamoeba histolytica (133.4)
Authors: Kim Kyeong Ah , Shin Myeong Heon , Lee Young Ah
Published: January 2023

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in human beings. During the infection in the colonic tissues, amoebic trophozoites have a capability to kill host cells via apoptosis or necrosis, which triggers IL-8-mediated acute inflammatory responses. However, signaling pathways involved in host cell death by E. histolytica is not fully defined. In this study, we examined whether calpain is closely involved in cleavage of transcription factors and XIAP, which are responsible for cell survival, during cell death of colon epithelial cells induced by live trophozoites of E. histolytica. Incubation with amoebic trophozoites induced PS externalization in CaCo2 cells in a time and dose-dependent manner. In addition, E. histolytica activated m-calpain and caspase-3 in CaCo2 cells. Moreover, incubation with amoeba resulted in marked degradation of STAT proteins (STAT1, 2, 3, 5, and 6), NF-¿B (p65 or p50), and XIAP in CaCo2 cells. However, I¿B, an inhibitor of NF-¿B, was not cleaved in CoCo2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins, NF-kB, and XIAP was inhibited by pretreatment of cells with a cell permeable calpain inhibitor, calpeptin. Furthermore, calpeptin, but not z-VAD-fmk, reduced Entamoeba-induced DNA fragmentation in CoCo2 cells. These results suggest that calpain-mediated degradation of anti-apoptotic proteins, such as transcription factors and XIAP, is required for acceleration of cell death in colon epithelial cells induced by Entamoeba histolytica.
Influenza A Virus Infects Human Mast Cells and Increases Expression of Antiviral Genes (133.43)
Authors: Marcet Candy , St. Laurent Chris , Singh Nav , Befus A. Dean
Published: January 2023

Evidence is building for the role of mast cells (MC) in innate immune responses against various pathogens. In the lungs, MC exist close to epithelial cells (EC), where influenza A virus (FluA) primarily replicates. Viruses such as dengue virus and HIV are capable of replicating in MC, but no report of FluA replication in MC currently exists. Our aim was to characterize FluA infection in human MC and examine host factors in MC that might regulate virus replication.

Human MC lines (HMC1 and LAD2) and primary MC cultured from human peripheral blood (PBMC) were exposed to FluA (PR/34/8, 20HAU/ml). EC lines (MDCK and Calu-3) were used as comparative cell types. The presence of RNA viral transcripts by RT-PCR followed similar time courses in MC and EC. Supernatants were harvested at various time points after infection. Total FluA parcticles as determined by ability to agglutinate red blood cells was significantly lower (p<0.05) in MC (27 ± 13 HAU/ml) than in EC (480 ± 160 HAU/ml) at 4 days after infection. Infectious FluA particles as determined by ability of supernatants to infect Calu-3 were detected from EC but not MC.

Since FluA replication was diminished in MC compared to EC, we explored whether MC express and/or upregulate antiviral genes in response to FluA. We have data that RNA transcripts of antiviral genes, such as nitric oxide synthase 3 and protein kinase R, are increased in MC exposed to FluA. Our evidence suggests that MC have antiviral mechanisms to restrict FluA replication, which may contribute to host immunity.
Geriatric adults have a significant defect in interferon-alpha production in response to influenza (133.50)
Authors: Canaday David H , Amponsah Naa Ayele , Ramachandra Lakshmi
Published: January 2023

Ninety percent of deaths attributed to influenza are in the elderly. While age-related decline in T cell function has been demonstrated, much less is known about the innate immune responses during natural infection and vaccination that shape humoral and cell mediated responses.

We analyzed interferon alpha (IFN-alpha) production in peripheral blood mononuclear cells (PBMCs) isolated from younger adult and geriatric individuals that were stimulated with influenza or TLR ligands. A majority of geriatric PBMCs made significantly lower amounts of IFN-alpha, a signature anti-viral cytokine, in response to influenza. Surprisingly, geriatric PBMCs had normal production of IFN-alpha in response to the TLR9 ligand, CpG ODN, and the TLR7 ligand, guardiquimod. We determined that plasmacytoid dendritic cells (pDCs) are the main producers of IFN-alpha in response to influenza, CpG ODN and guardiquimod in PBMCs by cell depletion studies, and that influenza signals through TLR7. We are currently investigating the mechanism for this defect in IFN-alpha production in response to influenza by geriatric pDCs.

A better understanding of the specific innate immune responses to influenza in geriatric individuals may allow a better understanding of the pathophysiology of the host-influenza interaction and therefore allow a more targeted approach to vaccine design.

This work is supported by AI077056 and AI36219.
Structure and function of the activating receptor CLEC-2. (134.10)
Authors: O'Callaghan Chris A , Watson Aleksandra A , Fenton-May Angharad E , Christou Charita M
Published: January 2023

We are studying the immune recognition of diseased or damaged cells. In cytotoxic cells, the C-type lectin-like receptor NKG2D plays a key role in this distinction. A number of related receptors are expressed on immune cells and we are studying their function. CLEC-2 is an activating receptor which is also expressed on platelets. We have crystallized CLEC-2 and solved its structure. We have used recombinant CLEC-2 as a probe to identify an endogenous ligand for CLEC-2, which is known as podoplanin. The podoplanin-CLEC-2 interaction triggers an activating signal in the cell expressing CLEC-2. We have also solved the crystal structure of rhodocytin, an exogenous ligand for CLEC-2. The structures of both ligand and receptor allow us to produce molecular models of the interactions and its effects. These suggest that the ligand-receptor interactions will cluster CLEC-2 on the cell surface. We have used surface plasmon resonance to define the binding characteristics of the interactions of CLEC-2 with both its exogenous and endogenous ligands.
Control of NK cell activation by distribution and mobility of ligands on target cells (134.16)
Authors: Gross Catharina C , Martinez Emily , Long Eric O.
Published: January 2023

Natural killer (NK) cells are large granular lymphocytes that comprise 5-15% of peripheral blood mononuclear cells and function through cytokine production or cytotoxicity. The response of NK cells to contact with other cells is regulated through many different receptor-ligand interactions. However, it is not clear yet to what extent the distribution and mobility of ligands anchored into the plasma membrane of target cells influence NK cell activation and immune synapse formation during NK-target cell interactions. To study the role of the distribution and mobility of ligands on target cells, and to avoid the use of toxic inhibitors such as cyclodextrin, cholesterol-auxotroph Drosophila cell line S2 was transfected with GPI-linked CD48 (the ligand of NK cell receptor 2B4) and ULBP1 (the ligand of NK cell receptor NKG2D) and grown in serum-free conditions to eliminate cholesterol-enriched domains. 2B4- and NKG2D-dependent activation of NK cells was altered when the ligands where expressed in cholesterol-free S2 cells, suggesting a role of target cell membrane micro-domains in the response of NK cells. Experiments are underway to test how the mobility of ligands on the target cell may influence recognition by NK cells.
The chemokine receptor CxCR3 retains invariant natural killer T cells in the thymus (134.21)
Authors: Elewaut Dirk , Franki Ann Sophie , Dewint Pieter , Lane Thomas , Deforce Dieter , Drennan Michael
Published: January 2023

Invariant Natural Killer T (iNKT) cells are characterized by expression of a semi-invariant Vα14-Jα18 TCRα chain and NK lineage markers such as NK1.1. In the thymus, iNKT cell maturation coincides with NK1.1 up-regulation and paradoxically, the retention of the mature NK1.1+ subset. The signals involved in the retention of iNKT cells remain undefined. Here, we report that upregulation of the chemokine receptor CxCR3 on thymic iNKT cells serves to retain them in the thymus of adult mice. Strikingly, genetic deletion or neutralization of CxCR3 results in leakage of thymic iNKT cells into the peripheral circulation. These results suggest an unexpected role for CxCR3 in thymic iNKT cell homing and localization patterns.
Murine B-1 B lymphocytes have active phagocytic and microbicidal abilities (134.31)
Authors: Li Wei , Gao Jixin , Ma Xiaoying , Gu Weijie , Zhang Ke , Liu Yufeng
Published: January 2023

It has long been recognized that B cells lack phagocytic capabilities. Until recently, B cells from teleost fish were shown to have potent phagocytic abilities for particle antigens; however, whether mammalian B cells are able to accomplish phagocytosis is still not clear. Here we demonstrated that mouse peritoneal cavity (PerC) B-1 B cells, which are thought to be the counterpart of the teleost fish blood B cells, had in vivo and in vitro phagocytic activities to Staphylococcus aureus and polystyrene fluorescent microspheres. Ingestion of particles by B cells led to activation of degradative pathways, resulting in phagolysosome formation and intracellular killing of ingested microbes. About 10% of PerC B cells, mostly CD11b+ B-1 B cells, showed phagocytic activities, and there was no apparent difference between B-1a and B-1b cells in the capabilities for phagocytotisis. Spleen B cells showed a very low degree of phagocytosis. Our results revealed for the first time that mammalian B-1 B cells have phagocytic capacities, which may contribute to their role in innate and adaptive immunity.
Interleukin-12 p40 and p35 subunit production coincides with inverse relationship between innate and acquired immune responsiveness in mice initially dosed with cationic lipid DNA complexes (JVRS-100) (134.42)
Authors: Vaughn Marla Lay , Callejo Bernadette , Liang Lily , Fairman Jeffery
Published: January 2023

Closely spaced intravenous (IV) administration of a potent activator of innate immunity, cationic lipid DNA complexes (JVRS-100), induces a short (7-10) day refractory period during which re-treatment with the drug is associated with reduced innate immune activation measured by decreased circulating interferon-? levels compared to primary treatment. Since innate immune responses are thought to upregulate adaptive immune response and we observed a reduction, it was surprising to find that IV administration of JVRS-100 one day prior to a single influenza vaccine dose significantly enhanced the influenza-specific antibody response. Adoptive transfer experiments were conducted to dissect the mediator of this effect. Interferon-? response to JVRS-100 stimulation was reduced after transfer of 3 hour post treatment serum and 3 or 6 hour post treatment splenocytes. In contrast transfer of 6 hour post treatment serum resulted in a significantly increased interferon-? response to repeat immune stimulation. In addition adoptive transfer of 6 hour, but not 3 hour, post-treatment splenocytes conferred a vaccine enhancement nearly equivalent to that seen after IV pre-treatment. Cytokine studies of post treatment sera support an important role for IL12p40 and p35 subunits in these observations.

Supported by NIAID grant "Innate Immune Stimulation as a Pathogen Countermeasure"
Chronic alcohol ingestion alters zinc homeostasis along the gut-pulmonary axis and decreases bacterial clearance in the lung (134.43)
Authors: Joshi Pratibha C , Mehta Ashish , Guidot David M
Published: January 2023

Although it has long been recognized that alcohol abuse is associated with zinc deficiency and chronic lung infections, it is not known if these two observations are interrelated. We hypothesized that chronic alcohol abuse decreases zinc bioavailability in the alveolar space resulting in the dysfunction of alveolar macrophages and increases susceptibility to infections. Sprague-Dawley rats were fed an isocaloric liquid diet with or without alcohol for 6 wks. The expression of zinc transporters and zinc storage protein metallothionein (MT) was examined in small intestine and alveolar macrophages from control- and alcohol-fed rats. In some experiments, rats were inoculated with Klebsiella pneumoniae intratracheally and after 24h, bacterial burden in the lung was determined by quantifying the number of colony forming units (CFUs). The expression of zinc transporters ZIP4, ZIP1, ZNT1 and MT was significantly decreased in the small intestine of alcohol-fed rats. Zinc concentration of alveolar macrophages and bronchoalveolar lavage fluid, and the expression of ZIP4, ZIP1, ZNT1, and MT were significantly reduced in the macrophages from alcohol-fed rats. In addition, the alcohol-fed rats showed a decrease in the bacterial clearance (significantly higher CFUs/lung) as compared to the control-fed rats. These data suggest that chronic alcohol ingestion decreases zinc status of the gut resulting in the decreased bioavailability of zinc in the alveolar space, alters alveolar macrophage immune function, and decreases bacterial clearance in the lung.

Supported by P50 AA013757 and 2R01 AA011660-05A1
Live Imaging of Cytotoxic Immune Synapses Reveals LFA-1-dependent Ligand Distribution and Bidirectional Vesicular Traffic (134.5)
Authors: LIU DONGFANG , Bryceson Yenan T. , Meckel Tobias , Vasiliver Gaia , Dustin Michael L , Long Eric O
Published: January 2023

Cytotoxic lymphocytes kill target cells by releasing the content of secretory lysosomes at the immune synapse. To obtain information on the dynamics and control of cytotoxic immune synapses we imaged primary, live natural killer cells on lipid bilayers carrying ligands of activation receptors by total internal reflection fluorescence microscopy. In the presence of the LFA-1 ligand ICAM-1, ligands of co-activation receptors 2B4 and NKG2D segregated into central and peripheral regions, respectively. Exocytosed lysosomal protein LAMP-1 accumulated in a central and stable region of the synapse, which overlapped with a site of membrane internalization. Lysosomal compartments reached the plasma membrane at focal points adjacent to centrally accumulated LAMP-1. In the absence of ICAM-1, the distribution of receptor ligands, of exocytosed LAMP-1, and of lysosomal compartment contacts with the plasma membrane was dispersed. Thus, live imaging has revealed LFA-1-dependent organization of cytotoxic immune synapses, including a central region of bidirectional vesicular traffic.