bluuebunny/crossref_metadata_2025 huggingface dataset:
Exposure to low doses of chemically attenuated blood stage malaria parasites rapidly activates CD4+ T cells and induces strain and species transcending immunity (P4477)
Authors: Good Michael , Reiman Jennifer , McPhun Virginia , Rodriguez Bibiana , Ito Koichi , Raja Amber , De Sai , Engwerda Christian , Lee Moses , Stanisic Danielle
Published: January 2023

There has been a re-appraisal of the role of cell-mediated immunity in protection against malaria. We have developed a vaccine strategy using chemical attenuation of infected red cells to induce protective cellular immunity. Our focus has been on attenuation using a class of chemicals typified by centanamycin, which binds in AT-rich regions of the parasite genome. Chemically attenuated parasites (CAPs) do not grow in immunodeficient mice. We observe that low doses of Plasmodium chabaudi ring stage CAPs induce profound protection against different strains and species of Plasmodium. CAPs prepared using other species of Plasmodium are also effective. CAPs are rapidly removed from the peripheral blood following vaccination and are taken up by dendritic cells and macrophages in the spleen and liver. Vaccination does not induce detectable antibodies but protection is T-cell dependent. Following vaccination there is a rapid increase within the blood of activated CD4+ T cells (defined as CD49d+ CD11a+). Transfused ovalbumin-specific transgenic OTII cells are not activated following vaccination. This novel approach is now being applied to P. falciparum where we plan to vaccinate humans with low doses of P. falciparum CAP, determine their immune responses and then challenge vaccinees with viable parasite-infected human red cells to determine vaccine efficacy.
Antigen experienced and memory T cell correlates of protection in peripheral blood following administration of a whole parasite blood stage malaria vaccine (P4479)
Authors: Reiman Jennifer , McPhun Virginia , Ito Koichi , Lee Moses , Good Michael
Published: January 2023

One of the greatest obstacles to a malaria vaccine is the lack of peripheral blood correlates of protection for all candidate vaccines. We have developed a novel vaccine using chemically attenuated parasitized erythrocytes. Plasmodium chabaudi ring stages were treated in vitro with seco-cyclopropylpyrroloindole (CPI) analogs which bind parasite DNA. Mice were immunized and protection was assessed. T cell phenotypes in peripheral blood were monitored utilizing CD11a+ and CD49d+ as markers of antigen experienced T cells as well as additional memory markers and intracellular cytokine staining. Specific antibodies were assessed but were undetectable. Mice that received three vaccinations were all strongly protected when challenged 113 days after last vaccination. However, only some mice that received a single dose of vaccine were protected. We observed a direct correlation between levels of CD11a+CD49d+ cells at day 21 post vaccination and subsequent protection, for CD4+ (r2=0.704, p=0.002) and CD8+ (r2=0.888, p=0.005) T cells. Immunity was abrogated if CD4+ T cells were depleted prior to challenge. Subsequently, we have observed generation of effector memory cells in blood and the boosting of CD4+CD49d+CD11a+ cells with a second and third dose of vaccination. We have also detected IFNγ positive CD4 and CD8 T cells in response to vaccination. Additional experiments are being performed to better understand the immune phenotype and properties of these T cells.
Self-assembling peptide nanofiber vaccines elicit robust cytotoxic T cell responses (P4515)
Authors: Rudra Jai , Chesson Brent , Huelsmann Erica , Lacek Andrew , Zloza Andrew
Published: January 2023

Vaccines that elicit robust CTL responses are desirable for protection against infectious diseases and cancer. However, clinical vaccine adjuvants like alum elicit robust antibody responses but poor T cell responses. We recently reported that self-assembling peptides that form nanofibers in physiological buffers act as powerful immune adjuvants. When peptide antigens (OVA323-339) were linked to self-assembling peptides, strong adjuvant-free and antigen-specific antibody responses were observed in mice. The protective efficacy of antibodies induced with self-assembling peptide vaccines, was confirmed in a murine malaria model. In this study, we investigated whether self-assembling peptides bearing a CD8+ T cell epitope (OVA257-264) would elicit antigen-specific effector and memory T cell responses. Our results indicate that OVA257-264-nanofibers elicit significantly higher levels of antigen-specific T cells, IFN-γ, and protected against OVA-influenza compared to alum or IFA. These results suggest that self-assembling peptide nanofibers may be useful as adjuvants for vaccines where CD8+ T cell-mediated protection is desirable.
Temporal effects of intestinal ischemia/reperfusion in phagocytosis and inflammatory mediators release (P5054)
Authors: Vitoretti Luana , Ferraz-de-Paula Viviane , Gimenes-Júnior João , Acceturi Beatriz , Ligeiro-de-Oliveira Ana Paula , Breithaupt-Faloppa Ana Cristina , Lino-dos-Santos-Franco Adriana , Tavares-de-Lima Wothan
Published: January 2023

Intestinal ischemia/reperfusion (i-I/R) can induce acute lung inflammation. Furthermore, injuries seen in i-I/R may cause deterioration of the immune system. Studies have demonstrated that dendritic cells present lower responsiveness to bacterial components, while peritoneal macrophages reduce the levels of MHC-II and release less IL-12. Male Wistar rats were subjected to 45 min of ischemia of the superior mesenteric artery and then to 2 h, 3, 5 or 20 days of reperfusion. After these times, the phagocytosis and oxidative burst of peritoneal macrophages and blood monocytes were evaluated by flow cytometry. In addition, inflammatory mediators were quantified in lung culture. We observed that after 5 days of reperfusion the phagocytosis of monocytes was lower than in basal and in the other periods studied. On the other hand, the oxidative burst in this time increased. The phagocytosis of peritoneal macrophages increased after 3 and 5 days of reperfusion, while the oxidative burst increased only after 3 days. Lung explants culture from reperfused rats revealed that while the levels of IL-1β increased during the days of reperfusion, the levels of IL-10 decreased. Furthermore, the amount of IL-17A, TNF-α and IL-4 were higher after 5 days of reperfusion than in the others times. Our data indicate the i-I/R as a promoter of temporal changes in the immune system that alter its ability to maintain the homeostasis and to respond appropriately to pathogens.
Decreased expression of receptor for advanced glycation end-products (RAGE) on neutrophils following surgery necessitating cardiopulmonary bypass (snCPB) (P5058)
Authors: Nikolakopoulou Zacharoula , Creagh-Brown Benedict , Burke-Gaffney Anne , Quinlan Gregory
Published: January 2023

Surgery necessitating cardiopulmonary bypass (snCPB) is associated with systemic inflammation which can be severe. Systemic inflammation is common in the critically ill, is linked to adverse outcome and currently has no specific therapy. Insight into the pathogenesis of systemic inflammation may lead to therapies. The receptor for advanced glycation end-products (RAGE) is an ubiquitous receptor that is up-regulated in the presence of its ligands which are glycated proteins, S100 proteins and high mobility group box 1 (HMGB1). It causes a pro-inflammatory response via NF-κB and the MAP kinases. RAGE inhibition has been associated with improved outcomes in animal models of inflammation and may represent a novel target for intervention. In this study, blood was obtained pre- and post-operation from patients having cardiac surgery involving CPB. The surface and intracellular expression levels of RAGE in neutrophils and monocytes were assessed by flow cytometry and mRNA was obtained for RT-PCR. Preliminary results showed that although snCPB made little difference to intracellular RAGE, cellular RAGE decreased significantly, suggesting that the operative procedure could have initiated shedding of the receptor. There was no significant difference in the RAGE mRNA levels pre- and post-operation. This study provides a better understanding of the involvement of RAGE in systemic inflammation suggesting that shedding of the surface RAGE might contribute to the inflammation process.
Transient receptor potential melastatin 2 is a critical mediator of lipopolysaccharide-induced pulmonary inflammation and injury (P5076)
Authors: Botta Davide , Ballesteros-Tato André , Mudunuru Uma , Martin Kyle , Randall Troy , Stevenson Christopher , Lund Frances
Published: January 2023

Lipopolysaccharide (LPS)-induced production of Reactive Oxygen Species (ROS) leads to lung inflammation and damage associated with several respiratory diseases, including Acute Respiratory Distress Syndrome (ARDS). The link between oxidative stress and inflammation in ARDS led us to hypothesize that the non-selective, redox-sensitive TRPM2 cation channel contributes to the pathogenesis of this syndrome. Following intranasal LPS challenge, Trpm2-/- mice had attenuated alveolar hemorrhage compared to wild-type mice. Flow cytometric analyses showed a modest increase in PMNs and alveolar macrophages (AMs) in the lung tissue and airways of Trpm2-/- mice at the early phase (3-12 hours) of inflammation but significant reductions in PMNs, monocytes, monocyte-derived cells and lymphocytes at the mid (1-2 days) and late (3-7 days) phases. Furthermore, Trpm2-/- mice had decreased expression of proinflammatory/chemoattractant factors and matrix metalloproteases in the lung. Irradiated bone marrow chimeric mice revealed that the protective, TRPM2-deficient phenotype is mainly due to defects in irradiation-resistant lung cells, suggesting that TRPM2 may play an important role in non-hematopoietic cells (e.g. pulmonary endothelial and epithelial cells), as well as AMs. Our data support a harmful role for TRPM2 in ARDS and highlight its pharmacological inhibition as a potential therapeutic approach in treating acute pulmonary inflammation and injury in patients with bacterial infections.
A novel role for IL-15 in the regulation of redox state in stressed mouse skin (P5086)
Authors: Lee Tsung-Lin , Yie Jr-Chi , Chuang Che-Ming , Kung John , Ku Chia-Chi
Published: January 2023

The association of IL-15 with a range of inflammatory disorders has implicated its function in innate immune response. Several isoforms of IL-15 mRNA generated by alternative splicing have been found constitutively expressed in normal mouse tissues at low level. An ENU mutagenized mouse (pedigree 191) bears a mutation in the exon 7 of IL-15 mRNA. Although the transcriptional level of the canonical IL-15 mRNA in P191 was comparable with B6 wild type skin, a transcript generated from alternatively spliced exon 7 of IL-15 mRNA (IL-15_ASE7) was increased by 8 folds in the epidermis of mutant mice. Mechanical stimulation induced downregulation of IL-15 mRNA in B6 but less reduced in P191 abraded skin which also exhibited attenuated dermal cellular infiltration and reduced expression of IL-1β, TNF-α and IL-6 transcripts. Moreover, genome-wide transcriptional profiling of B6 and P191 abraded skin by DNA microarray analysis showed that genes involved in oxidative phosphorylation and glutathione metabolism were distinguishably expressed between B6 and P191 abraded skin. Particularly, transcriptional levels of Uqcrb gene encoding components of the mitochondrial respiratory chain complex III and anti-oxidant Sod-1 gene were significantly reduced in P191 skin at 48 hours after abrasion. Detailed mechanisms by which IL-15 controls the redox state in epidermal keratinocytes as well as the regulatory role of IL-15_ASE7 on IL-15 action will be elucidated.
Interleukin 16 induces intestinal inflammation via PepT1 upregulation in a pufferfish model (P5050)
Authors: Wang Ping , Lu Yi-qi , Shao Jian-zhong , Dong Wei-ren , Xiang Li-xin
Published: January 2023

Interleukin 16 (IL-16) is a chemoattractant cytokine with various effects on cellular activities and diseases. Inflammatory bowel disease (IBD) has long been a worldwide health care problem. Although its clinical features have been well described, its etiology and pathogenesis remains unclear. Moreover, the involvement of IL-16 in IBD is still far from comprehension. Here, we report for the first time, the mechanism by which IL-16 induces intestinal inflammation by up-regulating the expression of oligopeptide transporter member 1 (PepT1) in a Tetraodon nigroviridis fish model. A dextran sulfate sodium (DSS)-induced IBD model in this species revealed that the IL-16 level significantly increased accompanied by the elevation of PepT1 in the colon. Moreover, the signs of IBD were dramatically attenuated by IL-16 depletion using anti-IL-16 antibodies. In vivo administration of IL-16 induces remarkable intestinal inflammation with typical IBD features, including a series of histological damages, increased MPO activity and pro-inflammatory cytokines expression, which corresponds with significant PepT1 upregulation in the colon. Our study provides the first evidence of the connection between IL-16 and PepT1, which provides new insights into the molecular mechanism underlying IBD development. We hope that this study would provide a better understanding of IL-16 biology from fish to mammals, and the development of IL-16-based therapies for IBD.
CD1d-restricted invariant natural killer T cells mediate airway inflammation through IL-25 and IL-18. (P6257)
Authors: Watarai Hiroshi
Published: January 2023

CD1d-restricted iNKT cells are characterized by their expression of an invariant Vα14-Jα18 antigen receptor in mice and of an invariant Vα24-Jα18 in humans that recognizes glycolipid antigen presented by MHC class I-like molecule CD1d. Another distinguishing feature of iNKT cells is their promiscuous production of Th1, Th2, Th9 and Th17 cytokines. Here we identified iNKT cells consist from distinct functional subtypes based on the expression of IL-17 receptor B (IL-17RB), a receptor for IL-25: IL-17RB+ iNKT cells produce robust IL-13 and IL-9 with moderate IL-17A in response to IL-25, which is phenotypically and functionally distinct from classical IL-12-reactive IFN-γ-producing iNKT cells bearing CD122 without IL-17RB. In the in vivo experimental settings, IL-17RB+ iNKT cells play a crucial role in the induction of airway inflammation both of OVA and of RSV model. Through the elucidation of the mechanisms in the activation of IL-17RB+ iNKT cells, IL-18 instead of IL-25 is responsible for the activation of these cells in RSV model, resulting in an acute and robust production of Th2 and Th17 cytokines. Our findings reveal that IL-17RB+ iNKT cells and leading effector cells as target cells of IL-25 and IL-18 in the pathogenesis of airway diseases, illustrating the clinical potential of severe exacerbation of developing asthma by IL-17RB+ iNKT cell-dependent cellular cascade targeted therapy in humans.
The interplay of macrophages and dendritic cells with WNV-specific T cells during single-cycle flavivirus immunization. (P6332)
Authors: Winkelmann Evandro , Xia Jingya , Widman Douglas , Gorder Summer , Mason Peter , van Rooijen Nico , Bourne Nigel , Milligan Gregg
Published: January 2023

Innate immunity is important to control the early phase of infection. Macrophages and dendritic cells (DCs) play a critical role in the initial stages of immune responses to several viruses, serving as antigen-presenting cells (APCs), especially DCs, which are potent APCs and play an essential role in the induction and control of the cell-mediated immune response. Here we investigated the role of macrophages in dissemination of single-cycle flavivirus (SCFV) particles to draining lymph nodes (LN) following footpad (FP) inoculation and evaluated the APC function in the absence of macrophages. Our results demonstrated higher virus replication in the absence of macrophages and a wider spread of the particles from the initial site of infection, demonstrating that macrophages were important in the early control of the virus. On the other hand, we demonstrated that macrophages were not critical for the induction of WNV-specific CD8+ T cell response, since similar responses were observed in the presence or absence of macrophages. Further, we began to investigate the function of the TLR3, IRF3 and IFNαβR signaling pathways in the migration and activation of specific DC populations into the regional LN of mice inoculated with SCFV via FP injection. Although we found differences in the number of DCs recruited to the site of infection, our preliminary results suggest similar activation and APC function of DC from mice lacking these innate pathways.
Early events dependent on the suppression of IPS signaling are necessary for persistent viral infection (P6300)
Authors: Sullivan Brian , Teijaro John , de la Torre Juan Carlos , Oldstone Michael
Published: January 2023

Persistent infections are characterized by evasion of innate and adaptive immune responses controlled by the upregulation of immunosuppressive cytokines. However, little is known whether events during the first day of infection form the environment necessary for the establishment of a persistent infection. Using a novel in vivo competition approach by priming a persistent lymphocytic choriomenengitis virus (LCMV) infection with a non-propagating LCM virus, we found that suppression of the intracellular RNA sensing pathway and not the TLR7 pathway is necessary for establishing a persistent infection. Furthermore, signaling through IPS/MAVS up to six hours before LCMV inoculation changes the nature of the immune response within the first 24 hours. Increased cytokine expression, normally observed in the first 18-24 hours during a persistent LCMV infection, is markedly reduced upon priming. Reduced cytokine expression is associated with complete clearance of the virus within 2 weeks, suggesting that LCMV initiates persistence through immune disregulation. Interestingly, localization of viral antigen in the spleen is markedly different in primed and unprimed mice despite similar viral titers in the serum and organs three days post-infection. Thus, our data are the first to document how early events after LCMV inoculation serve to establish persistent infection and suggest that early intervention can modify the pathogenesis and course of a persistent infection.
Age-dependent decline of B cell-mediated immunity contributes to West Nile virus susceptibility in mice (P6338)
Authors: Richner Justin , Diamond Michael
Published: January 2023

With aging, the ability of the immune system to mount an efficient response declines, leading to the increased susceptibility of elderly individuals to succumb to infectious diseases. West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes fatal encephalitis in several vertebrate animals including humans. Despite similar infection rates across all ages, elderly individuals have a higher rate of WNV associated encephalitis and death, and similar results have been documented in laboratory mice. While defects in T cells have been implicated in increased WNV disease severity in aged mice, the contribution of B cell-mediated immune responses has not been addressed. In this study, we compared the immunological response in adult and old mice infected with WNV. We discovered that during the acute phase post infection old mice have delayed germinal center responses that include reduced number of T follicular helper cells and fewer activated germinal center B cells. Consequently, aged mice develop a blunted humoral response with lower serum antibody titers, altered epitope specificity, and reduced serum neutralization. These findings support a model in which decreased B cell-mediated immune responses in elderly animals failed to control viral replication at early times post infection, which resulted in rapid dissemination to the central nervous system, encephalitis, and death.
A combination of provirus stimulants with blockers of regulators of complement activation represents a novel approach for purging HIV-1 latently infected cells (P6342)
Authors: Lan Jie , Byrd Daniel , Amet Tohti , Yu Qigui
Published: January 2023

HIV-1 latently infected cells are recognized as the last barrier towards viral eradication and cure. To purge these latently infected cells, we developed an “activation-killing” approach by combining provirus stimulants (SAHA, prostratin or both) with a blocker of human CD59, a key member of regulators of complement activation (RCA), to facilitate anti-HIV-1 antibodies to trigger antibody-dependent complement-mediated lysis (ADCML) of latently-infected cells. We found that both SAHA and prostratin activated proviruses in ACH-2 cells, a well-characterized cellular model of HIV-1 latency, resulting in expression of HIV-1 gp120 on the cell surface. To prevent new rounds of infections by virus released from the stimulated cells, we treated ACH-2 cells with provirus stimulants plus antiretroviral drugs. We found that reverse transcription inhibitors had no effect on virion production, whereas protease inhibitors (PIs) significantly reduced the production of mature virions, but not gp120 expression on the cell surface. ACH-2 cells post-activation of provirus became sensitive to ADCML in the presence of a human CD59 blocker plus anti-HIV-1 gp120 antibodies or sera from HIV-1-infected patients. The lysis resulted from specific cell killing by anti-HIV-1 antibodies because the lysis did not occur using irrelevant antibodies or inactivated complement. Thus, a combination of provirus stimulants with RCA blockers represents a novel approach for purging HIV-1 latently infected cells.
Immune mediated Arenaviral Hemorrhagic disease in a novel mouse model (P6304)
Authors: Schnell Fred , Mourich Dan , Iversen Patrick
Published: January 2023

Viral hemorrhagic fever viruses (VHF) are a group of RNA viruses that can cause severe multisystemic illness in humans. Development of efficient countermeasures to this disease has been hampered by the lack of clinically relevant animal models. A better understanding of mechanisms behind disease progression would illuminate therapeutic targets. To this end, we have identified the FVB mouse strain which succumbs to a hemorrhagic fever like illness when infected with lymphocytic choriomeningitis virus (LCMV). Hemorrhagic FVB mice demonstrate increased IFN-gamma, IL-6 and IL-17, along with increased chemokine production, at early times after LCMV infection, which suggests that a viral-induced host immune response is the cause of the pathology. Antisense-targeted reduction of IL-17 cytokine responsiveness provided significant protection from hemorrhagic pathology. F1 mice derived from FVB x C57BL/6 mating were generated to better characterize immune-mediated hemorrhagic disease. LCMV-13 infected F1 mice displayed reduced numbers of LCMV specific T cells compared to both LCMV-Armstrong infected F1 and LCMV-13 infected C57BL/6 mice. This report reveals a possible immune component to Arenaviral Hemorrhagic disease and offers a potential therapeutic target in IL-17.
MHC Class I Dk expression in hematopoietic and non-hematopoietic cells is essential to NK cell licensing and murine CMV resistance (P6308)
Authors: Wei Hairong , Brown Michael
Published: January 2023

Ly49G2 inhibitory receptor-bearing NK cells are essential to viral control in murine (M)CMV infected MA/My and MHC class I Dk-expressing C57L mice. In bone marrow chimeric (BMT) mice, highly effective NK cell-mediated viral control requires Dk expression in both hematopoietic and non-hematopoietic cells. Because Dk is a licensing ligand for Ly49G2, we reasoned that Dk expression in discrete cell lineages modulates NK responsiveness and viral control. To address the question, we generated additional BMT mice and measured the effect of Dk expression on NK effector competency and virus control. As expected, G2+ NK cells in Dk-transgenic (Dk-Tg) mice reconstituted with Dk-Tg bone marrow were fully licensed and conferred MCMV resistance. Conversely, in BMT recipients with Dk expression restricted to only hematopoietic or non-hematopoietic cells, G2+ NK cells displayed reduced activation receptor stimulation and significantly less viral control. These data demonstrate that NK cell licensing is regulated by MHC class I expression in hematopoietic and non-hematopoietic cells and this corresponds to the extent of G2+ NK cell-mediated viral control. Whereas recent data has shown that licensing of adoptively transferred mature NK cells is sensitive to MHC I expression on non-hematopoietic recipient cells, ongoing adoptive transfer experiments with Dk-Tg and non-Tg mature NK cells further examines the relationship between NK cell licensing and their capacity for viral control.
T-bet: a key player in a novel type of B cell activation essential for effective viral clearance (P4333)
Authors: Rubtsova Kira , Rubtsov Anatoly , Kappler John , Marrack Philippa
Published: January 2023

IgG2a is known to be the most efficient antibody isotype for viral clearance. Here we demonstrate a novel pathway of B cell activation, leading to IgG2a production, and involving synergistic stimulation via B cell antigen receptors, TLR7 and interferon-gamma receptors. This leads to induction of T-bet expression in B cells, driving their differentiation into age-associated B cells (ABCs). T-bet positive ABCs appear during anti-viral responses and produce high titers of anti-viral IgG2a antibodies which are critical for efficient viral clearance. The results thus demonstrate a previously unknown role for T-bet expression in B cells during viral infections. Moreover the appearance of ABCs during anti-viral responses and during autoimmunity suggests a possible link between these two processes.
Monocyte and NK cell dysfunctional responses to Mycobacteria in the context of chronic HIV-1 infection (P4377)
Authors: Gasper Melanie , Sherman David , Sodora Donald
Published: January 2023

Coinfection with M. tuberculosis (Mtb) and other Mycobacteria is a leading cause of morbity and mortality in HIV+ patients. While neither monocytes nor NK cells are directly infected with HIV, several of their phenotypic and functional properties become altered during chronic infection, and both cell types are important to the control of Mtb. We hypothesized that HIV-related NK cell dysfunction may result from impaired monocyte dysfunction and impaired crosstalk with NK cells, which we will evaluate in the context of Mycobacteria infection. In fact, we have observed a decrease in IL-12, an important cytokine in NK cell activation, by blood monocytes from HIV+ individuals following a 6h stimulation with M. Bovis BCG (opportunistic pathogen), compared to uninfected donors (p=0.017). In addition, while developing a flow-based functional assay to measure NK cell killing of autologous Mtb-infected monocytes, we have established that monocytes from HIV+ patients exhibit decreased phagocytosis of fluorescently-labeled BCG (p=0.016) but similar phagocytosis of Mtb compared to uninfected donors. Further, we found that NK cells from HIV+ donors exhibit decreased killing of target cells (p=0.04). Our data demonstrate a reduction in both monocyte and NK cell functionality in the same HIV+ patients and is suggestive of synergism in the dysfunction of two innate cell types with a potential for identifying novel therapeutic targets to enhance innate cellular function in HIV+ patients.
γδ T cells' unique WC1 PPRs direct cellular responses (P4337)
Authors: Baldwin Cynthia , Yokota Payal , Chen Chuang , Flores Carlo , Hsu Hao-Ting , Telfer Janice
Published: January 2023

IFNγ-producing bovine γδ T cells are the major cell population responding in recall responses to Leptospira during the first month following vaccination. The responsive cells bear a subset of the co-receptors known as WC1 which are PRR in the SRCR CD163 superfamily. Annotation of the bovine genome showed there were 13 WC1 molecules coded for by individual genes which are distributed among cells to form a number of γδ T cell subsets. RNA silencing has shown that WC1 co-receptors contribute to the γδ T cell response to leptospira and that the responsive are found within the serologically-defined WC1.1+ γδ T cell subpopulation. Since our data indicate that the WC1 molecules expressed act as pattern recognition receptors interacting directly with bacterial components, we have now evaluated the variegated gene expression of the individual WC1 molecules by specific subsets of gamma delta T cells. We have found that individual cells express more than one WC1 gene product but only a restricted subset of the available 13, generally expressing only 2 or 3. Thus the expression of the WC1 molecules is likely to direct the response by the γδ T cells to pathogens in conjunction with the TCR which is particularly interesting given that the various subsets defined by their WC1 expression also differ functionally with some being pro-inflammatory while others express cytokines consistent with regulatory functions.
CpG ODN-induced suppressor of cytokine signaling-3 expression is required for the activation of MAP kinase and NF-κB via toll-like receptors-9. (P6303)
Authors: Lee Chuhee , Kim Kyung-Chan , Kim Nam-Yi , Lee Hwa-Young
Published: January 2023

Toll-like receptors (TLRs) recognize pathogen-derived structures which can activate the innate immune system and play an important role in the recognition of various microbial-derived molecules; lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), and bacterial DNA/synthetic CpG oligodeoxynucleotides (CpG ODN). In this study, we demonstrated that unmethylated CpG ODNs induces suppressor of cytokine signaling (SOCS-3) expression from murine macrophage RAW 264.7 cells. CpG ODN-mediated SOCS-3 expression is regulated at transcriptional level and requires de novo protein synthesis. Expression of SOCS-3 mRNA and protein was blocked by inhibitors of TLR-9, MAP kinases, and NF-κB, indicating that TLR-9 dependent MAP kinases and the subsequent NF-κB activation is required for the CpG ODN-induced SOCS-3 expression.
Kinetics of lymphocyte activation in cytokine expression studies (P6317)
Authors: Tran Kimvan , Clor Julie , Tyagarajan Kamala
Published: January 2023

Activation of lymphocytes leads to expression of cytokine receptors, cytokine secretion and the expression of specific cell surface molecules resulting in divergent immune responses. Correlation of cell surface activation with cytokine secretion levels can provide valuable information to evaluate secretion levels. In this study, we demonstrate the kinetics of CD69 and CD25 expression on human peripheral blood lymphocytes from multiple donors following activation with polyclonal activator, phytohaemagglutinin or phorbol myristate acetate and Ionomycin. The studies were performed using novel no-wash assays on the Muse™ Cell analyzer to assess CD69 and CD25 levels on total lymphocytes. Our data shows that the kinetics of CD69 and CD25 expression demonstrates that a high level of lymphocytes depict CD69 expression as early as 6 hours of stimulation, peaked at 24 hours and down regulated after 48 hours. The observed CD25 expression on lymphocytes was significantly less than that of CD69 at 6 hours, increased at 24 hours and peaked at 48 hours with differences between PHA and PMA-ionomycin activation with some variability in extent of activation between donors. Comparison of the level of cytokines secreted and lymphocyte activation will be presented. The combined measurements of activation markers at the cellular level and the secreted cytokines can provide a comprehensive analysis in monitoring lymphocyte response to polyclonal stimuli.
Cytokine/chemokine secretion profiles in differentiated mouse muscle cells (P6341)
Authors: Xiao Qiang , Sun Jingyu , Rose Tina , Samovski Dmitri , Stahl Phillip
Published: January 2023

Cytokines and Chemokines are a group of small proteins that are secreted primarily from immune cells and play essential roles in inflammation and infectious disease. Over the past decade, a great number of studies have demonstrated that some cytokines/chemokines are also secreted from adipocytes (adipokines), hepatocytes (hepatokines), and myocytes (myokines). These non-immune-cell secreted cytokines/chemokines are involved in the regulation of metabolic homeostasis as well as the development of metabolic diseases such as diabetes, obesity, and cardiovascular diseases. To further study muscle cell cytokines/chemokines secretion systematically, we collected mouse muscle cell (C2C12) culture medium samples at Day 0, 1, 3, 5 after cell differentiation and measured cytokine/chemokine concentration in these samples using MILLIPLEX® map Mouse Cytokine/Chemokine Panels I (32 analytes), II (12 analytes), and III (6 analytes) multiplex assays using the Luminex® xMAP® technology. Among the 50 cytokines/chemokines measured we found 9 cytokines/chemokines were easily detectable in C2C12 culture medium samples. Six of them, including IL-6, Eotaxin, IP-10, VEGF, Fractalkine, and TARC, showed a gradual concentration increase after cell differentiation. These data demonstrated that MILLIPLEX® map Mouse Cytokine/Chemokine panels are useful tools for cytokine/chemokine secretion profiling and indicate that muscle cells may secrete more myokines than have been previously reported.
A single population of tolerogenic APC induce two types of CD4+ Treg cells by two different mechanisms. (P1054)
Authors: Stein-Streilein Joan , Mathew Rose , Wu-Freeman Ying , Gordon Siamon
Published: January 2023

Anterior chamber associated immune deviation,ACAID, is an animal model developed to study immune privileged mechanisms of the eye. The in vitro correlate of ACAID is traditionally used to study the cellular and molecular mechanism of the Treg cell development. Tolerogenic antigen-presenting cells (TolAPC) are required to express F4/80 for induction of efferent CD8+ Treg cells. We wondered if F480 is required for the development of afferent CD4+ Treg cells subpopulations. CD4+ Treg cells from cultures containing F4/80 deficient TolAPC cells partially inhibited an in vivo proliferative response to antigen. The depletion of CD25+ T cells after (but not before) exposure to TolAPC in vitro removed all inhibitory activity of the T cells in vivo. Thus, CD4+CD25+ Treg cells generated in vitro are not dependent on F4/80 but do require PDL-1 expression by the TolAPC . Since the F4/80 dependent CD4+ CD25- Treg cells express LAP-1 and PD-1, but not FoxP3, and the F4/80 independent CD4+ CD25+ Treg cells express FoxP3, PD-1, PD-L1, but not LAP-1, they resemble the mucosally-defined Th3 and inducible (i)Treg cells, respectively. Flow cytometry analyses showed that rather than two subpopulations of TolAPC, a single population of F4/80+PD-L1+TolAPC was generated by TGFβ and antigen pretreatment. Together, these data suggest that two subpopulations of afferent CD4+ Treg cells are induced by a single population of TolAPC in vitro.
A novel mechanism of B-cell mediated immune suppression through adenosine production (P1084)
Authors: Kaku Hiroaki , Cheng Kai Fan , Al Abed Yousef , Rothstein Thomas
Published: January 2023

Immune suppression is a critical mechanism to limit excess inflammation and autoimmunity. Treg and Breg (also called B10) are both involved in this process. So far, IL-10 is believed to be a major factor of B cell mediated immune suppression. Here, we report a novel mechanism that B-1 cells suppress inflammation through adenosine generation by CD73. CD73 is an ecto-enzyme that catalyzes the extracellular dephosphorylation of AMP to adenosine. We identified a novel B-1 cell subset, CD73-high B-1 cells. CD73-high B-1 cells exist in both of C57 BL/6 (50-60 %) and BALB/c mice (30-40 %) whereas B-2 cells don’t express CD73. All of B cells express CD39 but its expression levels in B-1 cells are 2-3 fold higher than those in B-2 cells. B-1 cells produced adenosine in the presence of the substrate of these enzymes whereas B-2 cells didn’t, suggesting that coordinated expression of CD39 and CD73 on B-1 cells is important for adenosine generation. CD73 KO mice were more susceptible to dextran sulfate sodium salt (DSS)-induced colitis than WT, and transfer of WT B-1 cells ameliorated the severity of colitis judging from body weight loss, suggesting that CD73 on B-1 cells has a regulatory role on DSS-induced colitis. Thus, our findings suggest a novel regulatory role of B-1 cells on colitis through adenosine generation. We will discuss which cytokines are regulated by B-1 cell-derived adenosine and how adenosine works in cooperation with IL-10 for suppression.
Apoptotic cell-induced tolerance implicates pdc and shapes adaptive immunity (P1100)
Authors: Perruche Sylvain , Bonnefoy Francis , Kaminski Sandra , Saas Philippe
Published: January 2023

Plasmacytoid dendritic cells (pDC) are involved in innate immune response to viral infection. They can mount a strong anti-viral response with the release of high amount of type I IFN after recognition of pathogen-derived material. Since pDC demonstrate potent antigen (Ag) presentation capacity, they have been involved in adaptive immunity. Effector T cell polarization is highly dictates by the microenvironment created by the Ag presenting cell, itself affected by the microenvironment shaped during pathogen elimination. We demonstrated that pDC are critical cells in the tolerance mechanism to apoptotic cells. The microenvironment created by apoptotic cell favors pDC to induce Treg polarization. However, we demonstrated that sole TGF-β polarizes pDC to induce Th17 commitment. Thus we recently identified factors in the secretome of phagocytes eliminating apoptotic cells that prevent pDC to promote Th17 polarization but favor pDC to drive Treg commitment. Such factors implicate chemokine released by phagocytes eliminating apoptotic cells and chemokine receptor signaling in the pDC rendering pDC no longer sensitive to inflammatory stimulus. A lack in this pathway strongly induced inflammatory pDC and inhibited apoptotic cell-induced tolerance through pDC. Thus we identified a chemokine/chemokine receptor critical pathway that prevents inflammation to apoptotic bodies and therefore favor tolerance induction to apoptotic cells though the specific involvement of pDC.
Classifying malaria using multidimensional space maps (P3108)
Authors: Torres Brenda , Oliveira Jose , Schneider David
Published: January 2023

If the path to sickness differs from the path to recovery, we should treat each route differently. Current treatments that inhibit pathogen growth might not be useful when pathogens are already being cleared. To determine the trajectory of both disease and recovery we created a multidimensional map of a malaria model infection in mice, tracing the route from initial infection through recovery. We did this using physiological parameters like red blood cell counts, temperature, weight and blood sugar and included molecular markers from blood transcriptome that was serially collected daily. We then plotted transcript levels versus parasite load and anemia to create a map for the full course of the infection. We can now use this map, constructed from longitudinal data, to scaffold data obtained in human cross sectional studies where we do not know the point of the patient in the infection timeline. This reveals previously unseen structure to these data and should provoke a re-examination of past conclusions. As we add data from non-recovering mice we will build maps that predict outcomes. Our goal is to not simply use these maps as a way of identifying biomarkers but to measure how treatments warp this multidimensional disease space.
The role of the central region of YopD in Yersinia pseudotuberculosis virulence (P3144)
Authors: Adams Walter , Kwuan Laura , Duncan Miles , Auerbuch Victoria
Published: January 2023

Human pathogenic Yersinia express a type III secretion system (T3SS) critical for virulence. The T3SS proteins YopD and YopB form pores in host membranes and mediate injection of effector proteins inside host cells to disrupt innate immune defenses such as production of reactive oxygen species (ROS). Discrete functional domains within YopD have been identified, yet the YopD central region (amino acids 150-227) remains largely uncharacterized. We assessed the importance of the YopD central region by generating Y. pseudotuberculosis YopDΔ150-170 and YopDΔ207-227 mutants and analyzing their virulence in mouse infection models. Y. pseudotuberculosis YopDΔ150-170 and YopDΔ207-227 grew normally in mesenteric lymph nodes and Peyer’s patches following oral infection, but displayed a 20-29,000-fold defect in the spleen and liver. These mutants were also highly attenuated following intraperitoneal infection. These data indicate that the YopD150-227 central region is critical for Y. pseudotuberculosis disseminated infection. To identify the underlying cause of this virulence defect, we examined the ability of the YopDΔ150-170 and YopDΔ207-227 mutants to translocate T3SS cargo and inhibit ROS production in macrophages. While only the YopDΔ150-170 mutant exhibited translocation defects, both mutants failed to fully inhibit ROS production. We hypothesize that the YopD central region facilitates optimal T3SS effector protein delivery to effectively disarm innate immune defenses.
Controlling inflammation: the neurotrophic anti-inflammatory pathway in Chagas disease (P3110)
Authors: Salvador Ryan , PereiraPerrin Mercio
Published: January 2023

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease. The acute phase of infection is characterized by a strong inflammatory response, which subsides in the chronic indeterminate phase. Clinical manifestations emerge decades after initial infection in 30% of those infected, mainly affecting the heart, contributing to chronic chagasic cardiomyopathy. Several neurotrophic receptors of the Trk family and their ligands impact inflammation; nerve growth factor activates TrkA and induces monocyte chemotactic protein-1 (MCP-1) chemokine in cardiomyocytes. We showed that T cruzi activates TrkA and TrkC through a surface-bound neuraminidase named parasite derived neurotrophic factor (PDNF). We hypothesize T cruzi PDNF induces MCP-1 expression in the heart and modulate immune and reparative responses. In T cruzi infected mice, MCP-1 expression correlates with heart parasite burden. Recombinant sPDNF induces MCP-1 in a Trk-dependent manner in cardiac cells, in vitro. In naïve mouse hearts, intravenous sPDNF induces MCP-1 and inflammatory/anti-inflammatory cytokine expression, while multiple sPDNF injections increases MCP-1 receptor CCR2 and stem cell antigen-1 (Sca-1) marker transcripts. In chronically infected mouse hearts, sPDNF treatment yields decreased inflammatory cytokine expression, inflammation and fibrosis. T cruzi PDNF may modulate inflammatory and anti-inflammatory responses, which may reflect the pathogenesis of the parasite and be of therapeutic use.
Vitamin D receptor regulates mucosal immune responses to Clostridium difficile infection (P3076)
Authors: Viladomiu Pujol Monica , Hontecillas Raquel , Pedragosa Mireia , Michalak Pawel , Michalak Katarzyna , Lu Pinyi , Bassaganya-Riera Josep
Published: January 2023

Clostridium difficile is an anaerobic bacterium that has re-emerged as a facultative pathogen and can cause diarrhea, colitis or even death. Vitamin D deficiency contributes to a 5-fold increase in healthcare costs during C. difficile infection (CDI). We investigated the role of vitamin D signaling during CDI by using vitamin D receptor (VDR) -/- and wild-type (WT) mice. The loss of VDR increased disease activity, weight loss, and the severity of colonic inflammatory lesions following CDI. RNAseq analyses showed upregulation of sentrin/SUMO-specific proteases (SENPs), downregulation of phosphatidylinositol 3-kinase regulatory subunit and cAMP-specific 3’5’-cyclic phophodiesterase in colons of VDR-/- mice. SENPs mediate deSUMOylation reactions that can affect both the transcriptional activity of nuclear receptors such as VDR and peroxisome proliferator-activated receptor (PPAR) γ as well as their ability to antagonize NF-kB activity. These results provide a mechanistic basis for the upregulation of IFNγ, IL-6, IL-17 and IL-1β in colons of VDR-/- mice infected with C. difficile. In addition, miR-760-5p was down-regulated in VDR-/- mice and upregulated in WT mice after CDI. Of note, miR-760 has 128 mRNA targets, including lanthionine synthetase component C-like 2 (LANCL2), which is upstream of PPARγ. We provide novel molecular evidence in vivo suggesting novel mechanisms involving LANCL2 and PPARγ by which vitamin D regulates C. difficile-associated disease and immunopathology.
Co-infection with Nippostrongylus brasiliensis does not impair host resistance during chronic Mycobacterium tuberculosis infection: a protective role for granulomas? (P3094)
Authors: Rafi Wasiulla , Potian Julius , Bhatt Kamlesh , Gause William , Salgame Padmini
Published: January 2023

Helminths are known to exert immunomodulatory effects on the host, an effect largely mediated by their induction of generalized Th2 responses. In a recent study, using the murine nematode Nippostrongylus brasiliensis (Nb), we have shown that a prior infection with Nb makes the host more susceptible to tuberculosis (TB). In this study we sought to investigate whether coinfection would exacerbate chronic infection, a phase where Mycobacterium tuberculosis (Mtb) is contained within granulomas. Mice chronically infected with Mtb were coinfected with Nb. We report herein that despite a helminth induced Th2 component, coinfected animals had a similar bacterial burden in comparison with Mtb alone infected animals. This observation could be due to the fact that the Mtb is already established in the granuloma and any effect of the helminth induced Th2 response is unable to shift this balance. In order to further understand the kinetics of helminths influencing TB, we infected mice with Mtb and these were infected 5days later with Nb. Interestingly we observed a significant increase in lung bacterial burden in coinfected animals. These results suggest that the timing of a helminth infection in an Mtb infected individual could have completely different outcomes. Studies are underway to unravel the mechanisms behind these observations. This finding is significant as a helminth infection can occur even during adulthood in endemic areas of the world, regions which are also endemic with TB
SIGIRR limits colitic and epithelial homeostatic responses, but promotes microbiota dependent colonization resistance to enteric bacterial pathogens (P3067)
Authors: Sham Ho Pan , Yu Emily , Gulen Muhammet , Chan Justin , Bhinder Ganive , Brewster Lara , Morampudi Vijay , Gibson Deanna , Hughes Michael , McNagny Kelly , Li Xiaoxia , Vallance Bruce
Published: January 2023

Enteric bacterial pathogens such as Enterohemorrhagic E. coli (EHEC) and Salmonella typhimurium target the intestinal epithelial cells (IEC) lining the gastrointestinal (GI) tract. Despite expressing Toll like receptors (TLRs), IEC are generally hypo-responsive to invading bacteria and their products. One reason is Single Ig IL-1 Related Receptor (SIGIRR), a negative regulator of interleukin (IL)-1R/ TLR signaling expressed by IEC. To address whether SIGIRR expression impacts on enteric host defense, Sigirr deficient (-/-) mice were infected with the EHEC related pathogen Citrobacter rodentium. Sigirr -/- mice responded with accelerated IEC proliferation and strong pro-inflammatory and antimicrobial responses that were primarily IL-1R signaling dependent. Surprisingly the Sigirr -/- mice were highly susceptible to infection, carrying 100-1000x heavier pathogen burden at Day (D) 6 and D10 post infection (pi). Sigirr -/- mice were also found to be unusually susceptible to intestinal S. typhimurium colonization, developing enterocolitis without the requirement for antibiotic based removal of commensal microbes. Strikingly, the exaggerated antimicrobial responses in the Sigirr -/- mice led to the rapid loss of competing commensal microbes (~80%) from the infected intestine, reducing colonization resistance as early as D1 pi. Thus, despite limiting IEC responses to infection, SIGIRR aids host defense by promoting commensal based resistance to pathogen colonization of the GI tract.