bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous IL-6 protects exocrine gland integrity and restrains Th1 immune responses in a mouse model of primary Sjögren’s syndrome (HUM7P.309)
Authors: Zhou Jing , Jin Jun-O , Patel Ekta S , Yu Qing
Published: January 2023

IL-6 is a multi-functional cytokine that can either promote or suppress tissue inflammation depending on specific disease context. Previous reports showed that IL-6 is elevated in exocrine glands and sera of patients with Sjögren’s syndrome (SS), a prevalent systemic autoimmune disease primarily affecting tear and saliva secretion. However, the particular function of IL-6 in the SS disease setting is not defined. Here we show that IL-6 levels were increased during the development SS-like disease in C57BL/6.NOD-Aec1Aec2 mice, a well-defined model of primary SS. Interestingly, in vivo neutralization of endogenous IL-6 led to enhanced tissue apoptosis, leukocytic infiltration and Th1 immune responses in salivary and lacrimal glands. The tissue protective effect of IL-6 was also demonstrated in an anti-CD3 antibody-induced multi-organ inflammation model, as neutralization of IL-6 enhanced, whereas administration of IL-6 inhibited tissue apoptosis and caspase-3 activation in both salivary and lacrimal glands. Preliminary molecular analyses showed that the pro-survival effect of IL-6 was associated with altered Bcl-xL and Mcl-1 gene expression in the exocrine glands. Thus, IL-6 exerts tissue-protective and anti-inflammatory effects on exocrine tissues under inflammatory conditions such as primary SS, demonstrating the complex, tissue- and disease-specific function of IL-6 and the possibility of developing new therapies for SS by manipulating IL-6 expression or function.
Peripheral human blood lymphocytes are associated with metabolic risk factors based on glycemic states (HUM7P.285)
Authors: Rosendahl Alexander , Cucak Helena , Vistisen Dorthe , Philipsen Annelotte , Witte Daniel
Published: January 2023

Inflammation is considered a contributing factor to obesity-linked insulin resistance, hyperglycemia and hyperlipidemia leading to type 2 diabetes (T2D). Cytokines are increased in early T2D reflecting a phenotypic switch of the circulating immune cells. To identify cellular inflammatory markers to identify patients at risk to develop T2D, blood from subjects with normal and impaired glucose tolerance was analyzed by flow cytometry. The markers were correlated with known risk factors for T2D (FPG, 2 h PG, HbA1c and BMI). By random forest analysis for each metabolic risk factor the four highest ranked immune cell markers was identified. Foremost, the results showed a reduction of subpopulations of TLR4 expressing T and B cells and T cells expressing the IL-21 receptor with increasing BMI. These data demonstrate that circulating immune cells displayed an altered phenotype already in patients having increase of common diabetic risk factors. These data may guide and provide novel insights to the inflammatory mechanisms involved in diabetic subjects and could be considered as future biomarkers to identify patient groups at risk for disease exacerbation.
Deficiency of Foxp3/Helios co-expressing regulatory T-cells correlates with clinical acitvity in autoimmunity (HUM7P.316)
Authors: Chen Xiaohua , Vander Lugt Mark , Szabolcs Paul
Published: January 2023

Helios is expressed by thymus-derived natural CD4+Foxp3+ Tregs. There is limited knowledge of the role of Helios+ Tregs in human autoimmune disease (AD). In this study, we monitored the phenotype and activation status of these cells in 3 patients with AD pre/post autologous-CD34 selected hematopoietic stem cell transplant (auto-HSCT) and 7 controls on an IRB-approved protocol. Treg related phenotypes were tested with conjugated antibodies against CD4, CD25, CD127, Foxp3, Helios, CD45RO, CD62L. The gating strategy is shown in Fig1. In patients, the mean percentage of single Foxp3+ or single Helios+ cells among CD4+CD25+CD127low Treg was comparable or somewhat higher than in controls. However, the proportion of Foxp3/Helios-co-expressing Tregs (FH-Treg) were significantly lower in patients (21 vs 46%; p=0.01) (Fig2a). Amongst Foxp3+/Helios+ Tregs, the central memory (CD45RO+CD62L+) population was significantly lower in AD compared to control (5 vs 15%; p=0.03) (Fig2b). In one patient, the percentage of FH-Treg normalized post-auto-HSCT and decreased to pre-transplant value after relapse (Fig3c). To our knowledge, we are the first to report deficiency of FH-Treg in human AD. FH-Treg, rather than Foxp3 or Helios single positive Treg, may play an important role in regulating autoimmunity. Co-expression of Foxp3+Helios+ may represent a critical functional stage of Treg. This will be studied in a large cohort.
Maturation defects in NOD macrophages impacts beta cell regeneration (HUM7P.286)
Authors: Coudriet Gina , Criscimanna Angela , Esni Farzad , Piganelli Jon
Published: January 2023

In Type 1 Diabetes (T1D), CD4+ T cells drive autoimmune destruction of pancreatic β cells by activating M1 macrophages which hinders β cell repair. Endogenous β cell growth can occur if inflammation is tempered. A more controlled inflammatory response is necessary to induce the regenerative process, characterized by a transition of macrophage phenotypes from M1 to M2. Macrophages from NOD mice have several maturation defects. We hypothesize that upon β cell destruction in autoimmunity defective macrophages fail to transition from the M1 to M2 phenotype, rendering β cells unable to initiate the repair program. We adoptively transferred NOD.scid mice with BDC-2.5-TCR-Tg splenocytes to cause T1D. Mice receiving a CD4+ T cell-depleting antibody (GK1.5) had a restricted inflammatory response and delayed T1D onset. We observed low levels of M1 macrophage genes in pancreatic tissue after GK1.5, while M2-macrophage gene expression was maintained. Control mice demonstrated a persistent M1 phenotype overwhelming the M2 macrophage lineage. β cell maintenance is achieved though β cell expressed growth factors, facilitated through M2 macrophages. In pancreatic injury we observed that autoimmune mice have decreased growth factor expression, e.g. HGF, compared to non-autoimmune mice. These macrophage defects may render β cells unable to initiate replicative programs. We propose that addition of HGF to the islet will provide the necessary cues from macrophages for β cell growth and function.
Failure of human maternal immune tolerance at the uteroplacental interface in pre-eclampsia (HUM7P.322)
Authors: Loewendorf-Snead Andrea , Nguyen Tina , Yesayan Maria , Kahn Daniel
Published: January 2023

Background: Pre-eclampsia is an elusive disease specific to human pregnancy. Risk factors suggest an immunologic cause, but specific data is lacking. Methods: A case control study of normal to pre-eclamptic mothers was undertaken (UCLA IRB # 11-002962). Cells were harvested at the uteroplacental interface at the time of cesarean section using a surgical sponge and, together with peripheral blood lymphocytes, subjected to FACS analysis. Results: Comprehensive evaluation of the peripheral and local maternal cellular milieu demonstrates maternal immune activation during pre-eclampsia. Significant reduction of peripheral naïve CD4 T cells concomitant with increased central memory CD4 T cells is accompanied by a significant increase of HLA-DR+ Activated Treg in pre-eclampsia. At the uteroplacental interface, CD8 HLA-DR+ effector T cells and Fas+ CD56hi NK cells are significantly enriched. Discussion: Systemic and local maternal immune activation is a feature of pre-eclampsia suggesting a breakdown in maternal immune tolerance. The observed peripheral increase in Activated Tregs is not mirrored at the uteroplacental interface suggesting a potential deficit in Treg trafficking. In pre-eclampsia, maternal cellular immune activation is invoked at the uteroplacental interface involving both the T cell and NK cell compartments. The kinetics of peripheral blood CD4 immune activation suggests long-standing underlying inflammation potentially providing a diagnostic- and prognostic tool.
Monocyte-derived dendritic cells from cirrhotic patients retain similar capacity for maturation/activation and antigen presentation as those from healthy subjects (HUM7P.290)
Authors: Kaplan David , Tanoue Shiroh
Published: January 2023

Defects in dendritic cell function have been implicated in impaired T-cell responses against chronic hepatitis viruses but few studies have investigated the impact of liver cirrhosis on DC function. The purpose of this study was to compare the activation and APC capacity of MoDC from cirrhotic relative to healthy donors. CD14+ monocytes isolated from PBMC from cirrhotic or healthy donors were matured with GM-CSF/IL-4 x 24h, then activated with TNF-α/IL-1β/IL-6/PGE2/GM-CSF/IL-4 x24h with pre- and post-activation phenotyping. MoDCs were irradiated to 30Gy and pulsed with 15mer peptide pools for two liver tumor antigens alphafetoprotein (AFP) and glypican-3 (GPC3) as well as 9-10mer peptides for CMV/EBV/influenza (CEF). Autologous T-cells purified from PBMC were plated with irradiated/pulsed DC (5:1) and expanded with IL-2, IL-15 and IL-21, refeeding on d13 and d20. Expanded T-cells were evaluated by IFNγ ELISPOT and intracellular cytokine staining on d27. 13 patients with cirrhosis (CIR) and 5 healthy age-matched donors (HD) were studied. No significant difference were identified in MoDC maturation/co-stimulatory phenotype between HD and CIR examining CD40, CD70, CD83, CD86, CD137, OX40L, or HLA-DR. No significant differences were observed in MoDC expansion of AFP, GPC3 or CEF-specific IFNγ+ T-cells by Elispot or ICCS. Conclusion: MoDCs isolated from cirrhotic individuals retain similar capacity for activation, maturation and antigen-presentation as those from healthy donors.
Activated PD-1highCD127highCD27highCD45RAlowCD4 T cell phenotype is associated with increased susceptibility to HIV infection rather than exhaustion. (HUM8P.339)
Authors: Petrovas Constantinos , Paris Robert , Koup Richard
Published: January 2023

The role of PD-1 as regulator of CD4 T cells in HIV infection is not well understood. We found a differential expression of PD-1 on CD4 and CD8 T cells with the PD-1highCTLA-4lowCD127highCD27highCD45RAlowCD4 T cell population (PD-1highCD4TCM) declining with disease progression. Induction of PD-1highCD4TCM cells after HAART was accompanied by reduction of PD-1highCTLA-4highCD27highCD45RAlowCD4 T cells, a population enriched with HIV-specific CD4 T cells. Increased HIV-1 gag DNA was found in PD-1high compared to PD-1lowCD127highCD27highCD45RAlowCD4 T cells. This profile was associated with increased activation and expression of the HIV coreceptor CCR5 on PD-1highCD4TCM cells. We found increased telomerase activity indicating that PD-1 upregulation is due to a direct rather than a bystander activation of CD127highCD27highCD45RAlowCD4 T cells. In vitro stimulation of sorted populations revealed an increased production of IFN-g, MIP1-a, IL-4, IL-10, and IL-17 by PD-1high compared to PD-1lowCD127high CD27high CD45RAlow CD4 T cells. In line with our previous studies, the majority of CCR7highCXCR5highCCR6highCD4 T cells, a population with increased capacity for delivering in vitro help to autologous B cells, was found in the PD-1highCD4TCM cell compartment. Our data show that expression of PD-1 on early-differentiated CD4 T cells may represent a higher susceptibility to HIV infection associated with a recent activation and differentiation rather than exhaustion of these cells.
Investigating the role of inducible T cell kinase in CD8+ T-lymphocyte cytolytic effector function (HUM8P.343)
Authors: Kapnick Senta , Schwartzberg Pamela
Published: January 2023

CD8+ cytotoxic T lymphocytes (CTLs) are critical for eliminating virally infected cells, and defects in CTL responses are implicated in lymphoproliferative diseases. Patients with mutations in inducible T cell kinase (Itk), a tyrosine kinase that serves as an amplifier of T cell receptor (TCR) signaling, develop lymphoproliferative disease associated with ineffective responses against EBV, a virus that infects B cells. This phenotype is similar to X-linked lymphoproliferative syndrome, where mutations in the adaptor SAP lead to fatal EBV infections associated with specific defects in B cell cytolysis. Unlike SAP-deficient CTLs however, we found CTLs from Itk-deficient mice exhibit impaired killing of multiple targets, suggesting Itk-deficiency leads to global defects in cytolysis. Killing by CTLs begins when TCR signaling triggers adherence to targets, centrosome polarization, and release of secretory granules inducing cytolysis of targets. Although early events such as adhesion, actin ring formation, and polarization of lytic machinery were intact in Itk-deficient murine CTLs, we found defects in granule secretion suggesting Itk may play an unappreciated role in the final stages of killing. Exploration of the role of Itk in CTL function may provide new insight into the contribution of early TCR signaling to late stages of cytolysis, and why mutations in Itk lead to lymphoproliferative diseases.
Acute symptomatic influenza A virus (IAV) infection in humans leads to expansion of highly diverse CD8 T cell repertoires crossreactive with persistent Epstein Barr virus (EBV) (HUM8P.344)
Authors: Gil Anna , Mishra Rabinarayan , Aslan Nuray , Selin Liisa
Published: January 2023

The competence of T cell responses predominantly depends on how efficient T cell receptors (TCRs) are at recognizing antigenic epitopes. We show here that during acute severely symptomatic IAV infection there was an expansion of IAV-M1/EBV-BRLF1 and IAV-M1/EBV-BMLF1 double-tetramer+ cells directly ex-vivo in 5 HLA-A2+ patients. We questioned whether this expansion specific to these two different crossreactive responses would lead to alterations in the TCR repertoire of the IAV-M1-58, EBV-BRLF1-109 and BMLF1-280 from before, during and following acute IAV infection. Using staining with Vb MAb we found that T cell responses generated to these epitopes became surprisingly more polyclonal, with the sharing of Vb between M1, BMLF1 and BRLF1 populations which is not seen in healthy donors and which decreased 2 months later consistent with crossreactive expansion. Furthermore, by using single-cell analysis of TCRα and TCRβ repertoire of tetramer sorted IAV-M1 cells we showed dramatic changes in specific clonotype usage and in JA and JB family usage during acute IAV infection compared to before infection. In summary, these changes in TCR repertoire during acute symptomatic IAV infection suggest that during severe infection there is a preferential expansion of highly diverse crossreactive responses between IAV and the persistent virus, EBV, which leads to permanent changes in TCR repertoires to both of these two viruses.
Kinetics of antibodies against major pneumococcal proteins in mother and child (HUM8P.348)
Authors: Mendy Awa , Aderonke Adutola , Sutherland Jayne , Ota Matrin , Mendy Awa
Published: January 2023

Invasive pneumococcal diseases (IPD) are responsible for almost 1 million deaths per year among children under five years. Streptococcus pneumoniae has over 90 serotypes yet the current conjugate vaccine only includes 13 serotypes. Pneumococcal proteins are conserved across all serotypes and therefore hold potential as vaccine candidates. Study aimed to investigate the transfer of antibodies against major pneumococcal antigens from pregnant mothers to their infants, and to correlate antibody concentrations with nasopharyngeal carriage (NPC).We analysed plasma from mother’s (3 months post-birth) and infants(cord blood) to determine antibody concentrations and avidity to recombinant pneumolysin (rPly), Pneumococcal surface protein A (PspA) and choline-binding protein A (CbpA) using an in-house IgG ELISA. Antibody transfer was antigen-dependent with significantly higher levels of antibodies against PspA and CbpA in mothers compared to cord blood but similar levels for rPly. PspA and CbpA were better transferred than rPly from mother to child (p=0.0001 and p=0.0024 respectively). Antibodies to PspA and CbpA but not rPly were retained up to 3 months in mothers with no significant difference in the avidity index at any time-point. Our data shows that transfer of antibodies against major pneumococcal proteins from mother to child is antigen-dependent. Further work will explore the relationship with NPC.
Functional IGH V polymorphism is frequent in humans: implications for immunity and vaccination (HUM8P.349)
Authors: Carlson Chris , Luning Prak Eline , Dagloria Jeanne , Utsugi Heidi , Chen Christina , Alvendia Melissa , Rieder Mark , Makar Karen , Robins Harlan
Published: January 2023

Antibody heavy chain variable region genes (VH) are important for antigen binding, as evidenced by the dominant usage of particular VHs, such as the VH3b subfamily, in the immune responses to the capsular polysaccharide of H. influenza b. The presence or absence of particular VH alleles may be important for immunity. To screen for polymorphic VH alleles in humans, we sequenced functionally rearranged VH from 500K circulating B cells in each of 100 healthy young adults. We focused on the naïve repertoire (0-1 mutations compared to the closest known germline allele), and analyzed the proportion of rearrangements that were in-frame without stop codons (IF). In mice, low IF VHs have not undergone robust selection and are often pseudogenes. We confirmed several polymorphically functional VH genes with variable IF fractions, including V3-20, V5-a, V3-f, V1-8, and V3-9. We also identified homozygous individuals carrying novel, non-functional alleles at V7-4-1, V6-1, V3-66 and V2-70. Comparison of VH usage patterns between individuals revealed significant variation in the number of functional VH contributing to the naïve repertoire, with between 36 and 43 functional VH segments observed in each individual. These data demonstrate a significant level of functional allelic diversity and VH copy number variation between individuals, which could contribute to differential disease susceptibility, as well as influence vaccine efficacy.
Regulation of B7-H1 (PD-L1) expression by gastric epithelial cells and development of Treg cells during Helicobacter pylori infection (HUM8P.328)
Authors: Lina Taslima , Shatha Alzahrani , Pinchuk Irina , Reyes Victor
Published: January 2023

Helicobacter pylori (H. pylori) infects >50% of the world’s population and is linked to peptic ulcers and gastric cancer. We have previously shown that H. pylori upregulates B7-H1 expression on GEC, which, in turn, suppress T cell proliferation and induction of Treg cells in vitro, but the mechanism was unknown. Herein, we investigated the underlying mechanisms behind H. pylori-mediated upregulation of B7-H1 expression by GEC and its functional relevance to chronic infection. Using H. pylori wild type and isogenic mutant strains we showed that H. pylori requires its type 4 secretion system (T4SS) component cytotoxin associated gene A (CagA) and peptidoglycan for B7-H1 upregulation in GEC. In vivo confirmation was obtained when infection of C57BL/6 mice with H. pylori PMSS1 strain, containing a functional T4SS, but not with H. pylori SS1 strain lacking this delivery system, led to upregulation of B7-H1 expression, increased bacterial load, induction of Treg cell in the stomach and increased IL-10 in the serum. Interestingly, B7-H1 knock out mice showed less Treg cells and reduced bacterial loads. We also showed that H. pylori uses p38 MAPK pathway to upregulate B7-H1 expression in GEC. Our observations suggest that H. pylori T4SS contributes to the ability to evade immune-mediated clearance by modulating expression of B7-H1 in GEC. These observations may have important implications in vaccine efforts directed at H. pylori.
Chronic infectious exposures during pregnancy shift the distribution of cord blood fetal memory B cell subpopulations in a Kenyan cohort (HUM8P.332)
Authors: Dent Arlene , Yeo Kee Thai , Embury Paula , Anderson Timothy , Mungai Peter , Holding Penny , Malhotra Indu , King Christopher , Kazura James
Published: January 2023

Chronic infections during pregnancy can expose the fetus to foreign antigens leading to cytokine responses that affect fetal B cell development. We hypothesize that this exposure shifts the distribution of B cell subpopulations. Using flow cytometry, we examined the proportions of various B cell subpopulations in cord blood from North American (Nam) and Kenyan neonates. NAm neonates had no prenatal infectious exposures whereas Kenyan neonates examined had prenatal HIV, CMV, or P falciparum exposure, or no infectious exposures. We also measured the plasma levels of pro-inflammatory biomarkers and BAFF in NAm and Kenyan cord plasma. We found that Kenyan neonates exposed to prenatal infections had greater proportions of memory B cells (CD19+CD27+), especially the activated (CD19+CD27+CD21-IgD-) memory B cells compared to NAm neonates. HIV exposed neonates had greater proportions of atypical MBC compared to the other Kenyan infectious exposed groups. Kenyan neonates had higher levels of IP-10, TNFa, CRP, sCD14 and BAFF than NAm neonates. Among the Kenyan groups, those with prenatal infectious exposures had higher levels of INFg, IL-7, sTNFR1 and sTNFR2 compared to Kenyan neonates with no infectious exposures. These results suggest that the presence of chronic infections during pregnancy affects B cell development including activated memory B cells, some pro-inflammatory biomarkers, and BAFF levels. The functional significance of these differences needs to be further investigated.
Intranasal interleukin-12 treatment for protection against respiratory infection with methicillin-resistant Staphylococcus aureus. (INC7P.413)
Authors: Nguyen Quang Tam , Furuya Yoichi , Roberts Sean , Metzger Dennis
Published: January 2023

Methicillin-resistant Staphylococcus aureus (MRSA) represents the most common pathogen associated with nosocomial pneumonia. In the present study, the role of interleukin-12 (IL-12) in inducing protective immunity in the lungs against intranasal infection of mice with MRSA was investigated. It was found that IL-12-deficient mice showed a lower survival rate, a higher bacterial burden in both the lung and spleen, and a lower level of interferon gamma (IFN-γ) in the lung than wild-type (WT) mice. These effects were completely reversed by replacement therapy with recombinant IL-12. Furthermore, exogenous IL-12 treatment of WT mice 24 h before intranasal infection with a lethal dose of MRSA significantly improved bacterial clearance and resulted in protection from death. These IL-12 treated-mice had a greater number of the lung natural killer (NK) cells and neutrophils, and had higher levels of IFN-γ in the lung and serum compared to control mice. The majority of IFN-γ-producing cells detected in IL-12-treated mice were NK cells. IL-12-induced protection against MRSA infection was abolished in NK cell-depleted, IFN-γ deficient, and MIIG mice. In addition, we found that combination therapy with linezolid and IL-12 following intranasal infection with MRSA significantly increased survival compared to mice receiving linezolid or IL-12 alone. Our results indicate that IL-12 can play an important role in protection against MRSA infection.
Mannose receptor (CD206)-mediated signaling in human macrophages in the context of tuberculosis (INC7P.418)
Authors: Rajaram Murugesan , Ni Bin , Carlson Tracy , Schlesinger Larry
Published: January 2023

The mannose receptor (MR, CD206) is abundantly expressed on alveolar and monocyte-derived macrophages (MDMs), recognizes several pathogens, mediates phagocytosis and endocytosis, and regulates inflammatory signaling pathways. It is a major human macrophage phagocytic receptor for Mycobacterium tuberculosis (M.tb). We have shown that MR-mediated phagocytosis of M.tb by MDMs up-regulates PPARγ expression, a negative regulator of NF-kB activation. However, the associated receptor-specific downstream signaling molecules are unknown. Thus, we investigated the signaling molecules involved in MR-mediated phagocytosis and other immune functions in MDMs. M.tb infection of MDMs leads to phosphorylation of tyrosine residues in the MR cytoplasmic tail that enable the recruitment of tyrosine kinases (e.g. Syk) which transmit the signals downstream. To identify MR-mediated signaling proteins, we assayed for MR-associated proteins by MALDI-TOF and Western blot using a pull down assay with macrophage lysates and phosphorylated and un-phosphorylated MR cytoplasmic tail peptides. We have identified several proteins bound to the phosphorylated peptides and linked to signaling pathways involved in MR-mediated phagocytosis and related immune funtions. In conclusion, we are identifying key signaling molecules and pathways used by the MR to transmit signals that initiate phagocytosis and other immune-related functions in human macrophages.
NK cell differential activation induced by Mycobacterium bovis BCG substrains and nontuberculous mycobacteria. (INC7P.420)
Authors: Salazar-Lopez Luis , Castillo-Rodal Antonia , Llorente-Peter Luis , Lopez-Vidal Yolanda
Published: January 2023

Mycobacterium bovis BCG is the only available vaccine against tuberculosis. Although widespread used, it is effectiveness is variable and protection conferred might be limited as following factors; BCG substrains used for vaccination, exposure to Nontuberculous mycobacteria and different components innate immune response interaction are not yet completely understood. When NK cells, are stimulated with BCG substrains proliferate and IFNγ production is induced, but recognition pathway and actual activation profiles existence remain obscure. To evaluate the activation profile of human NK cells isolated from PBMC of 20 healthy individuals. The NK cells were cultured and stimulated with BCG substrains or Nontuberculous mycobacteria species to induce an activation profile, assessed by flow cytometry. The activated NK cells were differentially recognized and induced by each BCG substrains used. Five different BCG substrains and Nontuberculous mycobacteria were included but, only BCG Mexico, M. avium and M. scrofulaceum were able to induce NK cells activation. We have shown that the differential recognitions to BCG substrains or Nontuberculous mycobacteria on NK cells occurred this could imply highly tuned response regarding cytokines promoting and enhanced impact in the regulation of immune response.
Pro-inflammatory response and defective bactericidal activity by Mycobacterium tuberculosis/HIV-1 co-infected human macrophages (INC7P.421)
Authors: Huante Matthew , Nusbaum Rebecca , Vijayakumar Sudhamanthi , Calderon Veronica , Sutjita Putri , Goez-Rivillas Yenni , Hunter Robert Hunter , Actor Jeffrey , Cirillo Jeffrey , Ferguson Monique , Valbuena Gustavo , Endsley Janice
Published: January 2023

Mycobacterium tuberculosis (M.tb), the etiological agent of tuberculosis (TB), is the leading cause of death in people living with Human Immunodeficiency Virus (HIV). In contrast to most other opportunistic infections, susceptibility to M.tb occurs prior to significant loss of peripheral CD4+T cells in people with HIV. The molecular basis for pathogen synergy that leads to an aggressive course of TB in those with HIV-1 infection is poorly defined. The primary objective of this study is to determine how HIV-1 alters the host macrophage innate immune response to M.tb infection. Primary human monocyte-derived macrophages, alveolar macrophages from human donor bronchoalveolar lavage, and our recently developed humanized mouse model of M.tb/HIV co-infection were used for in vitro and in vivo studies. Preliminary results demonstrate that HIV-1 (JR-CSF)/M.tb (H37Rv) co-infection results in greater cell death and increased expression of pro-inflammatory cytokines and chemokines (e.g. IL-1β, IL-6, MCP) compared to mono-infection with either HIV-1 or M.tb. A corresponding increase in leukocyte recruitment into the lungs of M.tb-infected humanized mice was observed in animals with HIV-1 infection and coincided with greater mycobacterial dissemination. These studies suggest that inappropriate inflammatory responses by host macrophages may contribute to dysfunctional host immunity to M.tb in those with concurrent HIV-1 infection.
Escherichia coli Nissle 1917 colonization upregulates innate immunity and partially protects against human rotavirus infection in a neonatal gnotobiotic pig model (INC7P.422)
Authors: Saif Linda , Vlasova Anastasia , Kandasamy Sukumar , Shao Lulu , Rauf Abdul , Fischer David , Chattha Kuldeep , Kumar Anand , Rajashekara Gireesh
Published: January 2023

We evaluated how colonization with gram-positive or gram-negative commensal bacteria (alone or combined) modulates virulent human rotavirus (VirHRV) infection and innate immunity in a neonatal gnotobiotic (Gn) pig model. Gn pigs were colonized with Escherichia coli Nissle 1917 (EcN), Lactobacillus rhamnosus GG (LGG) or both (EcN+LGG) and challenged with VirHRV 15 days later. Non-colonized VirHRV challenged pigs served as controls. Colonization with each bacteria alone or combined, decreased VirHRV shedding and diarrhea scores post-challenge. Among the colonized pigs, those were lowest in the EcN-, intermediate in EcN+LGG- and highest in the LGG-colonized pigs. EcN colonization induced the highest plasmacytoid dendritic cell (DC) frequencies (EcN or EcN+LGG, all tissues), significantly increased natural killer (NK) cell activity (EcN, spleen and blood) and decreased frequencies of VirHRV-induced apoptotic and TLR4+ mononuclear cells (MNCs) (EcN, all tissues). Consistent with the highest NK cell activity, SWC3a+ MNCs (DC enriched fraction) from spleen of EcN colonized Gn pigs produced the highest levels of IL12 (essential for NK cell activation) in vitro. LGG colonization had less effect on VirHRV induced innate immune responses, and those of EcN+LGG colonized pigs were intermediate, suggesting that LGG and EcN modulate each other’s effects. Our results indicate that EcN partially protects against VirHRV infection and diarrhea by modulating innate immune responses.
TLR2 and ERK signaling control macrophage responses to Mycobacterium tuberculosis and the balance of inflammatory mechanisms and Th1 activation (INC7P.402)
Authors: Richardson Edward , Wearsch Pamela , Boom W. , Harding Clifford
Published: January 2023

Tuberculosis is one of the leading causes of death and morbidity worldwide. The causative agent, Mycobacterium tuberculosis, has co-evolved with humanity and developed numerous immune evasion strategies to establish chronic, latent infection. These strategies include modification of anti-microbial responses and antigen processing and presentation by cells that harbor the pathogen, such as macrophages. In these studies, we demonstrate that inhibition of MHC class II, expression of the anti-inflammatory cytokine IL-10, and inhibition of maximal production of the Th1-inducing cytokine IL-12 are dependent on TLR2 and ERK signaling in macrophages following M. tuberculosis infection in vitro. Furthermore, macrophages deficient in the TLR2-ERK signaling pathway (macrophages deficient in Tpl2, the kinase that activates MEK/ERK downstream of TLR signaling) are more potent at activating antigen-specific CD4 T cells, enhance T cell expression of T-bet, a marker of Th1 differentiation, and induce more IFN-γ from activated T cells. These results collectively suggest that M. tuberculosis uses the TLR2-ERK signaling pathway in macrophages to facilitate its intracellular survival and prevent efficient elimination by antigen-specific, IFN-γ-producing T cells.
The role of innate stimulation of CD4 T cells in multiple infection models (INC7P.404)
Authors: Pham Oanh , O'Donnell Hope , McSorley Stephen
Published: January 2023

Salmonella-specific CD4 T cells acquire the ability to produce IFN-γ in response to non-cognate innate stimuli. CD4-Cre/Myd88-floxed mice with CD4 T cells lacking the ability to generate non-cognate responses are more susceptible to Salmonella infection, demonstrating the importance of non-cognate stimulation in vivo. Treatment of Salmonella-infected mice with LPS caused increased production of IFN-γ, IL-12, IL-18, and IL-33 mRNA in the spleen and liver and significantly higher concentrations of IFN-γ and IL-18 were detected in the serum. These cytokines were responsible for inducing large non-cognate responses in Salmonella-specific CD4 and CD8 T cells. We are currently testing whether non-cognate activation of CD4 T cells can provide cross-protection in mouse models of Chlamydia, Listeria, and Plasmodium infection. Together these data suggest an unappreciated role for non-cognate T cell activation in response to bacterial infection.
MOV10 provides antiviral activity against RNA viruses by enhancing IFN induction through IKKε and IRF3. (INC8P.441)
Authors: Cuevas Rolando , Hornung Veit , Coyne Carolyn , Sarkar Saumendra
Published: January 2023

MOV10 (Moloney leukemia virus 10, homolog) is an interferon-inducible putative DEAG-containing RNA helicase, which has been known to be associated with the RNA-induced silencing complex (RISC). Although, it is known to inhibit HIV-1 replication, analysis of its border antiviral activity and the mechanism has not been explored. Here, we report that MOV10 exhibits antiviral activity against a number of RNA viruses including EMCV, CVB, SeV and VSV by enhancing IFN induction. The antiviral activity of MOV10 is dependent on IFN signaling, and stable expression of MOV10 enhances IRF3 dimerization and IFN/ISG induction. Using various knockout cells combined with siRNA knockdowns, we show that MOV10-mediated IRF3 activation and IFN/ISG induction is not dependent on RIG-I/MAVS-mediated RNA sensing. Upon virus infection MOV10 specifically interacts and activates IKKε to induce IRF3 activation without involving TBK1. The important role of MOV10 in mediating antiviral signaling is further supported by our findings that CVB 3Cpro protease specifically targets MOV10 for degradation as a possible innate immune evasion mechanism. Taken together, these results establish MOV10, an evolutionary conserved protein, as an antiviral gene that uses a novel signaling pathway to enhance IFN response.
Functionally distinct subsets of natural killer cells help shape and regulate the immune response during viral infection (INC8P.429)
Authors: Zamora Anthony , Sungur Can , Aguilar Ethan , Baumgarth Nicole , Murphy William
Published: January 2023

Natural killer (NK) cells are innate lymphocytes that play an important role in the immune response against certain viral infections and transformed cells. Historically, NK cells have been recognized for their direct antiviral defense; however, a more complex view of NK cell function has recently emerged highlighting their ability to direct the balance between viral infection and host immunity. While the diverse effector functions of NK cells are increasingly seen as vital components of host immunity, the specific populations of NK cells that mediate these differential effects have not been identified. In this study, we provide evidence for distinct NK cell subsets that differ in their effector and immunoregulatory functions during influenza infection. We show here that during the early stages of the host immune response to viral infection, a population of effector NK cells directly contributes to viral resistance at the site of infection, while a separate helper NK subset concurrently functions to prime the adaptive immune response by expanding dendritic cells in the lymph nodes. Conversely, at the later stages of viral infection, a subset of suppressor NK cells dampens the adaptive immune response by directly regulating antigen-specific T cells. These results suggest that adoptive transfer of specific NK subsets may serve as a useful therapeutic strategy to modulate the immune response during viral infection.
Role of inflammasome cytokines IL-1 and IL-18 during infection with neurotropic mouse hepatitis virus (INC8P.434)
Authors: Zalinger Zachary , Weiss Susan
Published: January 2023

The inflammasome is a cytosolic multiprotein complex that mediates secretion of pro-inflammatory cytokines IL-1 and IL-18. These cytokines have been found to play a role in restriction of a wide range of pathogens, and to contribute to the pathogenesis of many autoimmune conditions. IL-18 signaling has a known role in promoting production of interferon gamma from T and natural killer cells, while IL-1 polarizes Th17 responses and recruits neutrophils to sites of infection. However, work with influenza A and West Nile virus have shown that IL-1 signaling can also be necessary to promote strong anti-viral T cell responses via activation of dendritic cells. This effect was found to be specific to the site of infection. To further elucidate the roles of these cytokines we have infected mice lacking the IL-1 or IL-18 receptor with a dual neuro- and hepatotropic strain of mouse hepatitis virus (MHV). IL-18 receptor deficient mice succumb to normally non-lethal doses of MHV while IL-1 receptor deficient animals are somewhat protected from lethal infection, suggesting the cytokines play opposite roles to each other. The T cell response is similar in all genotypes, suggesting the cytokines act on innate populations of cells. We are currently investigating the behavior of the neutrophil and natural killer cell populations in inflammasome cytokine deficient mice. This work should elucidate the effect of inflammasome signaling on innate immune responses in differing sites of infections.
Diminished IL-12 production mediates impaired T-cell stimulating capacity of lymph node dendritic cells and macrophages in SIV infection (INC9P.444)
Authors: Wonderlich Elizabeth , Barratt-Boyes Simon
Published: January 2023

Mononuclear phagocytes are integral components of the innate immune system that sense invading microbes and expand antigen-specific immune responses. Thus far, their roles in progressive HIV infection remain to be clarified. We used the pathogenic simian immunodeficiency virus (SIV) rhesus macaque model to determine the immune stimulating capacity of plasmacytoid dendritic cells (pDC), myeloid DC (mDC), and macrophages (Mφ) after SIV infection, focusing on the lymph node compartment. By positive selection through live-cell sorting, we measured the ex vivo ability of each subset to respond to uridine-rich ssRNA oligos derived from the infecting strain of SIV and to subsequently drive CD4+ T cell proliferation and IFN-γ production. Isolated pDC and Mφ from infected macaques were defective at stimulating CD4+ T cells to divide and produce IFN-γ. Co-stimulatory marker upregulation on mononuclear phagocytes remained comparable in naïve and infected samples, whereas deficiencies in IFN-α and IL-12 production by pDC and Mφ were found concurrent with lower CD4+ T cell function. Furthermore, exogenous IFN-α and IL-12 supplementation was able to recover the CD4+ T cell stimulating capacity of isolated mDC and Mφ from SIV-infected lymphatic tissue. These novel findings suggest that defects in mononuclear phagocyte T cell stimulating capacity through loss of IL-12 production may contribute to the overall immune dysfunction in pathogenic SIV of rhesus macaques and HIV infection of humans.
Protein kinase D1 links IRAK1 and TRAF6 in the MyD88-dependent Toll-like receptor signaling pathway. (INM9P.457)
Authors: Yi Ae-Kyung , Park Jeoung-Eun , Kim Young-In
Published: January 2023

The initial proinflammatory responses to Saccharopolyspora rectivirgula (SR), which causes farmers' lung disease, is dependent on Toll-like receptor 2 (TLR2) and MyD88, and MyD88 is absolutely required for SR-induced protein kinase D1 (PKD1) activation. We found that SR-mediated proinflammatory responses and neutrophil infiltration in the lungs are ablated in mice treated with PKD inhibitor or lacking the PKD1 gene. However, the mechanisms by which PKD1 is activated and functions to regulate TLR signaling are incompletely understood. In the present study, we found that upon TLR ligand stimulation, the receptor signaling complex composed of MyD88, IRAK4, IRAK1, TRAF6, and PKD1 is formed in membrane lipid rafts and that IRAK4 and IRAK1, but not TRAF6, are required for recruitment of PKD1 to the receptor complex and for activation by TLR ligands. In addition, MyD88-dependent translocation of TRAF6 into lipid rafts, ubiquitination of TRAF6, degradation of IRAK1, and activation of TAK1 are dependent on PKD1. Our findings indicate that PKD1 is a downstream effector of IRAK1, but an upstream modulator of TRAF6. Together with the known ability of PKD1 to shuttle between membrane lipid rafts and the cytosol, our results further support the hypothesis that PKD1 functions as a molecular shuttle that brings TRAF6 to IRAK1 in membrane lipid rafts and then aids in translocation of the IRAK1/TRAF6 complex to the cytosol, where TRAF6 interacts with its downstream effectors.
Innate immune-induced neuronal cell apoptosis via a SARM1 mediated mechanism in the absence of viral replication. (INM9P.451)
Authors: Taylor Katherine , Woods Tyson , Peterson Karin
Published: January 2023

Neuronal cell death is a hallmark of multiple neurological diseases including viral infections. The innate immune response in the CNS can contribute to neuronal cell death indirectly through the production of cytokines and neurotoxins as well as via the recruitment of immune cells. However, the direct induction of neuronal cell death via innate immune mechanisms is less well characterized and often confused by confounding factors such as viral replication. We previously identified sterile alpha and HEAT/Armadillo motif containing 1 (SARM1) as a mediator of bunyavirus induced neuronal apoptosis. Currently, we have identified a new mechanism of SARM1-mediated neuronal cell death independent of viral infection of the cell. Treatment with TLR7 and TLR9 agonists induces significant levels of neuronal cell death in cortical neurons. Cell death was associated with induction of SARM1 and inhibition of SARM1 suppressed TLR7 and TLR9 mediated neuronal apoptosis. SARM1 localized to the mitochondria and induced oxidative damage leading to neuronal apoptosis. Mechanistic studies indicated that SARM1 co-localized with both PINK1 and UXT proteins indicating a putative mechanism of SARM1 mediated neuronal cell. Thus, activation of pattern-recognition receptors on neurons in the absence of active virus infection can lead to neuronal death and the mechanism of death is via production of SARM leading to mitochondrial damage.
Mechanisms underlying specificity of natural killer cell regulation of adaptive immunity (IRC8P.485)
Authors: Waggoner Stephen , Hatfield Steven , Brooks Taylor , Karmele Erik , Peppa Dimitra , Rydyznski Carolyn , Daniels Keith , Welsh Raymond
Published: January 2023

Viral persistence is determined in part by the regulatory activities of murine natural killer (NK) cells, including the lysis of activated CD4 T cells. We now find that NK cells also impair virus-specific memory T cell responses [and repress humoral immunity (separate abstract)] after acute Pichinde or lymphocytic choriomeningitis virus infections. Hence, limiting NK cell inhibition of adaptive immunity during vaccination could enhance long-lived protective immunity. To enable selective targeting of this NK cell activity in a vaccine, the molecular mechanisms of such suppression must be ascertained. Thus, we have explored changes in the expression of NK cell receptors (NKR) and their ligands (NKRL) on both NK cells and target T cells after infection. This analysis revealed that: 1) suppression of adaptive immunity corresponded with reduced inhibitory NKR expression on NK cells; 2) archetypical activating NKR such as NKG2D were dispensable in our system; and 3) susceptibility of activated CD4 T cells to NK cell lysis correlated with reduced expression of inhibitory NKRL (e.g. CD48) and corresponding higher levels of activating NKRL (e.g. Ly108), than were present on NK cell-resistant CD8 T cells. Further, the selectivity of NK cell killing of different T cells could be deregulated by altering NKR/NKRL expression. Our findings may facilitate development of innovative strategies to harness or inhibit NK cell lysis of T cells in the context of vaccination or immune therapy.
Consumption of plant-derived agonists that enhance NK cell activity is beneficial in infection- and DSS- induced colitis (IRC8P.486)
Authors: Snyder Deann , Hedges Jodi , Robison Amanda , Kimmel Emily , Jutila Mark
Published: January 2023

Inflammatory bowel disease (IBD) is characterized by chronic, relapsing inflammation for which there is no known cure and limited treatment and preventative options. Plant products in many nutritional supplements have been shown by multiple groups to be protective in IBD. We have identified polysaccharides from Acai berries (Acai PS) and polyphenols from apple peels (APP) that specifically stimulate γδ T cells, as well as NK cell and monocyte populations. The effects are similar in some respects to responses induced by patterns derived from the microbiota. Mice fed Acai PS, but not APP, had increased IL-12 expression in spleens, MLN and serum compared to water fed mice and displayed no adverse inflammatory effects in response to the fed supplements. Ingestion of both APP and Acai PS protected from dextran sulfate sodium (DSS)-induced colitis. Acai PS-, but not APP- fed mice with colitis had increased expression of NKG2A, an NK cell receptor implicated in protection from DSS-induced colitis. Acai PS also protected against Salmonella Typhimurium-induced enterocolitis. Novel oral innate agonists Acai PS and APP protect against colitis, but perhaps through distinct mechanisms. Augmented immune signaling is likely to contribute to maintenance of the epithelial barrier and underlying immunity. Considering the paucity of therapeutic and prophylactic options, these plant derived agonists may represent novel therapeutic options for patients with IBD.
2B4(CD244) is required for optimal activation of iNKT cells through CD48 ligation on iNKT cells. (IRC8P.489)
Authors: Shin Jung Hoon , Park Se-ho
Published: January 2023

iNKT cells are a specialized subset of T cells which can modulate immune responses in a diverse way and require fine regulation of their own reactivity. 2B4 (CD244) is a member of the SLAM family molecules and acts as both inhibitory and activating receptor in murine CD8+ T and NK cells depending on relative expression levels of CD48 on target cells, whereas human 2B4 is known to be an activating molecule. Although iNKT cells also express 2B4, the function of 2B4 on iNKT cells remains largely unknown. Here, we report that the optimal activation of iNKT cells requires 2B4. iNKT cells in 2B4 knock-out (KO) mice showed impaired activity and the symptoms of Con-A induced hepatitis were substantially weaker in 2B4 KO mice than those in wild type mice. 2B4 on iNKT cells also required CD48 engagement to exert its effect. Strikingly however, we found that CD48 providers were not necessarily target cells. These results suggest that 2B4 on iNKT cells is required for the optimal stimulation of iNKT cells and 2B4/CD48 interaction of iNKT cells can be mediated through homotypic cell-cell interaction