bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Susceptibility of S1PR3-Deficient Mice to Escherichia coli Sepsis Is Associated with an Impaired Bactericidal Activity in Macrophage
Authors: Fang XiangMing , Hou Jinchao , Shu Qiang
Published: January 2023

Background

Sepsis, a leading cause of death in critically ill patients worldwide, is characterized with imbalanced proinflammatory responses and compromised bacterial clearance in host immunity. Monocytes/macrophages play a crucial role in elimination of bacteria through innate immunity receptors.

Methods

We explored the effect of Sphingosine-1-phosphate receptor 3 (S1PR3) gene deficiency on mortality, organ injury, and bacterial clearance in cecal ligation and puncture (CLP) or Escherichia coli (E. coli)-induced sepsis. Signaling molecules responsible for S1PR3-mediated bactericidal function were dissected in peritoneal macrophages. Furthermore, S1PR3 specific agonist (GPS-725.017) was evaluated for treatment of sepsis.

Results

After sepsis onset, S1pr3−/− mice showed increased mortality and organ injury, which attributed to markedly decreased E. coli killing. Mechanically, S1PR3 regulated activity of the NADPH oxidase NOX2 complex and phagolysosomal maturation through recruiting the intracellular class III phosphatidylinositol kinase Vps34 to the phagosome. S1PR3 specific agonist (GPS-725.017) enhanced NOX2 activity in E. coli-containing phagosomes and bactericidal function in wild-type macrophages. Additionally, the survival of mice treated with GPS-725.017 was improved when compared with those treated with vehicle.

Conclusion

S1PR3 is an important receptor to connect bacterial phagosome to ubiquitous cellular machinery responsible for control of bacterial killing in macrophage. Interventions targeting S1PR3 signaling may serve as promising therapeutic approaches for sepsis.
Nicotine-containing e-cigarette exposure influences the inflammatory response in an animal model of heart failure.
Authors: Molina Megan Stanley , Gamboa Antonio , Lima Vanessa Souza , Cameron D. Joshua , O’Barr Stephen A
Published: January 2023

Heart failure is a leading cause of hospitalization and death worldwide, and it is known that the initiation and progression of the inflammatory response is highly indicative of long-term patient outcomes. Interestingly, while smoking is a well-known risk factor for cardiovascular disease, recent findings reveal that smokers have improved clinical outcomes, a shorter length of stay in the hospital, and lower in-hospital mortality and mortality risk compared to non-smokers. While the precise mechanisms behind this finding, deemed the “smoker’s paradox”, are currently unknown, the phenomenon raises questions regarding the possible therapeutic benefit of nicotine. Additionally, the health effects of electronic cigarettes (e-cigs) in the setting of heart failure also come into question. In order to investigate the possible therapeutic benefit of nicotine and the health effects of e-cigs we have emulated the smoker’s paradox by creating a chronic smoking regime in which animals are pre-treated with nicotine-containing e-cigarette vapor for 20 minutes per day for four consecutive days. On day four following a short washout period, heart failure is chemically induced and survival is recorded. Here we show that pre-treatment with nicotine-containing e-cigarette vapor significantly improves survivability in a concentration dependent manner. Furthermore, analysis of blood and heart tissue shows that pre-treatment with e-cigarette vapor induces changes in circulating levels of key cytokines and in gene expression within the myocardium. These data indicate that nicotine indeed influences the inflammatory response to heart failure and therefore influences the pathogenesis of cardiovascular disease.
Dendritic Cells from Aged Subjects Display Impaired Responses to Retinoic acid
Authors: Agrawal Anshu , Agrawal Sudhanshu , Ganguly Sreerupa , Tran Alexander
Published: January 2023

Aged subjects display increased susceptibility to mucosal diseases. Infections and diseases of the respiratory, gastrointestinal and urinary tract are prevalent in this population. Sterile inflammation of the mucosa is considered a major culprit. Retinoic Acid (RA) secreted by mucosal epithelial cells plays a major role in inducing tolerance in the mucosa. RA acts on Dendritic cells (DCs) to induce mucosal tolerance. Here we compared the response of DCs from aged and young individuals to RA with a view to understand the mechanisms underlying chronic sterile inflammation in aged mucosa. Our investigations revealed that compared to young DCs, RA stimulated DCs from aged subjects are defective in inducing T regulatory cells. Examinations of the underlying mechanisms indicated that impaired upregulation of CD141 expression on aged DCs by RA was a major factor in reduced induction of T regulatory cells. In summary, reduced response of aged DCs to RA enhances mucosal inflammation in the elderly, increasing their susceptibility to mucosal diseases. In addition, our data also suggests a novel mechanism of immunosuppression by RA in tumor microenvironment.
Controlling Leukocyte Migration in Autoimmune Encephalomyelitis by the TIPE Family of Phospholipid Transfer Proteins.
Authors: Sun Honghong , Li Ning , Porturas Thomas , Boggs Amanda , Chen Youhai H
Published: January 2023

Leukocyte migration is essential for immune organ development, immune surveillance, and autoimmune diseases. A common pathogenic mechanism of multiple sclerosis (MS) is the infiltration of the central nervous system (CNS) by activated leukocytes. The CNS is an immunologically privileged site to which access of circulating leukocytes is tightly controlled by the blood-brain barrier (BBB). How leukocytes cross the BBB during MS is not well understood. We report here that members of the TNFAIP8 (TNF-alpha-induced protein 8-like) family control leukocyte migration during experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Using single or double TIPE deficient mice generated in our lab, we found that TNFAIP8 and TIPE2 were required for the development of EAE. Although TIPE2-deficient myelin-specific TH1 and TH17 cells were hypersensitive to myelin antigens, they had an intrinsic defect in migration into CNS. Further analysis indicated that TIPE family members affected cell polarization. The molecular mechanisms underlying these abnormalities are under investigation. These findings establish that TIPE family plays important roles in EAE and could be a potential therapeutic target for treating MS. Supported by NIH grant.
Serotonin Receptor Regulation of Eosinophil Transendothelial Migration
Authors: Walker Matthew T , Green Jeremy , Ferrie Ryan , Cook-Mills Joan M
Published: January 2023

The tryptophan amino acid metabolite, 5-hydroxytryptophan (5HTP), is a metabolic precursor of serotonin. 5HTP reduces allergic inflammation in the lung by inhibiting eosinophil migration across endothelial monolayers. Blocking the metabolic conversion of 5HTP to serotonin decreases inhibition of eosinophil migration, indicating that its metabolism is required for inhibition. Currently it is unknown if serotonin receptors are involved in mediating 5HTP function. We determined which serotonin receptors are expressed by endothelial cells and eosinophils by western blot and qPCR. There was expression of the serotonin receptors 1A, 1B, 3A, and 6. Function of the receptors in transendothelial migration was addressed. Selective receptor inhibitors for serotonin receptors 1A and 1B blocked 5HTP inhibition of migration. Current data suggests serotonin receptors are involved in 5HTP inhibition of transendothelial migration and thus may serve as a target for intervention during allergic inflammation. These results will help design ongoing clinical studies addressing whether 5HTP supplementation reduces allergic asthmatic responses.
FCRL4 and FCRL5 expression distinguishes three human memory B cell subsets in tonsils.
Authors: Li Huifang , Tolnay Mate
Published: January 2023

FCRL4 and FCRL5 are two members of the human Fc receptor like family with the capacity to regulate antigen receptor signaling. FCRL4 was shown to bind IgA, whereas FCRL5 binds IgG. Although the expression of FCRL4 and FCRL5 on human B cell subsets has each been individually evaluated, no study thus far addressed their potential co-expression. Here we report that three distinct subpopulations of CD38lowIgD-memory B cells exist in human tonsils, one that co-expresses FCRL4 and FCRL5, another that is FCRL5 single-positive, and a third that lacks both proteins. We found that the FCRL5+FCRL4+ subset expresses the highest levels of surface FCRL5, CD11c, FAS, PD-1, CD32, CCR1, CCR5 and CCR6 protein, as well as the lowest levels of LAIR-1 and CD21 among the three memory B cell populations. Furthermore, microarray analysis demonstrated that the three memory B cells subsets each possess distinct gene expression profiles. The FCRL5+FCRL4− memory B subset overexpressed dozens of immunoglobulin variable genes compared to the other two subsets, in addition to the transcription factors XBP1, PRDM1 and IRF4 that drive plasmablast and plasma cell differentiation. These results suggest that the FCRL5+FCRL4− memory B subset is committed to become plasmablast. Using KREC assay we established that the FCRL5+FCRL4+ subset underwent more rounds of cell divisions than the other two subsets. The low number of cell divisions measured in the FCRL5+FCRL4− subset suggests that the cells bypassed the germinal center. The surprising association of FCRL4 and FCRL5 expression with distinct memory B cell subsets could point to the functional role of these FCRL proteins and their immunoglobulin ligands to drive B cell development.
Visualizing mucosal resident memory CD8 T cell reactivation dynamics reveals in situ division and bystander resident memory T cell development
Authors: Beura Lalit K , Thompson Emily A , Mitchell Jason , Fife Brian T , Masopust David
Published: January 2023

CD8 T cells survey tissues for the presence of abnormal cells. The parameters of CD8 T cell search patterns and immunosurveillance determines outcome of infections and cancer (elimination, controlled persistence, or death). We established a two-photon intravital microscopy model to visualize resident memory CD8 T cell (TRM) behavior and recall responses within the female reproductive tract (FRT) mucosa of living mice. Unlike what has been described in skin epidermis, FRT TRM were highly motile (10 μm/min). Local antigen recognition induced pausing and initiation of in situ TRM division, and this appeared CD4 T cell and dendritic cell independent. Expansion by dividing TRM dominated the local recall response compared to recruitment of recirculating cells or progeny of reactivated central memory T cells. TRM reactivation also triggered recruitment of Ag-specific and bystander recirculating memory cells, and these underwent a de novo TRM differentiation program in situ. These results demonstrate that 1) T cell migration patterns and differentiation may be permanently altered by unrelated infectious experience, 2) that cognate antigen within the FRT is not required to induce local TRM differentiation, 3) TRM undergo in situ division upon recall that 4) may dominate the local secondary effector and memory response.
Elucidating the role of the Mst1/Foxo1 axis in CD8+ memory T cell development
Authors: Hills L Benjamin , Huang Yina H
Published: January 2023

The Foxo1 transcription factor is required for naïve T cell homeostasis and trafficking, T regulatory cell development, and CD8 memory T cell differentiation. Two kinases, Akt1 and Mst1 differentially control Foxo1 activity. Akt1-induced Foxo1 phosphorylation at T24, S256, and S319 causes association with 14-3-3 adapters, nuclear export, and degradation, resulting in decreased transcription of Foxo1 targets. Akt1-mediated control of Foxo1 activity in CD8 T cells promotes terminal differentiation into effector cells. In contrast, Mst1 may prevent Foxo transcription factors from binding 14-3-3, promoting Foxo nuclear localization and transcriptional activity. These data suggest that Akt1 and Mst1 have opposing effects on Foxo1 activity in T cells. However, how Mst1 regulates Foxo1 activity to impact CD8 memory T cell differentiation has not been directly evaluated. We hypothesize that, while phosphorylation of Foxo1 by Akt1 causes cytosolic localization and promotes effector CD8 differentiation, phosphorylation of Foxo1 by Mst1 increases Foxo1 stability and promotes CD8 central memory differentiation. To test this hypothesis, we have generated Mst1 phospho-null and phospho-mimetic Foxo1 variants (S209A and S209D). Surprisingly, Foxo1 mutations at the Mst1 phosphorylation site differentially altered Foxo1 subcellular localization. Current studies utilizing Lm--Ova to study the antigen-specific memory response of OT-I CD8 T cells expressing WT, S209A, or S209D Foxo1 variants in vivo revealed skewed effector and central memory differentiation. Together, these studies indicate that coordinated regulation of Foxo1 by Akt1 and Mst1 kinases determine the extent of CD8 terminal effector and memory cell differentiation.
Crucial signaling pathways in regulatory B lymphocytes
Authors: Taitano Sophina Horne
Published: January 2023

Regulatory B cells are critical players in immune regulation, protecting against autoimmune and allergic diseases. Regulatory B cells possess a several mechanisms for this purpose, including production of the anti-inflammatory cytokine interleukin 10 (IL-10) and the expression of the death inducing molecule Fas ligand (FasL). Despite the importance of these cells, what governs their activation and regulatory abilities is not fully understood. Our lab has developed in vitro cultures using CD40L and IL-5 stimulation to grow mouse regulatory B cells which produce IL-10 and display FasL associated killing abilities. To elucidate the signaling pathways involved in regulatory B cell activation and function, cultures were treated with inhibitors to specific signaling molecules. Inhibitor effects on IL-10 production, FasL killing ability, and cell growth of the B cells were evaluated. Through these inhibitor studies we have identified certain pathways imperative to regulatory B cell growth and function. Interestingly, regulatory B cells seem to use unique signaling pathways to respond to CD40L and IL-5 stimulation. Regulatory B cells can be activated by multiple stimuli in addition to CD40L and IL-5, but which stimuli are most important in vivo is still to be determined. Using the results from the in vitro studies we are now examining these pathways as potential targets for expansion and activation of regulatory B cells in vivo.
Resident Memory T cells in the liver Receive the Local Survival factors from the Specialized microenvironments in Response to Acute Viral Infection
Authors: Kim Hyun Gyung , Jung Yong Woo
Published: January 2023

Memory T (TM) cells provide long-term protection against the same infectious agents by maintaining their cell number and responding vigorously upon rechallenge. However, it remains to be investigated which type of TM cells are formed in the liver and how they maintain their homeostasis in response to systemic acute infections. Here, we show that virus-specific TM precursors were clustered in specialized anatomic microenvironments, called lymphoid aggregates. These cells did not circulate in the steady state, suggesting that they are resident in the lymphoid aggregates. We confirmed that TM cells in the liver did not migrate into the secondary lymphoid organs during recall responses. We further characterized the phenotypes of cells found in the lymphoid aggregates using immunofluorescent microscope. TM cells localized in these microenvironments are CXCR3+ and surrounding macrophages (Mø) showed the expression of CX3CR1. These Mø were purified based on the intensity of CX3CR1 expression and examined for expression of potential factors important for the homeostasis of TM cells. Compared to CX3CR1lo cells, CX3CR1hi or CX3CR1int Mø expressed inhibitory receptors, programmed death (PD) protein 1 and PD-ligand 1, and higher mRNA levels of chemokines, CXCL9 and CXCL10, which can induce the recruitment of CXCR3+ cells. These Mø also transcribed high level of interleukin (IL)-15 which is well-defined survival factors for TM cells, and the size of lymphoid aggregates decreased in IL-15-deficient mice. Therefore, we propose that CXCR3+ resident TM cells develop in the liver during acute viral infection, and the specialized Mø in the lymphoid aggregates provide important factors for the homeostasis of these TM cells.
Reduced TGF‑βR signaling results in increased T cell activation without autoinflammatory disease
Authors: Weissler Katherine Ann , Kinard Jessica , Laky Karen , Guerrerio Pamela
Published: January 2023

Transforming growth factor-β receptor (TGF-βR) signaling is important in regulating the proliferation and differentiation of CD4+ T cells, but how modulating the level of signaling can affect T cell fate remains unclear. We have created a mouse model of Loeys-Dietz syndrome (LDS), an autosomal dominant disease caused by mutations in genes encoding the TGF-βR, by targeting a missense mutation (M318R) to the endogenous murine Tgfbr1 locus. LDS patients exhibit an increased propensity for allergic disease, but no overt immunodeficiency or autoimmune disease. A higher percentage of CD4+ T cells in Tgfbr1M318R/+ mice exhibited a memory phenotype and produced enhanced levels of cytokines including IFN-γ, IL-13, IL-17, and IL-10 upon stimulation, but no autoimmunity was observed. In contrast, mice lacking TGF-βR2 specifically in T cells demonstrate exaggerated IFN-γ production and widespread autoinflammatory disease. While naïve CD4+ T cells from Tgfbr1M318R/+ mice were less likely to upregulate Foxp3 during in vitro culture with recombinant TGF-β, paradoxically, increased percentages and numbers of suppressive Foxp3+ regulatory T cells (Tregs) were observed in vivo, which may indicate a role for increased proliferation or survival of thymically-derived Tregs. The effects of the Tgfbr1M318R/+on T cells appear to be cell intrinsic. We observed no differences in T cells derived from WT bone marrow used to reconstitute irradiated Tgfbr1M318R/+ or WT hosts, while Tgfbr1M318R/+ bone marrow co-transferred with WT bone marrow into WT hosts gave rise to more activated CD4+ T cells and a higher frequency of Foxp3+ cells. These data suggest that partially abrogated TGF-β signaling leads to unexpected effects on T cell development and function.
CD28 induces survival in long-lived plasma cells through NFκB mediated Irf4 upregulation and mitochondrial respiration-dependent reactive oxygen species production.
Authors: Cooper James J. M. , Utley Adam , Peng Peng , Mahpour Amin , Carlson Louise M , Carle Sigrid A , Lee Kelvin P
Published: January 2023

Durable humoral immunity is dependent upon sustained antibody production by long-lived plasma cells (LLPCs). We have published that CD28 is required for LLPC survival and antibody production in vivo; however, the mechanistic basis for this is unclear. In T cells, CD28 is known to induce glycolysis at the expense of respiration. Surprisingly, CD28 induces mitochondrial respiration and biogenesis in LLPCs while also increasing glucose uptake. Paradoxically, respiration-dependent reactive oxygen species (ROS) are required for CD28-mediated survival. Using knock-in mice with mutations in the CD28 cytoplasmic tail, we demonstrate that CD28-Grb2-Vav-Slp76 signaling is required for the survival of LLPCs with high mitochondrial mass and ROS in vivo. Mechanistically, CD28 induces Irf4 in an NFκB-dependent manner. CD28-ROS drive cRel translocation, an NFκB subunit, which can bind directly to the Irf4 promoter. We also describe a super enhancer element further upstream of Irf4 driven by the NFκB subunit p65, induced by CD28 specifically in LLPCs. Furthermore, inhibition of NFκB abrogates CD28-mediated glucose uptake, mitochondrial respiration and biogenesis. Irf4 heterozygous LLPCs have lower mitochondrial mass in vivo, and knock-down of Irf4 decreases the mitochondrial mass. This leads to a model wherein CD28, through Grb2-Vav-Slp-76, reinforces LLPC survival through the induction of mitochondrial biogenesis and respiration. Respiration-dependent ROS then augment NFκB-mediated Irf4 expression through direct promoter activity and a previously undescribed super enhancer element. CD28, therefore, makes an attractive target for the augmentation of vaccine design and in the alleviation of antibody-mediated autoimmunity.
TGFβ Receptor mutations alter mast cell development and function
Authors: Turfkruyer Mathilde , Dugan Philip A , Kinard Jessica L. , Frischmeyer-Guerrerio Pamela
Published: January 2023

Mutations in TGFBRI, encoding the receptor for TGFβ, cause Loeys-Dietz syndrome (LDS), a Mendelian disorder associated with an increased risk of developing nearly all forms of allergic disease, including food allergy. To better understand the mechanisms responsible for this association, knock-in mice harboring LDS mutations have been developed, which recapitulate nearly all aspects of the human phenotype. Mast cells are the main effector cells of allergic disease and several studies have identified TGFβ as a critical mediator of mast cell development and function. We hypothesized that LDS mast cells are altered in a manner that would promote greater susceptibility to allergic outcomes. To address this possibility, in vitro assays were performed with peritoneal mast cells (pMCs) cultured in IL-3 and stem cell factor and stimulated with IL-33, TGFβ, or IgE crosslinking agents. LDS pMCs expressed higher levels of ST2, the receptor for IL-33, and greater secretion of IL-9 following IL-33 stimulation. Furthermore, LDS pMCs were more resistant to apoptosis, exhibited greater degranulation following IgE receptor crosslinking, and showed less downregulation of FcɛRI and c-kit following exposure to recombinant TGFβ1. This was associated with reduced pSmad2/3 levels, a key signaling molecule in the TGFβ pathway. Furthermore, we demonstrated that following food allergy induction, LDS mice exhibited greater MC hyperplasia in the small intestinal lamina propria as well as in the peritoneum where LDS peritoneal mast cells showed enhanced secretion of IL-9. In vivo studies are ongoing to further examine the role of altered mast cell frequency and/or function resulting from altered TGFβ signaling in promoting food-induced anaphylaxis.
Sphingosine Kinase Inhibitors Effectively Inhibit Growth of Mast Cells with D816V-KIT Mutations
Authors: AGEITOS ARACELI TOBIO , BANDARA GEETHANI , MUNOZ-CANO ROSA , YIN YUZHI , DESAI AVANTI , METCALFE DEAN , OLIVERA ANA
Published: January 2023

Sphingosine-1-phosphate (S1P) is a lipid mediator that promotes cell proliferation and prevents apoptosis in a variety of cells and its increased synthesis by Sphingosine Kinase (SphK) has been associated with oncogenesis and cancer prognosis. Mastocytosis is a disorder characterized by excessive mast cell (MC) proliferation and accumulation in tissues. Treatment therapies for aggressive mastocytosis have to date been partially effective, and thus the search of new pharmacological targets is warranted. We thus investigated the involvement of SphK isoforms (SphK1 and SphK2) and generation of S1P in the regulation of abnormal MC growth. The specific SphK1 and SphK2 inhibitors, SKI-178 and ABC294640, respectively, were used in this study. The effect of SphK1 and SphK2 inhibition on human (LAD2, HMC1.1 and HMC1.2) and murine (P815) neoplastic MC proliferation and survival was evaluated. SphK2 inhibition retarded entry into the cell cycle in all MC lines with no significant effect on cell survival. However, SphK1 inhibition led to cell cycle arrest in G2/M and apoptosis predominantly in MCs carrying D816V-KIT mutation. The effect of SKI-178 was associated with an alteration in the damage response cascade. This included activation of checkpoint kinase 2 (chk2) combined with a depletion of chk1, leading respectively to p53- and caspase2-induced apoptosis and cell cycle arrest mediated by cdc25c depletion. Both SphK inhibitors reduced MC accumulation in an in vivo mouse model of aggressive mastocytosis and the number of cultured bone marrow MCs and MC progenitors from a patient with systemic mastocytosis. These results support further consideration for SphK inhibitors as a cytoreductive strategy in proliferative MC disorders.
Mast cells are required for allergy-driven changes in senstivity to touch and pressure in oxazolone challenged skin in sensitized ND4 female mice.
Authors: Chatterjea Devavani , Landry Jasmine
Published: January 2023

Vulvodynia is a chronic vulvar pain condition affecting up to 8–16% of 18–60 year-old women. Epidemiological studies have demonstrated an increased risk of developing vulvodynia for women with histories of seasonal and insect allergies. We show that 10 exposures to the hapten oxazolone on the labiar skin of previously sensitized outbred ND4 female mice provokes increased sensitivity to touch and pressure that lasts for 3 weeks or longer. This persistent sensitivity is accompanied by local labiar mast cell accumulation and overgrowth of cutaneous neurites both of which persist for at least 3 weeks after the final allergen exposure. Oxazolone-challenged mice also show local labiar increases in inflammatory cytokine and neuro-immune signaling transcripts, infiltration of resident memory CD8 cells and CD4+CD25+ regulatory T cells. Local depletion of mast cells via intra-dermal injections of basic secretagogue compound 48/80 reduces the sensitivity to touch and pressure suggesting that persistent accumulation of mast cells in allergen-exposed tissue is necessary for the development of long-lasting painful responses in these mice. Our findings suggest that mast cell-targeted therapies may be a novel area of intervention for the management and treatment of chronic pain conditions associated with allergic and hyper-inflammatory states.
Chronic Litomosoides sigmodontis infection protects against IgE-mediated anaphylaxis in sensitized mice
Authors: Kropp Laura E. , Mitre Edward E.S.
Published: January 2023

While numerous animal studies have reported that helminths can prevent allergic sensitization, few have evaluated the effects of infection on pre-existing allergy. We hypothesized that chronic infection would protect against the clinical symptoms of IgE-mediated anaphylaxis in previously sensitized mice. Mice were sensitized by weekly intraperitoneal (IP) injection of either ovalbumin (OVA)/alum or PBS/alum for 3 weeks. At 5 weeks, mice were infected with Litomosoides sigmodontis (L.s.), a rodent filarial parasite, or mock infected. Mice were challenged IP with OVA during the twelfth week of infection. Following challenge, serum levels of murine mast cell protease 1 (mMCP-1) were significantly lower in sensitized mice that were L.s.-infected as compared to mock infected (212,923 vs. 447,062 pg/mL, p-value= 0.0459). Additionally, L.s.-infected mice that had been previously sensitized exhibited an average drop in core body temperature of 3.5 °C after 30 min, which was significantly less than the 5.0 °C drop observed in sensitized and mock-infected mice. Anaphylaxis was IgE and mast-cell dependent, as mice deficient in FcɛRI or mast cells mice released little to no mMCP-1 or exhibited minimal drops in core body temperature in response to challenge. Experiments to date suggest that changes in mast cell functionality were central to the protective effect. First, serum levels of OVA-IgE were not altered by L. sigmodontis infection. Second, purified mast cells from OVA-sensitized and infected mice released less histamine than OVA-sensitized and mock-infected mice in response to OVA. These results suggest that chronic helminth infection protects against anaphylaxis in part by decreasing allergen-driven mast cell degranulation.
AddaVax vs. Freund’s adjuvant use for animal immunization in hybridoma development
Authors: Hawksworth David J
Published: January 2023

Use of a potent adjuvant is essential for stimulating a strong, antigen specific response from immunized animals. While Freund’s continues as the gold standard for use in research animals we continuously evaluate available adjuvants as part of our improvement efforts in developing mouse and rabbit hybridomas. We recently evaluated AddaVax adjuvant for use in developing a high titer, high affinity immune response. Animals were immunized with peptides or recombinant proteins emulsified with Adjulite Freund’s adjuvant or AddaVax adjuvant. Sera samples were collected following the final immunization and the antibody evaluated for titer and relative affinity. Results show that the average serum antibody titer and relative affinity for AddaVax immunized mice were equivalent to or better than that from Freund’s mice for 3/7 peptides and 5/7 recombinant proteins. Data from two proteins also suggest AddaVax animals may show binding to antigenic epitopes not recognized by Freund’s animals. Disappointingly, rabbits immunized using AddaVax adjuvant consistently developed a much weaker response vs. Freund’s. Based on results generated, we have modified our protocols to include the use of Freund’s and AddaVax when initiating new hybridoma development projects in mice.
Structural characterization of disulfide-stabilized peptide/H2-Dd complexes
Authors: Allen Frederick M , Jiang Jiansheng , May Nathan A , Boyd Lisa F , Natarajan Kannan , Margulies David H
Published: January 2023

MHC class I (MHC-I) molecules fold and bind high-affinity peptides with the help of chaperones in the endoplasmic reticulum before being transported to the cell surface as stable antigen-presenting proteins. In order to understand this process, it may be necessary to artificially stabilize MHC-I molecules to behave manageably in vitro but still retain pre peptide-loaded characteristics. Others have approached this by engineering a disulfide bond in H2-Kb between residues 84 and 139, resulting in a “stitched” peptide binding grove that bypassed cellular checkpoints for misfolded proteins. We have crystallized H2-Ddwith the same Y84C/A139C mutations, as well as with W51C/G175C mutations that stitch the opposite end of the binding groove, and with all four mutations, all with the high-affinity peptide P18-I10. We determined the crystal structure of each peptide-MHC complex at 2.3, 1.7, and 1.65 Å resolution respectively. The electron density shows disulfide linkages at each expected site, and despite the mutations, the α1α2 platform domains all align to WT with an RMSD under 0.25 Å. Further efforts to measure thermal stability of each construct with high- and low-affinity peptides as well as attempts to crystallize each construct with low affinity peptides are underway. This work should contribute to a structural and mechanistic understanding of MHC-I peptide loading.
HLA-F and MHC open conformers cooperate in antigen presentation during the inflammatory response.
Authors: Geraghty Daniel , Wang Kevin L , Lee Ni , Burian Aura , Goodridge Jodie P , Ishitani Akiko , McNevin John , McElrath Margaret J
Published: January 2023

HLA-F is expressed as a protein independent of bound peptide or β2-microglobulin and surface expression is upregulated upon activation in dendritic cells, monocytes and most lymphocyte subsets. MHC class I (MHCI) is also expressed on proliferating lymphoid cells as open conformers (OCs), in addition to the ubiquitously expressed complexed form. Previous studies showed that HLA-F binds most MHCI proteins as open conformers without peptide but not as peptide bound complex. These studies were extended to show both HLA-F and MHCI OC are ligands for a subset of killer Ig-like receptors (KIRs), defining a new paradigm for MHCI function in the innate and acquired immune responses. The HLA-F/MHCI physical interaction was further implicated in the function of HLA-F and MHCI OCs in a general mode of exogenous MHCI antigen uptake and antigen presentation by activated immune cells that differs from the canonical MHCI endogenous antigen presentation. We are currently testing the hypothesis that antigen entry is governed by a synergy between specific structural characteristics of the exogenous antigen and the MHC-I allele types of target cells. New evidence is presented using HIV-1 p24 gag derived long polypeptides suggesting a requirement for HLA class I peptide epitope specificity in antigen uptake, upstream of antigen presentation, which subsequently can be presented by either or both of MHCI and MHCII. These experiments suggested that the physical proximity of HIV-1 class I and class II peptide epitopes within a p24 polypeptide can influence antigen presentation of epitopes through either MHCI or MHCII or both. A goal is to manipulate these features in designing effective immunogens for directed stimulation of antigen-specific host responses.
A whole genome mouse siRNA screen to identify novel genes involved in lipid antigen presentation
Authors: Chandra Shilpi , Khurana Archana , Kiosses William B. , Gray James , Hitomi Kaori , Zhao Meng , Richardson Stewart K. , Besra Gurudayal , Howell Amy R. , Sharma Sonia , Peters Bjoern , Kronenberg Mitchell
Published: January 2023

Although most T lymphocytes recognize peptides presented by major histocompatibility complex (MHC)-encoded class I and class II molecules, there also are significant populations of T cells that recognize nonpeptide antigens. Prominent among these T lymphocytes are the type I or invariant natural killer T cells (iNKT cells). These T lymphocytes recognize lipids presented by CD1d, a nonpolymorphic, class I-like, antigen-presenting molecule. We have carried out a whole genome siRNA screen in a macrophage cell line for genes that affect the presentation of a potent glycosphingolipid antigen, GalGal Cer, to iNKT cells. In order to stimulate iNKT cells, this antigen requires internalization and lysosomal carbohydrate antigen processing to remove the terminal galactose. After several rounds of validation, functional classification and gene expression analysis, we have identified genes that lead to altered antigen presentation in macrophages. A majority of the identified genes do not perturb surface CD1d expression, but we can demonstrate they effect the formation of surface CD1d complexes with the stimulating glycolipid. Members of the HOPS and ESCRT complexes have been identified to play an important role in Cd1d dependent antigen presentation. Interestingly, our data show that ABCC1 and several other ABC family transporters are involved in lipid antigen presentation by CD1d. CD1d and MHC-II antigen presentation pathways both depends on antigen loading in the lysosome, therefore we also analyzed the role of these genes in MHC-II antigen presentation. Although the majority of genes identified doesn’t affect MHC-II antigen presentation, there are a few molecules that affect both antigen presentation pathways.
Immuno-proteomic interrogation of antigen presentation during Dengue and Zika infections reveals novel targets for the design of broad acting T-cell vaccines
Authors: Swaminathan Kavya , Majzoub Karim , Lund Peder J , Marceau Caleb , Olsson Niclas , Davis Mark M , Carette Jan , Elias Joshua
Published: January 2023

The recent outbreak of Zika virus (ZIKV) infection across the Americas has raised dire concerns about its potential long-term health effects including its negative impact on neuronal, psychological and motor development in affected individuals. Emerging evidence pointing to Alzheimer’s-like damage to the adult brain further highlights the urgent need for efficacious prophylactic measures. Zika-affected regions are often endemic to the related Dengue virus (DENV). Antibodies against the two viruses are crossreactive and cause antibody-mediated enhancement (ADE) of infection. This confounds the design of traditional vaccines that elicit humoral antibody-mediated responses. T cell vaccines mediated by cytotoxic CD8+ T cells that recognize conserved viral epitopes presented on the major histocompatibility complex (MHC-I) of infected cells are unencumbered by ADE and can supplement or substitute traditional vaccines. In order to identify robust T cell vaccine candidates, we first used an immunoproteomic approach and isolated over 100 novel DENV and ZIKA epitopes presented by infected B lymphocytes in vitro. These viral peptides were not predicted to be epitopes by computational programs typically used for epitope discovery, which discard >95% of all predictions. Focussing on epitopes derived from polyprotein regions strictly conserved between the two viruses, we validated the presentation of these epitopes in primary dendritic cells and assayed their ability to stimulate robust T cell responses in ex vivo ELISpot assays. Together these discoveries let us assay the immunogenic potentials of an unprecedented range of DENV and ZIKV antigens which have the potential to serve as broad acting and effective Zika vaccine candidates.
Conventional influenza vaccines influence the efficacy of a candidate universal influenza vaccine in mice
Authors: Rowell Janelle , Garcia Mayra , Lo Chia-Yun , Price Graeme , Misplon Julia , Epstein Suzanne
Published: January 2023

Universal influenza vaccines are designed to protect against diverse strains of influenza virus. In a pandemic, universal vaccines could be life-saving. Candidate vaccines are usually tested in naïve animals, despite intended use in the human population that has a varied immune history, including responses to vaccination. To be more relevant to humans, we tested a candidate universal influenza vaccine in mice with a history of conventional vaccination. Female BALB/c mice were given two intramuscular doses of inactivated influenza vaccine (IIV) or diphtheria and tetanus toxoids vaccine (DT), one month apart. Another group had two intranasal (i.n.) doses of live attenuated influenza virus (LAIV). One month after the second dose, mice were given the universal vaccine, recombinant adenoviruses expressing influenza A NP and M2 (rAd-NP+M2, i.n.). Antibody responses to universal vaccine antigens NP and M2 were assessed by ELISA. T-cell responses to antigens were determined by IFN-γ ELISPOT. Mice were challenged with mouse-adapted A/FM/1/47 (H1N1) and subsequent weight loss determined rAd-NP+M2 efficacy. Efficacy was enhanced, inhibited or unaffected by prior history. Mice given Afluria IIV and LAIV lost less weight and had a greater antibody and T-cell response to NP than mice without prior vaccination, which are examples of enhanced rAd-NP+M2 efficacy. Inhibited vaccine efficacy was observed in mice that received Fluvirin IIV and mice had low IgG2a antibodies to NP and M2. Fluzone IIV and DT had no effect on rAd-NP+M2 efficacy. This study demonstrates that the protection by the universal influenza vaccine may be affected by a history of conventional vaccination, and suggests that performance in humans would be influenced by immune history.
Intradermal active full-length DNA Aβ42 immunization via electroporation leads to high anti-Aβ antibody levels in wild-type mice with variability in T cell responses depending on pulse parameters applied
Authors: Lambracht-Washington Doris , Fu Min , Rosenberg Roger N
Published: January 2023

Aβ immunotherapy has high potential as possible prevention treatment for Alzheimer’s disease, and anti-Aβ antibodies will help in the removal of excess amyloid from brain. We have previously shown that active DNA Aβ1-42 immunization via gene gun delivery led to a non-inflammatory immune response with efficient antibody levels which resulted in decreased Aβ levels in brains of the immunized AD mouse model mice. To optimize the DNA delivery and make DNA vaccination more applicable for clinical use, we have investigated here the use of intradermal electroporation using a variety of different voltages and electropulse parameters in wild-type mice. In the direct comparison to DNA delivery via gene gun, higher levels of anti-Aβ42 antibodies were detected already after three DNA immunizations. The antibody isotype profile following DNA electroporation was mixed with IgG1 and IgG2a antibodies. With a fine tuning of the electropulse protocol the T cell responses could be adjusted to similar findings we published earlier for DNA Aβ1-42 immunization via gene gun delivery with low levels of inflammatory cytokines (IFNγ, IL-17) in the in-vitro Aβ42 peptide re-stimulation assays. We found that short pulse durations (5 ms) were better than long pulse durations (10 ms) in regard to obtaining a Th2 type of immune response, and that a higher rate of pulses (16–20 pulses) increased the antibody response in our sets of experiments. Full-length DNA Aβ1-42 immunization delivered via electroporation with a fine tuning of the electropulse conditions has potential to be used in the clinical setting.
mBM-Mesenchymal Stem Cells as a vehicle for immunization of mice against Bordetella pertussis
Authors: MAROLI SREE LAKSHMI VELANDI , Molla Touhidul , Llontop Peter , Abramson Tzvia
Published: January 2023

Mesenchymal Stem Cells (MSC) derived from bone marrow is well documented to lack immunogenic features and demonstrate anti-inflammatory characteristics. Additionally, in vitro treatment of MSCs with IFNγ was previously shown to increase expression of antigen-presenting molecules such as MHC class II. Therefore in this study, we hypothesize that MSC can serve as an immunization vehicle to deliver Bordetella pertussis (Bp) antigens.

MSC were isolated from bone marrow. To confirm MSC characteristics, cells were differentiated into chondrocytes, adipocytes and osteocytes. Flow cytometry and immunofluorescence were used to demonstrate that the propagated MSCs express high level of MHC II when pretreated with IFNγ. IFNγ treated MSC were then pulsed overnight with heat-killed B. pertussis and intranasally administered (one million/mouse) twice with one-week interval. Mice were then challenged with Bp infection. Mice immunized with MSC/Bp were 100% protected from the infection challenge as measured by the bacterial load in lungs at 5dpi.

Th17 are required for effective resolution of B. pertussis infection, but those immune responses are mounted only late p.i. In contrast, mice immunized with INFγ+/MSC/Bp, display a high frequency of Th17 cells in the lungs that was detected as early as 5dpi. These preliminary data suggest that BM-MSCs can serve as a vector to protect mice from Bp infection. We will further investigate for the precise immune responses induced by MSC/Bp vaccine.
Chitosan nanoparticle delivery of Influenza A Virus DNA vaccine enhances antibody class switching and abrogates weight loss post IAV challenge
Authors: Brown Deborah M , Lampe Anna T. , Farris Eric , Williams James A. , Pannier Angela K.
Published: January 2023

DNA vaccination presents a promising new approach for Influenza A Virus (IAV) vaccines, as they can be generated quickly in response to the viral antigenic shift and drift characteristic of IAV pandemic outbreaks. A DNA vaccination approach for IAV was examined using a plasmid encoding PR8 H1N1 hemagglutinin (HA) protein as a potential IAV vaccine. In addition to the HA antigen sequence the plasmid also encoded a sequence that when transcribed, activates the pattern recognition receptor RIG-I, improving innate immune activation. To increase the potential of this IAV DNA vaccine, our study focused on delivery of the plasmid to the respiratory tract using chitosan (CS) nanoparticles. CS, a mucoadhesive/mucopenetrating derivative of chitin, forms complexes with DNA to improve uptake and transfection of the plasmid by immune cells in the lung. In vitro, plasmid/CS complexes containing unbound CS induced bone marrow derived dendritic cell (BMDC) death. BMDC death was accompanied by a robust increase in inflammatory cytokine mRNA. Although BMDC death was abrogated by free CS removal, cytokine expression was also reduced. Alternative complexation methods are being explored to achieve an appropriate balance between cytokine production and cell death. In vivo intranasal prime-boost vaccination with low dose PR8 HA plasmid/CS nanoparticles resulted in PR8 specific IgM and total IgG antibody formation. Although weight loss was abrogated in plasmid/CS vaccinated mice, protection from lethal PR8 infection did not correlate with antibody level. By optimizing this vaccine approach, including dose, regime, and CS nanoparticle modification, critical insights into the development of rapidly deployable IAV vaccines could be gained.
Type III interferons restrict neutrophil activity in response to Staphylococcus aureus
Authors: Pires Silvia D , Parker Dane
Published: January 2023

Staphylococcus aureus is an important human pathogen, in particular the methicillin resistant S. aureus (MRSA) strain USA300 that is epidemic in the United States. Type III interferons (IFN-λ) signal through the IL-28 receptor (IFNLR) located on epithelial cells and neutrophils. Neutrophil-mediated killing is a crucial defence system against S. aureus. Compared to WT mice, Ifnlr−/− mice had significantly improved clearance of S. aureus from the airway and lung tissue (>90%; P<0.001) following a 24-hour infection. In a mortality model of infection, all Ifnlr−/− mice survived, whereas 75% of WT mice succumbed to infection (P<0.05). Despite a lack of difference in neutrophil recruitment to the airway in WT and Ifnlr−/− mice in response to S. aureus, Ifnlr−/− infected mice showed a significant increase in the neutrophil related cytokine G-CSF (38%; P<0.05). Moreover, expression of the neutrophil surface markers, Ly6C and Ly6G, were higher (39% and 20%, respectively; P<0.01) on infected Ifnlr−/− neutrophils compared to WT infected mice. Neutrophils isolated from Ifnlr−/− mice compared to WT neutrophils also displayed a 2-fold increase in MPO activity (peroxidation) (p<0.05) and a 2.1-fold increase in Ca2+ flux when compared to WT neutrophils (P<0.001), while WT neutrophils stimulated with purified IFN-λ displayed a 0.5-fold decrease in Ca2+ flux (p<0.005). In addition, stimulation of WT neutrophils with purified IFN-λ led to a 2-fold decrease of their S. aureus killing capacity (P<0.05). Controlling neutrophil recruitment and activation is important to provide a balanced response to infection. Our data suggests that type III IFN signalling can impair neutrophil activation and their killing capacity in response to S. aureus.
RLR-mediated immunity is unique and critical in the response to West Nile Virus
Authors: Stone Amy E.L. , Wilkins Courtney , Green Richard , Gale Michael J
Published: January 2023

RIG-I-Like Receptors (RLRs) include RIG-I, MDA5, and LGP2. RLRs are vital pathogen recognition receptors in the defense against RNA viruses. West Nile Virus (WNV) is an emerging, neurotropic flavivirus that infects the central nervous system to cause disease and death. RIG-I and MDA5 work in tandem to initiate innate immune responses against WNV infection, though the role of LGP2 in this process is not well defined. To gain a deeper understanding of the contributions of each RLR in the innate immune response to WNV, we conducted a systems biology approach to assess the role of each RLR in immune programming. We conducted genome-wide RNAseq and bioinformatics analysis of WNV infection in bone marrow derived macrophages from the RLR-deficient mice. Here we show that the RLRs drive distinct immune gene activation and polarization of the immune response. In our data, the RLR-dependent, WNV-induced immune response polarization overshadows the classical drivers of viral innate immune responses, interferon I (IFN) and IFN-stimulated genes, thus underscoring the importance of innate immune activation for channeling the adaptive immune system into specific effector pathways. Bioinformatics analysis and computational modeling was used to define a unique gene signature of each RLR following WNV infection. Further, we dissected the complex virus-induced cytokine response utilizing multiplex analyses to link gene induction and cytokine protein output. These analyses reveal that distinct RLR-driven transcriptional programs lead to differential functional output in immune response initiation against WNV infection. Our study demonstrates a clear, defined role for RIG-I, MDA5, and LGP2 in immune programming against virus infection.