bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional differences exist in contribution of VH and VL from polyspecific IgM and monospecific IgG antibodies in antigen recognition and virus neutralization functions.
Authors: Kaushik Azad Kumar , Pasman Yfke
Published: January 2023

We analyzed role of individual variable heavy (FdVH) and variable light (FdVL) domains in comparison with VH+VL pair (scFv) originating from a polyspecific bovine IgM, with an exceptionally long CDR3H (61 amino acids), and a monospecific IgG1 antibody in antigen (Ag) recognition and virus neutralization functions. To this end, recombinant FdVH, FdVL and scFv were constructed and expressed in Pichia pastoris from bovine polyspecific IgM and IgG1 encoding cDNA. The scFv1H12 showed polyspecific antigen recognition similar to parent IgM antibody with minor differences. Unlike variable light domain FdVL1H12, variable heavy domain FdVH1H12 recognized multiple antigens that differed from scFv1H12 and the parent IgM antibody. Nevertheless, role of FdVL1H12 in providing structural support to FdVH in antigen recognition is noted. By contrast, the individual FdVH073 and FdVL074, originating from induced BoHV-1 neutralizing IgG1 antibody, recognized target epitope on BoHV-1 relatively weakly when compared to VH+VL pair as scFv3-18L. Both VH and VL domains of induced IgG antibody are required to achieve BoHV-1 neutralization function. To conclude, there exist subtle functional differences in relative contribution of VH and VL from polyspecific IgM and monospecific IgG antibodies in antigen recognition and virus neutralization functions. [Supportd by NSERC Canada]
Genome wide association study identifies loci associated with immune cell influx following experimental mammary challenge with Streptococcus uberis
Authors: Pighetti Gina M , Siebert Lydia , Staton Meg E , Oliver Stephen P
Published: January 2023

Mastitis, an inflammation of the mammary gland, significantly impairs animal health, productivity, and milk quality. A common mastitis-causing pathogen, Streptococcus uberis, is observed in 14–26% of subclinical and clinical cases. To identify potential candidate genes and regulatory regions that influence immune cell influx and clearance from the gland, Holstein dairy cows (n=40) were challenged intramammarily with S. uberis. Somatic cell counts (SCC), a measure of immune cells present in milk, were determined prior to and for 28 days following experimental challenge. Traditional GWAS have used SCC (cells/ml) to identify loci of interest. Our approach uses SCC to generate three novel phenotypes: area under the curve (AUC) of SCC for 0–7 d, AUC for 0–28 d, and resolution of infection based on SCC return to less than 200,000 cells/ml in milk (grouped as <21 d, 21–28 d, or >28 d). SNP were identified using the BovineSNP50 v2 DNA Analysis BeadChip (Illumina) and associations with SCC phenotypes tested using Plink. Analyses revealed 16 highly significant (p<1.0×10−5) SNP. The BTA20 region associated with SCC AUC for the first 7 days corresponded to a previously identified QTL for SCC. This region contains 8 genes and 1 miRNA, with two genes linked to migration and apoptosis. A significant association with the recovery or return to SCC <200,000 cells/ml was observed on BTA9, with the greatest association (p=8.5×10−7) observed within an immune related gene. The identified loci will be evaluated for potential contribution to the observed phenotypes. Such investigations could lead to novel treatment or preventive strategies for controlling mastitis, as well as identify markers for genetic selection of cows with greater resistance to infection.
Expression of T cell genes in adult Xenopus laevis and TCR gene expression in the Xenopus tadpole tail.
Authors: Foulkrod Ashley M , Geibel Gretchen M , Kongprachaya Yuthana , Appasamy Pierette M
Published: January 2023

Xenopus laevis is a unique and useful model for studies of the evolution and development of the adaptive immune response. The Xenopus immune system shares much similarity with the immune system of higher animals, with some unique exceptions, is relatively easy to maintain, and it’s larval period can be manipulated since it exists outside the female. This study explored the distribution of T cell specific genes, as markers for the presence of T cells, in adult X. laevis as well as X. laevis tadpoles. Transcripts for all four TCR genes (γ, δ, α and β) were detected in the adult Xenopus thymus and spleen and TCR δ and β were detected in adult Xenopus skin, intestine and lung, using RT-PCR of total cellular RNA extracted from these tissues. These data suggest that γδ and αβ T cells are plentiful in X. laevis and that the development and distribution of these cells is similar to that of humans and mice. In addition, because epidermal T cells are increasingly appreciated as having a significant role in wound healing, the mammalian equivalent of regeneration, we have begun to examine the expression of T cell-specific genes in the parts of the tadpole that are able to regenerate, specifically the tadpole tail and limbs. We have observed the presence of TCR β transcripts in the tadpole tail, and are extending our studies to an evaluation of the other TCR genes and other T cell-specific genes. These findings point to the utility of X. laevis in studies of the role of T cells in regeneration.
A comparative survey of Gopherus polyphemus hemoparasites in four different South Florida habitats
Authors: Cooney Brian Thomas , Elhassani Dana , Frazier Evelyn , Caruso Joseph
Published: January 2023

The gopher tortoise (Gopherus polyphemus) is a keystone species, for its burrows house more than 300 species of animals. Habitat destruction and its consequent fragmentation have led to a decline in populations within southeastern ranges of the United States. Haemogregarina (intracellular protozoan parasites) have previously been identified in the blood of gopher tortoises. High levels of blood parasitaemia have shown to be a potential indication of stress resulting from overcrowding. The goals of this study are: (1) to characterize hemoparasite species through the use of Polymerase Chain Reaction (PCR) from tortoise blood samples and the ticks attached to tortoises and (2) to determine if free ranging gopher tortoises that live in poorly maintained habitats exhibit higher prevalence and parasitaemia levels within their blood when compared to tortoises inhabiting better maintained sites with prescribed fires. Research goals will be examined at four sites: Blazing Star Preserve (BSP), Pine Jog Preserve (PJP), Florida Atlantic University Preserve (FAUP), and Johnathan Dickinson State Park (JDSP), to determine both hemoparasite prevalence and parasitaemia levels. PJP and JDSP are sites currently practicing fire management and herbicides, whereas FAUP utilizes mechanical and chemical management while BSP is not managed. We hypothesize that parasitaemia will be higher in poorly managed sites as a result of crowding, when compared to well managed sites. This study will benefit current conservation and management practices for gopher tortoises in South Florida and help provide a baseline study for reptile hemoparasites.
Comparative analysis of expression profiles of cytokines in Riemerella anatipestifer infection in ducks and chickens highlights the role of IL-17A in bacterial susceptibility
Authors: Kim Woohyun , Lillehoj Hyun S , Fernandez Cherry P , Min Wongi
Published: January 2023

Riemerella anatipestifer (R. anatipestifer) cause severe infection in ducks at up to 75% mortality, but corresponding infection in chickens is comparatively mild. In order to understand differences in susceptibility, both species infected with R. anatipestifer, then analyzed mortality, bacterial burden and expression profiles of cytokines in infected birds. As expected, ducks exhibited increased susceptibility to R. anatipestifer infection compared to chickens, as determined by mortality rate and bacterial burden. Interestingly, comparative expression analysis of immune- related genes in R. anatipestifer-infected tissues and -stimulated splenic lymphocytes obtained from both species revealed that IL-17A is strongly associated with Riemerella infection in only ducks. Moreover, marked expression of IL-6 which is associated with Th17 cell differentiation, but not TGF-β, was identified in infected tissues from ducks, but not those from chickens. Furthermore, treatment with 3,3′-diindolylmethane (DIM) and berberine (BBR) in ducks ameliorated the detrimental effects of R. anatipestifer infection, as determined by decreases in mortality rate and bacterial burden, as well as downregulation of IL-17A and IL-6 mRNA expression. Taken together, these results suggest that upregulation of IL-17A influenced susceptibility to R. anatipestifer infection and that anti-IL-17A agents could prove to be promising therapies for alleviating Riemerella infection.
Immunomodulation of Host Immunity by Bacteriodes fragillis Polysaccharide A (PSA) Prevents Viral Encephalitis
Authors: Chandran Ramakrishna , Liu Hiutung , Mazmanian Sarkis K , Cantin Edouard
Published: January 2023

A major impact of commensal microbiota on many aspects of host physiology including regulation of host immunity extends beyond the gut to remote sites, including the brain. Studies in mouse models have shown that the commensal Bacteroides fragilis and its capsular PSA can prevent various inflammatory disorders including colitis, experimental autoimmune encephalomyelitis and asthma. However, little is known about the interaction of gut bacteria and their products with the immune system during viral infection. We assessed the immunomodulatory potential of PSA in a murine model of herpes simplex virus (HSV) encephalitis (HSE). Fatal HSE results from inflammation of the brain in susceptible 129 strain of mice due to uncontrolled invasion by Ly6Chigh inflammatory monocytes (IM) and neutrophils. Naïve 129 mice pretreated with PSA or PBS were then given a 1-week course of the anti-viral drug acyclovir (ACV) from day 4-post HSV infection to simulate a clinical setting. PSA, but not PBS-treated mice survived with dramatically reduced brain inflammation including fewer degranulating IM and altered cytokine and chemokine profiles. Mesenteric lymph node cellularity was increased and IL-10 secreting ICOS+ CD73+ CD4+ T cells and CD73+ CD8+ T cells were induced by PSA treatment. Importantly, IL-10 deficient and Rag knockout mice were not protected, suggesting T/B cells derived IL-10 as the protective mechanism. Interestingly, B cells were required for PSA mediated protection. We infer that the microbiota augment immunity to HSV using pathways distinct from those required for protection in chronic autoimmunity models, and that probiotics in combination with antivirals might act synergistically to limit HSV induced inflammatory diseases.
Human microglia cells participate in inflammatory responses, viral reproduction and transmission in Japanese encephalitis virus infection
Authors: Lannes Nils , Neuhaus Viviane , Kharoubi-Hess Solange , Walch Michael , Summerfield Artur , Filgueira Luis
Published: January 2023

Japanese encephalitis virus (JEV) is a neurotropic flavivirus and a major cause of mortality and morbidity in humans. JEV-infected patients exhibit strong inflammation of the brain and an accumulation of viral particles in the hypothalamus and hippocampus. Microglial cells (MG) are the brain-resident macrophages and are proposed to serve as reservoir for JEV. Using primary human MG and a model of human monocyte-derived MG, this study explores the influence of JEV on chemokine ligands/receptors of human MG and the role of MG in viral replication and transmission to susceptible cells. To achieve this work, the attenuated inactivated JEV as used in vaccines and two live JEV strains were employed. JEV induced morphological changes of human microglia, consistent with an activated state. Upon exposure to live JEV, human microglia adopted an inflammatory state characterized by increased levels of CCL2, CXCL9 and CXCL10. Furthermore, both inactivated and live treated human microglia transiently up-regulated the fraktalkine receptor CX3CR1 in a dose dependent manner. Interestingly, JEV vaccine but not live JEV remarkably enhanced levels of MHCII expression on MG. Importantly, human microglia supported JEV replication. Although JEV-derived microglia was not infectious in supernatants, virus particles were highly infectious if preserved inside the cells. Indeed, human microglia contributes to viral transmission in cell-cell conditions. In conclusion, JEV-infected cells express higher levels of CX3CR1, a relevant chemokine receptor involved in cell migration and inflammation in the central nervous system. In long term infection, human microglia may be a source of neuronal infection and sustain JEV brain pathogenesis.
HIV and HIV Tat inhibit LPS-induced IL-23 production in human macrophages by distinct intracellular signaling pathways
Authors: Kumar Ashok , Gajnayaka Niranjala , Angel Jonathan
Published: January 2023

Monocyte-derived macrophages (MDMs) from HIV-infected patients and MDMs infected in vitro with HIV manifest inhibition of IL-12. Whether HIV infection or HIV accessory proteins such as tat, impact production of IL-23, a member of IL-12 family cytokines, in macrophages remains unknown. Our results show that in-vitro HIV infection, intracellular HIV-tat and HIV tat peptides inhibit LPS-induced IL-23 production in MDMs suggesting impairment of TLR-4 signalling.

To study the mechanism governing HIV- and HIV-tat-mediated inhibition of LPS-induced IL-23 production, we first established that p38 mitogen-activated protein kinase (MAPK), the phosphoinositide-3-kinase (PI3K), and SRC homology region 2 domain-containing tyrosine phosphatase-1 (SHP-1) positively regulated whereas c-Jun N-terminal kinase (JNK) MAPK negatively regulated LPS-induced IL-23 production in MDMs.

HIV-Tat downregulated TNF-receptor associated factor (TRAF)-6 and inhibitor of apoptosis-1 (cIAP-1) and caused decreased phosphorylation of downstream PI3K, and p38 MAPKs. In contrast, HIV-tat-mediated inhibition of JNK MAPK negatively regulated IL-23 production. However, SHP-1 and Src kinases were not inhibited by HIV-Tat and hence were not implicated in tat-mediated inhibition of LPS-induced IL-23 production.

In contrast to HIV-Tat, in vitro HIV infection of MDM inhibited LPS-induced IL-23 production via inhibition of p38 MAPK activation. Overall, HIV and HIV-Tat regulate LPS-induced IL-23 production in human macrophages via distinct mechanisms: HIV-Tat through the inhibition of cIAP-1-TRAF-6, and subsequent inhibition of PI3K and p38 and JNK MAPKs whereas HIV through the inhibition of p38 MAPK activation.
Dengue virus-elicited tryptase breaks tight junctions to induce endothelial permeability
Authors: Rathore Abhay P.S. , Syenina Ayesa , Gubler Duane , St. John Ashley L.
Published: January 2023

Dengue virus (DENV) is a mosquito-borne virus that causes a characteristic pathology in humans involving dysregulation of the vascular system. In some patients with dengue hemorrhagic fever (DHF), vascular pathology can become severe, resulting in extensive microvascular permeability and plasma leakage into tissues and organs. The predominant view in the field is that vascular leakage during infection is the result of immune pathology that is induced by cytokines and other de novo transcribed vasoactive factors released from infected cells. Recent evidence has shown that mast cells (MCs), which are granulated immune cells that line blood vessels and regulate vascular function, are able to detect DENV particles in vivo and promote vascular leakage, however the molecular mechanism of this vascular leakage is unknown. Here, we identified the pre-stored MC protease, tryptase, to be a product that promotes endothelial cell permeability during DENV infection, through inducing breakdown of tight junctions. Tryptase alone, injected in mice at concentrations equivalent to those found in the serum of human DHF patients, is able to promote substantial vascular leakage. This study provides a new molecular mechanism by which DENV can induce vascular pathology in vivo.
Synaptogyrin-2 Promotes Replication and Immune Evasion of A Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs
Authors: Xing Zheng , Sun Qiyu , Qi Xian , Wu Xiaodong , Liang Mifang , Li Chuan , Cardona Carol J
Published: January 2023

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little has been known about the function of synaptogyrin-2. Severe Fever with Thrombocytopenia Syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with a high fatality and caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that viral nonstructural protein NSs formed inclusion bodies (IBs) which are involved in viral immune evasion as well as in viral RNA replication. In this report, we sought to elucidate the mechanism how NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and ADRP. We identified synaptogyrin-2 to be highly upregulated in response to SFTS bunyavirus (SFTSV) infection through a high throughput screening and be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which was in parallel to the reduced number of the large IBs restructured from regular lipid droplets and increased antiviral interferon induction. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs, to be virus factories for viral RNA replication and immune evasion through its interaction with NSs. The findings unveiled the function of synaptogyrin-2 as an enhancer in viral infection.
Distinct upstream role of type I IFN signaling in hematopoietic stem cell-derived and epithelial resident cells for concerted recruitment of Ly-6Chi monocytes and NK cells via CCL2-CCL3 cascade
Authors: Eo Seong Kug , Kim Jin Hyoung , Patil Ajit , Choi Jin Young , Kim Seong Bum , Uyangaa Erdenebileg
Published: January 2023

Type I interferon (IFN-I)-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV). However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6Chi monocytes and IFN-g/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6Clo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6Chi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I–dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6Chi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6Chi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I–dependent pathway that establishes orchestrated mobilization of Ly-6Chi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells.
Calcitonin gene related protein antiviral role in LP-BM5 infection of astrocytes and microglia
Authors: Grlickova-Duzevik Eliza , Chiem Daniel , Vaughn James , Cao Ling
Published: January 2023

CGRP (calcitonin gene related protein) is a neuropeptide expressed by primary sensory neurons upon infection. LP-BM5 retrovirus infection induces profound immunodeficiency in B6 mice and has been used as a murine model of HIV infection. Previously, we showed that LP-BM5 induced increased CGRP expression in the spinal cords of infected mice. In vitro studies indicated a microglia dependent, CGRP-induced reduction of LP-BM5 viral loads in primary mixed glial cells. To further delineate CGRP’s effects on glial cells, we treated specific murine cell lines (C8D1A astrocyte cell line and C8B4 microglia cell line) with LP-BM5 and CGRP. We found that LP-BM5 was capable of infecting both microglia and astrocytes individually and PCR detection for selected CGRP receptor components indicated the presence of CGRP receptor in both cell lines. CGRP’s antiviral effect was observed in both astrocytes and microglia, however a lower dose of CGRP was required for microglia. Western blot analysis suggested differential changes in CGRP downstream signaling pathways in astrocyte vs. microglia upon CGRP treatment. In addition, type I interferons known for their antiviral properties were elevated in astrocytes following CGRP treatment. We propose that CGRP mediated signaling pathways may mediate type I interferon production thus further promote antiviral responses.
The JAK/STAT signaling cascades of multiple cytokines are dysregulated during West Nile virus infection.
Authors: Roby Justin Alexander , Keller Brian C , Ramos Hilario J , Diamond Michael S , Gale Michael J
Published: January 2023

The neurotropic arthropod-borne flavivirus West Nile virus (WNV) is responsible for sporadic outbreaks of fever and encephalitis in infected humans, with an almost global distribution of circulating strains. During WNV infection in mammals Interferon-α/β (IFN-α/β) drives signaling through JAK/STAT pathways to enhance the expression of antiviral genes with an overall protective effect against WNV infection. However, WNV strains have been demonstrated to be able to antagonize IFN-α/β signaling within infected cells, interrupting this JAK/STAT cascade and promoting viral replication. We hypothesized that JAK/STAT signaling downstream of multiple cytokines would be broadly inhibited in WNV infected cells as a consequence of viral replication. To assess the degree of cytokine signaling in infected cells, A549 and PMA-differentiated THP-1 cells were infected with WNV for 24 hr prior to acute course treatment with various cytokines. We reveal that STAT3 and STAT5 phosphorylation, in addition to STAT1 and STAT2, is blocked in WNV-infected cells in response to IFN-β. Remarkably, JAK/STAT signaling cascades downstream of IFN-γ, IFN-λ3, IL-4, and IL-6 are also similarly dysregulated upon WNV infection, suggesting that global JAK/STAT signaling may be inhibited independent of the identity of the cytokine receptor. The breadth and mechanisms of the WNV-imposed JAK/STAT signaling blockade will be presented.
IL-27 suppresses CD4 and CD8 T cell cytotoxicity and viral control during cytomegalovirus infection
Authors: Wehrens Ellen J , Wong Kurt , Gupta Ankan , Benedict Chris , Zuniga Elina
Published: January 2023

Cytomegalovirus (CMV) is a large DNA β-herpes virus that infects the majority of humans and is endemic in wild mice. CMV establishes life-long latency with sporadic reactivation, and both primary infection and reactivation can cause serious disease in patients with compromised or naïve immunity. Horizontal CMV transmission commonly occurs through prolonged replication in the salivary gland (SG). In mice, CD4 T cells are absolutely required to curtail SG persistence, whereas CD8 T cells control acute systemic infection. Both CD4 and CD8 CMV-specific T cells from infected individuals display a strong cytotoxic phenotype and are able to kill target cells ex vivo. However, the immune factors that promote or regulate differentiation of these cytotoxic T cells remain largely unclear. Here we show that the pleiotropic cytokine IL-27 plays a key role in regulating CMV-specific T cell responses. Infection of mice deficient for the IL-27 receptor a subunit (Il27ra−/−) with mouse CMV (MCMV), resulted in enhanced expression of KLRG1 by both CD4 and CD8 T cells, a marker associated with terminal differentiation and cytotoxicity. Consistently, MCMV-specific CD4 and CD8 T cells from Il27ra−/− mice showed increased expression of both granzyme A and FasL. Importantly, this enhanced cytotoxic phenotype correlated with improved control of MCMV persistence in the SG. Taken together, our data reveal an unexpected and novel role for IL-27 in restricting T cell cytotoxicity and control of MCMV persistence, and suggest that targeting IL-27 has the potential to limit disease caused by persistent viral infection.
Interferon-stimulated gene LY6E enhances entry of diverse RNA viruses
Authors: Mar Katrina Bockying , Eitson Jennifer , Schoggins John
Published: January 2023

The type I interferon (IFN) response is the primary cellular defense mechanism against viruses. IFN protects against viral infection by activating the expression of hundreds of interferon-stimulated genes (ISGs), some of which have potent antiviral effector activity. Our recent screening efforts to identify novel antiviral ISGs unexpectedly uncovered a small subset of genes that enhanced viral infectivity. The goals of this project are to characterize the mechanism of LY6E, an ISG that enhanced unrelated viruses from multiple families. Here we show that ectopic expression of LY6E enhances cellular infection by a subset of enveloped RNA viruses from diverse families, including yellow fever virus, influenza A virus, vesicular stomatitis virus, and O’nyong’nyong virus. The enhancement phenotype is dependent on cell lineage, with the strongest effects seen in fibroblast and monocytic cell lines. Ablation of endogenous LY6E by CRISPR/Cas9 reduces but does not abrogate viral infectivity, indicating that LY6E is important for optimal infection. Loss of LY6E does not alter antiviral protection by IFN, suggesting that the enhancing effects may be direct. Mechanistic studies reveal that LY6E enhances infectivity at an early step in the viral life cycle, after viral binding but prior to the establishment of replication. Visualization of single-cell infections by ImageStream confirms that LY6E enhances viral entry. We conclude that LY6E enhances viral uptake at the population level, which may be important for initiation of the cell-mediated antiviral immune response. Understanding the mechanism and significance of LY6E to the antiviral interferon response may aid in the development of novel antiviral therapeutics.
Exploring a possible role for the MHC Class II accessory molecule H2-O in the development of experimental autoimmune encephalomyelitis
Authors: Welsh Robin A , Song Nianbin , Kim Aeryon , Poluektov Yuri , Sadegh-Nasseri Scheherazade
Published: January 2023

HLA-DO (DO), or H2-O in mice, is a non-classical, non-polymorphic MHC Class II accessory molecule. Expressed primarily in the thymic medulla and B cells, little is known about its physiological role in vivo. To gain insight of what role DO might have in CD4 T cell selection, our laboratory has previously used in vitro biochemical experiments to show that DO binds peptide-receptive DR1 and enhances binding of DM-insensitive peptides to the HLA-DR1 molecule, while inhibiting the binding of DM-sensitive peptides. These observations led us to hypothesize that in vivo, DO might serve to optimize presentation of immunodominant epitopes in the thymic medulla; thereby ensuring proper negative selection in the thymus. Lack of DO then could lead to faulty negative selection and a larger precursor pool of autoreactive CD4 T cells in the periphery. To test this hypothesis, we utilized H2-O knock-out (KO) mice and the well characterized autoimmune murine model of experimental autoimmune encephalomyelitis (EAE). We found that H2-O KO mice have a faster onset of EAE when compared to C57BL/6 wild-type mice. The rapid onset of the disease correlates with the recovery of larger numbers of MOG tetramer specific CD4 T cells from the CNS of H2-O KO mice at all stages of disease. A comparison of MOG tetramer specific CD4 T cell precursors in naïve H2-O KO and H2-O WT mice demonstrated larger numbers of MOG specific CD4 T cells in H2-O KO mice. Our data suggests that loss of DO correlates with an increase in the number of autoreactive MOG CD4 in KO mice likely due to faulty negative selection.
Adaptive humoral immune responses to type III capsular polysaccharide of Streptococcus pneumoniae.
Authors: Middleton Dustin Reed , Wantuch Paeton , Sun Lina , Avci Fikri Y
Published: January 2023

Capsular polysaccharides (CPSs) are important vaccine candidates given that they are located on the outermost surface of bacteria and have distinct structures. To induce CPS specific adaptive immune responses (i.e., T cell-mediated B cell responses), CPSs are conjugated with carrier proteins, to make glycoconjugate vaccines. The production of the current generation of glycoconjugate vaccines does not make use of specific scientific knowledge to maximize stimulation of critical immune cells (i.e., helper T cells) involved in producing protective IgG antibodies. Our previous discovery demonstrated that the mammalian CD4+ T cell repertoire contains a population of carbohydrate-specific T cells (i.e., Tcarbs) that recognize carbohydrate epitopes. Here, we investigate the interactions of carbohydrate epitopes with the key molecules of the adaptive immune system, MHCII proteins and T cell receptors (TCRs). This knowledge will have fundamental biological implications in our comprehension of how the adaptive immune system functions, and in future knowledge-based vaccine design. We employed a model bacterial polysaccharide antigen (capsular polysaccharide of type 3 Streptococcus pneumoniae, Pn3P) and its protein or peptide conjugates. We have collected evidence for the presence of a Tcarb repertoire that recognizes MHCII-presented Pn3P epitopes and drives Pn3P-specific IgG production by B cells. We have also generated Pn3 oligosaccharides through controlled depolymerization of Pn3P to be used in interation studies. Building on our expertise in these systems, we will define the MHCII and TCR interactions of processed Pn3P epitopes, and characterize immune responses that result from these interactions.
Characterization of the activation history and antigen specificity of B cells in meningeal clusters in Central Nervous System autoimmunity
Authors: Kerfoot Steven , Tesfagiorgis Yodit , Dang Amy K , Zhu Sarah , Craig Heather C
Published: January 2023

An important role for B cells in the pathology of Multiple Sclerosis (MS) is demonstrated by the therapeutic benefit of broad B cell depletion by anti-CD20 monoclonal antibodies. However, little is known about the subset(s) of B cells involved, the pathogenic mechanism(s), or the anatomical location from which they drive disease. Clusters of B cells have been observed in the meninges of some MS brains post mortem and it has been hypothesized that B cells drive pathology from these structures within the inflamed central nervous system (CNS). We used spontaneous and induced models of CNS autoimmune disease that incorporate B cell recognition of myelin autoantigen to investigate the identity and activation history of meningeal B cells in disease. B cells accumulated in the meninges in both models, often in clusters directly associated with demyelinating lesions. We found little evidence of organization into true lymphoid follicles nor evidence of typical B cell activation, despite clear ongoing inflammation and demyelination within the white matter. B cells had a largely naïve-like phenotype with little class-switch. Nevertheless, when compared to naïve lymph node B cells, these cells expressed more CD80 and less CD62L indicating some level of activation. To determine if meningeal B cells are specific for myelin antigen, we transferred fluorescent myelin-reactive T and B cells into wild-type recipient mice and induced EAE. While myelin reactive T cells dominated the T cell infiltrate of the spinal cord, as expected, almost no myelin reactive B cells were evident. This suggests that, unlike T cells, autoimmune B cells are excluded from the CNS in this model.
Autoantibody-mediated PAP in mice requires Myd88 in B cells
Authors: Ferretti Andrew P , Beedy Cady M , Barrington Robert A
Published: January 2023

Autoimmune pulmonary alveolar proteinosis (aPAP) is a lung syndrome caused by autoantibodies that neutralize granulocyte-macrophage colony-stimulating factor (GM-CSF, also known as Csf2). GM-CSF neutralization impairs alveolar macrophage function leading to the accumulation of surfactants in the alveoli. We recently discovered Rasgrp1-deficient mice (KO) are the first rodent model for this human disease, enabling the study of underlying immune mechanisms regulating the production of pathogenic GM-CSF-specific IgG autoantibodies. Because TLR signaling promotes breaks in B cell tolerance, and because GCs appear essential for autoantibody production in KO mice, we bred KO mice to mice conditionally lacking Myd88 in IgG1-expressing B cells (KO x Cγ1creMyd88fl/fl, referred to as KO Cγ1-Myd88) to test whether TLR signaling in mature B cells abrogates development of aPAP. As previously established, IgG1 was elevated in KO mice, and intriguingly KO Cγ1-Myd88 mice exhibited IgG1 titers similar to healthy control mice. Notably, GM-CSF-specific IgG serum autoantibody was also reduced in KO Cγ1-Myd88 mice in comparison to KO mice. Finally, histological analysis of H&E stained lung sections revealed that KO Cγ1-Myd88 mice do not develop aPAP. Taken together, this work has important implications for pathogenic anti-GM-CSF autoantibodies and aPAP, suggesting that 1) IgG1 is the predominant subclass and 2) TLR signaling in germinal centers is needed for break in tolerance of GM-CSF-specific B cells.
The expression of estrogen receptor alpha (ERα) by both the immune system and renal cells is required for induction of nephrotoxic serum-induced nephritis
Authors: Corradetti Chelsea , Madaio Michael , Caricchio Roberto
Published: January 2023

Glomerulonephritis (GN) is one of the most serious manifestations of systemic lupus erythematous (SLE). Lupus GN is characterized by severe inflammation and necrosis and it accounts for most morbidity and mortality. There is sex bias in SLE and estrogens may play an important role in GN susceptibility and progression. Interestingly, estrogen receptor alpha (ERα) in the renal tissue is highly expressed and the renal specific estrogen-induced gene activation is second only to that of reproductive organs. We have found that absence of estrogen receptor alpha protects female mice from developing nephritis, despite the presence of immune complexes in the kidneys and production of pro-inflammatory cytokines. To determine whether the ERα-expressing cells found in the immune system or kidney compartments were most important for inducing nephritis in this model. Bone marrow chimeras were generated from WT and ERαko mice and we used the nephrotoxic serum induced nephritis (NTN) model to induce GN. The results revealed that lack of ERα from both immune and renal cells was required to prevent disease in female mice; while males developed nephritis despite absence of ERα in either or both compartments. From these results, we conclude that ERα expression on immune and renal cells equally contribute to development of NTN and lack of ERα expression in both cell compartments is required to significantly reduce NTN in females.
Detection of anti-non bilayer phospholipids arragements antibodies and genotyping in patients with systemic lupus erythematosus and secondary antiphospholipid syndrome
Authors: Martínez María Teresa Hernández , Escamilla-Tilch Mónica , Reséndiz Albany , Wong-Baeza Carlos , Landa Carla , Jakez Juan , Barquera-Lozano Rodrigo , Hernández-Pando Rogelio , Granados Julio , Baeza Isabel
Published: January 2023

Phospholipids are the main structural constituents of all cell membranes, their form the smooth bilayer matrix that delimited cells in which membrane proteins are located. However, some anionic phospholipids can lead to the formation of nonbilayer phospholipid arrangements (NPA) within the bilayer. NPA are transient but when they are stabilized by the drugs chlorpromazine, procainamide or hydralazine, which produced lupus-like disease in humans, they induce a disease resembling human lupus in mice. Mice present IgM and IgG antibodies against NPA which appear 4 weeks before that autoantibodies against cardiolipin, histones, nuclear and lupus anticoagulant antibodies. Mice also present histopathological abnormalities in the skin and kidneys similar to those observed in systemic lupus erythematosus (SLE). We proposed that the recognition of NPA by anti-NPA antibodies and after by the classical complement pathway causes cell lysis exposing intracellular antigens with the consequent formation of autoantibodies. Antibodies against NPA have been described in patients with SLE, lepromatous leprosy and hypertensive disorders of pregnancy. The present investigation is a clinical study not randomized, prospective, in Mexican mestizos patients of any sex and age, with SLE or with secondary antiphospholipid syndrome in which we found that the elevated levels of anti-NPA antibodies could trigger autoimmunity in these diseases and we determined that high titers lead to increased activity and correlate with the expression of HLA-DR, HLA-DQ, HLA-A, HLA-B, IL-10 (-1082G/A, -819C/T, -592C/A) and PTPN22 (+1858C/T) alleles. All clinic assays were approved by the Committee of Bioethics of our Institutions.
The effect on transforming growth factor-beta-induced protein (beta ig-h3) expression by LPS and MDP stimulation to mice bone marrow derived macrophages
Authors: Kim Ha-Jeong , Rhe Su-Yeon , Nam Ju-Ok
Published: January 2023

The crosstalk between the inflammatory milieu and the immune response during inflammatory process is likely to be communicated in large part through the extracellular matrix (ECM). Transforming growth factor-beta-induced protein (beta ig-h3) is produced from several cells include fibroblasts, and exist extracellular matrix. big-h3 acts as a proinflammatory molecule. It is reported that blood concentration of big-h3 is high in CLP-induced septic mice, and the absence of big-h3 attenuated CLP-induced sepsis. MDP, is small peptide from Gram positive bacterial wall product peptidoglycan, is reported to suppress LPS induced immune response. Taken together, Here, we addressed how big-h3 is regulated by LPS and MDP costimulation in macrophages and what is its function on inflammatory site.
Differences in gene expression in human monocytes and macrophages from healthy and asthmatic donors in response to IL-4
Authors: Becerra-Diaz Mireya , McCormick Sarah M. , Fang Jessie Xi , Heller Nicola M
Published: January 2023

The lungs from asthmatics are in a state of Th2-driven inflammation with a shift in macrophage polarization toward an M2 profile induced by IL-4 and IL-13. The number of M2 macrophages in the lung correlates with asthma severity. Women have more lung M2 cells than men and are more likely to have asthma than men. We hypothesized that sex differences affect macrophage polarization in humans. First, we determined that M2-related gene expression was different between healthy and asthmatic donors in response to IL-4 and IL-13. Monocytes from asthmatics expressed more CCL17 in response to IL-4, while CCL22 and MMP12 expression was similar. We found that CD200R was highly expressed in response to IL-4 in monocyte derived-macrophages (MDM) from asthmatics compared with those of healthy donors. Furthermore, this increase was greater in MDM from women compared to men. Moreover, MMP12 and CCL22 expression were not different between the sexes. Interestingly, some genes (TGM2) were only up-regulated in MDM from asthmatics. Second, we analyzed the effect of male and female sex steroid hormones on the IL-4 signaling pathway. In the presence of IL-4, we found that both estrogen (E2) and dihydrotestosterone(active metabolite of testosterone) can induce changes in phosphorylation of Akt but not in STAT6 in MDM. Similar effects on signaling were results were obtained in U937, a human monocytic cell line. The stronger M2 response in female cells could be due to increased expression of estrogen receptors. We found that monocytes from women displayed greater expression of ERa isoforms. These results indicate a potential role for both intrinsic sex differences and E2 as inducers of M2 polarization in macrophages in asthma, leading to worse lung inflammation in women.
TGFβ inhibits IL-33-mediated mast cell activation in vitro and in vivo
Authors: Ndaw Victor S. , Kolawole Elizabeth Motunrayo , Abebayehu Daniel , Paez Patrick A , Baker Bianca , Barnstein Brian O , Taruselli Marcela T , Ryan John J
Published: January 2023

TGFβ is involved in many pathological conditions, including autoimmune disorders, cancer, and cardiovascular and allergic diseases. We have previously found that TGFβ suppresses IgE-mediated mast cell activation in human and mouse mast cells in vitro. IL-33 is a recently discovered member of the IL-1 family that functions as an “alarmin” stimulating mast cell responses and enhancing IgE-mediated activation. How TGFβ affects IL-33 signaling is now known. We find that mouse bone marrow-derived mast cells cultured in TGFβ -1, -2, or -3 showed reduced IL-33-mediated production of TNF, IL-6, IL-13 and MCP-1, in a concentration-dependent manner. Furthermore, TGFβ also reduced expression of the IL-33 receptor ST2, as well as IL-33-mediated TAK1, IKB and ERK phosphorylation. TGF-β1 injection suppressed IL-33-mediated production of systemic inflammatory cytokines in vivo. The role of IL-33 in the pathogenesis of allergic diseases is incompletely understood. These findings, consistent with our previously reported effects of TGFβ on IgE-mediated activation, demonstrate that TGFβ can provide broad and substantial inhibitory signals to activated mast cells. These data suggest possible therapeutic strategies to maintain mast cell homeostasis in autoimmune and allergic diseases.
A Novel, Quantitative, Multi-analyte Immunoassay to Detect Biomarkers Involved in Lung Disease
Authors: Anderson Michael
Published: January 2023

Lung disorders affect anywhere from 5–10% of the world’s population, and account for 1/6 of deaths worldwide. Pulmonary insult can arise from a multitude of factors, including those that are extrinsic, intrinsic, or iatrogenic-related. Manifestations of pulmonary disease include asthma, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD), interstitial pneumonia (UIP), lung cancer, and tuberculosis (TB). The array of symptoms associated with these lung conditions involve shortness of breath, cough, fever, pleural effusion, pleural thickening pulmonary fibrosis, necrobiotic nodules, and bronchiolitis obliterans organizing pneumonia (BOOP). A decline in the quality of life in patients with lung disease is experienced not only physically, but also mentally due to the severity of the symptoms. One example is lung cancer, which is the most common cause of death in developed countries. Patients diagnosed with stage III or IV lung cancer have a survival rate of only 15% with current therapies.

The study presented here looked at several cytokine markers involved in airway constriction including IL-13, IL-5, and IL-4 as well as select biomarkers known to be involved in the regulatory pathways for IFN-g and TNF-a. The Simple Plexä assay that was employed is a novel, quantitative, multi-analyte immunoassay platform that delivers high precision and accuracy from only 25 μL of sample. This platform measures up to four analytes simultaneously from a single sample with very high sensitivity. It is a microfluidic-based system that allows parallel single analyte detection, and reduces non-specific antibody interactions often observed in other traditional multiplex platforms.
Leukocyte Intracellular Cytokine and Chemokine Response Kinetics to Poly-Trauma and Resuscitation in Non-Human Primates
Authors: Paredes Ruth Madelaine , Sooter Amanda , Vernon Philip Joseph , Glaser Jacob , Sheppard Forest
Published: January 2023

Background

Traumatic injury induces a significant inflammatory response. Post-injury responses of leukocyte subtypes, to include activation kinetics remain to be fully characterized to effectively direct therapeutic immunomodulation. We sought to determine leukocyte response kinetics to poly-trauma and resuscitation in non-human primates (NHPs).

Methods

Rhesus macaques (n=32) were randomized into four groups: 30min hemorrhagic shock (30HS), 60min HS (60HS), 60HS + laparotomy, and 60HS + laparotomy + femur fracture/repair followed by resuscitation and 24 hours monitoring. Blood was collected at baseline (BSLN), end of shock (EOS), end of resuscitation (EOR), t=360min and t=1440min and stained with a pan-leukocyte panel for flow cytometric analysis. Samples were stained for intracellular markers to differentiate leukocyte subsets such as cytotoxic lymphocytes (CTL), T helper (Th), Macrophage and B-cells. Results reported as mean±SEM, statistical analysis: two-way ANOVA with p<0.05 significance.

Results

Levels of IL-10 in CTL, Th, B, NK-cells, and neutrophils increased over the experiment from BSLN. Hemorrhage induces a significant elevation of regulatory T-cells at 24hours, however, the addition of injuries abrogates this expansion. No other marker was found significantly different between groups/time points.

Conclusion

We have demonstrated that comprehensive flow analysis is effective for determining leukocyte subset kinetics in a poly-trauma model. Increases in T-cell IL-10 during resuscitation indicate nominal regulatory T-cell bursts. This was not observed in animals with additional injuries, suggesting animals with severe trauma may suffer from unfettered inflammatory complications downstream.
Disruption of Interleukin-17 receptor A (IL-17RA) gene in mice aggravates renal interstitial fibrosis in obstructive nephropathy
Authors: Ramani Kritika , Zhou Dong , Tan Roderick , Liu Youhua , Gaffen Sarah , Biswas Partha Sarathi
Published: January 2023

Renal fibrosis is the final outcome of obstructive nephropathy and a major cause of chronic renal failure. Renal fibrosis results from an imbalance between extracellular matrix (ECM) synthesis and degradation, which is regulated by multiple inflammatory mediators in the kidney. Recently, Interleukin-17 (IL-17) emerged as a key cytokine in lung and liver fibrosis. Although IL-17 producing cells were detected in the obstructed kidney, the role of IL-17 in renal fibrosis is unknown. We sought to define the contribution of IL-17 in renal fibrosis in obstructive nephropathy. Mice deficient in IL-17 receptor signaling (IL-17RA−/−) were assessed for renal fibrosis following unilateral ureteral obstruction (UUO), a well-characterized mouse model of obstructive nephropathy. We determined IL-17 receptor signaling is required for protection against renal fibrosis following UUO. IL-17RA−/− mice developed severe morphological injury and displayed increased deposition of collagen in the obstructed kidney compared to controls. We observed a significant decrease in the level of matrix degrading enzymes in the kidney of IL-17RA−/− mice, despite normal ECM synthesis, correlating with increased fibrosis. Overexpression of IL-17 increased the level of matrix degrading enzymes and protected mice from renal fibrosis. We also show that IL-17R signaling in tubular epithelial cells up regulated multiple genes of Kallikrein-kinin system, known to play a critical role in renal protection against kidney fibrosis. Our data clearly indicate a previously unappreciated role of IL-17 in renal protection against fibrosis. These results offer the opportunity for the development of potential therapies in the treatment of chronic kidney diseases.