bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-15 expression on B cells and myeloid cells is distinctly modulated by inflammatory factors in MS and healthy donors
Authors: Farzam-kia Negar , Moratalla Ana Carmena , Lemaître Florent , Beauseigle Diane , Haddad Elie , Larochelle Catherine , Prat Alexandre , Arbour Nathalie
Published: January 2023

Although the salience of the immune system in multiple sclerosis (MS) pathology has been established, the mechanisms involved are incompletely characterized. We have identified IL-15 as a relevant contributor to MS neuropathology; a greater proportion of antigen presenting cells (APC) (B cells and myeloid cells) from MS patients express IL-15 vs. controls. Our aim is to identify factors and mechanisms that contribute to elevated IL-15 levels in the context of MS. The addition of CpG, a TLR9 agonist, to CD40L-cultured human B cells increased the proportion of IL-15+ B cells. Whereas other TLR ligands (e.g. polyI:C) did not alter B cell IL-15 levels, they nevertheless increased CD40 expression. Monocytes from MS patients and age/sex-matched healthy controls (HC) were differentiated into M0 macrophages prior to M1 or M2 polarization. M2 polarization significantly enhanced the percentage of IL-15+macrophages; in contrast, M1 polarization did not. GM-CSF, which is produced by encephalitogenic T cells, induced a major increase in the proportion of IL-15+ M0 and M1 macrophages and decreased the proportion of IL-15+ monocytes from HC and MS donors. While IL-15+ M2 macrophages from HC are increased upon GM-CSF stimulation, IL-15+ M2 from MS donors are not. GM-CSF did not alter IL-15 expression on microglia. Pharmacological inhibitors of STAT5 (Pimozide) and JAK2 (AG490) phosphorylation decreased the GM-CSF-induced increase of IL-15 on M0 macrophages. Thus, GM-CSF is a potent trigger for IL-15 expression on human M0, M1 and M2 macrophages, but not monocytes or microglia. Our data suggest that specific stimuli mediate IL-15 expression on human APC and that cells from MS donors exhibit altered susceptibility to IL-15 modulators compared to HC.
NSG Mice Engrafted with Human PBMC Provide a Robust Platform for Preclinical Testing of Immunomodulatory Therapeutics Relevant to GvHD, Autoimmunity, and Oncology
Authors: Soper Brian , Rose Aaron , Hodel Ashley , Kaur Pali
Published: January 2023

NSG mice engraft human PBMC and allow efficacy testing of immunomodulatory drugs in a GvHD setting. Engraftment is progressive and at d5 PBL has 10% human cells with a CD4:CD8 T cell ratio of 3:1. The CD4 cells are 30% CD45RO− CCR7+ naïve (Tn), 40% CD45RO+ CCR7+ central memory (Tcm) and 10% CD45RO+ CCR7− effector memory (Tem). The CD8 cells at d5 contain 9% Tn, 3% Tcm, and 5% Tem. At d18 human cells are 70% with a CD4:CD8 ratio of 1:3, 0% Tn, 10% Tcm and are 22% CD4 Tem and 52% CD8 Tem. Engrafted mice were treated either prophylactically (day −1) or therapeutically (day 5) with Abatacept or an unknown test article. PBL at d10 post engraftment from mice treated at day −1 showed human cells at 14.6%, 2.2%, and 1.6% for vehicle, Abatacept, and test article, respectively. The Abatacept group were primarily CD4 Tn. Abatacept and test article treated mice showed 100% survival, almost no tissue infiltration, normal body weight, and were healthier based on disease activity index (DAI) scores. When dosed therapeutically, human cells at d18 were 67.3%, 16.9%, and 37.9%, respectively. Both treatments suppressed T cell expansion, maintained a 3:1 CD4:CD8 ratio and decreased development of the CD8 Tem pool (CD8 Tem 32.7%, 15.4%, and 4.9%, respectively). 100% of the d5 dosed Abatacept group survived, maintained body weight, and had improved DAI. 70% of the d5 dosed test article group survived and at d20 began losing body weight and accumulated poor DAI scores. The data presented here provide a detailed analysis of activated human T cell pools and progression of GvHD in NSG recipients of human PBMC. The model allows detailed analysis of in vivo modulation of T cell activity and therefore enables rapid evaluation of therapies relevant to not only GvHD, but to autoimmunity and oncology.
Detection and Characterization of T cells Reactive to Hybrid Insulin Peptides in Patients with Type 1 Diabetes
Authors: Baker Rocky Lee , Gottlieb Peter A , Delong Thomas , Haskins Kathryn
Published: January 2023

Our lab has recently established that Hybrid Insulin Peptides (HIPs) are antigenic ligands for diabetogenic T cell clones in the NOD mouse model of autoimmune diabetes. Furthermore, these peptides are detected in the islets of NOD mice, indicating that islet-infiltrating T cells encounter their antigen at the site of immune injury. Importantly, we have found that the phenotype of HIP-reactive T cells present in the peripheral blood of NOD mice is indicative of ongoing autoimmunity in the pancreas. In the human setting, we demonstrated that T cells reactive to HIPs are present in the residual islets of type 1 diabetic (T1D) organ donors. In this study, using an IFN-g ELISPOT assay, we investigated whether T cells reactive to a panel of 16 HIPs could be observed in the PBMCs of T1D patients or control subjects. We observed that about a third of the T1D patients responded to at least one HIP; 70% of the responding patients (but only one control subject) showed reactivity to multiple HIPs. Responses to 4 HIPs were significantly elevated in T1D patients but not in control subjects. Using a multiplexed cytokine assay, we determined that T cells from responder patients had a bias towards a Th1 phenotype upon stimulation with HIPs. We isolated 8 non-redundant, antigen-specific T cell clones reactive to 6 different HIPs. These T cell clones react to their target HIPs in the low nanomolar range and secrete IFN-g, TNF-a and GM-CSF. This is the first report that HIP-reactive T cells with an inflammatory phenotype can be detected in the peripheral blood of patients with T1D, indicating that the presence of HIP-reactive T cells might serve as a biomarker of disease activity in the human setting.
Galectin-3 promotes non-canonical inflammasome activation through binding to glycans on LPS intracellularly
Authors: Lo Tzu-Han , Chen Hung-Lin , Chen Huan-Yuan , Liu Fu-Tong
Published: January 2023

In response to infection, pyroptosis is a typical inflammatory form of cell death. Even though the various stimuli have been well elucidated in the regulation of host immune responses through caspase-1, the mechanism of caspase-4/5/11 is not defined. Lipopolysaccharides (LPS) from Gram-negative bacteria have been shown to induce non-canonical inflammasome activation by binding to caspase-4/5/11 through its lipid A moiety intracellularly and causing oligomerization and activation. Galectin-3, belonging to a beta-galactoside-binding protein family, can bind to LPSs from different Gram-negative bacteria, which are composed of beta-galactoside-containing polysaccharide chains. Galectin-3 is known to self-associate upon binding to multivalent glycans through its non-lectin domain. Therefore, we hypothesized that galectin-3 can cross-link LPS and form an ordered array of galectin-3-LPS complex, and consequently, amplify LPS-induced caspase-4/5/11 oligomerization and activation. To test this hypothesis, we transferred LPSs intracellularly by liposome-mediated transfection into galectin-3−/− bone marrow-derived macrophages (BMDMs) and wild-type cells. We noted galectin-3 deficiency resulted in a lower level of LPS-induced pyroptosis. Co-immunoprecipitation confirmed the interaction between galectin-3 and caspase-11 in RAW264.7 cells after LPS intracellular delivery. In a cell-free system, we showed that galectin-3 facilitated the assembly of LPS-induced caspase-4 oligomerization and activation. In conclusion, galectin-3 is a novel host factor in the induction of higher degree of caspase-4/5/11 oligomerization and activation through LPS binding, resulting in more intense pyroptosis.
Glycolytic regulation of neuroinflammatory response: Role of monocarboxylate transporter 4
Authors: Giri Shailendra , Singh Jaspreet , Suhail Hamid , Kumar Ashok , Rattan Ramandeep
Published: January 2023

Our study aimed to investigate the functional importance of the monocarboxylate transporter (MCT) 4 in vitro, in mouse primary mixed glial cells and in vivo, in mouse brain. Stimulation of primary mixed glial cells with LPS/IFNγ (LI)-induced the expression of MCT4. Inflammatory signals increase glycolysis and lactate production, which have been shown to sustain inflammatory response in immune cells. A real-time increase in glycolysis in LI-stimulated mixed glial cells was observed. We also found that IL-1β and NLRP3 expression was significantly increased in stimulated mixed glial cells, which are associated with chronic inflammatory diseases. MCT4 inhibitor, CHCA, significantly downregulated the proinflammatory response, glycolytic gene expression and real-time glycolysis in the primary mixed glial cells. As expected, CHCA blocked the release of lactate into the culture medium. Knockdown of MCT4 by using siRNA significantly attenuated the proinflammatory response suggesting that MCT4 could be a therapeutic target for neuroinflammatory diseases. Further, targeted metabolomics approach revealed that glycolytic intermediated, phosphoenolpyruvate and ribulose-1,5-bisphosphate, were significantly increased in primary mixed glial cells stimulated with LI, which were significantly decreased by CHCA treatment suggesting that intracellular accumulation of lactate attenuates glycolysis. CHCA treatment also diminished production of cytokines in the brain and spleen cells in an endotoxin mouse model. Importantly, we show that pharmacologic targeting of lactate transporters in glial cells might provide a novel and viable approach to resolve chronic inflammation in metabolic and neurodegenerative diseases.
Mature Tissue-resident Memory T Cells Lose the Responsiveness to Bystander Inflammation
Authors: Liao Wei , Zhang Nu
Published: January 2023

Circulating memory T cells respond to both antigen-specific stimuli and antigen–nonspecific inflammation. IL-18, together with IL-12 induces robust bystander responses from circulating memory T cells. Tissue-resident memory T (Trm) cells represent an essential component of local immunity that elicits immediate response upon cognate antigen re-challenge. However, whether Trm responds to bystander inflammation is not entirely clear. Stepwise induction of CD69 and CD103 marks distinct differentiation stages of mucosal Trms. Because the majority of non-mucosal Trm lacks the expression of CD103, the maturation of non-mucosal Trms is largely unknown. Here, we found that both mucosal and non-mucosal Trms downregulated IL-18 receptor during maturation and largely lost the response to bystander inflammation. Local cytokines including TGF-β and type I IFN control Trm maturation. These data indicate that distinct from circulating memory T cells, Trms limit the response to bystander inflammation to prevent unnecessary collateral tissue damage.
Recruitment of NEMO/IKKγ to TCR microclusters during T cell activation
Authors: Koylass Nicholas Robert , DeRiso Elizabeth A. , Szymczak-Workman Andrea L. , Montecalvo Angela , Murphy Joanne M. , Seminario Maria Cristina , Kane Lawrence P. , Bunnell Stephen C.
Published: January 2023

The IκB kinase (IKK) complex mediates the activation of canonical NFκB isoforms following T cell receptor (TCR) ligation. This complex consists of the kinases IKKα and IKKβ and an essential adaptor subunit, the NFκB essential modulator protein (NEMO). Most models suggest that the IKK complex is activated within oligomeric Carma1/Bcl10/Malt1 (CBM) signalosomes. However, we observed that NEMO enters TCR microclusters before CBM complexes are assembled, within ~70 seconds of TCR engagement. NEMO also entered mobile vesicles and in larger membrane-bounded structures (hereafter ‘macroclusters’). The recruitment of NEMO into TCR microclusters is prevented by Src kinase inhibitors and by the catalytic inactivation of ZAP-70, but occurs in the absence of either SLP-76 or Carma1. Further, NEMO fails to co-localize with TCR-induced CBM polymers. Thus, the recruitment of NEMO to the TCR occurs via a CBM-independent mechanism. The deletion the zinc-finger (ZnF) domain of NEMO disables NFκB signaling and eliminates NEMO from microclusters, while preserving NEMO macroclusters. Since the ZnF domain interacts with polyubiquitin chains, we generated point mutations impacting the ubiquitin-binding site in the ZnF domain and two independent sites within the NEMO ubiquitin-binding ‘NUB’ domain. These mutations impair the ability of NEMO to capture K63-linked and/or linear polymers, hinder NFκB signaling, and eliminate NEMO from microclusters without disrupting NEMO macroclusters. These findings suggest that NEMO is rapidly recruited to polyubiquitin chains associated with the TCR, rather than the CBM complex, and that the CBM complex augments IKK-dependent NFκB signaling via a distinct, recruitment-independent mechanism.
TCR-pMHC bond length controls TCR ligand discrimination
Authors: Huang Jun , Sasmal Dibyendu K.
Published: January 2023

T-cell receptors (TCRs) detect specifically and sensitively a small number of agonist peptide-major histocompatibility complexes (pMHCs) from an ocean of structurally similar self-pMHCs to trigger antigen-specific adaptive immune responses. Despite intense efforts, the mechanism underlying TCR ligand discrimination remains a major unanswered question in immunology. Here we show that a TCR discriminates between closely related peptides by forming TCR-pMHC bonds with different lengths, which precisely control the accessibility of CD3ζ immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation. Using in situ fluorescence resonance energy transfer (FRET), we measured the intermolecular length of single TCR-pMHC bonds and the intramolecular distance of individual TCR-CD3ζ complexes at the membrane of live primary T cells. We found that an agonist forms a short TCR-pMHC bond to pull the otherwise sequestered CD3ζ off the inner leaflet of the plasma membrane, leading to full exposure of its ITAMs for strong phosphorylation. By contrast, a structurally similar weaker peptide forms a longer bond with the TCR, resulting in partial dissociation of CD3ζ from the membrane and weak phosphorylation. Furthermore, we found that TCR-pMHC bond length determines 2D TCR binding kinetics and affinity, T-cell calcium signaling and T-cell proliferation, governing the entire process of signal reception, transduction and regulation. Thus, our data reveal the fundamental mechanism by which a TCR deciphers the structural differences between foreign antigens and self-peptides via TCR-pMHC bond length to initiate different TCR signaling for ligand discrimination.
Regulatory role of Ifit2 in mouse hepatitis virus induced neuroinflammation
Authors: Sarma Jayasri Das , Burrows Amy , Kessler Patricia , Hwang Mi-Hyun , Bergmann Cornelia , Sen Ganes C
Published: January 2023

Interferons (IFNs) are known to protect from virus dissemination and pathogenesis by means of IFN stimulated genes (ISG) but the mechanisms underlying the antiviral effects are not clearly understood. IFN induced tetratricopeptide repeats (Ifit) genes are a class of ISGs represented in mice by Ifit1, Ifit2 and Ifit3. Intracranial infection of mice with MHV significantly increases mRNA levels of all 3 Ifit genes in the CNS. Moreover, Ifit2 is beneficial in limiting encephalitis caused by neurotropic viruses like VSV, WNV, EMCV, and MHV. To provide better cellular insights into the protective mechanisms of Ifit2, we studied viral spread and inflammation in young Ifit2−/− mice (4–5 weeks) infected with MHV-A59. Compared to wildtype (wt) viral spread was rapid and uncontrolled in Ifit2−/− mice and mortality was reduced to 70% by day 6–8 p.i. at 2000 PFU. Also, the pattern of virus distribution in spinal cords was significantly different. Where virus was restricted prominently to the gray-white matter border in wt mice, viral antigen expanded to both white and gray matter in Ifit2−/− mice, but they are less inflamed and less encephalitic. Surprisingly, despite vastly elevated virus load, microglia/macrophage activation was limited compared to wt mice as examined by anti-Iba-1 staining. Moreover, leukocyte recruitment was reduced despite similar induction of inflammatory cytokines. By contrast, type I IFN induction was significantly reduced. At lower viral doses (500 PFU), Ifit2−/− mice developed severe clinical symptoms and myelin damage at the chronic infection stage. Given the homeostatic interactions between microglia and neurons, we suggest that Ifit2 deficiency may disrupt microglial activation and protective functions to neurons.
The RNA binding protein HuR is required for antigen-mediated activation and proliferation of CD4+T cells
Authors: Ellis Jason S , Taylor Tiffany , Atasoy Ulus
Published: January 2023

The RNA binding protein HuR directs helper T cell fate decisions by interacting with 3′ AU-rich elements within the untranslated region of multiple target mRNAs, leading to increased stability and transport to the heavy polysomes. We hypothesized that HuR plays a key role in antigen-induced T cell activation and expansion. HuR was conditionally ablated late during T cell development, immediately before thymic egress (distal Lck-Cre ROSA HuRfl/fl mouse). Upon activation in vitro, HuR KO T cells had significant reductions in Th2 cytokine production (IL-4, IL-5, and IL-13) compared to control cells and greatly increased IL-2. There was no difference in IFNgproduction between HuR KO and controls. Reductions in Gata-3, IL-4 and IL-2Ra (CD25) nascent transcription were also observed. An OVA challenge model of airway inflammation was used to ascertain the effects of HuR KO in vivo. While only half of CD4+T cells in HuR KO mice lack HuR, there was a lack of Th2 responses and these mice had complete amelioration of lung inflammation. OVA challenged HuR KO mice also had significantly reduced BAL IL-13. In parallel, mice were immunized with either OVA or KLH, boosted 10 days later, and antigen specific proliferative responses measured in vitro to investigate antigen-specific T cell responses in the absence of HuR. HuR KO T cells had profound proliferation defects compared to controls, and CD3/CD28 stimulation only partially restored proliferation defects. HuR KO T cells had reductions in STAT5/STAT6 signaling, pathways which are key for Th2 differentiation and function. We conclude HuR plays an indispensable role in regulating antigen-specific activation and proliferation in CD4+T cells, as well as controlling IL-2 homeostasis.
JUUL E-vapor Has Less Protective Effect in a Zebrafish Model of Heart Failure Compared to Nicotine Salt, Nicotine Agonists, and First-Generation ENDS
Authors: Mai Xuyen Kim , Chow Sheryl , Cameron Joshua , O’Barr Stephen A
Published: January 2023

Heart failure (HF) is defined by the impairment of ventricular filling that results in loss of oxygen and damage to the myocardium. HF affects about 2% – 3% of people worldwide in the United States. At the same time, e-cigarette use among young adults has been on the rise because of social norms. The correlation between HF and e-cigarette can be explained by the phenomenon called the “smoker’s paradox”, which states improvement in acute myocardial infarction (MI) and smokers via the interaction between three processes. These processes involve activation of the cholinergic anti-inflammatory pathway (CAP) via the vagus nerve and stimulation of the α7 nicotinic acetylcholine receptor (α7 nAChR). When these processes are activated, inflammatory properties of HF are inhibited. Our lab and others have shown that nicotine salt, α7 nAChR agonists, and first-generation ENDS vapor have potential therapeutic benefit for HF through activation of CAP. In this study, zebrafish were pretreated for 20 minutes over four consecutive days with e-vapor from a flavored JUUL pod, nAChR agonists GTS-21 and AR-R17779, or pure nicotine salt. On day four, MI was induced by chemical induction. Survival times were recorded, and heart tissue was collected for analysis. Regulatory gene and cytokine levels in the myocardium were measured by rtPCR. Results show that nicotine salt, nicotine agonists, and e-vapor from first-generation ENDS have more protective effect as measured by longer survival times and lower regulatory gene and cytokine expression levels, compared to JUUL e-vapor pretreated fish.
The role of the von-Hippel Lindau gene in osteocytes on B lymphocyte maturation and activation
Authors: Chicana Betsabel J , Manilay Jennifer O
Published: January 2023

The von-Hippel Lindau protein (VHL) regulates hypoxia-inducible factor (HIF) degradation, which is involved in cellular adaptation to low oxygen environments. Conditional deletion of Vhl in osteoblasts and in hematopoietic progenitors have demonstrated a role for VHL in these cell types, but how changes in bone homeostasis affects immune development are not fully understood. Osteoblasts (OBs) support B cell development and mature into osteocytes (OCYs). To test if OCYs also support B cell development, we generated OCY-specific (Dmp1-Cre) conditional VHL knockout (cKO) mice. We previously found that cKO mice display dysregulated bone growth, high bone density, smaller bone marrow (BM) cavity volume, and overall decrease in BM cellularity compared to controls. In line with this, the frequencies and numbers of B cell precursors (B220+ IgM−), immature B (B220+ IgMint) and recirculating B (B220+ IgMhigh) cells in the bone marrow are significantly decreased. Although the absolute numbers of B cells in the spleens of cKOs were normal, frequency of B220+ CD19+ cells are decreased, while B1a (IgM+ CD5+) cells are increased. To define the mechanisms by which changes in the bone microenvironment affect B cells, we are investigating oxygenation status, vascular architecture and metabolic profiles of bone and B cells. We hypothesize that loss of VHL in OCYs could permanently and adversely affect B cell functional response. To test this, responses in T-independent and T-dependent antigens will be assessed in cKOs. Taken together, our data suggest that OCY-specific VHL deletion might be helpful to increase bone density, but at the expense of B cells.
LSD1 epigenetically represses NF-κB target genes to regulate marginal zone B cell development
Authors: Haines Robert Richard , Scharer Christopher D. , Boss Jeremy
Published: January 2023

Marginal zone B cells (MZB) are a mature B cell subset that rapidly respond to blood-borne pathogens and are transcriptionally poised to differentiate. Although the transcriptional changes that occur throughout MZB development are known, the corresponding epigenetic changes and epigenetic modifying proteins that facilitate these changes are poorly understood. The H3K4 mono- and di-methyl demethylase LSD1, an epigenetic and transcriptional modifier that promotes plasmablast formation, represents a prime candidate for regulating MZB development because of the high degree of overlap between MZB and plasmablast transcriptomes. B cell-conditional deletion of LSD1 resulted in a two-fold decrease in MZB while follicular B cells (FoB) and bone marrow B cell populations were minimally affected. LSD1 repressed genes in MZB that were normally upregulated in FoB and are involved in adhesion, migration, interferon response, transcriptional regulation, and signaling. MZB lacking LSD1 exhibited alterations in chromatin accessibility at transcription factor family motifs, including NF-κB. The importance of NF-κB signaling was examined through an ex vivo MZB development assay, which showed that both LSD1-deficient and NF-κB-inhibited transitional B cells failed to undergo full MZB development. Gene expression and chromatin accessibility analyses of in vivo- and ex vivo-generated LSD1-deficient MZB indicated that LSD1 regulated the downstream target genes of non-canonical NF-κB signaling. Overall, these data reveal that the epigenetic modulation of the NF-κB pathway by LSD1 is an essential process during MZB development.
Understanding mechanisms of disease tolerance during intestinal helminth infection
Authors: Gentile Marilena , Li Yue , Robertson Amicha , Fontes Ghislaine , Kaufmann Eva , Khan Nargis , Munter Hans M. , Divangahi Maziar , King Irah L.
Published: January 2023

Intestinal helminths infect over two billion people worldwide and can cause significant morbidity. In many cases, however, individuals fail to develop resistance to these tissue-invasive pathogens, indicating that mammalian hosts have evolved unknown strategies to tolerate infection. To investigate the mechanisms underlying host tolerance to helminth infection, we examined the initial tissue invasive stage of Heligmosomoides polygyrus bakeri (Hpb), a natural parasitic roundworm infection of mice. Unexpectedly, we observed a rapid and robust Type 1 inflammatory response that preceded the expected Type 2 immune response. This early Type 1 response was associated with a gut-specific expansion of IFNγ-producing Eomesodermin+ Natural Killer (NK) cells with no increase in other innate lymphoid cell populations. Parabiosis and confocal microscopy studies indicated that Eomes+ NK cells are recruited from circulation and surround the larvae in association with the intestinal vasculature. The recruitment of NK cells to the small intestinal lamina propria is dependent on IFNγ-induced expression of CXCL9 and CXCL10, and the presence of αβ+ T cells. NK cell depletion did not alter worm burden or fitness, but resulted in increased incidence of intestinal bleeding, suggesting a role for NK cells in promoting intestinal vascular integrity. In summary, our work provides new insight into the cellular dynamics required for protection during intestinal helminth infection and will help inform strategies to maximize host fitness in the context of tissue injury and repair.
Interplay of Wnt β-catenin pathway and mTORC1 in pathogenesis of Ehrlichia-induced sepsis
Authors: Haloul Mohamed Abdalla , El Andaloussi Abdeljabar , Ismail Nahed
Published: January 2023

The Wnt/β-catenin pathway has been evolved in regulating cell proliferation, survival, and inflammation in cancer development. However, the role of β-catenin during infections remains elusive. Ehrlichia is obligate intracellular bacteria that cause life-threatening sepsis, characterized by liver injury and dysregulated inflammatory response with no effective therapy available. We examined here the contribution of Wnt pathway to the pathogenesis of Ehrlichia-induced sepsis employing murine models of mild and fatal ehrlichiosis caused by intraperitoneal infection with avirulent Ehrlichia muris (EM) and virulent Ixodes Ovatus Ehrlichia (IOE), respectively. We show that EM infection induces higher activation of the hepatic Wnt/β-catenin pathway and activates Wnt target genes; C-Myc and Cyclin D1 on day 7 post infection compared to uninfected controls. Notably, IOE infection attenuated Wnt/β-catenin pathway in liver tissue, which is associated with inflammasome activation, inhibition of autophagy in macrophages, host cell death, and increased number of replicating bacteria, suggesting that Wnt pathway is a host-protective mechanism that also enhances cell survival and attenuate inflammation during Ehrlichia infection. Notably, pharmacologic or genetic inhibition of mTORC1 activation in IOE-infected mice by rapamycin or MyD88 deficiency, respectively, enhanced Wnt signaling, induced autophagy, and decreased inflammation, suggesting that suppression of Wnt pathway during IOE infection is mediated by mTORC1. These novel findings unravel that virulent Ehrlichia may cause liver injury via mTORC1-mediated inhibition of Wnt pathway. Targeting of mTORC1 could provide a novel therapy for Ehrlichia-induced sepsis.
Renewal rates of CD4 and CD11b cells in mycobacterial granulomas
Authors: Herbath Melinda , Marcus Sarah , Fabry Zsuzsanna , Sandor Matyas
Published: January 2023

Granulomas are the central feature of mycobacterial infections. The majority of granuloma cells are monocytes/macrophages but T cells also play an important role in regulating these lesions. The effector cells in the granuloma have a relatively short life time. Therefore, they must be continuously replaced from the blood. However, the rate of monocyte and T cell recruitment into Mtb-induced lung granulomas is not known and the focus of our work is to measure this rate using intravascular staining.

C57Bl/6 mice were infected via the aerosol route with 200 CFU Mtb H37Rv, expressing tdTomato. Four weeks post infection, the mice were IV injected with anti-CD45 antibody conjugated with BV421, A488 and A647, 24 hours, 4 hours and 3 minutes prior to harvest, respectively. Lung sections were labelled for CD4 and CD11b to test granuloma homing of the stained cells from the blood. A significant fraction of granuloma cells was labelled indicating an access of blood cells to these lesions and a fast turnover of monocytes and CD4 T cells in the granuloma. The cellular turnover differs between different regions, with a higher replacement rate for CD4+ cells (23% in 24 h, from which 16% occurred in the last 4 h) and a slower renewal for CD11b+ cells (19% in 24 h). The rapid cell renewal we observed suggests a therapeutic potential for cell migration blockers in the reduction of Mtb-induced granulomatous pathology. We examine the effect of VEGFR1 and CCR2 blocker treatments on short-term changes in cellular composition and cell distribution in Mtb-induced lung granulomas to demonstrate that this technology offers an approach to study the mechanism of cellular replacement in granulomatous lesions.
Indoleamine 2,3-dioxygenase during acute toxoplasmosis and the hyporesponsiveness of splenocytes
Authors: Ufermann Christoph-Martin , Eller Silvia , Domröse Andreas , Spekker-Bosker Katrin , Däubener Walter
Published: January 2023

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite of medical and veterinary importance. Major mediators of defense to a T. gondii infection are interferon-gamma induced effector mechanisms, e.g. inducible nitric oxide synthase, immunity related GTPases and indoleamine 2,3-dioxygenase (IDO). Suppression of T cell responses – that diminish after several weeks post infection – is observed during acute toxoplasmosis as well as in other infectious diseases. IDO catalyzes the rate limiting step of the kynurenine pathway by utilizing tryptophan. Furthermore, IDO activity has been described to possess antimicrobial and immunoregulatory properties, but its role during acute toxoplasmosis and the altered T cell responsiveness is obscure.

To investigate this part, WT (C57BL/6) and IDO-deficient mice were infected intraperitoneally with T. gondii ME49 tachyzoites. Parasite burden, IDO expression as well as tryptophan and kynurenine concentrations were analyzed. During murine T. gondii infection tryptophan depletion and kynurenine detection was clearly measureable. Since the role of IDO in the T cell hyporesponsiveness during acute toxoplasmosis is incompletely understood, primed and unprimed splenocytes were isolated from infected and naïve mice, respectively. Proliferative responses of the splenocytes to ex vivo mitogen stimulation were measured by detection of incorporated 3H-thymidine. Cell sorting and subsequent cell mixing experiments as well as supernatant transfer experiments were used to break down the mode of action. The observed hyporesponsiveness was independent of IDO. We suggest that the hyporesponsiveness is influenced by nitric oxide synthase and regulatory T cells.
The gut microbiome can contribute to colon tumor susceptibility via an effect on CD8+ T cell responses
Authors: Yu Amy I-Wen , Ho Sharon , Chen Jiachen , Koumpouras Charles , Zhao Lili , Schloss Patrick D , Eaton Kathryn A , Chen Grace Y
Published: January 2023

There is increasing evidence that colorectal cancer patients have altered gut microbiomes compared to healthy controls, but the mechanisms by which the microbiota contribute to colon carcinogenesis remain to be fully elucidated. Using the azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model of inflammation-associated colon tumorigenesis, our lab discovered two colonies of specific pathogen free (SPF) C57BL/6J wild type (WT) mice housed in the same mouse room that develop differential tumor burdens. Mice from the “WT1” colony developed five tumors on average while mice from the “WT2” colony developed 15 tumors on average. The increased tumor susceptibility in WT2 mice can be directly attributed to the gut microbiome as germ-free (GF) mice colonized with WT2 bacteria developed more tumors compared to that of GF mice colonized with WT1 bacteria. Additionally, 16S rRNA gene sequencing of fecal bacteria from WT1 and WT2 mice revealed distinct microbiomes with certain bacteria consistently associated with high or low tumor numbers. Furthermore, naïve and acutely-inflamed (day 10 of AOM/DSS) WT2 mice have increased colon lamina propria CD8+ IFNγ+ T cells compared to WT1 mice as measured by flow cytometry. However, in tumor-bearing WT2 mice, there was decreased tumor-infiltrating CD8+ T cells with reduced IFNγ production, possibly due to T cell exhaustion. GF Rag1−/− mice as well as SPF CD8−/− mice inoculated with WT2 gut microbiota developed fewer tumors than SPF WT2 mice, suggesting that the WT2 gut microbiome increases tumor susceptibility, in part, through an effect on CD8+ T cells. Altogether, our data reveal a potential novel role of microbiota in altering colon CD8+ T cell function that ultimately impacts colon cancer risk.
HHLA2 Expression in Human Clear Cell Renal Cell Carcinoma is Significantly Associated with Patient’s Prognosis and Promotes Cancer Progression
Authors: Chen Lujun , Jiang Jingting
Published: January 2023

HHLA2, a recently reported member of B7 family, plays an important role in the functional inhibition of T cells. Herein, we found that higher HHLA2 mRNA expression level in human ccRCC tissues compared with that in adjacent normal tissues based on TCGA data, and the HHLA2 mRNA level was positively and significantly correlated with PD-L1, PD-L2, B7-H6, but negatively and significantly correlated with B7-H3. Moreover, our immunohistochemistry study showed that HHLA2 staining intensity in human ccRCC tissues was significantly higher than that in normal tissues, and higher HHLA2 expression was significantly correlated with poorer survival of the patients. Higher HHLA2 expression in ccRCC tissues was positively and significantly associated with larger tumor size, advanced pathological stage, and advanced TNM stage. COX model revealed that the parameters including patient’s age, TNM stage and HHLA2 expression level could serve as independent risk factors for prognostic prediction of the patients. Furthermore, our cellular study showed that upon knockdown of HHLA2 expression in human ccRCC cell lines, the cell viability, migration and invasion ability were significantly inhibited, while the cell cycle arrest at G1 phase was induced and the expressions of Cyclin D1, c-Myc and Cyclin E1 were decreased. In addition, based on the microarray analysis data, we also confirmed that the expressions of epithelia-to-mesenchymal transition markers, such as E-cadherin, N-cadherin and Vimentin, were significantly decreased after knockdown of HHLA2 expression. Collectively, our findings indicated that HHLA2 was involved in the progression of human ccRCC and could be used as an important prognostic predictor for this malignancy.
CD4 T Cells modulate the protective vs. pathologic response of CD8 T cells during viral infection
Authors: Luna Evelyn
Published: January 2023

Vaccines expressing pathogen T cell epitopes have been a widely pursued strategy for improving the efficacy of vaccines against persistent viral infections such as HIV. However, selectively engaging T cells also has the potential to increase rather than prevent pathology, consequently shifting a mild disease to a lethal one after subsequent infection. Thus, determining the immune and viral parameters that lead to protection versus those that mediate pathology is critical for developing a safe and effective vaccine against persistent viral infections. Using lymphocytic choriomeningitis virus (LCMV) and an in vivo adoptive transfer mouse model, we investigated how the precursor frequency of CD8 T cells affected the balance of protection vs. pathology during chronic viral infection. We found that the balance is based largely on the number of T cells responding to the pathogen. Intermediate numbers of responding CD8 T cells results in maximum pathology while high numbers provide full protection. We also looked at CD4 T cells and unlike CD8 T cells, increasing the number of pathogen specific CD4 T cells never provides a protective response and instead leads to high host mortality. We have now begun to study the interaction of CD8 and CD4 T cells simultaneously during viral infection to determine the effects of CD4 T cells on CD8 T cell protective versus pathologic vaccine-induced immunity and what exactly mediates such responses.
Raising Endogenous Anti-HMGB1 IgM Antibody for Athero-Protection in the Apoe−/− Mouse Model of Atherosclerosis
Authors: Hariharasubramanian Anjhana , Elangovan Ramya , Shankar Archana , Giacobazzi Mario , Broski Annalisa , Panicker Anjali , Shin Soomin , Chen Aoshuang , Zheng Guoxing
Published: January 2023

Atherosclerosis is driven partly by inflammation, mediated mainly by pro-inflammatory mediators/cytokines. Previous studies have revealed the role of HMGB1, a prototypic DAMP (damage-associated molecular pattern) molecule, in atherogenesis. Recently, we have found that there is a spontaneous production of a neutralizing anti-HMGB1 IgM antibody in the Apoe−/− mouse model of atherosclerosis and healthy humans. In the present study, we have determined whether raising the anti-HMGB1 IgM, via immunization targeting HMW4 (a dominant epitope of HMGB1), reduces atherosclerosis. We first showed that the immunization of 8-week-old Apoe−/− C57BL/6 mice increased the HMW4-specific B-1 cells (which produce anti-HMW4 IgM), identified by HMW4-tetramer staining/flow cytometry. Next, we assessed the anti-atherogenic efficacy of the immunization. To the end, 8-week-old Apoe−/− mice were divided into 4 groups (n = 10/group). Each group was treated with: 1) HMW4 (“test”); 2) raHMW4 (randomized control peptide) (“control”); 3) depletion of the HMW4-specific B cells (to prevent the production of anti-HMW4 IgM), followed by the immunization (control for B cell dependence); or 4) none. At 12 weeks of age, mice were fed a western type diet (WTD) for 12 weeks. Lesions in treated mice were then quantified, by both en face analysis of the aortic arch and morphometric analysis of ORO-stained sections of the aortic root. The “test” group showed reduction in atherosclerosis (by ~40%), when compared to the “control” group (p = 0.006) or non-treated group (p = 0.01). Such reduction was diminished when the anti-HMW4 IgM-producing B cells were depleted (p = 0.02). This study raises the possibility to amplify the anti-HMGB1 IgM response to reduce atherosclerosis.
CD40 ligand (CD40L) multi-trimer antigen fusion protein (MagaVax) as a vaccine design for high-level CD8+ T cell responses
Authors: Kornbluth Richard S , Adase Christopher , Hamilton Victoria Snarsky , Studer Sean
Published: January 2023

Dendritic cells (DCs) can take up antigen via a number of routes. Of these, several studies have shown antigen uptake via the CD40 receptor generates some of the strongest CD8+ T cell responses. At the same time, stimulation through CD40 activates DCs for cross-presentation and heightened antigen presentation. Consequently, it is desirable to combine the CD40 targeting of antigen and a CD40 stimulant into a single vaccine formulation. When the CD40 stimulant is CD40 ligand (CD40L, CD154), it is important to use a multi-trimer complex that clusters CD40 in the DC membrane, thereby engaging downstream signaling pathways. Such a multi-trimer form of CD40L can be provided by fusing the extracellular domain of CD40L with the body of surfactant protein D (SPD), a self-assembling 12-chain soluble molecule with 4 trimeric “arms.” When an antigen sequence is included within the SPD arm, the entire protein complex is termed “Multi-trimer AntiGen Adjuvant Vaccine (Vax)” or “MagaVax.” Studies in mice have shown that MagaVax elicits very high levels of antigen-specific CD8+ T cells with high TCR avidity and functional activity. MagaVax can be effectively delivered as a DNA vaccine i.m. and even more effectively using an adenoviral (Ad5) vector. For a MagaVax encoding HIV-1 Gag, vaccination with Ad5-MagaVax-Gag led to complete protection (“sterilizing immunity”) from Vaccinia-Gag challenge (i.e., 7 log reduction of tissue virus). Thus far, MagaVax formulations have been made for HIV-1 Gag, Ebola, malaria, hepatitis B virus (HBV), and tumor-specific neoantigens. In all of these cases, there may be a clinical benefit for a vaccine that elicits very strong CD8+ T cell responses.
Per oral inoculation with live or killed Chlamydia muridarum induces protection against per-vaginal C. muridarum challenge
Authors: Shillova Nita , Howe Savannah E , Konjufca Vjollca H
Published: January 2023

In addition to infecting the female reproductive tract (FRT), Chlamydia spp. infect the gastro-intestinal (GI) tract of animals and humans. In the GI tract Chlamydia spp. persists without causing pathology, is more resistant to azithromycin treatment, and can be a reservoir for reinfection of the FRT. To date no effective vaccines have been licensed for use in humans. We have found that sIgA responses in the FRT mirror the responses in the GI tract following feeding with a high dose soluble Ova, leading us to hypothesize that immunity in the FRT may be induced via per-oral (PO) antigen administration. To test this hypothesis, 6 week-old C57BL/6 mice were PO or sub-cutaneously (SC) primed with live or killed C. muridarum, then 28 days later SC boosted with killed C. muridarum. At day 42 mice were per vaginally challenged with 2×105 IFUs of C. muridarum and six weeks following challenge mice were euthanized for determination of hydrosalpinx severity and C. muridarum tissue loads. PO inoculation with live or killed C. muridarum followed by SC boosting significantly reduced hydrosalpinx pathology and C. muridarum titers in ovaries, uterus, and vaginal swabs compared to unimmunized controls and to mice primed and boosted SC with killed EBs. Neither form of immunization affected C. muridarum titers in the GI tract, in spite of relatively high Chlamydia-specific sIgA titers. Further analysis revealed that intestinal sIgA are almost exclusively specific for a single antigen (HSP60, a cytoplasmic antigen), while serum antibodies recognize multiple antigens. Our results indicate that while mucosal priming is essential for induction of protective immunity in the FRT, colonization of the GI tract by live Chlamydia is not necessary for mucosal priming.
HIV-1 envelope glycan composition influences virus-host interaction
Authors: Hioe Catarina E , Jan Muzafar , Feyznezhad Roya , Itri Vincenza , Liu Xiaomei , Upadhyay Chitra
Published: January 2023

HIV-1 envelope (Env) is densely shrouded with oligomannose- and complex-type glycans. These glycans interact with lectins, a family of carbohydrate-recognizing proteins. We evaluated lectins for the ability to block infection of HIV-1 isolates that differ in their sensitivity to neutralizing antibodies. The antibody-sensitive tier 1 viruses were blocked by lectins more easily than the resistant tier 2 viruses. When tier 2 viruses were produced with glycosidase inhibitors to express homogenously oligomannose-type glycans, they became more sensitive to mannose-binding lectins (GNA, GRFT, CVN), suggesting that glycan homogeneity increases virus sensitivity to antibodies and lectins. Further, we tested the effect of glycan content on HIV-1 capture and transmission via DC-SIGN, a C-type lectin expressed by macrophages and dendritic cells. While the capture of virus enriched with high-mannose glycans was enhanced, transmission was diminished due to increased degradation. Under a biologically relevant setting, glycan composition was influenced by the Env signal sequence (SS), and SS mutations altered DC-SIGN-mediated virus transmission. The SS mutants also displayed altered sensitivity to inhibition by lectins GNA and GRFT. Mutations in gp120 did not have these effects. Env SS sequences and glycan compositions also vary among HIV-1 strains, leading to differences in their interactions with and sensitivity to lectins. These phenotypes were associated with the amounts of terminating Manα1–3 vs Manα1–2 residues on the Env oligosaccharides and their recognition by DC-SIGN, GNA, and GRFT. These data demonstrate the importance of the Env sugar contents for HIV-1 interactions with lectins expressed by the host immune cells.
Filovirus glycoprotein glycosylation analysis and effects on immunogenicity
Authors: Martinez Britney , Bradfute Steven
Published: January 2023

Filoviruses are hemorrhagic fever viruses that are some of the deadliest pathogens in the world with a fatality rate up to 90%. Symptoms include fever, vomiting, diarrhea, headache, weakness and can lead to hemorrhaging, multiple organ failure and death. Experimental vaccines use filovirus Surface glycoproteins (GPs) as immunogens to induce protective immune responses. Our laboratory studies the glycosylation patterns of different filovirus GPs to develop improved vaccines. Mammalian 293T cells have more complex N-linked glycosylation machinery compared to Sf9 insect cells but both are capable of O-linked glycosylation. Differences in N- and O-linked glycans on filovirus GPs may induce different immune responses upon vaccination. However, the glycosylation patterns on the different filovirus GPs have not been thoroughly elucidated. Here we report purification and glycan analysis of GPs from filoviruses produced in 293T mammalian cells and Sf9 insect cells. We are currently assessing the effects of different glycosylation of the understudied Lloviu virus GP on immunogenicity in mice.
West-Nile virus replicon particles infect 293T cells expressing DC-SIGNR
Authors: Martinez Osvaldo , Kunkel Hannah , Ludwig Anika
Published: January 2023

West-Nile virus (WNV) is an arbovirus usually transmitted to humans via a mosquito vector. Infections commonly result in febrile symptoms while severe neuroinvasive cases may result in encephalitis or meningitis. Studies have shown that WNV infection efficiency is enhanced by expression of DC-SIGNR on target cells, which normally do not express DC-SIGNR. To investigate WNV tropism, we established 293T kidney epithelial cell lines that stably express vector or DC-SIGNR. We demonstrate uniformly high levels of DC-SIGNR expression in selected cells. Virus replicon particles (VRPs), replication-incompetent viral particles containing necessary structural proteins for infection and a viral plasmid including a GFP reporter are used to safely and conveniently study viral entry. Entry assays using WNV (NY99) VRPs as well as a variant of WNV (NY99) which contains the beta-lactamase enzyme show significant entry into DC-SIGNR expressing cell lines, but not in controls that do not express DC-SIGNR. Future studies will focus on discovering which regions of the DC-SIGNR surface glycoprotein are required for WNV entry.