bluuebunny/crossref_metadata_2025 huggingface dataset:
A high throughput genetic screen to uncover novel antiviral mechanisms of the interferon system
Authors: Chattopadhyay Saurabh , Subramanian Gayatri , Glanz Anna , Chawla Karan , Gartl Julie , Jay Bryanna
Published: January 2023

The innate immune responses provide the first line of host defense against virus infection. Type I interferon (IFN) system is a well-known mediator of the host innate antiviral responses. Virus infection is recognized by the cellular sensors, also known as pattern recognition receptors, which via a series of signaling pathways, induce the synthesis of IFNs and the IFN-stimulated genes (ISGs). The ISG-encoded protein products function as viral restriction factors, either by interfering with the stages of viral life cycle or by promoting cell death. We have shown that the IRF3, a key transcription factor responsible for inducing IFNs and ISGs, also provides antiviral responses by triggering apoptosis of the infected cells (Chattopadhyay et al Immunity, 2016). To investigate how the IRF3-inducible genes provide antiviral protection, we performed a high throughput genetic screen of human ISGs. Using an shRNA library of the human ISGs, we have identified a novel antiviral ISG, tudor domain containing 7 (TDRD7). TDRD7 inhibits virus replication by interfering with the cellular autophagy pathway, which is required for the replication a number of viruses. Mechanistically, TDRD7 inhibits the autophagy-initiating kinase, AMPK to suppress virus-induced autophagy pathway. We reported that multiple paramyxoviruses, including the clinically relevant human pathogens, respiratory syncytial virus (RSV) and human parainfluenza virus (HPIV3), are inhibited by the new antiviral mechanism of TDRD7 (Subramanian et al PLOS Pathogens, 2018). Using additional RNA and DNA viruses, which activate autophagy during infection, we are investigating whether the new antiviral mechanism similarly inhibits them.
Cell type-specific sensing of murine cytomegalovirus through MyD88 and STING control virus infection
Authors: Piersma Sytse J , Poursine-Laurent Jennifer , Yang Liping , Parikh Bijal A , Yokoyama Wayne M
Published: January 2023

Viruses are sensed by pathogen recognition receptors expressed in the cytosol and on plasma membranes. While toll-like receptors (TLRs) are expressed on membranes and signal through the adapter molecules MyD88 and TRIF, other pathogen-associated molecular patterns are recognized by the DNA sensor cGAS that signals through STING. Murine cytomegalovirus (MCMV) has been reported to stimulate the TLR-MyD88 and cGASSTING pathways, but it is unclear as to what extent both pathways intersect and contribute to viral control. Herein we analyzed survival, viral titers, IFN-I and NK cell responses in mice deficient in MyD88, STING or both. Mice deficient in both MyD88 and STING showed increased mortality and viral titers compared to deficiency in either alone. Bone marrow chimera studies indicate that both hematological and non-hematological compartments contribute to STING-mediated viral control, whereas previously published data indicate that MyD88 does not play a role in the non-hematological compartment. Consistent with these data, our in vitro results showed that stromal cells produce IFN-I in a cGAS-STING-dependent, but MyD88-TRIF-independent manner. Finally, we observed that MCMV-induced natural killer cytotoxicity was partially dependent on both MyD88 and STING-dependent pathways. Taken together, our results indicate that both TLR-Myd88 and cGAS-STING-dependent sensing of MCMV represent partially evolutionary redundant pathways to control virus infection.
The Epigenetic Profile of CD8+ T Cells During SIV Infection
Authors: Pillay Santhoshan T , Nag Mukta , Fogle Jonathan E , De Paris Kristina
Published: January 2023

CD8+ T cells are crucial in the control of HIV infection. Chronic HIV infection results in CD8+ T cell dysfunction that is manifested in reduced cytokine production (IL-2, IFN-g, or TNF-a) and/or reduced cytotoxic function.

We investigated whether epigenetic changes in CD8 T cells contribute to impaired effector mechanisms using the infant Rhesus Macaque model of SIV infection. Epigenetic changes (e.g. DNA methylation) alter the accessibility of promoter regions, thereby regulating gene expression. We analyzed the DNA methylation of the granzyme B, IL-2, IFN-g, and TNF-a promoters of matched PBMCs prior to and 12 weeks post SIV infection (n=4) and of CD8 T cells from lymph nodes (LN) (n=4) and the intestine (n=4). Bisulfite reduced DNA was analyzed by MiSeq Paired End 2×150 sequencing, and the percent methylation at classic CpG sites and at all sites was determined using Qiagen CLC Genome Workbench v.11 software. SIV-negative and -positive samples were compared applying Fisher’s Exact Test (p £ 0.05).

PBMCs (p = 7.77 × 10−16), LNs (p = 0.01) and intestinal (p = 3.33 × 10−16) CD8+ T cells of SIV-infected animals exhibited increased methylation at the promoter for TNF-a. The methylation of CpG sites within the IFN-g (p = 1.11 × 10−16) and granzyme B (p = 8.88 × 10−16) promoters was only increased in LN CD8+ T cells, but not in the intestine or in PBMC. Our results suggest that immune dysfunction during SIV infection can be partially attributed to methylation changes in immune cells. Further, detailed studies of multiple epigenetic changes in larger animal cohorts are needed to explore SIV-induced tissue-, cell-, and gene-specific epigenetic modifications.
Activation of microglia in CD8 T cell-initiated blood-brain barrier disruption induced during Theiler’s virus infection
Authors: Khadka Roman , Zheng Jiaying , Ayasoufi Katayoun , Jin Fang , Trtiz Zachariah , Wu LongJun , Johnson Aaron
Published: January 2023

Immune-mediated blood brain barrier (BBB) disruption is a prominent feature of various neurological conditions for which an emerging role of CD8 T cells is being realized. Our laboratory has developed a model of CD8 T cell-mediated BBB disruption which employs a variation of the Theiler’s murine encephalomyelitis virus (TMEV) infection. At seven days post TMEV infection, the majority of antiviral CD8 T cells recognize an immunodominant virus peptide, VP2121–130, in the context of the H-2Db class I molecule. Upon intravenous administration of this VP2 peptide at seven days post infection, Db:VP2121–130 epitope specific CD8 T cells induce BBB in a perforin dependent process. In this model, we addressed the role of microglia in tandem with CD8+ T cells. Using real time two-photon in vivo imaging, we demonstrate that microglia adopt distinct morphological features including enlarged cell body and fewer ramified processes as early as 6 hours post administration of VP2 peptide. Notably, perforin-deficient mice failed to display BBB disruption and had significantly diminished microglial expression of MHC-II and CD68 in this model. This study demonstrates CD8 T cell promote microglia activation through a perforin-dependent process during BBB disruption. Importantly, microglia are activated concurrently with the onset of vascular permeability and could serve as a critical cell type in this process.
Butyrophilin molecules govern γδ T cell reactivity against phosphoantigens
Authors: Rigau Marc , Ostrouska Simone , Fulford Tom , Johnson Darryl Neil , Woods Katherine , Ruan Zheng , McWilliam Hamish , Hudson Christopher , Tutuka Candani , Wheatley Adam K , Kent Stephen J , Villadangos Jose A , Pal Bhupinder , Kurts Christian , Simmonds Jason , Pelzing Matthias , Hammet Andrew D , Verhagen Anne M , Vairo Gino , Maraskovsky Eugene , Panousis Con , Gherardin Nicholas Anthony , Cebon Jonathan , Godfrey Dale Ian , Behren Andreas , Uldrich Adam Peter
Published: January 2023

Humans have a minor lymphocyte population of gamma-delta (γδ) T cells. The majority of these express a recombined Vγ9Vδ2 T cell receptor (TCR) attractive to immunotherapy. This distinct TCR conveys reactivity to phosphorylated antigens (pAg) that derive from pathogens or accumulate inside tumour cells. Such T cell responses are regulated by butyrophilin (BTN) 3A1 and other membrane-related proteins present on antigen-presenting cells. However, the activation mechanism and direct molecular ligand recognised by the γδ TCR remain a crucial unresolved question. Herein, we used pAg-reactive TCR probes in a whole-genome screen to identify BTN2A1 as an essential ligand. In further investigation, we elucidated its functionality working in cis with BTN3A1. Also, a mutational analysis unveiled critical regions of the γδ TCR are positioned at opposite sides. We locate germ-line encoded residues of the Vγ9 chain were responsible for BTN2A1 binding, whereas two amino-acids of the Vδ2 chain were necessary for a complete response to pAg. In conclusion, we propose a dual-ligand complex model that senses pAg to evoke immune responses, wherein BTN2A1 sets the framework to develop new opportunities on γδ T cell-based immunotherapies.
Investigating the role of Quaking in antigen presentation by Dendritic Cells
Authors: Li Yating , Hu Jian , Yee Cassian
Published: January 2023

Dendritic Cells (DCs) are considered the most potent antigen-presenting cells due to their superior capacity to cross-present exogenous antigens to CD8 T cells for effective adaptive immunity. They internalize foreign antigens by phagocytosis, receptor-mediated endocytosis or macropinocytosis, which are then processed in endo/lysosomal compartments and loaded on MHC class I. However, the molecular mechanisms regulating exogenous antigen uptake and cross-presentation by DCs remain unclear. We revealed a potential regulatory role of an RNA-binding protein, Quaking (QKI), in antigen presentation. Our previous studies in neural stem cells and microglia identified QKI as a novel regulator of phagosome maturation and endosome signaling via interaction with the transcription factor PPARβ/δ. Therefore, we hypothesize that QKI, together with PPARβ/δ enhances DC antigen uptake and cross-presentation via promoting phagosome and endolysosome signaling. We found QKI and PPARβ/δ to be significantly unregulated in human monocyte-derived DCs, and demonstrated that activating the QKI-PPARβ/δ complex in DCs upregulates QKI targets and significantly promotes DC phagocytosis activity, suggesting QKI cooperates with PPARβ/δ to enhance antigen uptake. The biological role of QKI in antigen cross-presentation and the effect of QKI enhancement on induction of CD8 T cell response by DC are currently under investigation. Understanding the role of QKI in DC may reveal how antigen presentation is regulated and provide new strategies to improve DC-based immunotherapy.
Inhibition of autoimmune enteritis in IFABP-Ova mice by LCMV infection
Authors: Bhela Siddheshvar , Stolley J. Michael , Weyu Eyob , Quarnstrom Clare , Nelson Christine , Vezys Vaiva
Published: January 2023

Epidemiological data indicate that infections play a role in preventing autoimmunity. Thus, the decline in the incidences of infectious diseases and the increase in the frequency of autoimmune diseases also known as the hygiene hypothesis suggest that there might be a link between the two phenomena. In this study using the iFABP-Ova transgenic mouse model, that expresses ovalbumin in the small intestine, we showed that prior LCMV infection dampened self-reactive effector CD8 T cell responses and protected mice from enteric autoimmunity. LCMV specific memory CD8 T cells, generated after infection, were not required for this protection as elimination of these cells using a depleting antibody still protected the mice from enteric autoimmunity. Moreover, we found that infection induced suppression is specific to self antigen reactive CD8 T cells without affecting foreign antigen specific CD8 T cell responses. Lastly, we found that a similar suppression of self-reactive CD8 T cells occurs in iFABP-Ova mice when tolerant CD8 T cells are previously present. Thus there might be some similarities in the mechanism of suppression of self-reactive CD8 T cells by viral infections and tolerance generation. Taken together these data demonstrate that prior viral infections or tolerant CD8 T cells have the ability to suppress self-reactive autoimmune responses and provide protective effects without compromising immunity to foreign pathogens.
The novel role of signal transducer and activator of transcription 5B (STAT5B) in mediating T cell differentiation and regulatory functions
Authors: HSIEH HAO , Shieh Chi-Chang
Published: January 2023

Autosomal-recessive mutations in signal transducer and activator of transcription 5B (STAT5B) have been reported to cause abnormality in growth and immune development in patients. We recently encountered a male patient (designated patient C) who suffered from post-natal growth retardation, severe skin inflammation and lymphoproliferative syndrome. A novel heterozygous STAT5B missense mutation was identified in this patient. The point mutation is alanine to threonine variant (exon9, p.Ala371Thr) which locates on the DNA binding domain of STAT5B. We investigated the role of heterozygous STAT5BA371T variant in the pathogenesis of his immune dysregulation in this study. We confirmed that the mutation did not interfere the STAT5B protein expression and tyrosine phosphorylation response to IL-2 stimulation. Remarkably, significant reduction of p-STAT5 levels in the PBMC from the patient after TCR activation were noted. We found that normal proportion of CD4+CD25+ T cells in the PBMC of the patient before TCR stimulation. Furthermore, significant decrease in CD4+CD25high T cells was found in the patient’s PBMC after T cell activation. Given that CD25 is one of the STAT5B target genes in T cells, lower CD25 expression implicates that novel STAT5BA371T variant is a loss-of-function mutation. Lower CD25 expression in turn leads to lower sensitivity to IL-2 hence ineffective induction of induced regulatory T (iTreg) cells. Our data showed that a dominate-negative heterozygous STAT5BA371T variant may cause the immune dysregulation syndrome by deterring iTreg differentiation, suggesting a critical role of STAT5B in iTreg differentiation.
Prosthetic joint wear particle analogs induce osteoblast apoptosis
Authors: Fisher Cody , Patel Robin
Published: January 2023

Loosening of prosthetic joint implants is a leading cause of arthroplasty failure and the need for surgical revision. Though the mechanisms that induce loosening are complex and not fully understood, implant-derived wear debris has been suggested to be an important player in the interaction between the prosthesis and surrounding cells needed for post-operative immune response, bone remodeling and implant retention. In this study, MC-3T3 pre-osteoblasts were grown in media supplemented with wear particle analogs [titanium (II) oxide (TiO), chromium (III) oxide (Cr2O3), or cobalt (II, III) oxide (Co3O4) particles] for up to seven days and analyzed for apoptotic marker regulation and cell viability. An increase in apoptotic marker staining and a decrease in viability was found in osteoblasts cultured with TiO and Cr2O3 at concentrations >10 μg/ml after just one day and lasting through seven days of exposure. Similar apoptotic and viability response was observed in Co3O4 treated osteoblasts, but only after seven days of particle exposure. Osteoblasts exposed to 100 μg/ml of TiO, Cr2O3, or Co3O4 for seven days had AnnexinV+ frequencies of 60.2%, 74.7%, and 13.4%, respectively. No difference was detected in osteoblasts exposed to particle concentrations ≤10 μg/ml at any time point. Additionally, particles were found localized to cell membranes and the cytoplasm within a single day of treatment; internalization, specific intracellular location, and downstream processing of the particles are under active investigation. This work provides insight into interactions between prosthetic joint arthroplasty wear particles and the osteoblasts necessary for proper bone homeostasis and joint retention.
The actin-bundling protein L-plastin regulates NLRP3 inflammasome activation in macrophages
Authors: Joshi Hemant , Anaya Edgar , Almgren-Bell Alison , Szasz Talyor P. , Todd Elizabeth M. , Morley Sharon Celeste
Published: January 2023

In macrophages, inflammasome pathways induce IL-1β release and pyroptotic cell death to clear infections. For example, NLRP3 inflammasome activation in lung resident alveolar macrophages (AMs) is crucial in controlling pneumonia. We have found that mice lacking the actin-bundling protein L-plastin (LPL−/−) mice are highly susceptible to Streptococcus pnuemoniae lung infection and produce less IL-1β. LPL is also required for podosome formation. Podosomes are integrin-based sites of adhesion that anchor the actin cytoskeleton of AMs to the extracellular environment. As such, AMs can sense their mechanical environment via podosomes. We have found that AMs lacking LPL exhibit defective NLRP3 inflammasome activation in ex-vivo stimulation. Specifically, LPL is required for ASC oligomerization and downstream IL-1β processing after NLRP3 assembly. Since LPL also supports mechanosensitive integrin signaling, we hypothesized that LPL might link contact-based mechanosensation to inflammasome activation in macrophages. To test this hypothesis, we challenged LPL−/− mice with bleomycin, as bleomycin-induced lung injury and fibrosis is thought to be dependent upon NLRP3 activation. LPL−/− mice were resistant to bleomycin-induced lung injury, suggesting a previously unrecognized mechanosensitive mechanism regulating inflammasome activation in macrophages. We propose that the increased lung stiffness in fibrotic lung exerts mechanical stress on AMs and thus may contribute in inflammasome signaling. Increased understanding how mechanical signaling regulates may thus aid in developing therapies to treat lung pathologies i.e. acute respiratory distress syndrome (ARDS), COPD and lung fibrosis.
Endocannabinoid hydrolysis inhibition unmasks that unsaturated fatty acids induce a robust synthesis of 2-arachidonoyl-glycerol and its congeners in human myeloid leukocytes
Authors: Archambault Anne-Sophie , Turcotte Caroline , Dumais Elizabeth , Martin Cyril , Blanchet Marie-Renée , Bissonnette Elyse , Ohashi Nami , Yamamoto Keiko , Itoh Toshimasa , Laviolette Michel , Veilleux Alain , Boulet Louis-Philippe , Di Marzo Vincenzo , Flamand Nicolas
Published: January 2023

The endocannabinoid 2-arachidonoyl-gycerol (2-AG) modulates immune responses by activating cannabinoid receptors or through its multiple metabolites, notably eicosanoids. Thus, 2-AG hydrolysis inhibition might represent an interesting anti-inflammatory strategy that would simultaneously increase the levels of 2-AG and decrease those of eicosanoids. Accordingly, 2-AG hydrolysis inhibition increased 2-AG half-life in neutrophils. Under such setting, neutrophils, eosinophils and monocytes synthesized large amounts of 2-AG and other monoacylglycerols (MAGs) in response to arachidonic acid and other unsaturated fatty acids (UFAs). Arachidonic acid and UFAs were ~1000-fold more potent than GPCR agonists at stimulating MAG biosynthesis. Triascin C and thimerosal, which respectively inhibit fatty acyl-CoA synthases and acyl-CoA transferases, prevented the UFA-induced MAG synthesis, implying glycerolipid remodeling is essential in this process. 2-AG and other MAG biosynthesis was preceded by that of the corresponding lysophosphatidic acid (LPA). However, we could not directly implicate LPA dephosphorylation in MAG biosynthesis. While the GPCR agonists PAF, fMLP and LTB4 poorly or did not induced 2-AG biosynthesis, they inhibited that induced by AA by 25–50%, suggesting that 2-AG biosynthesis is decreased when leukocytes are surrounded by a pro-inflammatory entourage. Our data conclusively indicate that human leukocytes use AA and UFAs to biosynthesize biologically significant concentrations of 2-AG and other MAGs and that hijacking the immune system with 2-AG hydrolysis inhibitors might diminish inflammation in humans.
Characterization of the local and systemic temporal changes to the lipid mediator and cytokine cascades following osteotomy and polytrauma
Authors: Muire Preeti Judith , Mangum Lauren H , Mangum Lee C , Garcia Gerardo R , Romano Desiree R , Wenke Joseph C
Published: January 2023

Polytrauma (PT) inducesglobal changes to inflammatory signaling, contributing to delayed fracturehealing. In a rat model of PT encompassing burn, blunt trauma, and osteotomy, we characterized the local and systemic temporal changes to the lipid mediator(LM) cascade, circulating white blood cells (WBCs), and local levels of growthfactors (GF)s and cytokines. Male rats received either a non-critical 3mmfemoral osteotomy (OST) or PT trauma procedures and were survived for 6, 24,48, 72, 96, 168, or 240hrs and blood, plasma, and fractured femurs werecollected. Whole femurs were homogenized to extract LMs, protein, and RNA, while blood and plasma were used for WBC and LM analysis, respectively. At the fracture site, prostaglandin (PG) E2 was significantly higher in OST at 24 and 72hrs, levels of PGF2α were higher in OST at 24hrs, while resolvin (Rv) D1 was higher in PTat 6hrs post injury. There were no differences in the expression ofcyclooxygenase-2 (COX-2), or lipoxygenase-5 (LOX-5) and LOX-15. In plasma, there were no significant differences in PG levels at any time. Plasma RvE1 was elevated in PT immediately following injury while maresin 1 (MaR1) was significantly lower in PT at 168hrs. Total WBCs and lymphocytes (103cells/uL) were lower in PT overall and significantly so at 72 and 96hrs, respectively. Additionally, the percent of neutrophils in PT was significantlyelevated and percent lymphocytes was decreased at 48 and 168hrs compared toOST. Protein analysis of the fracture site found early elevation of cytokines(IL-1β, IL-13, IL-6) and GFs in PT at 48 and 72hrs. These results indicate thatchanges to circulating LM and WBCs may provide predictive measures of non-unionand therapeutic targets to improve fracture healing.
Characterization of immune cells found in keloid versus normal skin tissue
Authors: Anderson Jaclyn B , Harrant Alexander B , Navarro-Alvarez Nalu , Wang Zhaohui , van Bokhoven Adrie , High Whitney , Chong Tae W , Huang Christene A
Published: January 2023

Keloids are abnormal scar formation that extends beyond the confines of the original injury. Immune cell infiltration is associated with keloid pathogenesis, but the exact mechanisms remain unknown. We aim to characterize the immune profile of keloid skin compared to normal skin in order to further understand the role of specific immune subsets in keloid formation. One abdominal normal skin sample and five keloid samples (four ear and one shoulder) were obtained from the Plastic Surgery clinics and the University of Colorado Biorepository Core Facility. Paraffin-embedded samples were stained with CD19+, CD20+, CD3+, CD8+ and FoxP3+ by the Human Immunology and Immunotherapy Initiative. The multiplex immunohistochemistry slides were scanned using Vectra 3 (PerkinElmer, MA, US) and captured images were analyzed with InForm Tissue Finder, phenotpr, and phenoptrReports (Akoya Biosciences, CA, US). CD20+ and CD19+ B cells were significantly increased in keloid tissue compared to normal skin (14.50% and 14.20% vs 6.47% and 7.56%), respectively. In contrast, no significant difference was observed in the percentage of CD3+ and CD8+ cell subsets in keloids compared to normal skin (12.92% and 4.98% vs 8.19% and 4.04%), respectively. Moreover, there was a significant increase in FoxP3+ positive cells found in keloid scars (9.68%) compared to normal skin (2.89%). Further studies examining the role of B cells and FoxP3+ cells in keloid pathogenesis are warranted.
PD-L1 expression at the single cell level in Myeloid Derived Suppressor Cells using a stimulatory functional assay to evaluate immunotherapy strategies in multiple myeloma
Authors: Bradford Jolene A , Rico Laura G. , Junca Jordi , Bardina Jorge , Bistue-Rovira Angel , Ward Michael D. , Petriz Jordi
Published: January 2023

Background

Multiple myeloma (MM) is characterized by malignant plasma cells in the bone marrow with an associated immunosuppressive microenvironment. PD-L1 suppresses immune response and is overexpressed in MM plasma cells and MDSCs. PD-L1 plays an important role in tumor immune evasion and drug resistance. PD-L1 is considered a therapeutic target, yet some MM individuals do not respond to treatments with PD-L1 or PD-1 inhibitors. The aim of this study was to design a screening assay to identify PD-L1+ MDSCs using flow cytometry.

Methods

Using no-lyse no-wash methods, samples were labeled with Vybrant DyeCycle Violet Stain to identify nucleated cells and with antibodies to identify MDSCs. PD-L1 surface expression was studied ± PMA stimulation. Kinetics of surface expression of PD-L1 were studied over time. PD-L1 cell surface and cytoplasmic expression were compared. Competitive experiments with Durvalumab were studied. Samples were analyzed using Attune NxT Flow Cytometer.

Results

PD-L1 surface expression increased after stimulation, but showed variation among subjects. PD-L1 surface expression was highest after 1-5 minute stimulation. Cytoplasmic levels of PD-L1 were undetectable. Co-incubation with Durvalumab resulted in variable inhibitory PD-L1 profiles.

Conclusions

Since cytoplasmic PD-L1 is not found, PD-L1 may reveal steric changes in response to stimulation, which may be associated with a PD-L1 immunoregulatory mechanism affecting therapies. Assessment of PD-L1 folding may help to develop better treatment strategies or to predict therapy resistance. No-lyse no-wash methodologies show promise as an emerging strategy to model conformational changes in the target site. For research use only.
Disease-associated dominant negative CARD11 mutations interfere with the CARD11 Opening Step during antigen receptor signaling to NF-κB and JNK2
Authors: Bedsaul Jacquelyn Renee , Pomerantz Joel
Published: January 2023

CARD11 mediates lymphocyte activation by functioning as a scaffold in antigen receptor signaling to NF-κB, JNK, and mTOR. Antigen-induced CARD11 signaling proceeds through an ordered series of steps, including Opening, Cofactor Recruitment, Enzyme Activation, and Complex Disassembly. Recently, several forms of Primary Immunodeficiency have been described that are due to germline CARD11 mutations. One of these syndromes, Combined Immunodeficiency (CID) with atopy, is inherited in an autosomal dominant manner and caused by loss-of-function (LOF) CARD11 alleles, which display dominant negative activity that poisons signaling from co-expressed wild-type CARD11. To investigate the mechanistic basis for disease-associated dominant negative activity we reconstituted CARD11-deficient T cells with a panel of LOF CARD11 variants in the absence and presence of co-expressed wild-type CARD11. In the absence of wild-type CARD11, the LOF variants were indistinguishable, such that each variant failed to promote the signal-induced 1) recruitment of the cofactors Bcl10, HOIP, and MALT1 to CARD11, 2) Bcl10 polyubiquitinylation, 3) MALT1 protease activation, 4) NF-κB activation, and 5) JNK2 activation. In the presence of co-expressed wild-type CARD11, the LOF variants surprisingly displayed a range of dominant negative activity from strong to intermediate to weak, indicating a specific potency of some mutations to disrupt signaling when heterozygous. Our results suggest that disease-associated dominant negative CARD11 mutations interfere with the Opening Step in the CARD11 signaling cycle and provide a mechanistic basis to explain why some LOF CARD11 mutations can cause disease when heterozygous while others do so when homozygous.
T-cells dysregulate hematopoiesis in Myeloproliferative Neoplasm through increased interferon-gamma production
Authors: Lai Hew Yeng , Ramanathan Gajalakshmi , Brooks Stefan , Craver Brianna , Fleischman Angela
Published: January 2023

Chronic inflammation is a key feature of myeloproliferative neoplasm (MPN), a hematological malignancy. MPN is characterized acquisition of a somatic mutation in hematopoietic stem cells, most commonly the JAK2V617F mutation, which leads to unrestrained expansion of myeloid cells. Although lymphoid compartments do not have the JAK2V617F mutation, these cells are key mediators of inflammation. The precise role of lymphoid cells in disease pathogenesis is unclear. We found that CD4+ T cells from MPN patients produce significantly more interferon gamma (IFNg) than normal CD4+ T cells. IFNg has been reported to suppress the growth of myeloid colonies. We found that removal of T cells from peripheral blood mononuclear cells of MPN patients enhance myeloid colony formation. Removal of T cell also increase fraction of wild-type myeloid colonies, demonstrating that wild-type cells are more sensitive to suppression by T cells.

In addition, MPN mouse model using transduction-transplantation of JAK2V617F in RAG2−/− mice, lacking in T and B cells, display an attenuated MPN phenotype. Moreover, transplantation of JAK2V617F RAG2−/− bone marrow with wild-type bone marrow into lethally irradiated wild-type recipient restored the MPN pathology, such as erythrocytosis and expansion of megakaryocytes. This implicates that lymphocytes play an important role in driving MPN pathogenesis. More importantly, inhibition of T cells in JAK2V617F transplanted mouse using cyclosporine attenuates spleen size and marrow fibrosis, demonstrating that suppression of T cells after disease has already been established is therapeutic. In sum, these data shows that T mediates pathogenesis in MPN, and that suppressing T cells in MPN may have therapeutic benefit.
Over-expression of Chitotriosidase Modulates ECM Distribution in Atherosclerotic Plaques of Mice
Authors: Yap Jonathan , Mccurdy Sara , Garo Jan , Irei Jason , Alcala Martin , Kitamoto Shiro , Boisvert William A
Published: January 2023

Macrophage is the predominant cell type in all phases of atherosclerosis and it plays a major role in disease progression. Although mammals have no endogenous chitin, macrophages produce chitotriosidase-1 (CHIT1) as part of the innate immune response in various inflammatory conditions including atherosclerosis. We aimed to investigate mechanisms by which CHIT1 may modulate the progression of atherosclerosis using CHIT1-overexpressing, Ldlr−/− mice, and also to determine whether overexpression of CHIT1 by macrophages affects the morphology of atherosclerotic plaques. We developed an atherosclerosis-prone, conditional CHIT1 over-expressing (CHIT1-OE) mouse model which allowed us to study the effects of macrophage CHIT1 over-expression on morphological characteristics as well as extracellular matrix (ECM) formation in vivo. Further, bone marrow-derived macrophages (BMDM) from CHIT1-OE mice and littermate controls were used to determine the cellular mechanisms of non-enzymatic CHIT1 signaling in vitro. Over-expression of CHIT1 in Ldlr−/− mice after 12 weeks of high-fat diet modulated the ECM by altering the accumulation and localization of hyaluronic acid (HA) and collagen in atherosclerotic plaques observed within the aortic sinus. We also found, in vitro, that macrophage invasiveness was significantly enhanced in BMDM from CHIT1-OE mice upon addition of IL-13 as an immunoregulator. Our findings indicate that CHIT1 over-expression modulates ECM properties in the atherosclerotic plaques of Ldlr−/− mice and macrophage behavior in vitro, which may be atheroprotective and promote plaque stability.
Soluble B7H3 induction in pulmonary fibrosis is associated with disease severity
Authors: Liu Tianju , Rinke Andrew E , Flaherty Kevin , Phan Sem Hin
Published: January 2023

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with limited therapeutic options. Both innate and adaptive immune mechanisms contribute to fibrogenesis. B7 family member B7H3, identified as important regulator of the immune system, has been implicated in cancer, graft-versus host disease, and allergy-related disease. Increased soluble B7H3 (sB7H3) was associated with enhanced lung fibrosis in mice receiving BLM-primed BM. Elevated sB7H3 in bronchoalveolar lavage fluid was associated with acute exacerbation IPF. To investigate whether induced sB7H3 could be used as a biomaker for IPF diagnosis and/or prognosis, plasma sB7H3 from control subjects and IPF patients was measured by ELISA. Its correlation with peripheral blood immune cells, lung function, and drug treatments were evaluated in this study. The results showed that the sB7H3 level was significantly elevated in IPF patients. This induction was associated with increased circulating B7H3+ cells, and significantly correlated with increased granulocytic myeloid-derived suppressive cells (G-MDSCs). This suggests a potential regulatory role of the induced B7H3 in the immune response in IPF. The plasma sB7H3 was negatively correlated with lung function measured as diffusing capacity of the lungs for carbon monoxide (DLCO), indicating the potential utility of plasma sB7H3 as an indicator of disease severity. The slightly elevated sB7H3 in the pirfenidone and/or nintedanib treated IPF patients were not significantly different relative to controls, which was in contrast to the significant elevation noted for the untreated patients. These findings suggest that sB7H3 may be a useful biomarker for clinical assessment of prognosis and response to drug treatment.
Treatment of Experimental Asthma by Lung Restricted Inhibition of Jak1
Authors: Rardin Hart
Published: January 2023

Preclinical and clinical evidence indicates that a subset of asthma is driven by type 2 cytokines such as IL-4 and IL-13 that are dependent on Jak1 for signal transduction. Because many of the asthma-related effects of these cytokines are manifested in the lung, we reasoned that combined targeting of these cytokines by means of an inhaled Jak1 inhibitor with lung restricted exposure may confer therapeutic benefit. In order to test this, we synthesized iJak-381, a novel, inhalable small molecule specifically designed for local Jak1 inhibition in the lung and tested dry powder inhaled delivery of iJak-381 in preclinical PK and efficacy studies. iJak-381 suppressed OVA induced lung inflammation and improved allergen induced airway hyper-responsiveness in mice. In a model driven by human allergens, iJak-381 had a more potent suppressive effect than a systemic corticosteroid on neutrophilic inflammation. While iJak-381 strongly inhibited lung pathology, it did not affect systemic Jak1 activity. Our data suggests that local inhibition of Jak1 in the lung might be effective for treating asthma in the clinic.
Uncovering the allergen-specific modulation in non-allergic laboratory mouse workers – understanding health versus disease
Authors: Yu Esther Dawen , da Silva Antunes Ricardo Filipe , Frazier April , Schulten Véronique , Pham John , Peters Bjoern , Sette Alessandro
Published: January 2023

Mouse allergies are prevalent in inner city households and laboratory workers. Mouse allergens trigger allergic symptoms from atopic dermatitis/conjunctivitis to rhinitis and asthma, thus their research is of great clinical value. The aim of this study was to characterize the mouse-specific T cell response changes in non-allergic laboratory workers before and after vacation. Peripheral blood mononuclear cells (PBMC) were collected pre-break, pre re-exposure (after break), and after re-exposure to mouse, and were stimulated with peptides identified from high molecular weight fractions of mouse allergen extracts (HiMO). Non-allergics had sizeable reacitivity and majority had decreased T cell responses upon re-exposure. In addition, the decreased magnitudes were positively correlated with time of re-exposure but not length of break. Interestingly, the suppressive activity was dominant and specific for mouse responses and did not affect bystander responses. CD4 T cell responses was down-regulated temporally for a period of time (40–160 days) and eventually went back to baseline. Ex vivo immunophenotyping revealed a trend towards down-regulation of B cells and classical Tregs. Mouse-specific CD4 effector cells were reduced while total CD4 effector cells were not. The results indicate that non-allergic status is an active process which involves allergen-specific modulation of responses upon re-exposure to allergens. Uncovering the molecular programs associated with this modulation by transcriptomic study will be the next step to better understand health and allergic diseases.
The role of NK cells during bacterial and viral/bacterial co-infection
Authors: Aguilera Elizabeth Renata , Clark Sarah E. , Lenz Laurel L.
Published: January 2023

Influenza virus is a large burden to the human population and a leading cause of death worldwide. Much of this high morbidity and mortality is due to co-infections with pulmonary bacterial pathogens, such as Streptococcus pneumoniae. Prior studies indicate that a primary influenza infection exacerbates secondary infection of S. pneumoniae in murine models. In part, this is due to increased bacterial burdens in the lung driven by pro- or anti-inflammatory cytokines such as IFNγ and IL-10. However, the cellular source(s) of these cytokines and their roles during infection is unclear. Previous work in our lab has shown that during systemic Listeria monocytogenes infection, NK cells are a critical source of early IFNγ with delayed IL-10 production and that IL-10 specifically facilitates pathogenesis. Here, we report that NK cells also increase susceptibility to pulmonary S. pneumoniae infection, as demonstrated by reduced bacterial burdens in organs following NK cell depletion. Using an in vitro BMDC/NK cell co-culture system, we further show that S. pneumoniae infection can directly elicit NK cell IL-10 production. Therefore, we hypothesize that NK cells play an important role during S. pneumoniae infection. To test whether NK cells also influence influenza/S. pneumoniae co-infection, we established a mouse model where S. pneumoniae bacterial burdens are increased in organs of co-infected mice. We are currently investigating how NK cells affect susceptibility to secondary S. pneumoniae infection and specifically the role of NK cell IL-10. Ultimately, this work has the potential to further define the mechanism(s) by which NK cells contribute to the control or pathogenesis of S. pneumoniae and influenza virus/S. pneumoniae co-infection.
Transcriptional reprogramming drives neutrophil phenotype and function in chronic lung disease
Authors: Margaroli Camilla , Giraldo Diego Moncada , Arafat-Gulick Dalia , Dobosh Brian , Giacalone Vincent , Kissick Haydn , Forrest Osric A , Gu Chunhui , Gibson Gregory , Tirouvanziam Rabindra M
Published: January 2023

Rationale

Neutrophils are often considered the first line of defense against bacterial infections. In cystic fibrosis (CF), massive neutrophil presence is concomitant with phenotypical changes and prolonged bacterial infections. Here, we sought to determine whether this paradox was dependent on transcriptional reprogramming of airway neutrophils.

Methods

Blood neutrophils were transmigrated in an in vitro model recapitulating the in vivo phenotype of CF airway neutrophils. Blood and airway neutrophils from the in vitro model and from CF patients were analyzed by flow cytometry, microarrays and RNA sequencing, as well as used for bacterial killing assays.

Results

Airway neutrophils in vivo and in vitro showed increased total RNA content and profound shifts in their transcriptional profile for survival, anabolic, and antimicrobial genes. Furthermore, transcriptional blockade by α-amanitin in airway neutrophils in vitro and ex vivo fully restored their bacterial killing capacity.

Conclusions

These findings show that some canonical functions in neutrophils, including bacterial clearance, depend on the active repression of RNA transcription, which can be modulated by the local microenvironment at the site of inflammation, challenging the paradigm holding these cells as terminally differentiated.
Neuropeptide coordination of mucosal immunity by regulating ILC3 activity
Authors: Belz Gabrielle T
Published: January 2023

Group 3 innate lymphoid cells (ILC3)-mediated production of the cytokine IL-22 is critical for the maintenance of immune homeostasis in the gastrointestinal tract. How this occurs is not well defined. We found that the function of ILC3s was not constant across the day, but instead oscillated between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide VIP. Intestinal ILC3s expressed high levels of the G protein–coupled receptor VIPR2 and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signalling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP–VIPR2 pathway in ILC3s.
High dimensional cellular profiling of the myeloid compartment in the geriatric mouse model
Authors: Krishnarajah Sinduya , Ingelfinger Florian , Friebel Ekaterina , Cansever Dilay , Becher Burkhard
Published: January 2023

Aging, and age-related physiological changes, have been heavily implicated in declining immune functions. Many of these changes to the immune system include changes to the myeloid subsets, which have been poorly studied so far. Myeloid cells play crucial roles in acute infection and are involved in antigen presentation to cells of the adaptive immune system. Consequently, deciphering these age-related mechanisms holds great potential for targeting age-related changes in immunity. Here, we developed a model to map age related phenotypic changes in the myeloid compartment in all immunologically relevant murine organs. Using mass cytometry analysis (CyTOF), we assessed over 35 cell surface parameters on myeloid cells using a geriatric healthy mouse model. Results indicates age-related changes affecting the frequency and cell surface density of lineage markers on myeloid cells. Concretely, we see significant changes to cellular frequencies and marker expression within various resident myeloid populations, and exemplar organs will be presented here. Such age-related patterns may contribute to the impaired immune decline observed in aging. We are currently continually expanding this study and will validate the implications of these findings using spectral flow cytometry analysis.
Inverse relationship between LPS concentration and monocyte and DC inflammatory response
Authors: Perros Alexis J , Flower Robert L , Dean Melinda M
Published: January 2023

Dendritic cells (DC) and monocytes, which link the innate and adaptive immune response, can be stimulated with lipopolysaccharide (LPS) via toll-like receptor 4 recognition. However, understanding of human cell-specific responses to different doses of stimuli (LPS) is limited. We investigated the monocyte and DC specific, as well as the overall inflammatory response following exposure to varying doses of LPS. Fresh peripheral whole blood (n=8) was used in an in-vitro whole blood culture model to assess DC and monocyte responses in co-culture with varying doses of LPS (0.25 μg/mL, 0.5 μg/mL, 0.75 μg/mL, 1 μg/mL). DC and monocyte cytokine responses were measured via flow cytometry. Supernatants collected from the in-vitro model were used to assess the overall inflammatory response using a cytometric bead array. Exposure to all doses of LPS tested increased monocyte and DC specific cytokine production, and the overall leucocyte response. DC and monocyte cytokine production decreased in a dose-dependent manner when exposed to higher LPS doses. Our data suggests LPS concentration impacts on cell-subset specific responses more than the overall inflammatory response. Assays that assess the overall inflammatory response may lack the sensitivity required to elucidate underlying pathophysiology of infection and inflammation.
Cross-reactive dengue immune complexes modulate innate antiviral responses to Zika virus infection in human placental macrophages
Authors: Zimmerman Matthew G , Eddins Devon , Muhammad Ali , Register Emery , Kauffman Robert , Cordes Sarah , Jean Valerie , Haddad Lisa , Wrammert Jens , Suthar Mehul S
Published: January 2023

Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently emerged in the Americas and is a pathogen of significant public health concern. Unique amongst flaviviruses, ZIKV can be vertically transmitted from an infected mother to the developing fetus in utero, in some cases resulting in adverse pregnancy outcomes, including spontaneous abortion and fetal brain abnormalities. We recently found that Hofbauer cells (HCs), a fetal-derived placental macrophage, are primary targets for ZIKV infection. Further, cross-reactive dengue antibodies facilitate ZIKV transcytosis across the placental barrier to seed infection within HCs. However, little is known about the innate immune mechanisms of HCs to limit virus replication and spread within the placenta. Here, we report that HCs express the RIG-I-like receptors, RIG-I, MDA5, and LGP2, and the cytosolic DNA sensor, cGAS, and can induce a potent innate immune response characterized by production of monocyte and macrophage chemoattractants, MCP-1, MIP-1α, and MIP-1β. In contrast, HCs infected in the presence or absence of ZIKV immune complexes induced little to no type I interferon (IFN), pro-inflammatory cytokines, or chemokines. Notably, HCs infected with ZIKV immune complexes displayed reduced antiviral gene induction as compared to HCs infected in the absence of immune complexes despite similar kinetics of virus replication. We are currently performing RNA sequencing analyses to determine how binding of viral immune complexes can alter the antiviral gene signature within HCs to enhance productive ZIKV infection. These studies will further our understanding of innate immunologic mechanisms and modulation during vertical transmission of flaviviruses in the placenta.
Analysis of PBMC Activation Using Parallel Bead and Cellular Assays
Authors: Clor Julie L. , Tyagarajan Kamala
Published: January 2023

Activation of human peripheral blood mononuclear cells (PBMCs) leads to expression of cytokine receptors, cytokine secretion and the expression of specific cell surface molecules resulting in divergent immune responses. The correlation of cell surface activation and cellular health markers from the lymphocyte subpopulations with cytokine levels can vary across donors and can provide valuable information to evaluate secretion levels. Bead-based multiplexed assays using Luminex xMAP® platforms provide a powerful tool for quantifying and studying several secreted cytokine levels in a single assay. Performing cellular assays performed in with flow cytometry provide information in immune cell population percentages, counts, and overall health. In this study, we present the combined cellular and secreted cytokine data on human PBMCs stimulated by the lectin phytohaemagglutinin (PHA) or the endotoxin lipopolysaccharides (LPS). Time course data from multiple donors will be presented. Combining data from these two approaches gives a comprehensive picture of the impact of stimulation on the various PBMC populations and the correlation of the secreted cytokine levels to the number of cells.