bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel B cell-dependent multiple sclerosis model using extracellular domains of myelin proteolipid protein
Authors: Boyden Alexander W. , Brate Ashley A. , Karandikar Nitin J.
Published: January 2023

Therapeutic success of B cell-targeting approaches in multiple sclerosis (MS) has intensified research into the role B cells play in pathogenesis and regulation of disease. Historical dissection of B cell function in the MS mouse model experimental autoimmune encephalomyelitis (EAE) has largely been confined to disease induced with either the immunodominant epitope of myelin oligodendrocyte glycoprotein (MOG35–55) or full length recombinant human MOG protein (hrMOG), the latter representing the only available B cell-dependent EAE model. We have demonstrated that myelin proteolipid protein (PLP178–191)-specific CD8 T cells possess striking EAE-suppression capacity. However, little is known regarding the role of B cells in these contexts. Interestingly, unlike MOG35–55, where genetic absence or depletion of B cells yields a more severe disease course, PLP178–191 yields identical EAE progression in both WT and μMT mice, suggesting suboptimal B cell engagement. Given the reports that hrMOG drives a B cell-dependent demyelinating disease versus its MOG35–55 counterpart, we hypothesized that a longer PLP antigen consisting of more epitopes may better engage B cells and provide an additional model for the field. To this end, we designed a novel peptide encompassing the extracellular domains (ECD) of PLP and compared its immunogenicity and disease-inducing capacity with PLP178–191 in μMT and WT mice. We demonstrate here that PLPECD-immunized B cell-deficient mice failed to exhibit EAE disease compared to WT counterparts. PLPECD also induced EAE in B cell-sufficient mice incapable of secreting antibodies, suggesting a predominant antigen presentation role. These results establish a novel, efficient B cell-dependent EAE model.
Novel shared antibody specificities in anti-Ro/ La antibody negative Sjögren’s Syndrome
Authors: Longobardi Sherri , Georgescu Constantin , Lawrence Christina , Moya Charmaine , Wren Jonathan , James Judith A , Sivils Kathy L , Farris A. Darise
Published: January 2023

INTRODUCTION

Sjögren’s syndrome (SS) is a rheumatic autoimmune disease characterized by focal lymphocytic infiltrates in the lacrimal and salivary glands, severe dry mouth and eyes, pain and debilitation. Diagnosis requires autoantibodies to ubiquitous Ro antigens or a lip biopsy positive for focal lymphocytic infiltrates. Here we used human proteome arrays to identify novel antibodies in plasma from Ro positive and Ro/La antibody negative SS patients compared with healthy controls.

METHODS

Anti-Ro positive (n=15) and anti-Ro negative (n=15) cases meeting 2016 ACR/EULAR classification criteria for SS were age, race, and sex matched with each other and healthy controls (n=15). Plasma IgG binding to human proteome arrays containing >19,500 recombinant human proteins representing >80% of the human proteome (HuProt v3.2 arrays, CDI Laboratories) was assessed. Data were normalized by the Robust Linear Model using the PAA Bioconductor Package in R and log intensity values for each protein generated. Thresholds of mean + 4SD were established using the controls. Antigens bound by IgG more frequently in cases compared to controls (p<0.05, one-tailed Fisher’s exact test) were considered significant.

RESULTS

IgG from Ro positive SS cases significantly bound 18 proteins, including the canonical SS antigens Ro60 and Ro52. IgG from Ro negative SS cases significantly bound 4 proteins compared to controls, 3 of which were shared with the Ro positive group. Binding to any one of 4 novel proteins identified 73% of the Ro negative SS cases.

CONCLUSION

A total of 17 novel antigen specificities were identified in SS, with 4 antigens being bound by plasma IgG from Ro/La negative SS cases. These antigens may be useful for diagnosing SS without a lip biopsy.
Expression of ANA patterns by Indirect Fluorescent Assay in a local Hong Kong population from private sector
Authors: Lau Kwun Hung , Hung Marco Hing-Lam , Kwan Po-Yi , Lam Tsoi-Hin , Kai Cheuk-Him , Lai Ho-Hin , Yau Peter Kin-Nam
Published: January 2023

Objective

Our aim was to investigate the association among age, sex and Antinuclear Antibody (ANA) patterns expressed in Hong Kong population for improving the autoimmune diagnosis by using ANA HEp-2 slides under fluorescent microscope.

Methods

A total of 65 blood samples from both male and female were collected and examined under fluorescent microscope. Haemolysed samples were excluded. In this research, a HEp-2 complete Kits with the methodology of indirect fluorescent antibody (IFA) was utilized for the detection and semi - quantification of antinuclear antibodies to distinguish positive ANA patterns classified in International Consensus on ANA Patterns (ICAP) for human body.

Results

In this experiment, we obtained that the number of positive samples was 6 (9%), the number of suspected (weak) positive samples was 4 (6%) and the number of negative samples was 55 (85%). In 6 positive samples, they were all females aged between 59 – 89. 5 in 6 positive samples in the result were over age 69 (83%). The positive ANA patterns included AC2 – Speckled, AC3 – Centromere, AC6 – Discrete Nuclear Dots, AC7 – Discrete Nuclear Dots, and AC8 – Nucleolar patterns separately. No AC-29 - DNA topoisomerase I was examined and resulted. We found that Hong Kong have a higher distribution of ANA positive frequency for female (9.23%) than male.

Conclusion

Our data indicated that ANA patterns were found in female aged above 69 is more likely to suffer from autoimmune disease related to ANA, providing an important foundation for considering and studying the trend in the elderly who are easier to have an ANA related autoimmune disease in Hong Kong.
The Use of Interactive Educational Tools to Engage Students in Microbiology and Immunology Classroom
Authors: Simmons Jazmyne Cymone Durrah , Fadhil Shams , Edquiban Marianne , Olk Sandra , Fazal Nadeem
Published: January 2023

Microbiology and Immunology is a required 4 credit hour course for pharmacy students to progress in Chicago State University College of Pharmacy program. Twenty lectures are offered to first-year pharmacy students to grasp fundamentals of Immunology. We surveyed this course for five weeks and observed the classroom lectures, student’s participation, and the learning materials that were provided to students to use in this course. We noticed the major hindrance in learning was lack of student engagement in the classroom. Our survey indicated 77.6% of the students could not focus during lectures for more than 10 minutes. In the classroom environment, traditional PowerPoint lectures were given, students were quizzed during the lecture to keep them attentive, but still there was an obvious lack in lively participation and learning process. The objective of this study was to use interactive educational tools to help engage students. Our hypothesis was that interactive educational tools will improve student’s engagement in the classroom and possibly enhanced student learning. We experimented with various interactive educational tools, such as Moodle (Millionaire game, Quiz, Adaptive Quiz), Facebook livestream, and a private YouTube channel to keep students engaged in the classroom. We observed that simply using various diverse educational tools during the lectures drew student attention and participation rather than sticking to only one method or tool of teaching. The results showed that students were not bored and seemed to enjoy learning and lively contributed to their own learning process. In conclusion, the use of interactive educational tools improved both student engagement in the class as well as boosted the morale of the Instructor.
Conditional abrogation of Ddx3x reveals its essential role in lymphocyte development and partial compensation by Y chromosome
Authors: Krishnamurthy Durga , Seelamneni Harsha , Ochayon David , Harley John B. , Waggoner Stephen N.
Published: January 2023

Ddx3x on the X-chromosome encodes a DEAD-box RNA helicase with putative roles in RNA metabolism, cell cycle progression, apoptosis, and viral immunity. We previously found that hematopoietic-specific hemizygosity of Ddx3x studies caused a marked reduction in the frequencies of natural killer (NK) and B cells in peripheral lymphoid organs of male mice (Liu et al. bioRxiv 2018). No homozygous Ddx3x-deficient female pups were ever obtained, suggesting that hematopoietic loss of this gene contributes to the embryonic lethality observed in germline Ddx3x knockout mice. To assess cell-lineage intrinsic roles for Ddx3x in B-cell and NK-cell development, we bred Ddx3x-floxed mice with Mb1-Cre and Ncr1-iCre mice, respectively. While hemizygous male Ddx3x-deficient mice display mild reductions in B-cell (Mb1-Cre) and NK-cell (Ncr1-iCre) frequencies, homozygous Ddx3x-deficient female mice from each were almost completely devoid of the relevant cell lineages. B-cell loss was evident from the pro-B cells stage, with B cells surviving beyond this stage showing evidence of having escaped deletion of Ddx3x. Ncr1-iCre-mediated deletion of Ddx3x completely abrogated development of both NK cells and other Ncr1-expressing innate lymphoid cells. Thus, DDX3 plays an essential role in the lymphocyte development, where Ddx3y may partially compensate for loss of Ddx3x in these lineages.
Enhanced development of functional human innate immune cells in a novel mouse FLT3null NSG mouse strain expressing human FLT3L
Authors: Vaidya Shantashri , St. Louis Pamela , Burzenski Lisa , Greiner Dale L , Brehm Michael A , Shultz Leonard D
Published: January 2023

Humanized mice are being applied widely to study human immune system homeostasis, function, and as a testing platform for therapeutics. A significant limitation for many humanized mouse models is the lack of mature human innate immune cells with full functionality. Previous studies have demonstrated that delivery of human FLT3L into immunodeficient mice that lack mouse FLT3, promotes the development of human innate immune subsets following engraftment with human HSC. Here we describe a NOD-scid IL2rgnull (NSG) mouse that lacks the expression of mouse FLT3 and expresses human FLT3L transgenically (NSG-mFLT3null Tg(HuFLT3L) or NSG-FLT3L. In these studies, NSG-FLT3L and NSG mice were engrafted with human UCB CD34+ HSC and compared for human immune system development and function. HSC-engrafted NSG-FLT3L and NSG mice show similar levels of total human CD45+ cells in blood over the course of 18 weeks post-engraftment. However, HSC-engrafted NSG-FLT3L mice show significantly increased levels of human immune cells reconstituting the gut mucosa. HSC-engrafted NSG-FLT3L mice show significantly higher levels of human CD141+ and CD1c+ DC subsets, CD123+ pDC, CD14+ monocytes, CD56+ NK cells and CD3+ T cells in the blood as compared to NSG mice. CD56+ NK cells and CD3+ T cells from HSC-engrafted NSG-FLT3L mice express granzyme A and granzyme B, indicating cytotoxic activity. Following treatment of HSC-engrafted NSG-FLT3L mice with LPS, significant levels of human cytokines were detected in serum samples, confirming innate immune system function. Overall these results demonstrate that the NSG-FLT3L model supports enhanced development of functional, human innate immune cells and is a novel tool to study human immunobiology.
The Transcription Factors Thpok And Runx3 Identify Two Distinct CD4−CD8− Double Negative T Cell Populations And Respectively Define Their Commitment In The Helper Or Cytotoxic Fate
Authors: Thiault Nicolas , Verstichel Greet , Seumois Gregory , Vijayanand Pandurangan , Kronenberg Mitchell , Cheroutre Hilde
Published: January 2023

In the small intestine epithelium, the major TCRab population is the double negative (DNT) cells lacking the expression of either CD4 or CD8. In contrast to the conventional selected cells, the DNT cells undergo an agonist selection, which leads to their functional education and capacity to migrate to intestine.

DNT cells also possess the capacity to reach the liver, where we observed a heterogeneity of the DNT cells populations, based on expression of ThPOK and Runx3. In the gut, the latter is only expressed by the DNT cells. GSEA analysis and cytokines intra-cellular staining have shown that ThPOK+ DNT cells and Runx3 DNT cells are strongly divergent in their biological function. ThPOK+ DNT cells signature is enriched in genes expressed by CD4 T helper cells with a strong production of the inflammatory cytokine Il-17 whereas Runx3+ DNT cells signature is enriched in gene upregulated in CD8 T cytotoxic cells. In the thymus, based on the expression of these two transcription factors, we also found distinct DNT populations. Adoptive transfer, as well as hanging drop fetal thymus culture have demonstrated that solely Runx3+ DNT cells in the thymus are the precursors of the Runx3+ DNT cells in the gut. Furthermore, we demonstrated in the thymus both DNT cells populations are selected upon an agonist interaction with a self-antigen presented by developing thymocytes, for the development of ThPOK+ DNT cells, and TECs for Runx3+ DNT cells. RNA-seq experiment of thymic DNT populations revealed that the agonist selection process results in their differential functional education since T helper pathways are upregulated in ThPOK+ DNT cells whereas pathways involved in function and differentiation of cytotoxic cells are upregulated in Runx3+ DNT cells.
Quantitative Expression of GATA3 Specifies Lineage Fates and Functions of Innate Lymphoid Cells
Authors: Zheng Mingzhu , Zhong Chao , Cui Kairong , Martins Andrew , Hu Gangqing , Li Dan , Tessarollo Lino , Kozlov Serguei , Keller Jonathan , Tsang John , Zhao Keji , Zhu Jinfang (Jeff)
Published: January 2023

Innate lymphoid cells (ILCs) are the innate counterparts of the CD4 T helper cells of the adaptive system. While the CD4 T cell development and differentiation have been well studied, the development of ILC subsets is far from clear. Lymphoid tissue inducer (LTi) population is the founding member of ILCs, however, recent study has shown that these cells are not derived from a PLZF-expressing ILC common progenitor that generates other ILCs. The transcription factor(s) determining the fate of non-LTi progenitor versus LTi progenitor are unknown. Here we report that GATA3 is absolutely required for the generation of PLZF-expressing non-LTi progenitors, which express high level of GATA3, but not for the generation of RORgammat-expressing LTi progenitors consistent with low levels of GATA3 expression in these progenitors. Nevertheless, low level of GATA3 expression by LTi progenitors is critical for the generation of functional LTi cells. Thus, quantitative expression of GATA3 functionally determines the fates and functions of distinct ILC progenitors.
Inflammation-Induced Tumorigenesis: Diminished Noncanonical NF-κB Signaling is Associated with Pathogenesis of Colitis-associated Colorectal Cancer
Authors: Morrison Holly , Mounzer Christina , Eden Kristin , Baumgarner Katherine , Brown Stephan , Holl Eda , Allen Irving C
Published: January 2023

Colitis-associated colorectal cancer (CAC) is mediated by inflammation-induced tumorigenesis, causing DNA damage in susceptible cell populations. Dysregulation of inflammatory pathways in the colon is attributed to cancer pathogenesis and such signaling is attributed to NF-κB signaling. Transcription factor NF-κB is the master regulator of gene transcription and is activated through one of two pathways. Our focus is on the understudied noncanonical NF-κB signaling pathway, including NF-κB inducing kinase (NIK). NIK must be stabilized for activation of this pathway. Noncanonical signaling is attributed to the production of pro-inflammatory cytokines, recruitment of immune cells, and cell proliferation. We hypothesize that this pathway maintains gut homeostasis and when dysregulated causes cell populations to become tumorigenic. The objective of this study is to validate the clinical relevance of noncanonical signaling. We observe that diminished noncanonical signaling via whole-body Nik knockout mice results in reduced stem cell marker expression, enhanced proliferative capacity, altered microbiome composition, and increased susceptibility towards inflammation-induced tumorigenesis in the colon. Using CAC mouse models, conditional knockout strains show deletion of noncanonical signaling in epithelial cells results in increased susceptibility to colorectal tumorigenesis. Likewise, human colonic biopsy samples collected from CAC patients show significantly decreased expression levels of genes related to the noncanonical NF-kB pathway. This data suggests that the noncanonical NF-κB pathway has a protective role against colorectal cancer by regulating immune system homeostasis in the GI tract.
Influence of proton pump inhibitor or rebamipide use on gut microbiota of rheumatoid arthritis patients
Authors: Kim Ji-Won , Jeong Yunju , Yoon Jun Sik , Ji Geun Eog , Park Sung-Hwan
Published: January 2023

Objective

Patients with rheumatoid arthritis (RA) commonly use gastrointestinal (GI) protective drugs for treatment and prevention of peptic ulcer disease. However, how these drugs affect gut microbiota is unknown in RA patients. The objective of this study was to examine the gut microbiota of RA patients according to their use of GI protective drugs, such as proton pump inhibitors (PPIs), H2-receptor antagonists, and rebamipide.

Methods

Fecal samples were obtained from 15 healthy controls (HCs) and 32 RA patients who were receiving PPI, H2-receptor antagonist, or rebamipide. Bacterial DNA was extracted from the fecal samples and 16S rRNA sequencing was performed. Microbial composition and function were analyzed using QIIME and PICRUSt.

Results

RA patients showed reduced diversity and altered composition of the gut microbiota compared with HCs. Gut microbiota of RA patients receiving acid-suppressing drugs, particularly PPI, was distinct from that of RA patients receiving rebamipide (PPI versus rebamipide, p=0.005). Streptococcus was enriched in RA patients receiving PPI, whereas Clostridium bolteae was enriched in RA patients receiving rebamipide. Gut microbiota of PPI-users was highly expressing the microbial functional pathway involved in virulence factors production. This featured microbial function was positively correlated with the relative abundance of Streptococcus, the differentially abundant taxa of PPI-users.

Conclusions

Gut microbiota of RA patients receiving PPI was distinguished from those receiving rebamipide. Enriched virulent function in gut microbiota of PPI-users suggests that PPIs should not be overprescribed in RA patients.
Comparative Profiling of the Immune System in Sarcoidosis via CITE-Seq and Flow Cytometry
Authors: Magallon Roman E. , Knapp Jennifer R. , Harmacek Laura D. , Tu Ting-Hui Clair , Vestal Brian , Gillespie May , MacPhail Kristyn , Li LI , Elliot Jill , Barkes Briana , Maier Lisa , Sommer Anne , Grewal Pineet , Koth Laura , Arger Nicholas , Werner Brenda , Powers Linda , Hamzeh Nabeel , Breslin Linda , Chen Edward , Danhorn Thomas , Leach Sonia M , Fingerlin Tasha E. , O’Connor Brian P.
Published: January 2023

CITE-Seq enables simultaneous single cell transcriptome and proteome analysis via combining single cell RNA-seq with oligo-labeled antibodies. Conventional techniques such as flow or mass cytometry have caveats including the number of epitopes accurately detected or an inability to recover samples for transcriptome analysis. These limitations are prohibitive for multivariate analysis of limited clinical samples. We developed a CITE-seq assay that enables comprehensive immune cell profiling of Sarcoidosis. Sarcoidosis is a granulomatous lung disease characterized by abnormal CD4+ T cell Th1 activity. However, the disease etiology and course are variable and the underlying molecular drivers remain unknown. The long-term goal of this study is to utilize CITE-seq to identify immune molecular pathways of Sarcoidosis pathogenesis. In our initial studies, we analyzed PBMC’s by CITE-seq vs. flow cytometry and observed similar cell profiles. However, the synergy of protein detection coupled with transcriptome analysis via CITE-seq enhanced cell subset identification vs. flow or scRNA-seq alone. We utilized CITE-seq in an ongoing longitudinal study of Sarcoidosis subjects to enhance resolution of the immune components contributing to disease. We compared CITE-seq to a flow cytometry panel analyzing the differential contributions of various CD4+ T cell lineages. The enhanced granularity provided by CITE-seq elucidated molecular pathways associated with disease pathogenesis. Thus, moving forward CITE-seq can provide the resolution and multivariate data collection required to identify the inflammatory drivers of Sarcoidosis.
Adipose Tissue-Derived Mesenchymal Stem Cells’ Acellular Product Extracellular Vesicles as a Potential Therapy for Crohn Disease
Authors: Li Yan , Altemus Jessica , Lightner Amy
Published: January 2023

The breakdown of gastrointestinal tract immune homeostasis leads to Crohn’s disease (CD). Mesenchymal stem cells (MSCs) have demonstrated clinical efficacy in treating CD in clinical trials, but there is little known about the mechanism of healing. Considering the critical roles of macrophage polarization and T cell activation in CD, and immunomodulatory properties of MSCs, we sought to decipher the interaction between adipose derived MSCs, macrophages and T cells including their cytokine production, regulation of differentiation and pro-/anti-inflammatory function.

When MSC derived EVs from inflamed Crohn’s mesentery were added to monocyte culture, monocytes rapidly differentiated into M1 macrophages (CD68+) from myeloid progenitors. When MSC derived EVS from control patients, regardless of the adipose source of MSCs (subcutaneous fat, omentum, normal mesentery), monocytes selectively differentiated into M2 macrophages (Arg-1+). Adoptive transfer of these differentiated M2 macrophages, effectively reversed T cell activation, proliferation, and cytokine production. Control patient isolated MSC derived EVs significantly suppressed T cells function with significantly decreased levels of inflammatory cytokines, as compared to diseased mesentery MSC derived EVs. This T cells inhibition was a result of NO synthase, arginase, and reduced ERK induced inflammatory signaling.

MSCs alter macrophage polarization and subsequent T cell activation. Healthy MSCs have the ability to drive monocytes to a M2 subset which then effectively reverses T cell function. This mechanism supports why MSCs are effective for regulating CD progression and offers pathways for the evaluation of various MSC sources for optimal efficacy.
Hemagglutinin as potential in vivo driver of DQ6-restricted clonal expansion of CD4+ T cells cross-reactive with hypocretin in narcolepsy
Authors: Jiang Wei , Birtley James R. , Hung Shu-Chen , Wang Weiqi , Chiou Shin-Heng , Davis Mark M , Stern Lawrence J. , Mellins Elizabeth D
Published: January 2023

Narcolepsy is a sleeping disorder, caused by selective loss of neurons secreting a wake-promoting hormone, hypocretin (HCRT). Increased narcolepsy incidence was noted in China after the peak of 2009 H1N1 pandemic influenza (pH1N1) cases and in Europe after Pandemrix vaccine administration. The causes of HCRT neuron depletion and its relationship with pH1N1 are unknown. As narcolepsy susceptibility is associated with HLA-DQ6 and with a TCR α gene (J24) polymorphism, we hypothesized that abnormally expanded J24+/CD4+ T cells in DQ6+ subjects mediate autoimmunity against HCRT neurons and that molecular mimicry (epitopes in pH1N1 and self-antigens) drives T cell expansion via DQ6 presentation. We reported the in vivo expansion of DQ6/HCRT87–97–reactive J24+/CD4+ clones using a J24 allele and an un-conventional phenotype in patients compared to related clones in DQ6+ controls. Here, DQ6/peptide binding and crystal structure identify an epitope, HA274–287 from pH1N1 hemagglutinin, with homology to HCRT87–97 and another peptide, HCRT1–13. DQ6/HA274–287 tetramers isolated >2000 tetramer+/CD4+ T cells in DQ6+ subjects. Single-cell sequencing of TCRs and 25 phenotypic transcripts identify a DQ6/HA274–287 tetramer+/J24+ clone with the identical TCR clonotype (TCR27) and phenotype as the expanded DQ6/HCRT87–97–reactive J24+ clones previously found in the same patient. Control-derived TCR27+ clones identified by DQ6/HCRT1–13 or DQ6/HCRT25–37 tetramers are unexpanded and lack expression of any of the phenotypic genes. Our findings show that DQ6+ subjects carry J24+/CD4+ cells, both public and cross-reactive (e.g TCR27). TCR27 expansion in patients argues for molecular mimicry as a factor in narcolepsy immunopathogenesis.
Inflammatory environments disrupt both bone formation and bone resorption
Authors: Chu Pei-Wen , Chen Yu-Hsu , Chen Chien-Hui , Chen Shau-Kwaun
Published: January 2023

Inflammation has been associated with bone diseases such as osteoporosis and osteoarthritis. Bone loss were reported in the patients of several inflammatory diseases, such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease. However, how inflammation influence bone metabolism remains elusive. The bone loss in inflammatory environments are widely considered as the results of osteoclast overactivation which leads to excessive bone resorption. We previously discovered that osteoclasts induced from RAW macrophage treated with RANKL exhibited different cell properties and gene expression profile with undifferentiated macrophage. In this research we examined the excessive bone resorption hypothesis in in vitro systems. RANKL stimulated differentiation of RAW cells into bone-resorptive osteoclasts, and induction of pre-osteoblasts (MC-3T3 E1) into mature osteoblasts are utilized in this research. Inflammatory environments are mimic by treating cultured osteoclast or osteoblast with conditioned medium collected from bone marrow derived macrophage primed with LPS or interferon-γ. The pro-inflammatory cytokines inhibit the proliferation and disrupt the expression of genes that are needed for bone formation, such as osteocalcin and collagen. On the other hand, inflammatory environments did not activate osteoclast, nor promote bone resorption. Instead, pro-inflammatory cytokines inhibit osteoclastogenesis and bone resorption, induce mitochondrial dysfunctions and lead to apoptosis of osteoclast. These results indicated that the bone loss developed in the inflammatory environments might be due to the disruption of both bone formation and bone resorption.
HERV1-env dependent unfolded protein response activation is a potential initiator of autoreactivity in autoimmune liver disease
Authors: Ekong Udeme D , Yao Jie , Knight James , Mehta Sameet , Avitzur Yaron , Martinez Mercedes , Lobritto Steve J , Mason Andrew
Published: January 2023

Regulatory T cells are not terminally differentiated but can acquire effector properties. Here we report Human Endogenous Retrovirus 1 (HERV1-env) induction of endoplasmic reticulum (ER) stress with Unfolded Protein Response (UPR) activation, through its interaction with ATF6. UPR activation cleaves ATF6 to its α and β isoforms. ATF6α up-regulates RORC, STAT3 and TBX21 and induces IL-17A and INF-γ production in regulatory T cells by binding to promoter sequences. Silencing of HERV1-env results in partial recovery of regulatory T cell suppressive function and abrogation of apoptosis. These findings identify ER stress and UPR activation as key factors driving regulatory T cell plasticity.
Characterizing Infection of B Lymphocytes with Measles Virus
Authors: Melot Logan , Coughlin Melissa , Rota Paul , Bankamp Bettina
Published: January 2023

Wild-type (w.t.) measles virus (MeV) infection generates a state of immunosuppression, which does not occur after vaccination with live attenuated vaccine. MeV has been shown to alter the function of immune cells such as dendritic cells and lymphocytes. Though altered B cell functions contribute to immune suppression, the pathogenesis of measles infection of B cells is not understood. Here, we characterized the differences in viral replication and cytokine expression in human B cells infected with vaccine (Edmonston-Zagreb [EZ]) or w.t. MeV. To measure viral replication, human B cells were infected with an EZ virus expressing GFP and the number of GFP expressing cells and mean fluorescence intensity (MFI) were measured after 24 hours. To measure differences in cytokine responses following infection of B cells with EZ or w.t., transcription of IL-1β, IL-6, IL-10, TNFα, IFNβ, and IFNγ was measured at 24, 48, and 72 hours after infection by quantitative RT-PCR. The number of B cells expressing GFP and the MFI increased 3-fold from 0 to 24 hours after infection. Preliminary data suggested that IL-1β transcription is higher in w.t. infected B cells, while IL-6 and IL-10 transcription were higher in EZ-infected B cells. Measles virus replication was detected 24 hours after infection of isolated, human B cells. The differential expression of cytokines in B cells suggest differences in the B cell response to infection with w.t and vaccine strains of MeV. Differential cytokine expression in w.t. infected B cells could play a role in the development of immune cell dysregulation, and the mechanism could be elucidated through further functional studies of infected B cells.
The dual role of alkylated trehalose copolymers as C-Type Lectin Receptor agonists and excipients
Authors: Mancini Rock , Davaritouchaee Maryam , Hendricksen Aaron , Nielsen Amy
Published: January 2023

In nature, trehalose plays many roles, from bacterial signaling and immune cell activation through C-Type Lectin Receptors (CLRs) activated by trehalose-6,6′-dimycolate (cord factor), to the excipient properties of trehalose, which stabilizes organisms against extreme environmental conditions. To-date, our molecular understanding of trehalose has enabled the creation of synthetic trehalose analogues that are superior to the natural systems: trehalose-6,6′-dibehenate is an optimally active motif that retains CLR agonism, and polymers of 4,6-O-(4-vinylbenzylidene)-α,α-trehalose far out-perform the natural excipient abilities of trehalose. Here we report our results from combining these benefits to create synthetic copolymers of 4,6-O-(4-vinylbenzylidene)-trehalose and 4′,6′-O-(4-vinylbenzylidene)-trehalose-6-behenate by Reversible Addition-Fragmentation Chain Transfer Polymerization. The resulting alkylated trehalose copolymers are well defined (PDI < 1.4) with incorporation of behenic acid dispersed throughout the entire length of polymer as measured by DOSY-NMR. Overall, the copolymers retain the excipient properties of trehalose and enhance their ability to act as CLR agonists. A systematic exploration of the macromolecular parameters that drive CLR agonism (DP, PDI, and end-group chemistry), particularly through Dectin-1 and MINCLE, will be presented. These results suggest that alkylated trehalose copolymers could find use as dual vaccine adjuvants and stabilizing excipients.
Macrophage production and activity of innate immune proteins C1q, C1r, and C1s are modulated in response to molecular patterns
Authors: De Silva Angele M , Gallardo Ary , Fraser Deborah A
Published: January 2023

Pattern recognition receptors of the complement system, such as C1q, are vital for clearance of foreign and altered-self targets. C1q has a dual role in vivo: activating the inflammatory classical complement pathway via C1 complex formation (C1qC1r2C1s2) and opsonization in the absence of C1r and C1s which is anti-inflammatory. We hypothesize that macrophages encountering foreign targets will increase levels of C1 subunits to activate C1 and mount an inflammatory response while macrophages encountering damaged-self targets may only increase C1q production to promote anti-inflammatory responses and clearance. Human monocyte derived macrophages (HMDM) were incubated with LPS, E. coli bioparticles, oxLDL, apoptotic cells or polarized into M1 or M2 HMDMs. RNA was isolated for qPCR and supernatant for Luminex protein analysis, ELISA, and C1(q) hemolytic titers. Data show that M1 HMDM increase C1r and C1s mRNA, and produce higher levels of active C1 than M0 HMDM. In contrast, there is no change in gene or protein expression of C1 subunits in M2 HMDM. HMDM treatment with foreign targets revealed a trend of increased C1r and C1s mRNA, and decreased C1q mRNA, but not at the protein level. Treatments with damaged-self targets varied; as hypothesized, C1q gene and protein expression increases in response to apoptotic cells, however there are no substantial changes in treatments with oxLDL. Significant levels of active C1q, but not C1, were detected in response to LPS, oxLDL, and apoptotic cells. Data suggests there is modulation of C1 subunit production and activity in response to different targets. C1q production in the absence of C1r and C1s may be important in resolving inflammation during clearance of apoptotic cells, thus avoiding autoimmunity.
TLR2-Tpl2-dependent ERK Signaling Drives Opposite IL-12 Regulation in Dendritic Cells and Macrophages
Authors: Groft Sarah Grace , Nagy Nancy , Shukla Supriya , Harding Clifford V , Boom W Henry
Published: January 2023

This study investigated responses to TLR2-driven ERK signaling in dendritic cells versus macrophages. TLR2 signaling was induced with Pam3Cys, and the role of ERK signaling was interrogated pharmacologically with a MEK1/2 inhibitor (U0126) or genetically using bone-marrow-derived macrophages or dendritic cells from Tpl2−/− mice. We assessed cytokine production via ELISA and mRNA levels by qRT-PCR. In macrophages, blockade of ERK signaling by pharmacologic or genetic approaches inhibited IL-10 production and increased IL-12p40 production significantly. In dendritic cells, blockade of ERK signaling similarly inhibited IL-10 production but decreased IL-12p40 production, opposite to the effect of ERK signaling blockade in macrophages. This difference in IL-12p40 regulation correlated with differential expression of transcription factors cFos and IRF1, which are known to regulate IL-12. Thus, the impact of ERK signaling in response to TLR2 stimulation differs between macrophages and dendritic cells, potentially regulating their distinctive functions in the immune system. ERK-mediated suppression of IL-12p40 in macrophages may prevent excess inflammation and associated tissue damage following TLR2-stimuation, while ERK-mediated induction of IL-12p40 in dendritic cells may promote priming of Th1 responses. Greater understanding of the role that ERK signaling plays in different immune cell types may inform the development of host-directed therapy for a number of infectious pathogens.
Activated platelet-mediated induction of CD16 expression and its role in human monocytes
Authors: Lee Su Jeong , Yoon Bo Reum , Kim Bonah , Lee Ga Hye , Kang Yeon Jun , Kang Seong Wook , Lee Eun Bong , Lee Won-Woo
Published: January 2023

Monocytes are important cellular effectors of innate immune defense. In humans, circulating monocytes are heterogeneous and can be divided into three distinct subsets based on CD14 and CD16 expression. The expansion of “intermediate” CD14+CD16+ monocytes is well reported in many chronic inflammatory conditions such as rheumatoid arthritis (RA). However, the mechanism underlying induction of CD16 and the role of CD16 in CD14+CD16+ monocytes remain unclear. ADP-treated autologous platelets induced CD16 expression by highly purified “conventional” CD14+CD16− monocytes. Given that platelet activation causes the release of several soluble factors, we examined an effect of cytokines, which are involved in monocyte-platelet aggregation (MPA), on CD16 induction. MPA led to significantly increased production of TGF-β and IL-6. Exogenous TGF-β and IL-6 in platelet-free culture induced CD16 expression, whereas neutralizing antibodies for TGF-β and IL-6 largely suppressed CD16 induction. Furthermore, selective inhibitors of SMAD3 and STAT3 reduced CD16 expression, indicating a critical role of TGF-β and IL-6 for CD16 induction by CD14+CD16− monocytes. Functionally, induced CD16 participates in IgG-mediated phagocytosis and the level of CD16 expression in activated platelet-treated monocytes is correlated with uptake of beads coated with FITC-labeled IgG. Lastly, CD14+CD16− monocytes treated with activated platelets differentiate into M2-like macrophage. Our findings suggest an important role of activated platelets for modulating phenotypical and functional features of monocytes in humans. This will be helpful for understanding immunological role of CD14+CD16+ in chronic inflammatory diseases.
Immune sensing of synthetic, bacterial and protozoan RNA by TLR8 requires coordinated processing of RNA substrates by RNaseT2 and RNase2
Authors: Bartok Eva Sarah , Ostendorf Thomas , Zillinger Thomas , Andryka Katarzyna , Guimaraes Thais Marina , Schmitz Saskia , Marx Samira , Bayrak Kübra , Linke Rebecca , Salgert Sarah , Grasser Tatjana , Baersachs Sonja , Hübner Marc , Soltesz Leon , Kettwig Matthias , Nastaly Maximilian , Roehl Ingo , Henneke Marco , Barchet Winfried , Gärtner Jutta , Schlee Martin , Hartmann Gunther
Published: January 2023

Human TLR8 is an essential sensor of bacterial RNA which induces proinflammatory and Th1 cytokines. Crystallography revealed that TLR8 binds both uridine and short single-stranded RNA, but the RNases that process the RNA are still unknown. Herein, we demonstrate that two endosomal endoribonucleases, RNaseT2 and RNase2, can process RNA for TLR8 recognition. In the endosome, RNase2 and -T2 act synergistically to release uridine from oligoribonucleotides, with RNaseT2 cleaving preferentially before and RNase2 after uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic ligands all required RNase processing for TLR8 activation, and uridine supplementation restored RNA recognition in RNASE2−/− or RNASET2−/− but not double knockout cells. Strikingly, peripheral blood mononuclear cells from RNaseT2 hypomorphic patients did not respond to bacterial RNA but did to small-molecule TLR8 agonists. Our data provide a novel insight into TLR8 activation and its differences between cell types and species.
Role of Dual Oxidase 1 in severe malaria
Authors: Sarr Demba , Pathak Ashutosh K. , Murdock Courtney , Rada Balazs
Published: January 2023

Despite important advances in malaria control, the disease is still a major public health issue in sub-Saharan Africa. Recent reports indicate over 400,000 deaths annually. Parasite resistance to known antimalarial drugs delays control and elimination. Dual oxidase 1 (Duox1), a member of the NADPH oxidase enzyme family, generates hydrogen peroxide that is used by its partner enzyme lactoperoxidase to generate antimicrobial hypothiocyanite. Malaria parasites are susceptible to hypothiocyanite in vitro. Duox1 is required for the generation of hypothiocyanite in vivo. We hypothesized that malaria parasites are susceptible to the Duox1-mediated antimicrobial system in vivo. Duox1-deficient and wild-type, Duox1-expressing mice were infected with Plasmodium berghei by either intravenous injection of sporozoites or exposure to infected mosquitos. The results indicate that Duox1-deficient mice either infected with intravenous injection of P. berghei sporozoites (p<0.004) or by exposure to P. berghei-infected mosquito bites (p<0.002), have significantly higher blood parasite density at peak parasitemia compared to wild-type mice. Survival data indicated that Duox1-deficient mice are more susceptible to P. berghei infection than control animals (p<0.03 for intravenous infection, p<0.02 for exposure to mosquito bites). Histopathological analysis of brain tissues revealed a slightly higher number of indices of cerebral pathology including cerebrovascular infected red blood cells and hemozoin accumulation, hemostasis, and hemorrhage. Overall, these data propose a novel role of Duox1 in delaying the onset of parasitemia and clinical outcomes in malaria infection.
The transcription factor BACH1 promotes tissue damage and host susceptibility in Mycobacterium tuberculosis infection by reducing expression of Gpx4, a major negative regulator of ferroptosis
Authors: Amaral Eduardo Pinheiro , Namasivayam Sivaranjani , Costa Diego L. , Fisher Logan , Bomfim Caio Cesar B. , Andrade Bruno B. , Sher Alan
Published: January 2023

Ferroptosis is a type of regulated necrotic cell death induced by the acumulation of free iron and lipid peroxides. We have previously described a role for ferroptosis in Mtb-induced macrophage necrosis based on its biochemical requirements and its blockade both in vitro and in vivo by a lipid peroxidation inhibitor (fer-1). To help validate these findings and further delineate the mechanism involved we have analyzed Mtb-induced cellular necrosis and host resistance in mice deficient in or overexpressing glutathione peroxidase (Gpx4), an essential regulator of ferroptosis. To do so we generated conditional-knockout mice for Gpx4 by using creERT2, CD45cre and LysMcre systems targeting different cell compartments. After aerosol low dose Mtb infection these conditional-knockout mice showed both increased lung necrosis and elevated pulmonary and splenic bacterial burdens. In the opposite direction, Gpx4-overexpressing mice infected at high dose with Mtb were found to display decreased bacterial burdens as well as reduced pulmonary necrosis. Genetic deletion of BACH1, a transcription factor known to repress antioxidant genes including Gpx4, increased the levels of intracellular glutathione as well as enhanced expression of Gpx4 during Mtb infection in vitro and in vivo. Moreover, Bach1−/− mice infected at high dose with Mtb displayed a significant reduction in bacterial loads and lung necrosis along with lowered levels of lipid peroxides as well as enhanced survival. Together, these findings provide genetic evidence further delineating the role of ferroptosis as a mechanism of host cell death and tissue necrosis in Mtb infection and implicate both Gpx4 and Bach1 as potential targets for host-direct therapy of tuberculosis.
Dual RNA-seq as an effective tool to simultaneously identify transcriptional changes in host macrophages and invading intracellular pathogens
Authors: Choudhury Sreetama , Akhade Ajay Suresh , Subramanian Naeha
Published: January 2023

Intracellular pathogens have evolved strategies to subvert the host immune response and establish infection leading to disease. For example, Mycobacterium tuberculosis (MTB) has been recently reported to effectively counteract immunological and anti-tubercular challenges by activating resistance mechanisms that enable the pathogen to attain an immune tolerant state. A systems scale approach using dual RNA Sequencing revealed key transcriptional factors that enabled researchers to discover a drug combination that delivers more effective killing of MTB. On the basis of our preliminary findings we hypothesize that similar to MTB, Salmonella spp. have evolved to escape host macrophage responses by down-regulating the expression of flagellin, a potent activator of the NLRC4 inflammasome, by taking advantage of a natural host negative feedback mechanism that allows the pathogen to reside intracellularly within macrophages. However, how Salmonella and host transcriptional networks rewire over the course of infection to permit development of an immune-evasive phenotype in the pathogen remains unknown. Using dual RNA-Seq and regulatory network analysis, we propose to simultaneously identify the molecular changes that occur in both the host and the pathogen during infection. We aim to conduct a temporal study to find transcriptional changes at different time points post infection that lead to development of a permissive host environment and allow Salmonella to undergo immune escape within macrophages. Our study will help identify the key regulators that shape host-pathogen crosstalk over the course of Salmonella infection with potentially important implications for bacterial pathogenesis and immunity.
Gasdermin D Promotes AIM2 Inflammasome Activation and Is Required for Host Protection against Francisella novicida
Authors: zheng Min , Zhu Qi Fan , Balakrishnan Arjun , Karki Rajendra , Kanneganti Thirumala-Devi
Published: January 2023

The DNA sensor absent in melanoma 2 (AIM2) forms an inflammasome complex with ASC and caspase-1 in response to Francisella tularensis subspecies novicida infection, leading to maturation of IL-1b and IL-18 and pyroptosis. AIM2 is critical for host protection against F. novicida infection in vivo; however, the role of pyroptosis downstream of the AIM2 inflammasome is unknown. Recent studies have identified gasdermin D (GSDMD) as the molecule executing pyroptosis by forming pores on the plasma membrane following activation by inflammatory caspase-1 and -11. In this study, we report that GSDMD-deficient mice were susceptible to F. novicida infection compared with wild type mice. Interestingly, we observed that GSDMD is required for optimal caspase-1 activation and pyroptotic cell death in F. novicida–infected bone marrow–derived macrophages. Furthermore, caspase-1 activation was compromised in bone marrow–derived macrophages lacking GSDMD stimulated with other AIM2 inflammasome triggers, including poly(dA:dT) transfection and mouse CMV infection. Overall, our study highlights a function, to our knowledge previously unknown, for GSDMD in promoting caspase-1 activation by AIM2 inflammasome.
Runx1 is a key regulator of T cell anergy
Authors: Wilfahrt Drew Nicholas , Hsu Fan Chi , McCue Shaylene , Rajcula Matthew , Shapiro Virginia Smith
Published: January 2023

T cell anergy is a state of cellular hyporesponsiveness that prevents the propagation of an immune response to self. Consequently, the development and maintenance of the anergy program in T cells is tightly controlled. We found in wild type (WT) mice that anergic T cells, defined by co-expression of the cell surface markers CD73 and FR4, express lower expression of the transcription factor Runx1. To test if Runx1 acts as a negative regulator of the anergy program, we utilized a 1:1 mixed bone marrow chimera (BMC) system with ER-Cre Runx1 cKO and WT B6.SJL bone marrow to delete Runx1 in mature CD4+ T cells. Upon tamoxifen treatment and deletion of Runx1, ER-Cre Runx1 cKO CD4+ T cells develop a CD73+FR4+ anergic phenotype. This effect is cell-intrinsic since WT T cells from the same bone marrow chimera do not exhibit the anergic phenotype. Functionally, ER-Cre Runx1 cKO T cells fail to proliferate and produce less IL-2 after TCR/CD28 stimulation, consistent with anergy. Interestingly, WT B6.SJL T cells from the ER-Cre Runx1 cKO:B6.SJL BMC mice fail to proliferate in response to TCR/CD28 stimulation, suggesting Runx1 additionally regulates a cell-extrinsic suppressive mechanism. Using depletion experiments, we found this extrinsic suppression requires FR4+ anergic T cells, but not CD25+ Tregs. Interestingly, the loss of Runx1 leads to the generation of CD73+FR4+ anergy in OT-II T cells, which do not engage their TCR. Thus, the loss of Runx1 drives anergy in the absence of TCR signaling. We conducted RNA-sequencing on ER-Cre Runx1 cKO and WT anergic T cells, and have identified several genes regulated by Runx1 that could play a role in anergy. Further analysis will serve to define the molecular mechanisms Runx1 employs to regulate T cell anergy.