bluuebunny/crossref_metadata_2025 huggingface dataset:
Single cell analyses of human antibody responses to Vibrio cholerae
Authors: Kauffman Robert C. , Bhuiyan Taufiqur , Adekunle Oluwaseyi , Ryan Edward , Qadri Firdausi , Harris Jason , Wrammert Jens
Published: January 2023

Cholera is a severe diarrheal disease affecting 2.9 million people annually resulting in an estimated 95,000 deaths. Infection induces long lasting protective immunity, likely mediated by intestinal antibodies. To further understand humoral immunity against cholera, we used single cell expression cloning to generate panels of human monoclonal antibodies (mAbs) from acutely induced plasmablasts isolated from patients in Bangladesh. mAbs largely targeted the dominant antigens cholera toxin and lipopolysaccharide (LPS). Notably, while LPS responses targeted the O-specific polysaccharide moiety, mAbs varied in serotype specificity and functional characteristics. Unexpectedly, despite all patients being infected by the Ogawa serotype, isolated mAbs from one patient preferentially bound to the Inaba serotype, which had been almost undetectable in circulation for 5 years. Inaba selective mAbs were characterized by high levels of somatic hypermutation. These findings suggest that cholera can generate immunologic memory and induce significant somatic hypermutation in response to this polysaccharide antigen. To expand on these findings we generated a panel of isotype and subclass variants from six LPS specific mAbs, representing a range of affinity and serotype specificity. mAbs were expressed as IgG1, IgG2, IgG3, IgG4, pentameric IgM, and the monomeric and dimeric forms of IgA1 and IgA2. This analysis provided detailed insight into how immunoglobulin isotype/subclass impacts functional characteristics such as agglutination and vibriocidal activity. Moreover, using live-cell microscopy and soft-agar migration assays, we provide novel insight into the impact of these antibody variants on bacterial propulsion.
Pathophysiology of rheumatoid arthritis is associated with polymorphisms in Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) and susceptibility to Mycobacteria
Authors: Sharp Robert C. , Beg Shazia A. , Naser Saleh A.
Published: January 2023

Single nucleotide polymorphisms (SNPs) in T-cell regulator genes such as Protein Tyrosine Phosphatase Non-receptor type 2 and 22 (PTPN2/22) have been associated with many inflammatory autoimmune disorders including rheumatoid arthritis (RA). Along with these SNPs and an environmental trigger of a mycobacterial infection from Mycobacterium avium subspecies paratuberculosis (MAP), we hypothesis that this will lead to chronic inflammation of the synovial joints of RA. We gathered 132 consented subjects blood samples, where genotyping was done for nine SNPs found in PTPN2/22 using TaqMan™ genotyping. Quantification of the effect of the SNPs on gene expression of PTPN2/22 and IFN-γ expression was determined through real time PCR. T-cell proliferation assays were done to examine the response of phytohematoagglutonin (PHA) and mycobacterial antigens in subject samples by the use of BrdU proliferation assays. Detection of MAP bacterial IS900 DNA was done by nested PCR. Out of the nine SNPs examined, PTPN2:rs478582/PTPN22:rs2476601 were found significant in RA compared to the healthy controls (p-values < 0.05). Gene expression of PTPN2/22 was downregulated in RA overall compared to healthy controls. IFN-γ expression was upregulated in samples with significant SNPs. T-cell proliferation increased when T-cells tested had significant SNPs (p-values < 0.05). IS900 MAP DNA was detected more significantly in RA than in healthy controls, where samples with either significant SNPs had higher presence of MAP (p-values < 0.05). This study suggests that SNPs in PTPN2/22 dysregulates T-cell regulation and increases susceptibility to Mycobacteria, leading to increased proliferation of T-cells and higher amounts of IFN-γ expression in RA.
Differences in the male and female immune response to Cryptococcus neoformans infections<
Authors: Guess Tiffany , McClelland Erin
Published: January 2023

Introduction

Cryptococcus neoformans (Cn) is responsible for an estimated 220,000 cases of cryptococcus, resulting in more than 181,000 deaths each year worldwide. Prevalence of this disease is skewed between males and females. Numerous studies show differences in Cn infection rates, with males having a higher incidence of disease and death (7:3, m:f). Sexual dimorphism in infections is not uncommon. However, these differences in Cn have not been well elucidated. Our goal is to unravel the complexity of sex-specific, host-pathogen interactions by examining the effect of sex on the immune response to Cn and whether sex hormones contribute to those differences. We hypothesize that males have an increased incidence of disease due, at least in part, to differences in immune response during a Cn infection.

Methods

Peripheral blood mononuclear cells were isolated from 40 healthy volunteers and infected with Cn. Physiological levels of testosterone and estrogen were added to subsets of all infections. Infected cells were incubated for seven days after which they were harvested and B- and T-cell responses assessed via flow cytometry. Analytes measured include: CD3, CD4, CD8 (T-cell markers), and CD19 (B-cell marker). Serotyping was done on all samples and correlated with immune response.

Results

Subjects with previous exposure to Cn exhibited an increased CD19 response. Data show lower numbers of all T-cell markers in males during infection. There was no difference in hormonal treatment groups.

Discussion

The sexual dimorphism seen in Cn infections is commonly observed, but understanding what accounts for these differences is unknown. These results could aid in understanding why males have increased disease in Cn infections.
Differences of C-type lectin receptors and their adaptor molecules in the peritoneal fluid of patients with endometriosis and gynecologic cancers
Authors: Park Dong Choon , Kim Young Il , Lee Jin-Woo , Yeo Seung Geun
Published: January 2023

Objective

The immunological differences between endometriosis and malignant gynecologic tumors were analyzed by assessing C-type lectin receptors, which are associated with innate immunity, and immunoglobulin secretion, which is associated with B cell adaptive immunity, in peritoneal fluid of these patients.

Methods

The levels of expression of C-type lectin receptors mRNAs were measured by real time PCR in peritoneal fluid of 35 patients with benign masses, 36 patients with endometriosis, and 35 patients with gynecologic cancers. In addition, the concentrations of IgG, IgA and IgM were measured by ELISA in peritoneal fluid of the same patients.

Results

The level of galectin mRNA was significantly lower, and the level of MR2 and DAP 12 mRNA significantly higher, in the endometriosis than in the control group (p<0.05 each). Compared with the gynecologic cancer group, the level of Bcl 10 mRNA was significantly lower, and the levels of MR1, MR2, Syk, Card 9, Malt 1, Dec 205, Tim 3, and DAP 12 mRNAs significantly higher, in the endometriosis group (p<0.05 each). The level of MR2 & DAP 12 mRNA in the endometriosis group was significantly higher than in the control groups (p<0.05), whereas the level of galectin mRNA in the endometriosis group was similar to that in the gynecologic cancer group (p<0.05). IgA and IgG concentrations in peritoneal fluid were significantly lower in the gynecologic cancer than in control group (p<0.05 each).

Conclusions

C-type lectin receptors and immunoglobulins act cooperatively and are closely associated in the pathogenesis of endometriosis.
Characterization of the Thymic Hypoplasia in Mouse Models of 22q11.2 Deletion Syndrome (DiGeorge Syndrome)
Authors: van Oers Nicolai S. , Du Qiumei , de la Morena M. Teresa , Dozmorov Igor , Khan Shaheen , Cleaver Ondine
Published: January 2023

Patients with 22q11.2 deletion syndrome have variable, multi-system disorders including a thymic hypoplasia, cardiac anomalies, and hypoparathyroidism. Over 90% have a deletion of 2.5 Mb on chromosome 22q11.2, affecting protein coding genes, microRNAs, long noncoding RNAs, and pseudogenes. 50%~70% have some degree of thymic hypoplasia (DiGeorge syndrome), resulting in a T cell lymphopenia. Successful transplantation of thymic tissue in patients with a thymic aplasia suggests stromal tissue abnormalities. The thymic stroma consists of thymic epithelial cells (TEC), mesenchymal cells, and endothelial populations. We are exploring the TEC-mesenchyme-endothelial interactions during thymus organogenesis in the mouse model of 22q11.2 deletion syndrome (Df1/+). Comparative transcriptome analyses of hypoplastic and normal thymic lobes from embryos revealed a unique mesenchymal cells mRNA expression signature, including reduced levels of the PDGFRa. PDGFRaH2B-EGFP heterozygous mice that have lower level of PDGFRa expression are being crossed with the Df1/+ model to ascertain whether the hypoplasia is influenced by this receptor. Additional mouse models with prominent penetrance of thymic hypoplasia are being used to determine how Tbx1 (encoded on 22q11.2) influences pharyngeal pouch mesenchymal-TEC development. The results from the study will likely lead to novel approaches for thymus reconstitution in various clinical settings.
Compound Heterozygous Mutations in Forkhead Box N1 (FOXN1) Lead to a Severe Immunodeficiency but Normal Hair and Nail Development in Patients
Authors: van Oers Nicolai S. , Khan Shaheen , Hunyh Larry K. , Du Qiumei , Padron Grace T. , Molina Erika , Dozmorov Igor , Markert M Louise , de la Morena M. Teresa
Published: January 2023

Patients with mutations in the Forkhead Box N1 (FOXN1) transcription factor are born with a severe T-cell lymphopenia in conjunction with alopecia and nail dystrophy (OMIM # 600838). The T-cell lymphopenia results from the impaired development and/or function of the thymic epithelial cells (TECs). TECs are essential regulators of positive and negative selection of developing thymocytes. We report on 3 independent patients with compound heterozygous mutations in FOXN1. All 3 patients had a severe T cell lymphopenia. However, each had normal hair growth and nail bed formation, suggesting that the compound heterozygous mutations result in clinical presentations distinct from classic cases. To determine how the mutations impact murine Foxn1 function, transcriptional reporter assays and protein expression studies were done. Only one of the mutations affected the transcriptional activity of murine Foxn1, with Western blot analyses indicating that this mutation caused production of a truncated protein. CRISPR/Cas9 technologies were used to create mouse lines with compound heterozygous mutations in Foxn1. The mice are currently being intercrossed. The impact of the compound heterozygous Foxn1 mutations on T cell development in the thymus will be presented. Findings from this study may suggest novel therapeutic strategies at restoring thymopoiesis in different clinical settings.
Skin Microbiota as Antigenic Triggers for Cutaneous T Cell Lymphoma
Authors: Dehner Carina Anja , Ruff William Edwards , Foss Francine , Girardi Michael , Kriegel Martin A.
Published: January 2023

Background

Cutaneous T Cell lymphoma (CTCL) is a debilitating malignancy of lymphocytes that home to the skin, thereby manifesting in a variety of chronic skin lesions. CTCL is thought to arise from a combination of genetic, epigenetic and environmental factors, but specific triggers remain unknown. We propose a model in which adaptive immune responses to particular skin commensals evolve into clonal proliferation of T cells in genetically predisposed patients.

Methods and Results

10 CTCL patients with active skin lesions were sampled at 3 time points (blood, stool and lesional vs non-lesional skin). Skin swabs of SLE patients and healthy donors served as control. Dual-index V1–3 and V4 16S rRNA sequencing was performed on the MiSeq platform and revealed significantly more Firmicutes in patient skin compared to controls. In addition, single bacterial colonies were cultured from 3 subjects. Full-length 16S rRNA sequencing demonstrated unique bacterial strains in lesional compared to non-lesional cultures. Malignant cutaneous T cells from patient biopsies were extracted and cultured in vitro for antigenic challenge with patient-isolated skin commensal candidates. One patient’s T cells showed significant proliferation to lesionally enriched B. safensis, while not responding to non-lesional bacteria.

Discussion

Dissecting cancer cell-microbiota interactions revealed potential skin commensal candidates involved in the pathogenesis of CTCL. Pathobionts enriched in lesional skin might act as antigenic drivers for progression to clonality similar to processes involved in H. pylori-induced MALT lymphoma, a paradigm that may be applicable to skin lymphomagenesis.
Abnormal T follicular helper cell subsets in Chronic Lymphocytic Leukemia
Authors: Marshall Aaron J. , Zhang Christine , Hou Sen , Wu Xun
Published: January 2023

The B cell malignancy Chronic Lymphocytic Leukemia (CLL) is a slow progressing disease of monoclonal B cell expansion and immune dysfunction associated with recurrent infections and second malignancies. CD4+ CXCR5+ T cells, known as follicular helper T cells (Tfh), have specialized properties in promoting B cell activation but their role in CLL is unknown. Here we examine the phenotype, activation status, clinical relevance and potential function of circulating Tfh cell subsets in CLL patients. We find that the percentage of Tfh cells is significantly increased in CLL patients compared to healthy age-matched controls. Tfh frequencies are significantly increased in advanced stage CLL patients compared to early stage disease. CLL patients also exhibited different composition of Tfh1, Tfh2 and Th17 subsets and show differential expression of activation markers compared to healthy controls. Our results indicate that the Tfh1 subset is particularly activated and expanded in CLL patients with more progressive disease. Tfh subset expansion and activation correlated with specific cytokine and chemokine levels and with decline in serum immunoglobulin levels. A large proportion of CD4+ T cells in CLL bone marrow express activated Tfh1 phenotype, suggesting that these cells are present within lymphoid tissues where CLL cells proliferate. Lastly, we found that treatment of CLL patients with the Btk inhibitor Ibrutinib lead to normalization of Tfh populations and reduced Tfh activation markers. Our results indicate that Tfh subset profiles could serve as useful biomarker to evaluate the progression of CLL and suggest selective expansion of Tfh1 cells may help drive CLL proliferation and immune dysfunction.
Sustained secretion of immunoglobulin by long-lived plasma cells from human nasal biopsies
Authors: Song Jia , Zhang Ya-na , Wang Hai , Wang Zhe-zheng , Liu Zheng
Published: January 2023

Background

During secondary immune responses, plasma cell survival and antibody production is mediated by extrinsic factors provided by the local environment. It is unknown whether there are long-lived plasma cells (LLPCs) in the human nasal mucosa.

Objective

To identify LLPCs and characterize functionally specific survival niche in the human nasal mucosa.

Methods

Nasal polyp (NP) biopsies were used for ex vivo air-liquid interface culture. Protein levels of immunoglobulins in tissue homogenates were detected by Bio-Plex. The expression of LLPCs related markers including NGF, TrkA, P75, IL-6, APRIL, CD138, CD20, CD4, CD8, ECP, Bcl2, Ki-67, CD3 were detected by means of quantitative RT-PCR, immunohistochemistry (IHC) and ELISA.

Results

Using an ex vivo biopsies culture, we found sustained IgG, IgA, IgE, and IgM secretion >4 wk of culture. IL-6, APRIL NGF, TrkA, and P-75 were up-regulated in CRSwNP compared with controls by ELISA or IHC. We assessed TrkA+ cells, ECP+ eosinophils, and tryptase+ mast cells represent the major interstitial cell sources of NGF, while tryptase+ mast cells and CD138+ plasma cells represent the major sources for TrkA in CRSwNP by immunohistochemistry. We performed histological analysis of fresh nasal polyp tissues, and of tissue fragments harvested 10, 20, 32 days after cultured respectively and found that there’s a small portion of plasma cell in CRSwNP expressed Bcl2 pre- and post-culture. However, post-cultured plasma cells do not express Ki67.

Conclusion

This study demonstrates that the human nasal mucosa harbors a population of nonproliferating plasma cells that are instructed by the microenvironment for prolonged survival and Ab secretion.
Mouse Models for Immunology Research available from The Jackson Laboratory Repository.
Authors: Valenzuela Alicia , Fancher Karen , Rockwood Stephen , Lutz Cathleen ,   The Genetic Resource Science Team
Published: January 2023

The mouse continues to provide researchers a means by which to address complicated immunological questions. The emergence of disruptive genetic engineering technologies have proven to be very applicable to the mouse, spawning a new generation of promising preclinical models. To facilitate access to this flourishing resource, The Jackson Laboratory (JAX) Mouse Repository offers one of the largest sets of well-characterized, genetically engineered mutant mice. Hundreds of new strains are added annually to the collection.

Our newest strains include conditionally targeted interleukins, often combined with Cre recombinase and/or reporters. The conditional Il33 knock-out/in (KO/KI) carries an EGFP reporter, a KO/KI to the Il5 gene results in Il15-specific expression of tdTomato and humanized cre, a similar KO/KI to the Il22 gene results in tdTomato/cre expression in intestinal lymphoid tissues, and a KI to Il21 results in expression of Verde green (VFP). Another new strain expresses cre and GFP within peripheral CD8+ T cells.

MMRRC program mice include a suite of strains expressing and responding to influenza virus hemagglutinin (HA) neo-self antigen for the study of immune regulation, tolerance and autoimmunity. A Pad4 KO, exhibits absent histone deimination in neutrophils and a defective response to acute infection. KOMP mice, also offered via the MMRRC portion of the JAX Mouse Repository, provide a growing number of targeted immunological mutations. Donating a strain to the Repository fulfills NIH’s requirements for sharing mice. Researchers wishing to have mice considered for inclusion in the Repository are encouraged to submit strains: www.jax.org/donate-a-mouse.

This work is supported by NIH, HHMI and private foundations.
Heterogeneity of plaque macrophages derived from CX3CR1+ monocyte precursors in atherosclerosis progression and regression at a single-cell level
Authors: Lin Jian-Da , Nishi Hitoo , Poles Jordan , Mccauley Caroline , Rahman Karishma , Hine Ashley , Vozhilla Nikollaq , Fisher Edward A. , Loke P’ng
Published: January 2023

Atherosclerosis is a leading cause of death worldwide in industrialized countries. We have previously shown that disease progression and regression are strongly associated with the M1/M2 activation status of macrophages derived from inflammatory monocytes in the plaques with large differences in transcriptional profiles. However, the features of monocyte to macrophage transitions during either plaque progression or regression are poorly understood. Here, we use a combination of single-cell RNA sequencing and tamoxifen-inducible fate mapping approaches to profile 3157 and 2198 arterial cells derived from CX3CR1+ precursor in atherosclerotic mice during plaque progression and regression. There was surprising heterogeneity among the cell populations, despite derivation from a common precursor. We compared the distinct cell clusters with the IMMGEN database to define each population and then used pseudo-time analysis to model the differentiation process from monocyte precursors to activated macrophages under progression and regression conditions. Notably, distinct cell clusters with a type 1 interferon (IFN) signature was observed during progression, which was significantly rare in regression. Whereas a unique cluster in regression was marked by increased expression of various heat shock proteins (HSP). Overall, this study highlights previously unidentified genes and programs in macrophages associated with atherosclerosis progression and regression, uncovering the unexpected plasticity of CX3CR1+ monocyte derived cells.
Clonally expanded alpha-chain T-cell receptor (TCR) transcripts are present in aneurysmal lesions of patients with Abdominal Aortic Aneurysm (AAA).
Authors: Lu Song , White John V. , Judy Raquel Ines , Merritt Lisa , Lin Wan Lu , Solomides Charalambos C. , Zhang Xiaoying , Nwaneshiudu Ifeyinwa , Monos Dimitrios S. , Gaughan John P. , Oleszak Emilia L. , Platsoucas Chris D.
Published: January 2023

AAA is a life-threatening vascular disease and one of the ten leading causes of death of 55–74 years old men and women. Although autoimmunity may be responsible for the initiation and/or progression of the disease, limited information is available about the pathogenesis of AAA. Mononuclear infiltrates comprised mostly of T and B cells and monocytes are present in AAA lesions. We have reported (J. Immunol. 192:4897, 2014) that clonally expanded beta-chain TCR transcripts are present in AAA lesions, providing evidence suggesting that AAA is a specific antigen-driven T-cell disease. In addition to the beta-chain TCR, there are several reasons to study the clonality and the repertoire of the alpha-chain TCR in AAA. Alpha-chain TCR transcripts from AAA lesions from these patients were amplified by the non-palindromic adaptor-PCR (NPA-PCR)/Vα-specific PCR and/or the Vα-specific PCR and the amplified transcripts were cloned and sequenced. Sequence analysis revealed substantial proportions of identical alpha-chain TCR transcripts in 4 of 5 AAA patients, demonstrating that clonally expanded T cells are present in these AAA lesions. These results are statistically significant. These T-cell clones very likely have undergone proliferation and clonal expansion in vivo in response to specific, although, as yet unidentified, antigen(s) (self or non-self) recognized by the T-cell clones. Three of 5 patients with AAA were typed by DNA-based HLA-typing and all expressed DRB1 alleles positive for the DRbGln70 amino acid residue, reported to be associated with AAA. Clonally expanded T cells in AAA lesions were found in all these 3 patients. These findings provide additional evidence suggesting that AAA is a specific antigen-driven T-cell disease.
A Role for DPP4 Expression via Oxidized LDL/TLR4/TRIF/CD36 Pathways in Human Obesity and Atherosclerosis
Authors: Zhong Jixin , Rao Xiaoquan , Duan Lihua , Xia Chang , Varghese Juliet , Mihai Georgeta , Rajagopalan Sanjay
Published: January 2023

Objective

Dipeptidyl peptidase IV (DPP4) is well-known for its regulatory role in incretin biology. Although prior studies have suggested a role for DPP4 in atherosclerosis, the regulation of DPP4 in diabetes and atherosclerosis has not been investigated. In this study, we investigated the relationship of DPP4 with human aortic atherosclerosis and the role of obesity.

Methods and Results

We tested the expression of membrane bound DPP4 on circulating monocytes and plasma DPP4 activity (cleaved) in patients with atherosclerosis and their relationship with measures of metabolic health. In atherosclerosis, both cleaved and membrane bound DPP4 were increased. DPP4 expression on monocytes was positively correlated with atherosclerotic plaque volume assessed by high-resolution MRI with expression positively correlating with non-HDL cholesterol level (P=0.0242) but not fasting blood glucose or insulin. Oxidized low-density lipoprotein (oxLDL) up-regulated DPP4 expression on macrophages with a preferential increase in CD36+ cells. OxLDL mediated increase in DPP4 was abolished by siRNA-mediated Toll-like receptor 4 (TLR4) knock-down and considerably diminished by CD36 deficiency. Loss of TRIF but not MyD88 attenuated DPP4 increase with oxLDL.

Conclusions

DPP4 expression on monocytes correlates with atherosclerotic plaque volume with a key role for oxidized LDL in regulating DPP4 expression via CD36/TLR4/TRIF pathways. Increased DPP4 in response to oxidized lipids may represent an integrated mechanism by which post-prandial glucose metabolism is linked to lipoprotein abnormalities potentiates atherosclerosis.
Human anti-dopamine receptor 1 monoclonal autoantibody (mAb) identifies potential mechanisms of neuronal signaling in post-infectious autoimmune-mediated neuropsychiatric disease
Authors: Menendez Chandra M. , Zuccolo Jonathan , Zuccolo Amir , Ben-Pazi Hilla , Cox Carol , Swedo Susan , Cunningham Madeleine W.
Published: January 2023

Neuropsychiatric and movement disorders are associated with infections and anti-neuronal autoantibodies against dopamine receptors D1R and D2R. Most characterized of these disorders is Sydenham chorea (SC), an autoimmune neurologic manifestation of streptococcal-induced rheumatic fever where autoantibodies target the basal ganglia. Pediatric autoimmune neuropsychiatric disorder associated with streptococci (PANDAS) have similar autoantibodies that react with D1R and D2R. SC-derived human mAb 24.3.1 or SC derived IgG signal D2R in transfected cells by decreasing cyclic AMP-dependent kinase (cAMP) activity, reflective of an inhibitory receptor. Anti-D2R 24.3.1 increases dopamine release from human neuronal cells. However, signaling of D1R, an activating receptor, can have different biological effects. Our PANDAS-derived anti-D1R mAb, B4C, and PANDAS sera signaled D1R by activation of cAMP in transfected cells. IgG titers against D1R were significantly elevated in SC and PANDAS sera. To further show IgG recognized the D1R receptor, peptides of the extracellular loop of D1R were constructed and a defined sequence reacted significantly with human mAb B4C and PANDAS serum IgG. Human anti-D1R mAb B4C induced cAMP activity levels comparable to dopamine in a dose-dependent manner. Combined with dopamine, mAb B4C sensitized D1R to at least 50X more responsive to dopamine. In neuropsychiatric diseases where both anti-D1R and anti-D2R autoantibodies may be in combination, anti-D1R autoantibodies could sensitize D1R making it more sensitive to excess dopamine induced by anti-D2R autoantibodies; thus, contributing to dopamine signaling abnormalities and neuropsychiatric pathogenesis.
Translational profiling of microglia in Alzheimer’s disease models
Authors: Fryer John D. , Kang Silvia S.
Published: January 2023

Alzheimer’s disease (AD) is a neurodegenerative disease characterized pathologically by amyloidosis, tauopathy, and activation of microglia, the resident innate immune cells of the brain. Using a translational RiboTag profiling approach, we found that cellular isolation methods (enzymatic digestion, myelin removal, and CD45/CD11b sorting) altered the microglial transcriptome due to microglia activation and potential cellular contamination from sorting. Additionally, lipopolysaccharide-induced systemic inflammation generated completely different “top hits” from microglial RiboTag vs cellular isolation RNAseq datasets, likely due to microglial activation during the isolation process. RiboTag profiling of mouse models of AD revealed many significantly altered microglial transcripts that were shared between amyloidosis and tauopathy models, forming a network containing apolipoprotein E (APOE), CCL3, and CCL4. Inflammation and neutrophil chemotaxis pathways were also significantly enriched amongst the shared transcripts. This study has broad implications for microglial transcriptomic approaches and provides insights on microglial pathways associated with different pathological aspects of AD.
Autoantibodies to myelin proteins as biomarkers to identify nervous system injuries in Gulf War Illness and other neuromuscular disorders
Authors: Brahmajothi Mulugu V. , Abou-Donia Mohamed Bahie
Published: January 2023

A study on veterans with Gulf War Illness (GWI) from Madison VA Hospital demonstrated a widespread disruption of damages to the white matter using magnetic resonance brain imaging suggesting a cause of prolonged chronic musculoskeletal pain. We hypothesized that exposure to various chemicals could cause delayed neurotoxicity and that myelin is an important white matter component, which play a critical role in neural function and plasticity. Any disruption to myelin may result in reduced brain connectivity, musculoskeletal pain, and cognitive dysfunction. Since myelin is composed of various proteins, dysregulation or damage over a period will form a distinguishable biomarker in CSF and in serum/plasma. Further, continued damage could elicit an immune response. With these reasoning, we measured serum/plasma levels of myelin proteins and autoantibodies specific to myelin proteins by ELISA in Veterans with Gulf War Illness (GWI) and other patients with neuronal disorders (Multiple Sclerosis (MS), Parkinson’s disease (PD), mild Traumatic Brain Injury (TBI), Flight Crews with chronic pain and veterans with Chronic Fatigue Syndrome (CFS) and Lupus (SLE)). We quantitated several myelin proteins (MPZ, MAG, MBP MOG, MOBP, PMP2, and NF155) to determine the intensity of disruption. Autoantibody levels in MS patients were used as positive control and since all the samples (n=10) tested showed the highest level of IgG specific autoantibodies compared to other cohorts. Significantly increased levels of NF155, MOG, MOBP, and MAG were observed among all groups compared to controls (p<0.001). Elevated levels of MPZ and PMP2 characterized PD and SLE (p<0.01) and GWI veterans showed elevated levels of MBP compared CFS and healthy cohorts.
Anti-neuronal and anti-microbial immunity link CaMKII and autism spectrum disorder with pediatric acute-onset neuropsychiatric syndrome
Authors: Chain Jennifer L. , Cox Carol J. , Alvarez Kathy M. , Menendez Chandra M. , Reim Sean M. , Shawler Paul M. , Richmand Brian , Stoner Julie , Frye Richard E. , Swedo Susan , McBride Bonnie , Cunningham Madeleine W.
Published: January 2023

Autism spectrum disorders (ASD) may be associated with neuropsychiatric symptoms such as tics, obsessive-compulsive behaviors, and other symptoms characteristic of pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). Abnormal anti-neuronal autoantibody responses against the basal ganglia are elevated in neuropsychiatric disorders such as PANDAS. Our study suggests that anti-neuronal autoantibodies in ASD may be associated with the presence of PANDAS and investigates microbial polysaccharides as potential inducers of anti-neuronal autoantibodies in children with confirmed ASD or ASD/PANDAS. Functional anti-neuronal autoantibodies which signal CaMKII and induce excess dopamine release were found in ASD/PANDAS serum. Autoantibody signaling or reactivity was inhibited by 3 common microbial antigens, by a group of neuronal antigens, or by anti-IgG beads which significantly reduced CaMKII activation in ASD/PANDAS sera. Interestingly, a CamKII mutation has recently been associated with autistic symptoms in animal models and also in ASD in humans. Children with ASD/PANDAS had significantly elevated anti-neuronal autoantibodies by ELISA, and strong IgG responses against the group A streptococcal epitope GlcNAc were observed in ASD/PANDAS. Our study links three common microbial antigens to anti-neuronal antibody responses in ASD/PANDAS and suggests that molecular mimicry between host and pathogen may play a role in development of the autoantibodies and potentially lead to neuropsychiatric symptoms in ASD/PANDAS. Thus, ASD may be complicated by anti-neuronal antibodies which can develop during infections and may contribute to the co-morbid symptoms of PANDAS in ASD.
The Benzathine Penicillin G (BPG) Reformulation Preferences Study - Tonga
Authors: Sika-Paotonu Dianne , Aho George , ‘Ulufonua Lisiate Kilipeni , ‘Ofanoa Malakai , ‘Akau’ola Siale , Vaea Kolinisiupeli , Tuipulotu Amelia Afuha’amango , Toumoua Seventeen , Lilo Filimone , Fotu Latu , Tiatia Ramona , Sung Yun K. , Zonneveld Rebecca , Poot Betty , Norris Pauline , Betty Bryan , Thornley Craig , Eddie Barbara , Wineera-Parai Ranei , Baker Michael , Carapetis Jonathan
Published: January 2023

Acute Rheumatic Fever (ARF) is an autoimmune condition caused by untreated Group A Streptococcal (GAS) infection of the upper respiratory tract (and possibly skin). Multiple or severe attacks of ARF can cause cardiac damage, known as Rheumatic Heart Disease (RHD) which remains a significant cause of morbidity and mortality globally and is rare in developed countries. In Australia, New Zealand (NZ) and the Pacific Region, the disease burden of ARF and RHD amongst Indigenous and Pacific communities is one of the highest in the world, usually affecting children and young adults. A GAS vaccine remains years from being available and until then, the most effective recommended preventative measure for ARF requires painful monthly intramuscular injections of Benzathine Penicillin G (BPG) for 10 years or more, known as secondary prophylaxis (SP). Adherence to SP remains a challenge in many settings and improved understanding of barriers and novel approaches to BPG delivery are urgently needed. To create a reformulation of BPG that encourages SP adherence, insight is needed into the reformulation preferences of those with ARF/RHD. This work seeks to explore the experiences and investigate children/teen on SP, family, and healthcare professional and practitioner preferences for potential BPG reformulation characteristics in Tonga. Three software applications will be adapted from pre-developed applications already piloted and pretested, and optimized for use to ensure age, cultural appropriateness and efficient data collection. This is the first time software applications have been successfully developed and utilized to collect qualitative and quantitative data on individual preferences for BPG formulations and dosing regimens in Tonga.
Cereblon tunes c-Myc protein expression and regulates the metabolic function of activated T cells
Authors: Epling-Burnette Pearlie , Hesterberg Rebecca , Akuffo Afua Adutwumwa , Beatty Matthew S. , Goodheart William E. , Fernandez Mario , Cleveland John L.
Published: January 2023

Immunomodulatory drugs, such as lenalidomide, pomalidomide, and CC122 are glutarimide derivatives that alter T cell effector functions, reverse tolerance, and increase cytokine production. We hypothesized that cereblon (CRBN), the molecular target of these drugs, is a negative regulator of T cell function. Therefore, germline Crbn knockout mice were used to investigate its role in T cell regulation. Similar to human T cells treated with immunomodulatory drugs, stimulated Crbn−/− T cells exhibit increased proliferation and cytokine production and maintain this phenotype in the absence of CD28 co-stimulation. Using Gene Set Enrichment Analysis (GSEA) to identify transcriptional drivers of differentially expressed genes, we found that Myc and its family member Max may drive the observed activated phenotype in Crbn−/− T cell. Crbn−/− T cells have Myc-related phenotypic changes, such as increased cell size, CD98 expression, glucose uptake, glutamine and arginine uptake, increased intracellular polyamine biosynthesis, and oxygen consumption. Similar increases in Myc-related processes were observed in activated human T cells treated with immunomodulatory drugs. While c-myc mRNA is similar, Crbn−/− T cells and drug-treated human T cells have prolonged c-Myc protein expression. CRBN, a substrate receptor for the DDB1/Cul4A/Rbx1 E3 ubiquitin ligase complex, has only one known endogenous substrate which is glutamine synthetase (GS). GS interacts with CRBN through acetylated lysine residues yielding a protein that is succeptable to polyubiquitinlyation. Collectively, this suggests that CRBN tunes c-Myc-regulated pathways in activated T cells and that this process is blocked by immunomodulatory drugs.
Programmed cell death ligand 1 (PD-L1) regulates tumor initiating cell (TIC) generation by controlling the stemness gene Oct4 through mTORC1
Authors: Gupta Harshita B. , Deng June , Clark Curtis A. , Drerup Justin Michael , Wu Bogang , Sareddy Gangadhara , Hurez Vincent , Vadlamudi Ratna , Li Rong , Curiel Tyler J.
Published: January 2023

TIC mediate tumor formation, drug resistance and relapse. We reported that PD-L1 promotes tumor mTORC1, and TIC number and functions. Knocking down PD-L1 reduced expression of stemness genes Oct4 and Nanog and the mTORC1 signaling component Raptor in B16 melanoma and ID8agg ovarian cancer (OC) cells. To understand how PD-L1 controls stemness, we treated control and PD-L1 KO (KO) B16 cells with rapamycin (mTORC1 inhibitor), PI3K inhibitor wortmannin, Notch inhibitor Compound E, and c-Myc inhibitor JQ1. Only rapamycin affected control and KO CD133+CD44+CD24+ TIC differentially (50% reduced TIC in control versus 0% in KO, p=0.0075). We over-expressed Oct4 in B16 but failed to get clones, so over-expressed it in ID8agg PD-L1lo cells (KI) and found that TICs (CD44+CD24+) had improved proliferation and 2-fold more TIC than PD-L1lo cells (p=0.015, p=0.018). We sorted ID8agg, PD-L1lo and KI TIC and cultured them in stem cell medium. KI TICs formed four-fold bigger spheres than PD-L1lo TIC (p<0.0001), consistent with improved self-renewal. The KI spheres were also 1.5-fold larger than those of ID8agg (p=0.032). ID8agg, PD-L1lo and KI cells were then treated with rapamycin. KI TIC were resistant to rapamycin treatment (3 fold reduction in control, 2.9 fold in PD-L1lo versus none in KI), suggesting that PD-L1 drives stemness through mTORC1 driven Oct4 expression. Thus, PD-L1 regulates stemness genes through mTORC1.
A mechanistic base for alternative splicing of human immune system-derived thyroid stimulating hormone (TSH) β-subunit.
Authors: Klein John R.
Published: January 2023

Thyroid stimulating hormone (TSH) is a glycoprotein hormone consisting of an α and a β chain. Within the hypothalamus-pituitary-thyroid axis, TSH is produced by thyrotropic cells in the anterior pituitary. Upon release into the circulation, TSH binds to receptors in the thyroid, resulting in the release of triiodothyronine (T3) and thyroxine (T4). T3 and T4 regulate nearly every aspect of mammalian physiology, including basal metabolism, growth and development, mood, and cognition. Although TSHβ has been known for years to be produced by cells of the immune system, the functional significance of that has remained unclear. My laboratory identified a splice variant of human TSHβ (TSHβv) that consists of a truncated but biologically active portion of TSHβ made from exon 3 with the deletion of exon 2. The signal peptide of TSHβv is coded for by a portion of the 3′ end of intron 2. TSHβv is primarily produced by bone marrow cells and peripheral blood leukocytes, particularly though not exclusively by cells of the myeloid/monocyte lineage. An unresolved question pertains to the molecular mechanisms that account for TSHβv alternative splicing, Here, a model is presented whereby TSHβv is made through a series of unique genetic reorganization steps. These findings have implications not only for understanding how the TSHβv isoform is made, but also for gaining insights into the involvement of immune system TSHβ in health and disease.
MRP14 as an important regulator of macrophage function in type 2 diabetes mellitus
Authors: xia chang , Rao Xiaoquan , Wang Yunmei , Simon Daniel I. , Rajagopalan Sanjay , Zhong Jixin
Published: January 2023

Background

Myeloid-related protein14 (MRP14) plays an essential role in myeloid cell-mediated innate immune function and a number of inflammatory diseases. However, the role of MRP14 in obesity-induced inflammation and insulin resistance is not well defined. This study investigated the role of MRP14 in macrophage inflammation and obesity-induced insulin resistance.

Methods and Results

WT and MRP14−/− mice on C57BL/6 background were fed a high-fat diet or normal chow for 12 weeks. Tissue-resident macrophages in both adipose tissue and liver from obese mice expressed higher levels of MRP14. MRP14−/− mice showed a significantly improved insulin sensitivity, as measured by intraperitoneal glucose tolerance test and insulin tolerance test, compared to WT mice. There were reduced NLRP3 expression and caspase-1 activation in MRP14−/− macrophages after LPS stimulation compared to WT macrophages. In consistency, treatment of exogenous recombinant MRP8/MRP14 complex induced NFκB and caspase-1 activation. Glycolysis and mitochondrial respiration assay of macrophage were similar between WT and MRP14−/− mice. Activated MRP14−/− macrophages produced less T cell chemokines CCL5, CXCL9, and CXLL10, accompanied by reduced NFκB activation. Depletion of MRP14 from the supernatant decreased the activation of NFκB and production of chemokines (CCL5, CXCL9, and CXLL10) in WT macrophage but not MRP14−/− macrophage. Treatment of MRP8/MRP14 complex induced NFκB activation and increased the expression of CCL5, CXCL9, and CXLL10. In addition, T cells showed reduced migration towards the supernatant from MRP14−/− macrophages.

Conclusion

MRP8/14 regulates chemokine production from macrophage and T cell migration.
Statins regulates inflammatory macrophage phenotype through the activation of AhR
Authors: Manni Giorgia , Gargaro Marco , Turco Antonella , Scalisi Giulia , Matino Davide , Pirro Matteo , Fallarino Francesca
Published: January 2023

Statins are 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase competitive inhibitors, that lower blood cholesterol, but also exert potent anti-inflammatory effects. The xenobiotic receptor, aryl hydrocarbon receptor (AhR), plays crucial role in the control of inflammatory responses. Evidences suggest that selected statins promote the transcription of the main AhR target gene, cytochrome P450 family 1 (Cyp1a1). Macrophages are essential components of innate and adaptive immunity, characterized by AhR expression and activity. Based on these evidences, we investigated potential AhR-mediated immune regulatory properties of statins in Bone Marrow Derived Macrophages (BMDM).

We found that BMDM activation with the pro-inflammatory cytokine TNFa induced the inflammatory marker, inducible nitric oxide synthase (iNOS), which was completely prevented in BMDMs treated with TNFa in combination with specific statins, namely, atorvastatin and rosuvastatin. We confirmed high AhR expression in BMDM that was increased upon treatment with TNFa. We investigated the ability of different statins to activate AhR using a luciferase reporter assay cell lines and in BMDCs. We found that selected statins, such as atorvastatin and rosuvastatin, activated AhR in a dose dependent manner, an activation prevented in cells expressing selected AhR mutants. Notably, AhR deficient BMDMs, expressed undetectable levels of the main statin target protein HMG-CoA, which was instead highly expressed in wild type BMDMs.

These data demonstrate that natural or synthetic statins can play regulatory roles in macrophages. Moreover, our results suggest a potential involvement of AhR in statin-mediated anti-inflammatory effects in macrophages.
Distinct iron homeostasis in Th1- and Th2-biased mouse strains
Authors: Xiao Xia , Saha Piu , Yeoh Beng San , Kumar Matam Vijay
Published: January 2023

Iron is a proinflammatory transition metal ion, yet limited information is documented on the differences in iron homeostasis under Th1 and Th2 biased conditions. We used C57BL/6 (BL6) and Balb/C (BB) strains, which exhibit prototypical Th1 and Th2 immune responses, to investigate the extent to which Th1 and Th2 biased responses dictate iron homeostasis during healthy and inflammatory conditions. Systemic and tissue iron levels were assessed in age and gender matched BL6 and BB mice. Gene expression for iron absorption, transport, and storage proteins were quantified in duodenum and liver. Intracellular labile iron pool (LIP) was assessed in bone marrow-derived macrophages (BMDM) and neutrophils (BMDN). Hypoferremia of inflammation was assessed by measuring serum iron before and after LPS treatment. BB mice displayed significantly higher levels of systemic and storage iron than BL6 mice. Although the expression of hepcidin and ferroportin were comparable between the two strains, hepcidin-independent iron absorption is more prominent in BB mice, as indicated by higher expression of genes involved in iron absorption and transport: DcytB, DMT1, SFT, and hephaestin. Similarly, hepatic TfR1, TfR2, transferrin, and DMT1 expression was higher in BB mice. Moreover, the basal levels of LIP in BB BMDM and BMDN were 3.4 and 2.6 folds higher than BL6, respectively. The LPS-induced hypoferremia of inflammation is more pronounced in BL6 than BB mice. Taken together, iron homeostasis is distinct between these two strains during both healthy and inflammatory conditions. BB mice showed more iron load than BL6 mice, which could be explained, in part, by the higher expression of iron uptake genes and also reduced Th1 responses in BB mice.
Aggravated gut inflammation in mice lacking the taste signaling protein α-gustducin
Authors: Wang Hong , Feng Pu , Chai Jinghua , Redding Kevin , Margolskee Robert F. , Huang Liquan
Published: January 2023

Inflammatory bowel disease (IBD) is a debilitating immune-related condition that affects over 1.4 million Americans. Recent studies indicate that the taste system is involved in much more than sensing food flavor, and taste receptors have been localized in a variety of extra-oral tissues. One of the newly revealed functions of taste receptors and signaling proteins is modulation of immune responses to microbes and parasites. We previously found that components of the taste receptor signaling pathway are expressed in subsets of the intestinal epithelial cells. α-Gustducin, a key G-protein α subunit involved in sweet, umami, and bitter taste receptor signaling, is expressed in the intestinal mucosa. In this study, we investigated the role of α-gustducin in regulation of gut mucosal immunity and inflammation using gustducin knockout mice in the dextran sulfate sodium (DSS)-induced IBD model. DSS is a chemical colitogen that can cause intestinal epithelial damage and inflammation. We analyzed DSS-induced colitis in gustducin knockout versus wild-type mice after administration of 3% DSS in drinking water to mice. Our results show that the knockout mice had increased weight loss, diarrhea, intestinal bleeding, and inflammation over the experimental period compared to wild-type mice, concurrent with increased expression of TNF and IFN-γ but decreased expression of IL-13 and IL-5 in the colon. These results suggest that the taste receptor signaling pathway may play critical roles in regulating gut immune balance and inflammation.