bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoantibodies targeting apolipoprotein A-I are associated with cardiovascular disease outcomes
Authors: Venditto Vincent J , Pitts Michelle , Henson David
Published: January 2023

Cardiovascular disease (CVD) is characterized by an inflammatory immune response associated with disease progression. Apolipoprotein A-I (ApoA-I), the major protein of high-density lipoprotein (HDL), has been identified as the target of antibodies in patients, but their role in disease progression is poorly understood. Recently, we identified immune complexes composed of ApoA-I and IgG (ApoA-I/IgG ICs) in human plasma, which are associated with delayed disease progression. Studies in a large prospective cohort of cardiovascular disease are currently underway. The mechanistic impact of these observations and the dichotomy between free and bound antibodies urge continued exploration of the anti-ApoA-I antibody response in mouse models of inflammation. To complement our efforts with plasma from human subjects, we evaluated anti-ApoA-I antibody induction in mice in response to immune stimuli including western diet, immunization with ApoA-I epitopes, induction of hypercholesterolemia, or combinations of all three. Anti-ApoA-I antibody profiles and subclass composition were characterized by ELISA and compared between groups. Most notably, mice exhibit elevated levels of free antibody responses toward ApoA-I with age, irrespective of gender or diet composition. These data indicate that autoantibodies targeting ApoA-I are naturally occurring in both mice and humans and prompt continued investigation into their role in cardiovascular disease progression.
Autoantibodies targeting components of neutrophil extracellular traps are elevated in cystic fibrosis
Authors: Yadav Ruchi , Fejes Zsolt , Li Quan-Zhen , Nagy Bela , Rada Balazs
Published: January 2023

Cystic fibrosis (CF) lung disease is characterized by the presence of neutrophils and released neutrophil extracellular traps (NETs). NETs are composed of a genomic DNA scaffold decorated with histones and neutrophil granule proteins. The long-term presence of NETs could lead to an autoimmune response in CF. This is supported by our published results showing elevated levels of anti-PAD4 autoantibodies in a U.S. CF cohort. To confirm and expand this observation, here we hypothesized that anti-PAD4 autoantibodies are also elevated in another CF cohort from Hungary, and additional, NET-targeting autoantibodies can be identified in CF.

Fifty-seven CF patients (class I–III CFTR mutations, stable lung function, 26±−5.4 years of age (mean±SD), 54% female) and forty healthy subjects (26.2±3.6 years of age, 57% female) were recruited into this study in Hungary. Anti-PAD4 and anti-dsDNA autoantibodies were measured by ELISA while additional autoantibodies were determined by autoantigen microarray. Increased levels of anti-PAD4 autoantibodies were confirmed in this CF cohort. Serum levels of anti-dsDNA IgA autoantibodies were significantly higher in CF patients compared to control subjects. The autoantigen microarray analysis revealed significantly elevated levels of several, novel, NET-targeting autoantibodies in CF: anti-chromatin IgA, anti-core Histone IgA and IgE, anti-Histone H1 IgA, anti-Histone H2A IgA and IgM. No significant correlations of these autoantibodies with lung function (FEV1%pred), age or inflammatory markers (human epididymis protein 4, C-reactive protein) were found. Overall, these results indicate that chronic presence of NETs leads to a systemic autoimmune response in CF.
Conditional deletion of the actin-regulatory protein hematopoietic protein-1 alters B cell maturation, migration and antibody production
Authors: Avalos Alan Vinicio , Park Heon , Suwankitwat Nutthakarn , Iwata Terri , Iritani Brian M
Published: January 2023

Hematopoietic protein-1 is a hematopoietic cell specific member of the actin-regulatory WAVE complex 2, which acts downstream of multiple immune receptors to stimulate F-actin nucleation. Loss-of-function mutations in the gene encoding Hem-1 (NCKAP1L) have recently been found to result in Primary Immunodeficiency Disease (PID) in humans, characterized by recurrent bacterial and viral respiratory infections, asthma, skin infections, otitis media, and bacteremia. However, the cellular and molecular mechanisms of how loss of Hem-1 results in PID are not known. In this study, we generated constitutive and B cell specific Nckap1l knockout mice using the Cre-LoxP system to dissect the importance of Hem-1 in B cell development and functions. Mice with constitutive deletions of the NCKAP1L gene are severely immunodeficient, characterized by reduced total B and T cells, dysregulated cytokine production and impaired neutrophil migration. To determine why B cells are reduced in the mouse, we bred Nckap1lfloxed mice to Mb1Cre which expresses Cre throughout B cell development. Utilizing flow cytometry, we found that B cell specific deletion of Hem-1 in Nckap1lfl/fl Mb1Cre mice resulted in a reduction in mature B cells in the bone marrow, reduced follicular and marginal zone B cells in the spleen, and reduced innate-like B1a cells. Peripheral B cells from Hem1fl/fl Mb1Cre mice had decreased abilities to migrate to lymphoid tissues in vivo, and altered antibody production following immunization with T-dependent and T-independent immunogens. These results suggest that Hem-1 is essential for normal B cell development and Ab production, and provide novel information regarding why mutations in NCKAP1L result in severe immunodeficiency in humans.
Neonatal exposure to commensal bacteria-derived antigens directs polysaccharide-specific B-1 B cell repertoire development
Authors: New James Stewart , Dizon Brian L.P. , Fucile Christopher , Rosenberg Alexander F. , Kearney John F , King Rodney G
Published: January 2023

Mouse B-1 B lymphocytes play essential roles in defense against infectious microorganisms and maintenance of self-tolerance by producing natural antibodies. However, whether exogenous factors influence B-1 B cell repertoire formation remains poorly understood. Herein, we demonstrate that early immunization with N-acetyl-D-glucosamine (GlcNAc)-bearing Streptococcus pyogenes elicits GlcNAc-reactive B-1 B cells in C57BL/6 mice expressing immunoglobulin heavy chains distinct from those of naïve mice and mice immunized with S. pyogenes in adulthood. Moreover, germ-free mice exhibit greatly reduced GlcNAc-reactive serum antibodies and deficiencies in oligoclonal GlcNAc-reactive B-1 cell clonotypes dominant in conventionally raised mice. These phenotypes are also observed in Myd88-deficient mice and reversed following conventionalization of adult germ-free mice. We show that colonization by the microbiota promotes development of GlcNAc-reactive B cells and IgA-secreting plasma cells in the small intestine that are clonally related to PerC and splenic B-1 B cells. These results indicate that environmental and microbiota-derived antigens influence B-1 clonotype selection into the mature repertoire.
Altered 3D chromatin structure permits inversional recombination at the IgH locus
Authors: Du Hansen , Qiu Xiang , Zhao Mingming , Singh Amit , Sen Ranjan
Published: January 2023

Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombinase activating gene products (RAG) 1 and 2. Diversity gene segments (DH) rearrange first, followed by variable (VH) gene rearrangements. Here we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments. DH gene segments, that recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination. In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on WT IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA. We propose that VH gene segments find their targets by diffusion-controlled mechanisms. These distinct mechanisms may underlie differential allelic choice associated with each step of IgH gene assembly.
Early AID expression in developing B cells marks a population with a restricted IgH repertoire favoring D-proximal VH gene usage
Authors: von Beck Troy Alexander , Dale Gordon , Jacob Joshy
Published: January 2023

Activation-induced cytidine deaminase (AID) is well characterized in germinal center reactions as a driver of secondary antibody diversification through the processes of somatic hypermutation and class-switch recombination, but recent reports from clinical and murine studies suggest an additional role during primary diversification. Specifically, inhibition or knockout of AID activity during B cell development results in increased IgM autoantibody production. Furthermore, the expression of AID during development appears to be driven by B cell receptor mediated recognition of autoantigens, including RNA and dsDNA. To examine the pattern of early AID expression, we used lineage tracing in an AID-Cre x R26R-EYFP mouse model to identify a subpopulation of AID experienced (AID-exp) pre-B cells. Sequencing of the IgH repertoire from the AID-exp subpopulation reveals a stark reduction in the diversity of VH gene usage compared to AID-naïve pre-B cells. The reduction in diversity is accompanied by enrichment for specific VH genes, including D-proximal elements associated with B1 cells and autoantibodies. Thus, we discern skewing within the AID-exp repertoire suggestive of antigen-dependent selection in the bone marrow compartment. Our results support the hypothesis that AID is induced in pre-B cells carrying an autoreactive heavy chain and effects tolerization to specific self-antigens.
Depletion of SCFA-fermenting gut bacteria alters the epigenome of hematopoietic stem and progenitor cells
Authors: Hughes Michael R , Cait Alissa , Bilenky Misha , Moksa Michelle M. , Yip William , Li Kristen , Canals-Hernaez Diana , Hirst Martin , Mohn William W , McNagny Kelly M
Published: January 2023

Gut dysbiosis alters the development and severity of atopic disease. We previously demonstrated that nursing dams and newborn mice treated with low-dose vancomycin alters gut microbial diversity with a marked loss of bacteria that produce short-chain fatty acids (including butyrate). Vancomycin-induced gut dysbiosis enhances the TH2 response to lung allergens due to altered dendritic cell trafficking and activation in addition to modifying the behavior of other mature leukocyte lineages. Butyrate supplementation reverses the vancomycin-induced TH2 pro-inflammatory phenotype.

Butyrate is known to exert some of its effects on target cells by inhibiting histone deacetylases (HDACs) with consequent effects on gene expression. Consistent with a role for epigenetic skewing of the hematopoietic compartment, we found that engraftment of total bone marrow from dysbiotic mice transferred enhanced TH2 proclivity in normobiotic recipients. Strikingly, we found unique regulatory states (H3K27ac marks) in purified hematopoietic stem and progenitor cells (HSPC) of TH2-skewed recipient mice. Single cell RNA sequence analyses identified a distinct transcriptomic signature in HSPC of dysbiotic mice that was reversed by butyrate supplementation. Together, these data suggest that the gut microbiome alters gene expression in blood progenitor cells with long term consequences on the immune response to peripheral allergens.
A single-cell multiomics approach to study tumor-driven perturbations during hematopoiesis in mice
Authors: Kara Nihan , Shi Xiaoshan , Samusik Nikolay , Widmann Stephanie , Tyznik Aaron J
Published: January 2023

Cancer is associated with concomitant myeloid cell responses that are characterized by an expansion of tumor associated myeloid cells, including myeloid derived suppressor cells, tumor associated macrophages and neutrophils. The prevailing paradigm is that cancer interferes with normal hematopoiesis by skewing the generation of myeloid cells with tumor promoting functions and promoting extramedullary hematopoiesis in organs such as the spleen. Advances in single cell multiomic technologies now enable the analysis of high dimensional protein and mRNA expression from thousands of cells simultaneously. Using the power of the BD Rhapsody™ Single-Cell Analysis System, we have carefully investigated melanoma associated extramedullary hematopoiesis in mice. To do this, we isolated 4 hematopoietic stem and progenitor cell populations simultaneously, including Lin− Sca-1+ c-Kit+, common myeloid progenitor, granulocyte-macrophage progenitor, and megakaryocyte-erythrocyte progenitor cells from the spleen and bone marrow of melanoma bearing mice using the BD FACSMelody™ cell sorter in combination with a multiplex BD® AbSeq Oligos panel for surface protein expression and a single-cell whole transcriptome analysis. Using advanced analysis plugins in SeqGeq™ software, we were able to delineate the developmental trajectories of these hematopoietic stem and progenitor cells in this systemic immuno-suppressive myeloid environment. These advanced technologies are critical to uncover tumor mediated abnormalities in hematopoiesis.

Disclaimers: For Research Use Only.

Class 1 Laser Product.

BD, the BD Logo, FACSMelody and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates.

© 2019 BD. All rights reserved.
Metabolic pathways that mediate expansion and homeostasis of hematopoietic lineages
Authors: Pizzato Hannah A , Wang Yahui , Lam Wing Y. , Wolfgang Michael J. , Finck Brian N. , Patti Gary J. , Bhattacharya Deepta
Published: January 2023

Due to the rarity of hematopoietic stem cells, their differentiation into progenitors then on to mature cells must occur with proliferation, leading to an expansion of each downstream population. This process, called transit amplification, is required to produce a sufficient mature cell population and must be balanced with survival to maintain homeostasis and prevent hematological malignancies. How transit amplification and subsequent homeostasis is controlled is not well understood but may involve metabolic switches. Various groups have demonstrated that hematopoietic expansion requires a switch from glycolysis to oxidative phosphorylation, but what induces this switch and the specific carbon sources that fuel it remain unclear. To address these questions, we employed mice conditionally deficient in mitochondrial pyruvate import, fatty acid oxidation, or glutamine hydrolysis and examined the requirements of these pathways in hematopoietic homeostasis. Ablation of carnitine palmitoyltransferase II (Cpt2) or glutaminase (Gls) had no effect on lineages examined, suggesting fatty acid oxidation and glutaminolysis are not required for the production or survival of various immune cells. In contrast, upon deletion of mitochondrial pyruvate carrier 2 (Mpc2), peripheral myeloid cell numbers declined dramatically. Over time after Mpc2 ablation, a recovery of myeloid cells was observed. This recovery was associated with increased proliferation of Mpc2-deficient progenitors relative to wild type counterparts immediately following genetic deletion. Further studies are being done to elucidate what metabolic pathway may allow myeloid progenitors to rapidly proliferate and recover after mitochondrial pyruvate ablation.
Human TLR8 Adversely Affects Placental Development and Induces Spontaneous Pregnancy Loss in a model of Systemic Lupus Erythematosus
Authors: Maria Naomi I. , Martinez Shani , Raparia Chirag , Diadhiou Catherine M.M. , Davidson Anne
Published: January 2023

SLE is associated with adverse pregnancy outcomes, particularly in the presence of antiphospholipid antibodies (aPL). Pathogenic mechanisms damaging the fetal-maternal unit, however, remain poorly understood. A role for endosomal TLR8 was recently hypothesized in aPL induced placental damage. The role of TLR8 in systemic autoimmunity has remained elusive as TLR8 function differs from mouse to man. We investigated the effect of human (hu)TLR8 expression on pregnancy outcomes in Sle1 mice that develop aPL antibodies.

huTLR8 transgenic(tg) Sle1 mice were generated and huTLR8 DNA copy number and mRNA expression was confirmed by qDigital- and qRT-PCR. Pregnancies were closely followed and terminal C-sections performed at signs of dystocia/prolonged labor. Pup and placental weight was assessed. Placental tissues were characterized by H&E and Myeloperoxidase (MPO) staining and inflammatory cytokine expression was assessed in placental tissue. Splenic and bone marrow immune populations were analyzed using flow cytometry.

Female Sle1.huTLR8tg mice developed aPL and sm/RNP autoantibodies earlier than Sle1 controls and ±50% loss of litters was observed, compared to 0% in controls. IL-6, TNFa and MPO mRNA expression was significantly increased in placentas of affected litters and negatively correlated with placental weight. Abnormal placental morphology included placental infarcts, loss of placental lakes, arterial wall thickening and increased netting neutrophils.

Here we identify a new model to study adverse pregnancy complications in SLE. In the presence of both Sle1 and huTLR8, we observed early onset of aPS autoantibodies and pregnancy loss attributed to placental developmental abnormalities, infarcts and inflammation.
Pro-fibrotic Activation of Human Macrophages in Systemic Sclerosis
Authors: Bhandari Rajan , Ball Michael S. , Martyanov Viktor , Popovich Dillon , Schaafsma Evelien , Han Saemi , Eltanbouly Mohamed , Carns Mary , Arroyo Esperanza , Aren Kathleen , Hinchcliff Monique , Whitfield Michael L , Pioli Patricia A
Published: January 2023

Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in Systemic sclerosis (SSc), and we have shown that macrophages constitute the dominant inflammatory signature in SSc tissue. However, little is known about how these cells contribute to fibrotic activation in SSc. Using a bioinformatics approach, we show that the gene expression profile of blood-derived human SSc macrophages is significantly enriched in SSc patient skin. We characterize the immunophenotype of human SSc macrophages as pro-fibrotic, demonstrating that these cells are activated under basal conditions, expressing elevated levels of surface markers associated with activation and releasing CCL2, IL-6, and TGF-beta. We also show that STAT3 is phosphorylated in macrophage from SSc patient compared to the healthy donor, and the phosphorylation is attenuated after IL-6 blockade, suggesting that increased expression of IL-6 by SSc macrophages accounts, at least in part, for increased signaling. Moreover, our results suggest that activation of SSc macrophages arises from soluble factors, as differentiation of healthy donor monocytes in SSc patient-derived plasma confers the immunophenotype of SSc patient macrophages. For the first time, we show that co-culture of human SSc macrophages with SSc fibroblasts induces fibroblast activation. Collectively, these studies implicate macrophages as likely drivers of fibrosis in SSc and suggest therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.
Connecting metabolism to inflammation: the role of LACC1 in inflammatory diseases
Authors: Li Yingcong , Chandra Vivek , Vijayanand Pandurangan , Kronenberg Mitchell
Published: January 2023

Genome wide association studies (GWAS) identified several single nucleotide polymorphisms (SNPs) in inflammatory diseases. However, it is difficult to determine the gene that GWAS identified SNPs affect and in which cell type(s). RNA-seq analysis of immune cells from 89 healthy humans, led to the identification of the gene laccase domain containing 1 (LACC1), as one of the most promising targets for investigating the role of human gene polymorphisms in inflammatory bowel disease (IBD). LACC1 is a critical immunometabolic mediator in macrophages that drives fatty-acid oxidation. Surprisingly, we found that polymorphisms near LACC1 that have been linked to IBD correlated with significantly lower LACC1 expression in naïve and activated CD4 and CD8 T cells. No difference in expression was observed in macrophages, where it’s highly expressed and has been shown to influence macrophage function. Thus, we hypothesized that these polymorphisms contribute to the genetic risk for IBD by reducing LACC1 expression and, subsequently, inducing T cell dysfunction. As LACC1 plays an important role in macrophage fatty acid metabolism, we tested if this held true in T cells. In our study, knockdown of LACC1 in human CD4 T cells increased mitochondrial mass and lipid droplet formation. This change in metabolism was associated with a significantly reduced production of several cytokines, including IL-2 and IFNg, following anti-CD3/CD28 stimulation. Further, levels of expression of the T cell activation marker CD25 were also attenuated. In summary, our studies provide insights into new mechanisms controlling T cell metabolism and suggest that LACC1 may provide a target for regulating T cell function to prevent inflammatory and autoimmune diseases.
Genetic immunodeficiency and autoimmune disease reveals a WRC-independent role for Hem1 in mTORC2 regulation
Authors: Cook Sarah
Published: January 2023

Inborn errors of immunity (IEI) are genetic disorders that cause life-threatening infections, lymphoproliferation, autoimmunity, or combinations of these features. We describe five patients from four families with a novel IEI characterized by recurrent infections, atopic disease, lymphoproliferation, cytokine overproduction, and autoimmunity. All patients harbored loss-of-function (LOF) variants in the gene NCKAP1L, which encodes the hematopoietic-specific Hem1 protein. Hem1 is a required component of the WAVE regulatory complex (WRC), a critical mediator of Arp2/3-dependent actin polymerization. Three LOF mutations impair interactions between Hem1 and other WRC components, resulting in degradation of the complex. A fourth mutation in Hem1 (M371V) abrogates activation of the WRC by Arf1 GTPase, resulting in WRC deficiency. Loss of Hem1 function disrupts Arp2/3-dependent actin polymerization, immune synapse formation, lamellipodia formation, and migration. Diminished cortical actin barriers in Hem1-deficient cells enable increased degranulation, cytokine secretion, and immune hyperresponsiveness leading to lymphoproliferation. Surprisingly, Hem1 deficiency causes reduced mTORC2-mediated AKT phosphorylation independently of its role in the actin polymerization. Dysregulation of the mTORC2/AKT axis leads to impaired T cell proliferation and effector cytokine production upon T cell activation. Our data show that Hem1 governs two key regulatory complexes, the WRC and mTORC2, and provide a cellular explanation for the combined immunodeficiency and immune hyperresponsiveness observed in Hem1-deficient patients. This research was supported by the Intramural Research Program of the NIH, NIAID.
Intestinal commensal bacteria promote AT2 self-renewal and AT1 differentiation in an IL-22 dependent fashion and prepare the newborn to fight potentially fatal respiratory pathogens
Authors: Nadeem Amraha , Deshmukh Hitesh , Gray Jerilyn , Wang Timothy
Published: January 2023

We demonstrated that disruption of intestinal commensal bacteria with antibiotics (ABX) severely diminished the levels of cytokine interleukin (IL)-22 in the newborn lung and rendered the ABX-exposed newborn mice susceptible post challenge with Streptococcus pneumoniae (S. pneumoniae). These experimental data suggest that colonization by intestinal commensal bacteria is vital for newborn’s lung defense against respiratory pathogens. Pregnant mouse dams were treated with ABX, and newborn mice were challenged with intratracheal S. pneumoniae (on postnatal day 5). Lungs were harvested and stained. AT2 cells and fibroblast harvested from ABX-treated or ABX-free newborn mice were cultured in matrigel to generate alveolar organoids. After harvest, the organoids were stained. To test the role of IL-22 signaling, Il22rafl/fl mice were bred with SftpcCreERT2 mice. The progeny was treated with tamoxifen and then challenged with S. pneumoniae. We found increased alveolar injury, loss of AT1 cells and reduced the frequency of proliferating AT2 cells in ABX-treated newborn mice compared to ABX-free newborn mice post-challenge with S. pneumoniae. Reconstitution of intestinal commensal bacteria or treatment with recombinant IL-22 restored the numbers of proliferating AT2 cells, reduced the injury score, and improved survival in ABX-treated newborn mice. Organoids derived ABX-exposed newborns demonstrated reduced growth and diminished AT1 differentiation. Using a developmentally appropriate and clinically relevant model, we report that intestinal commensal bacteria promote AT2 self-renewal and AT1 differentiation in an IL-22 dependent fashion, thus preparing the newborn to fight potentially fatal respiratory pathogens.
Cytomegalovirus coinfection is associated with increased vascular-homing CD57+ CD4 T cells in HIV infection
Authors: Freeman Michael L , Chen Bonnie , Morris Stephen R , Panigrahi Soumya , Michaelson Gillian M , Wyrick Jonathan M , Komissarov Alexey A , Potashnikova Daria , Lebedeva Anna , Younes Souheil Antoine , Harth Karem , Kashyap Vikram S , Vasilieva Elena , Margolis Leonid , Zidar David A , Sieg Scott F , Shive Carey L , Funderburg Nicholas T , Gianella Sara , Lederman Michael M
Published: January 2023

Cytotoxic CD4 T cells are linked to cardiovascular morbidities and accumulate in both human immunodeficiency virus (HIV) and cytomegalovirus (CMV) infections, both of which are associated with increased risk of cardiovascular disease. Here we identify CMV coinfection as a major driver of the cytotoxic phenotype – characterized by elevated CD57 expression and reduced CD28 expression – in circulating CD4 T cells from people living with HIV infection (PLWH). We find that CD57+ CD4 T cells express high levels of the costimulatory receptor CD2 and that CD2/LFA-3 costimulation results in a more robust and polyfunctional effector response to T cell receptor (TCR) signals, compared to CD28-mediated costimulation. CD57+ CD4 T cells also express the vascular endothelium-homing receptor CX3CR1 and migrate toward CX3CL1-expressing endothelial cells in vitro. IL-15 promotes the cytotoxic phenotype, elevates CX3CR1 expression, and enhances the trafficking of CD57+ CD4 T cells, and may therefore be important in linking these cells to cardiovascular complications. Finally, we demonstrate the presence of CD57+ CD4 T cells and expression of IL-15, CX3CL1, and LFA-3 in atherosclerotic plaque tissues from HIV-uninfected donors. Our findings are consistent with a model in which cytotoxic CD4 T cells contribute to cardiovascular disease in HIV/CMV coinfection and in atherosclerosis via CX3CR1-mediated trafficking and CD2-mediated costimulation, and may help bridge the gap in understanding how CMV infection and immunity are linked to increased cardiovascular risk in PLWH.
Defects in the Macrophage Galactose-type Lectin Pathway due to HIV Infection
Authors: Bharaj Preeti , Lisinicchia Joshua , Naqvi Kubra Fatima , Fan Xiuzhen , Huante Matthew B. , Chahuan Sadhana , Martinez Yazmin , Endsley Mark , Endsley Janice , Gelman Benjamin
Published: January 2023

In the era of highly active antiretroviral therapy, HIV infection still produces 800,000 deaths and 1.2 million new infections per year. People living with HIV (PLWH) are vulnerable to several co-morbidities and co-infections, due to depletion of CD4+T cells and other incompletely characterized immune disturbances. To address the mechanisms involved, lung mRNAs from humanized mice co-infected with HIV and Mycobacterium tuberculosis (Mtb) were screened. Macrophage Galactose-type Lectin (MGL) was differentially regulated in co-infected animals. When MGL protein was quantified in biobanked frozen human lung specimens from 47 HIV-infected decedents, it was lower in the lung specimens with actively replicating HIV-1, as compared to those lacking detectable viral load. Lung MGL levels were negatively correlated with HIV RNA copies in the lung, and in the spleen, which is a key lymphoid organ that influences systemic immunity. Peripheral blood leukocytes from HIV infected patients showed that MGL activation was abnormal in those cells; comparable results were obtained in cultured HIV-infected macrophages. Molecular silencing of MGL in human monocyte-derived macrophages in vitro produced increased growth of Mtb, suggesting that lower MGL expression in PLWH could compromise their innate immunity to opportunistic pathogens such as Mtb. Macrophage MGL is a novel target to elucidate how HIV infection disrupts innate immunity and increases vulnerability to tuberculosis.
A host innate immune circuit licenses downregulation of Salmonella flagellin expression inside macrophages
Authors: Akhade Ajay Suresh , Atif Shaikh M , Lakshmi Bhavna S , Dikshit Neha , Qadri Ayub , Subramanian Naeha
Published: January 2023

While the effect of bacterial molecules on host immune system is well studied, how host factors affect expression of bacterial molecules is not understood in great detail. Inflammasomes are instrumental in detection and clearance of bacterial pathogens, but little is known about whether there is an active cross-talk between the host sensing mechanism and the expression of stimulatory ligands by the pathogen. We used Salmonella-macrophage infection model to assess the impact of inflammasome activation on the expression of flagellin as a candidate Salmonella protein. We discovered that early during infection, host lysophospholipids released upon rapid NLRC4-mediated pyroptosis of macrophages increase flagellin expression by extracellular bacteria which leads to even greater pyroptosis when these bacteria infect healthy macrophages, thus facilitating NLRC4-mediated detection and clearance of Salmonella. A TLR-dependent host response, however, later inhibits expression of NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids pool that permits downregulation of flagellin by intracellular Salmonella. Our findings reveal a novel mode of host-pathogen cross-talk wherein expression of a bacterium-derived inflammasome ligand is temporally controlled by the very process of inflammasome activation, and identify a host mechanism that controls switching of Salmonella to a flagellin-low phenotype inside macrophages with important implications for bacterial pathogenesis and immunity. Future investigations will utilize dual RNA-Seq studies to understand how Salmonella gene expression networks rewire to adapt to different host intracellular microenvironments.
The role of novel E3 ubiquitin ligase in the regulation of TLR3 signaling pathway
Authors: Lin You-Sheng , Chang Yung-Chi , Lai Ting-Yu , Lee Chih-Yuan , Chuang Tsung-Hsien , Hsu Li-Chung
Published: January 2023

Toll-like receptors (TLRs) are critical players in the host’s defense against infection by recognizing pathogen-associated molecular patterns (PAMPs) derived from microbes, and subsequently induce inflammatory responses, which eventually eliminate pathogens and repair damage tissues. However, excessive inflammation is detrimental to the host, and has been associated with the pathogenesis of various inflammatory and autoimmune diseases. Ubiquitination is a crucial strategy to alter protein function, expression, or cellular localization at the post-translational level, and has been shown to participate in the regulation of innate immune responses. We previously found that a novel E3 ubiquitin ligase was induced upon endosomal TLRs (TLR3/7/9) activation. In this project, we demonstrate that this E3 ubiquitin ligase negatively regulates TLR3-driven immune responses, which is dependent on its ubiquitin ligase activity. This novel E3 ubiquitin ligase controls TLR3 trafficking from the endosomes to the lysosomes for degradation. In addition, this E3 ubiquitin ligase interacts with TLR3 and promotes the ubiquitination of TLR3. We also found that lysosomal activity and intracellular pH value were altered in macrophages lacking this E3 ubiquitin ligase after poly(I:C) stimulation. Furthermore, we showed that mice with this E3 ubiquitin ligase deficiency were resistant to encephalomyocarditis virus (EMCV) infection due to enhanced type I interferon production. Our study together reveals this novel E3 ubiquitin ligase as a negative regulator of TLR3-induced inflammatory responses and proposes a novel mechanism for the termination of TLR3 signaling and immune response.
DNA damage responses modulate macrophage transcriptional networks during microbial infection
Authors: Morales Abigail
Published: January 2023

In response to inflammatory stimuli, macrophages integrate myriad signaling pathways that influence the overall response to the pathogenic threat. Specifically, signals emanating from pattern recognition receptors (PRRs) activate several transcriptional networks that promote the expression of inflammatory mediators. Among the transcription factors activated downstream of PRR engagement is NF-κB, which promotes the production of pro-inflammatory cytokines and chemokines. Previous work has established that NF-κB is also activated downstream of DNA damage. In contrast to NF-κB activation following PRR engagement, this DNA damage-elicited signaling cascade originates in the nucleus and depends, in part, on Serine 85 phosphorylation of IKK regulatory subunit NEMO. During infection, macrophages produce genotoxic intermediates that are highly microbicidal. Recent work demonstrated that in macrophages, these agents damage macrophage DNA. We hypothesize that this DNA damage may modulate NF-κB activation downstream of PRR engagement. Indeed, we find that in activated macrophages, NEMO is phosphorylated at Serine 85, suggesting DNA damage-elicited NF-κB activation. Though previous reports have established that ATM is the kinase responsible for this signaling event, our data suggests that in macrophages, optimal NEMO S85 phosphorylation depends on the related DNA damage response kinase DNA-PKcs. Indeed, in the absence of functional DNA-PKcs, inflammatory cytokine production is markedly reduced in macrophages after exposure to bacterial stimuli. These findings underscore a previously underappreciated role for DNA-PKcs in modulating macrophage transcriptional networks during innate immune responses to infection.
Identifying critical immune effectors of the dermal wound healing response by innate cells using microporous-annealed-particle (MAP) hydrogel
Authors: Lempke Samantha of , Roosa Colleen , Griffin Donald , Ewald Sarah
Published: January 2023

The wound healing response is critical to maintain tissue function after injury. Inflammation is needed to sterilize the wound site, but this results in scar formation and impaired tissue function. Topical anti-microbial use, fetal wound healing, and limited studies in germ-free animals suggest that the degree of microbial contamination at a wound site positively correlates with scar formation and recovery time. However, tools to identify the molecular signals and cell types that alter tissue repair verses have been limited. Here we test the hypothesis that Danger-Associated-Molecular Patterns (DAMPs) released during sterile injury guide a fundamentally distinct wound healing response than Pathogen-Associated-Molecular-Patterns (PAMPs) using a novel microporous annealed particle hydrogel (MAP-gel). MAP-gel is an immunologically inert, injectable tissue matrix that is easily linked to a broad range of small signaling molecules. We show that subcutaneous injection of MAP-gel is a robust platform to identify innate immune signaling molecules and infiltrating cell types that control wound healing. Conjugating the model PAMP, LPS, to MAP-gel is sufficient to induce a fibrotic encapsulation response and alter infiltrating immune cell populations. To identify the candidate modulators, proteins enriched in MAP-gels isolated from non-inflammatory or LPS-driven inflammatory responses were identified by LC-MS. These candidates are being conjugated directly to MAP-gel to identify immune effectors that promote wound healing with the absence of fibrotic encapsulation. The modified MAP-hydrogel will be used to gain insight into the molecular mechanisms behind scaring and the unique role DAMPs play in tissue resolution.
Macrophages activate a MLK3/MEK signaling axis to remove pore forming toxins by microvesicle shedding
Authors: Ray Sucharit , Keyel Peter A
Published: January 2023

The innate immune system is critical to promoting host survival during bacterial necrotizing soft tissue infections (NSTIs). NSTI causing pathogens like Streptococcus pyogenes and Clostridium perfringens use pore-forming toxins to kill cells, including macrophages. Toxins cause rapid accumulation of Ca2+ and ceramide at the site of damage, which triggers repair responses. Ca2+ sensitive repair proteins like Annexins may clog the injured site and are spontaneously shed with toxin pores on microvesicles. However, the molecular mechanism orchestrating this repair is unknown. Pore formation activates sphingomyelinases, which generate ceramide. Ceramide signaling may promote repair, potentially via the Mixed Lineage Kinase 3 (MLK3) Mitogen Activated Protein Kinase (MAPK) pathway. Long term toxin exposure activates stress responses via the MAPK pathway but the short-term signaling during repair is unknown. We hypothesized that MLK3 activates a pro-survival kinase cascade (MEK/ERK) to activate membrane repair by microvesicle shedding in macrophages. We tested cell viability and repair responses to toxins after blocking proteins in the MAPK pathways using inhibitors or siRNA in mouse bone marrow derived macrophages and cell lines. We found MLK3 or MEK inhibition significantly reduced toxin resistance. Protective MLK3/MEK activity was downstream of Ca2+ and ceramide signaling. Inhibiting this axis reduced ANXA1/6 translocation to the site of membrane damage and decreased microvesicle shedding. Overall, these findings suggest that a MLK3/MEK axis protects macrophages from pore-forming toxins. Modulation of this axis could promote resistance to NSTIs.
Therapeutic targeting of intracellular Toll-like and interleukin-1/18 receptor (TIR) resistance domain containing proteins for protection against infection, inflammation and disease
Authors: Snyder Greg A , Lai Wendy , Brown Lindsey , Blanco Jorge , Beadenkpf Robert , wang Yajing , Vogel Stefanie , Shirey Kari Ann
Published: January 2023

TIR domain containing proteins are important immune associated proteins shared among Toll-like and interleukin-1/18 receptor family members. In a recent discovery select bacterial, plant and human TIR proteins exhibit enzymatic activity in binding and processing nicotinamide adenine dinucleotide (NAD+). Based on their abilities to facilitate signaling across biology, we hypothesize that TIR proteins represent unique therapeutic targets for modulating infection, inflammation and disease. Our previous structural studies of bacterial-host TIR proteins B. melitensis (TcpB) and uropathogenic E. coli CFT073 (TcpC) with human host TIRAP and MyD88 characterized peptides that negatively regulate signaling and infection. From these studies we identified interactions that are at or near reported biological TIR protein interfaces. In particular, we identified a functionally important motif found conserved on the C helix of most bacterial, human host and NAD+ consuming TIR proteins. As proof of concept for select targeting of TIR proteins and this region in particular we have used the TLR4 antagonist, TAK-242, which selectively binds within this motif. Treatment with TAK-242 or TLR4-C747S blocks LPS signaling. Additionally, TAK-242 protected mice from lethal influenza challenge similar to an extracellular TLR4 antagonist, Eritoran. Bioinformatic analysis of the region targeted by TAK242 show that it is located within the WxxxE structural motif identified to be important for protecting against microtubule destabilization and includes a catalytically essential glutamic acid (E) residue conserved among nearly all NAD+ consuming TIR proteins. These studies provide a framework for future studies targeting TIR protein function.
Physiologic Media Alters Macrophage Phenotype and Activation State
Authors: Bader Jackie E , Rathmell Jeffrey C
Published: January 2023

Traditional cell culture media such as RPMI is consistently used in in-vitro studies in order to inform in-vivo translation. However traditional media poorly simulates physiological conditions which can lead to inaccurate interpretations of data and failed in-vivo applications. The recent development of human plasma like media (HPLM) mirrors physiologic conditions with more realistic levels of glucose, amino acids and other nutrients. We investigated bone marrow derived macrophage phenotype and activation in either RPMI or HPLM to better understand the effects of physiologic conditions on macrophages behavior. Classically activated M1 (100ng/mL LPS + 10ng/mL IFNγ) or alternatively activated M2 (20ng/mL IL-4) were investigated for metabolic activity and behavior. Macrophages cultured in HPLM exhibit a unique phenotype compared to RPMI with increased iNOS expression in response to LPS treatment and reduced CD206 after treatment with IL-4. These data suggest physiologic media can alter the behavior and function of macrophages in-vitro. Following more investigation, these unique activation states in physiologic conditions may uncover differential metabolic requirements and behavior not previously seen in traditional culture media.
Lactoperoxidase-generated hypothiocyanite efficiently kills Streptococcus pneumoniae
Authors: Rada Balazs , Gingerich Aaron , Doja Fayhaa , Thomason Rachel , Toth Eszter , Bradshaw Jessica L , McDaniel Larry S
Published: January 2023

Streptococcus pneumoniae (pneumococcus) infections affect millions of people worldwide, cause serious mortality and represent a major economic burden. Airway epithelial cells, the primary responders to pneumococcal infection, orchestrate an extracellular antimicrobial system consisting of lactoperoxidase, thiocyanate anion and hydrogen peroxide (H2O2). Lactoperoxidase oxidizes thiocyanate using H2O2 into the final product hypothiocyanite. While hypothiocyanite has known antimicrobial effects against a wide range of microorganisms, its effect on pneumococcus has never been studied. To test this, pneumococci were exposed to hypothiocyanite generated enzymatically in a cell-free in vitro system. Bacterial killing was determined by colony forming units on agar plates while bacteriostatic effects were measured by changes in optical density of liquid cultures. We demonstrate hypothiocyanite exerted robust killing of several pneumococcal strains. Nonencapsulated pneumococcal mutants were killed to the same extent as their parental strains, indicating no role of the capsule in protection against hypothiocyanite. Catalase, an H2O2 scavenger, inhibited pneumococcal killing by hypothiocyanite under all circumstances. Interestingly, a pneumococcal mutant deficient in pyruvate oxidase, the main bacterial H2O2 source, had enhanced susceptibility to hypothiocyanite indicating the role of this gene in partial protection of the bacterium against this oxidizing agent. Overall, numerous pneumococcal strains were found to be susceptible to lactoperoxidase-generated hypothiocyanite in vitro, which presents an opportunity to explore hypothiocyanite as a potential novel anti-pneumococcal therapy.
Cholesterol-dependent cytolysins promote immune evasion by clustering host membrane lipids
Authors: Thapa Roshan , Keyel Peter A
Published: January 2023

Pathogens causing necrotizing soft tissue infections, such as Streptococcus pyogenes and Clostridium perfringens, use cholesterol dependent cytolysins (CDCs) to evade immune responses by blocking pro-inflammatory TNFα production in macrophages. This evasion may be an unintended consequence of membrane repair responses to the CDC. CDCs trigger lipid-dependent sequestration and shedding of CDC pores on microvesicles. However, the molecular mechanisms by which CDCs exploit membrane repair to disrupt immune signaling is unknown. One target may be cholesterol-rich microdomains (CRMs) because they are focal points for toxin binding and immune signaling. We hypothesized that when CDCs oligomerize, they cluster membrane lipids in CRMs, along with immune receptors, into microscale assemblies that are spontaneously shed as microvesicles. We tested this hypothesis by artificially clustering sphingolipids using the sphingolipid-binding protein Ostreolysin A (OlyA), fused with tandem FKBP dimerization domains. We challenged murine bone marrow derived macrophages with and without FKBP homodimerizer and OlyA prior to LPS challenge and measured intracellular TNFα by flow cytometry. We found that oligomerization reduced OlyA binding, consistent with shedding. OlyA oligomerization further suppressed TNFα production, suggesting that microscale lipid assemblies interfere with cellular signaling. Overall, these results suggest that one cost to repair by shedding toxins on microvesicles is the loss of cellular signaling needed to promote inflammation. This may represent one strategy CDC-producing pathogens use to promote immune evasion and cause disease.
BRAF-V600E causes intrinsic hyper-responsiveness of dendritic cells to TLR4 stimulus
Authors: Minichino Danielle E , Behrens Edward M
Published: January 2023

Background

The origin of inflammation in Langerhans Cell Histiocytosis (LCH), an inflammatory disease of dendritic cells (DCs) in children, remains elusive. Over 85% of patients harbor somatic, gain-of-function mutations in the Ras-ERK signaling pathway in DCs, which leads to hyper-pERK activity. We hypothesize that DCs with hyper-pERK signaling are intrinsically hyper-responsive to TLR stimuli, resulting in increased levels of cytokines that contribute to LCH etiology.

Methods

To determine the TLR response in hyper-pERK DCs, we generated bone marrow derived DCs (BMDCs) from wild-type and CD11c-Cre:BRAF-V600E flox mice (V600E-DCs). Cells were pre-treated with or without a V600E-inhibitor and stimulated with TLR ligands. Cytokine levels were measured by ELISA and qPCR. TLR levels were measured by qPCR.

Results

V600E-DCs secreted increased LPS-induced TNFa and IL-6 compared to wild type, which was reversible with acute inhibition of V600E. Intriguingly, this was despite V600E-DCs having decreased tnfa, il-6, and tlr4 transcripts. Osteoponton (OPN) secretion was irreversibly increased in V600E-DCs both at baseline and after stimulation with LPS, CpG, and Poly(I:C); however, its gene, spp1, was not increased.

Conclusions

DCs harboring the BRAF-V600E allele have a reversible increase in LPS induced inflammatory cytokine expression, suggesting that increased pERK directly caused a hyper-response to TLR4 stimulation. The irreversible increase in OPN secretion suggests a permanent defect in V600E-DCs independent of pERK levels at time of stimulation. These data taken together suggest that the increased pool of pERK in V600E-DCs contributes to a complex disruption of cytokine secretion that may play a role in LCH etiology.
Lack of PD-1 suppressed the growth of E. multilocularis in mice
Authors: Lin Renyong , Li Zhide , Zhang Chuanshan
Published: January 2023

The cestode Echinococcus multilocularis (E. multilocularis) infection, a serious health problem worldwide, causes alveolar echinococcosis (AE), a tumor-like disease predominantly located in the liver. Until now, there have been few studies to explore how PD-1 contributes to parasite’s escape from immune attack and how it might be reversed. In this study, we found that liver PD-1 expression was significantly enhanced in AE patients. Compared to E. multilocularis infected wild-type mice, we found lesion’s weights in the liver were significantly deceased (101.20±43.12mg vs 393.70±123.5 mg, P<0.05) and lesion’s volumes were significantly lower (82.17±28.43 mm3 vs 429.30±148.30 mm3, P<0.05) in E. multilocularis infected PD-1 knockdown mice. This study demonstrates blocking PD-1 may suppress the growth of E. multulocularis in mice, and represent a possible approach to E. multilocularis immunotherapy.