Biology subjects

Boothby, M. R.

Publications and source records attributed to Boothby, M. R..

9 recordsLinked to original sources

bioRxiv2025

The interferon response signature links to increased expression of transposable elements in neutrophils of systemic lupus erythematosus patients

BackgroundSystemic lupus erythematosus (SLE) is a spontaneous systemic auto-immune condition for which the inciting factors and genetic basis are generally unknown. Although heterogeneous in its manifestations and severity, SLE involves chronic inflammation along with sustained autoantibody production. The root causes and pathophysiology of the inflammation and breaches of tolerance are incompletely understood, but neutrophils are thought to be important elements of the pathophysiology. Type I interferons (IFN) in the bloodstream and an IFN-stimulated gene (ISG) signature in circulating leukocytes, including neutrophils, are common features in many patients. Earlier work has provided evidence of increased levels of transcripts derived from transposable elements (TEs) in peripheral blood cells of SLE patients. Using six leukocyte types, including neutrophils, we tested the correlation of TE expression with disease severity and explored the relationships between increased ISG and TE expression with attention to the genomic locations of the expressed TEs. ResultsWe reanalysed previously published data from neutrophils and other leukocytes of SLE patients sub-divided into ISG-high (termed IFNpos, n=12) and ISG-low (termed IFNneg, n=11) patients in the original study, examining RNA-seq data from B and T lymphocytes, conventional and plasmacytoid dendritic cells (DC), monocytes and PMN of IFNpos and IFNneg SLE patients compared to healthy controls. SLE patients pre-stratified as IFNneg showed no significant increase in TE expression. All IFNpos cell types had similar amounts of total TE-encoded RNA, but among the 6 cell types, PMN had the highest number of differentially expressed TEs and ISGs in IFNpos SLE patients compared to healthy controls. There was a strong correlation between expression of several specific TE families and disease activity assessed at the time of the visit. Most upregulated TEs ([~]80%) were present in introns of upregulated genes, and [~]67% of these were ISGs. By mapping expressed TEs in ISGs, we found that high intronic TE expression correlated strongly with increased ISG expression as well as with splicing alterations in annotated exons flanking expressed TEs. Consistent with autonomous TE expression, upregulated TEs were also observed at intergenic sites distant from annotated genes, perhaps due to weakening of heterochromatin integrity. ConclusionsOur findings show a strong association and suggest mechanistic relationships between increased TE expression and IFN responses in multiple types of leukocytes centrally involved in SLE pathogenesis. Although limited by short-read RNA-seq technology, our analyses support selective upregulation of some TEs independent from the regulation of conventional genes, concurrent with many intron-localized TEs whose expression tracks with ISGs. The data emphasize the need for long-reads sequencing to understand the causes and consequences of high TE expression in SLE and other autoimmune/inflammatory disorders. Important questions include whether TE expression in introns of ISGs and other genes is independently regulated or reflects exonization or partial intron retention, and how frequently it correlates with splicing variations in adjacent exons.

immunology↗

bioRxiv2025

Synthetic auxotrophy reveals metabolic regulation of plasma cell generation, affinity maturation, and cytokine receptor signaling

The efficiencies with which activated B lymphocytes proliferate and develop into antibody (Ab)-secreting plasma cells are critical determinants of adaptive humoral immunity and sustain certain autoimmune diseases. Specific pathways in intermediary metabolism, or their substrate supply, influence lymphocyte differentiation and function. We now show that although stringent restriction of glutamine supply decreases proliferation and differentiation of B cells into plasma cells, glutaminolysis - a major means of metabolism of this amino acid - was only conditionally crucial in B cells and the Ab responses derived from them. Strikingly, Gls, the gene encoding the main glutaminase of lymphocytes, promoted anti-NP Ab responses at the primary and recall phases if either glucose uptake into B cells or pyruvate into their mitochondria was also impaired but otherwise was dispensable. This synthetic auxotrophy, i.e., conditional requirement of glutaminase for processes in addition to survival and proliferation, involved support to a progressive expansion of mitochondrial respiration followed by plasma cell differentiation. Surprisingly, impairment of glutaminase and the mitochondrial pyruvate channel decreased IL-21 stimulation of STAT3 phosphorylation as well as interferon stimulation of STAT1 activation. Together, our findings establish not only a powerful collaboration of metabolic pathways in programming increased respiration and the development of Ab-secreting cells, but also reveal modulation of cytokine receptor signaling by metabolism.

immunology↗

bioRxiv2024

B cell expression of the enzyme PexRAP, an intermediary in ether lipid biosynthesis, promotes antibody responses and germinal center size

The qualities of antibody (Ab) responses provided by B lymphocytes and their plasma cell (PC) descendants are crucial facets of responses to vaccines and microbes. Metabolic processes and products regulate aspects of B cell proliferation and differentiation into germinal center (GC) and PC states as well as Ab diversification. However, there is little information about lymphoid cell-intrinsic functions of enzymes that mediate ether lipid biosynthesis, including a major class of membrane phospholipids. Imaging mass spectrometry (IMS) results had indicated that concentrations of a number of these phospholipids were substantially enhanced in GC compared to the background average in spleens. However, it was not clear if biosynthesis in B cells was a basis for this finding, or whether such cell-intrinsic biosynthesis contributes to B cell physiology or Ab responses. Ether lipid biosynthesis can involve the enzyme PexRAP, the product of the Dhrs7b gene. Using combinations of IMS and immunization experiments in mouse models with inducible Dhrs7b loss-of-function, we now show that B lineage-intrinsic expression of PexRAP promotes the magnitude and affinity maturation of a serological response. Moreover, the data revealed a Dhrs7b-dependent increase in ether phospholipids in primary follicles with a more prominent increase in GC. Mechanistically, PexRAP impacted B cell proliferation via enhanced survival associated with controlling levels of ROS and membrane peroxidation. These findings reveal a vital role of this peroxisomal enzyme in B cell homeostasis and the physiology of humoral immunity.

immunology↗

bioRxiv2024

Regulation of fatty acid delivery to metastases by tumor endothelium

Tumor metastasis, the main cause of death in cancer patients, requires outgrowth of tumor cells after their dissemination and residence in microscopic niches. Nutrient sufficiency is a determinant of such outgrowth1. Fatty acids (FA) can be metabolized by cancer cells for their energetic and anabolic needs but impair the cytotoxicity of T cells in the tumor microenvironment (TME)2, 3, thereby supporting metastatic progression. However, despite the important role of FA in metastatic outgrowth, the regulation of intratumoral FA is poorly understood. In this report, we show that tumor endothelium actively promotes tumor growth and restricts anti-tumor cytolysis by transferring FA into developing metastatic tumors. This process uses transendothelial fatty acid transport via endosome cargo trafficking in a mechanism that requires mTORC1 activity. Thus, tumor burden was significantly reduced upon endothelial-specific targeted deletion of Raptor, a unique component of the mTORC1 complex (RptorECKO). In vivo trafficking of a fluorescent palmitic acid analog to tumor cells and T cells was reduced in RptorECKO lung metastatic tumors, which correlated with improved markers of T cell cytotoxicity. Combination of anti-PD1 with RAD001/everolimus, at a low dose that selectively inhibits mTORC1 in endothelial cells4, impaired FA uptake in T cells and reduced metastatic disease, corresponding to improved anti-tumor immunity. These findings describe a novel mechanism of transendothelial fatty acid transfer into the TME during metastatic outgrowth and highlight a target for future development of therapeutic strategies.

cancer biology↗

bioRxiv2024

The nutrient-sensing Rag-GTPase complex in B cells controls humoral immunity via TFEB/TFE3-dependent mitochondrial fitness

During the humoral immune response, B cells undergo rapid metabolic reprogramming with a high demand for nutrients, which are vital to sustain the formation of the germinal centers (GCs). Rag-GTPases sense amino acid availability to modulate the mechanistic target of rapamycin complex 1 (mTORC1) pathway and suppress transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3), members of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors. However, how Rag-GTPases coordinate amino acid sensing, mTORC1 activation, and TFEB/TFE3 activity in humoral immunity remains undefined. Here, we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs, produce antibodies, and generate plasmablasts in both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically, the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells, which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development, GC formation in Peyers patches and TI humoral immunity, but not TD humoral immunity in the absence of Rag-GTPases. Collectively, our data establish Rag-GTPase-TFEB/TFE3 pathway as an mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells. One sentence summaryRag-GTPases restrain TFEB/TFE3 to prevent abnormal mitophagy, maintain mitochondrial fitness, and support B cell development, Peyers patch germinal center formation, and T-independent humoral immunity.

immunology↗

bioRxiv2023

Plasma cell differentiation, antibody quality, and initial germinal center B cell population depend on glucose influx rate

Antibody secretion into sera, selection for higher affinity BCR, and the generation of higher Ab affinities are important elements of immune response optimization, and a core function of germinal center reactions. B cell proliferation requires nutrients to support the anabolism inherent in clonal expansion. Glucose usage by GC B cells has been reported to contribute little to their energy needs, with questions raised as to whether or not glucose uptake or glycolysis increases in GC B cells compared to their naive precursors. Indeed, metabolism can be highly flexible, such that supply shortage along one pathway may be compensated by increased flux on others. We now show that elimination of the glucose transporter GLUT1 after establishment of a pre-immune B cell repertoire, even after initiation of the GC B cell gene expression program, decreased initial GC B cell population numbers, affinity maturation, and PC outputs. Glucose oxidation was heightened in GC B cells, but this hexose flowed more into the pentose phosphate pathway (PPP), whose activity was important in controlling reactive oxygen (ROS) and ASC production. In modeling how glucose usage by B cells promotes the Ab response, the control of ROS appeared insufficient. Surprisingly, the combination of galactose, which mitigated ROS, with provision of mannose - an efficient precursor to glycosylation - supported robust production of and normal Ab secretion by ASC under glucose-free conditions. Collectively, the findings indicate that GC depend on normal glucose influx, especially in PC production, but reveal an unexpected metabolic flexibility in hexose requirements. KEY POINTSO_LIGlucose influx is critical for GC homeostasis, affinity maturation and the generation of Ab-secreting cells. C_LIO_LIPlasma cell development uses the Pentose Phosphate Pathway, and hexose sugars maintain redox homeostasis. C_LIO_LIPCs can develop and achieve robust Ab secretion in the absence of glucose using a combination of hexose alternatives. C_LI

immunology↗

bioRxiv2023

CXCR2 expression during melanoma tumorigenesis controls transcriptional programs that facilitate tumor growth

BackgroundThough the CXCR2 chemokine receptor is known to play a key role in cancer growth and response to therapy, a direct link between expression of CXCR2 in tumor progenitor cells during induction of tumorigenesis has not been established. MethodsTo characterize the role of CXCR2 during melanoma tumorigenesis, we generated tamoxifen-inducible tyrosinase-promoter driven BrafV600E/Pten-/-/Cxcr2-/- and NRasQ61R/INK4a-/-/Cxcr2-/- melanoma models. In addition, the effects of a CXCR1/CXCR2 antagonist, SX-682, on melanoma tumorigenesis were evaluated in BrafV600E/Pten-/- and NRasQ61R/INK4a-/- mice and in melanoma cell lines. Potential mechanisms by which Cxcr2 affects melanoma tumorigenesis in these murine models were explored using RNAseq, mMCP-counter, ChIPseq, and qRT-PCR; flow cytometry, and reverse phosphoprotein analysis (RPPA). ResultsGenetic loss of Cxcr2 or pharmacological inhibition of CXCR1/CXCR2 during melanoma tumor induction resulted in key changes in gene expression that reduced tumor incidence/growth and increased anti-tumor immunity. Interestingly, after Cxcr2 ablation, Tfcp2l1, a key tumor suppressive transcription factor, was the only gene significantly induced with a log2 fold-change greater than 2 in these three different melanoma models. ConclusionsHere, we provide novel mechanistic insight revealing how loss of Cxcr2 expression/activity in melanoma tumor progenitor cells results in reduced tumor burden and creation of an anti-tumor immune microenvironment. This mechanism entails an increase in expression of the tumor suppressive transcription factor, Tfcp2l1, along with alteration in the expression of genes involved in growth regulation, tumor suppression, stemness, differentiation, and immune modulation. These gene expression changes are coincident with reduction in the activation of key growth regulatory pathways, including AKT and mTOR.

cancer biology↗

bioRxiv2021

Selective pharmaceutical inhibition of PARP14 mitigates allergen-induced IgE and mucus overproduction in a mouse model of pulmonary allergic response

The type 2 cytokines IL-4 and IL-13, which share use of an IL-4 receptor alpha chain and its nuclear induction of the transcription factor STAT6, are crucial in elicitation and maintenance of allergic conditions that include asthma. Prior work has shown a physical and functional association of STAT6 with PARP14, an ADP-ribosyl monotransferase. Moreover, elimination of all PARP14 expression by gene targeting led to altered recall antibody responses and attenuation of ovalbumin-specific allergic lung inflammation with no apparent health issues for mice lacking this protein. However, an unanswered question is whether or not inhibition of the catalytic function has any biological consequence since PARP14 has multiple functional domains apart from the portion that catalyzes ADP-ribosylation. As reported separately, iterative structural analyses and medicinal chemistry fostered the generation of a compound, RBN2759, that is highly selective in its inhibition of PARP14 with negligible impact on other members of the PARP gene family. We show here that administration of this compound to mice previously sensitized to the allergen Alternaria alternata achieved biochemically active levels and altered physiological responses to the antigen. These results show for the first time that in vivo administration of a specific inhibitor of the ADP-ribosyltransferase activity encoded by PARP14 is sufficient to alter biological responses. Specifically, the orally absorbable pharmaceutical compound decreased allergen-induced mucus, blunted the induced increases in circulating IgE, and prevented suppression of IgG2a. We conclude that the catalytic activity can contribute to pathogenesis in allergic processes and propose that other biological endpoints that depend on ADP-ribosylation by PARP14 can be targeted using selective inhibition.

immunology↗

bioRxiv2021

Generalizing about GC (hypoxia): Dys- & Dat-InformaticaComment on Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis

Steadily accumulating evidence supports the concept that the outputs of immune responses are influenced by local nutrient and metabolite conditions or concentrations, as well as by the molecular programming of intermediary metabolism within immune cells. Humoral immunity and germinal center reactions are one setting in which these factors are under active investigation. Hypoxia has been highlighted as one example of how a particular nutrient is distributed in primary and secondary follicles during an antibody response, and how its sensors could impact the qualities of antibody output after immunization. Based on a bio-informatic analysis of mRNA levels in germinal center and other B cells, recently published work challenges the concept that there is any hypoxia or that it has any influence. In this perspective, we perform new analyses of published genomics data to explore potential sources of disparity and elucidate aspects of what on the surface might seem to be conflicting conclusions. In particular, the replicability and variance among data sets derived from different naive as well as germinal center B cells are considered. The results of the investigation highlight several broader issues that merit consideration, especially at a time of heightened focus on scientific reports in the realm of immunity and antibody responses. From one finding of this re-analysis, it is proposed that a standard should be expected in which the relationship of new data sets compared to prior "fingerprints" of cell types should be reported transparently to referees and readers. In light of the strong evidence for diversity in the constituencies within germinal centers elicited by protein immunization, it also is proposed that a core practice should be to avoid overly broad conclusions about germinal centers in general when experimental systems are subject to substantial constraints imposed by technical features.

immunology↗