bluuebunny/crossref_metadata_2025_split huggingface dataset:
Delineate the effects of cannabidiol on type 1 IFN mediated inflammation during HIV infection
Authors: Tomer Shallu , Mu Wenli , Ng Hwee , Wang Li , Wennerberg Wally , Razek Valerie , Martin Heather , Zhen Anjie , Kitchen Scott 
Published: January 2023

Abstract

Methods
THP-1 and HMC3 cells were exposed to CBD/vehicle with and without stimulation with 2’3’-cGAMP (cGAMP), LPS, or PMA/ionomycin. qPCR was carried out to investigate the expression of interferon stimulatory genes (ISGs). RNA-Seq was performed to investigate gene expression in cGAMP-stimulated THP1 cells treated with vehicle/CBD. Western blot was performed with THP1 cells treated with vehicle/CBD then cGAMP to investigate the change of expression of phosphorylated STING (pSTING) and autophagy markers (LC3B, p62). Lastly, humanized mice (Hu mice) were constructed, infected with HIV-1 for 12 wks then treated with vehicle/CBD for 2 wks. Flow cytometry and qPCR were performed to examine changes in T cell activation and IFN-1 signaling in peripheral blood before and after CBD treatment.

Results
We observed significant down regulation of ISGs in stimulated, CBD-treated THP-1 and HMC3 cells. RNA-seq and qPCR confirmatory analysis showed that after stimulation IFN-1 genes were down regulated in CBD group compared to vehicle. We observed significantly higher LC3B/p62 and lower pSTING levels in CBD treated, stimulated cells as compared to vehicle treated, stimulated cells. Lastly, we observed a reduction in PD-1, Tim-3 and ISG MX1 expression in HIV-1 infected Hu mice treated with CBD.

Conclusion
We observed down regulation of ISGs in stimulated CBD treated cells. CBD treatment leads to induction of cellular autophagy, responsible for regulating/degrading pSTING after cGAS-STING pathway activation and lower ISGs as observed in our study. Lastly, we observed reduced ISGs, and decreased activation/exhaustion marker expression on T cells in HIV infected Hu mice after CBD treatment, suggesting the potential of CBD as an immunomodulator.
Supported by grants from NIH/NIDA RFA-DA-20-022
Cholesterol, LXR activation, and myelin uptake in the macrophage response to diverse signals
Authors: DeLong Jonathan Howard , Velazquez Sofia Evelyn , Sun Leonardo Shiuchi , Landreneau Margaret J , Sansing Lauren Hachmann 
Published: January 2023

Abstract

Introduction
Monocyte-derived macrophages contribute to neurotoxic inflammation during hemorrhagic stroke. Cholesterol is an immunosuppressive molecule present at high levels in the brain, primarily as a component of myelin. Uptake of myelin and activation of the transcription factor LXR downstream of myelin and cholesterol uptake limits the macrophage response to LPS. However, the roles of these molecules in macrophage responses to the endogenous signals present during sterile inflammation are poorly understood. The present study seeks to determine what role myelin, cholesterol, and LXR activation play in modifying macrophage responses to diverse signals.

Methods and Results
Murine bone marrow-derived macrophages were treated with myelin, cholesterol, or the LXR agonist T0901317 18h before stimulation with S100A9, IFN-g, TNF-a, IL-4, IL-10, TGF-b, p(I:C), Pam3CSK4, or LPS. Production of TNF-a, IL-6, CCL2, and IL-10 were measured by multiplex ELISA. Transcriptome-wide effects were measured by RNA sequencing. Cholesterol limited inflammatory cytokine production in response to LPS but not to other stimuli. The LXR agonist T0901317 did not suppress cytokine production. Principal component analysis revealed that cholesterol had strong effects on the transcriptomic responses to LPS but not to the other stimulations. Myelin affected the transcriptomic response to all inflammatory stimulations.

Conclusions
Cholesterol limits the macrophage response to LPS, partially through activation of LXR, but does not broadly limit macrophage activation. Uptake of myelin has broader effects than cholesterol, suggesting that non-cholesterol components of myelin modulate the macrophage response.
Supported by grants from NIH (R01 NS097728, T32 HL007950) and AHA (Postdoctoral Fellowship 830877)
Vagus nerve stimulation leads to contraction of the spleen and dampened IFNγ production in response to TLR stimulation
Authors: Nash William T , Tanaka Shinji , Okusa Mark D 
Published: January 2023

Abstract
Vagus nerve stimulation (VNS) generates an anti-inflammatory state and limits the pathology of inflammatory diseases, such as sepsis and acute kidney injury. Interestingly, splenectomy prior to stimulation results in a loss of this protection. Thus, the spleen is a central organ for converting VNS signals into protective anti-inflammation. To better understand the interplay between VNS and the spleen and gain insight into the mechanisms of anti-inflammation, we performed a broad query of the spleen post-VNS. Mice received either electrical stimulation of the left-cervical vagus nerve or sham surgery. Spleens were harvested and assessed for mass, immune cell populations, and splenocyte in vitro cytokine production in response to TLR4, 3, and 9 agonists (LPS, poly I:C, and CpG, respectively). We discovered that by 48 hours post-VNS, spleen mass was reduced and the number of CD45+ cells was significantly decreased compared to sham mice. Analysis of CD45+ cells revealed that many individual immune cell populations were diminished. B cells, T cells, NK cells, dendritic cells, and basophils were all significantly reduced and eosinophils and monocytes showed strong trends towards reduction, nearing significance. In response to TLR agonists, our Luminex analysis revealed a strong impact on TLR4 stimulation, with 12 of the 32 measured cytokines showing a significant reduction in supernatants from VNS samples. TLR3 and TLR9 stimulation also exhibited significant reductions in a subset of the measured cytokines, but only IFNγ exhibited strong signs of reduced production in all TLR stimulation conditions. Thus, VNS has profound impacts in the spleen and some of the observed protection could be due to tighter control of immune cell IFNγ secretion.
Supported by grants from the NIH (R01 DK123248)
The DEAH-box helicase RHAU regulates immunoglobulin class switch recombination
Authors: Thavornwatanayong Thongthai , Zheng Simin , Guillaume Sabine Jean , Vuong Bao Q 
Published: January 2023

Abstract
During a humoral immune response, B cells produce alternative immunoglobulin isotypes (IgG, IgA, IgE) through class switch recombination (CSR). Initiation of CSR requires AID-mediated deamination of switch (S) regions in the immunoglobulin heavy chain (IgH) locus. Although G-quadruplex(G4)-forming S region RNAs bind AID and localize AID to S region DNA sequences, the molecular mechanism that regulates the transition of AID binding from RNA to DNA remains uncharacterized. Highly stable G4 structures necessitate helicases to unwind the G-G hydrogen bonds, which in turn may permit the base-pairing of the S transcript to the complementary DNA sequence in the IgH locus during CSR. An S region RNA pull-down assay identified the RNA Helicase associated with AU-rich element (RHAU), a DEAH-box RNA helicase, as an S transcript binding protein. To examine the role of RHAU in CSR, we genetically deleted a floxed RHAU allele in B cells of mice using CD23-Cre. Conditional deletion of RHAU in B cells reduced CSR in vivo and in vitro to approximately 50% of wild-type controls. This data suggests that RHAU plays an important role in CSR. We hypothesize that RHAU unwinds G4 in S transcripts to facilitate the handoff of AID from G4-RNA to S region DNA during CSR.
Supported by The National Cancer Institute (2U54CA132378) and The National Institute of General Medical Sciences (1SC1GM132035-01)
The plasma cell proteome initiates early in B cell activation in advance of Blimp-1.
Authors: Gaudette Brian T , Allman David 
Published: January 2023

Abstract
Plasma cell (PC) differentiation is a highly regulated process that radically changes the morphology and function of a B lymphocyte to that of a professional protein secretor. It is becoming clear that this process precedes increase in immunoglobulin synthesis rather than reacting to it. We have shown previously that large segments of the PC transcriptome are initiated in an mTORC1-dependent manner prior to both Blimp-1 and Xbp1s expression. Still, the kinetics of induction of the PC proteome remain largely unknown. We performed a whole proteome kinetic analysis of activated Blimp-1-reporter follicular B cells in PC-inductive conditions every 12 hours for three days and observed distinct patterns in induction of the PC proteome. To elucidate transcriptional networks, we performed single cell RNA-seq with the same conditions and correlated transcription factor expression with genes coding for these proteome modules within single cells. We found distinct subgroups within proteome modules corresponding to transcriptional patterns each with a highly correlated transcription factor set. These included robust, transient, Myc-associated factors required for translation initiation; rapid, sustained expression of protein synthesis machinery; and gradual, sustained increases in proteins associated with ER function and secretion. Each of these preceded the induction of Blimp-1 and increase in immunoglobulin protein. Finally, using inducible deletion of Blimp-1 in conjunction with the reporter allele we show that these unique transcriptional and proteomic programs are initiated independently of Blimp-1 expression. Thus, activated B cells prepare for protein secretion by initiating the PC proteome prior to antibody secretion.
Supported by grants from the NIH: R01 AI113545, AI139123, AI154932, R21 AI133998 (PI:Allman) and BTG was supported by training grant T32 CA009140 (PI:Pear)
Function of ATM and MSH2 during nonhomologous end joining in DNA repair
Authors: Sible Emily , Attaway Mary , Fiorica Giuseppe , Choi Jee Eun , Chwat-Edelstein Tzippora , Haughton Lyric , Bedasee Donna , Vuong Bao Q 
Published: January 2023

Abstract
Class switch recombination (CSR) produces secondary immunoglobulin isotypes and requires AID-dependent DNA deamination of intronic switch (S) regions within the immunoglobulin heavy chain gene locus. Non-canonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates DNA breaks that permit recombination between distal S regions. ATM-dependent phosphorylation of AID at serine-38 (pS38-AID) promotes its interaction with APE1, a BER protein, suggesting that ATM regulates CSR through BER. However, pS38-AID may also play a role in MMR during CSR, although the mechanism remains unknown. To examine whether ATM modulates BER- and/or MMR-dependent CSR, ATM−/− mice were bred to mice deficient for the MMR gene MSH2. Surprisingly, the predicted Mendelian frequencies of ATM−/− MSH2−/− adult mice were not observed. To obtain ATM- and MSH2-deficient B cells, ATM was conditionally deleted on an MSH2−/− background using a floxed ATM allele [ATMF] and B cell-specific Cre recombinase expression (CD23-cre) to generate a deleted ATM allele (ATMD). As compared to the ATMD/D and MSH2−/− mice and B cells, the ATMD/D MSH2−/− mice and B cells display a reduced CSR phenotype. Interestingly, Sμ-Sγ1 junctions from ATMD/D MSH2−/− B cells that were induced to switch to IgG1 in vitro revealed a significant loss of blunt end joins and an increase in insertions as compared to wildtype, ATMD/D, or MSH2−/− B cells. This data suggests that the absence of both ATM and MSH2 blocks NHEJ and leads to inefficient end joining and the reduced CSR. We identify complementary roles for ATM and MSH2 in NHEJ and A-EJ during CSR and propose a model whereby ATM and MSH2 function cooperatively to regulate end-joining during CSR through pS38-AID.
This work was supported by The National Institute on Minority Health and Health Disparities (5G12MD007603), The National Cancer Institute (2U54CA132378), and The National Institute of General Medical Sciences (1SC1GM132035-01).
Defining the cis- and trans-regulatory architecture for human PD-1 in T Follicular Helper Cells
Authors: Powell Michael Duane , Rose James R , Neeld Dennis , Rahmberg Andrew R , Scharer Christopher D , Boss Jeremy M 
Published: January 2023

Abstract
The surface immune checkpoint inhibitor Programed Death-1 (PD-1), encoded by PDCD1, is expressed throughout the immune system. Notably, PD-1 expression is amongst its highest in a specialized subset of CD4+ T follicular helper (TFH) cells. TFH cells primarily function by engaging with cognate B cells, facilitating the generation of high affinity vaccine- and pathogen-specific antibodies. Despite the proven clinical benefit of therapies targeting the PD-1 pathway, very little is known regarding the mechanisms by which human PDCD1 is transcriptionally regulated in humans. Here, we seek to elucidate both the cis- and trans-regulatory mechanisms that govern PDCD1 expression in TFH cells. Human TFH cells were sourced from discarded tonsil tissue, circulating blood, or through an ex vivo differentiation model to generate ‘TFH-like’ cells. RNA-seq analysis revealed that ex vivo-derived ‘TFH-like’ cells resembled bona-fide in vivo TFH cells, including induction of PDCD1. Furthermore, ATAC-sequencing experiments identified 8 conserved regions in the PDCD1 locus that exhibited altered accessibility in TFH cells. These cis-regulatory elements were determined to have distinct functions in controlling PDCD1 expression. Additionally, bioinformatic analysis revealed a core TFH gene program, implicating the transcription factors BCL-6, ASCL2, TOX, TOX2, and AIOLOS as regulators of PDCD1 and TFH fate. Taken together, these findings uncover the molecular mechanisms that control PDCD1 expression, informing the design of future therapies aimed at manipulating PD-1.
Supported by National Institute of Health grants: RO1 AI113021 to J.M.B, F32 AI161857 to M.D.P
A temporal switch in T follicular helper cells controls the output of the GC response to influenza
Authors: Arroyo Nicole M , Bachus Holly , Papillion Amber , Rosenberg Alexander F , Bradley John Edwin , Leon-Ruiz Beatriz , Ballesteros-Tato Andre 
Published: January 2023

Abstract
T follicular helper (Tfh) cells are a specific subset of CD4 T cells that localize in the B cell follicle and are essential for the Germinal Center (GC) response. Under polarizing environments, Tfh cells produce effector cytokines, including IFN-γ and IL-4. Beyond promoting class switching, the role played by the different subsets of cytokine-producing Tfh cells remains largely unexplored. Using an influenza infection model, we show here that Tfh cells switch from producing IFN-γ early after infection to mainly producing IL-4 at later time points. Importantly, IFN-γ and IL-4-producing Tfh cells differently control the GC B cell response to influenza. Whereas IL-4-producing Tfh cells help GC maintenance late after infection, interaction with IFN-γ producing Tfh cells skews the GC B cell response towards the memory differentiation pathway early after infection. Consequently, lung-memory B cells fail to differentiate in the absence of IFN-γ producing Tfh cells. Collectively, our results support a model in which temporary changes in cytokine production by Tfh cells dynamically control the outcome of the GC response. Hence, our data provide new insights into GC fate decisions following influenza infection.
Supported by grants from NIH (R56 AI162698, R01 AI110480)
Human Circulating Plasmablasts Are Not Blasting: A Probable Misnomer
Authors: Nguyen Doan C , Saney Celia , Woodruff Matthew C , Sanz Ignacio , Lee F Eun-Hyung 
Published: January 2023

Abstract
Following infection or vaccination, antibody-secreting cells (ASC), also called plasmablasts, appear in circulation transiently; a small fraction of them then migrate to the spleen or bone marrow (BM) to mature into long-lived plasma cells (LLPC). While LLPC, by definition, are quiescent or non-dividing, early-minted blood ASC were mostly thought to be “blasting” or proliferative. In this study, we show &lt;3% of LLPC are Ki-67+ with none taking up BrdU. In contrast, nearly all (&gt;95%) nascent blood ASC express Ki-67 but only 10–15% actually incorporate BrdU. Due to the limitations of traditional flow cytometry, we integrated an optofluidic technology capable of single-cell visualization into a unique ASC survival system to directly evaluate both cell division status and ongoing antibody secretion. In agreement with the literature, LLPC displayed no proliferative potential. Surprisingly, only 11–13% of the blood ASC displaying ongoing antibody secretion had divided by day 10–14. Thus, while newly-generated ASC in the blood have recently undergone proliferation, the majority have exited the mitotic cell cycle and are not actively “blasting”. As they further mature into terminal LLPC, they undergo complete cell cycle arrest. Therefore, the term “plasmablast” may actually be a misnomer.
Supported by NIH/NIAID 1R01AI121252, 1P01AI125180, U01AI141993, U54CA260563, U19AI110483, T32AI070081 and the Bill & Melinda Gates Foundation Grant INV-002351.
Modulating Immunoglobulin Secretion of a Single Human Plasma Cell by Glycolysis Inhibition
Authors: Nguyen Doan C , Woodruff Matthew C , Sanz Ignacio , Lee F Eun-Hyung 
Published: January 2023

Abstract
Early-minted antibody-secreting cells (ASC) undergo further maturation to become long-lived plasma cells (LLPC) in the bone marrow (BM) microniche to afford life-long humoral protection. As terminally-differentiated cells, ASC are thought to be antibody factories that secrete immunoglobulins (Ig) constitutively. This model suggests that Ig secretion is linked with survival and loss of Ig secretion is associated with apoptosis. Here, we show that the Ig secretory function of human ASC is inhibited after treatment with an GAPDH inhibitor as measured by IgG Elispot assays and by direct visualization of IgG secretion from a single ASC. Although Ig secretion ceases quickly with the initial treatment, ASC promptly recover secretory function upon removal of the inhibitor. The modulation of Ig secretory function was directly visualized in both human early-minted blood ASC and BM LLPC by single-cell analysis using an optofluidic technology. Thus, we conclude that ASC Ig secretion is not constitutive but a dynamic process which can be modulated by metabolic processes such as glycolysis.
Supported by NIH/NIAID 1R01AI121252, 1P01AI125180, U01AI141993, U54CA260563, U19AI110483, T32AI070081 and the Bill & Melinda Gates Foundation Grant INV-002351.
B-cell-intrinsic MHCII signaling limits bacteremia and systemic invasion by Citrobacter rodentium
Authors: Roland Mary Melissa , Hall Nia , Peacock Tori , Mohammed Ahmed , Jolly Amy , Kubinak Jason L 
Published: January 2023

Abstract
B-cell-intrinsic major histocompatibility complex class II (MHCII) antigen presentation is central to the development of T-cell-dependent antibody responses. However, the physiological significance of B-cell-intrinsic MHCII signaling during homeostatic conditions and infection are unknown. Here, using a B-cell-intrinsic MHCII knockout mouse model, we demonstrate that B-cell-intrinsic MHCII signaling is important for the development of systemic IgG1 and mucosal, high-affinity IgA responses. We also show that the conditional ablation of MHCII signaling on B cells results in a change in the microbiota composition, exhibiting a significant increase in the mucin-degrading bacteria Akkermansia muciniphila. This change in the microbiome is also associated with elevated bacterial dissemination from the gut into the systemic compartment under homeostatic conditions. Additionally, using a Citrobacter rodentium model of acute gastrointestinal infection, we also demonstrate the relevance of B-cell-intrinsic MHCII signaling in limiting invasion of the systemic compartment by an opportunistic enteric pathogen. Results from these experiments demonstrate that B-cell-intrinsic MHCII signaling is an important factor minimizing the physiological cost of microbial colonization of the gut.
Supported by NIH R21AI142409, R01AI155887
IgA B cell receptor signaling protects from FasL counterselection during germinal center reaction in Peyer’s patches and shapes humoral mucosal response.
Authors: Reboldi Andrea , Raso Fiona , Berthellete Alyssa , Sagadiev Sara , Moses Stephanie , Acharya Mridu , Barton Gregory , Muppidi Jagan , Marshak-Rothstein Ann 
Published: January 2023

Abstract
Immunoglobulin A (IgA) is the main antibody isotype found in the intestine: deficiency in IgA alters microbiome composition, leads to increased susceptibility to enteric pathogens and reduces tissue response to oral vaccine. IgA can bind multiple unrelated bacterial taxa, but mechanistic insights into the generation of such cross-species reactivity are lacking. IgA+ B cells arise during germinal center (GC) reaction in Peyer’s patches: however, the role of IgA B cell receptor (BCR) in shaping the humoral response at the intestinal interface remains unknown.
Here we showed that GC B cells lacking IgA are rapidly outcompeted by IgA+ B cells during GC reaction in Peyer’s patches and they are unable to contribute to memory B cell and intestinal-homing plasma cell compartments. IgA BCR did not impact cell proliferation in dark zone, and ectopic Bcl-2 expression was unable to rescue IgA-deficient GC B cells in mixed bone marrow chimeras. In contrast, IgA-deficient GC B cells underwent increased apoptosis in the light zone. In line with these findings, we observed that IgA BCR mediated faster and stronger intracellular Ca2+ signaling and led to increase BCR-dependent phosphorylation events. Mechanistically, IgA BCR signaling conferred resistance to Fas ligand (FasL)-dependent cell death in vitro, and genetic inhibition of Fas-FasL pathway restored IgA-deficient B cell fitness during GC reaction in vivo.
Thus, our results showed that IgA-expressing GC B cells are protected from FasL counterselection in the light zone via enhanced BCR intracellular signaling. This process might allow low affinity B cell clones to participate to the GC reaction and assure a comprehensive humoral response to poorly immunogenic commensal species.
Supported by grants from NIH (T32 AI132152, T32 AI007349 and R01 AI155727-02)