bluuebunny/crossref_metadata_2025_split huggingface dataset:
Somatosensory regulation of macrophage function during inflammatory pain
Authors: Hakim Sara N. , Woolf Clifford J. 
Published: February 2023

Abstract
Pain is a protective mechanism that alerts the organism against dangerous stimuli, much like the innate immune system. These stimuli range from noxious chemical, mechanical, and thermal stimuli to regional inflammation caused by wounds or infections. During an inflammatory pain condition, such as post-operative skin incision, the inflammatory milieu communicates directly with sensory neurons via signaling molecules to either promote or resolve the sensation of pain. The sensory neurons that initiate pain, nociceptors, release neuropeptides in the tissue they innervate which can act on neighboring immune cells and regulate their function. Here, I investigated how neuropeptides released by nociceptors during inflammatory pain regulate macrophage function. In LPS-activated macrophages, I found that the neuropeptides CGRP and PACAP but not Substance P and Galanin reduced secretion of IL-6 and TNF-α suggesting a neuropeptide-specific effect. Notably, CGRP promoted a reparative macrophage signature, by enhancing the release of IL-10 and the transcription of reparative genes such as Arginase I. When macrophages are only transiently exposed to LPS, they acquire a reparative phenotype. Interestingly, CGRP promoted Arginase I induction even further in these reparative macrophages, suggesting that CGRP could act on reparative as well as inflammatory macrophages to promote an anti-inflammatory milieu. These findings suggest that, during an inflammatory condition, when sensory neurons relay the sensation of pain, they also regulate macrophage function locally by release of CGRP and PACAP. By promoting an anti-inflammatory milieu, CGRP release may accelerate both the healing of tissue injury as well as the resolution of pain.
Neutrophil Extracellular Traps promote joint damage in hemophilia.
Authors: Kaminski Tomasz W. , Brzoska Tomasz , Tutuncouglu Egemen , Ragni Margaret V. , Sundd Prithu 
Published: February 2023

Abstract
Hemophilic arthropathy (HA) is the pathophysiology resulting from recurrent joint bleeding in hemophilia. HA leads to joint damage, chronic pain and poor quality of life. Despite advances in the treatment to prevent joint bleeding, HA continues to be a major morbidity affecting hemophilia patients and the mechanism contributing to HA remains elusive. Recent evidence suggests that joint-bleeding may promote the release of erythrocyte and tissue-derived damage-associated-molecular-pattern molecules (DAMPs) that can trigger the sterile inflammation in the joints, however, the innate immune pathways remain unclear. We used a model of knee joint injury-induced HA in FVIII-total knockout (F8TKO) mice and samples from hemophilia patients diagnosed with HA. In vivo multiphoton-excitation fluorescence imaging of injured synovial cavity in live F8TKO or control mice was used to assess NETs formation within joint capsule. Imaging-flow-cytometry and ELISA assays were used to evaluate circulating NETs in HA patients and mice with knee-injury. Bleeding severity scoring, histology, IHC and confocal imaging of joints were conducted to assess the joint injury in mice. F8TKO but not control mice manifested knee-joint injury and severity of bleeding 5-days post knee-injury. Knee-joint injury was associated with increased neutrophil accumulation and NETs shedding within the synovium of F8TKO mice. Circulating NETs were abundant in the plasma of patients diagnosed with HA and F8TKO following injury but not plasma of controls. These findings are the first to suggest that NETs contribute to pathogenesis of HA. Currently, study is underway to identify the innate immune pathways that promote NETs shedding, leading to joint-damage in hemophilia.
In vitro co-culture approach reveals multi-modal control of dendritic cell functions by nociceptors
Authors: Hanc Pavel , von Andrian Ulrich H 
Published: February 2023

Abstract
Nociceptors – sensory neurons that respond to noxious stimuli by eliciting the sensation of pain or itch – are increasingly beginning to be appreciated as controllers of the immune response. Notably, dendritic cells (DCs) – myeloid cells of the immune system – were previously found to be impacted by nociceptors, however, while some functional outcomes of the interaction have been described, our mechanistic understanding of the underlying molecular communication network is rudimentary at best and a bigger picture pertaining the modalities and outcomes of the interaction is missing entirely.
Consequently, here, we have established a novel in vitro co-culture system for DCs and nociceptors, which allowed us to explore in detail the modalities and outcomes of their communication. We show that DCs and nociceptors engage in a tight physical interaction and that nociceptors have the ability to attract DCs by production of the chemokine CCL2. Additionally, nociceptors enhance pro-inflammatory cytokine production by activated DCs in a contact-dependent, neuropeptide-independent manner, and nociceptive activity induces membrane depolarization and calcium flux in the interacting DCs. Next, using RNA sequencing, we identified genes that are controlled by nociceptors in steady-state DCs in the absence of immune stimulation, and uncovered a nociceptor-induced program of enhanced sentinel function, characterized by an increased expression of pro-IL1b but no overt immune activation of DCs both in vitro and in vivo. Finally, we identified the CGRP family of neuropeptides as nociceptor-derived signals mediating this effect.
Using CRISPR/cas9 in primary natural killer cells to study antibody dependent cellular cytotoxicity signaling.
Authors: Dahlvang James D. , Sangala Jules A. , Hart Geoffrey T. 
Published: February 2023

Abstract
In contrast to conventional natural killer (NK) cells, adaptive NK cells lack expression of the Fc receptor γ chain and transcription factor PLZF. A higher proportion of adaptive NK cells is correlated with protection from malaria. Adaptive NK cells have shown enhanced degranulation in response to opsonized targets, as measured by CD107A and interferon-γ (IFN-γ) expression. We hypothesize that people with a higher frequency of adaptive NK cells are protected from malaria because of the enhanced degranulation of adaptive NK cells. However, it is unclear why adaptive NK cells degranulate better than conventional NK cells. To answer this question, we developed a CRISPR/cas9 protocol to ablate genes associated with the antibody-dependent cellular cytotoxicity (ADCC) signaling pathway and evaluated the effect of these ablations on NK cell degranulation. Individual gene targets included the Fc receptor γ chain, CD3ζ, Syk, ZAP70, and PLZF. Combinations of these targets were also ablated. After ablating gene(s) of interest, mutant NK cells were incubated with an opsonized target and evaluated five hours later for CD107A and IFN-γ expression. As a control, we found that the combined ablation of the Fc receptor γ and CD3ζ chain severely diminishes NK cell degranulation. Our results from other gene targets will be shown. Furthermore, details of our CRISPR/cas9 method will be shared. The method is the first to effectively ablate multiple primary NK cell targets using the Amaxa 4D nucleofector. On average, we obtained 82.8% protein ablation across all targets. Overall, this work will give insight into why adaptive NK cells function better at ADCC and help the field effectively ablate genes in primary NK cells.
HMGB1 dimerization could protect nuclear DNA damage and cell death from oxidative stress
Authors: Kwak Man Sup , Rhee Woo Joong , Lee Yong Joon , Kim Hee Sue , Kim Young Hun , Kwon Min Kyung , Shin Jeon-Soo 
Published: February 2023

Abstract
Oxidative stress can induce covalent disulfide bond formation between protein-protein thiol groups and generate hydroxyl free radicals that damage DNA. HMGB1 is a DNA chaperone and damage-associated molecular pattern molecule. As a redox-sensitive protein, HMGB1 contains three cysteine residues: Cys23, Cys45, and Cys106. In this study, we focused on the relationship between HMGB1 dimerization and DNA stabilization under oxidative stress conditions. HMGB1 dimerization was positively modulated by CuCl2 and H2O2. Mutation of the Cys106 residue blocked dimer formation. Treatment of HEK293T cells with CuCl2 and H2O2 enhanced the oxidative self-dimerization of HMGB1, whereas this dimerization was inhibited in mutant HMGB1C106A cells. Furthermore, we performed a bimolecular fluorescence complementation assay to visualize Cys106 oxidation-induced HMGB1 dimerization in live cells exposed to oxidative stress and were able to reproduce the dimerization effect of HMGB1 in fluorescence resonance energy transfer analysis. Interestingly, dimerized HMGB1 bound to DNA with higher affinity than monomeric HMGB1. Dimerized HMGB1 protected DNA from damage due to hydroxyl free radicals and prevented cell death. In conclusion, dimerized HMGB1 may play a regulatory role in DNA stabilization under oxidative stress.
Identifying innate immune regulators of Mycobacterium tuberculosis pathogenesis in macrophages
Authors: Choudhury Sreetama , Peterson Eliza , Maciag karolina , Akhade Ajay Suresh , Rostomily Clifford , Urdahl Kevin , Baliga Nitin , Subramanian Naeha 
Published: February 2023

Abstract
Tuberculosis (TB) is a leading infectious disease that contributes to approximately 1.4 million deaths annually worldwide. In the present situation of the COVID-19 pandemic and disrupted healthcare services, mathematical modelling predicts that there will be an additional 6.3 million new cases and 1.4 million more TB deaths by the end of 2025. Innate immune cells like macrophages are the frontline responders in host defense against Mycobacterium tuberculosis (MTB) and may also play a major role in disease establishment. A major roadblock in developing effective strategies against TB is the inability of current anti-TB drugs to target both replicating and non-replicating bacteria. We have found that lack of specific components of the host innate sensing machinery has a major impact on intracellular bacterial growth and the production of IL-1β and IFN-β during MTB infection of macrophages; the latter cytokines play major roles in host-protective and host-detrimental outcomes of infection respectively. RNA seq reveals that these innate immune modules differentially control the expression of genes implicated in host protein synthesis and MTB dissemination and escape from the granuloma. Ongoing work is utilizing Path-Seq for quantitative profiling of the MTB transcriptome within infected macrophages and investigating macrophage-dependent mechanisms of MTB restriction and persistence in vivo. Our study will potentially identify immune regulators that make the macrophage environment either permissive or restrictive for intracellular replication of MTB with important implications for developing combinatorial targeting approaches for host-directed treatment of TB.
Tregs facilitate obesity and insulin resistance via a Blimp-1-IL-10 axis
Authors: Beppu Lisa , Mooli Raja Gopal Reddy , Qu Xiaoyao , Marrero Giovanni , Finley Christopher , Fooks Allen , Mullen Zackary , Frias Adolfo , Sipula Ian , Xie Bingxian , Helfrich Katherine , Watkins Simon , Poholek Amanda , Ramakrishnan Sadeesh , Jurczak Michael , D’Cruz Louise 
Published: February 2023

Abstract
Adipose-resident Tregs protect against systemic inflammation and metabolic disease by limiting expansion of pro-inflammatory cells, preserving insulin sensitivity and maintaining glucose tolerance. Although their basic markers and roles have been studied, less is known about the transcriptional machinery regulating their differentiation and function.
B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional regulator known to be involved in development, polarization, and maintenance of various immune cells including CD4+ T cells. Using Blimp-1 reporter mice, we discovered that Blimp-1 is constitutively expressed in a subset of visceral adipose tissue (VAT) Tregs, and that Blimp-1+ VAT Tregs are phenotypically distinct from their Blimp-1-counterparts. We also found that Treg-specific Blimp-1 deletion led to altered differentiation and function of VAT and inguinal adipose tissue Tregs. Surprisingly, during diet-induced obesity, Blimp-1 Treg deficient mice gained less weight, had reduced body fat percentage, and exhibited improved insulin sensitivity compared to wild type mice. Furthermore, this was accompanied by upregulation of thermogenic genes such as Ucp1, Prdm16 and Dio2 in inguinal adipose tissue, and increased overall fatty acid oxidation. It has previously been shown that IL-10 can induce thermogenesis. Therefore, we repeated these experiments utilizing mice with Treg-specific deletion of IL-10 and found that they phenocopied the Blimp-1 Treg deficient mice. Based on these results, we hypothesize that cross-talk between Tregs and adipocytes via a Blimp-1-IL-10 axis suppresses thermogenesis, and that absence of Blimp-1+ Tregs is metabolically protective during diet-induced obesity.
Reconstituting T Cell Receptor Selection In-Silico
Authors: Ostmeyer Jared L , Cowell Lindsay , Greenberg Benjamin , Christley Scott 
Published: February 2023

Abstract
Each T cell receptor (TCR) gene is created without regard for which substances (antigens) the receptor can recognize. T cell selection culls developing T cells when their TCRs (i) fail to recognize major histocompatibility complexes (MHCs) that act as antigen presenting platforms or (ii) recognize with high affinity self-antigens derived from healthy cells and tissue. Here, we show that we can accurately predict from the TCR if any T cell is pre- or post-selection (and by extension if that T cell would be culled).
First, we analyze millions of TCR genes sequenced from mouse spleen using high-throughput receptor sequencing. To get examples of TCRs pre-selection, we develop a way to translate non-productive TCR genes that otherwise cannot express protein, obtaining candidate TCR protein sequences never subjected to T cell selection. To get examples of TCRs post-selection, we translate productive TCR genes sequenced from a peripheral location such as spleen, obtaining TCR protein sequences representing T cells that survived T cell selection. Next, we train a machine learning model to find sequence level patterns that discriminate between these two categories of TCR protein sequences, achieving a balanced classification accuracy of 73% (AUC of 0.8) on de-novo TCR β-chain protein sequences. This classification accuracy may be near the achievable limit using only the β-chain representing half of the TCR heterodimer.
To verify our approach, we show our machine learning model accurately predicts that (i) productive TCRs from thymus (where T selection occurs) are predominantly pre-selection and (ii) productive TCRs from peripheral tissues like skin are post-selection.
Pleiotropic adipose Treg subsets, defined by Atf3 expression, modulate adipocyte energy expenditure and weight loss
Authors: D’Cruz Louise M , Marrero Giovanni , Fooks Allen , Finley Christopher , Hyzny Eric , Frias Adolfo B , Beppu Lisa , Sipula Ian , Xie Bingxian , Jurczak Michael 
Published: February 2023

Abstract
Chronic obesity occurs when there is imbalance between caloric intake and energy expenditure. It is now clear that immune cells, through their communication with adipocytes, can drive or suppress adipocyte energy expenditure, thus regulating weight gain and obesity. Adipose tissue resident regulatory T cells (aTregs) are a dominant, unique CD4+ T cell population in the adipose tissue depots of mice and humans. Here we show that the transcription factor Activating Transcription Factor 3 (Atf3), defines a population of aTregs that secrete opioid-like peptides called Methionine-Enkephalins (Met-Enk). We show that aTreg-derived Met-Enk increases adipocyte function and energy expenditure by upregulating Uncoupling Protein 1 (Ucp-1) and other genes associated with adipocyte thermogenesis. Loss of Atf3 expression in aTregs, resulted in loss of Met-Enk expressing aTregs and conversely, resulted in increased Interelukin-10 (IL-10)-secreting aTregs. Physiologically, Atf3+ aTregs are required to maintain metabolic homeostasis and loss of Atf3+ aTregs increased insulin resistance in mice. Taken together, our data reveal that distinct aTreg populations with unique gene expression and cytokine secretion profiles, work together to modulate adipocyte function and energy expenditure.
Interleukin-2 inducible T cell kinase functions as a molecular switch to fine tune differentiation of naive T helper cells in pro/anti-inflammatory effector T cell lineages
Authors: Anannya Orchi , August Avery 
Published: February 2023

Abstract
Naïve CD4+ T helper cells differentiate into effector CD4+ T cells with pro/anti-inflammatory functions upon receipt of signals from the T cell receptor (TCR) in presence of cytokines in the environment. Interleukin-2 inducible T cell kinase (ITK) has been shown to control the strength of signals downstream of the TCR. Here we have investigated the potential of ITK to act as a molecular switch in controlling T cell differentiation fate. Our results demonstrate in the absence of ITK expression/activity, naïve CD4+ T cells activated under conditions that promote differentiation into pro-inflammatory T helper type-17 (Th17) cells fail to differentiate into Th17 cells and instead switch into T cells expressing the T regulatory (Treg) lineage specific transcription factor Forkhead Box P3 (FoxP3). Similarly, we found that naïve CD4+ T cells activated to differentiate into anti-inflammatory Type 1 regulatory (Tr1) cells in the absence of ITK expression/activity fail to differentiate into Tr1 cells and instead switch into expressing the T helper type-1 (Th1) lineage specific T-box transcription factor (T-Bet). The switched FoxP3 expressing T cells resemble Tregs by their expression of Treg specific markers and have anti-inflammatory properties in suppressing effector T cell proliferation. In addition the switched T-Bet expressing T cells resemble Th1 cells by their expression of Th1 specific markers and the Th1 effector cytokine Interferon γ (IFNγ). This work suggest that signals regulated by ITK may function as a molecular switch to control Th17/Treg and Tr1/Th1 axes, highlighting the potential of manipulating ITK to control the balance of pro/anti-inflammatory T cells in immune disorders.
HOP homeobox (HOPX) represses activation-induced human T cell proliferation
Authors: Yang Qian , Patrick Michael , Lu Jian , Weng Nanping 
Published: February 2023

Abstract
Aging is associated with the decline function of T cells including reduced activation-induced proliferation. HOP homeobox (HOPX), a homeodomain protein, is reported to regulate functions of T helper type 1 and regulatory T cells in murine model. However, its role in human T cells has not been examined. Here, we report that HOPX mRNA was detected in human CD4+ and CD8+ T cells and increased via anti-CD3/28 activation. Additionally, HOPX level was 1.5-fold higher in isolated CD4+ T cells from old health subjects (≥70 years) than from young subjects (≤40 years). Expression of HOPX in Jurkat T cells via lentiviral transduction significantly reduced the proliferation compared to sham viral transduced control. Furthermore, the reduced proliferation was also observed in HOPX induced primary human CD4+ and CD8+ T cells as measured by dilution of CFSE tracking dye and cell number. The overexpression of HOPX did not appear to increase cell death as measured by cell viability dye. Currently, we are investigating the mechanism underlying the HOPX mediated suppression of T cell proliferation and its role in T cell aging.
Pro-survival IL-7-stimulated weak strength of mTORC1-S6K signaling controls T cell memory via transcriptional FOXO1 and metabolic AMPK pathways
Authors: Xiang Jim 
Published: February 2023

Abstract
Distinct strengths of stimuli (DSS) is a well-known model for T cell memory. Two common γ-chain (γc)-family cytokines IL-2 and IL-7, which trigger the same mTORC1-S6K pathway, distinctly induce effector T (Te) cells and memory T (Tm) cell differentiation. However, their underlying mechanisms are elusive. In this study, we want to elucidate molecular mechanism for IL-7-induced Tm (IL-7/Tm) cells by performing in vitro and in vivo experiments using genetic and pharmacological tools. We show that IL-2 and IL-7 stimulate strong and weak S6K signals leading to IL-7R−CD62L−KLRG1+ Te and IL-7R+CD62L+KLRG1− Tm cell formation in our study model of adoptive transfer of T cells derived from wild-type and IL-7R knockout (KO)/OTI mice and L. monocytogenes rLmOVA infection. We further show that IL-7/Tm cells upregulate transcriptional factors (FOXO1, Id3 and TCF1) for regulating Tm’s differentiation and promote expression of autophagy/metabolic molecules (AMPK, ULK1 and ATG7) for controlling Tm’s fatty acid oxidation (FAO) metabolism by flow cytometry, Western blotting, confocal and electron microscopy and Seahorse assay analyses, and vice versa for IL-2-induced Te (IL-2/Te) cells. IL-7/Tm cells derived from AMPK KO/OTI mice abolish expression of ULK1 and ATG7, FAO metabolism and long-term survival, while IL-2/Te cells with S6K deficiency show Tm’s differentiation, metabolic/transcriptional pathways and prolonged survival. Taken together, our data demonstrate that pro-survival IL-7-stimulated weak S6K signaling controls T cell memory via transcriptional FOXO1 and metabolic AMPK pathways. The novel finding provides a new potential mechanism for DSS model in T cell memory and has a great impact on vaccine development and immunotherapy.
Receptor interacting protein kinase 1 (RIPK1) pathways regulate innate B cell developmental checkpoints and delay effector function
Authors: Parthasarathy Raksha , Hagglof Thomas , McLennan Alexandra , Mattke Aiden , Dudley Elizabeth , Kumagai Abigail , Leadbetter Elizabeth A 
Published: February 2023

Abstract
Mutations in RIP kinases underlie the recently described human autoimmune syndrome, CRIA, characterized by lymphadenopathy, splenomegaly, and autoantibody production. While disease mechanisms for CRIA remain undescribed, RIP kinases work together with Caspase-8 to regulate cell death, which is critical for normal differentiation of many cell types. Here, we describe a key role for RIPK1 in facilitating innate B cell differentiation and subsequent activation. By comparing RIPK1, RIPK3, and Caspase8 triple deficient and RIPK3, Caspase 8 double deficient mice, we identified selective contributions of RIPK1 to an accumulation of murine splenic Marginal Zone (MZ) B cells and B1-b cells. We used mixed bone-marrow chimeras to determine that innate B cell commitment required B cell-intrinsic RIPK1, RIPK3, and Casp8 sufficiency. RIPK1 regulated MZ B cell differentiation rather than development and RIPK1 mediates its innate immune effects independent of the RIPK1 kinase domain. NP-KLH/alum and NP-Ficoll vaccination of RIPK3, Casp8, and RIPK1 triple deficient mice revealed uniquely delayed T-dependent and T-independent IgG responses, abnormal splenic germinal center architecture, and reduced extrafollicular plasmablast formation compared to double deficient and WT mice. RIPK1 deficiency was beneficial for survival following lethal, systemic Streptococcus pneumoniae infection, confirming the functionality of innate B cell populations which increased in the absence of RIPK1. Thus, RIPK1 orchestrates B cell fate, and delayed effector function, through a B cell-intrinsic mechanism.