bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of (+)-naloxone blocking Toll-like Receptor 4 in depression and cognition
Authors: Medina-Rodriguez Eva Maria , Rice Kenner C , Beurel Eleonore , Jope Richard S
Published: February 2023

The immune system has been shown to affect the brain, and consequently behaviors. In particular, increasing evidence indicates that inflammation contributes to susceptibility to depression, which is a leading cause of disability, and for which current treatments are often inadequate. We hypothesized that targeting Toll-like receptor 4 (TLR4), one of the main signaling pathways for triggering an inflammatory response, would lessen stress-induced depression-like behaviors in mice. TLR4 inhibition with the CNS-penetrating drug (+)-naloxone that is a TLR4 antagonist but is inactive at opiate receptors, reversed chronic restraint stress-induced impairments of various depression-like behaviors, as well as cognitive tests in male mice but had no effect in female mice. These effects might be mediated by interferon-β (IFNβ) pathway in microglial cells. Altogether, our results confirmed the critical role of TLR4 in mediating depressive-like behaviors and cognitive impairments and demonstrated that (+)-naloxone might be a promising therapeutic strategy for depression.
Visualization of leukemic B cell clonal dynamics during therapy using tSNE.
Authors: Welch Baustin M
Published: February 2023

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+ CD5+ clonal B lymphocytes in the blood, bone marrow, and peripheral lymphoid organs. Many patients with CLL never receive treatment for the leukemia and therapy is typically only initiated upon progression of disease-related clinical complications. Treatment options for progressing patients range from chemoimmunotherapy (CIT) to small molecule inhibitors that target pro-survival signaling pathways in leukemic B cells, such as the BTK inhibitor ibrutinib. Although therapy regimens have improved, there is still no cure for CLL, in part due to clonal evolution of the leukemic B cell. The goal of this study is to document the clonal dynamics of CLL B cells at baseline and at response evaluation(s) for alterations in clonal architecture and expression of cell surface markers associated with therapy resistance using the single cell flow cytometry analysis tool, t-distributed stochastic neighbor embedding (t-SNE). Biobanked peripheral blood mononuclear cells were obtained from patients that underwent frontline CIT or ibrutinib, or secondary ibrutinib therapy. Fourteen parameter flow cytometry was performed and subjected to tSNE analysis to document CLL B cell clonal architecture and evolution in patients undergoing the various therapies. Profound differences in treatment responses and leukemic cell clonal dynamics were observed which may inform the discovery of novel biomarkers associated with therapy resistance.
NLRP7 interacts with EBP1 providing evidence for its role in molar pregnancies and development.
Authors: So Matthew , Lupfer Christopher R
Published: February 2023

Recurrent hydatidiform molar pregnancies are characterized by uncontrolled proliferation of trophoblasts resulting in the failure of the embryo and placenta to develop properly. Previous studies genetically mapped a mutation in NLRP7 as a cause for recurrent molar pregnancies with abnormal methylation patterns. The goal of this study is to identify human proteins that interact with NLRP7, especially those that are involved in DNA acetylation and methylation, which could lead to the development of molar pregnancies. A yeast two-hybrid system using a human cDNA library was used to screen for interacting proteins and protein interactions were verified by co-immunoprecipitation in HEK 239T cells. Additional experiments are being performed to determine the change in global methylation when NLRP7 and EBP1 interact. Yeast screening results indicated several proteins that interact with NLRP7, however further testing will be focused on the interaction with EBP1, a human protein involved in growth regulation. Having potentially identified a protein involved in development through acetylation/deacetylation that interacts with NLRP7, we hope to shed more light on the molecular pathways that lead to the formation of recurrent molar pregnancies.
An Assay to Monitor the Engagement and Modulation of CD6 on T cells as a Clinical Biomarker of Treatment with Itolizumab
Authors: Bisconte Angelina R , Kisselbach Lynn , Ng Cherie , Ampudia Jeanette , Chu Dalena Ngoc , Belkina Natalya , Connelly Stephen , Phippard Deborah
Published: February 2023

Itolizumab is a novel first-in-class monoclonal antibody that selectively targets the co-stimulatory molecule CD6, a receptor that is highly expressed on CD4 and CD8 T cells and plays an important role in activation and migration. Monitoring target engagement and changes in receptor levels is critically important to interpreting clinical data. To evaluate the pharmacodynamic properties of itolizumab treatment on T cells in patients including those with graft versus host disease (GvHD), Precision for Medicine has developed and validated a 10-color flow cytometry assay to assess the engagement and modulation of cell-surface CD6.

The assay was validated using whole blood from healthy donors. Blood was spiked with five concentrations of drug selected based on expected PK in the clinical study. The assay conditions were optimized for sensitivity, signal: noise ratio, detection of free receptor, receptor-bound itolizumab and total surface CD6. Itolizumab was detected using a fluorochrome conjugated α-human IgG1 antibody while total surface CD6 expression was assessed using an antibody to a non-competing site on CD6. For validation, pre-set criteria were used to assess inter-assay, intra-assay, inter-operator precision and post-staining stability. Technical validation was successfully met; and the assay performs within acceptable precision parameters.

Measuring cell-based receptor engagement and fate in patients on immuno-modulatory therapies is very challenging. This assay was designed and validated to be both sensitive and selective in the quantification of CD6 receptor occupancy and modulation to facilitate the determination of an optimal therapeutic dose in autoimmune and inflammatory diseases.
Fasting-induced FOXO4 blunts human CD4+ T helper cell responsiveness
Authors: Han Kim K , Singh Komudi , Rodman Matthew J , Hassanzadeh Shahin , Wu Kaiyuan , Huffstutler Rebecca D , Dagur Pradeep K , Saxena Ankit , McCoy J. Philip , Teague Heather J , Mehta Nehal N , Pirooznia Mehdi , Sack Michael N
Published: February 2023

Intermittent fasting blunts inflammation in asthma and rheumatoid arthritis, suggesting that fasting may be exploited as an immune-modulatory intervention. However, mechanisms underpinning anti-inflammatory effects of fasting remain poorly characterized. Here, we show that fasting in humans is sufficient to blunt CD4+ T helper cell responsiveness. RNA-seq and flow cytometric immunophenotyping of peripheral blood mononuclear cells (PBMCs) from volunteers subjected to overnight or 24-hour fasting, and 3-hours of refeeding implicate that fasting blunts CD4+ T helper cell activation and differentiation. Transcriptomic analysis reveal that the longer fast-duration has a more robust effect on CD4+ T cell biology. Through bioinformatic analyses, we identify the transcription factor FOXO4 and its canonical target FKBP5 as a potential fasting-responsive regulatory axis. Genetic gain- or loss-of-function of FOXO4 and FKBP5 is sufficient to modulate Th1 and Th17 cytokine production. Moreover, we find that fasting-induced or genetic overexpression of FOXO4 and FKBP5 is sufficient to downregulate mTORC1 signaling and suppress STAT1/3 activation. Our results identify FOXO4-FKBP5 as a novel fasting-induced, STAT-mediated, regulatory pathway to blunt human CD4+ T helper cell responsiveness.
Synergistic induction of TNF-α by palmitate and oxidative stress in human monocytic cells involves the NF-κB/p38 MAPK signaling
Authors: Sindhu Sardar T.A.K. , Akhter Nadeem , Wilson Ajit , Al-Rashed Fatema , Al-Madhoun Ashraf , Arefanian Hossein , AL-Mulla Fahd , Ahmad Rasheed
Published: February 2023

Tumor necrosis factor (TNF)-α is a key proinflammatory cytokine expressed in obesity/ type-2 diabetes. In metabolic disease conditions, TNF-α is co-expressed with increased circulatory levels of free fatty acids, such as long-chain unsaturated fatty acid palmitate. The hypoxic milieu in the adipose tissue is known to exert oxidative stress. Although it is known that palmitate may induce TNF-α in different cell types, it still remains unclear whether the oxidative stress could augment TNF-α expression in human monocytic cells, and thus exacerbate metabolic inflammation. To test the hypothesis, THP-1 monocytic cells were stimulated with palmitate, in presence or absence of hydrogen peroxide (H2O2) or 1% hypoxia. TNF-α, CHOP, ERN1, and HIF-1α mRNA expression was assessed by qRT-PCR; TNF-α protein expression was detected by flow cytometry/ immunohistochemistry; reactive oxygen species (ROS) expression was measured by DCFH-DA assay; and NF-κB/p38 MAPK phosphorylation was detected by western blotting. We found that TNF-α mRNA and protein expression were significantly upregulated in THP-1 cells co-stimulated with palmitate and H2O2/hypoxia. TNF-α expression associated positively with intracellular ROS, transcripts of CHOP, ERN1, HIF-1α, and phosphorylation of NF-κB/p38 MAPK. Synergistic TNF-α expression co-induced by palmitate and H2O2/hypoxia was diminished by cell treatments with curcumin, apocynin, or N-acetyl cysteine (P<0.01). In conclusion, palmitate and oxidative stress promote the TNF-α expression in human monocytic cells via the mechanisms involving ER stress, HIF-1α stabilization, and NF-κB/p38 MAPK signaling. These findings may have pathophysiological significance in metabolic inflammatory conditions.
The Wnt antagonist Dickkopf1(DKK1) promotes pulmonary fibrosis via M2-like macrophage polarization
Authors: Chae Wook-Jin , Sung Eun-Ah , Hur Brian , Park Min Hee
Published: February 2023

Tissue fibrosis is a progressive pathological process induced by repeated tissue injury and inflammation resulting in excessive collagen deposition. The Wnt signaling pathway regulates cell proliferation and differentiation for tissue repair processes. Dickkopf1 (DKK1) is a quintessential inhibitory ligand of the Wnt pathway. M2-like macrophages, induced by type 2 cytokines such as IL-13, play a central role in tissue fibrosis. We found that DKK1 protein expression markedly increased in both lung tissues of human idiopathic pulmonary fibrosis (IPF) and lungs from the murine bleomycin (BLM)-induced fibrosis model. To investigate the role of DKK1 in lung fibrosis, we used the DKK1 hypomorphic doubleridge (Dkk1d/d) mouse model. Collagen mRNA and protein expressions were markedly decreased in the lungs of Dkk1d/d mice compared to those of WT mice upon BLM challenge. The infiltration of macrophages was significantly decreased in the lungs of Dkk1d/d mice upon lung injury. We tested whether DKK1 modulates the polarization of M2-like macrophages in vitro using mouse bone marrow-derived macrophages (BMDMs). Interestingly, DKK1 induced M2-like macrophage marker CD206 (mannose receptor) and Arginase 1 (Arg1) protein expression. The expression of CD206 and Arg1 was synergistically increased by DKK1 and IL-13. We demonstrated that DKK1 utilizes STAT6 and JNK to induce Arg1 and CD206. Taken together, our study demonstrates that DKK1 promotes pulmonary fibrosis by activation of M2-like macrophages. Our study will provide new mechanistic insights into DKK1-mediated lung fibrosis pathogenesis.
Antigen presenting group 3 innate lymphoid cells orchestrate neuroinflammation
Authors: Grigg John B , Shanmugavadivu Arthi , Regen Tommy , Parkhurst Christopher N , Ahmed Anees , Joseph Ann M , Mazzucco Michael , Eberl Gerard , Vartanian Timothy , Waisman Ari , Sonnenberg Gregory F
Published: February 2023

Pro-inflammatory T cells in the central nervous system (CNS) are causally associated with multiple demyelinating and neurodegenerative diseases, but the pathways controlling these responses remain unclear. Here we define a unique population of group 3 innate lymphoid cells (ILC3s) that infiltrate the CNS in a mouse model of multiple sclerosis. These ILC3s are derived from the circulation, localize in proximity to infiltrating T cells in the CNS, function as antigen presenting cells that restimulate myelin-specific T cells, and are elevated in the blood of individuals with multiple sclerosis. Critically, antigen presentation by ILC3s is required to promote pro-inflammatory T cell responses in the CNS and the development of multiple sclerosis-like disease in mouse models. In contrast, conventional and tissue-resident ILC3s in the periphery do not appear to contribute to disease induction, but rather remain tolerogenic with the potential to limit autoimmune T cells and prevent multiple sclerosis-like disease when experimentally targeted to present myelin antigen. Collectively, our data define an inflammatory ILC3 population that is essential for directly promoting T cell-dependent neuroinflammation in the CNS and reveal the potential to harness peripheral tissue-resident ILC3s for the prevention of autoimmune disease.
Atherosclerotic plaque T cells produce cytokines but do not exhibit signs of T-cell receptor-mediated activation
Authors: Mulholland Megan , Jakobsson Gabriel , Sundius Lena , Ljungcrantz Irena , Rattik Sara , Bengtsson Eva , Schiopu Alexandru , Nilsson Jan , Herter Jan , Björkbacka Harry , Lichtman Andrew H , Engelbertsen Daniel
Published: February 2023

T-cell activation in the atherosclerotic plaque has been proposed to be a key event promoting lesion inflammation and destabilization. Although T cells and antigen-presenting cells are present in the plaque, it is unclear whether primary activation or secondary re-activation of T cells occurs in the lesion. We sought to quantify and characterize activated plaque T-cells in experimental atherosclerosis utilizing transgenic reporter mice of TCR-signalling (Nur77-GFP) or cytokine production (IFN-γ-YFP). As has been previously described, splenic Nur77hi T cells displayed markers consistent with a recently activated phenotype (PD-1+CD44+). Importantly, we observed very low levels of Nur77hi CD4+ or CD8+ T cells in the atherosclerotic aorta of Nur77-GFP-Apoe−/− mice compared to other tissues. In line with these findings, adoptive transfer of splenic Nur77-GFP CD4+ T cells to lymphodeficient atherosclerotic Rag1−/−Apoe−/− mice revealed markedly lower levels of Nur77hi T cells in plaque compared to lymphoid organs 10 weeks post-transfer, suggesting limited TCR-mediated activation in plaques. Conversely, analysis of atherosclerotic plaques from Apoe−/−IFN-γ-YFP reporter mice demonstrated robust IFN-γ production by CD4+ and CD8+ T cells. Altogether these results indicate that levels of cytokine release are above that of TCR-mediated T-cell activation, suggesting that antigen-driven activation of T cells in the plaque is not key for mediating T-cell driven atherosclerosis.
Electrical activation of vagal efferent or afferent neurons elicits two distinct mechanisms of anti-inflammatory actions.
Authors: Murray Kaitlin M , Rude Kavi M , Sladek Jessica , Reardon Colin
Published: February 2023

Inhibition of inflammation by reducing macrophage activation and pro-inflammatory cytokine production by electrical vagal nerve stimulation is well documented. Despite this ability to regulate inflammation, the mechanisms and intermediaries in this process are not completely understood. Vagal efferent neurons are thought to be the primary source of anti-inflammatory activity, despite the composition of the vagus nerve, which is predominantly vagal afferent neurons. Activation of vagal efferent neurons ameliorates lipopolysaccharide (LPS)-induced sepsis and is dependent on acetylcholine (ACh) derived from choline acetyltransferase (ChAT+) T-cells. Recent studies have identified vagal afferent fibers as anti-inflammatory as well, however the circuitry, including whether these vagal-mediated anti-inflammatory mechanisms converge, is unknown. Here, we show that VNS is capable of eliciting two distinct, anti-inflammatory mechanisms orchestrated by vagal efferent or afferent neurons. Using ChAT+ T-cell conditional KO mice, we show that afferent VNS reduced LPS-induced inflammation independently of T-cell derived ACh by measuring serum and lymphatic organ TNF-α and IL-6 levels. Using a highly selective β2-adrenergic receptor (β2-AR) antagonist, we show that intact, efferent, and notably afferent VNS regulates immunity in a β2-AR-dependent fashion. Together, these findings demonstrate that VNS is capable of eliciting two unique neuro-immune circuits with distinct mechanistic elements. Continued development of these circuits will inform the design of future vagal nerve stimulators, that may improve the efficacy of these devices in the regulation of immunity.
Single-cell RNA Sequencing Reveals Heterogeneity Amongst Immune Cell Subpopulations in LPS-Induced ALI Mice treated with I3C
Authors: Holloman Bryan L , Nagarkatti Mitzi , Nagarkatti Prakash , Wilson Kiesha
Published: February 2023

Traditionally, macrophage M1-vs-M2 dichotomy have remained fundamentally unchanged since their identification. The oversimplification of immune cell subpopulations using flow cytometric analysis fails to capture the full heterogeneity of these cells in response to disease state and treatment. However, single-cell RNA sequencing (sc-RNAseq) serves as a diagnostic tool that clusters cells based on the whole transcriptomics of individual cells, without the limitation of surface markers and transcriptional factors. In the current study, we aimed to better understand the immunomodulatory mechanism of indole-3-carbinol (I3C) on cell state. To that end, 5mg/kg of LPS was intranasally injected in C57BL/6 mice to induce ALI, and the mice were treated with 80mg/kg I3C three hours after disease induction. After 72 hours mice were euthanized. Whole lung cells were utilized for flow cytometry and sc-RNAseq. Strikingly, the traditional M1-vs-M2 separation was inadequate in classifying all macrophage clusters. LPS+I3C treated-mice had a decrease in Isg15, Ccl5, and Ly6c2 expression in M1 cluster; genes linked to inflammation. Also, the expression of monocyte chemoattractant genes (Cxcl2 and Cxcl3), arginine-associated transport gene (Slc7ac), and major histocompatibility complex (MHC) genes (H2-Ab1 and H2-K1) in M2 cluster was decreased in the diseased group following I3C treatment. In conclusion, several alterations in gene expression in myeloid immune cell subpopulations were identified following treatment with indole-3-carbinol. Thus, shedding light on several new macrophage subpopulations not identified using traditional flow cytometric analysis may offer novel mechanistic insights into disease induction.
Immunological Age Prediction in HIV-Infected, ART-Treated Individuals
Authors: de Armas Lesley , Pallikkuth Suresh , Pan Li , Rinaldi Stefano , Pahwa Rajendra , Pahwa Savita
Published: February 2023

Anti-retroviral therapy (ART) improves life expectancy in people living with HIV (PWH), but it remains unclear how chronic HIV infection affects normal aging of the immune system.

Plasma cell-free protein expression and immune phenotypes were assessed in blood from ART treated PWH (19–77yrs, n=106) and age-matched, HIV-negative controls (HC, n=103). Age-associated parameters were identified in HC and PWH independently by univariate Spearman correlation (p<0.05) to obtain a reduced dataset for downstream statistical modeling. Machine learning algorithms were applied to generate predictive models for age as a continuous variable.

Plasma biomarker analysis resulted in 8-parameter inflammatory index that was higher in PWH compared to HC and correlated with age. Combining flow and plasma data generated 277 and 491 age-associated parameters (out of total 1,357) in HC and PWH, respectively. PWH exhibited different immunological profiles associated with age compared to HC. However, CD38 expression on T cells declined with age in both HC and PWH. Predictive modeling using age-associated parameters in HC generated a 14-parameter signature with 76.9% accuracy to predict chronological age in HC, however this model failed to predict age in PWH (18.6% accuracy) highlighting the effect of HIV infection on immunological aging. A 25-parameter model (IMmunological Age Prediction, IMAP-25) however showed 70% and 53% accuracy in HC and PWH and was used to show an accelerated aging rate in PWH by 5.6 yrs compared to HC.

We applied a statistical approach to generate immune signatures, easily monitored in human blood samples, that could be used as an indicator of one’s ‘immunological age’ during ART-treated HIV infection.
Analysis of the role of helminth TGF-β mimic in tissue repair.
Authors: Lothstein Katherine E , Smyth Danielle J , Maizels Rick , Gause William C
Published: February 2023

Intestinal parasites express excretory/secretory (ES) molecules, which modulate the type-2 immune response, including anti-inflammatory and tissue repair mechanisms. TGF-β mimic (TGM), an ES molecule secreted by Heligmosomoides polygyrus (Hp), binds TGF-β receptors yet lacks structural homology to TGF-β and exhibits distinct receptor interactions. Studies suggest TGM generates higher expression of immunoregulatory Foxp3+ Tregs and decreased fibrotic markers in vitro compared to TGF-β. Host TGF-β also influences wound healing. In cutaneous wounds, effective tissue repair requires rapid suppression of inflammation and production of wound healing factors. We investigated whether TGM modulates wound healing using an in vivo wound biopsy model in which TGM is injected beneath a Tegaderm® layer to mimic cutaneous wound treatment. We showed that TGM accelerates wound healing when applied to the wound at multiple doses and modulates the associated inflammatory response. LC-MS/MS analysis of TGM-treated wound exudate showed increases in proteins mediating leukocyte chemotaxis and proliferation, collagen deposition and fibrotic regulation. Gene expression analysis of TGM-treated wounds revealed enhanced immune cell infiltration of alternatively activated CD206+F4/80+ macrophages and epithelial cells concurrent with accelerated wound closure. These macrophage cell subsets are known to be essential for effective wound healing. Our findings indicate that TGM can enhance skin wound healing and associated recruitment of specific macrophage subsets, type 2 innate immune cells and their cytokine expression. This study will investigate TGM as a potential novel and effective therapeutic option for enhanced wound healing.
Mononuclear phagocytes drive Mertk-dependent T cell regulation at autoimmune and tumor sites
Authors: Whitesell Jennifer C , Lindsay Robin S , Dew Kristen , Rodriguez Erika , Tracy Dayna , Sandor Adam , Yannacone Seth , Jacobelli Jordan , Friedman Rachel Sharon
Published: February 2023

Understanding mechanisms of immune regulation is key to developing effective immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes (MNPs) in regulating effector T cells in type 1 diabetes and melanoma. MNPs in the islets impaired T cell responsiveness to antigen by preventing T cell arrest. MNPs in the autoimmune lesion express the TAM family receptor tyrosine kinase Mertk which functions in efferocytosis. Inhibition or deficiency of Mertk led to a release from T cell regulation characterized by enhanced T cell arrest in pre-diabetic islets and at the tumor site. T cell arrest was accompanied by increased T cell-antigen presenting cell interactions in the islets. Antigen experience and effector function by T cells was increased with Mertk inhibition in the islets, but not in lymph nodes. Single cell RNA-seq analysis of islet MNPs identified multiple myeloid subsets. Inhibition of Mertk induced the most significant changes in gene expression among the islet resident macrophages including upregulation of inflammatory cytokine and adhesion genes. In the NOD mouse model, inhibition of Mertk-dependent T cell regulation culminated in the rapid acceleration of autoimmune pathology and disease. In human islets, the number of Mertk-expressing cells were increased in remaining insulin-containing islets from type 1 diabetic patients, suggesting that they might have a protective role in human disease. These data indicate that Mertk signaling in MNPs drives T cell regulation that functions specifically at the disease site in peripheral tissues through a mechanism that prevents T cell arrest and hinders response to antigen.
CD82 promotes CD8+ T cell immune responses by mediating T cell polarization and immunological synapse formation
Authors: Kim Sang-Hoon , Park Areum , Yu Jihyun , Kim Jiyoung , Song Hyun-Sup , Bae Jihong , Chae Seung Yun , Kim Seongryong , Jung Woomi , Kim You-Me
Published: February 2023

Tetraspanin family proteins form a rigid membrane structure called tetraspanin-enriched microdomain (TEM) on the plasma membrane and regulate multiple cellular processes. CD82, a tetraspanin protein, is highly expressed in various immune cells, but its role in immune responses has been poorly understood. In this study, we investigated the role of CD82 in T cells using CD82 knock-out (KO) mice. At steady state, CD82 KO mice had less memory CD8+ T cells in secondary lymphoid organs compared to wild type mice, which became more pronounced in aged mice. Upon in vitro T cell receptor (TCR) stimulation, CD82 KO CD8+ T cells showed defects in phosphorylation of TCR downstream signaling molecules, activation marker expression, proliferation, and cytokine production. By confocal immunofluorescence imaging, we found that TCR stimulation-induced translocation of the microtubule organizing center and formation of the immunological synapse was impaired in CD82 KO T cells. CD82 seems to recruit T cell polarity-regulating molecules such as Scribble via the PDZ domain-binding motif located in its C-terminus and mediate the T cell polarization. In association with the defective CD8+ T cell activation caused by CD82 deficiency, CD82 KO CD8+ T cells show less efficient anti-tumor immune responses than wild-type cells when transferred to tumor-bearing mice. Collectively, our study demonstrates that CD82 mediates the T cell polarization and is required for the optimal CD8+ T cell effector function.
IL-15 priming alters the regulation of IFN-γ production in NK cells
Authors: Cimpean Maria , Keppel Molly P. , Gainullina Anastasiia , Swain Amanda , Andhey Prabhakar Sairam , He Diana , Young Howard A. , Artyomov Maxim N. , Cooper Megan A.
Published: February 2023

Natural killer (NK) cells are innate lymphocytes important for the control of infections and tumors. Their effector functions can be triggered by inflammatory cytokines or engagement of ITAM-bearing activating receptors. We previously reported that IFN-γ production through ITAM signaling is metabolism-dependent in naïve NK cells. However, cytokine stimulation of naïve cells or priming with high-dose IL-15 prior to ITAM stimulation leads to metabolism-independent IFN-γ production. While both cytokine and receptor stimulation leads to similar IFN-γ protein production, only cytokines upregulate Ifng transcript, suggesting that protein production is translationally regulated in the receptor condition. Based on these observations, we hypothesized that IL-15 priming leads to transcriptional regulation of IFN-γ production in response to ITAM signaling. Indeed, IL-15 primed NK cells strongly upregulated Ifng transcript after receptor stimulation, rendering this receptor stimulation metabolism-independent. Inhibition of glycolysis with 2DG or mTOR with rapamycin during priming inhibited Ifng upregulation upon receptor stimulation and returned metabolism-dependent IFN-γ production. RNA-seq analysis of cytokine-, receptor-stimulated, and IL-15 cultured NK cells identified an association between glycolysis, mTORC1 signaling, and MYC targets with Ifng upregulation. Ongoing work aims to identify how ITAM signaling differs in IL-15 primed versus naïve NK cells. Our studies demonstrate distinct mechanisms of IFN-γ protein production in response to ITAM-mediated signaling between naïve and IL-15 primed NK cells.
TET2 controls differentiation of terminally exhausted CD8 T cells
Authors: Jordan Martha S , Drury Sydney , Giles Josephine R , Manne Sasikanth , Huang Hua , Chen Zeyu , Oldridge Derek , Wherry E. John , Baxter Amy E
Published: February 2023

CD8 T cells are critical for clearing infections and immune surveillance of cancers. However, in the setting of chronic viral infections and tumor growth, CD8 T cells become exhausted. T cell exhaustion (TEX) is characterized by a progressive increase of inhibitory receptor expression and concomitant diminishment of effector function. This diminished function renders the host unable to clear the virus or cancer. The TEX population is heterogenous and can be divided broadly into TEX progenitor and TEX terminal populations. TEX progenitor are self-renewing, responsive to PD-1 blockade and give rise to the later TEX subsets, including short-lived TEX terminal cells. Control of DNA demethylation is required in multiple settings for maintenance or loss of stem-like characteristics. We report that TET2, a methylcytosine dioxygenase that mediates active DNA demethylation, is required for TEX progenitors to differentiate fully into TEX terminal cells. TET2-deficiency during persistent viral infection resulted in a numerical loss of TEX terminal cells, while maintaining the TEX progenitor population. The TEX terminal cells that escaped this differentiation block retained some features of TEX progenitors, including decreased expression of several inhibitory receptors. TET2-deficient TEX progenitors responded to PD-1 blockade but were unable to establish expansion of TEX cells thought to be required for viral control. These data highlight the role of DNA methylation and TET2 in establishing TEX diversity.
Single-cell proteogenomics reveals that SLAM/SAP signaling regulates the development of innate-like γδT cell subsets with distinct TCR repertoires
Authors: Mistri Somen K , Hampel Kenneth J , Sidiropoulos Nikoletta , Andretta Emma , Dienz Oliver , Boyson Jonathan
Published: February 2023

Innate-like T cells are unusual T cells that are enriched in mucosal tissues, and which constitute a prominent source of pro-inflammatory cytokines like IL-17 and IFN-γ. While it is known that SLAM/SAP signaling is required for the development of innate-like iNKT and MAIT αβ T cells, far less is known about the role of this pathway in the development and function of γδ T cells. Here, we utilized a single-cell proteogenomics approach coupled with γδ V(D)J profiling to define the transcriptional landscape and developmental checkpoints of SAP-dependent γδ T cells. We found that SAP-dependent γδNKT TCRs utilized TRGV1 paired with TRAV15N-1 or TRAV15-1/DV6-1, and we identified two distinct developmental γδNKT stages in the neonatal thymus that were distinguished by SLAMF6 and SLAMF7 expression. Moreover, we found that a significant fraction of SLAMf1+innate-like γδT17 cells utilized TRGV4 and TRGV6, γ-chains paired with TRDV2 or TRDV5 δ-chains of limited diversity. Examination of SAP-deficient neonatal thymus revealed decreased numbers of mature SLAMF1+ γδT17 cells, which was associated with lower numbers of an invariant germline-encoded TRGV4/TRDV5 clonotype. Accordingly, we observed significant alterations in the SLAMF1+ γδT17 TCR repertoire in adult lung. We found that lung γδTIFN were CD44+CD45RB+CD27+ cells that co-expressed SLAMF6and SLAMF7, and that these cells predominantly utilized a diverse TRDV7 paired with TRGV4. Interestingly, we observed a specific decrease of these TRDV7+ γδTIFN cells in SAP-deficient mice, which we confirmed using qPCR. Altogether, these data indicate a crucial link between SLAM/SAP signaling and the development of functionally distinct innate-like γδ TCR clonotypes.
Mitochondrial calcium uniporter positively regulates phagocytosis-dependent activation of NLRP3 inflammasome
Authors: Dong Hong , Zhao Bao , Wen Haitao
Published: February 2023

Mitochondria have been shown to play an essential role in the innate immune function and inflammatory response. Mitochondrial Ca2+ signaling, which is controlled by mitochondrial Ca2+ uniporter (MCU), is a fundamental mechanism regulating mitochondrial metabolism. Accumulative evidence suggests MCU-dependent mitochondrial Ca2+ signaling may bridge the metabolic reprogramming and regulation of immune cell function. However, the mechanism by which MCU regulates inflammation and its related disease remains elusive. Here we report a critical role of MCU in promoting phagocytosis-dependent activation of the NLRP3 inflammasome by dampening phagosome membrane repair. Myeloid deletion of MCU (McuΔmye) resulted in decreased caspase-1 cleavage and IL-1β maturation in response to the challenge with silica and alum, but not ATP and nigericin, in lipopolysaccharide (LPS)-primed macrophages. Stimulation with poly(dA:dT) or flagellin induced a similar IL-1β release between McuΔmye and wild-type (WT) macrophages. Mechanistically, we detected an enhanced phagosomal recruitment of ESCRT (endosomal sorting complex required for transport)-III complex in McuΔmye macrophages after silica or alum stimulation compared to similarly treated WT cells. ESCRT-III complex is known to be essential for repair of phagosomal membrane after damage, suggesting that deletion of MCU might lead to improved phagosomal membrane repair to limit NLRP3 inflammasome activation. Furthermore, McuΔmye mice showed attenuated inflammatory response and tissue damage in a chemically induced colitis model. In sum, our results identify a novel function and mechanism of MCU in regulating phagocytosis-dependent NLRP3 inflammatory response.
Recognition of gasdermins by proteases
Authors: Xiao Tsan Sam , Liu Zhonghua , Wang Chuanping
Published: February 2023

The recognition and cleavage of gasdermin family members by proteases trigger the activation of the pore-forming activities of gasdermins. A prominent example is the targeting of gasdermin D (GSDMD) by inflammatory caspases-1/4/5/11 as an essential step in initiating pyroptosis following inflammasome activation. Previous work has identified cleavage site signatures in substrates such as GSDMD and inflammatory cytokines, but it is unclear if these are the sole determinants for caspase engagement. Here we describe structural studies of a complex between caspase-1 (CASP1) and the full-length GSDMD, which reveals that the cleavage site-containing linker in GSDMD adopts a long loop structure that engages the CASP1 active site. In addition, an exosite is observed between the caspase-1 L2 and L2′ loops and a hydrophobic pocket within the GSDMD C-terminal domain distal to its N-terminal domain. The exosites endows a novel function for the GSDMD C-terminal domain as a caspase-recruitment module, in addition to its role in autoinhibition. The dual site recognition may allow stringent substrate selectivity while facilitating cleavage and pyroptosis upon inflammasome activation. The residues forming the hydrophobic pocket are conserved between human and murine GSDMD, but not in GSDME, suggesting that the exosite interface may underlie the specific recognition of GSDMD but not GSDME by inflammatory caspases. Such mode of dual site recognition may be applicable to other physiological substrates of caspases.
Defining intracellular signaling pathways that dictate a universal antiviral response to conserved viral architectures in TLR2/6-dependent manner.
Authors: Hatton Alexis Alexandria , Luu Abby , Shepardson Kelly , Goodall Cheri , Wang Yang , Elkins Morgan , Rynda-Apple Agnieszka , Douglas Trevor
Published: February 2023

Previously we found that conserved viral architecture, namely the repeating protein subunit pattern (RPSP) of virus capsids is recognized by the cell-surface receptor TLR2/6. This RPSP is common to most, if not all virus capsids (vc) potentially serving as a conserved mechanism of extracellular recognition for viral pathogen-associated molecular patterns (PAMPs). TLR2/6-dependent recognition of vcRPSP induced an antiviral state that resulted in recruitment of immune cells and induction of type I IFN which we found to be required for reduced susceptibility to subsequent bacterial infections (BSI) in murine model of respiratory S. aureus infections. The improved BSI clearance after RPSP recognition was also seen in vcRPSP-treated PBMC-derived human macrophages, indicating the RPSP response is likely similar in humans. TLR2 is unique in that it recognizes an array of PAMPs, signals from the cell surface or endosome (proinflammatory or type I IFN response, respectively), and interacts with co-receptors, including CD14. Indeed, using bone marrow macrophages (BMMs) deficient in CD14, we found that CD14 was required for improved BSI clearance post-vcRPSP recognition. Interestingly, while blocking endosome maturation and acidification did not reduce RPSP-dependent improved clearance of BSI, using knockout BMMs we also found that MyD88, a canonical cell surface TLR signaling molecule, but also TRAM that is associated with endosomal signaling, were both required for improved BSI clearance post-vcRPSP recognition. This indicates that CD14, and the TLR signaling adaptors, MyD88 and TRAM play a role in the recognition and response to RPSP for a TLR2/6-vcRPSP mediated antiviral response but that endosome is not involved in the response.
TurboID biotin-tagging mass spectrometry identifies specific caspase-11-associated proteins regulating non-canonical inflammasome activation
Authors: Rabinovich-Ernst Orna , Bradfield Clinton , Yoon SungHwan , Armstrong Anthony , Katz Samuel , Nita-Lazar Aleksandra , Fraser Iain
Published: February 2023

While it has been demonstrated that cytosolic LPS can directly activate caspase11, the cellular processes regulating the non-canonical inflammasome response remain poorly defined. Caspase11 and caspase1 show substantial structural similarity, however, unlike the activation of caspase1 by NLR inflammasomes, there are no sensor or adaptor proteins known to be involved in transmitting cytosolic LPS signal to caspase11. Also, while caspase11 has been shown to associate with LPS, it lacks a characteristic LPS binding domain as observed in many other LPS binding proteins such as MD2 and LBP. Thus, we hypothesized that other effectors may be required to facilitate cytosolic LPS recognition. Moreover, the pathway is likely to be tightly regulated as caspase11 activation leads to highly inflammatory cell death.

To identify novel regulators of caspase11, we generated immortalized macrophages expressing a caspase11-TurboID-DHFR chimeric protein. The destabilizing domain was included to avoid cell death induced by caspase11 over-expression. We used a TurboID biotin-tagging MS assay to detect proteins in close proximity to caspase11 pre and post cytosolic LPS introduction. Importantly, the TurboID assay permits recognition of transient interactions, typically missed by traditional IP. To validate relevance of putative hits, we used siRNA knockdown in BMDM.

We’ve identified novel regulators specific for cytosolic LPS triggering. Several proteins interact with caspase11 only in the resting state, suggesting negative regulation to prevent pyroptosis. Among the identified regulators are kinases and proteins with pyrin and LRR domains, both common NLR features.

This work was supported by the Intramural Research Program of NIAID, NIH.
-Itaconate confers tolerance to late NLRP3 inflammasome activation
Authors: Bambouskova Monika , Potuckova Lucie , Paulenda Tomas , Kerndl Martina , Mogilenko Denis A. , Lizotte Kate , Swain Amanda , Hayes Sebastian , Sheldon Ryan D. , Kim Hyeryun , Kapadnis Unnati , Ellis Abigail E. , Isaguirre Christine , Burdess Samantha , Laha Anwesha , Amarasinghe Gaya K. , Chubukov Victor , Roddy Thomas P. , Diamond Michael S. , Jones Russell J. , Simons Donald M. , Artyomov Maxim N.
Published: February 2023

Itaconate is a unique regulatory metabolite that is induced upon toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLRP3 inflammasome activation after long LPS priming which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with iNOS and the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation which, if uncontrolled, can result in pyroptotic cell death and tissue damage.
Citrin, a mitochondrial transporter, interacts with NLRP3 and regulates inflammasome activity
Authors: Shuvarikov Andrey A , Davis Michael , Esser-Nobis Katharina , White Sean , Gale Michael
Published: February 2023

The NLRP3 protein is a key initiator of inflammation in humans. NLRP3 becomes activated by a multitude of danger signals, including microbial infection, metabolic dysfunction, and cell-internalized particulates. Upon activation, NLRP3 nucleates formation of a multiprotein complex called the inflammasome, in which caspase-1 activity mediates processing of the pro-inflammatory cytokines IL-1β and IL-18 and induces pyroptosis, a pro-inflammatory form of cell death. While multiple regulators of the NLRP3 inflammasome have been described, specific ligands of NLRP3 and its mechanism of activation remain largely unknown. We performed a proteomics screen using co-immunoprecipitation (IP) and mass spectrometry to identify cellular proteins that bind NLRP3. Using this screen, we identified multiple NLRP3 interactors, including a family of mitochondrial solute carrier proteins. Co-IP experiments verified that four of these carriers, namely CTP, ANT3, OGCP, and Citrin, specifically interact with NLRP3. Because of its linkage with known metabolic disease, we further assessed the role of Citrin, a calcium-binding aspartate/glutamate carrier, in inflammasome function. When ectopically expressed, Citrin induced IL-1β release in cells, while CRISPR/Cas9 knockout of Citrin from THP-1 macrophages significantly abrogated NLRP3-dependent pyroptosis, IL-1β release, and ASC speck formation. This regulatory activity of Citrin was dependent on its calcium-sensing N-terminal domain. Importantly, this regulation was specific to NLRP3 and did not affect the AIM2 inflammasome or other cell death pathways. Altogether, our studies reveal a novel role for Citrin in regulation of the NLRP3 inflammasome and inflammatory cytokine release.
Liver-localized CD8 T cells do not need Levy walks to be efficient killers
Authors: Ganusov Vitaly V , Rajakaruna Harshana , O’Connor James , Cockburn Ian A
Published: February 2023

Pathogen-specific CD8 T cells face the problem of finding rare cells that present their cognate antigen either in the lymph node or infected tissue. To optimize the search for rare targets it has been proposed that T cells might perform a random walk with long displacements called Levy walks enabling superdiffusive behavior and shorter search times which may be evolutionary selected. We examined the behavior of activated CD8 T cells in the liver where both the movement of the cells and the underlying structural constraints can be clearly defined. We show that Plasmodium-specific liver-localized CD8 T cells perform short displacement, Brownian-like walks and yet display transiently superdiffusive displacement, the cardinal feature of efficient Levy walks. Because liver-localized CD8 T cells are mainly associated with liver sinusoids, simulations of Brownian or Levy walkers in structures derived from the liver sinusoids illustrate that structure together with preferred forward movement results in superdiffusive movement. Interestingly, Brownian walkers require less time to find a rare target when T cells search for the infection in physiologically-derived liver structures. Our results strongly suggest that observed patterns of movement of CD8 T cells are likely to result from a combination of a cell-intrinsic movement program, physical constraints imposed by the environmental structures, and other environmental cues. Future work needs to focus on quantifying the relative contributions of these factors to the overall observed movement patterns of biological agents.
Immunopathology and Meningitis During Late-Onset Gut-Originating Neonatal Sepsis
Authors: Yokanovich Lila T , Greenfield Kara , Knoop Kathryn
Published: February 2023

Late-onset neonatal sepsis (LOS) is a bacterial bloodstream infection that occurs during the first two months of life, and remains a significant contributor to neonatal mortality. In addition to the number of clinical challenges these patients face in the short term, neonates who survive LOS can experience a number of long-term adverse outcomes, such as impaired cognitive development. A major obstacle in LOS research remains the lack of an animal model that successfully recapitulates clinical LOS. We have developed a mouse model that replicates the clinical finding that a subset of LOS infections results from enterally-derived pathogens translocating from the intestine. In this model, pups develop sepsis-like disease upon oral gavage of virulent E. coli species. This translocation results in immune cell infiltration and bacterial presence in many peripheral sites, including the meninges. Specifically, we found infiltration of monocytes, and neutrophils into the meninges, along with increased numbers and activation status of both αβ+ and γδ+ T cells. Meningitis could be replicated by LPS alone, suggesting systemic bacterial presence contribute to infiltration into the meninges. Interestingly, mice older than 10 days did not develop meningitis in response to LPS, suggesting neonatal pups are uniquely susceptible to bacterial-initiated meningitis. Furthermore, this meningitis is induced partially by adaptive immune cell components, as no neutrophil or monocyte infiltration was seen in RAG KO mice following LPS injection. This study provides novel insights into the clinical reported complication of meningitis during LOS and suggests a unique contribution of adaptive lymphocytes in the neonate during this systemic infection.