bluuebunny/crossref_metadata_2025_split huggingface dataset:
UBL5 Modulates Expression of Crucial TLR Pathway Genes and Inflammasome Activation in Human Macrophage Cells
Authors: John Sinu P , Jean-Pierre Makheni , Katz Samuel , Fraser Iain D C 
Published: August 2023

Abstract
We conducted a genome-wide siRNA screen for regulators of LPS-driven TNF expression in macrophages. We used a splicing-independent reporter for the screen, and while we identified very few core spliceosome components among the strongest screen hits, we observed a clear enrichment for peripheral splicing regulators. Among these, a novel putative splicing regulator, UBL5, gave a strong screen phenotype, and a UBL5 shRNA-expressing cell line showed a selective defect in LPS-driven gene expression. While many genes were induced normally by LPS in the UBL5 deficient cells, a subset of genes, including core components of the LPS-activated TLR4 signaling pathway, were substantially diminished in UBL5-deficient cells. We conducted both Illumina and Pacbio long-read sequencing, and we observed marked defects in the LPS-induced splicing of numerous genes, however the primary impact on known components of the TLR4 pathway was observed at the level of overall gene expression rather than alternative splicing. Our data suggests that the putative splicing regulator UBL5 also has key roles in the maintenance and induction of gene expression for several core components of the TLR4 signaling pathway. Key role of UBL5 in the induction of TLR4 signaling pathway was further evidenced by the defective NLRP3 inflammasome activation and protection of OC43 coronavirus infection in UBL5-deficient cells.
This work was supported by the Intramural Research Program of NIAID, NIH
Intramural Research Program of NIAID, NIH
Characterization of Calcium signals and oscillations in dendritic cells in vivo in response to pathogens
Authors: Groot N , Sathuvalli Pranav , Barbet Gaëtan 
Published: August 2023

Abstract
Dendritic cells (DCs) are disseminated throughout organisms and serve as sentinels, organizing immune responses to infection, tissue damage, and cancer. DCs play a crucial role bridging innate and adaptive immune systems and elicit a tailored adaptive immune response to the types of threats they encounter. Manipulation of DCs is critical for vaccination, and DC-based immunotherapies have been tested in clinical trials as anti-cancer therapeutics and for treatment of infectious diseases. Calcium, a ubiquitous secondary messenger, regulates many critical effector functions of DCs like phagocytosis, maturation, migration, and cytokine production. However, regulation and mobilization of calcium in DCs remains poorly understood. Using microscopy, our preliminary experiments unveiled for the first time an oscillatory calcium signal in DCs in response to whole bacteria; our in vitro studies show that oscillatory calcium signaling patterns are specific to the type of bacteria. To decipher calcium signals induced by bacterial stimuli in DCs intravitally, we generated CD11c-Salsa6f reporter mice that express the fluorescent protein (FP) construct tdTomato fused to GCaMP6f in CD11c expressing cells. Hence, expression of TdTomato, a red FP, highlights cell shape, and GCaMP6f, which fluoresces green upon calcium binding, visualizes intracellular calcium levels. We are using CD11c-Salsa6f mice to observe calcium signals in DCs in the periphery and in secondary lymphoid organs using two photon microscopy. This model, perfectly suited for intravital two photon microscopy, allows us to unveil bacteria-specific calcium signals in DCs in in their natural microenvironment.
Supported by a grant from the NIH (1T32GM139804-01)
Pharmacological activation of STING agonists blocks schistosome egg-mediated pro-inflammatory cytokine production
Authors: Liu Pengyu , Jump Kaile , Kalantari Parisa 
Published: August 2023

Abstract
Infection with the helminth parasite Schistosoma mansoni causes morbidity and mortality via a pathogenic host CD4 T cell-mediated immune response directed against parasite egg antigens. Here we demonstrated that low-pathology BL/6 mice lacking Stimulator of Interferon genes (STING) exhibited markedly enhanced hepatic granulomatous inflammation associated with significantly increased Th17 and diminished Th2 cytokine responses. Additionally, CBA mice tend to have a severe form of liver granulomatous inflammation due to a profound decrease of type I Interferon production. Since CBA bone marrow-derived dendritic cells have lower expression of STING, we hypothesized that STING agonists such as diABZI-3 and DMXAA would control the pro-inflammatory cytokine production and inflammation by activating STING expression and activation. Both diABZI-3 and DMXAA induced robust Interferon (IFN)β production, while IL-1β and IL-17 induced by schistosome eggs were impaired in egg-stimulated BMDCs. Our study suggest that STING agonists may serve as a novel therapeutic strategy to restrain schistosome immunopathology. This work is supported by NIAID grant R01 AI148656 to PK.
This work is supported by NIAID grant R01 AI148656 to PK.
Distinct regulation of CD4+ T-cell function in cancer and autoimmunity by canonical NF-kappaB subunits
Authors: Lautraite Raphaelle , Lalle Guilhem , Klein Ulf , Ghosh Sankar , Grinberg-Bleyer Yenkel 
Published: August 2023

Abstract
Whereas CD4+ effector T cells (Teff cells) are critical to limit tumor progression, uncontrolled Teff cell activation can also lead to autoimmunity; however the molecular basis of these functions are elusive. Here we investigated the selective functions of distinct NF-kappaB transcription factors in the function of Teff cells.
Using mice carrying conditional ablation of the canonical NF-kB subunits RelA and c-Rel, in Teff cells, we found that RelA, rather than c-Rel, shaped the transcriptome of Teff cells in the steady-state and their ability to polarize toward the TH17 lineage, by directly binding to genes encoding cytokines and other transcription factors. Similar conclusions could be reached using CRISPR/Cas9-edited primary human Teff cells. Consistently, RelA – but not c-Rel-deficient mice were fully protected against neuro-inflammation in a murine model of multiple sclerosis. Mechanistically, TH17 priming and in situ reactivation, were impaired in RelA-deficient mice. Next, we investigated the roles of NF-kB in cancer immunity. Strikingly, Teff-restricted ablation of c-Rel, but not RelA, impaired their accumulation and function in the microenvironment of melanoma, resulting in enhanced cancer burden. In addition, c-Rel-deficiency abrogated the therapeutic effect of PD-1-blockade. Accordingly, in melanoma patients, the expression of c-Rel-dependent genes was associated with prolonged survival and better response to immunotherapy.
Together, our data highlight novel, context-dependent, regulators of Teff cell function. We propose a division of labor between the different subunits of the NF-kB pathway, paving the way to subunit-targeted immunotherapies in autoimmunity and cancer.
LncRNA U90926 is induced in activated macrophages, is protective in endotoxic shock, and encodes a novel secreted protein
Authors: Sabikunnahar Bristy , Varnum Stella , Caldwell Sydney , Hogan Tyler , Cousens Phoebe , Ballif Bryan , Krementsov Dimitry 
Published: August 2023

Abstract
The most commonly transcribed non-coding RNA class is long non-coding RNAs (lncRNAs) in mammalian genomes, yet the majority of them remain understudied. In our studies, we have identified a mouse lncRNA of unknown function named U90926, whose expression is greatly induced upon TLR-stimulation in macrophages in a Myd88- and p38 MAPK-dependent manner. To characterize this gene, we have generated a global U90926-knockout (U9-KO) mice in C57BL/6J background using CRISPR-Cas9. Surprisingly, we found minimal effects of U90926 deficiency on gene expression and basic immune effector function in cultured LPS-stimulated macrophages. Given the lack of macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol and associates with ribosomes. Using an epitope-tagged construct, we found that U90926 contains a functional open reading frame that encodes a novel glycosylated protein (U9-ORF), which is secreted from the cell. We have confirmed the identity of U9-ORF protein by mass spectrophotometry. We generated a U9-ORF-specific monoclonal antibody which detected the endogenous U9-ORF protein in LPS-stimulated macrophages. In further characterization, we found that U9-KO mice exhibited increased sickness responses and mortality in an in vivo model of LPS-endotoxemia. Mechanistically, serum levels of IL-6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that inflammation induced expression of U90926 decreases endotoxic shock susceptibility, potentially mediated by the novel U9-ORF protein secreted by activated macrophages.
NIH NIAID (R21AI151116)
LKB1 regulates macrophage metabolism and functional polarization in immunomodulation
Authors: Yang Jing , Xu Chengxian , Lechner Joseph , Walls Haley , Yang Kai 
Published: August 2023

Abstract
Macrophages play critical roles in maintaining tissue homeostasis and modulating immune responses. In response to microenvironmental cytokines, macrophages coordinate cellular signaling networks and diverse transcriptional programs to dictate their phenotypical and functional heterogeneity. For instance, LPS/IFNγ and IL-4 induce classically (M1) and alternatively (M2) activated macrophages, respectively. Remodeling cellular metabolism has been highlighted a key process underlying macrophage functional polarization. However, the precise mechanisms coordinating macrophage metabolism and polarization remain elusive. We report here that the kinase LKB1, a well-known negative regulator of mTOR signaling pathway, serves as a metabolic checkpoint that connects cellular metabolic reprogramming to functional polarization in macrophages. We found that genetic depletion of LKB1 in macrophages polarized their differentiation towards M2-like macrophages, characterized by enhanced expression of M2-associated markers, independently of mTOR and STAT6 signaling pathways. In contrast, loss of LKB1 had no substantial impact on M1-like macrophages. Moreover, LKB1-defienct macrophages orchestrated reprogramming of mitochondrial metabolism to facilitate M2 polarization and tumor immune evasion. Collectively, our studies establish a previously unappreciated role for LKB1 in connecting macrophage metabolism and functional polarization in modulating tumor immunity.
This work was partially supported by the Herman B Wells Center and Riley Children’s Foundation.
T cell expansion is regulated by mutual regulation of ribosome biosynthesis and mTORC1 activity
Authors: Obst Reinhard , Rosenlehner Teresa , Pennavaria Stefanie , Akcabozan Batuhan , Kadam Pallavi , Parlato Rosanna , Straub Tobias , Kranich Jan 
Published: August 2023

Abstract
The clonal expansion of T cells entails a swift and substantial repurposing of cellular physiology towards anabolic pathways to allow for a massive increase of cellular biomass for rapid cell division. Ribosomal biosynthesis is an essential and highly energy demanding process driven by the actvities of all RNA polymerases and ~300 proteins processing and modifying the rRNA and its precursors. How ribosomal biosynthesis is integrated into the process of T cell activation is incompletely understood. We have investigated how the mTOR complexes affect ribosomal biosynthesis, T cell proliferation and TCR signaling. We found that the proliferation of mTORC1-but not mTORC2-ablated T cells is slowed from three divisions per day to one with each consecutive division being delayed, mostly at the G1/S transition. mTORC1 signaling is affected by conventional TCR signaling pathways via PI3K while TCR sensitivity is hampered by mTORC1 inhibition. Upon activation the amount of RNA per cell increases at least 10-fold, which is reduced by half in mTORC1-ablated cells. RNA-seq data showed that this reduction affects all RNA biotypes and that rRNA processing is ineffective in activated T cells. Using 5’ ETS FISH probes of nascent pre-rRNA in imaging cytometry revealed that mTORC1 but not mTORC2 ablation reduces the number and the expansion of nucleoli while translation was not yet affected. Inhibiting RNA Polymerase I by chemical and genetic means blocked not only proliferation but mTORC1 signaling as well. Our data show that TCR signaling, mTORC1 activity and ribosomal biosynthesis are tightly interlinked and regulate each other.
Supported by grants from the German Research Council (CRC1054-B07, OB150/7-1).
Tumor cell-macrophage crosstalk promotes immunotherapy resistance via lipid metabolism
Authors: Liang Zhixian , Cao Jianquan , Tang Wenshu , Wu Haoran , Tu Yalin , Xiong Zhewen , Zhang Lingyun , Zhou Jingying , Chan Stephen L , Cheng Alfred S L 
Published: August 2023

Abstract
Immune-checkpoint blockade (ICB) therapies have transformed the treatment landscapes of hepatocellular carcinoma (HCC). However, the immunosuppressive tumor microenvironment (TME) constructed by tumor cells restricts the responsiveness of ICB therapies to a minority of patients. Triggering receptor expressed on myeloid cells-2 (TREM2) counteracts inflammation and maintains metabolic fitness in myeloid cells. Single-cell RNA-sequencing analysis of our pembrolizumab study (NCT03419481) identified a subset of tumor-associated macrophages over-expressing TREM2 in non-responders. Consistently, our syngeneic ICB-resistant HCC mouse models verified a group of Trem2+ myeloid cells, which were accumulated in the lipid-rich TME of ICB-resistant tumors. Compared to the parental ICB-sensitive tumor cells, the conditional medium (CM) of ICB-resistant tumor cells significantly increased Trem2 expression in macrophages, which was abolished when lipids in the CM were depleted. Notably, lentivirus-mediated Trem2 ablation reduced lipid accumulation in TME and overcame anti-PD-1 resistance with increased cytotoxic CD8+ T cell infiltration. In conclusion, our study highlights TREM2 as an immuno-metabolic target to enhance HCC immunotherapy.
This study is supported by Collaborative Research Fund (C4045-18W) and Li Ka Shing Foundation. Z.L. is supported by Hong Kong PhD Fellowship Scheme.
TGFβ trained immunity in mast cells: do inflammasomes matter?
Authors: Ashbaugh Alexandria M , Lyons David O , Weisen Arianna L , Rasé Viva J , Haughian James M , Pullen Nicholas A 
Published: August 2023

Abstract
Mast cell (MC) contributions to solid cancer microenvironments are controversial and seem tissue dependent. TGFβ is a potent MC chemokine abundantly expressed by high-grade cancers. Previous work reported that TGFβ alone induces biologically relevant expression of IL-6 from MC, which is potentially important for reciprocal paracrine interactions with cancer cells and other leukocytes. Therefore, the objective of the present study was to examine MC in an immunocompetent, high-grade model of breast cancer. Using the 4T1 system in BALB/c mice, we compared mast cell infiltration into primary tumors of wild-type versus 4T1 cell-specific knockouts of IL-6. We also hypothesized that TGFβ institutes a training effect on MC like other metabolically driven mechanisms involving the NLRP3 inflammasome. Using a mixture of flow cytometric and qPCR assays we explored potential MC training in the context of TGFβ.

Results
Contrary to our prediction, MC infiltration into IL-6KO tumors was significantly increased compared to WT. Studies of classic intracellular training mechanisms in response to TGFβ were mixed. Glucose uptake, glycolytic capacity, mitochondrial potential, and ASC speck formation were unaffected by TGFβ, but IL-6 expression increased as previously seen. Additionally, we were unable to detect substantive NLRP3 expression in MC regardless of condition. Despite these results, we were surprised to observe robust increases in expression of IL-1β, IL-18, and caspase-1 with TGFβ stimulus. We conclude that MC are actively recruited to 4T1 tumors dependent on IL-6 (and TGFβ), MC produce select cytokines in response to TGFβ, yet the mechanism explaining this does not involve the NLRP3 inflammasome and remains unresolved.
Reprogramming of tumor-induced emergency granulopoiesis overcomes immune checkpoint therapy resistance
Authors: Tang Hui , Xie Ping , Ahn Ji Hae , Choi Mi Ran , Fan Jie , Orpano Rachel , Zhang Bin 
Published: August 2023

Abstract
In established cancer in mice and humans, “emergency” myelopoiesis usually occurs to facilitate malignancy progression. Neutrophils and immature myeloid cells accumulated in the bone marrow, periphery and tumor are potent mediators of immunosuppression and tumor promotion. Here, we demonstrated that Cysteinyl leukotriene receptor 1 (CysLTR1), a key player for asthma, and other inflammatory conditons, sustained tumor-induced emergency granulopoiesis. Genetic ablation and pharmacological inhibition of CysLTR1 resulted in diminished tumor growth with enhanced antitumor immunity. The anti-tumor effect of CysLTR1 inhibition was linked with transcriptomic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor immune phenotype; this process required the distinct transcriptional factors to specifically dictate neutrophil progenitor commitment and differentiation in association with controlled degranulation. Furthermore, single-cell RNA sequencing analysis provided a comprehensive transcriptional landscape of neutrophil maturation, function and fate decision in tumor bearers. CysLTR1-mediated changes in the differentiation and degranulation also occurred in human neutrophils and correlated with the survival and ICT responsiveness in cancer patients. Consistently, targeting CysLTR1 with clinically available antagonists overcame resistance to immune checkpoint PD-1 inhibitors in several mouse tumor models. These findings thus have important implications for understanding the abnormal myelopoiesis during cancer progression and suggest new therapeutic approaches to improve ICT.
Supported by grants from NCI (R01CA250101) and DoD (W81XWH-20-1-0024).
NCG-M humanized mice - an excellent model for human immune reconstitution of myeloid lineages
Authors: Chen Santi Suryani , Wang Huiyi , Fan Jialu , Guo Shiying , Zhang Mingkun , Li Shuai , Li Zhiying , Moore Mark , Zhao Jing , Xiang Gao , Ju Cunxiang 
Published: August 2023

Abstract
Severely immunodeficient mice engrafted with human hematopoietic stem cells (HSC) have been extensively used in immuno-oncology studies to evaluate the efficacy of cancer therapies. However, because of species differences in the immune system between human and mouse, murine cytokines provide limited support to human immune cells, thus human immune system mice have limited human immune cell engraftment. Increasing evidence has shown that myeloid cells, especially macrophages and dendritic cells, are critical for the induction of anti-tumor immunity. We developed a mouse model, NCG-M, that can support human T, B, NK, and various myeloid and granulocyte cells such that in vivo evaluation of agents that require the interplay between these immune cells can be examined. This model was genetically engineered on the severely immunodeficient strain NCG and can produce human granulocyte/macrophage colony-stimulating factor 2 (GM-CSF, also known as CSF2), interleukin-3 (IL-3) and stem cell factor (SCF, also known as KITLG). Upon human CD34+ HSC cell engraftment, a significant increase in myeloid lineage cells, such as granulocytes, monocytes, neutrophils, macrophages, dendritic cells, and mast cells, was observed in the NCG-M cohort compared to NCG mice. The NCG-M mouse also supports the development of human T cells, and preliminary data showed increased B cells and NK cells. Importantly, the increased efficiency of immune cell reconstitution did not affect the morbitity and mortality of this mouse. The NCG-M is an appropriate mouse model for studying the efficacy of therapeutic agents that require human T cells and myeloid cells.
Cancer promotes immune escape via transdifferentiating B cells into macrophage-like cells
Authors: Biragyn Arya , Chen Chen , Park Bongsoo , Bodogai Monica , Lee Jung-Min , Beerman Isabel 
Published: August 2023

Abstract
Previously we reported that breast cancer uses the generation of regulatory TGFb+ B cells to facilitate its metastasis via inducing FoxP3+Tregs and educating MDSCs 1,2. To do this, the cancer secretes TSLP to cause premature emigration of B-cell precursors from the bone marrow (BM) and accumulation in the spleen as a source of tBregs 3. However, it remains unclear why other cancers similarly cause premature emigration and accumulation of B-cell precursors, although they do not generate tBregs. Here, we report that this is to use these B-cell precursors to generate macrophage-like cells (termed B-MF) 4. We found that cancer transdifferentiate a small but bona fide Csf1R+Pax5Low subsets of BM B cells(pre-B and immature IgM+ B cells) into B-MF in the tumor. This alternative pathway of the generation of tumor-associated macrophages (TAM) is phenotypically and functionally distinguishable from that of BM monocyte-derived TAMs, as B-MFs more efficiently phagocytize apoptotic cells, suppress proliferation of T cells, and induce FoxP3+regulatory T cells. Our modeling studies in mice with cancer reveal that B-MFs support tumor progression and metastasis presumably by retarding tumor-infiltrating IFNγ+CD4+ T cells. Since B-MF-like cells and their transcriptional signature can be detected in patients with breast and ovarian cancers, we think that this transdifferentiation pathway is clinically relevant and hence could serve as an immunotherapeutic target.
This work was supported by the Intramural Research Program, NIA/NIH.
Inhibition of TCR-MHC-II interactions abrogates Treg-mediated immune suppression and augments anti-tumor immunity
Authors: Panda Abir Kumar , Shevach Ethan M 
Published: August 2023

Abstract
CD4+Foxp3+ Tregs play a non-redundant role in controlling autoimmunity. Continuous TCR signaling and the availability of exogenous IL-2 from CD4+ effectors regulate the size of the Treg pool and their suppressive function in the steady state. Homeostatic proliferation of Tregs and memory phenotype (MP) CD4+Foxp3−T cells depends on CD80/CD86-CD28 signaling. Acute blockade of TCR-MHC-II interactions in vivo by anti-MHC-II mAb significantly enhances memory phenotype (MP) Treg, CD4+, and CD8+ T cell expansion by a CD28-dependent pathway. IL-15 trans presentation by APC also contributes to Treg, MP CD4+ and MP CD8+ proliferation following blockade of TCR-MHC-II interaction by a CD122-dependent pathway. Combined CD28 activation and IL-15 stimulation together with IL-2 deprivation secondary to MHCII-TCR blockade result in decreased expression of CD25, but enhancement of Foxp3 expression by Treg. In addition, Treg exhibit decreased pAKT/NFAT signaling, but enhanced pSTAT5/mTOR signaling. Mice transplanted with several different transplantable tumors manifest enhanced tumor immunity in the presence of MHCII-TCR inhibition. Our results demonstrate that in the absence of TCR-MHC-II interactions, Treg proliferate, but lose their identity and manifest disabled suppressor function resulting in enhanced innate and adaptive immunity against cancer progression.
This work was supported by the Intramural Research Program of NIAID, NIH.
This work was supported by the Intramural Research Program of NIAID, NIH.