bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) of peripheral blood to dissect the effect of different spondyloarthropathies
Authors: Gill Tejpal , Davin Sean , Ogle Kimberly , Ahuja Jasvinder , Choi Dongseok , Martin Tammy , Rosenbaum James T
Published: August 2023

Spondyloarthritis is a family of immune mediated inflammatory disorders with overlapping articular and extraarticular clinical manifestations and a strong association to human leukocyte antigen (HLA)-B27. Despite a strong association of HLA-B27 with prototypic spondyloarthropathies such as axial spondyloarthritis (axSpA) and acute anterior uveitis (AAU), the underlying mechanisms are not known. Here, we aim to determine whether HLA-B27 associated axSpA and AAU are associated with common or divergent immunological markers in the blood. Previously, we did single cell RNA-seq analysis on a small number of patients with AAU and axSpA, which revealed increase in T cell, MAIT cells and NK cell clusters in AAU patients, whereas axSpA patients had increase in MAIT cells, gd-T cells, ILCs and Tregs and decrease in naïve CD8 T cell clusters, suggesting that distinct signatures maybe associated with axSpA and AAU. Changes in ILCs or MAIT cells may suggest involvement of the gut mucosal surface in pathogenesis. This is consistent with our previous studies showing gut microbial perturbation in clinical samples and in rat model of spondyloarthritis.

Leveraging on our preliminary data, we are performing CITE-seq to capture paired transcriptome and cell surface epitope expression in individual PBMCs from 5 subjects each with axSpA, AAU and axSpA+AAU in comparison with healthy individuals (5 HLA-B27 negative and 5 HLA-B27 positive). Since, HLA-B27 allele is also present in healthy population, these analyses will allow us to dissect the effect of host genetics from the disease. These studies represent potential advances in the understanding of spondyloarthritis and may reveal genetic and disease biomarkers associated with axSpA and AAU.

TG (principal investigator) is a recipient of research awards from the Spondylitis Association of America, SPARTAN (Spondyloarthritis Research and Treatment Network), GRAPPA (Group for Research and Assessment of Psoriasis and Psoriatic Arthritis), The Collins Medical Trust, Medical Research Foundation of Oregon and, Innovation Award from OHSU. This work was funded in part by the Grandmaison Fund for Autoimmunity Research. JTR is a recipient of the Innovative Research Award, Rheumatology Research Foundation.
Regulation of respiratory CD8+ T cell immunity by suppressive monocyte-like dendritic cells
Authors: Oliva Kimberly , Reagin Katie , Klonowski Kimberly D
Published: August 2023

Active immune suppression can mediate the balance between protective cellular immunity and harmful immunopathology. This suppression can occur locally, at an infection site, or in regional draining lymph nodes (dLN). Immune regulation is of particular importance in sites such as the lung where aberrant immunopathology can result in loss of tissue function. We have recently identified a novel population of CD11b+CD103+CCR2+ monocyte-like dendritic cells (moDCs) which directly suppress CD8+ T cell proliferation in vitro. Respiratory infection with RNA viruses recruits these moDCs either exclusively to the dLN (after vesicular stomatitis virus infection) or both the dLN and site of viral replication (after influenza infection). Exclusive depletion of moDCs from the dLN using CCR2-DTR bone marrow chimeras results in enhanced respiratory CD8+ lung tissue resident memory cell formation. By contrast, depletion of moDCs from both the dLN and respiratory tract following influenza infection results in enhanced respiratory CD8+ T cell responses coupled with fatal immunopathology. Together, these data suggest that suppressive moDCs govern key aspects of respiratory immunity by regulating the function and development of long-lived CD8+ T cells, thereby balancing immunity and adverse pathology in the context of viral infection.

Supported by grant from NIH (R21 AI131093)
Resilient CD8+ T cells maintain a high cytotoxic capacity by balancing ROS and energy needs via ME1 upregulation
Authors: Gicobi Joanina K , Mao Zhiming , DeFranco Grace , Li Ying , Liu Xin , Hirdler Jacob H , Vianzon Vianca , Dellacecca Emilia R , Hsu Michelle A , Barham Whitney , Kim Yohan , Abraha Feven , Harmsen William , Yan Yiyi , Dronca Roxana S , Zhu Mojun , Markovic Svetomir , Mansfield Aaron , Lin Yi , Wu Xiaosheng , Owen Dawn , Grams Michael P , Orme Jacob J , Lucien Fabrice , Zeng Hu , Park Sean S , Dong Haidong
Published: August 2023

Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignancies. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+ CD8+ T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+ CD8+ T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysis-independent manner while concurrently curtailing excessive ROS in activated CD8+ T cells; and expands CX3CR1+NKG7+ effector CD8+ T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8+ T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion.

Supported by grants from Mayo Clinic Foundation and NIH (R01CA 256927-2)
Investigation of the effect of 5’UTR on immunogenicity in coxsackievirus VP1 mRNA vaccine
Authors: Li Jiali , Liu Longding
Published: August 2023

Hand, foot and mouth disease (HFMD) is a group of common infectious diseases caused by various enteroviruses, which are transmitted through gastrointestinal tract, respiratory tract and contact. The mRNA vaccine is safe, effective, simple to produce and easy to promote, and has attracted much attention in recent years. Starting from the screening modification of the 5’UTR of antigen nucleic acid sequence, the mRNA translation efficiency was improved by introducing the antigen epitope cleavage processing signal through the untranslated region screening modification, and the nucleic acid vaccine design with stable translation characteristics and effective control of inflammation homeostasis was studied to provide a reference for the molecular design of future mRNA vaccines. In this study, 5’UTRs of different lengths of human β-globin origin were successfully constructed in front of the gene sequences of CA6-VP1 and CA10-VP1; the target gene sequences were transcribed in vitro with capping and tailing reactions to obtain structurally complete mRNA sequences; 293T cells were transfected and Western Blot assays were performed to find that the target genes with the modified 5’UTRs attached The mRNA was able to express the protein correctly and stably, and the protein expression was more compared with the original sequence without the modified 5’UTR. The human β-globin 5’UTR is useful for improving the translation efficiency and protein stability of the target gene mRNA. Further experimental evidence is needed to assess its induction of immune responses in vivo.
Reducing PI3Kδ signaling in a dose-dependent manner promotes human T cell stemness
Authors: Li Matthew , Rivera Guillermo Omar Rangel , Paulos Chrystal Mary
Published: August 2023

Adoptive T Cell Therapy (ACT) is a promising type of highly personalized immunotherapy against cancer. However, during the clinical production of T cell products for ACT, the cells experience cues that lead to early senescence and poor quality. Thus, ways to improve T cell quality for ACT is warranted. Previous mouse data indicate that inhibiting the PI3Kδ pathway in T cells using Idelalisib, an FDA-approved selective inhibitor of the δ subunit of PI3K, produces less differentiated T cells with enhanced efficacy against cancer. We hypothesized that Idelalisib treatment in human T cells would recapitulate a dose-dependent acquisition of stemness features. Mouse T cells, bulk donor human peripheral blood mononuclear cells (PBMCs), and isolated human CD3+ T cells were cultured in vitro for seven days with dose escalation of Idelalisib. We counted the number of T cells to track expansion and assessed the phenotype through flow cytometry. Reducing PI3Kδ signaling produced a less differentiated phenotype in both murine and human CD3+ T cells. Human CD3+ T cells had lower expression of TCF-1 and LEF-1, transcription factors regulating stemness, due to Idelalisib. However, other markers (CD62L and CD45RA) still suggested dose escalation up to 25 μM led to less differentiation. PBMCs and CD3+ T cells treated with dosages of Idelalisib above 10 μM had limited expansion and decreased viability compared to mouse T cells. Altogether, our data demonstrate that blocking PI3Kδ with Idelalisib in human T cells supports acquisition of stemness features.
The role of specific IL-27-producing DC subsets on the magnitude of the CD8 T cell response to subunit vaccines
Authors: Moreno Miguel Guerrero , Manalastas John J , Kedl Ross M , Klarquist Jared
Published: August 2023

The generation of long-lasting, adjuvant-elicited cellular immunity is a significant unmet medical need. Specifically, the use of adjuvants to induce clinically relevant CD8 T cells responses would have great utility in chronic infectious diseases and cancer. Our previous data has shown a unique requirement for T cell intrinsic IL-27 signaling in their response to adjuvanted subunit vaccination. Indeed, the magnitude of IL-27p28 production by cDC1s (CD3/B220−, CD11c+, XCR1+) actually predicts the magnitude of the CD8 T cell response generated. cDC1s are also required to present subunit vaccine-derived antigens to responding CD8+ T cells. However, whether IL-27 needs to be produced by the same cDC1s presenting antigen, or whether non-antigen presenting APCs can produce IL-27 in trans, for the maximal CD8+ T cell response to subunit vaccination is as of yet unknown. Here, we confirm that IL-27p28 is required for maximal adjuvant-elicited CD8 T cell responses. Furthermore, in mice with Il-27p28 deleted in only CD11c+ cells (IL-27p28fl/fl x CD11c-cre) the CD8 cell response to vaccination phenocopied the defective response seen in mice fully deficient in IL-27p28, EIB3, and IL-27 receptor. Interestingly, the distribution of MPECs (CD127hi) and SLECs (KLRG1hi, CD127lo) in these mice as compared to WT (C57BL/6) controls did not vary. Numerous myeloid subsets (cDC2, monocytes) as well as B cells also make IL-27. Utilizing cell-specific cre expression transgenes in conjunction with the floxed IL27p28, we have characterized the cellular requirements for IL-27 in the adjuvant elicited CD8+ T cell response.
Elucidation of regulatory mechanisms in the activation of dendritic cells by combined stimuli with PRR ligands and sulfated polysaccharide fucoidan
Authors: Miyazaki Yoshiyuki , Satoyama Toshiya , Nakano Hayato , Takeuchi Shugo , Takeuchi Hideaki , Tachikawa Daisuke
Published: August 2023

Fucoidan, a series of natural high-molecular weight sulfated polysaccharides derived from brown algae, has been reported to have various physiological effects such as antitumor, antiviral, and immunomodulatory activities. In a previous study, we demonstrated that fucoidan from Cladosiphon okamuranus (Okinawamozuku) was effectively activate murine macrophage-like cell line RAW264 in synergistic action with dectin-1-stimulating beta-glucan from Saccharomyces cerevisiae. In this study, we further examined their synergistic mode of action of Okinawamozuku-derived fucoidan in cooperate with pathogen stimulation in terms of dendritic cell activation. After myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were differentiated by culturing bone marrow cells isolated from C57BL/6J mice with Flt3 ligand, cytokine production upon stimulation with fucoidan and various pathogen components (pattern recognition receptor, PRR ligands) was measured. As the results, fucoidan alone enhanced the production of interferon (IFN)-gamma by the Flt3-L-induced mixed BMDCs, and besides, fucoidan synergistically augmented IFN-gamma production with Pam3CSK4 (bacterial TLR1/TLR2 ligand) or Poly(I:C)/LyoVec (viral RIG-I ligand). On the other hand, their IFN-alpha productions were reinforced with Poly(I:C) (viral TLR3 ligand), ODN1585 (viral TLR9 ligand) and Poly(I:C)/LyoVec, although no synergic enhancement by fucoidan was observed. These results suggested that fucoidan was extremely effective in activating dendritic cells, which play a central role in the regulation of immune function to eliminate infectious pathogens.

Supported by grants from JSPS KAKENHI (JP21K05423)
Combined IL-12, IL-15 and IL-18 activation induces epigenetic changes in human NK cells during the memory-like state transition
Authors: Tran Jennifer , Wong Pamela , Fan Changxu , Russler-Germain David , Cubitt Celia C , Marin Nancy D , Schappe Timothy , Berrien-Elliott Melissa , Wang Ting , Foltz Jennifer A , Fehniger Todd A
Published: August 2023

NK cells exhibit memory properties after activation with haptens, viral infection, or cytokines with each stimulus having distinct biology. Memory-like (ML) NK cells differentiate over one week after brief activation with combined IL-12/15/18, and have augmented responses upon restimulation by cytokines, activating receptor, or tumor targets. While much is known about their functional characteristics, there are large gaps in our understanding of the molecular mechanisms involved in programming conventional (c)NK cells into ML NK cells. As ML NK cells have enhanced ability to produce IFN-γ after multiple cell divisions, this suggests that the IFNG gene, and others, may be epigenetically poised. Therefore, we hypothesize that activation with IL-12/15/18 triggers epigenetic remodeling of cNK cells to ML NK cells that drives enhanced functionality. To test this, we performed ATAC-seq on baseline cNK cells, IL-12/15/18 activated NK cells, and in vitro differentiated cNK and ML NK cells after one week. Activation with IL-12/15/18 induces dramatic early epigenetic remodeling with >10,000 differentially accessible regions (DARs) compared to cNK cells. Moreover, ML differentiation generates epigenetically distinct ML NK cells (>1,000 DARs) compared to in vitro differentiated cNK cells, including increased IFNG locus accessibility, suggesting epigenetic mechanisms regulate their enhanced functionality. Also, ML NK cells are markedly different from IL-12/15/18 activated NK cells (>15,000 DARs) suggesting not all epigenetic changes are immediately induced, and time is required for full implementation of the memory-like state. Together, this supports that ML NK cells rely on epigenetic programming for enhanced function.

Supported by grants from NIH (P50CA171963, R01CA205239, P30CA91842, 1F31GM146361-01).
Targeting S100A9 to prevent Streptococcus pneumoniae-induced allergic asthma exacerbation
Authors: Boko Mèdéton Mahoussi Michaël , de Campos Fraga-Silva Thais Fernanda , Martins Núbia Sabrina , Cipriano Ualter Guilherme , Veras Flávio Protásio , Melo Bruno Marcel Silva , Ramalho Leandra Naira Zambelli , Cunha Fernando Queiroz , Alves-Filho José Carlos , Bonato Vânia Luiza Deperon
Published: August 2023

The alarmin S100 calcium-binding protein A9 (S100A9) plays a role in the immunopathology of lung diseases such as asthma and pneumonia. We hypothesized that acute pneumonia induced by S. pneumoniae exacerbates asthma through S100A9 induction that increase neutrophilic inflammation and NET production. C57BL/6 Wild Type (WT) animals and mice deficient for the expression of S100A9 (S100A9−/−) were sensitized and challenged with ovalbumin (OVA) and infected or not with S. pneumoniae during the challenge. In WT mice, the comorbidity resulted in a significant increase in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) accompanied by an increased perivascular and peribronchial leukocyte infiltration, S100A9 secretion and NET production in the lungs compared to the WT mice exposed only to the allergen. During the comorbidity, S100A9−/− mice exhibited significant reduction in the levels of neutrophil chemoattractant CXCL1 and neutrophilic inflammation in the BALF as well as perivascular and peribronchial leukocyte infiltration into the lungs compared to their WT counterpart. Moreover, during the comorbidity, S100A9−/− mice showed an increase in neutrophil death. Pharmacological intervention with tasquinimod, an inhibitor of S100A9 signaling, or BB Cl-amidine, an inhibitor of NET, protected mice from neutrophilic inflammation and NET production and reduced lung inflammation during the asthma and acute pneumonia comorbidity.Our results provided evidence that S100A9 might be a therapeutic target in severe asthma triggered by S. pneumoniae infection.

Financial support: The Sao Paulo Research Foundation (FAPESP), grants 2017/21629­5 (VLDB) and 2019/09881-6 (TFCF-S), and the Brazilian National Council for Scientific and Technological Development (CNPq), grant 142139/2019-0 (MMMB).
Role of mechanosensitive Piezo1 receptor expressed on macrophage in Periodontitis
Authors: Shindo Satoru , Kumagai Tomoki , Nakamura Shin , Pastore Maria Rita , Leon Elizabeth , Huang Shengyuan , Han Xiaozhe , Kawai Toshihisa
Published: August 2023

Objectives

The mechanosensitive Ca2+ ion channels, Piezo1, converts the mechanical stimulus into cell signals in a variety of immune cells. However, the pathophysiological property of Piezo1 in the oral polymicrobial infectious disease of periodontitis is largely unknown. This study investigated the role of Piezo1 in the inflammatory and phagocytic reactions by RAW264.7 macrophages in response to the exposures to periodontal pathogen, P.gingivalis.

Experimental methods

Short hairpin RNA (shRNA) vector for Piezo1 or control scrambled sequence was transfected to RAW264.7 cells. Then, RAW264.7 cells were incubated for 24 hours in the presence or absence of whole P.gingivalis (MOI; 10) or outer membrane vesicles (OMVs, 1 μg/ml) isolated from P.gingivalis, w or w/o Yoda1 (synthetic Piezo1 activator), or shear stress. qPCR was used to monitor the mRNA expressions of Piezo1, TNF-α, TLR-2, and TLR-4, whereas TNF-α protein was detected by ELISA. RAW264.7’s phagocytic ability was evaluated by cytoplasm-internalization of pHrodo-labeled whole P.gingivalis or OMVs using a flow cytometer.

Results

OMVs induced more TNF-α production and phagocytosis by RAW264.7 cells than whole P.gingivalis, both of which were further upregulated by Yoda1 as well as shear stress (p<0.05, ANOVA). Shear stress combined with OMV-stimulation upregulated the gene expression of TLR-4, while downregulating TLR-2 gene in RAW264.7 cells. The shRNA-targeting Piezo1 significantly suppressed the TNF-α production and internalization of OMVs by RAW264.7 cells compared control scramble shRNA (p<0.05, ANOVA).

Conclusion

The present study indicated that mechanical stress may dysregulate the macrophage-mediated innate immune responses in periodontitis.

NIH NIDCR grants, DE-027851, DE-028715 and DE-029709
The alarmin interleukin-33 promotes the expansion and preserves the stemness of Tcf-1+ CD8 T cells in chronic viral infection
Authors: Marx Anna-Friederike , Pinschewer Daniel David
Published: August 2023

T-cell factor 1 (Tcf-1) expressing CD8 T cells exhibit stem-like self-renewing capacity rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8 T cell (CD8SL) remain poorly defined.

Studying CD8 T cell differentiation in mice with chronic viral infection we identify the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8SL as well as for virus control. IL-33 receptor-(ST2-) deficient CD8 T cells exhibit biased end-differentiation and premature loss of Tcf-1. Intriguingly, ST2-deficient CD8SL responses are restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8SL formation in chronic infection. IL-33 signals broadly augment chromatin accessibility in CD8SL and determine these cells’ re-expansion potential.

Our study identifies the IL-33 – ST2 axis as an important CD8SL-promoting pathway in the context of chronic viral infection.

Swiss National Science Foundation (grant No. 310030_185318/1) Swiss National Science Foundation (grant No. 310030_200898) Swiss National Science Foundation (grant No. 310030_185226/1) Swiss National Science Foundation (grant No. CRSII3_160772/1) DFG grant LO 1542/5-1 Willy Robert Pitzer Foundation Dr. Rolf M. Schwiete Foundation
Indol-3-Carbinol ameliorates colitis in mice by AhR-mediated production of antimicrobial peptides and mucins
Authors: Palrasu Manikandan , Kakar Khadija , Aladhami Ahmed , Carter Tayler , Wilson Kiesha Maria , Zhong Yin , Yang Xiaoming , Singh Narendra P , Busbee Philip Brandon , Nagarkatti Prakash , Nagarkatti Mitzi
Published: August 2023

Indole-3-carbinol (I3C) is a dietary compound that acts as a ligand for the aryl hydrocarbon receptor (AhR), an important sensor for environmental polyaromatic chemicals and a regulator of the immune response. The pathogenesis of colitis involves multiple interactions between microbial dysbiosis, dysregulation of the host immune response, environmental factors, and host genetic profile. Indeed, studies have shown defective expression of AhR and antimicrobial peptides (AMPs) particularly defensins in inflammatory bowel disease (IBD) patients. Here, we investigated how I3C influences the production of AMPs, particularly b-defensins, through AhR and colonic microbiota composition in Anti-CD40 or dextran sulfate sodium (DSS)-induced colitis. Our analysis found that I3C attenuates colitis through AhR activation leading to increased expression of murine b-defensins (mBDs) by Colonic Epithelial Cells, resulting in the restoration of healthy gut microbiota and prevention of colonic inflammation. We analyzed the colonic expression of AMPs and mucins. Our analysis found that I3C significantly increased the mRNA expression of AhR, AMPs such as mBDs, Reg4, and mucins, when compared to vehicle-treated mice with colitis. Similarly, the mRNA levels of AhR, AMPs, and mucins were significantly increased in DSS+I3C-treated colon adenocarcinoma cells MC-38 (murine) and Caco2 (human) when compared to DSS-treated cells. To conclude, our findings demonstrate that I3C inhibits colitis primarily through AhR-mediated induction of AMPs and protective mucins, resulting in the enrichment of healthy gut microbiota (This work was supported in part by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896).

This work was supported in part by NIH grants R01ES030144, P01AT003961, P20GM103641, and R01AI123947, R01AI160896
Immunoresponsive Gene 1: an important regulator of autoreactive CD4+ T Cells in mouse model of multiple sclerosis
Authors: Nematullah Mohammad , Ahmed Mohammad Ejaz , Zahoor Insha , Rashid Faraz , Waters Jeffery , Ayasolla Kameshwar , Mir Sajad , Hoda Nasrul , Rattan Ramandeep , Giri Shailendra
Published: August 2023

Th17 and Th1 CD4 T cells play important roles in the pathophysiology of human MS and the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). As per our findings, metabolic changes in MS alter the phenotypic and functionality of immune cells, which may influence their effector activities as disease progresses. IRG1 (Immuno response gene 1), encodes for cis-aconitic acid decarboxylase in TCA cycle & enhances the manufacture of mitochondria-selective metabolite, itaconic acid from cis-aconitate, is one such gene linking metabolism to immune effector function. It modulates inflammation in myeloid cells in addition to its antibacterial activity. The relevance of IRG1 in CNS autoimmunity, however, is still unknown. We used IRG1 KO mice to know what role it plays in EAE. We observed that global IRG1 KO showed significantly worsened clinical disease severity compared to their WT at the peak of the disease (3.3+0.50 vs. 2.1+0.50, p< 0.05). We found an evident early onset of disease induction & severity in the IRG1 KO as compared to the Wt (D9 vs. D13). Furthermore, to examine the mechanism of EAE pathogenesis, found a significantly higher infiltration of CD45hi cells (mainly Th17/Th1 cells) in the CNS of IRG1 KO mice compared to Wt mice. Adoptive transfer of CD45+ cells from immunized IRG1 KO augmented the disease progression in RAG1 deficient mice suggesting a possible role of IRG1 in a hyperactivated Th17 response. Also, itaconate treatment at the dose of 15mg/kg bw attenuated EAE disease relapse in SJL mice. Our findings suggest that IRG1/itaconate negatively regulates the Th17 immune response & moDCs’ function to attenuate the EAE disease progression, which may open a novel therapeutic avenue in the development of MS-related therapies.
The effect of tolerogenic PLG nanoparticles on transgenic CD8+ T cells
Authors: Neef Tobias , Miller Stephen D
Published: August 2023

Antigen-loaded, biodegradable PLG nanoparticles have demonstrated tolerance induction in models of autoimmune disease. Here we report on the effect of these particles on transgenic CD8+ T cells. Wild-type mice received transgenic cells, were treated with nanoparticles, and then had their spleens and/or pancreases removed for flow cytometric analysis. Relative to controls, mice treated with these particles exhibited a larger percentage of transgenic CD8+ T cells in spleens, suggesting that a sequestration of autoreactive T cells prevents their trafficking to inflamed tissues. Furthermore, nanoparticle treatment led to a higher percentage of cells with surface markers indicative of a regulatory phenotype. These results imply that altered trafficking and an expansion of regulatory T cells are two mechanisms by which PLG nanoparticles induce tolerance in CD8+ T cell - mediated autoimmune disease.

Supported by a grant from the JDRF
CARD-only proteins (COPs) inhibit uric acid crystal-induced inflammasome activation and ameliorate gout
Authors: Devi Savita , Indramohan Mohanalaxmi , Jager Elisabeth , Carriere Jessica , Chu Lan H , Almeida Lucia , Greaves David , Stehlik Christian , Dorfleutner Andrea
Published: August 2023

The NLRP3 inflammasome mediates the response to monosodium urate (MSU) crystals and is responsible for the debilitating symptoms of gout. ASC is the essential inflammasome adaptor that bridges NLRP3 to caspase-1 and inflammasome assembly is via sequential homotypic PYRIN domain (PYD)-PYD and caspase recruitment domain (CARD)-CARD interactions between NLRP3 and ASC and ASC and caspase-1, respectively. While controlled inflammasome activation is essential for host defense and wound healing, dysregulated and excessive inflammasome responses cause inflammatory diseases. Hence, proper control is key for a balanced inflammasome response. Humans, but not mice, encode a family of 3 small CARD-only proteins (COPs): CARD16, CARD17 and CARD18, but their role in controlling inflammasome responses in vivo is unknown, which is the focus of our study. COP transgenic mice show reduced MSU crystal induced release of IL-1b and consequently ameliorated gout symptoms. Transgenic macrophages and COP expressing THP-1 cells show impaired MSU crystal-mediated NLRP3 inflammasome and caspase-1 activation, cytokine release and pyroptosis, while COP KO results in a hyper response to MSU. Also, COPs show unique binding pattern to the CARD of caspase-1 and ASC in naïve, primed and MSU-activated macrophages and interfere with the essential CARD-CARD-mediated caspase-1 self interaction and the ASC-caspase-1 interaction by a competitive binding mechanism. We demonstrate that COPs are functional in mice and inhibit NLRP3 inflammasome assembly by a competitive binding mechanism that prevents cytokine release and pyroptotis and ameliorates inflammatory disease. Altogether, our findings illustrate that the COPs have a role in maintaining homeostasis.

National Institutes of Health. (AI099009 and AR064349 to C.S, AI134030, AI140702 and AI120625 to C.S. and A.D.)
TLR-mediated immune activation is a strong suppressor of the Treg-attracting chemokine CCL22
Authors: Gärtig Jan , Grün Juliane , Perleberg Carolin , Schnell Konstantin , Beimert Antonia , Knott Maximilian Martin Ludwig , Röhrle Natascha , Bahner Kevin , Layritz Patrick , Endres Stefan , Anz David , Piseddu Ignazio
Published: August 2023

The immunoregulatory chemokine CCL22 is mainly secreted by dendritic cells (DC) in lymph nodes and plays a crucial role in maintaining the balance between immune tolerance and efficient immune response by induction of intercellular contacts between DC and regulatory T cells (Treg) expressing the only so far known CCL22 receptor CCR4. In the absence of CCL22, the immune response to vaccination and cancer has been proven to be enhanced. In this work, we aimed to understand how activation of Toll-like receptors (TLR) modulates CCL22 levels mechanistically in vitro and in vivo. We observed that a bacterial infection with salmonella typhimurium and treatment with different TLR agonists such as Poly (I:C), LPS, R848 and CpG led to a potent and long-lasting suppression of systemic CCL22 in vivo. Furthermore, using in vitro co-cultures of DC, B cells and T cells, we identified TLR-mediated CCL22 suppression to be mediated by soluble factors derived from non-DC. Additionally, we pursued to investigate the underlying mechanism and could show in vitro that CCL22 suppression is particularly driven by redundant action of IL-10, IFN-α and IFN-γ. Moreover, inhibition of NF-κB reverted the CCL22-suppressing effect of CpG on splenocytes. In CpG-treated mice, Treg/DC contacts were diminished to the level known from Ccl22−/− mice, an observation which links TLR-mediated CCL22 suppression to an immunological relevance. Taken together, we demonstrate that TLR signaling results in CCL22 downregulation via different cytokines, a mechanism which is potentially important to enable the induction of a strong immune response and efficient pathogen clearance in the setting of microbial infection.
Lithium chloride attenuates sepsis-induced acute respiratory distress syndrome via decreasing the expressions of TNF-α, IL-6, CXCL1, and pulmonary edema
Authors: Lin Pei-Yun , Shih Fu-Chia , Yang Chi-Hsuan , Chen Chia-Ling
Published: August 2023

Sepsis is a life-threatening illness caused by an excessive host response to infection. Acute respiratory distress syndrome (ARDS), a serious complication of sepsis, is characterized by pulmonary edema and alveolar barrier injury. However, there is no effective drug proven to reduce the mortality of ARDS yet. Lithium chloride (LiCl) is an inhibitor of glycogen synthase kinase-3β (GSK-3β). Recent studies reveal that GSK-3β inhibition exhibits anti-inflammatory effects in sepsis thereby ameliorating lipopolysaccharide (LPS)-induced acute lung injury. Given that GSK-3β inhibition has been investigated for various diseases, whether LiCl exerting a novel therapeutic effect for treating endotoxemia-induced ARDS remains unclear. Here, we investigated the effects of LiCl on endotoxemia-induced lung inflammation, respiratory failure, and the pulmonary barrier integrity of respiratory membrane. Mice intraperitoneally injected with LPS could induce a significant lymphopenia, lung inflammation with amount immune cells infiltration, and high mobility group box 1 (HMGB1) expression in lung supernatants. LiCl treatment ameliorated lung inflammation and lung function decline in LPS mice. In addition, LPS-induced TNF-α, IL-6, as well as CXCL1 were significantly lessened after LiCl treatment. The measurements of E-cadherin/VE-cadherin expression in lung tissues and IgM in bronchoalveolar lavage fluids indicated that LiCl could partly ameliorate LPS-mediated loss of pulmonary barrier integrity. Accordingly, LiCl effectively attenuated sepsis-caused lung inflammation, lung function decline, and alveolar barrier injury, which suggested that LiCl might act as a potential repurposing drug for treating ARDS in future.
Anti-rheumatic effect of KMU-11361 in various rheumatoid arthritis-related cells targeting multiple protein kinase
Authors: Baek Hye Suk , Hong Victor Sukbong , Lee Hyun-Su , Son Chang-Nam , Kim Sang-Hyon , Jeong Gil-Saeng , Jeon Jieun , Lee Jinho , Kim Shin
Published: August 2023

Protein kinases important for regulating protein phosphorylation, which is a promising therapeutic target for inflammatory disease, such as rheumatoid arthritis (RA). In this study, we synthesized a novel multi-protein kinase inhibitor, KMU-11361, a derivative of indolin-2-one and investigated the mechanisms of anti-inflammatory and anti-rheumatic effects using THP-1 cells, RAW264.7 cells, Jurkat T cells, and human RA fibroblast-like synoviocytes (RA-FLS). Our results revealed that KMU-11361 inhibited LPS-induced upregulation of inducible iNOS, and COX-2, and suppressed LPS-induced phosphorylation of TAK1, JNK, inhibitor of NF-κB kinase α/β (IKKα/β), IκBα, and NF-κB p65 as well as nuclear translocation of NF-κB p65 in THP-1 cells. Furthermore, KMU-11361 attenuated proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, and, remarkably, inhibited LPS-induced activation of the NLRP3 inflammasome in THP-1 cells. Additionally, in activated Jurkat T cells, KMU-11361 inhibited the TAK1-IKKα-mediated NF-κB pathway as well as the phosphorylation of MAPKs. Moreover, KMU-11361 inhibited anti-CD3/CD28 antibodies-induced upregulation of proinflammatory cytokine IL-2. Moreover, it suppressed LPS-induced upregulation of proinflammatory cytokines, p-TAK1, p-IKKα/β, and p-NF-κB p65 in RA-FLS. Surprisingly, KMU-11361 also inhibited the receptor activator of NF-κB-induced osteoclast differentiation and up-regulation of nuclear factor of activated T-cells, NFATc-1 and c-Fos in RAW264.7 cells. Taken together, our results suggest that KMU-11361 has anti-inflammatory and anti-rheumatic effects in various RA-related cells and could be developed as a potent anti-rheumatic drug.
FoxO6 attenuates neuroinflammation by the regulation of anti-inflammatory factors
Authors: Kim Mi Eun , Lee Jun Sik
Published: August 2023

Forkhead transcription factor (FoxO) plays key roles in the induction of various downstream genes involved in the inhibition of cellular metabolism, cell death, cell cycle, and oxidative stress responses. FoxO6 is expressed in the liver, skeletal muscle, and the hippocampus of the brain. FoxO6 also has been proposed to play a role in the promotion of insulin resistance, and inflammation. In this study, we tried to determine the neuroinflammation inhibitory response and signaling mechanism for FoxO6. In order to confirm the inhibitory function of neuroinflammation, FoxO6 knock out cell line and mice were prepared and their functions were confirmed. We have confirmed that neuroinflammation is suppressed in FoxO6 knock out cell line and mice. These findings may suggest that FoxO6 is a novel factor in the regulation of neuroinflammation.

Supported by grants from NRF (NRF-2020R1A2C1012984)
Extracellular vesicles derived miRNAs from bovine milk modulate the inflammatory response to lipopolysaccharide in vitro
Authors: Rosa Fernanda , Santos Rafaela , Lugo Tomas , Brown Audrey , Bickel Ulrich
Published: August 2023

Bovine milk is composed of a variety of biologically active compounds including mi-croRNAs (miRNA), which are found within extracellular vesicles (EVs). Extracellular vesicle-derived miRNAs may play a crucial role in the regulation of the immune response. Thus, this study aimed to identify specific miRNAs present in bovine milk that modulate the immune system’s response to infection. To address this, RAW-Blue™ Cells, a mouse macrophage reporter cell line was challenged with E. coli lipopolysaccharide (LPS) and incubated with a pool of miRNAs isolated from bovine milk through a 24-hour period. After incubation, QUANTI-Blue™ assay was used to detect the activation of the transcription factor NF-κB by the different treatments. Raw-Blue™ cells treated with miRNAs induced to some extend the signaling pathways leading to the activation of NF-κB relative to control cells (P<0.01). Bovine-milk derived miRNAs suppressed LPS-induced NF-κB activity in a dose-dependent manner (P=0.01). To identify the top expressed miRNAs that are driving such effects in vitro, real-time qPCR assays were performed in the milk samples. The miRNA-profiling revealed the presence of miRNAs involved in tumor suppression and cell apoptosis. Overall, our preliminary findings indicated that miRNAs derived from bovine milk have the potential to modulate the immune response to LPS in vitro.

Texas Tech University, School of Veterinary Medicine
Trogocytic-molting of T-cell microvilli controls T-cell clonal expansion
Authors: Jun Chang-Duk , Park Jeong-Su , Kim Jun-Hyeong , Soh Won-Chang , Kim Na-Young , Lee Kyung-Sik , Kim Chang-Hyun , Chung Ik-Joo , Lee Sunjae , Kim Hye-Ran
Published: August 2023

Internalization of the T-cell antigen receptor (TCR) is intimately linked to T-cell activation: a phenomenon thought to be related to the “exhaustion” of T-cell responses. To date, however, no report has considered that during physical interaction with cognate antigen-presenting cells, T cells release many TCRs via T-cell microvilli particles, which are derived from finger-like membrane structures (microvilli) in a combined process of trogocytosis and enzymatic vesiculation and correspond with the loss of membrane TCRs and many external membrane components. Surprisingly, in contrast to TCR internalization, this event leads to rapid upregulation of surface TCRs and remarkable metabolic reprogramming of cholesterol and fatty acids synthesis to meet the demands of clonal expansion, which drives multiple rounds of division and cell survival. We called this event “trogocytic-molting,” which represents an intrinsic molecular basis of T-cell clonal expansion by which T cells gain increased sensitivity to low antigen concentrations.

This work was supported by the Creative Research Initiative Program (2015R1A3A2066253); Bio-Synergy Research Project (2021M3A9C4000991); Bio & Medical Technology Development Program [2020M3A9G3080281] through National Research Foundation (NRF) grants funded by the Ministry of Science and ICT (MSIT), the Basic Science Program (2019R1C1C1009570 & 2022R1A2C4002627) through National Research Foundation (NRF) grants funded by the Ministry of Education (MOE), and supported by Global University Project (GUP), GIST Research Institute (GRI) IBBR grant funded by the GIST (in 2021-2022), and the Joint Research Project of Institutes of Science and Technology (2021–2022), Korea.
3D co-culture models demonstrate radiation-mediated activation of tumor-associated macrophages
Authors: Kaur Aanchal Preet , Baird Jason , Alice Alejandro F , Ball Megan , Kramer Gwen , Crittenden Marka R , Gough Michael J
Published: August 2023

Type I IFN signaling mediated by STING pathway is important for the induction of radiation-stimulated adaptive immune responses in responsive tumors. Macrophages recruited to tumors following radiotherapy (RT) can influence the response to RT. Reprogramming immunosuppressive M2-like macrophages in tumors to exhibit a more activated M1-like phenotype can help improve response to therapies. Here, we hypothesize that a 3D co-culture model of cancer cells and macrophages will be more contextually relevant to the events occurring in tumors and can be used to study the crosstalk that occurs between these cells in vivo. We initially optimized seeding densities of murine MC38 colorectal carcinoma cells to develop spheroids of consistent size. Using GFP+ macrophages and Hoechst stained MC38, we imaged the development and localization of these cells during spheroid formation. Next, co-culture spheroids were exposed to 12 Gy radiation on day 1. Viability and phenotype of macrophages in spheroid co-cultures was assessed on day 4 using flow cytometry by staining for CD80, CD86, MHC Class II, CD69 and CD206 expression. STING ligand, cyclic di-AMP (CDA) treated spheroid co-cultures were used as positive controls. CDA treatment significantly increased CD80/86 expression in 3D co-cultures. Treatment with 12 Gy radiation showed similar effect. Interestingly, macrophages derived from MyD88 Lyz2Cre, MyD88fl/fland STING knockout mice also showed a significant increase in CD80/86 and CD40, suggesting that macrophage activation was independent of MyD88 or STING signaling. Ongoing studies aim to explore the pathways activated on irradiation in these cells and further cross-validate with the macrophage phenotypes observed in vivo in response to RT.
PIEZO1 Deletion in CD11b+ Cells Suppresses Rhabdomyosarcoma Tumor Rejection and Generates a Tumor-Permissive Immune Landscape
Authors: Bonner Melissa Romain , Tomchuck Suzanne , Askew David , Abiff Muta , Huang Alex Y
Published: August 2023

In several cancers, including the aggressive sarcoma, rhabdomyosarcoma (RMS), tumor-associated myeloid cells (TAMCs) are the largest immune populations and are correlated with poor survival outcomes. TAMCs are a mixed and ‘plastic’ population that can sense changes in their environment, including intra-tumoral ECM stiffness, and change their functioning accordingly. Despite the clinical influences these cells have and the detrimental effects that elevated tumor microenvironment (TME) stiffness has on immune cells’ phenotype, little is known about the proteins that sense and mediate these changes. We propose inhibition of mechanosensitive cation channel, PIEZO1, promotes a pro-tumor phenotype in TAMCs while its activation skews this population to anti-tumor functioning thereby enhancing tumor rejection. PIEZO1’s influence on tumor rejection was assessed by inoculating a novel murine CD11b-conditional homozygous PIEZO1 knockout strain with syngeneic RMS cells and performing several analyses. Tumor volume and survival analyses found that RMS-bearing CD11b-conditional PIEZO1 KO mice developed larger tumors and had diminished overall survival as compared to wild-type mice. Flow cytometric analysis of tumors revealed increased pro-tumor macrophage frequency in tumor and expansion of MDSCs in the TME and tumor-draining lymph node (TDLN). Interestingly, despite PIEZO1 KO tumors having increased T cells, they had lower PD-1+ CD4 and CD8 T cell frequencies; a trend also observed in the spleen and TDLN. These findings suggest without PIEZO1 expression, TAMCs predominantly polarize to a ‘pro-tumor’ state, thereby preventing T cell and adaptive immune cells’ activation, leading to lower survival and increased tumor burden.

NCI T32 (5T32CA059366-27)
The ubiquitin ligase Itch skews light zone selection in germinal centers
Authors: Renshaw Lindsay R , Kim Peter , Crici Macaul , Fazelinia Hossein , Spruce Lynn , Oliver Paula M , Moser Emily
Published: August 2023

Immunoglobulin diversification occurs in peripheral lymphoid organs, after the central tolerance checkpoints during B cell development. High levels of somatic hypermutation (SHM) of Ig genes occurs in germinal center (GC) B cells, followed by selection of mutated clones. This process is essential for generation of high affinity antibody to pathogens, but can also produce autoreactive Ig receptors. Selection mechanisms that govern antigen-driven Ig diversification are still incompletely understood. The E3 ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop debilitating, multi-faceted, potentially fatal, autoimmune disease; yet how Itch regulates GC B cell fate or function is not well understood. By studying Itch deficient mice, we have recently shown that Itch directly limits GC B cell activity to shape antibody responses. Proteomic profiling of GC B cells uncovered that Itch deficient cells exhibit high mTORC1 and Myc activity, hallmarks of positive selection. Mixed bone marrow chimeras revealed that Itch acts within B cells to limit proportions of light zone GC cells, and Itch deficient light zone GC cells were mainly in G1/G0 rather than in cell cycle. These results support a novel role for Itch in skewing selection of GC B cells to restrict LZ accumulation and shape GC-derived humoral immunity. Determining how B cells integrate cues within GCs to navigate through LZs and DZs will aid in understanding how autoreactive clones emerge from GCs in those suffering from autoimmune disease.

Supported by a grant from NIH (K22AI148597)
Deletion of Vβ3+ CD4+ T-cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8+ T-cells
Authors: Driver John P , Ye Cheng , Clements Sadie , Gu Weihong , Geurts Aron , Mathews Clayton , Serreze David V , Chen Yi-Guang
Published: August 2023

In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T-cells. Interactions between both helper CD4+ and cytotoxic CD8+ T-cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T-cells arise from deleterious interactions between relatively common genes which regulate aspects of T-cell activation/effector function (Ctla-4, Tnfrsf9, Il2/Il21), peptide presentation (H2-Ag7, B2m), and T-cell receptor signaling (Ptpn22). In this study, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded Mtv3 provirus as a genetic element affecting CD4+/CD8+ T-cell interactions through an additional mechanism, altering the T-cell receptor (TCR) repertoire. Mtv3 encodes a superantigen that deletes the majority of Vβ3+ thymocytes in NOD mice. Ablating Mtv3 and restoring Vβ3+ T-cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2g7 MHC haplotype (B6.H2g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8+ T-cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifest by Vβ3+ CD4+ T-cells, which unless deleted by Mtv3, accumulate in insulitic lesions and trigger in B6 background mice the pathogenic activation of diabetogenic CD8+ T-cells. Our findings provide evidence that endogenous Mtv superantigens can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vβ repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human disorders.

Supported by grants from the American Diabetes Association (1-14-BS-051) and NIH (AI130656)