bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of nano-particulate carbon black in lung cancer development
Authors: Chang Cheng-Yen , You Ran , Kheradmand Farrah
Published: January 2023

Chronic inflammation induced by inhalation of airborne contaminants can contribute to cancer development. We have found that the anthracotic pigment in lungs of smokers is composed of nano-particulate carbon black (nCB), which accumulates in the antigen presenting cells (APCs) and promotes inflammation. Other occupational nCB exposures, such as coal-mining and carbon-black manufacturing, also raise the risk of chronic inflammatory lung diseases. No direct evidence establishes that nCB is a carcinogen; however, several cohort studies have shown a strong correlation between lung inflammation in carbon black industry workers and cancer. We examined whether nCB can promote non-small cell lung cancer (NSCLC) development. Using a novel non-Kras model of NSCLC, in which mice have airway epithelia-specific deletion of tumor suppressors Pten and Smad4 (Ptsd/d), we have found that intranasal challenge with nCB accelerates NSCLC progression. nCB exposure results in myeloid-derived suppressor recruitment and lactate accumulation to paralyze anti-tumor effects. Further, adoptive transfer of APCs from nCB exposed wild type mice (nCB-APC) to Ptsd/d mice showed increased incidences of lung tumor and metastasis, indicating a critical role for APCs in NSCLC progression. nCB-APC-induced immune suppression is in part mediated by expression of immunosuppressive enzymes and lactate production. Additionally, nCB-APCs express both pro-inflammatory and immunosuppressive cytokines to promote tumor growth. These findings suggest that nCB-APCs foster an immunosuppressive microenvironment, and indicate a mechanism responsible for environmental and occupational exposure to nCB in accelerating NSCLC progression.
CD146 contributes the metastatic properties and antitumor immunity of human colon adenocarcinoma cells
Authors: Kato Kazunori , Shimizu Yui , Yamazaki Takumi
Published: January 2023

CD146 (MCAM) expressed on not only vascular endothelial and smooth muscle cells but also various malignant tumor cells. It has been shown that overexpression of CD146 predicts poor prognosis of solid tumor. Several reports by using transfectants or gene silencing technique have shown that CD146 is involved in cell adhesion and inflammatory cell migration. However, contribution of CD146 in tumor metastasis and antitumor immunity are still unknown. To investigate the effect of CD146 on tumor metastatic properties, we establish two subclones from DLD-1 (human colon adenocarcinoma cells) expressing CD146 or not. Both cell lines express epithelial tumor marker EpCAM and TROP2 equally, while CD146 (+) DLD-1 could only express CD44 variant v8–v10 and EphA2. Using metabolic assay, we found enhanced metabolism and decreased lactate production in CD146 (+) DLD-1. Consist with the morphological changes to mesenchymal features CD146 (+) DLD-1, the expression of E-cadherin and claudin-3 in tight junction were decreased in CD146 (+) DLD-1. In addition, CD146 (+) DLD-1 significantly increased the production of VEGF and the induction of various genes (slug, twist and zeb1) which are involved in epithelial to mesenchymal transition (EMT). Interestingly, the expression of immune checkpoint molecules including B7-H1, B7-DC (both PD-1 ligands), CD155 and IDO were up-regulated in CD146 (+) DLD-1 inducing the resistance of cytotoxic effect of activated lymphocytes. These results indicate that CD146 (+) DLD-1 strongly induces immune escape mechanism, thus demonstrating a new relationship between CD146 and EMT and anti-tumor immunity, which would be a new treatment and diagnostic target for immunotherapy.
Therapeutic Induction of Tertiary Lymphoid Structures in Melanoma using STING Agonists
Authors: Chelvanambi Manoj , Taylor Jennifer L , Fecek Ronald J , Storkus Walter J
Published: January 2023

Tertiary lymphoid structures (TLS) are non-encapsulated functional aggregates of T cells, B cells, dendritic cells (DC) and high endothelial venules (HEV) located in peripheral sites of chronic inflammation. TLS may serve as sites of local antigen presentation and immune priming that may protect against tumor progression. Homeostatic chemokines CCL19, CCL21 and CXCL13 produced by DCs and/or stromal cells are known to support secondary lymphoid organogenesis and also play key roles in TLS formation. Other DC-associated pro-inflammatory cytokines including lymphotoxin α, IL-36γ and type I interferons also appear to aid in organizing TLS. Hence, we hypothesize that appropriate therapeutic activation of DCs within the tumor microenvironment may serve to nucleate TLS in vivo, leading to slowed tumor progression. We found that activation of the cytosolic DNA sensor STING using ML-RR-S2-CDA, a murine and human STING agonist, leads to not only an expected increased production of type I interferons by CD11c+ DCs in vitro but also leads to increased transcript levels of TLS-associated cytokines/chemokines via activation of pIRF3 and pIRF7. In mice harboring B16.F10 melanomas, provision of STING agonists slows tumor growth in a host STING dependent manner. Further using immunofluorescence microscopy, we observed increased infiltration and clustering of CD3+ T cells and CD11c+ DCs and the induction of HEVs in treated tumors within tumor-associated TLS as early as 5 days post-treatment. Future studies will determine how TLS shape the local versus systemic protective T cell repertoire in treated animals.
Targeting the tumor microenvironment with the monoclonal antibody Tocilizumab, reduces IgM secretion in Waldenström macroglobulinemia
Authors: Han Weiguo , Matissek Stephan Josef , Jackson David , Sklavanitis Brandon , Elsawa Sherine F.
Published: January 2023

The tumor microenvironment (TME) plays an important role in cancer and has been shown to play a role in resistance to therapy and is therefore important in cancer cell biology. In Waldenström macroglobulinemia (WM), a B cell malignancy characterized by the overproduction of a monoclonal IgM protein, the TME secrets cytokines that promote malignant phenotype. In previous work, we have shown that TME-IL-6 promotes WM cell growth and IgM secretion in WM. Tocilizumab/Actemra is an anti-IL-6R antibody, which can competitively block IL-6 binding to the IL-6R. Here we investigated the efficacy of Tocilizumab in a preclinical mouse model of WM that considers the role of the TME in disease biology. Hairless SCID mice were injected subcutaneously with BCWM.1 or RPCI-WM1 and bone marrow stromal cells (HS-5) at a ratio of 5:1. Groups of mice with treated with Tocilizumab or control antibody three times/week for 5 weeks and the effect on tumor burden and disease biology were evaluated. Although Tocilizumab had no effect on mice survival, there was a reduction in tumor growth rate in mice injected with RPCI-WM1 cells + stromal cells and treated with Tocilizumab (p=0.0394). In mice injected with BCWM.1 + stromal cells, there was a significant reduction in human IgM secretion in mice sera with Tocilizumab treatment (p=0.0099). There was no significant change in mice weight suggesting Tocilizumab induced no toxicities to the mice. Taken together, our data suggests that administration of Tocilizumab to tumor bearing mice, results in a significant reduction in tumor volume and IgM secretion. Therefore, the evaluation of the role of Tocilizumab in WM patients may provide therapeutic efficacy by reducing IgM production and slowing the rate of tumor growth.
Hypoxia-Inducible Factor-1α is upregulated in Natural Killer cells by Interleukin-2 and hypoxia via PI3K/mTOR signaling pathway
Authors: Cluff Emily R , Nolan Joshua , Collins Cortney , Varadaraj Archana , Rajasekaran Narendiran
Published: January 2023

Natural killer (NK) cells have cytotoxic functions that aid in the recognition and killing of tumors. Hypoxia is a major characteristic of the tumor microenvironment (TME) and cellular adaptive responses are mediated by hypoxia inducible factors (HIFs). This study investigates how activated NK cells adapt to hypoxia through the upregulation of HIF-1α. Both NK cell lines, NKL and NK92, upregulated HIF-1α when exposed to hypoxia but only when stimulated with IL-2. NKL cells activated with IL-2 and exposed to 5% O2 did not increase expression of HIF-1α. Treatment with translational inhibitor cycloheximide (CHX) blocked HIF-1α expression indicating that the IL-2 mediated signaling stimulates HIF-1α protein synthesis under hypoxia. The synthesis of HIF-1α through IL-2 induced signaling via the PI3K/mTOR pathway was essential for HIF-1α expression by NK cells under hypoxia. Treatment of PI3K inhibitor LY294002 or mTOR inhibitor Rapamycin blocked the expression of HIF-1α, while MAPK inhibitor PD98059 had no effect on IL-2 stimulated NKL cells. Importantly, NKL cells exposed to hypoxia demonstrated a reduced anti-tumor cytotoxicity against the colorectal cancer cell line DLD-1 in a real time impedance-based cytotoxicity assay (xCelligence RTCA). Similar results were obtained with NK-92 cells and primary NK cells derived from human PBMCs. Increase in HIF-1α expression under hypoxia were observed in NK cells treated with IL-15, another common cytokine receptor gamma chain cytokine. Together, these data illustrate that the activation of the PI3K/mTOR signaling pathway through the common cytokine receptor gamma chain is critical for HIF-1α upregulation in NK cells under hypoxia and provide a molecular basis for reduced NK cell functions.
Immune cell phenotype and cytokine secretion in mouse breast cancer responding to anti-CD47 antibody treatment
Authors: wen Chiao Kai , Elia Jeanne , Loter Lorraine , RAFIZADEH TANNAZ , Jiang Weiping , Zhang Hong , Yang Xifeng
Published: January 2023

Anti-CD47 antibodies have been shown to inhibit the progression of several types of cancer by blocking the “don’t eat me” signals to macrophage through CD47/SIRPα interaction, causing phagocytosis of tumor cell. However, mechanistic details in the response of the cancer cells still remain to be explored. In this study, we investigated different cytokine levels in 4T1 mammary carcinoma induced mouse breast cancer model with or without anti-mouse CD47 monoclonal antibody treatment. The data showed that this cancer model presented significantly higher percentages of CD11b+, CD43+, CD80+, CD86+ and CD371+ cells after 1 week of 4T1 cells injection, CD11b+ cells percentage has been reached to 40% and 90% in spleen and bone marrow respectively at 2 weeks after 4T1 cells injection. After 4 doses of anti-mouse CD47 antibody treatment (i.p. injection per 2 days started from one week after 4T1 challenge), anti-CD47 antibody reduced significantly not only tumor size but also the levels of IL-1α in spleen (P=0.026) and of MCP-1/CCL2 in tumors (P=0.029). Interestingly, fresh isolated 4T1 induced breast cancer mouse spleen and bone marrow cells appeared 5%-10% CD11b+/Sca-1+ populations whereas in vitro culture of 4T1 cells had 5% of cells of Sca-1+/CD326+. When the freshly isolated 4T1 induced breast cancer mouse spleen and bone marrow cells were continuously cultured for 2–3 weeks, CD326+ cells became dominant and about 10% – 30% of population showed Sca-1+/CD11b+ positive. This study provided inside thoroughly investigated the response of cytokines and 4T1 cells induced mouse mammary carcinoma. Our data may further discover the mechanism for tumor growth and metastasis.
Quantification of A Spectrum of Adhesion Molecules in Metastatic Cancer Patient Samples Using a Multiplex Bead-Based Immunoassay Panel
Authors: Zhao Amy , Lehmann Jason , Sun Binggang , Jiang Weiping , Ji Shaoquan
Published: January 2023

Metastasis to distant organs is responsible for ~90% of cancer-related deaths. Adhesion molecules are cell surface proteins that mediate the interactions between disseminated tumor cells and the endothelium of blood vessels present in the tumor microenvironment, and thus play a major role in tumor metastasis. Serum and plasma samples from patients with metastatic disease (head, neck, breast, pancreatic, gastric, and lung cancer patients) or healthy controls were assayed with a bead-based multiplex immunoassay which measured the concentration levels of the following adhesion molecules: ICAM-1, ICAM-2, ICAM-3, VCAM-1, PECAM-1, ALCAM-1, EpCAM, NCAM, E-selectin, P-selectin, L-selectin, PSGL-1, and CD44. Although limited by sample size, differential concentrations of assay targets were detected between healthy and disease-state samples. We have demonstrated that this multiplex panel is a robust tool for measuring the concentration of a spectrum of adhesion molecules with great efficiency and convenience. This panel will help screen and identify the adhesion molecules that may be universally or specifically involved in metastasis of different types of cancer.
Virus-specific memory T cells populate tumors and can be repurposed as a tumor immunotherapy
Authors: Rosato Pamela , Wijeyesinghe Sathi , Stolley Michael , Nelson Christine , Davis Rachel L , Manlove Luke S , Pennell Christopher A , Blazar Bruce R. , Chen Clark C , Geller Melissa A , Vezys Vaiva , Masopust David
Published: January 2023

The immunosuppressive tumor microenvironment limits the success of current immunotherapies. The host retains memory T cells specific for previous infections throughout the entire body that are capable of executing potent and immediate immunostimulatory functions. Here we show that virus-specific memory T cells extend their surveillance to mouse and human tumors. Reactivating these antiviral T cells can arrest growth of checkpoint blockade-resistant and poorly immunogenic tumors in mice after injecting adjuvant-free non-replicating viral peptides into tumors. Peptide mimics a viral reinfection event to memory CD8+ T cells, triggering antigen presentation and cytotoxic pathways within the tumor, activating dendritic cells and natural killer cells, and recruiting the adaptive immune system. Viral peptide treatment of ex vivo human tumors recapitulates immune activation gene expression profiles observed in mice. Lastly, peptide therapy renders resistant mouse tumors susceptible to PD-L1 blockade. Thus, re-stimulating known antiviral immunity may provide a unique therapeutic approach for cancer immunotherapy.
Adipocyte PD-L1 Modulates PD-1/PD-L1 Checkpoint Blockade Cancer Immunotherapy Efficacy
Authors: Wu Bogang , Sun Xiujie , Gupta Harshita B. , Curiel Tyler J , Li Rong
Published: January 2023

PD-L1 expression in both tumor and host cells correlates with antitumor therapeutic efficacy, but the specific contribution of PD-L1 in various cell compartments to antitumor immunity remains to be fully elucidated. Here we show that PD-L1 expression is significantly elevated in human and mouse mature adipocytes versus preadipocytes. When co-cultured with mouse splenocytes, adipocytes reduce αPD-L1 antibody-mediated CD8+T cell activation. Genetic ablation of adipose PD-L1 obliterates, while enforced PD-L1 expression in preadipocytes confers, the immune-inhibitory effect of adipocytes. Pharmacologic inhibition of adipogenesis by the PPARγ antagonist GW9662 reduces adipose PD-L1 expression and enhances the antitumor efficacy of αPD-L1 and αPD-1 immunotherapies in female mice bearing syngeneic melanoma or mammary tumors. Combo treatment with GW9662 and αPD-L1 increased antitumor lymphocytes infiltration versus control or single agent treatment. In diet-induced obese female mice, combo treatment elicited suppressed melanoma growth, although less effectively versus lean females. In contrast to females, melanomas in either lean or obese male mice exhibited no applicable response to combo treatment. More strikingly, obese males lost αPD-L1 single treatment response versus lean males, with lower CD45+and CD3+T cell infiltration in tumors. However, castration in lean males rescued efficacy of combo treatment. These data suggest an antagonistic effect of male hormones in this combination. The potential impact of sex and obesity warrants consideration in future development of immunotherapy-related combination therapy.
B7H3 regulates tumor-mediated Th1 type immune responses
Authors: Lee Jun Sik , Kim Mi Eun , Cho Jun Hwi
Published: January 2023

B7H3 is a new molecule that can regulate T cells-mediated immune responses. Several studies suggested a costimulatory function for B7-H3 in murine models of cancer. In contrast, clinical association of B7-H3 expression suggested a coinhibitory role. The physiological role of B7-H3 is still unclear. To investigate the B7-H3 functions on T cell activation, we used the B7-H3 peptide, B7-H3 negative and overexpressed tumor cells. To establish the T cell inhibitory gene database by B7-H3, we performed the microarray. In the results, B7-H3 inhibited the T cell proliferation and production of cytokines including IL-2 and IFN-γ. We identified the 12 significant effective genes, and confirm the mRNA expression by real-time PCR. These results suggest that B7-H3 showed the co-inhibitory role on T cell functions. And further study is needed to identify the receptor and mechanism of B7-H3 on T cell function.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2017R1D1A1B03034673), Korea.
MyD88 dimerization inhibitors for targeting Diffuse Large B-cell Lymphomas
Authors: Snyder Greg A , Brown Lindsey , Faupel Ciara , Taylor Savannah , Sherman Matthew , Beadenkopf Robert , Montague Justin , Saikh Kamal , Wang Yajing
Published: January 2023

A recurring single amino acid somatic mutation associated with human diffuse large B cell lymphomas (DLBCLs), correlates with tumor cell proliferation and survival involving spontaneous and sustained activation of MyD88-dependent NF-κB and Janus Kinase (JAK) signaling pathways. MyD88 acts as a central signaling adapter for mediating innate and cytokine driven inflammation for the Interleukin-1 (IL-1R) and Toll-like receptors (TLRs). Computer aided molecular modeling of MyD88 and in silico screening have identified and functionally characterized MyD88 specific small molecule compounds shown to protect against Staphylococcal enterotoxin B (SEB) induced death in animal models. We hypothesize that MyD88 specific small molecule compounds may also be useful in treating DLBCLs bearing the oncogenic mutation MYD88L265P. Using in vitro and in vivo studies we evaluate MyD88 specific small molecule compounds for the ability to inhibit tumor cell proliferation and signaling in human patient cancer cells OCI Ly3 bearing the oncogenic mutation MYD88 L265P and OCI Ly19 DLBCLs. Previously we identified differences in the ability of MyD88 small molecule compounds to inhibit cell proliferation in activated human B cell lymphoma cells bearing the MyD88 L265P mutation. We now correlate these differences with a reduction of MyD88 interaction with IRAK in small molecule treated OCI-Ly3 cells bearing the MYD88 L265P mutation in comparison to OCI-Ly19 (wt-MyD88) and treated controls, as measured by CoIP. We continue to characterize MyD88 specific small molecule compounds that target MyD88 dimerization for their ability to reduce MyD88 containing signaling complexes in DLBCLs bearing MYD88 L265P mutation.
Host genetic background affects response to immune checkpoint inhibitors in a B16 melanoma model using Diversity Outbred mice
Authors: Hackett Justin , McCarthy Claire , Muniz Maria , Malysa Agnes , Wiesend Wendy , Gibson Heather
Published: January 2023

Historically metastatic melanoma has been associated with poor prognosis. The advent of immune checkpoint inhibitors targeting CTLA-4 and PD-1 have greatly improved patient survival in metastatic melanoma, but a significant proportion of patients fail to respond to therapy. To explore the effect of genetic background on immune checkpoint inhibitor efficacy, we have developed a tumor immunotherapy model using genetically heterogeneous Diversity Outbred (DO) mice and the C57BL/6 syngeneic B16F0 melanoma model. To implement genetic diversity, DO mice were crossed with C57BL/6 to generate (C57BL/6xDO)F1 mice. Untreated (C57BL/6xDO)F1 mice (n=17) reliably develop B16F0 tumors after subcutaneous inoculation, with some variation in tumor onset (8.2 +/− 4.1 days, CV 49.9%). In a pilot immunotherapy study, 20 (C57BL/6xDO)F1 mice were treated with anti-PD-1/anti-CTLA4 on days 12, 14, and 16, after tumors were palpable (mean onset 6.8 +/− 4.6 days, CV 68.2%). Tumor tissue was collected on day 19 and CD8 T cell infiltration was analyzed by IHC and qPCR. Mice with reduced tumor growth showed an increase presence of CD8+ cells and IFN-g expression. To further test the role of genetics in response, a second cohort of C57BL/6xDO)F1 mice (n=95) was treated with combined anti-PD1/anti-CTLA-4 on days 3, 6, and 10 after B16F0 inoculation. In this larger trial, mice receiving therapy show wide variation in tumor onset up to 61 days (mean 13.6 +/− 8.9, CV 65.2%), with 10 never developing tumor by day 88. This data further suggests that the genetic background significantly influences response to treatment and validates further testing of this model and subsequent Quantitative Trait Locus analysis. Supported by NIH R37 CA220482.
TSLP modulates anti-tumor Th2 immunity to melanoma via regulation of the PD-L2/PD-1 axis
Authors: Lai Jen-Feng , Thompson Lucas , Ziegler Steven F
Published: January 2023

The cytokine Thymic Stromal Lymphopoeitin (TSLP) has generally been ascribed with both suppressive and stimulatory roles in the context of tumor immune responses. Typically, TSLP-mediated tumor immunosuppression has been attributed to Th2 skewing of T cell responses; here we show that overexpression of TSLP in skin enhanced Th2 anti-tumor immunity to melanoma while deficiency of host expressed TSLP resulted in increased tumor growth. The effect of TSLP-mediated tumor immunity requires TSLP signal in host cells, resulting in elevated infiltration of PD-1+ Th2 CD4 T cells required for the response. Additionally, expression of PD-L2 on tumor-infiltrating antigen presenting cells (APCs) was enhanced by TLSP overexpression; conditional knockout of TSLP receptor on CD11c-expressing cells negated this effect. Disruption of PD-L2 signaling by genetic manipulation or antibody-mediated blockade of PD-L2 reduced infiltration of functional Th2 cells and abolished TSLP-mediated enhanced anti-tumor activity. These results suggest that anti-melanoma Th2-PD-L2/PD-1 axis is potentiated by TSLP to provide effective anti-tumor immunity. These findings provide novel cellular mechanistic insights for potential immunotherapeutic strategies.
MHCII density only weakly impacts autoreactive germinal center B cell fitness
Authors: Wittenborn Thomas Rea , Hagert Cecilia , Ferapontov Alexey , Jensen Lisbeth , Degn Søren Egedal
Published: January 2023

B cells generate high-affinity antibodies within induced microanatomical structures called germinal centers (GCs). Each GC is, in essence, a Darwinian microcosm, in which B cells undergo consecutive rounds of division, hypermutation, and selection based on antigen affinity. The consensus has been that this selection is governed by T follicular helper (Tfh) cell signals, determined by the amount of cognate peptide presented by B cells on major histocompatibility complex (MHC) class II. This view was recently challenged by observations in a foreign antigen model system suggesting that peptide-MHCII density dictates the degree of initial GC entry, but does not impact B cell selection once in the GC. GCs are central to the development of pathogenic autoantibodies in a breadth of autoimmune diseases, but it is unclear to what extent autoreactive GC behavior mimics that of foreign-antigen-reactive GCs. The question of peptide-MHCII in autoreactive GC fitness is particularly relevant because the linked recognition requirement provides a safeguard against inadvertent autoreactivity. Accordingly, we investigated the role of peptide-MHCII density in self-reactive GC B cell selection in a chimeric mouse model of epitope spreading. Bone marrow chimeras were generated, in which either MHCII null/b or MHCII null/null cells were competing with MHCII b/b cells for participation in chronic autoreactive GCs. Flow cytometry and confocal analysis of GCs revealed that whereas complete MHCII deficiency blocked B cell participation in the GC, partial MHCII deficiency had only a minor impact on GC fitness. Thus, our results confirm, in an autoreactive setting, the recently reported observation of MHCII haplosufficiency in foreign antigen responses.
Defining altered regulome structured in response to antigen co-receptors, toll-like receptors, and cytokine receptors stimuli to predispose early developing B cells for pathogenic fate in Systemic lupus erythematosus
Authors: Saini Ankur , Duan Meixue , Mi Tian , Hicks Sakeenah , Lee Eun-Hyung , Scharer Christopher D. , Boss Jeremy M , Gibson Greg , Sanz Ignacio
Published: January 2023

A TLR7-IFNg driven IL-21 mediated unique extra-follicular pathway of ASC differentiation of activated naïve B cells through an intermediary DN2 B cell subset has been characterized in pathogenesis of SLE. It has been correlated to distinct priming of naïve B cells characterized by the presence of SLE disease signatures which indicates towards the convergence of TLR, and cytokine signaling along with BCR signaling in the expansion and differentiation of peripheral B cells in SLE. Thus, we hypothesize that distinct combinations of innate, cytokines and antigen driven stimuli cause pathogenesis of SLE by maneuvering disease-related epigenetic programs and molecular networks during B cell development resulting in perturbed activation and differentiation. Indeed, initial data from bulk RNA-seq show enrichment of Type I interferon production and signature genes in HSCs to transitional B cells in SLE BM. Confounded by scRNA-seq, these populations also show profound type II interferon signature genes in SLE BM. Interestingly, BCR devoid Pro-B cells show reduced expression of BCR signaling intermediates with enhanced proliferation. However, expression of Pre-BCR or BCR on the surface of Pre-B cells or Immature B cells respectively are accompanied by increased expression of BCR co-receptors in SLE BM. On the other hand, metabolically SLE early BM B cells are less dependent on oxidative phosphorylation with reduced protein synthesis and processing. Together, these initial findings provide evidence that the proinflammatory microenvironment in SLE BM exerts its effect by differentially regulating BCR signaling factors and co-receptors in early B cell populations and thereby could define the pathogenic fate of B cell in periphery in SLE.
Metformin enhances the immunomodulatory potential of adipose-derived mesenchymal stem cells through STAT1 in an animal model of lupus
Authors: Jang Se Gwang , Lee Jaeseon , Hong Seung-Min , Kwok Seung-Ki , Cho Mi-La , Park Sung-Hwan
Published: January 2023

Mesenchymal stem cells (MSCs) are considered potential therapeutic agents for treating autoimmune disease because of their immunomodulatory capacities and anti-inflammatory effects. However, several studies have shown that there is no consistency in the effectiveness of the MSCs to treat for autoimmune disease. In this study, we investigated whether metformin could enhance the immunoregulatory function of MSCs, what mechanism is relevant, and whether metformin-treated MSCs could be effective in an animal lupus model. To examine the effects of metformin, adipose-derived (Ad)-MSCs were cultured for 72 h in the presence of metformin. Immunoregulatory factors expression was analyzed by real-time PCR and ELISA. MRL/lpr mice weekly injected 1×106 metformin-treated Ad-MSCs for 8 weeks. Proteinuria, serum anti-dsDNA antibody, lupus nephritis, immune complex, cellular subset were analyzed in each group. Metformin enhanced the immunomodulatory functions of Ad-MSCs including IDO, IL-10, and TGF-β. Metformin upregulated the expression of p-AMPK, p-STAT1, and inhibited the expression of p-STAT3, p-mTOR in Ad-MSCs. STAT1 inhibition by siRNA strongly diminished IDO, IL-10, TGF-β in metformin-treated Ad-MSCs. As a result, metformin promoted the immunoregulatory effect of Ad-MSCs by enhancing STAT1 expression, which was dependent on the AMPK/mTOR pathway. Administration of metformin-treated Ad-MSCs resulted in significant disease activity improvement in MRL/lpr mice. Moreover, metformin-treated Ad-MSCs inhibited CD4−CD8− T cell expansion and Th17/Treg cell ratio. These finding suggest that metformin optimized the immunoregulatory properties of Ad-MSCs and may be a novel therapeutic agent for the treatment of lupus.
Dnase1L3 levels from macrophages in providing protection against SLE development
Authors: Hernandez Colton J , Engavale Minal B , Keyel Peter A
Published: January 2023

A hallmark symptom of SLE is elevated autoantibodies, especially anti-dsDNA antibodies in multiple organs and tissues. Deficiency of Dnase1L3, a serum endonuclease, allows the accumulation of self-DNA leading to autoantibody generation. The source of Dnase1L3 is primarily expressed by macrophages and dendritic cells. Complete Dnase1L3 deficiency is known to promote the SLE phenotype, however, the dosage required to prevent disease onset is unknown. We hypothesize that macrophages provide sufficient Dnase1L3 expression to regulate autoantibody levels in the host’s system. We generated conditional, macrophage specific Dnase1L3 knockout mice to assess disease progression. We collected serum samples from 6–50 weeks of age and measured total IgG and anti-dsDNA levels by ELISA to develop a longitudinal analysis of autoantibody elevation. At sacrifice, we observed pathology and measured IgG deposition in the kidney and heart by immunofluorescence. We observed that the elimination of Dnase1L3 in macrophages increased anti-dsDNA antibodies in the conditional knockout mice. In addition, we observed minor abnormalities in the glomeruli size for the conditional knockout mice as compared to the wild type mice. Our results indicate that macrophages are capable of providing protection against the development of SLE.
Human gingiva-derived mesenchymal stem cells ameliorate rheumatoid arthritis through directly targeting of rheumatoid arthritis synovial fibroblasts
Authors: Zheng Song Guo , Zhang Ximei , Jarjour Wael , Wang Julie
Published: January 2023

Rheumatoid arthritis (RA) is a systemic-autoimmune-mediated disease characterized by synovial hyperplasia and progressive destruction of joint. The aim of this study is to evaluate whether human gingiva derived MSCs (GMSCs) can alleviate rheumatoid arthritis through directly targeting of rheumatoid arthritis synovial fibroblasts (RA-SF) and underlying mechanism(s).

GMSCs or Primary Dermal Fibroblast (PDF) were co-cultured with LPS-stimulated RA-SF. The proliferation, migration and invasion abilities of RA-SF or GMSCs treated RA-SF were examined for determining the suppressive effects of GMSCs on RA-SF. To learn whether GMSCs suppress RA-SF migration and invasion in vivo, GMSCs were subcutaneously co-transplanted with RA-SF and health cartilage into SCID mice to develop a humanized synovitis model, H&E or immunofluorescence staining were employed to determine the effects. Moreover, GMSCs were transferred into collagen induced arthritis model (CIA), proliferation, migration and invasion capacities of synovial fibroblasts from model mice or GMSCs treated mice were compared.

We found that the proliferation, migration and invasion capacities of GMSCs treated RA SF were significantly declined. Importantly, the expression of IL-6, IL-8, MMP1, MMP2, MMP3 and MMP13 were dramatically reduced in GMSCs treated RA-SF (qPCR results). Administration of GMSCs was therapeutic in collagen induced arthritis synovial fibroblasts pathology (proliferation, migration and invasion). Furthermore, GMSCs directly suppress RA-SF invasion in a humanized synovitis model.

We conclude that the manipulation of GMSCs can ameliorate rheumatoid arthritis (RA) through directly targeting of rheumatoid arthritis synovial fibroblasts.
IRAK-M Down-regulates the Activation of Microglial NLRP3 Inflammasome and GSDMD-Mediated Pyroptosis in Microglial through Inhibiting IRAK1/TRAF6 Combination During Experimental Autoimmune Encephalomyelitis
Authors: Zhang Jun , Pei Shanshan , Ding Yuewen , Peng Yu , Chen Jinyu , Wang Haitao , Wang Honghao
Published: January 2023

The NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) is a protein complex which mediates pro-inflammatory response and pyroptosis. The activation of NLRP3 inflammasome triggers pyroptosis via cleavage of the pore-forming protein gasdermin D. Previous studies have demonstrated that NLRP3 inflammasome activation in microglial contributes to the pathogenesis of EAE, while Inhibition of microglial NLRP3 inflammasome and the subsequent pyroptosis attenuates the severity of EAE. In addition, Interleukin-1 receptor-associated kinase (IRAK)-M, mainly expressed in microglial in the central nervous system, negatively regulates the TLR4/IL-1R signaling pathway through inhibiting the phosphorylation of IRAK1 and dissociation of IRAK1 from MyD88/IRAK-4/IRAK-1 complex as well as the formation of IRAK-1 and TRAF6 complex. Our recent study suggests the protective effect of microglial IRAK-M in EAE mice. Therefore, we hypothesize that IRAK-M may alleviate IRAK1-induced NLRP3 inflammasome activation and pyroptosis through inhibiting dissociation of IRAK1 from MyD88/IRAK-4/IRAK-1 complex and the following association of IRAK1 and TRAF6. Here, we used IRAK-M knockout mice and their microglia to clarify the role of IRAK-M in EAE. We demonstrated that IRAK-M decreased the incidence rate and improved the clinical symptoms accompanied by mild demyelination in EAE mice, and which was parallel to the inhibition of microglial NLRP3 inflammasome activation, pyroptosis, and IRAK1/TRAF6 formation in EAE. Our findings fill the gap in knowledge of IRAK-M on activation of NLRP3 inflammasome and pyroptosis and provide a potential novel target for the therapeutic strategy of multiple sclerosis and NLRP3-related diseases.
Retention and egress of Th1 cells in the inflamed skin
Authors: Salama Noah Alexander , Fowell Deborah J
Published: January 2023

The inappropriate trafficking and retention of immune subsets in tissue is a hallmark of many diseases prevalent in the developed world. These issues include autoimmunity, chronic inflammation, and unbalanced tissue repair responses which represent a significant disease burden: diabetes, coronary artery disease, inflammatory bowel disease, hepatitis, rheumatoid arthritis, and asthma. Although knowledge about cell trafficking to inflamed sites has grown in the past decade, there is a current gap in knowledge in identifying when and how immune cells decide to stay or leave these tissues.

To determine phenotypic and functional differences between those Th1 cells that remain at the inflamed site and those that exit, we employed the photoconvertible Kaede protein system to enable the labelling of Th1 cells in time and space. Kaede+OT-II Th1 T cells were adoptively transferred into WT mice immunized with CFA/OVA. Th1 cells recruited to the inflamed ear skin were photoconverted from Kaede green to Kaede red. Kaede red Th1 cells retained at the inflamed site could be distinguished from newly recruited Kaede green cells, while Kaede red Th1 cells that subsequently exited the inflamed skin were readily detected in the draining lymph node. Using this system, we are able to analyze the phenotypic (multicolor flow cytometry) and transcriptional signatures (RNA-seq) of Th1 cells that are retained at the inflamed site and those Th1 cells that exit (tissue-migrants). Given the temporal component of our model, we are gaining new insight into when and where expression of conventional markers of trafficking and activation are regulated (CD62L, CD44, and CD69) and seek to identify new pathways that dictate T cell retention and egress at inflammatory sites.
Epithelial cytokine IL-33 and sub-epithelial matrix protein tenascin C compartmentalize eosinophil phenotype and function
Authors: Nagasaka Reina , Doan Ton , Coden Mackenzie , Jeong Brian , Berdnikovs Sergejs
Published: January 2023

In allergic inflammation, eosinophils interacting with epithelial barriers experience exposure to epithelial cytokines and the extracellular milieu of airway remodeling. To better understand eosinophil activation biology in allergic tissue, we modeled the synergistic and opposing regulation of naïve eosinophils by both sub-epithelial extracellular matrix protein tenascin C (TNC), a key player in epithelial regeneration, and IL-33, an epithelial damage-induced cytokine. IL-5-differentiated bone marrow-derived murine eosinophils were cultured with recombinant TNC (25 ug/ml), IL-33 (100 ng/ml), or both for 24 hours and profiled by flow cytometry, qPCR, and DiffQuick staining. IL-33 alone significantly induced IL-4 and integrin CD11b expression, which was antagonized by co-culture with TNC. Conversely, TNC alone significantly promoted CD34 and intra-epithelial docking integrin CD103 expression, which was suppressed by IL-33 addition. Both treatments synergized in promoting expression of trans-epithelial migration marker CD11c. TNC promoted resident morphology while IL-33 induced an activated phenotype. This illustrates how microenvironmental cues may progressively shape eosinophils trafficking from sub-epithelial space across epithelial barriers.
Formin-like-1 mediates effector T cell extravasation through restrictive endothelial barriers enabling T cell entry into peripheral tissues and autoimmunity induction
Authors: Jacobelli Jordan , Thompson Scott B. , Sandor Adam M. , Lui Victor , Chung Jeffrey W. , Waldman Monique M. , Friedman Rachel S.
Published: January 2023

To perform their functions, T cells migrate from the blood vasculature into tissues by traversing the vascular wall through a process known as transendothelial migration (TEM). While many of the extracellular cues that guide this process have been identified, the role of downstream cytoskeletal effectors that mediate force generation and morphological changes required for TEM remains an area of active research. Formin-like-1 (FMNL1) is a cytoskeletal effector that mediates cytoskeletal remodeling. However, the role of FMNL1 in T cell functions, and migration in particular, is mostly unknown. The goal of our study was to examine the role of FMNL1 in T cell extravasation and trafficking. We generated a new FMNL1 knock-out (KO) mouse and identified a novel role for FMNL1 in promoting T cell migration through restrictive endothelial barriers during TEM. In vitro, FMNL1-deficient T cells had reduced actin polymerization in response to chemokine signaling. Furthermore, FMNL1 KO T cells had a selective impairment in chemotaxis through restrictive 3μm pores vs permissive 5μm pores. Using time-lapse microscopy, we determined that FMNL1-deficient T cells are severely impaired in their capacity to complete TEM through endothelial barriers. In vivo, we found that FMNL1 was dispensable for T cell extravasation through permissive high endothelial venules and homeostatic trafficking to lymphoid organs. Instead, FMNL1 regulated the ability of activated T cells to extravasate into inflamed peripheral tissues and enabled self-reactive T cells to induce autoimmune disease. Overall, our data identify FMNL1 as a key regulator of lymphocyte migration through restrictive barriers and as a potential target for modulating effector T cell trafficking.
Unraveling the role of β2 integrins in neutrophil migration to the lung and subsequent acute lung injury in sepsis
Authors: Koutsogiannaki Sophia , Kho Alvin T. , Yuki Koichi
Published: January 2023

Sepsis is the leading cause of death in intensive care units but not specific therapy is yet available. Acute lung injury (ALI) is a major complication in sepsis, resulting from massive migration of neutrophils to the lung, but the exact mechanism is not known. β2 (CD18) integrin family [consisting of αLβ2 (CD11a/CD18), αMβ2 (CD11b/CD18), αXβ2 (CD11c/CD18) and αDβ2 (CD11d/CD18)] and its counter-receptor on the endothelium, ICAM-1 (CD54), are critical for neutrophil migration, but their role in ALI is not yet delineated. Using a murine model of sepsis induced by cecal ligation and puncture (CLP) surgery, we showed decreased neutrophil levels and attenuated injury in the lung of β2−/− and ICAM-1−/−mice at 12h post-CLP, suggesting the importance of β2 integrins in this process. In addition, we observed decreased neutrophil levels and attenuated injury in the lung of αDβ2−/− mice but not in the lung of αLβ2−/− and αMβ2−/− mice, suggesting that among β2 integrins, αDβ2 has an orchestrating role in neutrophil migration to the lung and subsequent ALI in sepsis. In support, we found increased αDβ2 expression levels on neutrophils in the lung and blood of WT mice as sepsis progressed. RNAseq analysis in neutrophils from blood and lung of WT and αDβ2−/− mice showed that αDβ2 mediates neutrophil migration to the lung through pathways associated with antigen representation, maturation, apoptosis and NOD-like receptor signaling. αDβ2−/− neutrophils had also less Cxcr2, Ltb4r1 and Dhrs9 expression, associated with neutrophil migration and the retinoic acid pathway. Together, our results suggest a mechanism for neutrophil migration to the lung and subsequent ALI in sepsis in which αDβ2 has a major role and could be a novel target for therapeutic intervention.
BCG-Infected Dendritic Cells Induce TNF-alpha Dependent Cell Cluster Formation that Damage Brain Endothelial Cells in an In Vitro Model of the Blood Brain Barrier
Authors: Gilpin Trey E , Walter Fruzsina R , Herbath Melinda , Wigand Kelsey , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

Central nervous system tuberculosis (CNSTB) is the most serious manifestation of extrapulmonary tuberculosis infections representing 5–10% of all cases. How Mycobacterium tuberculosis (Mtb) crosses the highly regulated blood-brain barrier (BBB) and enters the CNS, is still not well understood. We showed previously that mycobacterium infected dendritic cells adhere to brain microvessel endothelial cells. Here, we applied a novel model of in vitro BBB consisting of primary brain astrocytes and microvessel endothelial cells to test the mechanisms of how mycobacterium infected dendritic cells (DCs) interact with peripheral blood mononuclear cells (PBMC) and affect the BBB. We show that infected DC migration was hindered but infected DCs induce cellular cluster formations containing PMBC and T cells in addition to DCs. These clusters contained 24% CD11c+, 57%CD11b+, and 16% CD4+ cells, while B220+ B cell number was negligible mimicking the cellular composition of pulmonary granulomatous-like lesions. PBMC and infected DC clusters strongly adhered to the in vitro BBB and induced cluster associated damage (CAD). CAD correlated with mitochondrial disorganization and the degradation of ZO-1, claudin-5 tight junction associated proteins, upregulation of cellular adhesion molecules VCAM-1 and ICAM-1 in endothelial cells. Anti-TNF-a inhibition reduced cluster formation as well as ICAM-1 expression on brain microvessel endothelial cells and mitigated CAD. This research will lead to better knowledge in understanding how mycobacteria affects the BBB that could lead to better therapeutic treatments for CNSTB infection.
Combining RNA in situ hybridization and spectral flow cytometry to investigate the leukocyte glycocalyx in autoimmunity
Authors: Marshall Payton L , Kaber Gernot , Linde Miles H , Barlow Graham L , Haddock Naomi L , Wagar Lisa , Nagy Nadine , Bollyky Paul L
Published: January 2023

The glycocalyx surrounding leukocytes has been studied for its role in immunomodulation and its potential as a therapeutic target. 4-methylumbelliferone (4MU) is a small molecule that inhibits production of the glycocalyx’s primary polysaccharide component hyaluronan (HA), by targeting the HA synthase (HAS). Oral treatment with 4MU has proved efficacious in preventing the development and progression of autoimmunity, though the mechanism is contested.

We have previously shown that 4MU strongly inhibits T-cell mediated autoimmunity, and report here that it also attenuates formation of antigen-specific plasmablasts and subsequent autoantibody production. This suggests that the HA glycocalyx plays a role in promoting the humoral component of autoimmunity.

The HAS proteins are deeply membrane embedded, preventing the use of traditional flow cytometry antibodies to define expression levels across leukocyte compartments. This limitation has made it difficult to identify cellular targets of 4MU. Alternative measurements must be used to study the HA glycocalyx. Surface HA content of a cell can be measured with labelled HA binding protein (HABP), though this does not explain whether HA was synthesized or scavenged with HA-binding receptors. RNA measurement can also be used to look at transcript levels of the HAS proteins.

Here, we combine RNA in situ hybridization, fluorescently labeled HA binding protein (HABP), and conventional flow cytometry antibodies to define the HA glycocalyx on leukocytes during autoimmunity. Utilizing Spectral Flow Cytometry, we can assess parameters in numbers comparable to Mass Cytometry (CyTOF) to identify which cells are most likely targeted by 4MU to prevent antibody formation during autoimmunity.
GPR56 inhibits chemotaxis of human Natural Killer and TEMRA lymphocytes
Authors: Janks Laura , Habiel David , Litvak Vladimir , Lin Lih-Ling , Kwon Hakju
Published: January 2023

Within the human immune system, the expression of GPR56, an adhesion G-protein couple receptor, is restricted to cytotoxic Natural Killer (NK) and T lymphocytes, including T effector memory re-expressing CD45RA (TEMRA) cells. In primary NK cells, GPR56 acts as an inhibitory receptor, as it diminishes their ability to eliminate target cells. Overexpression of GPR56 in immortal NK-92 cells also leads to robust inhibition of cell chemotaxis. GPR56 is speculated to promote retention of cytotoxic lymphocytes in inflamed peripheral tissues. Thus, GPR56 has the potential to regulate immune responses that may lead to excessive inflammation and autoimmunity. The aim of the present study was, therefore, to examine whether GPR56 mediates cytotoxic immune cell functions and whether GPR56 expression is altered in autoimmune diseases. To tackle these questions, we first compared expression of GPR56 in healthy and autoimmune disease patient tissues using scRNA-seq datasets. Next, we tested whether stimulation of GPR56 with a monoclonal antibody modulated the migratory capacity of NK and CD8+ TEMRA cells. We found that chemotaxis toward CXCL12, IL-8, and CX3CL1 was strikingly inhibited by GPR56. Using bulk RNA-seq, we found GPR56-stimulation impacted a multitude of signaling cascades that regulate cell chemotaxis. Taken together, our results confirm that GPR56 is a marker for NK and TEMRA cells in healthy and autoimmune patient tissues and establish GPR56 as a key regulator of lymphocyte chemotaxis.