bluuebunny/crossref_metadata_2025 huggingface dataset:
Induced MHCII expression on breast cancer cells broadens the responding T cell repertoire, delays tumor-specific T cell exhaustion, and impairs tumor growth
Authors: McCaw Tyler R , Li Mei , Starenki Dmytro , Cooper Sara , Meza-Perez Selene , Buchsbaum Donald , Forero Andres , Randall Troy D.
Published: January 2023

We recently reported that aberrant expression of Major Histocompatibility Class II (MHCII) on human triple negative breast cancer cells correlates with increased tumor infiltrating lymphocytes and prolonged progression free survival. This observation led us to hypothesize that expression of MHCII enhanced the intratumoral CD4+ T cell response, thereby bolstering the tumor-specific CD8+ T cell response, leading to impaired tumor growth. To test this, we transfected the murine breast cancer line TS/A with human class II transcriptional activator (hCIITA) or empty vector, creating MHCII-expressing and MHCII-negative cell lines, respectively. We found that MHCII-expressing tumors grew slower than controls in immunocompetent recipients, but that this difference was abrogated in CD4-depleted and nullified in SCID mice. CD4+ T cells within hCIITA-transfected tumors produced more IFNγ for longer times than those from control tumors. Similarly, CD8+ T cells in MHCII-expressing tumors displayed a more activated phenotype and produced more IFNγ and granzyme B for longer times. Nevertheless, both CD4+ and CD8+ T cells eventually became exhausted in both groups. In addition to boosted effector function, TCR repertoire analysis demonstrated an increase in both breadth and amplitude of T cell response to MHCII-expressing tumors. We next investigated the possibility of inducing MHCII on non-expressing tumors using the histone deacetylase inhibitor Entinostat. Indeed, Entinostat turned on MHCII expression, which correlated with significantly reduced tumor burden. Thus, epigenetic modifiers capable of inducing MHCII expression on tumor cells could avail this augmented T cell response to all patients for enhanced tumor control.
Combining impedence-based viability measurements and flow cytometric analyte quantitation to evaluate effector cell killing of T lymphocytes
Authors: Guenther Garret , Tang Wei , Ye Peifang , Le Ming , Cerignoli Fabio , Xi Biao , Zhou Leyna , Abassi Yama
Published: January 2023

Cancer immunotherapy is increasingly being evaluated in the lab, as well as the clinic as an approach for cancer treatment by harnessing and enhancing the immune system to attack cancer cells. Moving this research from the bench to the clinic is critically important, but we need reliable tools to be able to translate the in-vitro protocols and results for use in-vivo. Here, we have used a combination of impedence-based technology and flow cytometry to evaluate both the target and effector cells in an immune-cell based killing assay. The impedence based Real Time Cell Analysis technology, provides us with a continuous readout of target cell viability and flow cytometry allows us to measure responses using specific labels, such as effector cell viability and cytokine production in the supernatant of these assays. Here, we demonstrate cell killing assays measuring the cytotoxic effect of T-lymphocytes on a B-cancer cell line in conjunction with cytokine concentration measurements of IL-2, IL-4, IL-5, IL-10, TNF-a, IFN-g, IL-8, IL-1b, IL-6, IL-10, and IL-12p70 to simultaneously understand the T-lymphocyte response in these assays. Using both of these methods allows us to thoroughly evaluate immune cell killing efficacy and kinetics. The potency assays described in these studies have great potential for use in-vivo models and clinic.
Immunization with Human Papillomavirus type-18 E1 protein induce CD8+ T cells with cytotoxic activity against cells expressing E1 from HPV-11, 33 and 53.
Authors: Amador Alfredo , Romero Damaris , Lizano Marcela
Published: January 2023

CD8+ mediated immune response plays a major role in the clearance of Human Papillomavirus (HPV) infected cells. Although HPV vaccines are effective in preventing HPV infections through humoral response, they fail to protect against some HPV types and established infections. In the natural history of an HPV infection, the expression of E1 helicase during viral replication makes it a potential immunological target. The early expressed E1 protein is one of the most conserved proteins among papillomaviruses. We demonstrate in previous work, that immunization of HPV type 18 E1 protein along with α-GalactosylCeramide (α-GalCer) adjuvant elicited CD8 T cell-immune response, able to eliminate tumor cells expressing HPV18 E1 protein. For this study, we compared the amino acids sequence of E1carboxi-terminal (CT) domain from HPV18, 6, 11, 16, 30, 31, 35, 45 and 53. The sequences have 60% of identity and preserve two motifs previously described for being common between many Helicase types. We cloned E1-CT domain of HPV11, HPV33 and HPV53, and its expression in B16-F0 cells was determine.

We show that CD8 T cells from mice immunized with HPV18 E1 protein plus α-GalCer, are actively cytotoxic in co-culture with B16-F0 cells expressing E1-CT of HPV11, HPV33 and HPV53 in comparison with unimmunized mice. Cytotoxicity activity was evaluated by detection of CD107a and CD107b on the surface of CD8 T cells as part of cytotoxic granule exocytosis. Ongoing experiments will examine if these CD8 T cells from mice immunized with HPV18 E1 protein plus α-GalCer are also actively cytotoxic in presence of other E1 HPV types.

The characterization of new immunological targets against HPV, could help to develop new strategies to clearance HPV established infections.
CD8+ T cell-independent mechanisms of PD-1-mediated suppression of anti-tumor immunity in mice
Authors: Pauken Kristen E , Juneja Vikram , McGuire Kathleen , LaFleur Martin , Kuchroo Juhi , Sage Peter , Chevrier Nicolas , Freeman Gordon J , Sharpe Arlene H
Published: January 2023

Over expression of the inhibitory receptor PD-1 commonly occurs in cancer, and PD-1 signaling suppresses protective anti-tumor immune responses. While early clinical trials with PD-1 inhibitors in cancer have been promising, the majority of patients have not shown durable clinical benefit, and the mechanisms underlying both successes and failures remain poorly defined. CD8+ T cells can directly kill tumor cells, and CD8+T cell infiltration of human tumors positively correlates with responses to PD-1 blockade. CD8+ T cells in tumors (TILs) often show high expression of PD-1; however, PD-1 is also expressed by other cell types, including CD4+ T cells, regulatory T cells (Treg), B cells, and some myeloid cells. Consequently, we hypothesized that loss of PD-1 only on CD8+ T cells may not be sufficient to control tumor burden. To address this question, we compared tumor responses in mice completely deficient for PD-1 (referred to as cKO) or selectively deficient for PD-1 on CD8+ T cells (E8i-cre PD-1f/f mice, referred to as CD8ΔPD-1). Following subcutaneous implantation of MC38 adenocarcinoma tumor cells, 100% of PD-1 cKO mice cleared the tumor. In contrast, only 70% of CD8ΔPD-1 mice controlled the tumor. Moreover, in CD8ΔPD-1 mice that controlled tumor, the average time to clearance was delayed compared to PD-1 cKO mice. In both PD-1 cKO and CD8ΔPD-1 mice, CD8+ TILs showed elevated granzyme B levels and an increased CD8 to Treg ratio, suggesting that CD8+ T cell-independent mechanisms contribute to immunity following loss of PD-1 in MC38 tumors. On-going work examining both tumor cells and infiltrating immune cells is focused on identifying these CD8+T cell-independent mechanisms of PD-1-mediated suppression.
SETD1B compensates loss of IRF8 expression to activate iNOS expression in myeloid-derived suppressor cells through an epigenetic mechanism
Authors: Redd Priscilla S , Ibrahim Mohammed L , Klement John D , Sharman Sarah K , Paschall Amy V , Yang Dafeng , Nayak-Kapoor Asha , Liu Kebin
Published: January 2023

Inducible nitric oxide synthase (iNOS) generates nitric oxide (NO) in myeloid cells that acts as a defense mechanism to suppress invading microorganisms or neoplastic cells. In tumor-bearing mice, elevated iNOS expression is a hallmark of myeloid-derived suppressor cells (MDSCs). MDSCs use NO to nitrate the T cell receptor and STAT1 to inhibit T cell activation and the anti-tumor immune response. The molecular mechanism for iNOS expression regulation in tumor-induced MDSCs is unknown. We show that IRF8 deficiency results in diminished iNOS expression in mature CD11b+Gr1− and immature CD11b+Gr1+ myeloid cells in vivo, indicating that IRF8 is an essential transcriptional activator for iNOS expression in myeloid cells under physiological conditions. Though IRF8 is silenced in tumor-induced MDSCs, iNOS expression is dramatically elevated in tumor-induced MDSCs suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathological conditions. Tumor-induced MDSCs exhibit diminished STAT1 and NF-κB Rel protein level, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSCs show increased SETD1B expression compared to their equivalent in tumor-free mice. Chromatin immunoprecipitation revealed that H3K4me3, a product of SETD1B enzymatic activity, is enriched at the nos2 promoter in tumor-induced MDSCs. Inhibition/silencing of SETD1B diminished iNOS expression in tumor-induced MDSCs. Our data determine that IRF8 is an essential transcriptional activator of iNOS in myeloid cells under physiological conditions. Tumor cells use the SETD1B-H3K4me3 epigenetic axis to compensate for loss of IRF8 expression to activate iNOS expression in MDSCs under pathological conditions.
Delineating the epigenetic regulation of myeloid derived suppressor cell generation in hepatocellular carcinoma associated with cirrhosis
Authors: MAN LIU , Zhou Jingying , Wong John , Chan Anthony W.H. , Chen Zhiwei , Cheng Alfred S.L.
Published: January 2023

Hepatocellular carcinoma (HCC) is the most common liver tumor with dismal prognosis. Since most liver tumors arise in a context of chronic inflammation associated with fibrosis, tumor microenvironment plays a critical role in liver carcinogenesis. Myeloid derived suppressor cell (MDSC) is a population of immature myeloid cells that undermine immune surveillance and facilitate tumor immune escape. As a key driver of liver fibrosis, activated hepatic stellate cell (HSC) has been recently shown to induce MDSC generation through cell-cell contact and/or secreted soluble factors. Given the crucial role of epigenetics in cell lineage specification, we aim to elucidate the chromatin regulation of MDSC in the immunosuppressive liver tumor microenvironment. Using human PBMCs from healthy donors, we have successfully recapitulated the immunomodulatory effect of HSCs on monocytic MDSC generation. Comprehensive epigenetic drug screening and expression analysis indicated that accumulation of histone H3 lysine 27 acetylation (H3K27ac), an active enhancer histone mark, is involved in the process of HSC-induced MDSC generation. We further demonstrated that inhibitors of BET family, which disrupts BRD4 association with H3K27ac-enriched active enhancers, abrogates MDSC generation. These results highlight the importance of epigenetic regulation in MDSC accumulation in the fibrotic microenvironment. Ongoing work is directed towards the identification of HSC-activated enhancers that orchestrate MDSC identity and functions. As we showed high level of monocytic MDSC accumulation in patients with cirrhotic HCC, our study may provide novel therapeutic targets to restore effective anti-tumor immune response in this dreadful disease.
TIPE2 is a novel direct target of STAT3 in MDSC and inhibition of its expression on MDSC enhanced T cell activation in tumor
Authors: Yan De hong , Wang Jing hui , Wan Xiao chun
Published: January 2023

MDSCs represent an immature population of myeloid cells that inhibit both innate and adaptive immunity in most cancer patients and mice bearing various tumors through several inflammatory mediators. TIPE2, a member of tumor necrosis factor (TNF)-alpha-induced protein 8 (TNFAIP8 or TIPE) family, is a negative regulator of innate and adaptive immunity. In this study, we investigated the influence of tumor-derived factors on TIPE2 expression in MDSCs. We demonstrate that tumor-derived IL-6 induced TIPE2 up-regulation expression of MDSCs. This effect was dependent on phosphorylation signal transducer of activator of transcription 3 (STAT3) and blockade of STAT3 signaling also reversed the effect of tumor-derived IL-6 on TIPE2 expression in MDSCs. Chromatin immunoprecipitation and luciferase reporter assay revealed direct binding of STAT3 to a transcriptionally active STAT3-response element (SRE) in the TIPE2 proximal promoter. Inhibition of TIPE2 expression on MDSCs by shRNA technology enhanced T cell activation and abrogated the suppressive activity of MDSCs. More importantly, downregulation of TIPE2 level on MDSCs by stattic, a STAT3 inhibitor, significantly delayed tumor growth in tumor-bearing mice. Targeting of TIPE2 therapy may represent a novel approach for cancer immunotherapy.
In vitro model of Rich foci: mycobacterium-induced granuloma formation in the central nervous system
Authors: Walter Fruzsina R , Gilpin Trey E , Mergaert Aisha , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

Central nervous system (CNS) involvement is the most devastating manifestation of tuberculosis (TB) infection. 5–10 % of the extrapulmonary cases affect immunocompromised individuals and children. Although CNS TB is a global phenomenon with extremely high mortality, understanding how bacteria disseminate into the brain parenchyma is not well understood. In the 1930s Rich and McCordock proposed that bacteria travel via the blood and form a tuberculoma (Rich foci). These foci may rupture resulting in diffuse meningitis. Previously our laboratory showed that bone marrow derived dendritic cells (DCs) migrate across the blood-brain barrier (BBB) into the CNS during inflammation in vitro and in vivo. We also described Mycobacterium bovis (BCG) and M. tuberculosis (Mtb) dissemination from pulmonary granulomas by DCs. We hypothesize that DCs play a main role in the dissemination of mycobacteria and formation of Rich foci. Our goal was to understand the mechanisms and cellular interactions between brain endothelial cells and leukocytes in the formation of the foci in the CNS. Here we show that using an in vitro, primary mouse cell based BBB co-culture system, cellular aggregates resembling granulomas form on the endothelial cell layer, which after four days consisted of infected cells including DCs that were surrounded by leukocytes. More importantly aggregates migrated together to the other side of the monolayer as a migrating island of cells showing that cellular migration of mycobacterium infected cells across the BBB involves relocalization of cellular aggregates. The potential interference through the formation of inflammatory foci on the surface of the brain endothelial layer may provide a way to decrease Mtb dissemination.
Early disruption of the intercelullar junction proteins leads to alveolar-capillary barrier dysfunction and pulmonary edema in a mouse ricin intoxication model of Acute Respiratory Distress Syndrome.
Authors: Sapoznikov Anita , Ehrlich Sharon , Gal Yoav , Falach Reut , Kronman Chanoch , Sabo Tamar
Published: January 2023

Acute respiratory distress syndrome (ARDS), charactarized by an intense pulmonary inflammation involving neutrophil recruitment, is accompanied by a severe disruption of the alveolar-capillary barrier leading to life-threatening pulmonary edema. Ricin, a plant-toxin derived from the seeds of Ricinus communis, irreversibly inactivates ribosomes by site-specific depurination, thereby arresting cell protein synthesis, while its clinical manifestation following pulmonary exposure is that of rapidly developing severe ARDS. We evaluated the role of the members of tight, adherens and gap junction proteins in pulmonary edema formation in a clinically relevant mouse model of ARDS induced by ricin intoxication, since the respiratory endothelium and epithelium barrier function depends on the integrity of intercellular junctions. We found a significant reduction in Claudin5, Claudin18, VE-Cadherin and Connexin43 in mice lungs 3 hours after ricin intoxication. Neutrophil depletion markedly attenuated pulmonary edema and in combination with a matrix metalloproteinase (MMP) inhibitor restored the expression of VE-Cadherin and Connexin43, providing an evidence for MMP-dependent proteolysis of these proteins. Unlike the aforementioned proteins, disruption of Claudin18 following ricin intoxication was found to be neutrophil-independent and involved tyrosine phosphorylation. These results provide an in vivo mechanism for early impairment of the alveolar-capillary barrier by intercellular junction protein disruption, which at later time points, is enhanced by the specific loss of type II alveolar epithelial cells that no longer provide resistance to edema formation and altogether lead to respiratory failure and death.
A potential target for methotrexate in macrophages : AMP-activated protein kinase.
Authors: Cudrici Cornelia D , Pelletier Martin , Siegel Richard
Published: January 2023

Background

Methotrexate (MTX) remains a cornerstone of treatment in multiple forms of inflammatory arthritis. AMPK is a highly conserved protein kinase present in all eukaryotic cells and is activated by an increasing intracellular ADP/ATP levels. Once activated, AMPK will promote ATP production by switching on catabolic and turning off anabolic pathways. We hypothesize that AMPK activation mediates a major portion of the anti-inflammatory effects of MTX.

Methods

We investigated the role of the anti-inflammatory effect of MTX via AMPK in human monocytes-derived macrophages (MDM) and mouse bone marrow-derived macrophages (BMDM) along with AICAR and A769662 (AMPK activators) and compound C (a selective AMPK inhibitor). AMPK phosphorylation and total AMPK were measured by Western blotting. Cells were then stimulated with LPS or TNF-α, and production of pro-inflammatory cytokines were measured in the supernatant.

Results

MTX induced AMPK activation with effects comparable to the AMPK activators (A769662, AICAR ) in hMDM and BMDM. MTX-induced AMPK activation was associated with a reduction in the production of IL-6, IL-1 β, and TNF-α in response to LPS and TNF stimulation. Compound C is able to partially reverse the effects of MTX.

Conclusions

MTX is able to induce AMPK activation in both MDM and BMDM, and suppress pro-inflammatory cytokines in a manner dependent on AMPK activity. These results have been confirmed genetically in macrophages deficient in AMPK subunits and in serum transfer arthritis. Our findings raise the possibility that some anti-inflammatory effects of MTX are mediated by AMPK, suggest that AMPK may be a target for the anti-inflammatory agents and a target for the development of new anti-inflammatory drugs.
Conditioned medium from tonsil-derived mesenchymal stem cells promotes adiponectin production
Authors: Kim Yu-Hee , Cho Kyung-Ah , Park Minhwa , Woo So-Youn , Ryu Kyung-Ha
Published: January 2023

Mesenchymal stem cells (MSCs) and conditioned medium (MSC CM) are now considered to be a good source for the development of regenerative medicine. Previously, we reported that tonsil-derived MSC (T-MSC) CM produces visceral fat reducing effects. As reduction in visceral adiposity is closely related to the increase of adiponectin in circulation, we sought to extend our previous findings and explore the effects of T-MSC CM on adiponectin production. T-MSC CM was collected from previously isolated and characterized T-MSCs and injected into a senescence-accelerated mouse prone 6 (SAMP6), which exhibits characteristics of aging and obesity. We observed reduction of mouse weight and epididymal adipose tissue (eAT) mass following injection of T-MSC CM. Significant increase in adiponectin expression in eAT and total and high molecular weight (HMW) adiponectin in circulation were determined in the T-MSC CM-injected mice compared to the control. In 3T3-L1 adipocytes, T-MSC CM treatment increased adiponectin secretion and multimerization. Furthermore, glucose oxidase was used to induce oxidative stress in 3T3-L1 adipocytes and effects of T-MSC CM on the reduction of reactive oxygen species (ROS) production and oxidative stress markers expression were observed. Restoration in the HMW adiponectin production was also shown which suggests that T-MSC CM may enhance adiponectin multimerization via amelioration in the oxidative stress. Further studies are guaranteed in a purpose of elucidation of anti-oxidants secreted from T-MSCs and these findings highlight the potential therapeutic relevance of T-MSC CM for treatment of obesity or obesity-related diseases. (NRF-2016R1A6A3A11933360)
Platelet TGFBIp induces foam cell formation after platelet phagocytosis
Authors: Kim Ha-Jeong , Rhe Suyeon , Jung Haeuk
Published: January 2023

Foam cells play a critical role in the atherosclerosis development. It is reported that platelets phagocytosis induces foam cell formation by producing more inflammatory cytokines. However, platelet induced foam cell formation mechanism is not clear. TGFBIp is a secreted extracellular matrix protein that is consisted by 4 fasciclin-1 domains. TGFBIp is produced by several cells include macrophages and works as chemoattractant to monocytes. TGFBIp is also in the platelet granule and it is secreted and bound on platelets when platelets are activated. Platelets are removed by macrophages when they are activated. However, the role of TGFBIp on activated platelets is not clear. In this report, we used TGFBIp lacking platelets to investigate the role of TGFBIp in platelets. When macrophages uptake TGFBIp lacking platelets, foam cell formation is less than WT platelets are uptaken.
TLR4 Activation of Small GTPase RhoA Signaling Mediates Endotoxin-Induced Endothelial Barrier Dysfunction
Authors: Tauseef Mohammad , Newaz Mohammad , Al-Ghoul Walid , Aqil Madeeha , Fazal Nadeem
Published: January 2023

Sepsis is an uncontrolled activation of immune response, leads to multi-organ failure and high rate of mortality. Studies show the role of increase in endothelial activation and barrier dysfunction significantly contribute to the pathophysiology in sepsis. Endothelial cells form a tight barrier between blood and underlying tissue, and thus maintain an anti-inflammatory milieu. Here, we investigated the role of small GTPase, RhoA activation in regulating the increase in human coronary artery endothelial (HCAE) permeability in response to TLR4-receptor agonist, endotoxin lipopolysaccharide (LPS). Endothelial permeability was determined by determining the influx of Evans blue-labeled albumin across endothelial monolayer on 0.4μ Transwell filters. Our data show that LPS induced RhoA activation and resulted in increase in HCAE cell monolayer permeability. Thus, we tested the hypothesis that LPS mediated induction of RhoA activates pro-inflammatory signaling and eliciting endothelial barrier dysfunction. Knockdown of RhoA using siRNA approach abrogated LPS induced increase in endothelial permeability. Rho-associated kinase (ROCK) is an effector of the RhoA. To further determine the molecular mechanism involved downstream of RhoA activation, we pretreated the cell with ROCK inhibitor (Y-27632) and we found that LPS failed to induce endothelial permeability in cells treated with Y-27642. In conclusion, our study shows that LPS induces activation of the RhoA - ROCK signaling pathway, which leads to increase in endothelial permeability and barrier dysfunction. Therefore, ROCK emerges as a potential drug target to treat sepsis induced endothelial permeability and inflammation.
The role of innate lymphoid cells in the cardiac inflammation
Authors: Choi Hee Sun , Bracamonte-Baran William , Hou Xuezhou , Diny Nicola , Talor Monica , Cihakova Daniela
Published: January 2023

To investigate the role of innate lymphoid cells (ILCs) in the heart, we have established a pericarditis model in which mice develop pericarditis upon IL-33 injections. Studies have shown the role of group 2 innate lymphoid cells (ILC2s) mostly in mucosal organs such as lung and gut, however, the role of ILC2s in the heart has been overlooked. We found that the number of ILC2s increases in the heart after IL-33 treatment. The aim of this work is to examine the role of ILC2s in inflammatory heart disease using the IL-33-induced pericarditis model. We hypothesize that IL-33 induces cardiac resident ILCs to become activated ILC2s to produce large amounts of IL-5 and IL-13, driving the development of pericarditis. To determine whether ILCs are required for pericarditis development, we compared WT mice to Rag2−/− mice and to Rag2−/−γc−/− mice which lack all of lymphoid cells including ILCs. Whereas Rag2−/− mice developed pericarditis, Rag2−/−γc−/− mice were completely protected. This indicates that the adaptive immune response is not required for the development of IL-33-induced pericarditis, but ILCs may play a pathogenic role. We assessed cytokine production by ILC2s in pericarditis to determine the function of ILC2s in the heart. Cardiac ILC2s were a major source of IL-5 and IL-13. The number of eosinophils in the heart was significantly reduced by anti-IL-5 treatment, which prevented the development of pericarditis. In addition, IL-13−/− mice developed less severe pericarditis compared to WT mice. These data suggest that ILCs play a critical role in inflammatory heart diseases. The pathogenic mechanism of ILC2s in the heart could provide insight into novel therapeutic strategies for treating cardiac inflammation including pericarditis.
CD300f-expressing dendritic cells are critical for controlling chronic gut inflammation
Authors: LEE HA-NA , Linjie Tian , Krzewski Konrad , Coligan John E.
Published: January 2023

Pro-inflammatory cytokine overproduction and excessive cell death, coupled with the impaired apoptotic cell clearance, have been implicated as causes for failure to resolve gut inflammation in inflammatory bowel diseases. CD300f (CLM-1), a cell surface receptor that recognizes phosphatidylserine exposed on apoptotic cells, is important for immune homeostasis. In this study, we show that CD300f -expressing dendritic cells play a crucial role in regulating gut inflammatory responses. CD300f-deficient mice fail to resolve colonic inflammation, due to defects in dendritic cell function associated with abnormal apoptotic cell build-up in the gut. CD300f-deficient dendritic cells display hyperactive phagocytosis of apoptotic cells, which stimulates them to excessive TNF-α secretion. This, in turn, induces secondary IFN-γ overproduction by colonic T cells, leading toprolonged gut inflammation. Our data highlight a previously unappreciated role of dendritic cells in controlling gut homeostasis, and show that CD300f regulation of apoptotic cell uptake is essential for suppressing overactive dendritic cell-mediated inflammatory responses, thereby controlling the development of chronic gut inflammation.
Sex and the Nba2 lupus susceptibility interval are differentially associated with specific costimulatory molecule expression profiles on dendritic cells in mice.
Authors: Bupp Melanie R Gubbels , Jorgensen Trine N , Thoner Timothy
Published: January 2023

Systemic lupus erythematosus (SLE), an autoimmune disorder that primarily affects women, is characterized by the production of autoreactive antinuclear autoantibodies and elevated levels of serum interferon-alpha (IFN-α). Female B6 mice congenic for the Nba2 lupus susceptibility interval develop a lupus-like disease with elevated serum IFN-α secreted by plasmacytoid dendritic cells (pDC). We hypothesized that genetic and/or sex-specific differences in DC activation states may contribute to differential susceptibility to lupus. Therefore, we compared the expression levels of CD80, CD86, CD40, CD40L, ICOSL, and MHC Class II with flow cytometry on DCs in male and female control (B6) and lupus-prone (B6.Nba2) mice. pDCs from pre-disease, 8-week old, B6.Nba2 lupus-prone mice expressed greater levels of CD80 than did those from B6 controls, while levels did not vary by sex. Additionally, CD19+ Siglec H-pDCs were found to express greater levels of costimulatory molecules as a whole than other pDC subsets. Interestingly, a greater proportion of male conventional dendritic cells (cDCs) expressed ICOSL as compared to female cells, regardless of genetics. These data indicate that Nba2 pDCs exist in a more activated state as compared to control cells, while male cDCs (regardless of lupus-susceptibility) may maintain a larger regulatory T cell population, via increased expression of ICOSL, as compared to female cells. It will be important to test these and other possible functional consequences of sex- and Nba2-specific costimulatory profiles and to determine if they are reflective of developmental events unique to each sex and strain.
‘The role of leukocyte NADPH oxidase in the regulation of IL-1 family cytokines and innate lymphoid cells in serum-induced arthritis.’
Authors: CHAN TZU-YI , Shieh Chi-Chang K
Published: January 2023

Innate lymphoid cells (ILCs) have been recently identified in different tissues as an important class of innate immune cells involved in the inflammatory response during health and disease. Our previous studies showed that reactive oxygen species (ROS) act as negative feedback to regulate IL-1β-mediated inflammation, accounting for the more severe arthritis in the absence of leukocyte NADPH oxidase (NOX2), which is responsible for producing most of the ROS in inflammatory tissues. In this study, we set out to identify and characterize innate lymphoid cells in the arthritic joints based on the hypothesis that leukocyte-produced ROS are critical for modulating innate lymphoid cells activation and IL-1 family cytokines production in serum induced arthritis. We found that serum-induced joint inflammation may be more severe and induce higher IL-1β, IL-18 but less IL-33 cytokines expression and a population of lineage-negative CD45 positive cells was identified in the synovial fluid and tissue of diseased joints. Comparison of ILC populations between WT and Ncf1−/− arthritic mice showed that ILC2 and NCR posetive ILC3 were more abundant in the joint of NOX2 deficient mice. Moreover, the phagocytic NADPH oxidase was an essential regulator of ILC3 populations switching in the inflammatory tissues of arthritis. Given the fact that these cells are capable of rapid cytokine production and leukocyte-produced ROS appear to affect these ILC populations, our ongoing investigations promise to reveal novel redox-sensitive immunoregulatory mechanisms mediated by ILC through IL-1 family cytokines.
Assessment of endocrine molecular entities targeted by inflammation that are involved in the onset of autoimmune diabetes.
Authors: Mora Conchi , Saavedra-Ávila Noemí Alejandra , Sala Ester , Sengupta Upasana , Verdaguer Joan , Stratmann Thomas , Sicinski Peter , Lahti Jill
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease caused by pancreatic beta cells demise due to the attack of self-lymphocyte repertoire. We aim to identify molecular entities targeted by the autoimmune assault to pancreatic beta cells that are causally related to T1D progression. The proinflammatory biological niche in which beta cells are immersed during the autoimmune insult promotes deep phenotypical changes crucial for the pathogenic process. These changes lead mainly to beta cell fitness impairment, cell cycle intervention and apoptosis triggering.

By using the Microarray technology we identified a series of genes the expression of which is altered in the islet endocrine cells prior to diabetes onset in the NOD (Non Obese Diabetic) mice. One of those genes encodes for cyclin D3 that triggers cell cycle progression through G1 phase towards the S phase. Cyclin D3 can also bind certain transcription factors and activate inflammation process and development of the T cells (NFκB, GATA). The cyclin D3 promoter has binding sequences to NFκB a transcription factor linked to the action of T1D-related proinflammatory cytokines such as IL-1beta and TNFalpha. We found that cyclin D3 is the only D-type cyclin the expression of which is regulated by inflammation in NOD endocrine islet cells. Moreover we found that cyclin D3 protects beta cells against cytokine-induced apoptosis and is required for proper beta cell function. Moreoever, we observed that CDK11, another cell-cycle related gene that interacts with cyclin D3, is also regulated by inflammation in endocrine islet cells. We have assessed whether there is a causal relationship between the coordinated differential expression of both genes during the insulitic assault and diabetes onset
Taking a ‘Toll’ on Skin: A novel TLR9-deficient Model of Cutaneous Lupus
Authors: Mande Purvi , Taravati Keyon , Rosenblum Michael , Rothstein Ann Marshak
Published: January 2023

Endosomal Toll-like receptors (TLRs), TLR7 and TLR9 have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). However, their role in cutaneous lupus (CL) is not well established, in part due to lack of appropriate mouse models for CL. We developed a novel murine model of CL that depends on T cell recognition of an ovalbumin (OVA) peptide on MHC class II+ cells and closely mimics human CL. These mice develop epidermal hyperplasia associated with mononuclear interface dermatitis and IgG deposition at the dermal-epidermal border within 4 weeks of administration of “pseudo-alloantigen” (OVA)-specific DO11 T cells. Importantly, cutaneous disease is entirely dependent on expression of TLR7 and the loss of TLR9. TLR9KO recipients have fewer DO11 Tregs, more germinal center B cells and more plasma cells than TLR9-sufficient recipients. They make autoantibodies specific for Ro52, a common self-reactivity in human CL. Additional clinical manifestations include extensive accumulation of IFNγ-producing DO11 T cells, elevated class II expression by keratinocytes (KC) and epidermal dendritic cells and extensive KC death. Preliminary data point to a mechanistic role of FasL in KC death that presumably drives skin lesions in this model. Intriguingly, radiation chimeras identify a critical role for radio-resistant TLR9KO cells in the development of cutaneous lesions in this model, suggesting a role for KC or Langerhans cell expression of TLR9 in constraining the development of CL. Our model thus provides a novel, rapid and reproducible system for investigating the opposing roles of TLR9 in CL and SLE. Findings from this study will have important applications in designing TLR-targeting molecules as therapeutic agents for CL.
Identification of perturbations in macrophage polarization in active systemic lupus erythematosus.
Authors: Labonte Adam C , Bachali Prathyusha , Catalina Michelle , Robl Robert , Geraci Nicholas , Lipsky Peter , Grammer Amrie
Published: January 2023

SLE is characterized by abnormalities in B cell and T cell function, but the role of disturbances in the polarization of macrophages (Mφ) into functional subpopulations has not been delineated. Gene expression profiles were analyzed to identify differentially expressed (DE) genes between healthy controls and patients with either inactive (SLEDAI < 6) or active (SLEDAI ≥ 6) SLE. While hundreds of DE genes were identified in B and T cells of active SLE patients (760 and 164 genes respectively), there were no DE genes found in B or T cells from patients with inactive SLE compared to healthy controls. In contrast, large numbers of DE genes were found in myeloid cells from both active (2,135) and inactive (1,260) SLE patients. Many of these DE genes were known to play roles in Mφ polarization, including the M1 genes STAT1 and SOCS3 and the M2 genes STAT3, STAT6, and CD163. M1-associated genes were far more frequent in data sets from active versus inactive SLE patients. To further characterize the relationship between M1 phenotype and disease activity, Weighted Gene Cluster Network Analysis (WGCNA) was used to identify modules of genes associated with clinical activity in SLE patients. Among these were disease activity-correlated modules containing polarization signatures of predominantly M1-associated genes. No disease activity-correlated modules were enriched in M2-associated genes. In summary, altered Mφ gene expression is characteristic of both active and inactive SLE. However, disease activity is associated with an alteration in the functional differentiation of Mφ to the M1 proinflammatory phenotype. This switch in Mφ phenotype is related to the altered expression of genes by T cells and B cells characteristic of active SLE.
The absence of estrogen receptor alpha (ERα) protects females during nephrotoxic serum-induced nephritis by preventing up-regulation of lipid metabolism.
Authors: Corradetti Chelsea , Madaio Michael , Caricchio Roberto
Published: January 2023

Glomerulonephritis (GN) is one of the most serious manifestations of systemic lupus erythematous (SLE). Lupus GN is characterized by severe inflammation and necrosis, and it accounts for most morbidity and mortality. There is sex bias in SLE and estrogens may play an important role in GN susceptibility and progression. Interestingly, estrogen receptor alpha (ERα) in the renal tissue is highly expressed and the renal specific estrogen-induced gene activation is second only to that of reproductive organs. We have found that absence of estrogen receptor alpha protects female mice from developing nephritis, despite the presence of immune complexes in the kidneys and production of pro-inflammatory cytokines. Timecourse microarray analysis of glomeruli isolated from kidneys during nephrotoxic-induced nephritis (NTN) revealed significant up-regulation of genes related to lipid metabolism, such as Acsl1 and Cyp4a14, in WT females compared to ERαko females. Oil red-O staining of the nephritic kidney sections revealed increased lipid presence in the WT females compared to the ERαko females, supporting the microarray data. From these results, we conclude that during NTN, the up-regulation of lipid metabolism occurs downstream of ERα activation and is essential for the progression to end-stage renal failure. Therefore, females lacking ERα have impaired capacity of increasing their lipid metabolism under inflammatory stress, do not exacerbate pro-inflammatory responses, and this results in protection from severe glomerulonephritis. Our results demonstrate that lipid metabolism via the activation of ERα in the renal tissue contributes to local inflammation and disease progression.
Spontaneous secretion of peptidyl-arginine deiminase 2 and cell surface exposure of peptidyl-arginine deiminase 4 by neutrophils
Authors: Zhou Yebin , Chen Bo , Mittereder Nanette , Chaerkady Raghothama , Strain Martin , Rahman Saifur , Ma Wenting , Low Choon Pei , Chan Denice , Neal Frances , Bingham Clifton O , Sampson Kevon , Darrah Erika , Bowen Michael , Andrade Felipe , Vousden Katherine , Sims Gary P , Mustelin Tomas Mikael
Published: January 2023

Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA) and elevated levels of protein citrullination can be found in the joints of RA patients. Although the pathophysiological mechanisms and the cell type(s) responsible for the increase in protein citrullination remain incompletely understood, the neutrophil is emerging as a prime suspect. Here we report that fully viable resting neutrophils from healthy donors have enzymatically active PAD4 exposed on their surface and that they spontaneously secrete enzymatically active PAD2. Activation of the neutrophils by several stimulatory agents, such as immune complexes, tumor necrosis factor a, phorbol myristate acetate, and agonists of Toll-like receptors1, 5, 7, and 9, increased the immunoreactive amount of PAD4 on the cells. However, immune complex, LPS and PMA stimulation reduced PAD2 secretion. The presence of extracellular PAD2 and PAD4 was not caused by NETosis, apoptosis, or lysis of the cells, as verified by gating on live cells and adding NETosis inhibitor in flow cytometry experiments. Neutrophils from the blood of RA patients also exposed PAD4 on their surface at levels similar to those from healthy subjects. Our data add a novel pathway for the extracellular citrullination of proteins by PAD2 and PAD4 in RA and suggest that this pathway is part of normal neutrophil biology also in the absence of disease.
TACI-deficient macrophages alleviate metabolic disease symptoms in obese mice
Authors: Liu Lunhua , Allman Windy R , Coleman Adam S , Siddiqui Shafiuddin , Akkoyunlu Mustafa
Published: January 2023

TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) is one of receptors for the TNF superfamily cytokines BAFF (B cell activating factor) and APRIL (A Proliferation Inducing Ligand). In this study, we investigated the role of TACI in high fat diet induced obesity and metabolic disease. Compared to WT mice, TACI KO mice exhibited less weight gain, decreased body fat, enhanced glucose tolerance and insulin sensitivity. Improved metabolic changes in TACI KO mice were accompanied by significantly less accumulation of CD8+ T cells and F4/80+CD11c+ inflammatory macrophages in visceral fat pad. In addition, TACI KO mouse adipose tissue contained elevated numbers of ILC2, eosinophils and M2 macrophages. Underscoring the protective phenotype of M2-skewed macrophages, the transfer of TACI KO macrophages into obese WT mouse significantly improved insulin sensitivity and glucose tolerance in WT mouse. Interestingly, adoptively transferred TACI KO macrophages into WT mice also increased numbers of the host M2 macrophages and eosinophils in the adipose tissue. We identified IL-10 secreted by TACI KO macrophages was majorly responsible for the improved insulin sensitivity because not only TACI KO macrophage culture media contained high levels of IL-10 but also an IL-10 blocking antibody abolished the enhancement of insulin signaling provided by TACI KO macrophage culture media. These findings indicate that macrophage TACI mediated inflammatory signals promote obesity induced metabolic changes. Consequently, TACI blocking reagents may offer therapeutic benefits to obese patients suffering from metabolic complications.
Eosinophil-derived IL-4 drives inflammatory dilated cardiomyopathy
Authors: Diny Nicola , Barin Jobert G , Talor Monica , Rose Noel R , Cihakova Daniela
Published: January 2023

Inflammatory dilated cardiomyopathy (DCM) is a major cause of heart failure in children and young adults. DCM develops in up to 30% of myocarditis patients, but the mechanisms involved in disease progression are poorly understood. Patients with eosinophilia frequently develop cardiomyopathies. We determined the role of eosinophils in myocarditis and DCM using the experimental autoimmune myocarditis (EAM) model. In wildtype (WT) BALB/c mice, EAM progresses to DCM and heart failure. Eosinophil-deficient ΔdblGATA1 mice developed myocarditis but showed no signs of heart failure. Thus, eosinophils were dispensable for myocarditis induction but required for progression to DCM. EAM in hypereosinophilic IL5Tg mice resulted in eosinophilic myocarditis with severe atrial inflammation and progressed to severe DCM. Atrial inflammation was also observed in 2 of 3 patients with eosinophilic myocarditis. The DCM phenotype required eosinophils but not IL5. IL5TgΔdblGATA1 mice were protected from DCM while IL5−/− mice exhibited DCM comparable to WT mice. Eosinophils drove progression to DCM through their production of IL4. In our experiments, eosinophils were the major IL4 expressing cell type in the heart during EAM. Like ΔdblGATA1 mice, IL4−/− mice were protected from DCM and eosinophil-specific IL4 deletion resulted in improved heart function. In conclusion, eosinophils drive progression of myocarditis to DCM, cause severe DCM when present in large numbers, and mediate this process through IL4.
Modulation of human innate lymphoid cell function by IL-10
Authors: Bonne-Annee Sandra , Nutman Thomas B
Published: January 2023

Although present in extremely low numbers in the human circulation at steady state, innate lymphoid cells (ILCs) are expanded in human helminth infections. Despite their low frequency (accounting for 0.21% (range 0.023–3.58%) of lymphocytes in the peripheral blood under homeostatic conditions), ILCs release extremely large quantities of cytokines following activation. If left unchecked, these cytokines can have deleterious effects on the host. We have observed that circulating ILCs demonstrate surface expression of IL-10R under homeostatic conditions. To examine how the expression of IL-10R regulates cytokine production by ILCs, ILC subsets were isolated from the peripheral blood of healthy donors by flow cytometry-based sorting and activated in the presence or absence of the immunoregulatory cytokine IL-10. ILCs stimulated in the presence of IL-10 had a marked reduction (range from 2–3.6 fold) in cytokines (IL-4, IL-5, IL-13, IL-17A, INF-γ, and TNF-α) when compared to ILCs activated without IL-10. These findings demonstrate that IL-10 signaling can inhibit cytokine production by activated ILCs. Furthermore, studies are currently underway to examine ILC subsets in the context of chronic helminth infections, where IL-10 has a modulatory effect on innate and adaptive immune cells and to identify additional mechanisms that regulate ILC cytokine production, including TGF-β.
Characterization of an alternatively activated IL-10 producing innate lymphoid type-2 effector cell population
Authors: Kaye Jonathan , Seehus Corey Ray , Kadavallore Asha , de la Torre Brian , Yeckes Alyson , Wang Yizhou , Tang Jie
Published: January 2023

Type-2 innate lymphoid (ILC2) cells share cytokine and transcription factor expression with CD4+ Th2 cells, but it is unknown if this ILC subtype undergoes functional diversification. Here, we report in vivo induction of a molecularly distinct subset of activated lung ILC2 cells, termed ILC210 cells, that produce IL-10 and downregulate genes associated with inflammation. Signals that generate ILC210 cells were distinct from those that induced IL-13 production and, along with transcriptome data, suggested an alternative pathway of activation. In vivo, IL-2 greatly enhanced ILC210 cell generation by IL-33 and was associated with decreased eosinophil recruitment to the lung. Unlike the majority of activated ILC2 cells, the ILC210 cell population underwent contraction following cessation of IL-33 stimulation in vivo, but with maintenance of a subpopulation of cells that can be recalled by restimulation, analogous to T cell effector cell and memory cell generation. Together, these data demonstrate previously unappreciated heterogeneity in the ILC2 cell response.