bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of an Ultra-Sensitive Multiplex (6-plex) Cytokine Digital Immunoassay with fg/ml Sensitivity
Authors: Lambert Jeremy , Shan Dandan , Chang Lei , Rissin David , Nie Shuai , Hanlon David
Published: January 2023

The future of immuno-oncology treatment and autoimmune diagnostics will involve the detection and quantification of inflammatory biomarkers. As immunotherapy becomes a more common approach for treating cancer and inflammatory disease, cytokines will not only play a role in determining a patient’s disease diagnosis, prognosis, and potential treatment options, but also may serve as a primary index of a therapy’s effectiveness.

Incorporating these biomarkers into clinical practice, however, has been a challenge. Only a few cytokines have been studied in relation to inflammatory disease and immunotherapy, mainly because the biomarkers for these conditions are typically present in very small concentrations, and current research methods aren’t sensitive enough to detect them. It is critical that physicians have the ability to quantify these cytokines to track disease progression before and after treatment.

We present development and characterization of a multiplex digital immunoassay to simultaneously detect and quantify six inflammatory biomarkers (IL-10, IL-12p70, IL-17A, IL-6, TNFa, IFNg) with fg/ml sensitivity. This advanced approach reduces assay volume requirements for multiple sample types and may be sensitive enough to detect circulating disease biomarkers before symptoms appear. As a result, it could potentially provide physicians with a new capability to track disease progression, and the effectiveness of treatments against diseases, which they previously could not monitor with such precision.
Tristetraprolin family of RNA binding proteins post-transcriptionally regulate inflammation and metabolic responses in the liver
Authors: Patial Sonika , Blackshear Perry
Published: January 2023

Tristetraprolin family of RNA binding proteins, including tristetraprolin (TTP), zinc finger protein 36 like 1 (ZFP36L1), and zinc finger protein 36 like 2 (ZFP36L2) regulate mRNA levels by binding to AU-rich elements on the 3′untranslated regions of specific mRNAs and enhancing their turnover. Global loss of TTP results in systemic inflammatory syndrome in mice; however, the physiological role of the other two TTP family members is unclear due to embryonic lethality of global knockout of ZFP36L1 and early post-natal death of global knockout of ZFP36L2. Here, using Cre-loxP technology, we independently and simultaneously (triple KO) deleted the three TTP family members in mouse liver and characterized the phenotype of the resulting mice strains using histopathological, immunohistochemical, clinical, and transcriptomic approaches. The triple KO mice exhibited chronic active peri-portal hepatitis, bile duct hyperplasia, and portal fibrosis. Immunohistochemically, increased numbers of T cells, B cells, macrophages, and neutrophils were detected within the peri-portal areas. Increased expression of heme-oxygenase-1, a marker of reactive oxygen species was also found. Clinically, serum levels of bile acids, bilirubin, and liver function enzymes were markedly elevated. Transcriptomic and bioinformatics analysis of liver RNA demonstrated novel potential liver specific inflammatory and metabolic mRNA targets of TTP family proteins that are post-transcriptionally regulated by TTP family proteins in both specific and redundant manner. In summary, our study provides strong evidence that TTP family of RNA binding proteins maintain immune and metabolic homeostasis in the liver.
Metabolomics analysis reveals differential T cell serine metabolism as a target in autoimmunity.
Authors: Andrejeva Gabriela , Wolf Melissa M. , Johnson Marc O. , Rutledge Alexandra C. , Codreanu Gabriela S. , Sherrod Stacy D. , Gutierrez Danielle , Rose Kristie L. , Norris Jeremy L. , Schey Kevin L. , McLean John A. , Rathmell Jeffrey C.
Published: January 2023

Inflammatory cytokines are key players in the pathogenesis of autoimmune disorders where their signaling leads to inappropriate balance between effector CD4 T-cells (Teff), Th1 and Th17, and the immunosuppressive regulatory T-cells (Treg). Each subset utilizes a distinctive metabolic program but how cytokines induce differential metabolic rewiring and what aspects of it are critical for the functioning and lineage stability of Teff subsets and Treg is not understood.

To delineate pathogenicity-associated metabolic programs, we performed global untargeted metabolomics mass spectrometry (MS) in in vitro cytokine-differentiated mouse Th1, Th17 and Treg cells with varying degrees of pathogenicity over time. Integrating our data-driven metabolic network with global proteomics profiling and cytokine secretion allowed us to examine metabolic pathways at metabolite-enzyme level. Metabolites at the interface of amino acid metabolism, particularly glutamine and serine, glycolysis, the TCA cycle and nucleotide synthesis were the most differentially changed. Serine limitation or pharmacological inhibition indicated differential coupling of serine/one-carbon metabolism to T-cell proliferation, lineage choices and the production of cytokines IL-17 or IFNγ. Furthermore, targeting this pathway promoted Treg lineage development in the presence of pathogenic cytokines and prevented loss of Treg transcription factor FoxP3 upon re-stimulation. In vivo, spinal cord infiltrating T-cells in murine experimental autoimmune encephalomyelitis had altered serine metabolic pathway. Our findings identify key areas of T-cell metabolism that may offer a new category of therapeutic targets for autoimmune and inflammatory disease.
Plasmacytoid dendritic cells inhibit Toxoplasma gondii infection induced cytokine storm
Authors: Zhou Wei , Wang Ding Ding , Tang Hua
Published: January 2023

Cytokine storm has serious harmfulness in infectious diseases, but its regulatory mechanism is not clear. The immunopathology is mediated by local overproduction of Th1-type cytokines during acute infection with Toxoplasma gondii (T. gondii). Because dendritic cells (DCs) are essential in the elicitation of these immune responses, we investigated the presence the role of plasmacytoid dendritic cells (pDCs) upon T. gondii infection. Towards this goal, we examined T. gondii infection of BDCA2-DTR mice, which specifically depleting this cell population. Here, we found that T. gondii infection reduced the number of pDCs, and depletion of pDCs in BDCA2-DTR mice were extremely susceptible to T. gondii infection, with decreased serum levels of IL-12, but increased serum levels of IFN-γ, IL-6 and TNF-α. In addition, depletion of pDCs also increased IFN-γ production by CD4+T cells, and resulted in an exacerbation of immunopathological damage caused by T. gondii infection. These results reveal that pDCs play a protective role during T. gondii infection by modulation of cytokine triggers immune response.
Natural Secretory Immunoglobulins Enhance Norovirus Infection
Authors: Turula Holly , Pry Alexandra , Wilke Carol A. , Fava Julianne , Wobus Christiane
Published: January 2023

Noroviruses are highly prevalent, enteric pathogens that cause significant morbidity, mortality and economic losses world-wide. Murine norovirus (MNV) exploits M-cells to cross the follicle-associated epithelium (FAE) and infect underlying populations of immune cells. Secretory immunoglobulins (SIg) are a first line of mucosal defense. However, whether natural, non-specific SIg can protect against norovirus infection remains unknown. SIg are secreted into the gut lumen via the polymeric immunoglobulin receptor (pIgR) where they bind to antigen and are transported back across the FAE via M-cells. To determine if natural SIg influence MNV pathogenesis, we used pIgR-deficient animals which lack SIg in the intestinal lumen. Contrary to other enteric pathogens, acute MNV replication was significantly reduced in the gastrointestinal tract of pIgR-deficient animals compared to controls, despite increased target cells (DC, Macrophages, B cells). Also, natural SIg did not alter MNV FAE binding or FAE crossing into the lymphoid follicle. Further analysis revealed enhanced baseline levels of inflammatory molecules known to inhibit viral infection, interferon gamma (IFNɣ) and inducible nitric oxide synthase (iNOS), in the small intestine of naive pIgR-deficient animals compared to controls. Removing the microbiota equalized IFNɣ and iNOS levels as well as MNV infection in germ-free pIgR KO mice compared to germ-free controls. Future studies will determine if neutralization of IFNɣ is sufficient to enhance MNV infection in pIgR KO mice. Taken together, these findings suggest a model whereby SIg sensing reduces pro-inflammatory, antiviral molecules which facilitates intestinal homeostasis but thereby promotes MNV infection.
Exploring anti-viral and immunomodulatory activities of Regenerating Islet-Derived proteins
Authors: De Luna Xavier , Hsieh I-Ni , White Mitchell , Hartshorn Kevan L.
Published: January 2023

The Regenerating Islet Derived Protein 3 (REG3) family are classified as antimicrobial peptides typically expressed in the gut epithelia of mammals, and help maintain host-bacteria homeostasis in our gastrointestinal tract. However, recent data has shown that the peptides are expressed in respiratory epithelia as well and play an important role in host defense in the lung against gram positive bacteria. The expression of one of the REG peptides in the lung is suppressed by IAV infection in mice and this contributes to bacterial superinfections which complicate influenza. REG3 peptides are classified as C-type lectins and our lab focuses on anti-microbial peptides as well as C-type lectins (mannose-binding lectin and surfactant protein D) and their function against Influenza A virus. Here I will be describing the previously unknown effects REG3 family of peptides on IAV infection. Preliminary data I have generated indicate that REG3 peptides inhibit the infectivity of IAV. I have compared the activity two different REG3 peptides and gained preliminary evidence that they not only inhibit viral infectivity but disrupt viral membranes (using electron microscopy) and alter viral interactions with epithelial cells using confocal microscopy. Of interest, the peptides also increase uptake of IAV by neutrophils and also increase neutrophil respiratory burst responses to the virus. These data suggest that REG proteins have significant and previously unknown effects to inhibit viral infectivity but also can modulate responsiveness of phagocyte to viruses
TLR3 of murine astrocytes is cleaved into several forms upon stimulation of cells with synthetic dsRNA
Authors: Mielcarska Matylda Barbara , Bossowska-Nowicka Magdalena , Gregorczyk Karolina Paulina , Wyzewski Zbigniew , Toka Felix Ngosa
Published: January 2023

TLR3 (Toll-like receptor 3) belongs to the subfamily of TLRs that reside in the endosomal compartment of the cell. It recognizes dsRNA (double-stranded RNA) of viruses and initiates a signaling pathway leading to generation of antiviral molecules such as type I interferons. Precursor TLR3 present in mammalian cells has a size of approximately 110 kDa, however, recent findings show that the TLR3 may also occur in two progeny forms: C-terminal and N-terminal, appearing after cleavage of TLR3 in specific conditions by cysteine proteases – cathepsins. Furthermore, cleaved and/or associated TLR3 molecules may represent the primary form of the receptor and are capable of binding the TLR3 ligand. Our results show that in C8D1A murine astrocyte cell line, cleavage of TLR3 is time- and dose-dependent upon stimulation with poly(I:C) (polyinosinic:polycytidylic acid, synthetic dsRNA analogue). The cleavage process promotes and increases expression of the progeny receptor forms. Presentation of TLR3 in such manner may modulate the level of response to viral dsRNA, especially in central nervous system cells, however, this requires further investigation.
STAT reporter cell line systems as a tool for cancer therapeutic target screening.
Authors: Lee Hyun-Ku , Singh Gita , Singh Sujay
Published: January 2023

Signal transducer and activator of transcription (STAT) proteins as cytoplasmic transcription factors respond to cytokines and growth factors that mediate downstream signaling. STATs are closely related with cancers as they are frequently found to be dysregulated in primary tumors, leading to enhanced survival of tumors and increased angiogenesis. Among seven STAT family members, STAT3, STAT4 and STAT5 are considered to primarily promote cancer development and progression, while STAT1 may function either as a tumor suppressor or tumor promoter. STAT3, 4 and 5 are persistently activated in many human cancer cell lines, leading to increased cancer cell survival. Several studies demonstrate that inhibition of STAT3 or STAT5 signaling decreases cancer cell proliferation leading to apoptosis. Taken together, these indicate that STAT proteins can be ideal targets for anti-cancer therapy, and so it is crucial to develop assay systems that can identify inhibitors targeting those pro-tumorigenic STATs. Hence we developed the four reporter cell lines, each of which stably expresses STAT1-, STAT3-, STAT4-, or STAT5-response element that controls an optimized Renilla luciferase reporter gene upon stimulation. Functional activity of each cell line was evaluated through dose response of various cytokines such as IFNs, IL-3 and IL-6. One of the STAT inhibitors, curcumin as well as other putative STAT suppressors were utilized to characterize how each STAT can differentially respond to each test molecule and how its response can affect the host cells.
Epigallocatechin-3-gallate prevents Non-alcoholic fatty liver disease by modulating liver function, lipid profiles and M1/M2 macrophage polarization
Authors: Paglicawan Laura Q. , Du Yong , Soomro Sanam , Baig Sahar , Vanarsa Kamala , Mohan Chandra
Published: January 2023

Background

Here, we examine the therapeutic efficacy of Epigallocatechin-3-gallate, the most potent catechin in green tea, on nonalcoholic fatty liver disease (NAFLD), a growing epidemic in humans.

Methods

NAFLD was induced by high-fat diet (HFD) with 30% fructose in drinking water for 8 weeks. Mice were divided into 3 groups: model group (MD; no EGCG), low dose EGCG group (LE, 20 mg/kg/d) and high dose EGCG group (HE, 50 mg/kg/d). Liver histology, serum lipid prolife, liver function as well as liver M1/M2 polarization were evaluated.

Results

At the end of the experiment, the mean body weight of LE and HE mice were 39.7g and 45.7g, while it was 51.9g in the MD group (P< 0.05). Similarly, liver weights in the LE and HE groups (means = 1.44g, and 1.68g, respectively) were lower than that in MD (2.94g, P < 0.01). Liver dysfunction observed in the MD group (mean AST=133.2 U/L and ALT=183.4 U/L), was significantly improved in both LE (44.8 U/L, 86.8 U/L, respectively, P < 0.001) and HE groups (83.63 U/L, 117.6 U/L, respectively P < 0.05). Serum cholesterol, HDL, triglyceride levels were increased in the MD group, but lowered in both LE and HE mice (p<0.01). Hepatic FACS, RT-PCR and IHC studies demonstrated that the HE group had lower F4/80+ CD86+ (0.3 ×103 vs 1.5 × 104), F4/80+ MHC II+ (0.7 ×104 vs 1.3 × 104), and higher F4/80+ CD206+(2.96 ×104 vs 0.3 × 104), F4/80+ CD23+ (1.27 ×104 vs 0.65 × 104) hepatic infiltration, accompanied by down-regulated CD68 and up-regulated CD163 mRNA expression.

Conclusion

EGCG subdued NAFLD despite consumption of HDF and high fructose water, marked by improved liver function and serum lipid profile, as well as M1/M2 polarization. EGCG should be investigated as a therapeutic modality in human NAFLD.
The intracellular target of the antimicrobial peptide thanatin
Authors: Jackson Karen E. , Miller-White Tiffany
Published: January 2023

Thanatin is an inducible 21-residue antimicrobial peptide (AMP) derived from the insect Podisus maculiventris. Thanatin and its analogs have bactericidal activity against gram-positive and gram-negative bacteria, including antibiotic resistant strains. Due to the lack of hemolytic action, thanatin is a good candidate for therapeutic use, as evidenced by its ability to fight sepsis in mice. However, the mode of action of thanatin remains unexplored. Introduction of the respiratory poison carbonyl cyanide m-chlorophenyl hydrazone (CCCP) inhibits AMP induced death, suggesting that the bacterial respiratory pathway is targeted. In this study, biotinylated S-thanatin was used to determine a minimum inhibitory concentration (MIC) in Escherichia coli. Biotinylated S-thanatin showed comparable killing activity to the non-biotinylated peptide, 8μg/mL vs. 4μg/mL, respectively. Next, the killing activity of biotinylated S-thanatin was determined for cultures of E. coli grown under anaerobic, aerobic and respiratory dependent conditions. Anaerobic growth was shown to inhibit thanatin-induced microbial death, and respiratory dependent increased sensitivity. Currently, we are using biotinylated S-thanatin in an affinity purification assay to isolate the intracellular target to be analyzed using matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectroscopy.
Nitric oxide dictates the reprogramming of carbon flux during M1 macrophage polarization
Authors: Palmieri Erika M. , Baseler Walter A. , Davies Luke C. , Gonzalez-Cotto Marieli , Ghesquiere Bart , Fan Teresa W. M. , Lane Andrew N. , Wink David A. , McVicar Daniel W.
Published: January 2023

Classical pro-inflammatory activation of macrophages is characterized by profound intracellular metabolic changes, with increased glycolytic usage of carbon, away from Oxidative Phosphorylation (OXPHOS). We previously demonstrated that Nitric Oxide (NO) levels induced in Bone Marrow Derived Macrophages (BMDMs) from Wild Type (WT) mice are necessary and sufficient for the repression of OXPHOS. Here we demonstrate that NO is also responsible for the “break” in the mitochondrial TCA cycle and citrate accumulation during LPS/IFNγ stimulation; macrophages that lack NO maintain indeed substantial levels of Oxygen Consumption Rates (OCR) and TCA cycle intermediates. Carbon tracing experiments in the presence of U13C-glucose show almost undetected labelled α-ketoglutarate from citrate in WT but a conserved pattern of heavy carbon fate in Nos2−/− macrophages. Moreover we found that mitochondrial respiration elicited through citrate was decreased in WT M1 macrophages, but isocitrate was a full substrate for complex I-dependent OCR suggesting suppression of metabolism at mitochondrial Aconitase (ACO2). Consistent with this data, we found ACO2 enzymatic activity blunted in WT vs Nos2−/−. In addition we observed that M1 macrophages reroute pyruvate away from Pyruvate Dehydrogenase (PDH) in an NO dependent manner since only WT show halted flux through PDH. Surprisingly, we demonstrate this mechanism to be independent on the activation of Hif1α and its suggested effect on limiting acetyl-coA for the TCA. With these data together we hypothesize that NO orchestrates macrophage metabolism during inflammation inhibiting OXPHOS by blocking Krebs Cycle, therefore depriving of substrates the mitochondrial electron transport chain.
mTOR induces lysosome expansion by selective translation of lysosomal transcripts during phagocyte activation
Authors: Hipolito Victoria E. B. , Diaz Jacqueline A. , Tandoc Kristofferson V. , Saric Amra , Toposiviric Ivan , Botelho Roberto J.
Published: January 2023

The molecular mechanisms that govern and adapt organelle number, size, morphology and activities to suit the needs of many cell types and the conditions that a cell may encounter remain poorly defined. Lysosomes are organelles that degrade cargo from a variety of routes including endocytosis, phagocytosis and autophagy. Lysosomes have emerged as a signalling platform that senses and couples stress signals such as nutrient deprivation to regulatory kinase hubs like mTOR and AMPK to modulate metabolic activity. For phagocytes and antigen-presenting cells like macrophages and dendritic cells, lysosomes are a kingpin organelle since they are essential to kill and process pathogens, and present antigens. During phagocyte activation, lysosomes undergo a striking reorganization, changing from dozens of globular structures to a tubular network, in a process that requires the phosphatidylinositol-3-kinase-Akt-mTOR signalling pathway. Ultimately, lysosome tubulation is thought to promote pinocytic retention and antigen presentation. We show that lysosome tubulation is accompanied by a rapid boost in lysosome volume and holding capacity during phagocyte activation with lipopolysaccharides. Unexpectedly, lysosome expansion was paralleled with the induction of lysosomal proteins, which was independent of TFEB and TFE3, transcription factors known to scale up lysosome biogenesis. Instead, we demonstrate a hitherto unappreciated mechanism of lysosome expansion via mTOR-dependent increase in translation of mRNAs encoding key lysosomal proteins including LAMP1 and V-ATPase subunits. Collectively, we identified a mechanism of rapid organelle expansion and remodelling driven by selective enhancement of protein synthesis.
Sympathetic neural control of inflammation by ADRB2-mediated IL-10 secretion
Authors: Agac Didem , Estrada Leonardo , Farrar John David
Published: January 2023

The nervous and immune systems reciprocally regulate their functions through the release of chemical messengers. Norepinephrine (NE), a neurotransmitter released by catecholaminergic nerve endings, allows the sympathetic nervous system to communicate with immune cells through adrenergic receptors (ADR). We identified a novel role for the β2-ADR (ADRB2) in controlling inflammation. NE suppresses pro-inflammatory cytokine secretion from primary macrophages in response to multiple TLR agonists, and ADRB2 signaling enhances early induction of IL-10. We assessed the in vivo role of this pathway using a LPS endotoxemia model. ADRB2 −/− animals rapidly succumbed to a sub-lethal LPS challenge, which correlated with elevated serum levels of TNFα and reduced IL-10. The lethality in the ADRB2 −/− animals was rescued by administering exogenous IL-10. Additionally, the ADRB2-specific agonist salmeterol rescued wild-type animals from a lethal LPS challenge, which was reversed by neutralizing anti-IL-10 antibody. Additionally, ADRB2 −/− mice are unable to control inflammation models of infection and colitis. These observations suggest that ADRB2 signaling is critical for controlling inflammation through the rapid induction of IL-10. Through transcriptome analysis, the NR4A nuclear orphan family members were induced by NE, and the presence of several putative NR4A binding sites within the IL-10 promoter suggests that these factors may directly regulate IL-10 expression in response to ADRB2 signaling. Further studies will test the control of IL-10 expression by the NR4A family of transcription factors. Understanding this pathway will provide new insights into how the nervous and immune systems communicate through ADRB2 signaling.
Isoflavones increase radiation-induced apoptosis and modulate pro-inflammatory signaling in EA.hy926 endothelial cells.
Authors: Fountain Matthew Donovan , McLellan Laura Ann , Cannon Alexa C. , Hillman Gilda G.
Published: January 2023

Background

The therapeutic use of x-ray ionizing radiation (XIR) for cancer treatment is limited by off target injury to normal tissues resulting in organ-specific toxicities with perpetual inflammation and fibrosis. XIR causes different forms of cell death including apoptosis, necrosis, and mitotic catastrophe that can dictate the resulting inflammatory response. Our lab has shown that soy isoflavones (SIF) reduce XIR-induced inflammation and fibrosis of thoracic organs. We now investigated the effect of SIF and XIR on endothelial cell death in vitro, which could play a role in SIF mitigation of inflammatory response.

Methods

EA.hy926 endothelial cells were treated with or without 30μM Novasoy 400 SIF for 24 hours prior to 3 Gy XIR and assessed at various time points. Cells were utilized for cell viability assays, stained for g-H2AX, Annexin-V, and NF-κB-p65, and lysed for western blot analysis of proteins involved in apoptosis and inflammatory signaling.

Results

Endothelial cell viability was significantly decreased for cells treated with SIF+XIR at 72 hours compared to XIR alone (p <0.05). Endothelial cells treated with SIF+XIR had increased staining of Annexin-V and g-H2AX staining at 48 hours compared to XIR alone (p <0.05). Western blot analysis showed increases in pro-apoptotic caspases 9 and 3 as well as Bax, IL-1b, ICAM-1 and VCAM-1 after cell treatment with SIF+XIR.

Conclusions

SIF enhances endothelial cell susceptibility to XIR-induced pro-apoptotic pathways resulting in greater apoptotic cell death. These findings suggest a mechanism of SIF-mediated radioprotection caused by increased apoptosis of normal cells and could limit the uncontrolled XIR-induced inflammatory signaling leading to organ fibrosis.
Docosahexaenoic acid as a prophylactic treatment on particle-exposed Balb/c mice
Authors: Fletcher Paige , Hamilton Raymond F. , Pestka James J. , Holian Andrij
Published: January 2023

Rationale

There is a rising trend in the use of nanomaterials in various consumer products; increasing the risk of human exposures. Inflammation is a vital contributing factor to pulmonary diseases which can be triggered by exposure to airborne toxicants such as nanomaterials. However, there are no effective treatments for acute inflammation. One potential treatment shown to have anti-inflammatory effects is the dietary supplement docosahexaenoic acid (DHA); an omega-3 polyunsaturated fatty acid found in fish oil. This study investigated DHA as a prophylactic treatment for particle-induced inflammation.

Methods

Balb/c mice were fed a control diet or a 1% DHA-containing diet for five weeks. At week four the mice were anesthetized and oropharyngeal instilled with dispersion media (50 μl) or a highly inflammatory multi-walled carbon nanotube (FA21; 50 μg). One week after instillation lung histology, macrophage NLRP3 inflammasome activation (IL-1β release) and lung lavage fluid (LLF) levels of high-mobility group box 1 (HMGB1), protein levels and inflammatory cytokines were evaluated. DHA’s impact on macrophage phenotype was also examined.

Results

Macrophage production of IL-1β and LLF levels of HMGB1 and protein decreased in mice fed the DHA diet. Mice exposed to FA21 on the DHA diet had less pro-inflammatory M1 macrophage phenotype gene expression compared to the control diet-fed mice in lung tissue and decreased pro-inflammatory cytokines in the LLF. The lung tissue airway thickness was also decreased in the DHA-fed mice.

Conclusion

DHA could potentially be used as a prophylactic treatment since it was demonstrated to have a number of anti-inflammatory effects on Balb/c mice exposed to a common nanomaterial.
TCR affinity influences helper T cell differentiation by biasing dendritic cell interactions
Authors: Kotov Dmitri Ivanovich , Mitchell Jason S. , Pengo Thomas , Kotov Jessica Amy , Ruedl Christiane , Way Sing Sing Y. , Langlois Ryan A. , Fife Brian T. , Jenkins Marc K.
Published: January 2023

Naïve CD4+ T lymphocytes differentiate into various subsets when their TCRs detect microbial peptide-MHCII complexes presented by dendritic cells. However, the mechanism by which TCR signaling influences differentiation is unknown. We found that low affinity TCRs biased naïve T cells to become B cell helpers while high affinity TCRs promoted the phagocyte helper fate. We created software for high-throughput, multicolor, quantitative imaging called Chrysalis to determine if differentiation was influenced by the type of dendritic cell that the naïve T cell interacted with. Our results show that T cells with high affinity TCRs had prolonged interactions with XCR1+ dendritic cells and tended to become phagocyte helpers, while T cells with lower affinity TCRs preferentially interacted with SIRPα+ dendritic cells and tended to become B cell helpers. Thus, TCR affinity influences T cell differentiation by dictating interactions with distinct dendritic cell types.
Salt-Inducible kinases are critical regulators of terminal B-cell differentiation
Authors: Robinson Emily , Care Matthew , Tooze Reuben , Doody Gina
Published: January 2023

The generation of antibody-secreting plasma cells is a step-wise process involving initial B-cell activation and expansion, followed by the persistence of end-stage effector cells that reside in supportive niches. The transition through these stages requires reprogramming for high levels of secretion and adaptation to the bioenergetic demands of producing immunoglobulin. To improve our understanding of the pathways involved, we applied gene co-expression network analysis to temporal data derived from in vitro differentiating human plasma cells. We identified salt-inducible kinase 1 (SIK1) as a highly connected hub gene involved in the transition from a cycling plasmablast to a quiescent plasma cell. SIK1 is part of a kinase family previously linked to the control of metabolism and the negative regulation of TLR signaling, in part through the inactivation of cAMP-regulated transcriptional co-activators (CRTCs). To assess the contribution of SIK family kinases during plasma cell differentiation, we utilized the SIK inhibitors HG-9-91-01 and YKL-05-099. In contrast to macrophages where SIK inhibition enhances anti-inflammatory polarization, exposure of primary human or murine B-cells that have activated plasma cell differentiation to the SIK inhibitors lead to a profound reduction in cell viability. These effects were also replicated in a subset of multiple myeloma cell lines. The results are consistent with the described requirement for CRTC2 inactivation to exit the germinal center reaction and initiate plasma cell differentiation. Our findings extend these earlier observations and provide scope for identification of additional mechanisms that control plasma cell quiescence and secretory capacity.
Ocular exocrine glands are infected, harbor T cells, and express select cytokines and chemokines following corneal HSV-1 infection.
Authors: Montgomery Micaela Lee , Carr Daniel J. J.
Published: January 2023

Herpes simplex virus (HSV)-1 is a pathogen that infects a large portion of the human population and establishes a life-long, latent infection. Reactivation of HSV-1 can manifest as “cold sores” or more severe pathologies, including corneal herpes stromal keratitis. The potential involvement of ocular glands following corneal HSV-1 infection is not well described, despite their significant contribution to eye health. CD-1 outbred mice were ocularly infected with HSV-1, and ocular exocrine glands, including harderian glands (HGs) and extraorbital lacrimal glands (ELGs), were evaluated for susceptibility to infection and immunological activity. HSV-1 was found to infect ocular glands in a time-dependent fashion with HGs infected earlier than ELGs. Surgical removal of HGs prior to infection resulted in a significant increase in infectious virus recovered from corneas, but not ELGs or brainstems, when compared to mice with intact HGs (p≤0.01). Bidirectional flow was shown to occur between ocular surface contents and HGs in less than 4 hours, which might contribute to HSV-1 trafficking to HGs. CD4+ and CD8+ T cell infiltration/expansion occurs within HGs and ELGs in a time-dependent fashion post-infection (pi) compared to B cells that began to infiltrate HGs, but not ELGs, by day 5 pi. Cytokines, including IL-4, IL-6, IL-22, and IFN-γ, were found to be significantly (p≤0.05) elevated in ELGs, but not HGs, at day 7 pi. Chemokines, including CCL2, CCL5, and CXCL10, were significantly (p≤0.05) elevated in both ELGs and HGs at day 7 pi. These results underscore a dynamic response of ocular exocrine glands to corneal HSV-1 infection and the potential contribution these glands make to viral resistance.
Deficiency of neutrophil extracellular traps increases susceptibility to colonic inflammation
Authors: Singh Vishal , Yeoh Beng San , Xiao Xia , Wang Yanming , Vijay-Kumar Matam
Published: January 2023

Neutrophil extracellular traps (NETs), DNA structures released from neutrophils, are primarily enriched with bactericidal proteins. We envision that the antimicrobial nature of NETs would be an appropriate immune response in the gut, which is continuously exposed to trillions of bacteria. NETs formation is peptidyl arginine deiminase-4 (PAD4)-dependent, therefore, we employed Pad4KO mice and their WT littermates in two well-established models of murine colitis, i.e., loss of IL-10 signaling (immune hyperactivation) and chemical-induced injury (DSS). Upon treatment with αIL-10R mAb, Pad4KO mice developed robust chronic colitis as exemplified by colomegaly, colonic crypt elongation, and increased immune cell infiltration. In comparison, αIL-10R-treated WT mice exhibited modest colitis. We theorized that NETs may function as a ‘safety net’ which confine the pro-inflammatory neutrophil granule proteins (NGP) and prevent them to cause further damage. Indeed, the concentration and activity of NGP [i.e. lipocalin 2 (Lcn2), myeloperoxidase (MPO) and neutrophil elastase (NE)], in serum and in colons, were strikingly elevated in colitic Pad4KO mice, when compared to WT mice. The level of colonic secretory leukocyte protease inhibitor (SLPI) was reduced in the colitic Pad4KO mice. Such impaired SLPI response coupled with the ‘spillover’ of NGPs may, in part, explain the aggravated colitis in mice lacking NETs. Similarly, upon DSS treatment Pad4KO mice developed more severe chronic colitis than WT mice. Taken together, our findings advance the notion that gastrointestinal NETs are beneficial, and elucidation of NETs-mediated signaling, during colitis, may yield a novel therapeutic approach to mitigate intestinal inflammation.
Resveratrol increases efferocytosis via both Mertk-GAS6/PROS and Integrin-TG2-Mfge8 pathway
Authors: Tsay Gregory J.
Published: January 2023

The impairment of clearance of apoptotic cells (efferocytosis) leads to local inflammatory response and tissue damage. Recent studies have found the defective mechanism in apoptotic clearance is related to the pathogenesis of autoimmune disease such as systemic lupus erythematosus (SLE).

There have been many studies about the positive effects of resveratrol, including anti-diabetic effect, anti-inflammation, anti-oxidation, and anti-aging. However, the effect of resveratrol on clearance of apoptotic cells by macrophage is still remaining unknown. We investigated the effects of resveratrol on efferocytosis.

Methods

HaCaT and the thymocytes from C57BL/6 were induced apoptosis by UVB and stained with Deep red mitotracker. CFSE stained RAW 264.7 was treated with 1, 5, 10 mM resveratrol for 24 hrs then co-cultured with apoptotic HaCaT to investigate the effect of resveratrol on efferocytosis. To measure the mRNA expression, RAW 264.7 RNA was extracted by RNAzol and then progress reverse transcription and real time PCR.

Results

The results showed that 10 mM resveratrol increased efferocytosis. We further investigated the molecular mechanism of efferocytosis. We found that ITGa5, ITGb3, TG2, Mfge8, and Mertk mRNA were up-regulated by resveratrol after co-cultured with or without apoptotic HaCaT; GAS6 and PROS mRNA expression were up-regulated by resveratrol only after co-cultured with apoptotic cells. The results revealed that resveratrol might enhance efferocytosis by regulating Mfge8-Integrin and GAS6/PROS-Mertk signaling pathway.

Conclusion

Resveratrol enhances efferocytosis via both Mertk-GAS6/PROS and Integrin-TG2-Mfge8 pathway. Resveratrol may be used for the therapy of autoimmune diseases.
Modulating T cell function through elongation of the mitochondrial network to improve cytotoxic CD8+ tumor infiltrating lymphocyte function
Authors: Gonzales Marissa A. , Bullock Timothy N. J.
Published: January 2023

For a number of tumor types, including melanoma, positive clinical outcomes correlate with infiltration of T-cells into the tumor. However, failure to completely control disease progression often occurs, even with immune responses enhanced by checkpoint blockade or other immunotherapy. When T-cells fail to control tumor growth, the tumor infiltrating T lymphocytes (TIL) exhibit characteristics of exhaustion such as poor proliferation, poor effector function, and an increase in inhibitory receptor expression. Metabolic activity of T-cells contributes to differentiation and effector function, leading us to investigate the metabolic state of TIL. By flow cytometry, we find that CD8+ cytotoxic TIL have lower mitochondrial mass, as well as lower mitochondrial membrane potential, compared to effector CD8+ T-cells that respond to vaccination. We hypothesize that TIL are unable to effectively regulate their mitochondrial network, and it is likely that one or more fusion or fission machinery proteins has altered expression or can be manipulated to improve TIL function. We find that TIL have punctate mitochondria as compared to functional effector cells and hypothesize that elongation of the mitochondrial network will improve TIL function such as increasing the capacity for OXPHOS, which is seen in memory T cells with elongated mitochondrial networks. Using mice lacking the fission protein, Drp1, in T cells as a model of elongated mitochondria, we see increased mitochondrial membrane potential, spare respiratory capacity and OXPHOS as compared to WT cells with fission capability. We continue to investigate the effects of mitochondrial elongation of CD8+ cytotoxic TIL.
T cell co-stimulation reprograms the metabolic fitness of highly differentiated CD8+ T cells for improved adoptive T–cell therapy
Authors: Srivastava Abhishek K. , Paria Biman C. , Chandran Smita , Kammula Udai S.
Published: January 2023

Adoptive T cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) can result in durable complete cancer remission in select patients with metastatic melanoma. These clinical responses have strongly correlated with the ability of the transferred cells to engraft and persist in these patients. Preclinical murine studies have further supported that T cells with superior anti-tumor efficacy have higher proliferative capacity, reduced AICD and enhanced in vivo persistence. Thus, in an effort to further improve ACT, we sought to exploit ex vivo 4-1BB co-stimulation to generate T cells with enhanced in vivo anti-tumor activity. We used an artificial APCs genetically engineered to express 4-1BBL to provide ex-vivo 4-1BB co-stimulation to human tumor specific CD8+ T cells. Our results demonstrate that ex-vivo 4-1BBL co-stimulation enhanced proliferative capacity and reduced AICD of the CD8+ T cells without loss of functional tumor avidity. Mechanistically, 4-1BB co-stimulation demonstrated enhanced glycolysis (ECAR) and mitochondrial respiration (OXPHOS) further suggested that 4-1BB co-stimulation could induce significant reprogramming of the bioenergetic pathways used by highly differentiated T cells. The 4-1BB co-stimulation further restored the ability of the highly differentiated T cells to persist and survive after adoptive transfer in NSG mice and resulted in improved survival of established tumor bearing NSG mice. These findings shed light on a fundamental role of 4-1BB co-stimulation and further suggest a practical approach to reprogram the metabolic fitness of highly differentiated T cells for adoptive immunotherapy
MicroRNA-92 expression in CD133+ melanoma stem cells regulates immunosuppression in the tumor microenvironment through integrin-dependent TGF-β activation
Authors: Shidal Chris , Nagarkatti Mitzi , Nagarkatti Prakash S.
Published: January 2023

The existence of cancer stem cells (CSC) accounts for the high degree of chemoresistance and heterogeneity characterizing refractory melanomas. Yet, the degree to which CSCs modulate immune cells in the tumor microenvironment has yet to be revealed. The present study aims to establish a novel role for miR-92 and its associated gene networks in immunomodulation. We employed syngeneic mouse models utilizing B16-F10 melanoma cells to observe primary tumor and metastatic growth. CSCs were isolated from the parental line based on expression of the putative CSC marker, CD133. CD133+ cells were functionally distinct from CD133− cells with CD133+ cells showing increased proliferation in vitro and enhanced tumorigenesis when administered subcutaneously. Our data indicated that compared to CD133− cells, CD133+ CSCs had a greater capacity to recruit immunosuppressive cell types during tumor formation. Using microarray technology in order to expose disparities in microRNA expression between CSC and non-CSC compartments, we identified several miRs that were significantly downregulated in CD133+ cells including miR-92. We hypothesized that lower levels of miR-92 in CSCs led to higher expression of integrin subunits as predicted by gene alignment software and confirmed using qRT-PCR. Flow cytometry analysis of dissociated tumors demonstrated that tumors initiated by CD133+ cells displayed significantly higher levels of TGF-β compared to CD133− cells. Additionally, qRT-PCR for TGF-β associated genes confirmed a significant increase in expression in CD133+ cells. These data suggest that CSCs efficiently induce intratumoral immunosuppression which is potentially regulated by a miR-92 driven axis involving integrin activation of TGF-β.
Targeting FAAH in hematological and non-hematological cancers
Authors: Doan Thao , Sun Jeff , Henderson Jared , Du Yong , Mohan Chandra
Published: January 2023

Background

FAAH is an enzyme that degrades endocannabanoids (eCBs), which have been shown to have pro-apoptotic effects on cancer cells. The body naturally produces eCBs, but they are degraded by endogenous FAAH. FAAH presence was investigated in diffuse large B-cell lymphoma (DLBCL) and breast cancer. By inhibiting the effects of FAAH through small molecule inhibitors, we hope to increase the effects of natural eCBs, such as AEA and PEA, in inducing apoptosis in cancer cells.

Methods

FAAH presence was measured through western blot in 8 DLBCL lines and 3 human breast cancer cell lines using anti-FAAH antibodies. Flow cytometry and MTT assays were used to measure apoptosis. Twenty nude mice were injected to 2M MDA-MB-231 GFP/luciferase cells in the mammary fat pad. After 7 days, tumors were imaged using IVIS and split into two groups. At 14 days, treatment with URB 597 (10mg/kg, 3 times a week) was commenced.

Results

Elevated FAAH was observed in DLBCL lines and breast cancer cell lines. Inhibition of FAAH and addition of exogenous eCBs resulted in decreased proliferation and increased necrosis of the tumor lines. The addition of exogenous eCBs and inhibition of FAAH resulted in a decrease in total EGFR, Akt, and Erk1/2 in MDA 231 tumor cells. There was also a decrease in p-Akt and p-Erk1/2. Inhibiting FAAH and eCB addition resulted in decreased cell viability (p < 0.01) when compared to control with MDA 231 with AEA/URB. By tumor size (2.13 fold decrease, p < 0.001), there was a reduction in in-vivo tumor growth over time with treatment using URB 597.

Conclusions

Inhibition of FAAH in vivo results in the buildup of eCBs, which activates apoptosis and results in decreased tumor growth. FAAH inhibition is a novel therapeutic approach for DLBCL and breast cancer.
Platelet activation alters the immune landscape of the tumor microenvironment
Authors: Riesenberg Brian P. , Rachidi Saleh , Metelli Alessandra , Gutierrez Jennifer , Liu Bie , Li Zihai
Published: January 2023

In a cancerous setting, the normal hemostatic role for platelets can be hijacked to promote tumor growth, survival and metastasis. The most well-established role for platelets in this regard occurs in the bloodstream where activated platelets coat and protect malignant tumor cells from immune attack allowing for successful metastases. Recently, platelet activation was shown to suppress the primary immune response to cancer, indicating a functional role for platelets in the tumor. However, the extent to which this phenomenon is generalizable or the degree to which platelets influence the immune signature of the tumor microenvironment has yet to be fully elucidated. We found that genetic inhibition of platelet activation was capable of slowing primary tumor growth in multiple murine tumor models. Prophylactic treatment with pharmacological platelet inhibitors, aspirin and clopidogrel, slowed tumor growth similar to the genetic model. Antiplatelet therapy mediated tumor control was dependent on the adaptive immune system, more specifically functional CD8+ T cells. Finally, we found therapeutic intervention targeting platelets in established tumors significantly altered the immune landscape of the microenvironment by increasing the percentage and altering the phenotype of CD8+ T cells. These data show that platelet activation can suppress the immune response to multiple cancers. Furthermore, this is the first study to demonstrate targeting platelet activation changes the immune landscape of tumor infiltrating lymphocytes suggesting a potential synergy with other immunotherapeutic strategies to improve tumor control.
Development of an Orthotopic Colorectal Cancer Mouse Model for Radiation Therapy Studies
Authors: Rodriguez Carlos J. , Battaglia Nicholas Griffin , Gerber Scott A. , Lord Edith M.
Published: January 2023

Radiation is commonly used before surgery to reduce tumor burden in rectal cancer. It has been suggested that radiation therapy induces an immune response against the tumor. Previous work using mice ectopic models has shown a correlation between tumor regression and the production of IFN-g and the induction of cytotoxic T cells after radiotherapy. Ectopic models, such as the use of colon cell lines in leg muscle, provide us with some information about the response to radiation, but this response may differ if the tumor is grown orthotopically in the tissue from which it originates. To assess this difference in response, we have developed an orthotopic model to study the growth of colorectal cancer in mice and the response to radiation treatment. In our model, MC38 adenocarcinoma murine cells expressing luciferase are implanted in the rectal wall of C57BL/6 mice through the serous membrane. Tumor growth, assessed using IVIS, revealed tumor masses that steadily increase in size reaching approximately 1 gram 21 days post-injection of 50,000 cells. Importantly, tumor growth in most mice was limited to the site of injection, as mesenteric and omental sites were free of tumor burden. The cellular composition and the morphology of these tumors is being examined using flow cytometry and immunohistochemistry, respectively. Furthermore, studies are underway to determine the response of these tumors to fractionated radiation and to determine if there are differences from the ectopic models of colon cancer. This more clinically relevant model will improve our understanding of response to radiotherapy in colorectal cancer and help develop more effective treatments for this disease.