bluuebunny/crossref_metadata_2025 huggingface dataset:
A New Melanoma Vaccine Based on Conditioned tumor Cell Lysates and Adjuvants Inhibits Tumor Growth
Authors: Onfray Flavio A Salazar
Published: January 2023

Clinical strategies using immune-checkpoint blockers, such as anti-CTLA4, (Ipilimumab) or anti-PD1 antibodies (Nivolumab), have demonstrated durable survival benefits in patients with melanoma and other solid tumors. Nevertheless, an important percentage of patients treated with anti-checkpoint proteins remain refractory. In this context, cancer vaccines become again a complementary alternative as cancer treatment. The optimal delivery of antigens and the use of adequate adjuvants are crucial for vaccine success. Here, a prototype of a generic therapeutic vaccine for the treatment of malignant melanoma named TRIMELVax™ was tested in an experimental model. This vaccine is based on conditioned melanoma tumor lysates combined with a specific adjuvant already tested in patients in the context of a DC vaccine, without adverse effects. The vaccine is intended to directly activate in vivo response against tumors inhibiting their growth. TRIMELVax was evaluated, in terms of safety and efficacy at pre-clinical level, in C57Bl/6 murine model of melanoma (B16). In short, immunocompetent mice were vaccinated with three doses of TRIMELVax, and then challenged with B16 melanoma cells. Alternative, immunization was also tested therapeutically in tumor bearing mice. Our results showed that only TRIMELVax was capable to reduce drastically the occurrence of tumor, inhibiting tumor growth in immunized mice. In contrast the separated use of tumor lysate, or adjuvant did not impact tumor growth. This response was associated to CD8+ T cells mediated immune response and antibody production. Immune-checkpoint blockers therapy may be strongly potentiated by the combined use of TRIMELVax, which need to be proved in future clinical trials.
Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) increases IL-10 via Bruton’s tyrosine kinase and STAT3: Effect on T cell function
Authors: Swanson-Mungerson Michelle Anne , Nelson Theodore , Stone Amanda , Incrocci Ryan
Published: January 2023

Data from our laboratory demonstrate that Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) promotes B cell lymphoma survival by enhancing IL-10 production, which has important implications in the treatment of EBV-associated B cell tumors. To determine the pathway used by LMP2A to increase IL-10, pharmacological inhibitors were added to LMP2A-expressing B cells and IL-10 was assessed. Our findings indicate for the first time, that LMP2A activates BTK to phosphorylate STAT3 in B cell tumors, which mediates the LMP2A-dependent increase in IL-10. Additionally, we assessed whether the LMP2A-mediated increase in IL-10 is immunosuppresssive to cytotoxic T cells that are critical for controlling the growth of EBV-associated tumors. Purified human CD8+ T cells activated with anti-CD3/CD28 mAbs were exposed to supernatants from either LMP2A-negative or –positive B cell lines for 48 hours. Our findings indicate that there is no difference in proliferation and IFN-γ secretion by activated CD8+ T cells exposed to supernatants from LMP2A-positive B cell lymphomas when compared to CD8+ T cells exposed to supernatants from LMP2A-negative B cell lymphomas. Although the LMP2A-mediated increase in IL-10 does not significantly influence the CD8+ T cell response, these findings highlight the possibility of using BTK and STAT3 inhibitors in clinical trials to treat EBV-associated lymphomas that express LMP2A. This work was supported by NIH grant 1R15CA149690-01.
Immune and metabolic regulation mechanism of Dangguiliuhuang decoction against insulin resistance
Authors: Cao Hui , Xiang Ming
Published: January 2023

Insulin resistance (IR) is recognized as a chronic inflammatory and metabolic disease, playing a pivotal role in the development of diabetes. Obesity and abnormal lipids metabolism increase infiltration of inflammatory cells, affect immune homeostasis, and reduce insulin sensitivity, consequently leading to the occurrence of insulin resistance. In this study, we found that Dangguiliuhuang decoction (DGLHD), a traditional Chinese medicine formula used for the treatment of diabetes, presented a beneficial effect on the progression of IR in ob/ob mice characterized by leptin gene deficiency. DGLHD exerted anti-inflammatory and immunosuppressive actions on ob/ob mice through altering function and metabolism of DCs and T cells with activated PPAR-γ molecule or inhibited PI3K/Akt signaling pathway in cells. Additionally, DGLHD also ameliorated glucolipid metabolism in liver and adipose tissue via regulating the expression of metabolic genes and activating PI3K/Akt signaling pathway. Importantly, metabolomics analysis revealed that DGLHD could decrease glutamate, phenlalanine, branched-chain fatty acids and glyoxylate as well as cholesterol and triglyceride in adipose and hepatic tissues of ob/ob mice, thereby improving the insulin sensitivity of target organs. Therefore, we first elucidated that DGLHD has anti-insulin resistant effects by maintaining immune and metabolic homeostasis.
Negative regulation of Th17 cell development and function by the REV-ERBs
Authors: Solt Laura A , Amir Mohammed , Chaudhari Sweena M. , Wang Ran , Campbell Sean
Published: January 2023

A significant amount of work has identified key factors that drive Th17 cell development, including the nuclear receptors (NRs) RORα and RORγ. However, cell-intrinsic mechanisms that negatively regulate Th17 cell development and inflammation have received less attention. Two other members of the NR superfamily, the REV-ERBs (REV-ERBα and REV-ERBβ), are often co-expressed in the same tissues as the RORs, bind the same DNA response elements, and co-regulate their shared target genes. The REV-ERBs have well described roles in the regulation of the circadian rhythm and metabolism, but their roles in immune cell function are poorly understood. Therefore, we hypothesize that the REV-ERBs are key negative regulators of Th17 cell development and function.

We we aim to understand the role of the REV-ERBs in Th17 cell development as well as determine whether targeting these NRs is a viable therapeutic option for the treatment of Th17-mediated autoimmune diseases. Our results indicate that the REV-ERBs are differentially expressed during Th17 cell development. Overexpression and pharmacological modulation of REV-ERB activity suppresses Th17 cell development whereas genetic deletion of the REV-ERBs perturbs Th17 cell development in vitro and induces autoimmunity in vivo. Using REV-ERB-specific small molecules that we have developed, we have found that pharmacological modulation of REV-ERB activity suppresses Th17 cell development in vitro and development of autoimmunity in vivo. Collectively, our data suggest that the REV-ERBs are key negative regulators of Th17 cell development and function and represent a novel therapeutic target for the treatment of Th17-mediated autoimmune diseases.

Funding support – NIH (R01AI116885-01A1)
The resolutive factors issued from apoptotic cell clearance allow the termination of ongoing inflammation
Authors: Perruche Sylvain , Bonnefoy Francis , Couturier Mélanie , Martin-Rodriguez Omayra , Gauthier Thierry , Saas Philippe
Published: January 2023

Immune-mediated inflammatory diseases have in common a dysregulation of the immune response. Resolution of inflammation is a powerful process involving a numerous of factors including specialized pro-resolutive lipid mediators and proteins such as TGF-β, resulting in inflammation termination and the initiation of tissue healing. A critical step of inflammation resolution is efferocytosis of apoptotic effector cells by phagocytes which produce factors allowing the termination of inflammation and initiating tissue healing. TGF-β has been shown has mandatory in such process since it is produced by apoptotic cell and phagocytes clearing them. We hypothesize whether the reintroduction of pro-resolutive factors issued from apoptotic cell efferocytosis would allow a termination of ongoing inflammation. Injection of such resolutome in ongoing collagen-induced experimental arthritis allowed the long term reduction of clinical symptoms and was accompanied by the emergence of highly collagen-specific Tregs and antigen presenting cells (APC) reprograming through a tolerogenic state. When injected in ongoing T cell-transfer-induced Inflammatory Bowel Disease experimental model, again, clinical symptoms and colon mucosa lesions were severely reduced, accompanied by a higher proliferation, migration and wound healing capacities of both epithelial cells and fibroblasts. TGF-β demonstrated a prominent role in the reprogramming of APC and regulatory T cell emergence. So far the pro-resolutive factors issued from apoptotic cell efferocytosis demonstrated the potential to terminate ongoing inflammation. This opens clinical drug development opportunities in the treatment of refractory chronic inflammatory diseases.
Temporally controlled B cell depletion with universal chimeric antigen receptor (CAR) T cells for pemphigus vulgaris (PV) therapy
Authors: Ellebrecht Christoph T , Mao X , Melenhorst J J , Lacey S F , Zhao Y , Milone M C , Payne A S
Published: January 2023

Therapy of PV relies on chronic immunosuppression, which results in significant morbidity. Complete, transient B cell depletion should cure PV, since autoreactive clones do not recur upon regeneration of the B cell repertoire. Engineered CAR T cells (CARTs) are the most potent means for total B cell depletion, but for therapy of autoimmunity, temporal control of CART activity is necessary to prevent lasting immunosuppression. Here we validate 3 novel strategies to control CART survival and function. We combined a B cell targeting CAR with an inducible caspase 9 suicide gene (sCAR), a reverse (constitutively active) suicide gene (revCAR), or a molecular on-switch that permits CAR surface expression (onCAR). sCAR, revCAR and onCARTs showed potent in vitro killing equivalent to conventional CARTs. Activation of the respective regulatory system resulted in in vitro depletion of >95% of sCART and revCARTs and loss of CAR expression in onCARTs. In an in vivo leukemia model, >90% of sCARTs were eliminated (p<0.01) as compared to vehicle treated mice, while preserving their efficacy before suicide gene activation (p<0.001). RevCARTs showed complete loss of leukemia control in vivo in the absence of a suicide-preventing compound (p<0.01), indicating their functional depletion in vivo. Finally, to allow universal (allogeneic) CART therapy, we used CRISPR genome editing to disrupt endogenous T cell receptor and MHCI expression. Universal sCARTs caused total B cell depletion in a humanized allogeneic BLT mouse model (p<0.05) with complete in vivo depletion of sCARTs. In summary, we present 3 novel strategies to regulate CARTs, which provide a platform for curative, universal, and large-scale applications in PV and other autoimmune diseases.
A pharmacological protein kinase C/D inhibitor Gö6976 ameliorates collagen-induced arthritis
Authors: Yoon Taewon , Cho Hongsik , Kim Young-In , Brand David , Hasty Karen , Stuart John , Yi Ae-Kyung
Published: January 2023

Toll-like receptor (TLR) signaling has been implicated with the initiation and progression of rheumatoid arthritis (RA). Disruption of TLR signaling at early stages of RA might thereby provide an opportunity to halt the RA progression and ameliorate outcomes. We previously found that protein kinase D1 (PKD1) is essential for proinflammatory responses mediated by MyD88-dependent TLRs, and TLR-dependent PKD1 activation is inhibited by Gö6976. However, it is currently unknown whether PKD1 plays a role in pathogenesis of RA and whether Gö6976 has ameliorating effects on arthritis. Here, we investigated whether PKD1 contributes to the TLR-mediated proinflammatory responses in human synovial cells, and to development and progression of type II collagen (CII)-induced arthritis (CIA; an experimental animal model for human RA). We found that PKD1 is constitutively hyperactivated in synoviocytes of RA patients, and is rapidly activated in response to TLR/IL-1R ligands. Silencing PKD1 expression in RA synoviocytes resulted in substantial inhibition of both spontaneous and TLR/IL-1R-mediated expression of cytokines/chemokines. Although Gö6976 had no effect on the TCR-induced cell proliferation and cytokine production, synergistic effects of TCR and TLR2, as well as TLR2 alone, on the spleen cell cytokine production and proliferation were significantly inhibited in the presence of Gö6976. In addition, daily treatment with Gö6976 significantly reduced serum levels of IFNg and anti-CII IgG, and the incidence and severity of arthritis in humanized HLA-DR1 transgenic mice immunized with CII. Our data suggest that PKD1 might play an important role in proinflammatory responses in RA and CIA, and could be a therapeutic target for arthritis.
Glucocorticoid induced leucine zipper in Alzheimer’s disease.
Authors: Srinivasan Mythily , Lahiri Debomoy K
Published: January 2023

Aging and stress are significant risk factors for Alzheimer’s disease (AD). Stress induced glucocorticoids accelerate beta amyloid (Aβ) plaque formation and tau phosphorylation, aggregates of which upregulate reactive oxygen species in vulnerable neurons. The oxidative stress negatively regulates glucocorticoid receptor, preventing nuclear translocation and suppression of the transcriptional factor, nuclear factor-κB (NF-κB) by glucocorticoids. The ensuing inflammatory distress enhance further Aβ accumulation initiating a vicious cycle. Glucocorticoid induced leucine zipper (GILZ) is a recently identified protein that mediates many of the cellular effects of glucocorticoids. Exogenous GILZ ameliorated disease in models of chronic inflammation. Mechanistically GILZ inhibits NF-κB by binding the p65 subunit of NF-κB in the cytoplasm and thereby preventing transactivation of inflammatory and apoptosis mediators. Microglial GILZ expression has been shown to correlate negatively with inflammatory cytokines. Increased p65, upregulated inflammatory cytokines and apoptotic markers are frequently observed in postmortem AD tissues. Hence selective targeting of activated p65 is an attractive strategy to modulate neuro-inflammation and suppress neurodegeneration. Recently we reported the design and functional potential of structural analogs of GILZ that selectively blocked activated p65 and suppressed Aβ induced cytotoxicity in mixed brain cell cultures. Here we evaluated the efficacy of GILZ mimics in lipopolysaccharide induced mouse model of AD. Our data suggest that mice treated with select mimics of GILZ exhibited reduced inflammation and Aβ deposits suggesting a therapeutic potential.
Teaching students how to think like an immunologist: flipping a classroom without flipping out.
Authors: Parkin Kirstin
Published: January 2023

Study

A traditional lecture-based 400 level immunology course was converted into a team-based learning environment. Activities were used to teach students how to analyze published data from research labs at MSU and thus, learn how to “think” like MSU scientists.

Objectives

For the main concepts covered in the course, the goal was to improve students’ ability to discuss the concept orally and in written format, diagram the concept, use drawings to portray the concept, graph the concept, and identify techniques and measurements used in current research related to the concept.

Methods

Learning objectives, guided textbook readings, weekly on-line formative assessments with feedback, in-class team based activities, Top Hat in-class response questions, and in-class summative assessments were used to meet the course objectives.

Assessments

35 on-line formative assessments, 35 Top Hat in-class response questions, and 4 in-class summative assessments were provided during the semester. Online student feedback regarding exam questions and course satisfaction were also used.

Results

Student performance on short-answer questions on formative assessments improved throughout the semester. Students identified multiple choice questions as the least likely to assess course objectives. Students with low attendance earned the lowest scores.

Implications

By providing students with examples of how MSU scientists design experiments, display data, and analyze data, students learn how to “think” like a scientist rather than learning a large number of scientific facts. The active learning environment also provides an opportunity for students to improve teamwork skills that are sought by corporations hiring college graduates.
Aggregation and innate immune response modulating impurities
Authors: Polumuri Swamy kumar , Haile Lydia , Rao Roshni , Verthelyi Daniela
Published: January 2023

Therapeutic proteins can induce immune responses that affect the safety and efficacy even if derived from human sequences. The factors that contribute to the immunogenicity of this therapeutics are poorly understood, but protein aggregation is thought to facilitate it. Previously, our lab showed that trace levels of product or process derived innate immune response modulating impurities (IIRMIs) can also increase product immunogenicity. In this study we hypothesized that protein aggregates and IIRMIs may synergize to facilitate product immunogenicity and explored it using clinical grade Human Serum Albumin (HSA) and Intravenous Immunoglobulin (IVIG) proteins as model. Protein aggregations formed by mechanical stress (shaking and stirring) were characterized by SDS-PAGE, turbidity, MFI and FlowCAM. Cellular responses to aggregated proteins with trace levels of IIRMs were tested in human PBMC. We show that stirring and shaking resulted in the formation of stable aggregates with size ranges of 2 – 50 μm for stirred HSA; and 2–100 μm for IVIG. Despite the similarity in size, the functional assays showed that aggregates of IVIG result in increased mRNA expression of IL-8, IL-6, IL-1β and CCL-2 whereas HSA aggregates did not. Further, we analyzed how the aggregated product changed the transcriptome using Nanostring and showed that aggregated IVIG (shaken or stirred) significantly increased the mRNA of CCL2, CCL7, CCL3, CCL24, CSF1, CXCL2, IRAK1, EGR2, CEBPβ, PPARg, TNFSF15 and whereas HSA aggregates did not. Similarly MAPKs (pp38, pERK and pJNK) and pAKT were activated when PBMC were stimulated with IVIG aggregates but not HSA aggregates. Lastly, we examined whether the effect was mediated by Toll like receptors.
Human astrocytes and microglia-like cells express cGAS-STING viral sensing components
Authors: Jeffries Austin , Marriott Ian
Published: January 2023

NeuroAIDS is a major health problem in AIDS patients and long-term HIV survivors, and evidence is accumulating that the innate immune responses of glial cells to this virus play a critical role in disease pathology within the central nervous system (CNS). While astrocytes and microglia are uniquely positioned to respond to CNS pathogens and these cells have been shown to produce key inflammatory and antiviral mediators following exposure to retroviruses including HIV, the molecular mechanisms by which glia perceive such viruses remain incompletely understood. It has become increasingly apparent that innate immune sensing of nucleic acids underpins many physiological and pathological responses to viral pathogens, and the recent identification of a key role for cyclic GMP-AMP synthase (cGAS) as a cytosolic DNA sensor has been one of the most exciting developments in viral immunology in recent years. Importantly, cGAS has been demonstrated to contribute to the sensing of replicative HIV-1 DNA intermediates and eliciting cytokine responses in a human monocytic cell line. Here, we demonstrate that human glial cell lines and primary human astrocytes constitutively express robust levels of cGAS, and we show that such expression is maintained in these cells following challenge with either viral ligands or a laboratory strain of HIV-1. Furthermore, we show that human glial cells express the critical cGAS downstream adaptor molecule STING, and confirm that the robust levels of expression of this molecule are similarly maintained following viral challenge. The data therefore support a potential role for the cGAS-STING cytosolic viral sensing pathway during acute and/or chronic HIV infection of the CNS.
An Essential Role for TAGLN2 in Phagocytosis of Lipopolysaccharide-activated Macrophages
Authors: Jun Chang-Duk , Kim Hye-Ran
Published: January 2023

Activated macrophages have a greater ability of phagocytosis against pathogens that is mediated by large-scale actin rearrangement. However, molecular machineries that conduct this task have not been fully identified. Here, we demonstrate an unanticipated role of TAGLN2, a 22-kDa actin-binding protein, in Toll-like receptor (TLR)-stimulated phagocytosis. TAGLN2 was greatly induced in macrophages in response to lipopolysaccharide (LPS), a ligand for TLR4, partly via the NF-κB pathway. TAGLN2-deficient macrophages (TAGLN2−/−) showed defective phagocytic functions of IgM- and IgG-coated sheep red blood cells as well as bacteria. Cell signaling pathways involved in actin rearrangement—PI3 kinase/AKT and Ras-ERK—were also down-regulated in LPS-stimulated TAGLN2-deficient macrophages. Moreover, TAGLN2−/− mice showed higher mortality after bacterial infection than wild-type littermates. Thus, current results provide a novel function of TAGLN2 as a molecular armament required for host defense.
Role of Natural Killer Cells in Sonic Hedgehog driven Medulloblastoma
Authors: Schiavone Francesco , Carlino Claudia , Ascenzi Sonia , Po Agnese , Sanseviero Emilio , Ferretti Elisabetta , Gismondi Angela , Santoni Angela
Published: January 2023

Medulloblastoma (MB) is the most common paediatric neoplasm of the CNS. Standard therapy is mainly curative; however, the severe side effects make immunotherapy an attractive therapeutic option. Natural Killer (NK) cells are innate lymphoid cells that can be used for adoptive cell therapy. Herein, we studied the role of NK cells in MB growth control, focusing on Sonic Hedgehog (SHH) subgroup, using the MB cell line DAOY.

The contribution of SHH pathway on NK cell recruitment to MB was analysed performing an orthotopic xenograft of DAOY in nude mice treated with a pharmacological inhibitor of the SHH pathway. FACS analysis revealed higher percentage of NK cells in cerebella of treated mice compared to control group.

Based on this in vivo evidence, we characterized the mechanism underlying NK cell recruitment. Pharmacological inhibition of the SHH pathway in DAOY raised the expression and secretion of chemokines involved in NK cell migration. Chemotaxis assay confirmed that these secreted molecules were functional.

Successively, we assessed the ability of NK cells to recognize SHH-MB. By FACS analysis we determined the tumor line expression of ligands for the NKG2D and DNAM1 activating receptor. In particular, ULBP3 and ULBP2 were expressed at high levels.

Moreover, we found that NK cells co-culture with DAOY induced degranulation that was reduced in the presence of anti-NKG2D and anti-DNAM1 blocking antibodies, suggesting an involvement of these receptors in triggering degranulation.

Besides, DAOY increased the production of IFNγ in NK cells pre-activated of with suboptimal doses of IL12 and IL15.

Our data provide evidence for a role of NK cells in the control of SHH-MB growth.
Cell surface PCNA is associated with expression of cancer stem cell transcription factors and inhibit NK cell effector function.
Authors: Malaer Joseph D. , Mathew Porunelloor Abraham
Published: January 2023

Natural killer (NK) cell function is regulated through a delicate balance of signals received from activating and inhibitory receptors interacting with ligands on the surface of target cells, playing a vital role in the innate immune response against cancer and infection. Cancer cells may evade NK-mediated killing by expressing ligands for NK cell inhibitory receptors. NKp44, a member of the Natural Cytotoxicity Receptor (NCR) family, functions as an activating or inhibitory receptor. Proliferating cell nuclear antigen (PCNA) associates with Human Leukocyte Antigen (HLA) I and forms the inhibitory ligand for NKp44, resulting in the inhibition of NK function. In this report, we have identified diffuse B cell lymphoma (DB) cells with cell surface PCNA have higher expression of cancer stem cell transcription factors as determined by cell sorting and qRT-PCR. We further demonstrate that the expression of cell surface PCNA is restricted to tumor cells exhibiting a cancer stem cell phenotype. Blocking the NKp44-PCNA interaction enhanced the killing of DB cells displaying cancer stem cell characteristics by NK cells. Characterization of stem cell transcription factors and cell surface PCNA may provide novel immunotherapeutic targets for cancer stem cells.
Ex-Vivo Expanded Human Vγ9Vδ2 T Cells Efficiently Suppress Epithelial Ovarian Cancer Development
Authors: Liu Chao Lien Liu , Miao Carol H. , Yeh Chia Yu
Published: January 2023

The γδ-T cells attract attentions because of their potent cytotoxicity towards tumors. Most γδ-T cells facilitate the major histocompatibility complex (MHC)-independent activation by the interaction between their receptor Natural Killer Group 2 Member D (NKG2D) and MICA/MICB molecules expressed on the tumor cells, leading to tumor death. However, despite of the potent antitumor effects, their therapeutic efficiency and molecular mechanisms remain largely elusive. Ovarian cancer is one of the most lethal and challenging female malignancies worldwide because of the delayed diagnosis and the resistance to traditional chemotherapy, prompting us to investigate the immunotherapeutic effects of γδ-T cells as a new treatment. We successfully expanded γδ-T cells up to ~ 78% from peripheral blood mononuclear cells (PBMCs) with mostly Vγ9Vδ2-T cell subtype. We showed that the expanded γδ-T cells exerted significant cytotoxicity activities towards OVCAR3 and HTB75 cells in-vitro. Furthermore, in the tumor xenografts of NSG mice, γδ-T cells not only suppressed tumor growth but also completely eradicated the preexisting tumors with initial size ~5mm. Thus, we conclude that γδ-T cells possess dramatic cytotoxicity activities towards epithelial ovarian cancers both in-vitro and in-vivo, which strongly support the potential clinical immunotherapeutic application of γδ-T cells to treat this dreadful female malignancy.
Level of expression of vimentin mRNA in 9L gliosarcoma cells compared to microglial cells, astrocytes, and oligodendrocytes isolated from Fischer 344 rats
Authors: Hillman Tyler C , Redd Joan M
Published: January 2023

Gliosarcomas are an aggressive brain cancer that can become metastatic. Previously, we used a monoclonal antibody, designated H5, to isolate a tumor-associated antigen (TAA) expressed by 9L, a gliosarcoma line isolated from Fischer 344 (F344) rats. This TAA was tentatively identified as vimentin via mass spectrometry. In this project, we compared the level of vimentin mRNA in 9L cells to that of three types of glial cells: microglial cells, astrocytes, and oligodendrocytes isolated from F344 rats via qPCR. The level of vimentin mRNA in 9L cells was significantly upregulated as compared to the expression levels in all three types of glial cells. Similar research on carcinomas has identified vimentin as being involved in metastatic tumors and is now used as a genetic marker of this specific type of cancer. This research suggests that vimentin mRNA in 9L undergoes similar upregulation to that of carcinomas.
Tumorous niche drives NK exhaustion by disordering metabolic program
Authors: Zheng Xiaohu , Sun Rui , Tian Zhigang , Wei Haiming
Published: January 2023

Natural killer (NK) cells are important effectors of the immune system that can lyse malignancy-transformed cells. In the human liver, NK cells can account up to 50% of the total lymphocyte population. How NK cells lose immune surveillance occurs in hepatocellular carcinoma progression.

In this study, 75 cases from hepatocellular carcinoma (HCC) patients were analyzed by RNA sequence, pathology, flow cytometry and cell culture in vitro. We found a significant enrichment of infiltrating T cells within the tumor tissues of HCC patients but not NK cells, as compared to the adjacent peritumoral tissues. It is interesting that the infiltrating NK cells enrich CD56brightCD49a+ NK subset, like liver-resident NK. Nearly 50% of the entire healthy liver NK cell population is composed of functionally relevant CD56bright CD49a-NK cells, and almost all of the subset was lose. The infiltrating CD56brightCD49a+ NK cells present exhausted phenotype and downregulated expression of efficacy molecules of CD11b, CD226 and IFN-γ. We also found a few of CD56dimCD16+ NK cells were infiltrating, which were similar to their peripheral circulating counterparts in transcription and phenotype, but the lower effect function and viability of infiltrating NK cells compared with normal liver or peripheral circulating counterparts.

Dynamic reprogramming of metabolism is essential for immune cell stable liveness and effector function. Gene Ontology profiling and metabolism analysis revealed the infiltrating NK cells bioenergetic insufficiencies and enrichment of metabolic oxygen specis leading to its subset variations and immune surveillance insensitivity.
Mycoplasma pneumoniae CARDS toxin is a unique virulence factor targeting the Th1/Th2 response for persistent infection
Authors: Segovia Jesus A , Chang Te-Hung , Cagle Marianna P , Winter Vicki T , Coalson Jacqueline J , Kannan Thirumalai R , Baseman Joel B
Published: January 2023

Mycoplasma pneumoniae is a leading cause of bacterial community-acquired pneumonia and is implicated in the initiation and exacerbation of asthma. Over 2 million cases of M. pneumoniae infection occur in the United States each year. However, with no surveillance system in place for reporting infections, the true scope of the problem may be vastly underestimated. One of the hallmark immunological effects of M. pneumoniae infection is an imbalanced Th1/Th2 response. A recently identified M. pneumoniae virulence factor is the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin, an ADP-ribosylating and vacuolating protein produced abundantly during infection that is capable of provoking a Th2 cytokine phenotype in mice. We hypothesize that CARDS toxin is utilized by M. pneumoniae in vivo to dampen the Th1 response and influence the Th2 response in order to establish a strong, persistent infection.

The clinical isolate strain M. pneumoniae S1 abundantly produces CARDS toxin during infection and causes severe pathology. We utilized S1 as a template to generate a CARDS-deficient mutant we designated M7. M7 and S1 were used in vitro and in vivo to identify Th1- and Th2-specific cytokine changes during active infection. We observed significant differences in pathology between M7- and S1-infected mice, indicating that CARDS toxin directly impacted M. pneumoniae disease and persistence. Our findings provide insights to understand the elusive pathological mechanisms behind M. pneumoniae infection and implicate CARDS toxin as a potentially important target for therapeutic intervention during M. pneumoniae infection.
Macrophage galactose-type lectin 1 (MGL-1) regulates neutrophil mobilization during pulmonary Gram negative pneumonia
Authors: Jondle Christopher Ned , Mishra Bibhuti B. , Sharma Jyotika
Published: January 2023

Macrophage galactose-type lectin 1 (MGL-1) is a type 2 transmembrane C-type lectin receptor. We recently reported that MGL1-mediated responses are required to mitigate disease severity in Klebsiella pneumoniae (KPn) induced murine pneumosepsis. Here we investigated the role of MGL-1 in neutrophil development and homeostasis during KPn infection. We found that MGL-1−/− mice exhibit severe neutrophilia in their lungs upon KPn infection. Adaptive transfer experiments showed that MGL-1 deficiency on neutrophils significantly increased their capacity to infiltrate the lungs. Analysis of neutrophil numbers and their progenitors in various compartments showed that increased mobilization of mature neutrophils from bone marrow storage pools, but not the progenitor proliferation, accounted for the increased systemic neutrophil counts. This was correlated with elevated levels of neutrophil mobilizing factors G-CSF and MCP-2 in MGL-1−/− mice. Collectively, our findings demonstrate that MGL-1 has a profound role in neutrophil homeostasis during acute inflammatory condition.
The role of ecSOD in phagosomal containment of Listeria monocytogenes by neutrophils
Authors: Okunnu Busola , Witter Alexandra R , Swanta Naomi K , Berg Rance E
Published: January 2023

Extracellular superoxide dismutase (ecSOD) is an enzyme associated with the extracellular matrix that plays a protective role during reactive oxygen species mediated inflammatory responses. Previously, we reported that neutrophils are essential for protection during infection with Listeria monocytogenes (LM), and that high ecSOD activity is detrimental to host resistance during infection, while lack of ecSOD activity is beneficial. Furthermore, using depletion studies, it was determined that neutrophils from ecSOD KO mice are more protective than neutrophils from ecSOD expressing mice during LM infection. To understand how ecSOD modulates neutrophil function during LM infection, bacterial uptake and phagosomal escape were measured. A higher percentage of neutrophils from the spleen and liver of ecSOD KO mice associated with and allowed for phagosomal escape of LM. However, using MFI as an indicator of the relative number of bacteria per neutrophil, we observed that the ratio of cell associated to cytosolic bacteria was higher in ecSOD KO neutrophils in comparison to neutrophils with ecSOD activity. These data suggest that the absence of ecSOD activity enhances neutrophil association with LM and simultaneously limits LM escape from the phagosome. Future studies utilizing neutrophils obtained from other organs as well as from infected mice should help conclusively determine how the lack of ecSOD activity enhances the protective capabilities of neutrophils.
Nociceptor neurons regulate lung infections and bacterial host defense
Authors: Chiu Isaac M , Baral Pankaj
Published: January 2023

The sensory nervous system densely innervates the respiratory tract, mediating breathing, cough and bronchoconstriction. However, the role of the nervous system in lung host defenses has not been defined. Nociceptor neurons sense noxious/harmful stimuli and protect organisms from danger. Here, we find that nociceptor neurons play a critical role in regulating innate immunity against bacterial lung infections and lethal pneumonia. Staphylococcus aureus is a major cause of human disease and pneumonia in hospital acquired infections. We find that S. aureus directly induces nociceptor neuron ionic influx and activation. Nociceptor neurons released the neuropeptide CGRP upon bacterial activation, which suppressed neutrophil recruitment and bacterial clearance. Targeting nociceptor neurons by genetic and chemical strategies led to significantly enhanced innate immunity and survival in a mouse model of S. aureus lethal pneumonia. Therefore, the nervous system plays an important role in lung infections, and targeting sensory neurons could lead to better treatments for bacterial pneumonia.
Eosinophils actively participate in the host response to Mtb infection
Authors: Tocheny Claire , Senbagavalli Prakash , Hu Zhidong , Kauffman Keith , Sakai Shunsuke , Yan Bo , Song Yanzhen , Wong Ka-Wing , Barber Daniel , Klion Amy , Mayer-Barber Katrin
Published: January 2023

Mycobacterium tuberculosis (Mtb) is the leading cause of mortality worldwide due to a single infectious agent. Mtb resides in pulmonary macrophages, neutrophils and phagocytes, which are also key effector cells that limit bacterial growth. The role of other granulocytic phagocytes during Mtb infection is largely unknown. Eosinophils have comparable phagocytic function to neutrophils and an overlapping repertoire of granular contents capable of limiting bacterial growth. Here we characterized eosinophil responses to Mtb in vitro and in vivo. Eosinophils from healthy human donors responded to Mtb in vitro by CD69 up-regulation and CD62L down-regulation. Mtb induced the release of the eosinophil-specific granule proteins EDN and ECP. Mtb exposure resulted in secretion of IL-1a, TNFa, MIP-1a, MIP-1b, and IL-8. These results suggest that Mtb directly affects eosinophil degranulation, and that eosinophils are able to contribute to Mtb-driven inflammation. To investigate whether eosinophils actively participate in the cellular immune response to Mtb in vivo we followed the eosinophilic response after pulmonary Mtb infection in non-human primates. Eosinophils were significantly increased in the BAL fluid of Mtb infected rhesus macaques, providing evidence that eosinophils are being actively recruited to the lung in response to Mtb in vivo. Indeed, when we analyzed Mtb infected lung tissue from patients who had undergone lung resection, eosinophils were enriched in fibrotic TB consolidations, cavity wall and necrotic caseum.

We are currently investigating the biological relevance of the eosinophilic response during Mtb infection in the mouse model.

This work was supported by the intramural research programs of NIAID, NIH.
CD4+ T cells induce homeostatic expression of the Treg-attracting chemokine CCL22 in dendritic cells
Authors: Piseddu Ignazio , Kühnemuth Benjamin , Knott Maximilian , Vetter Viola , Layritz Patrick , Endres Stefan , Anz David
Published: January 2023

Regulatory T cells (Tregs) are highly potent modulators of immune responses by various mechanisms including direct interaction with dendritic cells. Thereby, Tregs play a critical role not only in prevention of autoimmunity but also in tumor immune escape. By binding to its only known receptor CCR4 the chemokine CCL22 is a potent chemoattractant for Tregs, influencing their localization and activity. However, the regulation of CCL22 expression is still poorly understood.

Here we demonstrate that under homeostatic conditions cells of the dendritic cell lineage are the main producers of CCL22. In vitro, coincubation of dendritic cells with CD4+ CD25− T cells strongly induced CCL22 expression in a paracrine manner. Using neutralizing antibodies we identified the cytokine family IL-3, IL-5 and GM-CSF, as well as IL-4 to be the main inducers of CCL22 in dendritic cells. In vivo, transfer of CD4+ T cells into Rag−/−-mice lacking mature T and B cells was able to increase CCL22 levels in several lymphoid organs.

In summary, we identified the aforementioned T cell-derived factors as the main inducers of the Treg-associated chemokine CCL22, in vitro and in vivo. Therefore, our data suggests that paracrine signalling of T cells might indirectly enhance Treg activity by induction of CCL22 expression in dendritic cells. In conclusion, the data presented here potentially shows a self-limiting mechanism of adaptive immune responses.
Vasoactive intestinal peptide induced epithelial to mesenchymal transition activities in PC3 prostate cancer cells
Authors: Laney Caleb N , Bains Manpreet , Dorsam Glenn
Published: January 2023

Epithelial to mesenchymal transition (EMT) is a fundamental cellular process preceding movement. Malignant transformation of epithelial cells also undergo EMT leading to metastasis and a worse prognosis for human cancer patients. Understanding molecular pathways that are linked to EMT may provide additional drug targets to treat epithelial cancers like prostate cancer. Vasoactive intestinal peptide signaling through one of its naturally occurring G-protein coupled receptors, termed VPAC1, induces non-tumor and tumorigenic epithelial cell lines (RWPE-1, LnCaP and PC3 cells) to undergo EMT and downregulate E-cadherin, a hallmark for EMT. Curcumin, an anti-inflammatory molecule, blocked these VIP EMT effects suggesting a role for NF-kB signaling. Our group has uncovered EMT-associated molecular mRNA targets induced by VIP treatment of primary, resting CD4 T cells, including the EMT master regulator transcription factor, SNAIL1. We hypothesize that EMT transition effects by VIP signaling may also involve the upregulation of SNAIL1 protein. To this end, we plan on treating PC3 cells with varying levels of VIP over time (0–24 hr) and measure by immunoblots the extent to which Snail1 protein, and up to 10 additional EMT-associated proteins, are differentially altered. Successful completion of this research is expected to uncover important VIP protein targets that is expected to better explain its epithelial tumorigenic potential.
The allergic T cell response against mouse proteins
Authors: Schulten Véronique MJ , Westernberg Luise , Peters Bjoern , Sette Alessandro
Published: January 2023

Mouse allergy is rising in prevalence, affecting inner-city households and laboratory workers. Increased exposure to mice in inner-city households is a strong predictor for asthmatic disease. Despite a strong link between mouse exposure and asthmatic disease, the allergic immune response to mouse has been significantly understudied. To date, only one major allergen in mouse, Mus m 1, has been identified and very little is known about the targets of the allergic immune response against mouse.

Using a proteomic/transcriptomic approach, we sought to identify T cell targets in 24 mouse allergic and asthmatic patients. Mouse urine and epithelial extracts were analyzed by 2D-IgE/IgG immunoblots using pooled sera from mouse-sensitized donors. Mass spectrometry of selected protein spots identified 30 novel antibody reactive proteins. Predicted MHC binding peptides from these novel proteins and mouse homologs to mammalian allergens were screened for T cell reactivity in PBMCs from mouse allergic patients. Overlapping peptides from the major mouse allergen Mus m 1 and its major urinary protein isoforms were screened in parallel. T cell epitope mapping experiments showed that responses to Mus m 1 peptides were dominant overall. However, reactivity to mouse peptides homologous to other mammalian allergens, specifically guinea pig, was also detected. In summary, our data demonstrates that the cellular and serological targets of the allergic response overlap, with Mus m 1 being the major target for both T cells and IgE antibodies. However, on a serological level, other proteins are also recognized. To the best of our knowledge, this is one of the first comprehensive studies of T cell epitope targets in mouse allergy.
Transplantation of Human Skin Microbiota in Models of Atopic Dermatitis
Authors: Myles Ian Antheni
Published: January 2023

Atopic dermatitis (AD) is characterized by reduced barrier function, reduced innate immune activation, and susceptibility to Staphylococcus aureus. Host susceptibility factors are suggested by monogenic disorders associated with AD-like phenotypes, and can be medically modulated. S. aureus contributes to AD pathogenesis and can be mitigated by antibiotics. Recent work has revealed that the skin microbiome differs significantly between healthy controls and patients with AD, including decreased Gram-negative bacteria in AD. However, little is known about the potential therapeutic benefit of microbiome modulation. To evaluate if parameters of AD pathogenesis are altered after exposure to different culturable Gram-negative (CGN) bacteria collected from human skin. CGN bacteria were collected from healthy controls and patients with AD. Impacts on cellular and culture-based models of immune, epithelial, and bacterial function were evaluated. Representative strains were evaluated in the MC903 mouse model of AD. We found that CGN bacteria taken from healthy volunteers but not from patients with AD were associated with enhanced barrier function, innate immunity activation, and control of S. aureus. Treatment with CGN from healthy controls improved outcomes in a mouse model of AD. These findings suggest that a live-biotherapeutic approach may hold promise for treatment of patients with AD.
Tyrosine Phosphorylation of NLRP3 by Lyn Suppresses NLRP3 Inflammasome Activation
Authors: Lin Guoxin , Tang Juan , Guo Hui , Xiao Yun , Gupta Neetu , Tang Na , Zhang Jian
Published: January 2023

The NLRP3 inflammasome is a multi-protein complex that triggers the activation of inflammatory caspase-1 and the maturation of IL-1β and IL-18 in response to microbes and danger signals in the cytosol of host cells. However, how the NLRP3 inflammasome is regulated is not well defined. Here, we showed that NLRP3 undergoes tyrosine phosphorylation in macrophages upon stimulation with ATP, a NLRP3 inflammsome stimulus, and the tyrosine phosphorylation of NLRP3 correlates with its ubiquitination. Pretreating the macrophages with a specific Src kinase inhibitor inhibits NLRP3 tyrosine phosphorylation and ubiquitination, which leads to enhanced production of IL-1β. Further we found that Src family PTK Lyn phosphorylates NLRP3 at Tyr918 which facilitates its ubiquitination and proteasome-mediated degradation. Consistent with these data, NLRP3 tyrosine phosphorylation and ubiquitination is abrogated in macrophages lacking Lyn, which correlates with heightened-activation of NLRP3 inflammasome. Therefore, our data demonstrates that the Lyn-mediated tyrosine phosphorylation of NLRP3 is prerequisite for its ubiquitination, thus dampening the NLRP3 inflammasome activity.
Purified type III secretion system needle proteins are novel TLR agonists that induce NF-κB signaling and stimulate NLRP3-dependent IL-1β production by innate cells in vitro
Authors: Alvine Travis D , Knopick Peter L , Nilles Matthew L , Bradley David S
Published: January 2023

Purified type III secretion (T3S) system needle proteins from a number of Gram-negative bacteria have been identified as protective antigens as well as strong adjuvants in vivo. These proteins have been shown to act as TLR2 and TLR4 agonists, producing robust pro-inflammatory cytokine release in a MyD88-dependant manner from both human and mouse cells in vitro. Additionally, it has been established that TLR agonists alone can stimulate IL-1β through the NLRP3 inflammasome. Interestingly, we have demonstrated that T3S needle proteins induce NF-κB signaling downstream of both TLR2 and TLR4. Given the vaccine potential of T3S needle proteins, it is necessary to further understand the signaling mechanisms underlying their adjuvant properties. Here we characterized the innate signaling pathways engaged by T3S needle proteins. We show T3S needle proteins induce NF-κB signaling and pro-inflammatory cytokine release from mouse macrophages more strongly through TLR4 as compared to TLR2. Utilizing primary cells from inflammasome component knockout mice, we demonstrate that IL-1β secretion from T3S needle protein stimulated cells was dependent on the NLRP3 inflammasome, as well as caspase-1. Interestingly, pre-treatment with cytochalasin D reduced IL-1β secretion and resulted in the accumulation of intracellular pro-IL-1β after T3S needle protein stimulation; suggesting that internalization of T3S needle proteins is critical for inflammasome activation and IL-1β secretion. These findings further identify critical innate signaling pathways engaged by T3S needle proteins. T3S needle proteins have tremendous vaccine potential due to their ability to act as protective antigens and their innate immunity stimulating properties.