bluuebunny/crossref_metadata_2025 huggingface dataset:
Keratinocyte-specific deletion of STAT3 promotes elevated serum IgE in response to Staphylococcus aureus exposure: relevance to hyper-IgE syndrome
Authors: Liu Haiyun , Archer Nathan K , Dillen Carly A , Wang Yu , Ashbaugh Alyssa G , Ortines Roger V , Lee Steven K , Kao Tracy , Miller Lloyd S
Published: January 2023

Autosomal dominant hyper-IgE syndrome is caused by STAT3 loss-of-function mutations, which results in a constellation of clinical features, including highly elevated serum IgE levels, a severe eczema-like skin eruption, and an increased susceptibility to Candida albicans and Staphylococcus aureus skin infections. However, the mechanism by which these individuals have elevated IgE levels is not well understood. To evaluate the role of defective STAT3 signaling in contributing to IgE production, we used a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous exposure to S. aureus in cre/lox mice with cell specific deletion of STAT3. Unexpectedly, STAT3 deletion in keratinocytes (K5-creERT2 × STAT3fl/fl mice) but not in T cells (CD4-creERT2 × STAT3fl/fl mice) resulted in a substantial elevation of serum IgE levels. The increase in serum IgE involved concomitant increase in IgG and IgA levels that were also significantly higher in the keratinocyte-specific STAT3-deficient mice compared with wildtype mice. CD4+ T cells were required for the IgE production, since CD4+ T cell depletion resulted in significantly reduced level of serum IgE in the K5-creERT2 × STAT3fl/fl mice. Taken together, these results indicate that keratinocytes can modulate serum antibody levels, especially in the absence of STAT3 signaling. These results provide an explanation of the increased serum IgE levels in autosomal dominant hyper-IgE syndrome and more broadly into the mechanisms that promote antibody production in response to epicutaneous exposure to microbes and inflammation.
Significance of VIP and VIP receptor interaction in promoting pathogenesis of Eosinophilic esophagitis
Authors: Verma Alok Kumar , Venkateshaiah Sathisha Upparahalli , Manohar Murli , Mishra Anil
Published: January 2023

Vasoactive intestinal peptide (VIP) is shown to have a role in the esophageal sphincter contraction and relaxation, and its role in pathogenesis of eosinophil associated allergic diseases like asthma and rhinitis is reported. Therefore, we tested the hypothesis that VIP may have an important role in the eosinophils accumulation in the esophagus that promote the pathogenesis of eosinophilic esophagitis (EoE). Accordingly, we performed flow cytometer analysis for VIP receptors expression in the blood eosinophils and real time PCR for VIP transcript analysis in the biopsies of human EoE. Herein, we show that human eosinophils highly express VIP associated receptor CRTH2, not VAPC1 or VAPC2. Further, VIP mRNA and protein is induced in esophageal biopsies of human EoE compare to the normal individuals. Additionally, we show that in vitro VIP has a similar chemoattractant activity for eosinophils as eotaxin. Further, we show that in vivo CRTH2 expressed eosinophils accumulated around the VIP producing nerves in the esophageal biopsies of EoE patients. Interestingly, we also observed VIP receptor VPAC2 on mast cells and their accumulation near the nerve cells in EoE biopsies. Taken together, we show the significance of VIP and its receptors interaction in the recruitment of eosinophils and mast cells that may have an important role in promoting chronic EoE pathogenesis in human.
Overexpression of tIK ameliorates DNCB-induced allergic contact dermatitis in BALB/c mice
Authors: Son JeHee , Jang Hyun-Young , Lee Sang-Myeong
Published: January 2023

Allergic contact dermatitis (ACD) is a delayed type hypersensitivity reaction that is mediated by hapten-specific T cells. Dinitrochlorobenzene (DNCB)-induced contact hypersensitivity of the skin is a commonly-used mice model for studying the pathogenesis of contact dermatitis. IK cytokine is known to suppress IFN-γ induced expression of MHC class II molecules on B cells by increasing cAMP. Recently, we reported that truncated IK (tIK) cytokine-expressed transgenic (Tg) mouse ameliorated inflammatory arthritis symptoms by suppressing the differentiation of TH1 and TH17 cells and the activation of macrophages. However, there are still limited studies showing the functionality of this cytokine in inflammatory diseases such as ACD. Here, we investigated whether tIK cytokine expressing Tg mice are less susceptible for DNCB induced ACD. DNCB treatment caused severe dermatitis symptoms in wild type (WT) including weight loss, increased ear thickness and scratching behavior but those symptoms are significantly reduced in tIK Tg mice. The weights of draining lymph node (dLN) and spleen were also much less in tIKTg mice. In addition, tIK overexpression decreased IgE serum level, mRNA transcript of mast cell associated factors in Tg mice compared to WT mice. Analyzing T cell subpopulation in dLN revealed that Th2 and Th17 cell differentiation were suppressed by tIK expression while Tregs and Th1 differentiation were not affected. Taken together, our results presented here suggest that tIK expression effectively attenuate DNCB induced allergic contact dermatitis lesions by modulation Th cell differentiation. Futher studies are needed to elucidate immunological and molecular mechanisms by which tIK functions on this disease model.
Regulation of IL-17-producing subsets of NKT and γδ-T cells by syndecan-1 and relevance to psoriasis
Authors: Jaiswal Anil Kumar , Sadasivam Mohanraj , Archer Nate , Miller Lloyd S , Hamad Abdel Rahim A
Published: January 2023

IL-17 is a potent proinflammatory cytokine that protects the host against extracellular bacteria and fungi and it also drives psoriasis in humans and in the imiquimod-treated mouse model of psoriasiform dermatitis. Although IL-17A contributes to disease pathogenesis in psoriasis as two IL-17A neutralizing mAbs have recently been approved for treatment of moderate to severe psoriasis, less is known about the mechanisms that regulate the numbers of IL-17A-producing cells. Production of IL-17 by innate-like T cells and Th17 cells provides a challenge and an opportunity for identification and selective targeting of appropriate cellular source(s). Here we show that the heparan sulfate proteoglycan syndecan-1 (sdc1) is a major regulator of IL-17A production by γδ-T cells and NKT cells. Sdc1 is selectively expressed on IL-17A-producing subsets of NKT (NKT17) and γδ-T cells in the thymus and in the periphery. Lack of sdc1 significantly increases frequency and absolute numbers of NKT17 and IL-17A+ γδ-T cells in the thymus and the periphery in sdc1 knockout mice. In Sdc1KO B6 mice, a population of IL-17A-producing γδ-T cells are significantly increased in the dermis and epidermis. Using the imiquimod-induced psoriasiform dermatitis model in C57BL/6 mice, we found that imiquimod treatment led to a significantly higher percentage of IL-17A+ γδ-T cells and Th17 cells and more severe skin disease. Insights into the underlying mechanisms and therapeutic implications are being investigated and will be presented at the meeting
Differential role of oxidative stress pathways and microbiota in the development of allergen specific IgE following chronic ingestion of low doses of cadmium
Authors: Kim Eunsoo , Lembert Melanie M. , Opiyo Stephen O. , Ahmer Brian M. , Cormet-Boyaka Estelle , Boyaka Prosper N
Published: January 2023

Chronic ingestion of low doses of cadmium is becoming a major public health issue due to the presence of this heavy metal in contaminated water and its accumulation in leafy vegetables, fish and grains. Environmental pollutants are believed to contribute to the increased incidence of allergy diseases, but underlying mechanisms remain poorly understood. We found that C57BL/6 mice chronically exposed to low doses of cadmium through drinking water develop higher titer of antigen-specific IgE responses upon oral sensitization. Cadmium-treated mice also exhibited higher IL-17 and Th1 responses in the airways and displayed more severe signs of airway allergic responses, upon nasal antigen challenge. Analysis of innate responses in the intestine revealed that ingestion of low doses of cadmium activated both the canonical and the non-canonical NF-κB pathway and promoted proinflammatory cytokine and antimicrobial responses through activation of oxidative signaling pathways including Duox2 and Nrf2. Chronic ingestion of low doses of cadmium also resulted in a dysbiosis of the intestinal microbial community, which persisted long after exposure was interrupted. Germ-free C57BL/6 mice exposed to cadmium and sensitized under the same conditions failed to develop elevated allergen-specific IgE responses. Finally, when transferred into untreated mice, the fecal microbe of cadmium-treated mice increased the allergen-specific IgE responses in the recipients confirming the central role the dysbiosis in the induction of allergic responses. Microbial products mediating the allergy-promoting effect of cadmium are being identified in ongoing studies.
Distinct immune and clinical responses to alcohol-soluble vs. salt-soluble wheat proteins in mice
Authors: Gangur Venugopal , Jin Yining , Ebaugh Sarah , Martens Anna , Olson Eric , Ng Perry KW
Published: January 2023

Food hypersensitivity is a growing global public health problem. Wheat has been linked to distinct types of human hypersensitivities with incompletely understood mechanisms. Although wheat proteins (WP) differ in solubility, it is unknown whether alcohol-soluble (AS) and salt-soluble (SS) WP differ in their immune and disease activation properties. Here we tested the hypothesis that AS and SSWP extracted from soft-wheat will elicit distinct types of immune and clinical responses in inbred mice. Groups of BALB/cJ mice weaned onto a plant-protein free diet received IP injections with AS and SSWP (0.01 mg/mouse) along with alum. The WP-specific (S) IgE, IgG1, IgG2a, IgA and total (T) IgE responses were analyzed. Systemic anaphylaxis and mucosal mast cell degranulation response (MMCR) upon IP challenge were quantified by hypothermia shock response (HSR) and elevation of plasma mMCP-1, respectively. Mice were monitored for dermatitis and alopecia. Spleen tissue extracts were used in measuring immune-activation biomarkers. Whereas the SSWP elicited higher titers of SIgA, the ASWP elicited higher titers of SIgE, SIgG1, SIgG2a and higher TIgE levels. The ASWP elicited significantly higher HSR and MMCR. Only ASWP but not SSWP elicited dorsal neck dermatitis lesions in 44% of mice. Alopecia lesions, without inflammation, were noted in 22% of ASWP- and 33% of SSWP-injected mice. Furthermore, ASWP and SSWP elicited distinct patterns of activation of acute-phase response proteins, chemokines, cytokines, growth factors, matrix proteins and signaling proteins. These data provide the first direct evidence that WP fractions that differ in solubility elicit qualitatively distinct immune and clinical reactions in genetically identical mice.
Tumor-derived indoleamine 2,3- dioxygenase regulates density of tumor infiltrating CD8+ T cells and myeloid derived suppressor cells in a murine model of ovarian cancer
Authors: Amobi Adaobi , Odunsi Kunle
Published: January 2023

The catabolism of the essential amino-acid tryptophan by indoleamine 2,3-dioxygenase (IDO1) is a central pathway that contributes to an immunosuppressive microenvironment in many types of cancer. Our lab has previously shown that IDO1 expression in human ovarian tumor correlates with poor prognosis and poor tumor infiltration by CD8+ T cells, and that improved survival is associated with increased tumor infiltrating lymphocytes. Thus, IDO1 inhibition represents an attractive target for cancer immunotherapy.

To establish the mechanism by which IDO1 inhibition augments immune responses in a murine model of metastatic ovarian cancer, we generated a stable IDO1-overexpressing cell line (IE9mp1-mIDO1) and confirmed functional IDO1 enzyme activity. C57BL/6 mice were challenged intraperitoneally with IE9mp1-mIDO1 or IE9mp1-Empty Vector as a control. IE9mp1-mIDO1 tumor-bearing mice displayed earlier onset of tumor burden and decreased overall survival.

To delineate the role of host- and tumor-derived IDO1 on immune cell infiltration in the tumor, IDO1 genetic knockout and C57BL/6 mice were challenged with IE9mp1-EV or -mIDO1 tumor cells. All mice challenged with IE9mp1-mIDO1 demonstrate decreased CD8+ T cells in the tumor compared to IDO1KO mice challenged with IE9mp1-EV tumor cells. Moreover, tumor-derived IDO1 mediates increased CD11b+Gr1+ myeloid derived suppressor cells (MDSC) in tumor ascites.

From these data, we conclude that regulation of IDO1 will promote anti-tumor immune responses by permitting increased effector T cells in tumor tissues and decrease MDSCs in ascites. Ongoing experiments will characterize the mechanism by which IDO1 inhibition may augment vaccine-induced immune responses in ovarian cancer.
Microbiota metabolites derived from tryptophan modulate macrophage inflammation and migration in non-alcoholic fatty liver disease
Authors: Ding Yufang , Krishnan Smitha , Lee Kyongbum , Jayaraman Arul
Published: January 2023

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in western countries. While it is established that the gut microbiota plays a role in NAFLD, the identities of microbiota metabolites involved and their target pathways in host cells is poorly understood. Based on prior work showing that the microbiota metabolite indole attenuates indicators of inflammation in intestinal epithelial cells, we hypothesized that microbiota metabolites modulate macrophage inflammation in the liver. Mass spectrometry analysis of microbiota metabolites in serum and liver of mice identified tryptamine (TA) and indole-3-acetate (I3A) as depleted in mice on a high-fat diet compared to low-fat diet. Therefore, we investigated the effect of I3A and TA on macrophage inflammation and migration. RAW 264.7 murine macrophages were treated with different concentrations of I3A and TA for 4 hours, stimulated with palmitate for 18 hours followed by LPS for 6 hours in the presence of metabolites. Our results show that treatment with I3A significantly attenuated the induction of the pro-inflammatory cytokines TNFa, IL-1b and MCP-1 by palmitate and LPS in a dose dependent manner at both mRNA and protein levels. Unlike I3A, TA only decreased the gene expression of IL-1b but significantly decreased TNFa and MCP-1 protein levels. Both I3A and TA significantly decreased bone marrow derived macrophage migration towards MCP-1 in a dose dependent manner. Together, our results suggest that microbiota metabolites can modulate NAFLD by directly acting on macrophages. Ongoing work focuses on investigating the mechanisms underlying the anti-inflammatory activity of I3A and TA, which could provide new strategies to treat NAFLD.
Comparison of cytotoxic dynamics between cytokine-induced killer cells and natural killer cells at the single cell level
Authors: Lee Jae Hee , Kim Ji Sung , Lee Hong Kyung , Kim Ki Hun , Choi Jeong Eun , Ji A Young , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Although much has been learned about the cytotoxic mechanisms of cytokine-induced killer (CIK) and natural killer (NK) cells, little is known about how they kill cancer cells at the single-cell level. In the present study, we examined the contact dynamics of CIK and NK cells at the single-cell level by using time-lapse imaging. CIK cells killed MHC-I-negative and -positive cancer cells, but NK cells destroyed MHC-I-negative cells only. Moreover, CIK cells moved in all directions and showed longer tracks than did NK cells. CIK cells showed higher displacement and straightness scores than did NK cells, which indicates long-distance random migration of CIK cells. CIK and NK cells moved at 6.7 mm/min and 4.5 mm/min on average, respectively. These data suggest that CIK cells are likely moving more actively than NK cells. The average threshold number of CIK cells required to kill an individual cancer cell was 6.7 for MHC-I-negative cells and 6.9 for MHC-I-positive cells. That of NK cells was 2.4 for MHC-I-negative cells. Likely due to the higher threshold numbers, killing by CIK cells was delayed in comparison with NK cells: 40% of MHC-negative target cells were killed after 5 h when co-cultured with CIK cells and after 2 h with NK cells. Our data have implications for the rational design of CIK or NK cell–based immunotherapy of cancer patients
A murine model of suicide gene therapy in the brainstem demonstrates efficacy without toxic inflammation, suggesting possible use as a treatment for diffuse intrinsic pontine glioma (DIPG)
Authors: Schuelke Matt , Evgin Laura , Thompson Jill , Daniels David , Rao Amulya Nageswara , Bram Richard J , Kasahara Noriyuki , Vile Richard
Published: January 2023

Diffuse intrinsic pontine glioma (DIPG) is a rare pediatric brainstem tumor that is the leading cause of death among pediatric brain malignancies. Despite over 200 clinical trials, no significant progress has been made, leading our lab to consider suicide gene therapy. The brainstem has particularly sensitive anatomy, creating the potential for toxic inflammation. Furthermore, the clinical standard of care for DIPG includes conventional radiation therapy (RT) followed by dexamethasone (DEX), each of which can alter the efficacy of gene therapy. In this study we found that a variety of gliomas, including human DIPG and murine glioma cell lines, are infectable in vitro with a replication-competent Moloney murine leukemia virus (MoMLV) (currently in Phase 2/3 clinical trials) and susceptible to ganciclovir (GCV)-mediated killing via the HSV thymidine kinase (tk) suicide gene. A B16 melanoma cell line expressing HSV tk implanted into the brainstem of mice, extended median survival with GCV therapy from 13 days to 38.5 days (p=.0006) with no signs of toxic inflammation. Pre-treatment with RT and DEX did not affect therapeutic efficacy. We have shown that GCV/TK therapy is mediated by NK and CD8 cells in subcutaneous tumors, and we are investigating whether a similar immunologic mechanism is involved in this brainstem model. These preclinical data will pave the way for novel clinical trials for the treatment of DIPG.
Granzyme B: a biomarker of influenza infection in older adults
Authors: Merani Shahzma , McNeil Shelly , McElhaney Janet E
Published: January 2023

The importance of a T-cell mediated response against influenza infection in older adults has growing support. It has been previously shown that effector cells in older adults challenged with influenza virus produce granzyme B (GrB) in the absence of perforin. This lack of perforin could impair CTL directed killing and result in high extracellular GrB levels.

GrB has been associated with various inflammatory and age related conditions and could also play an important role in influenza infection severity. We postulate that GrzB is a key mediator of inflammatory processes involved in severe influenza illness and could be used as a marker of influenza infection in older adults.

Subjects over the age of 65 admitted to hospital with influenza like symptoms were recruited and grouped into cases of laboratory confirmed influenza illness (LCII) and influenza like illness (ILI). PBMCs were isolated from blood samples collected from participants on hospital admission and one month post-discharge. PBMCs were challenge with live influenza virus. Real-time PCR was used to quantify IFNγ, GrB and IL10 in PBMCs and isolated T-cells.

We found that in vivo GrB levels in T-cells at the time of influenza infection in older adults were significantly higher in the LCII than the ILI group. Furthermore, ex vivo influenza virus challenge resulted in higher IFNγ, GrB and IL10 responses in subjects with LCII than ILI.

Our study supports the association of elevated GrB and influenza infection status as well as the importance of T-cell mediated responses to infection. Future work will characterize GrB levels in older adults with varying severities of influenza illness with the aim to develop a point of care test to identify patients likely to develop serious outcomes.
Combination treatment with erlotinib and ampelopsin overcomes erlotinib resistance in NSCLC cells via the Nox2-ROS-Bim pathway
Authors: Noh Minhye , Hong Seung-Woo , Park Nam-Sook , Shim Ju A , Ahn Byul-Nim , Kim Yeong Seok , Kim Daejin , Lee Hyun-Kyung , Hur Dae Young
Published: January 2023

Erlotinib, a tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR), has been shown to have a dramatic effect in non-small cell lung cancer (NSCLC) patients with EGFR mutation. However, the presence of primary resistance or acquired resistance to EGFR-TKI is the most common reason for switching to other anti-cancer agents. Even though there are newer agents that have activity in the presence of the T790M mutation, identification of potential agents that could overcome resistance to EGFR-TKI is still needed for the treatment of NSCLC patients. In this study, we used erlotinib-resistant NSCLC cell lines to investigate the effects of combination treatment with erlotinib and ampelopsin. To determine the effect of Nox2 and Bim on the combined treatment with erlotinib and ampelopsin-induced cell death, we transfected with Nox2 or Bim specific siRNA and performed with western blot assay for evaluation of its expression. Combined treatment with erlotinib and ampelopsin at non-cytotoxic concentrations significantly induced caspase-dependent cell death in erlotinib-resistant NSCLC cells. Furthermore, cell death resulted in the accumulation of reactive oxygen species (ROS) through upregulation of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) expression, a direct source of ROS. The expression level of Bim increased with combination treatment, but not with either treatment alone. Here in this study, we demonstrate that the combination of erlotinib and ampelopsin induces cell death via the Nox2-ROS-Bim pathway, and ampelopsin could be used as a novel anti-cancer agent combined with EGFR-TKI to overcome resistance to erlotinib in EGFR-mutant NSCLC.
Radiation improved IL-21 cancer immunotherapy
Authors: Lee I-Jung , Wu Chia-Jen , Wu Ping-Yi , Tao Mi-Hua
Published: January 2023

Interleukin-21 is a pleiotropic cytokine which has shown promising antitumor activities in several preclinical models but the response rate in phase I–II clinical studies has been disappointing. Therefore, the optimal usage of IL-21 as an effective therapy against cancers remains to be studied. Radiotherapy, a well establish treatment modality for cancers, has shown its potential in immunogenic modulation by stimulating immune cell phagocytosis and inducing local tumor-associated antigens releasing. We hypothesize that a synergistic antitumor effect could be achieved by combining IL-21 with radiation through releasing neoantigens and amplifying adaptive antitumor immune responses against both primary and metastatic tumors. We treated mice bearing large established tumors with radiation, IL-21 or a combination of radiation and IL-21. Mice treated with radiation alone showed delayed tumor growth, while IL-21 treatment had very little effect. In contrast, a synergistic antitumor effect was achieved in the combination group, which not only led to complete tumor regression of primary tumors, but also significantly suppressed the growth of distant, untreated metastatic tumors. Mechanistic analysis revealed that combination therapy increased accumulation of tumor-infiltrating CD8+ T cells which showed enhanced functional activities, marked by increased expression of CD107a, IFN-γ and TNF-α. Moreover, mice that had been cured by the combination therapy developed memory responses against the subsequent tumor re-challenge. Our results demonstrate that combination of IL-21 and radiation has potent antitumor activity against both primary and metastatic tumors and could open a new perspective for cancer therapy.
Generation of

I

mmuno-

M

odulator

R

eceptors (IMR)-transduced human JY cell lines to test combo immuno-therapies.
Authors: Le Saux Sabine , Li Sophie , Shields Robert , Nourzaie Omar , Ugarte Fernando , De Waal Malefyt Rene
Published: January 2023

T cell activation is initiated by signal 1 through an antigen–specific TCR recognition and engagement, then a second co-stimulation signal is needed. This co-stimulation is provided by the interaction of IMR receptors and ligands expressed on the T cells and APC or tumor cells. These receptor-ligand interactions can either stimulate or inhibit T cell activation and they are the main targets for the new therapeutics developed in the oncology field.

The establishment of Ag-specific human T cell clones is essential to study IMR biology and even more to test different biologics targeting those molecules. In our laboratory, we developed primary-cell based assays to test such biologics. In those assays allo-Ag-specific human CD4+ T cell clones are activated by HLA-DR-positive JY cells, a human B cell line, to proliferate and to produce IFNγ. To analyze the efficacy of PD-1/Lag3 and PD-1/TIGIT biologics, we generated by lentivirus transduction, different JY cell line clones expressing PD-L1, CD155 or PD-L1/CD155. The JY clones isolated after infection showed a stable expression of the IMRs and they modulated activation of the CD4+ T cell clones.
Tumor Control Index: a novel tool to assess and compare tumor growth in experimental animals
Authors: Ebrahimi-Nik Hakimeh , Corwin William L. , Floyd Stephanie M. , Tavousi Pouya , Mandoiu Ion I , Srivastava Pramod K
Published: January 2023

Measurement of tumor diameters, tumor volumes, or area under the curve has been traditionally used to quantitate the effects of experimental manipulations in immune competent as well as immune-compromised mice and rats. However, using these parameters to measure the kinetics of tumor growth fail to reveal several complexities of growing tumors. Here, by means of tumor growth data from a large number of mice challenged with live tumor cells, we describe the use of a new composite parameter, Tumor Control Index (TCI) as an alternative method to do the same. This index, comprised of three distinct values, the Tumor Progression Score, Tumor Rejection Score, and Tumor Stability Score, provides a complete picture of nearly every aspect of tumor growth in large numbers of animals, can be deduced automatically from tumor diameter or volume data, and can be used to compare several groups of animals in different experiments. This automatically derivable index also corresponds neatly to the use of complete and partial responses and tumor stability data generated in human tumors, and can be used to assess the efficacy of interventions to be used in clinical studies.
Obesity-induced changes in baseline immune responses to pre-clinical breast cancer
Authors: Gibson Justin Tyler , Nagareddy Prabhakara , Norian Lyse A
Published: January 2023

Obesity is known to increase morbidity and mortality in breast cancer patients; however, the immunological mechanisms behind these changes remain unclear. The long-term goal of our study is to investigate the broad impact of obesity on anti-tumor immunity, with a particular focus on better understanding immune alterations that may influence the use of immunotherapeutics in breast cancer patients. We began by utilizing the leptin-deficient ob/ob mouse model of obesity and non-obese age matched C57BL/6 controls, in combination with the syngeneic E0771 mammary carcinoma cell line. Tumor outgrowth was quantified by means of caliper measurements, endpoint tumor weight, and bioluminescent imaging (BLI) via luciferase-expressing E0771 (E0771-fLUC) cells; immune cell populations were evaluated via multi-parameter flow cytometry. Primary and metastatic tumor growth were not significantly different between lean and ob/ob mice. In primary tumors, we found that obesity caused significant decreases in the frequencies of tumor-infiltrating granulocytic myeloid derived suppressor cells (G-MDSCs) and CD19+ B cells, and trending decreases in the frequencies of T (CD3+, CD4+, and CD8+) cells. In spleens, we found that both obesity and breast cancer induced a significant decrease in the frequency of dendritic and natural killer (NK) cell populations. Additionally, obesity induced a significant increase in the frequency of splenic monocytic myeloid-derived suppressor cells (M-MDSCs). Our findings suggest several cellular candidates for future mechanistic investigations into obesity-induced dysfunctions in breast cancer immunity.
Melittin suppresses tumor progression by regulating tumor-associated macrophages in a Lewis lung carcinoma mouse model
Authors: Lee Chanju , Choi Dabin , Lee Sujin , Bae Sung-joo S. , Joo Hwansoo , Bae Hyunsu
Published: January 2023

Tumor-associated macrophages (TAM) are a major component of tumor stroma. It has been reported that TAMs have M2-like phenotype and facilitate tumor progression by promoting angiogenesis and immunosuppression. Melittin, a major polypeptide of bee venom, has been widely studied as an anti-cancer drug due to its cytotoxicity to malignant cells. However, very little is known regarding the effect of melittin on immune cells in the tumor microenvironment. We demonstrated here that melittin inhibited the rapid tumor growth compared to the control in vivo. Melittin increased the M1/M2 ratio of TAMs by selectively reducing the number of CD206+ M2-like TAMs, while not affecting the number of CD86+ M1-like TAMs. Melittin also preferentially bound to CD11b+ cells and this binding was not associated with phagocytosis. Gene expression of vascular endothelial growth factor (Vegf) and mannose receptor C type 1 (Mrc1/CD206) was reduced in M2-like bone marrow-derived macrophages by melittin treatment, but there was no significant change in the gene level of Vegf and FMS-like tyrosine kinase 1 (Flt1/VEGFR1) in tumor cells. Additionally, the levels of VEGF and CD31, markers of angiogenesis, were significantly decreased by melittin treatment in tumor tissues. This study revealed a novel role for melittin in tumor treatment and suggested that melittin could be a promising therapeutic agent for targeting M2-like TAMs.
Role of macrophages in the therapeutic effect of Regulatory T Cell ablation in breast cancer
Authors: Clark Nicholas M , Martínez Leandro M , Effi Comfort C , Bos Paula D
Published: January 2023

Tumor progression ensues when cancer cells escape the control of immune system. An important tumor immune evasion mechanism is the recruitment of regulatory T (Treg) cells. Treg cells, characterized by the expression of the transcription factor Foxp3, comprise a large proportion of the T lymphocyte population infiltrating mouse and human tumors. Treg cells correlate with poor prognosis in various tumors, such as breast carcinomas. Using the MMTV-PyMT-driven model of murine mammary gland cancer, our lab has demonstrated that short-term Treg cell ablation provides a significant therapeutic effect in vivo. Importantly, this effect does not require cytotoxic CD8+ and NK+ cells, but is dependent on INFg. The goal of this study is to identify the effector cell type responsible for this therapeutic effect, and characterize the mechanisms by which they lead to tumor growth inhibition. We discovered that bone marrow-derived inflammatory monocytes and macrophages play a critical role in the anti-tumorigenic effect observed after Treg cell ablation by adopting a pro-inflammatory phenotype, leading to increased tumor cell killing. Ongoing studies are addressing the role of INFg on this Treg cell induced phenotypic plasticity. Together, these results demonstrate an essential role for myeloid cells in the anti-tumor effect observed upon Treg cell ablation. Our studies have the potential to reveal promising targets for therapeutic intervention.
Myeloid-derived suppressor cell expansion in patients with trans-arterial chemoembolization history
Authors: Thevenot Paul T , Núñez Kelley , Gonzalez-Rosario Janet , Sandow Tyler , Galliano Gretchen , Wyczechowska Dorota Danuta , Ochoa Augusto C , Cohen Ari
Published: January 2023

Cancer-specific myelopoiesis causes peripheral myeloid cell expansion and accumulation at the tumor. Myeloid-derived factors negatively regulate anti-tumor immunity. Doxorubicin-eluting bead transarterial chemoembolization (DEB-TACE) can bridge hepatocellular carcinoma (HCC) patients with low stage tumors to liver transplantation (LTx). Unfortunately, HCC recurrence rates remain high, with 5yr tumor-free survival rates estimated at ~50%. Though myeloid-derived suppressor cell (MDSC) expansion has been associated with tumor progression and recurrence risk, the effect of DEB-TACE on MDSC levels is unclear. We observed DEB-TACE HCC patients with disease progression have elevated TGF-β and IL-6 with increased MDSCs at followup. In this study, we prospectively enrolled LTx-candidate HCC patients undergoing DEB-TACE treatment to track MDSCs before and after DEB-TACE treatment based on de novo (DN) verses prior embolization (PE) history. Blood was collected before and < 30 days after DEB-TACE (100–300μm LC Beads™ mixed with 50–75mg doxorubicin). Analyzed were 18 DN patients and 16 PE patients. Tumor diameter and TGF-β levels prior to treatment were similar. CD33+HLA-DRLOW MDSCs were elevated in PE patients both prior to (p<0.05) and following DEB-TACE (p<0.05), but not the number of prior DEB-TACE treatments or radiological treatment response. PE patients with elevated MDSCs (>10%) had elevated TGF-β levels compared to PE patients with <10% MDSCs (p<0.01). PE patients with >10% MDSC also had elevations in IL-6 (p<0.01) and the HCC-recurrence associated MMP-2 (p<0.05). MDSC expansion and elevated TGF-β in HCC patients with DEB-TACE history may identify patients at high risk of early HCC recurrence following LTx.
Non-oncogenic Acute Viral Infection Reduces Tumor Growth in Hosts with Established Cancer
Authors: Newman Jenna H , Li Shengguo , Chesson Brent , Schenkel Jason M , Silk Ann , Zloza Andrew
Published: January 2023

The ability of the immune system to recognize cancer cells as abnormal and to enact effector activity to destroy such cells is a delicate maneuver dependent upon multiple factors. Cancer patients exhibit higher rates of infection than the general population, prompting the question of whether non-oncogenic acute infections impact the ability of patients to mount an anti-tumor immune response, potentially affecting tumor growth and survival outcomes. To assess this, we challenged C57BL/6 mice with B16-F10 melanoma and A/H1N1/PR8 influenza. Influenza was administered upon development of a palpable tumor. We observed that mice bearing an established melanoma that were infected with influenza exhibited reduced tumor growth as compared to mice bearing melanoma alone. Tissues in tumor-bearing mice were interrogated via flow cytometry and a 9-fold reduction in myeloid-derived suppressor cells (MDSCs) was observed in mice concomitantly bearing melanoma and infected with influenza, relative to that observed in the mice bearing melanoma alone. These findings are in sharp contrast to our previous report of increased cancer incidence in cancer-prone mice infected with influenza prior to tumor emergence. These findings suggest that non-oncogenic acute viral infection may play a dual role (increasing the emergence of immune-controlled cancer but decreasing the progression of established cancer). Further, our findings suggest that viral infection in cancer-bearing hosts can be utilized as an immunotherapy for cancer. Future studies will focus on elucidating both the immune-related and cancer cell-related mechanisms that lead to altered tumor growth and survival outcomes in cancer-bearing hosts in the context of viral infection.
Development of an Ex Vivo 3D Functional Assay for Novel Drug Discoveries
Authors: Murali Vasanth Siruvallur , Danuser Gaudenz , Cobanoglu Murat Can
Published: January 2023

Cancer drug discovery is in crisis since only 5.1% of the total drugs entering Phase 1 clinical trials succeed to approval. A reason for the low success rate in clinical trials lies in the initial steps of drug screening using 2D cell cultures and use of immunocompromised mice. Cancer is not just a malignant outgrowth of cells, rather a complex malignancy where the clinical outcome is determined through crosstalk between the malignant, stromal, and immune cells. Furthermore the extracellular matrix is an integral and dynamic part of this crosstalk; examples include TAMs remodeling the ECM and the ECM in turn determining the invasiveness of the malignant cells. since ECM has been shown to be affected by these cells. Thus, a system needs to be designed that introduces all these factors into a drug discovery platform. My aim is to develop an ex vivo assay with humanized synthetic ECM embedded with patient tumor tissue and co-cultured with stromal cells, fibroblast and immune cells isolated from patient. To validate clinical relevance of this complex environment functional assays would be performed to compare drugs response between patients and patient derived tumor. Drug responses on malignant cells will be assessed by 3D high-resolution imaging and computational image morphometrics. My long term goal is to develop a medium throughput novel drug discovery platform that would be guided by machine learning to identify novel chemical compounds.
How do obesity and related inflammation decrease antibody responses in aging?
Authors: Blomberg Bonnie , Diaz Alain , Romero Maria , Vazquez Thomas , Frasca Daniela
Published: January 2023

Decreases in the immune response with aging, including B lymphocytes and antibody-secreting (ASC), are a major contributor to mortality and morbidity in the elderly with increased infections, cancer and autoimmunity. We have shown that the age-related decrease in B cell function is associated with chronic low-grade inflammation and here show is also associated with an increase in fat, visceral adipose tissue (VAT). Adipocyte-derived molecules may not only recruit immune cells but also contribute to the inflammatory process.

We hypothesize that the VAT is an important generator of inflammatory B (and T) cells which contributes to the dysfunction of the aged immune system. Our data in mice show infiltrating immune cells in the VAT have higher percentages of pro-inflammatory B cells (age-associated B cells, ABC) and less Follicular (FO) B cells than in the spleen. The presence of adipocyte inflammatory chemokines and VAT B cell ligands suggest B cell attraction to the VAT and a mechanism for increased pro-inflammatory B cell subsets there. VAT B cells express higher pro-adipogenic and inflammatory markers, including NF-kB, as compared to splenic B cells, and also secrete IgG2c antibodies, which have been shown to be autoimmune in other studies. We also found that in the presence of an adipocyte-conditioned medium, FO B cells differentiated into ABC. These results are the first to show a direct effect of adipocytes on the generation of pro-inflammatory B cells and should help to determine mechanisms for obesity-and age-related changes in inflammation and the immune system.
Lipocalin2, an innate immune protein, modulates iNOS and arginase-1 in macrophages to promote the anti-inflammatory function
Authors: Patterson Daryllynn C , Ignatowicz Aleksandra P , Samuels Janaiya S , Rayalam Srujana , Shashidharamurthy Rangaiah
Published: January 2023

Lipocalin 2 (Lcn2), an innate immune protein, is known to be dramatically up regulated in various inflammatory disorders including autoimmune arthritis, yet its biological role remains unclear. Recently our studies have shown an increase in the accumulation of macrophages in Lcn2 deficient mice compared to wild type (WT) mice in a serum transfer arthritic mouse model. Thus, indicating that the upregulation of Lcn2 might be necessary for the initiation and further resolution of inflammatory processes. In this study, we have investigated the anti-inflammatory function of Lcn2 using murine mouse macrophages. RAW 264.7 cells treated with INFγ and LPS both exhibited pro-inflammatory phenotype, M1, while cells treated with IL-4 displayed an anti-inflammatory phenotype, M2. To determine the anti-inflammatory effects of Lcn2 an IL-10 ELISA revealed that M2 macrophages in the presence of recombinant Lcn2 (rLcn2) significantly increased the expression of IL-10. In addition, arginase-1 expression was found to be significantly upregulated in M2 polarized macrophages in the presence of rLcn2 while inducible nitric oxide synthase (iNOS) expression was down-regulated in the M1 phenotype. However, RAW 264.7 cells treated with Lcn2 alone did not exert either pro- or anti-inflammatory effect. In conclusion, Lcn2 displays the ability to significantly increase the release of anti-inflammatory cytokine, IL-10, thus promoting the anti-inflammatory properties of M2 macrophages. Collectively, our data suggests a crucial role of Lcn2 in the resolution of arthritic inflammation via macrophage polarization, making it a promising target in designing better therapeutic strategies to control the ongoing inflammation during autoimmune arthritis.
β-hydroxybutyrate deactivates neutrophil NLRP3 inflammasome to relieve gout flares
Authors: Goldberg Emily Lauren , Asher Jennifer L , Molony Ryan , Shaw Albert , Zeiss Caroline , Wang Chao , Morozova-Roche Ludmilla A , Herzog Raimund I , Iwasaki Akiko , Dixit Vishwa Deep
Published: January 2023

Aging and lipotoxicity are two major risk factors for gout that are linked by the activation of the NLRP3 inflammasome. IL-1β produced by macrophages and neutrophils drives gouty flares that cause joint destruction, intense pain and fever. Small clinical trials have reported that IL-1R antagonists reduced pain in gout patients, but these biologicals are expensive and may compromise immune responses. Moreover, current treatment strategies for gout are limited in efficacy and do not specifically target the NLRP3 inflammasome. NLRP3 activation is regulated by numerous metabolic byproducts. However, metabolites that impact neutrophil inflammasome remain unknown. We identified that ketogenic diet (KD) increases beta-hydroxybutyrate (BHB) and alleviates urate crystal-induced gout without impairing immune-defense against bacterial infection. BHB inhibited NLRP3 inflammasome in S100A9 fibril-primed and urate crystal activated macrophages which serve to recruit inflammatory neutrophils in joints. Consistent with reduced gouty flares in outbred rats fed a ketogenic diet, BHB blocked NLRP3-dependent IL-1β secretion from neutrophils from both mice and humans, irrespective of age. Mechanistically, BHB inhibited the NLRP3 inflammasome in neutrophils by reducing priming and assembly steps. Collectively, our studies show that BHB, a known alternate metabolic fuel is also an anti-inflammatory molecule that may serve as a treatment for gout.
MSP-dependent Ron receptor tyrosine kinase signaling reduces white adipose tissue expansion and suppresses hepatic lipid accumulation through an LXR-dependent pathway in ApoE−/− mice fed a high fat/high cholesterol diet
Authors: Allen Joselyn Natasha , Dey Adwitia , Zhang Jingtao , Zhang Limin , Fraser James , Kennett Mary , Patterson Andrew , Hankey-Giblin Pamela
Published: January 2023

Non-alcoholic fatty liver disease (NAFLD) has become a global burden and the pathogenic mechanism is not well understood. The current study was undertaken to investigate a novel signaling pathway involved in attenuating the progression of NAFLD. To establish the role of Ron receptor signaling in NAFLD, Apolipoprotein E knockout (ApoE KO) and Ron receptor ApoE double knockout (DKO) mice maintained on a high fat/high cholesterol diet (HFHCD) for 18 weeks were evaluated. DKO mice showed increased epididymal white adipose tissue (eWAT) mass and type 2 diabetes. Gene expression profiling revealed that DKO eWAT had increased transcript expression of pro-inflammatory cytokines and hypoxia-regulated genes. 1H NMR analysis of blood serum from the HFHCD-fed animals demonstrated that DKO mice had higher levels of circulating free fatty acids which are known to trigger hepatocellular injury and the development of steatohepatitis. DKO mice demonstrated increased levels of circulating ALT and AST liver enzymes, which are key markers for liver injury. Additionally, livers obtained from DKO mice demonstrated increased hepatic lipid accumulation. Genes involved in fatty acid synthesis (srebp-1c, fas), reverse cholesterol transport (abca1), and bile acid synthesis (cyp7a1) were repressed in DKO mice which are all target genes of the liver X receptor (LXR) pathway. Lxr gene expression was also increased in HFHCD-fed ApoE KO mice. Furthermore, ApoE KO mice showed higher circulating levels of triglycerides. Altogether, Ron receptor signaling attenuates white adipose tissue dysfunction and provides insight into a possible mechanism underlying the ability of Ron to suppress hepatic lipid accumulation in mice challenged with dietary cholesterol.
Myeloid sirtuin 6 deletion delays wound healing in mice by modulating inflammation and macrophage phenotype
Authors: Koo Jeung-Hyun , Lee Youngyi , Yun Seok-Kweon , Park Byung-Hyun , Jang Hyun-Young
Published: January 2023

It has been reported that loss of sirtuin 6 (Sirt6) in skin wounds exhibit impaired healing. Given the heterogeneity of macrophage phenotypes during wound repair and the regulation of macrophage phenotypes by sirtuins, we here hypothesized that Sirt6 deletion in myeloid cells would affect macrophages phenotypes in skin wounds and delay wound closure. To address this question, floxed Sirt6 mice were bred with lysozyme M-Cre mice to generate myeloid Sirt6 knockout mice with reduced Sirt6 expression. A full-thickness excisional lesion was made on the dorsal skin of the mice. Compared with wild type mice, wound closure was delayed in KO mice with less collagen deposition, attenuated vWF expression and reduced expression of wound healing related genes. Using immunohistochemistry and RNA expression analyses on macrophage subpopulations from wound tissues, we identified an increased infiltration of M1 type macrophages in KO mice compared with wild type mice. Consistent with the in vivo defects in wound closure, the in vitro study demonstrated that keratinocytes and fibroblasts treated with KO macrophage conditioned medium migrated slower than those of wild type. These results suggest that an imbalance of macrophage phenotypes by Sirt6 deletion contributes to impaired wound healing in mice.
Circadian rhythm disruption promotes alcohol-induced liver and brain injury.
Authors: Fey Holger , Vogle Alyx , Grumish Eve , Dodiya Hemraj , Voigt Robin , Aloman Costica
Published: January 2023

Alcoholic liver disease (ALD) progression to cirrhosis and development of hepatic encephalopathy (HE) are characterized by hepatic and brain inflammation involving innate immune cells. Identifying the risk factors and immune mechanisms involved in susceptibility to liver and brain injury are significant unmet needs. Circadian rhythm disruption promotes alcohol-induced liver pathology. We aim to investigate whether circadian rhythm disruption also primes brain inflammation by impacting trafficking and inflammatory potential of recruited and resident mononuclear phagocytes (MP).

Alcohol (EtOH) is delivered to C57BL/6 mice per a modified NIAAA model of alcoholic liver disease whereby chronic alcohol consumption (20% EtOH v/v in drinking water, 4 wks (Meadows-Cook diet)) is combined with three once daily binges of alcohol. Tissue was assessed via H&E and IHC for α-smooth muscle actin (αSMA) and collagen I (Col I) (liver) and allograft inflammatory factor 1 (Iba-1) (brain). All features associated with human alcoholic liver injury including steatosis, hepatic inflammation and early fibrogenesis were present. Brain mononuclear phagocytes analysed by flow cytometry revealed an increase in recruited and resident MP in response to the alcohol treatment. Moreover, in the steady-state, hepatic and brain MP populations demonstrate circadian oscillations.

Thus, circadian rhythm disruption may modulate alcohol-induced alterations in mononuclear phagocyte trafficking and function similarly in brain and liver.