bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effects of Heat-killed Lactobacillus sakei CVL-001 on DSS-induced ulcerative colitis in mice
Authors: Kim Dong-Yeon , Lee Tae-Sung , Jung Do-Hyeon , Kim Yeong-Jun , Lee Yun-Ji , Seo In-Su , Song Eun-Jung , Jang Ah-Ra , Park Jong-Hwan
Published: August 2023

Recently, the incidence of Inflammatory Bowel Disease (IBD) is increasing due to westernized lifestyle and eating habits. IBD is a representative gastrointestinal immune disease, and casued by an excessive immune response in the gastrointestinal tract. In recent studies, lactic acid bacteria which known as beneficial intestinal microbiota have protective effects on IBD by various mechanisms. Here, we evaluated the protective effect of Lactobacillus sakei CVL-001 isolated from korean tradtional food kimchi against dextran sulfate sodium-induced colitis in mice. First, administration of Heat-killed (HK) CVL-001 showed protective effects against DSS-induced colitis. Also, colonic mRNA expression levels showed that pro-inflammatory cytokine was downlregulated and anti-inflammatory cytokine was upregulated. Nucleotide-oligomerization domain 2 (NOD2) is well known as innate immune receptor and deeply related to IBD. We found that HK CVL-001 induced a NOD2-dependent regulatory phenotype in mouse bone marrow derived dendritic cell (BMDCs). Especially, we demonstrated that the expression of CD103, which is known as a marker of tolerogenic DC, was expressed mediated by NOD2. In vivo studies, colon administered with HK CVL-001 favoured the expansion of mucosal CD103 DCs and regulatory T cells. However, protective effect was abolished in NOD2 knockout mice. This results indicated that Heat killed L. sakei CVL-001 might be used therapeutic agent and strategy in IBD.

This research was supported by the Mid-Career Researcher Programs (Grant No. 2022R1A2C2012287) of the National Research Foundation of Korea funded by the Ministry of Science and ICT (Information and Communication Technologies).
Akkermansia muciniphila acts synergistically with Btk-deficiency to prevent Type 1 Diabetes
Authors: Verma Sonam , Cohen Lucy S , Olin Ilana Rae , Kendall Peggy L
Published: August 2023

Btk-deficiency eliminates autoreactive B cells, but not normal ones, and protects against Type 1 diabetes (T1D). Surprisingly, we recently found that rederivation of Btk−/−/NOD mice into a cleaner, “barrier” facility resulted in loss of disease protection. This suggests that T1D protection is microbiome dependent in Btk−/− mice. Akkermansia muciniphila (A. muciniphila) was found to be increased in Btk−/−/NOD mice that were protected against disease compared to WT and Btk−/−/NOD that were not protected. A. muciniphila has previously been associated with disease protection in humans and mice under some conditions, although causality and mechanisms are unknown. We previously showed that Btk-deficient K/BxN mice have reduced intestinal IgA, and therefore hypothesized that A. muciniphila escapes suboptimal IgA-coating in Btk−/−/NOD and works synergistically with the loss of autoreactive B cells to protect against T1D. Germ-free female WT NOD and Btk−/−/NOD were gavaged with A. muciniphila at 3–4 weeks. Mice were tested weekly for diabetes to 30 weeks of age. Additional mice were treated in parallel and euthanized at 9–10 weeks to examine the effects on mucosal immunity and autoimmune attack on pancreatic islets. Monocolonization of Btk−/−/NOD with A. muciniphila protected significantly against T1D compared to monocolonized WT NOD. CD19+ GL7+ staining of Peyer’s patches showed small germinal centers in Btk−/−/NOD compared to WT NOD. Thus, A. muciniphila protects against T1D in Btk−/−/NOD but not WT NOD, indicating a synergistic effect. Altogether, the data show that Btk plays a previously unrecognized role in the maintenance of gut health that can have downstream effects on T1D outcomes.

National Institutes of Health Grants R01-DK-084246I Veteran’s Affairs I01-BX-002882 Washington University School of Medicine
Intestinal and skin colonization by Candida albicans contributes to disrupted regulatory T cells in early life
Authors: Greenfield Kara G , Joshi Avni , Knoop Kathryn A
Published: August 2023

Perturbations of the mucosal immune system and the microbiota can lead to allergic disorders with a lifelong impact. With up to 90% of cases diagnosed by age 6, atopic dermatitis represents a unique allergic disorder during early life. Diagnosis of atopic dermatitis is associated with an increased risk for other allergic disorders throughout life. The opportunistic fungal species Candida albicans is often found as part of a dysbiotic microbiota and its presence is associated with increased risk of atopy. While adult mice are unable to be stably colonized with C. albicans without the use of antibiotics, we have found that mice exposed to C. albicans prior to weaning maintain long term colonization in the gut and on the skin. In mice colonized in early life with C. albicans in the GI tract, FoxP3+ T cells were significantly decreased post-weaning in the mesenteric lymph node as well as in the small intestine and colon lamina propria. Colonization by C. albicans resulted in an increased Th17 response, with increased RORγt+ and c-Maf+ T cells in the colon lamina propria. In a clinical observation study, we found C. albicans colonization in the intestine of prematurely born infants, suggesting colonization of the human GI tract with C. albicans may frequently occur. As the microbiota develops with age, the presence of C. albicans may impact the normal course of increasing complexity and diversity in this ecosystem. The establishment of regulatory T cells in early life is vital to maintaining tolerance to commensal organisms throughout life; this disruption in regulatory T cells potentially links colonization by C. albicans to the development of atopy and allergic responses.

Supported by grants from Mayo Clinic Center for Individualized Medicine
Progressive accumulation of hyperinflammatory NKG2Dlow NK cells in early life defines a novel endotype of severe atopic dermatitis
Authors: Waggoner Stephen N , Ohayon David E , DeVore Stanley B , Chang Wan Chi , Krishnamurthy Durga , Seelamneni Harsha , Grashel Brittany , Spagna Daniel , Martin Lisa J , Biagini Jocelyn M , Khurana Hershey Gurjit K
Published: August 2023

Atopic dermatitis (AD) is a chronic inflammatory skin disease that often precedes the development of food allergy, asthma, and allergic rhinitis. The prevailing paradigm holds that a reduced frequency and function of natural killer (NK) cell contributes to AD pathogenesis, yet the underlying mechanisms and contributions of NK cells to allergic co-morbidities remain ill-defined. Herein, longitudinal analysis of circulating NK cells in a well-defined early life cohort of children with AD revealed a progressive accumulation of NK cells with low expression of the activating receptor NKG2D that was linked to more severe AD and sensitivity to allergens. This was most notable in children co-sensitized to food and aero allergens, a risk factor for development of asthma. Importantly, low expression of NKG2D on NK cells was paradoxically associated with depressed cytolytic function but exaggerated release of the proinflammatory cytokine TNF-a. These observations provide important new insights into a potential pathophysiological mechanism of atopic march involving altered NK cell functional responses and define a novel endotype of severe AD.

5U19AI70235 (GKKH, JBM, LJM); American Heart Association (DEO); R01AI148080, R01AR073228 (SNW), 5T32GM063483 (SBD)
Trends in current students learning-unknown unknowns!
Authors: Fazal Nadeem , Graefen Bahar , Aliyeva Leyla , Uzoma Emeakoroha , Arceo Adriel , Loya Egwin , Najafova Nurana
Published: August 2023

Never in the history of academics there has been such a wave of uncertainty, unpredictability, unrest, and unforeseeable challenges in learning methods adopted by academic departments. The traditional big barrier between instructor and students is becoming arbitrary. At one end instructors are experimenting all the modern tools and gadgets on the other end the students are responding with such a prominent variability making the statistical methods unreliable. Instructors are trying a plethora of modern available learning methods within a very short period of time and are equally met with a challenging students’ learning styles. The current teaching environment with millennial, generation x and z are further complicating the equation. We at Chicago State University College of Pharmacy surveyed both instructors and students over the last four years. Our hypothesis was that the academic stability and standardization has lost its value with frequent and untimely use of tsunami of new learning methods. Data was analyzed from two major departments of College of Pharmacy including basic sciences and clinical practice. This includes data of students from year one pharmacy to year four pharmacy. Our results suggest that instructors are becoming impulsive in changing their teaching methods without giving each method a deserved testing time and evaluation period. Likewise the students are becoming more self-directed, and are taking their learning in their own hands. This see-saw type of educational environment is taking root in academic settings and adding to frustrations and burnouts, both for Instructors as well as the students. We suggest that the instructors should bring the changes in their teaching styles in a slow, perpetual and steady way.

Funds from CTRE Chicago State University to Nadeem Fazal and to Bahar Graefen from Fulbright Association.
Bridging statistics and life sciences education for immunologists: Exploring significantly impactful teaching strategies and collaborations
Authors: Singh Jastaranpreet , White Bethany
Published: August 2023

The widespread misuse of statistics is one of the contributing factors to reproducibility concerns in immunological research. However, not all immunologists have the statistical expertise to choose the best methods to evaluate and represent their experiments, and the formal statistics training required by undergraduate and graduate immunology curricula is often quite limited. How can we prepare immunologists for statistical practice in research? We previously developed a second-year undergraduate course to integrate statistics instruction with research design to improve the quantitative training of life sciences students. In a Scholarship of Teaching and Learning (SoTL) study conducted within the course, student attitudes and self-efficacies for statistics, as well as their abilities to recognize and handle problems related to statistical practice in life sciences research, were assessed through surveys administered at the beginning and end of the course (n=126). Here, we present our team-teaching model and strategies for promoting meaningful connections between statistics and immunological research through course design, activities and assessments. We also highlight the results from our SoTL study, which have guided the development of several specialized immunology courses focusing on statistical practice and experimental design at the University of Toronto. We hope that insights from this teaching partnership and our SoTL findings will help inform future quantitative course offerings and training initiatives, and ultimately, better prepare students to effectively engage with statistics in immunological research.
Using lung-on-a-chip to characterize HIF-1α-dependent inflammatory responses during acute Streptococcus pneumoniae infection
Authors: Kang Hellen , Rosin Nicole , Sinha Sarthak , Biernaskie Jeff , Gillrie Mark
Published: August 2023

Current models used to study Streptococcus pneumoniae (SP) infections do not account for complex organ microenvironments or species-specific signaling pathways. To address this, we created a vascularized 3D lung-on-a-chip (LoC) that better recapitulates the physiologic lung, including cellular components, immune cell recruitment, blood flow, and oxygen tension. Hypoxia inducible factor 1-alpha (HIF-1α) is a transcription factor known to activate inflammatory pathways during infectious diseases, but its role in acute SP infection remains unclear. LoC was used to validate HIF-1α upregulation and characterize its role in immune cell recruitment. Whole human blood with fluorescently-labeled immune cells were perfused in LoC +/− BAY 87-2243 (HIF-1α chemical inhibitor). HIF1α-dependent inflammatory mediators involved in SP infection were identified by scRNA-seq and confirmed at the protein level by Western blot and ELISA. Preliminary studies of LoC stimulated with SP demonstrated HIF-1α early target gene activation. LoC showed increased CD15+-neutrophil recruitment from perfused whole blood in microvessels to tissue and airway during SP infection, partially mediated by HIF-1α. IL-17C was identified as the top differentially expressed gene activated by SP and 2D protein analysis showed increased IL-17C levels during HIF-1α knockdown, suggesting that HIF-1α is a negative regulator of IL-17C transcription. These preliminary findings suggest that HIF-1α mediates immune cell recruitment during acute SP infection and support the use of LoC as a pre-clinical human model that can identify key mediators during pneumonia.
Unraveling novel crosstalk between distinct neutrophil subsets and soluble plasma factors in sepsis and ARDS
Authors: Parthasarathy Upasana , Kuang Yi , Thakur Gunjan , Hogan John , Wyche Thomas , Norton James , Sana Theodore , Beakes Caroline , Shyong BaoJen , Zhang Rena , Filbin Michael , Christiani David , White Cory , Martinelli Roberta
Published: August 2023

Sepsis is a life-threatening condition caused by an aberrant immune response to infection and subsequent organ dysfunction. Further, due to shared mechanisms such as severe inflammation and endothelial dysfunction, sepsis remains the most common etiology in acute respiratory distress syndrome (ARDS) patients. Despite multiple efforts to identify a distinct signature to support the development of new therapeutics, sepsis remains a significant unmet clinical need. While singular components of the dysregulated immune response have been explored, studies linking different data layers are lacking. Using an integrated systems immunology approach, we evaluated neutrophil phenotypes and related changes in soluble plasma factors in sepsis versus healthy donors. Results from our studies identify differentially expressed mature and immature neutrophil subsets in sepsis patients. Further, we show that expression of neutrophil CD10 is associated with factors such as pentraxin-3, angiopoietin-2, and lysophosphatidylcholines, in the sepsis cohort. These findings detail a novel correlation between increased presence of immature neutrophils and endothelial dysfunction/metabolic alterations in sepsis patients. Additionally, we profiled the neutrophil landscape and evaluated levels of soluble plasma factors, in a subset of ARDS patients and healthy donors. Here, we present distinct findings that reveal a novel crosstalk between neutrophil subsets and parameters indicative of endothelial dysfunction, in sepsis and ARDS patients. Overall, our study reinforces the importance of data integration and provides novel insights that could help inform future mechanistic studies, early patient stratification and treatment options.
IRF3 in adipose tissue inflammation and metabolism
Authors: Zhang Yongliang
Published: August 2023

Dysfunction of adipocytes and adipose tissue is a primary defect in obesity and obesity-associated metabolic diseases. Interferon regulatory factor 3 (IRF3) has been implicated in adipogenesis. However, the role of IRF3 in obesity and obesity associated disorders still remains unclear. Here we show that IRF3 expression in human adipose tissue was positively associated with insulin sensitivity and negatively associated with type 2 diabetes. In mouse preadipocytes, IRF3 not only acts as a brake negatively regulating the adipogenic transcriptional network centered on the PPARg pathway, but also is important for the functionality of adipocytes. In macrophages, IRF3 inhibits inflammatory cytokine expression through IFNb/IL-10 axis, thereby restraining adipose tissue inflammation in obesity. Mice deficient in IRF3 developed obesity and type 2 diabetes spontaneously associated with increased adipose tissue inflammation. This study demonstrates that IRF3 regulates the biology of multiple cell types including adipocytes and macrophages to prevent the development of obesity and obesity-related complications, hence, could be a target for therapeutic interventions for prevention and treatment of obesity-associated metabolic disorders.

National Medical Research Council of Singapore
Dominant CD40 Receptor (CD40R) Antagonist Antibodies as Novel Approach to Treating Autoimmune Diseases
Authors: LaMontagne Russell W , Olivier Ken , Weaver Bruce
Published: August 2023

The CD40R-CD40 ligand pathway is a key co-stimulatory pathway for driving T cell-dependent B cell activation and humoral immune responses. This pathway has been implicated in the pathogenesis of several autoimmune diseases including autoimmune thyroiditis, type 1 diabetes, Crohn’s disease, inflammatory bowel disease, psoriasis, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and Sjogren’s syndrome. Although the use of anti-CD40 ligand antibodies have shown benefits in patients, their use has been linked to the development of thromboembolic events, ADCC issues and low efficacy with poor antagonism in vivo. Two novel IgG2 CD40R antagonists developed by BITT have differentiated mechanisms of action including the formation of inhibitory anti-parallel dimers of the surface receptor. Several unique features include: ligand independence, Fc receptor independence, potency with ligand present and selectivity to rapidly proliferating cells. Multiple BITT antibodies showed strong antagonist activity in B-cell proliferation assays. Two BITT CD40 antagonists were selected for testing in a humanized NSG Mouse Model of graft verse host disease. The BITT CD40R antagonists were compared to isotype control and a known IgG1 CD40R antagonist. The BITT antibodies showed better B cell depletion and better inhibition of IgG levels – including a unique ability to selectively deplete activated (CD86+ cells) and younger B cells making early IgM antibodies. This selective action could result in more potent immune-suppressive function at the site of inflammation and a reduced possibility of developing side effects.
β-defensin-1 regulates influenza virus infection in human bronchial epithelial cells through the STAT3 signaling Pathway
Authors: Othumpangat Sreekumar , Noti John D
Published: August 2023

To develop intervention strategies it is necessary to understand the host response to influenza A virus (IAV) infection. The primary barriers for invading respiratory pathogens are the airway epithelial cells of the respiratory tract and several antimicrobial peptides are produced by these cells. The antimicrobial peptide, β-defensin-1, has antiviral activity against both enveloped and non-enveloped viruses. Significant downregulation of β-defensin-1 gene (DEFB1) expression was observed when human bronchial epithelial cells (HBEpCs) were exposed to IAV. HBEpCs overexpressing DEFB1 caused a significant reduction in IAV, that was confirmed by IAV matrix gene analysis, plaque assay, and confocal microscopy. DEFB1 expression after transfection with two micro RNAs (miRNAs), hsa-miR-186-5p and hsa-miR-340-5p, provided evidence that DEFB1 expression could be modulated by these miRNAs and hsa-miR-186-5p had a higher binding efficiency with DEFB1. Overexpression of DEFB1 in IAV infected HBEpCs led to increased NF-κB expression. In a PCR array analysis of 84 transcription factors, either overexpressing DEFB1 or siRNA silencing of DEFB1 expression significantly modulated the expression of signal transducer and activator of transcription 3 (STAT3). In addition, Ingenuity Pathway Analysis (IPA) integrated with PCR array data showed that the JAK1/STAT3 pathway was significantly altered in cells overexpressing DEFB1, suggesting this to be one of the pathways by which defensin regulates IAV replication in HBEpCs. In conclusion, the reduction in IAV copy number in DEFB1 overexpressing cells suggests that β-defensin-1 plays a key role in regulating IAV survival through STAT3 and is a potential target for antiviral drug development.

No funding
Obesity is associated with impaired immune history to influenza
Authors: Abd Alhadi Marwa , Friedman Lilach M , Karlsson Erik A , Cohen-Lavi Liel , Burkovitz Anat , Schultz-Cherry Stacey , Noah Terry L , Weir Samuel S , Shulman Lester M , Beck Melinda A , Hertz Tomer
Published: August 2023

Obesity is a risk factor of severe disease and mortality for both influenza and SARS-CoV-2. While previous studies show that obese individuals generate antibody responses following influenza vaccination, infection rates within the obese group were twice as high compared to the healthy-weight group. To investigate the hypothesis that obesity impacts immune memory to infections and vaccines, we profiled the baseline immune history of obese and healthy-weight adults vaccinated with 2010–11 seasonal influenza vaccine to influenza antigens. Despite extensive heterogeneity in baseline immune history profiles in both groups, there were striking differences between obese and healthy controls, especially towards A/H1N1 strains and the 2009 pandemic virus (Cal09). Individuals with obesity had lower IgG and IgA magnitude and breadth towards a panel of A/H1N1 whole viruses and hemagglutinin proteins from 1933–2009, but increased IgG magnitude and breadth towards linear peptides from the Cal09 H1 and N1 proteins. Age was also associated with A/H1N1 baseline immune history, with young individuals with obesity more likely to have reduced A/H1N1 baseline immune history. We further found that IgG baseline immune history was associated with baseline and post-vaccination protective antibody responses, as measured by micro-neutralization and hemagglutinin inhibition assays. Taken together our findings suggest that increased susceptibility of obese subjects to influenza infection may be in-part mediated by obesity-associated differences in the memory antibody repertoire, which cannot be ameliorated by seasonal vaccination. Overall, these data have vital implications for the next generation of influenza and SARS-CoV-2 vaccines.
Inflammation perturbs NK cell phenotype and function in chronic myeloid leukemia (CML)
Authors: Kuznetsova Valeriya , Costa Amanda , Patel Sweta , Luca Francesca , Franceski Alana , Goel Paran , Crown Brittany , Camacho Virginia , Matkins Victoria , Welner Robert
Published: August 2023

Natural killer (NK) cells have the capacity to recognize and clear cancer cells by releasing cytotoxic granules. In fact, NK cells’ mature phenotype and abundance are associated with prolonged treatment-free remission in chronic myeloid leukemia (CML). However, NK cell anti-leukemic activity is suppressed during the disease, and the environmental triggers of this impairment are not fully characterized. Given the role of inflammatory cytokines in the progression of myeloid malignancies, we define their impact on NK cell function in a BCR-ABL1+ CML chimeric mouse model. Consistent with clinical observations, NK cells are reduced in counts in leukemic mice, display immature phenotype, low degranulation rate, and altered expression of activating and inhibitory receptors. Single-cell RNAseq analysis confirmed these observations at the transcriptional level, and revealed enrichment in genes and pathways associated with inflammatory cytokine response (e.g., GM-CSF, LTα/β, XCL1, IL-6st, IL-2Ra, SOCS1/2/3, CIS; IL-6/STAT3 and TNFa/NF-kB pathways) in CML-exposed NK cells. To validate the clinical relevance of our findings, we characterized NK cells from CML patients. As expected, NK cell frequencies are reduced in our cohort and display diminished K562-targeted degranulation. Mirroring mouse scRNAseq data, patient NK cells possess a pro-inflammatory gene signature with the activation of JAK-STAT, TNFa/NF-kB, and PI3K-Akt signaling pathways. As this is likely triggered by leukemic cytokines, we found that leukemic serum dampens ex vivo NK cell degranulation capacity. Thus, we speculate that NK cells are sensitive to CML-associated cytokines and that inflammation represents an optimal target for NK-boosting immunotherapies.
γδ T cells are significantly suppressed in glioma patients but retain their cytotoxic potential
Authors: Lehman Natalia , Mazurek Marek , Mrozowska Karolina , Michalski Adam , Rola Radosław , Bojarska-Junak Agnieszka , Zarobkiewicz Michał
Published: August 2023

Complex interactions between tumor and immune cells influence γδ T cells in the course of glioma. The study aimed to characterise the γδ T cells in glioma patients and assess their potential for γδ T-based immunotherapy. Peripheral blood from 52 glioma patients and 30 healthy controls was collected. γδ T cells and their subsets were assessed by flow cytometry and compared between stages i.e. LGG and HGG. Cytokine expression and cytotoxicity against U251MG was tested on Vδ2 in vitro cultures. The co-culture of healthy donors PBMC with glioma patient plasma or 4 hours stimulations with U251MG were performed.

Both total γδ T and Vδ1 cells were significantly expanded in glioma patients. While stimulatory molecules (CD226 and NKG2D) were downregulated, co-inhibitory molecules (PD-1, CTLA-4, TIGIT, TIM-3, LAG-3) were overexpressed. Suppression of γδ T cells seems to be promoted by circulating cytokines as PD-1, TIGIT, TIM-3, LAG-3 were upregulated in cultures with patient plasma. Healthy volunteer γδ T cells are usually significantly activated by U251MG as was demonstrated by an increase in CD69 and CD107a expression after short-term co-culture. Importantly, glioma patient-derived Vδ2 cells retained their cytotoxic potential against glioblastoma cells, even though they overexpressed anti-inflammatory cytokines (IL-10, TGF-β, IL-17).

The results highlight the profoundly immunosuppressive peripheral blood environment in gliomas. Most importantly, this may significantly affect the general fitness of γδ T cells, lowering their potential for immunotherapy. On the other hand, those changes seem to be at least partially reversible as patient-derived γδ T cells retain their cytotoxic potential after in-vitro activation and culture.

Supported by grant from Medical University of Lublin MG 25/2022.
Identification and characterization of selective FcγRIIb binders and their potential of IMPACTing immune-mediated diseases
Authors: Lutz Stephen , Cianci Mike , Grebinoski Stephanie J , Borah Minasri , Stanlie Andre , Goydel Rebecca , Visweswaraiah Jyothsna , Higginson-Scott Nathan , Otipoby Kevin , Cipolletta Daniela
Published: August 2023

Biologics targeting multiple pathways and/or multiple cell types simultaneously have the potential to restore normal immune homeostasis. Inhibitory checkpoint receptor agonism by antibodies benefits from higher order clustering for strong activation, which can be achieved by antibody Fc binding to Fc gamma receptors (FcγRs). However, many of the current agonist antibodies non discriminately bind activating FcγRs. This has the potential to trigger inflammatory cytokines by antigen presenting cells (APCs) or cause antibody dependent cellular cytotoxicity (ADCC).

Clustering by Fc binding to the inhibitory Fc receptor, FcγRIIb has the potential to provide superior clustering by avoiding inflammatory cytokine responses and limiting APC activation. However, FcγRIIb is 93% homologous to the activating receptor FcγRIIa, and designing molecules that specifically bind FcγRIIb presents a challenge that our IMPACT platform can address. Using integrated machine learning, structural biology and traditional antibody campaigns, we have identified selective FcγRIIb binding molecules, and characterized their potential for IMPACTing immune-mediated diseases.
The promise of machine learning: using Seismic’s IMPACT platform to design IgG cleaving enzymes for chronic treatment of autoimmune diseases
Authors: Pellerin Alex , Anderson Jordan , Rollins Nathan , Sanmarco Liliana , Newton Andita , Le Nam , Shin June , Hoel Chris , Cianci Michael , Hoyt Emily , Mojta Shanell , Hollfelder Melissa , Cipoletta Daniela , Peckner Ryan , Higginson-Scott Nathan , Otipoby Kevin , Mascanfroni Ivan
Published: August 2023

Proteases derived from human pathogens can specifically cleave IgG into F(ab′)2 and Fc fragments. This unique trait suggests a novel opportunity to use these molecules to treat auto antibody mediated disease. IdeS, an IgG cleaving enzyme derived from Streptococcus pyogenes has shown clinical proof of concept and is approved for use before kidney transplant. Due to the immunogenic nature of these proteases, the dosing regimen is impacted by pre-existing antibodies and the induction of anti-drug antibodies after dosing.

To mitigate the impact of the immune system on our novel enzyme, we employed our IMPACT platform leveraging machine learning to reduce T and B cell epitopes and to ensure that our molecule exhibits desirable drug like properties while maintaining enzymatic activity. To extend the pharmacokinetics (PK) of our molecule, we fused it with a Fc domain. To evaluate IgG protease PK and pharmacodynamics (PD), intravenous immunoglobulin (IVIg) was injected at different time points after protease treatment and IgG levels were quantified by MSD. As expected, the addition of the Fc increased the molecule’s half-life that resulted in a PD effect at later time points than observed with a control enzyme without a Fc. Taken together, our IMPACT platform leveraging machine learning demonstrates that we can optimize drug-like properties and reduce the immunogenicity of a molecule while maintaining function, resulting in a potential novel treatment for chronic autoimmune diseases.
Ineffective polarization characterizes CD4 T cell responses to persistent Borrelia burgdorferi infection
Authors: Hammond Elizabeth M , Tucker Inglis , Olsen Kimberly , Shah Priya , Baumgarth Nicole
Published: August 2023

Borrelia burgdorferi (Bb), the spirochetal agent of Lyme disease, evades immune clearance in infected mice and humans, suggesting ongoing immune suppression. Here we explored the functionality of Bb-induced CD4 T cell responses in infected mice, which are considered Th1 polarized. Surprisingly, in vitro restimulation of CD4 T cells from mice infected for 12 days failed to induce secretion of the hallmark Th1 cytokine IFNg. Consistent with that data, flow cytometry and RNAseq of CD4 T cells from lymph nodes (LNs) of Bb-infected mice at 6, 10, 14, and 21 days post infection (dpi) showed a transient Th1 and Th17 polarization that peaked at 10 dpi before declining, confirmed also by RT-PCR. While Th1 and Th17 gene expression profiles returned to baseline by 14 dpi, increased frequencies of CXCR4+ CXCR5+ PD-1hi ICOS+ BCL6+ T follicular helper (Tfh) cells emerged at that time, concomitant with germinal center induction. The lack of CD4 T cell effector polarization was noted also in the skin, an effector site after Bb infection, as well as in antigen-specific T cells, which were identified using a new MHCII tetramer to the Bb surface antigen arthritis related protein (Arp). Together, our results indicate a failure to sustain effector CD4 T cell polarization after Bb infection, identifying CD4 T cells as a potential target of Bb-mediated immune suppression.

This work was supported by grants from the Global Lyme Alliance, the Stephen & Alexander Cohen Foundation, NIH R01AI157007 (NB) and T32 AI060555 (EMH).

This work was supported by grants from the Global Lyme Alliance, the Stephen & Alexander Cohen Foundation, NIH R01AI157007 (NB) and T32 AI060555 (EMH).
IL-21 from IFN-g+ IL-21+ hybrid T cells promotes germinal center B cell proliferation
Authors: Gbedande Komi , Domingo Nadia N , Puebla-Clark Lucinda , Weinstein Jason D , Stephens Robin
Published: August 2023

Clearance of infection with Plasmodium spp. requires both antibody and Th1-type responses. While Th1-like cells control the peak in P. chabaudi infection, however specific IgG is delayed. Both Tfh and GC-Tfh can promote specific antibodies and long-lived plasma cells to fully clear infection. GC-Tfh numbers remain stable throughout P. chabaudi infection, while Th1/Tfh hybrid (IFN-g+/IL-21+CXCR5int) cells expand. Testing if triple cytokine reporter mice (Il21-Kat Il4-GFP Ifng/Thy1.1 Knock-In) report Tfh functionality accurately, we confirmed that Tfh-like Il-21+ Ifng− cells were enriched for GC Tfh (CXCR5hi PD-1hi CXCR6−) while the majority of Ifng+ Il21+ cells were CXCR5int CXCR6+. GC-Tfh appear functional sooner than Ifng+ Il21+ with expression of CCR7int and Il4+ as early as day 5p.i. Transfer of cytokine-expressing subsets from triple reporter and Il21−/− Ifng-Thy1.1 KI mice into T cell deficient recipients, which are then infected with P. chabaudi, also shows that Il21+ Ifng− Tfh-like cells from day 8 of P. chabaudi infection are functional. Il21+ Ifng− cell recipients generated more GC B cells, larger GCs and more parasite-specific IgG compared to other groups. Il21+ Ifng+ hybrid T cells, also promoted GCs, but Th1-like Ifng+ T cells from WT or Il21−/− mice did not. As GCs were larger, but not more numerous, in Il21+ Ifng− recipient spleens, we hypothesized that B cell proliferation was increased. Indeed, there were more BrdU+ dark zone (CXCR4+ CD86−) GCB cells in recipients of Il21+ Ifng− cells than other groups, particularly recipients of Ifng+ T cells from IL-21 KO. Combined, this data suggests that IL-21 from hybrid Th1/Tfh promotes GCB cells in vivo, though less than Il21+ Ifng− T cells.

R01AI13506101
Modeling phagocytosis resistance in Escherichia coli using Dictyostelium discoideum
Authors: Riddick Terra , Riaz Tahreem , Perez-Vazquez Marta , Snyder Michelle
Published: August 2023

Pathogenic Escherichia coli are responsible for morbidity and mortality around the world, especially in young children in developing countries. Though some strains of E. coli are not harmful, other strains are responsible for diarrhea, intestinal diseases, and urinary infections. E. coli and other bacteria have acquired virulence factors which have led to increased resistance to phagocytosis and other immune system mechanisms. Although sequencing has assisted in the determination of some genes and virulence factors associated with E. coli, many genes remain uncharacterized. Dictyostelium discoideum is a social amoeba that feeds on bacteria using phagocytosis. Its phagocytic mechanisms are conserved in mammalian immune cells like macrophages. D. discoideum is a good model to study host pathogen interactions because it is cost effective, easy to cultivate, and it has previously been used to identify virulence factors in bacteria. We hypothesize that D. discoideum can be used as a model organism to identify E. coli genes involved in resistance to phagocytosis. The aims of my project are to identify and characterize potential virulence genes granting resistance to D. discoideum grazing and phagocytic killing. Results show that extraintestinal pathogenic E. coli strains are more grazing resistant than intestinal pathogenic strains. We are currently working to characterize gipA (growth in Peyer’s patches gene), a gene found in strains of E. coli resistant to D. discoideum grazing. By identifying genes associated with phagocytosis resistance, our research can lead to better targeting of pathogenic bacteria and the development of more effective treatments for bacterial infections.

Supported by grants from NIH NIGMS R25 GM119970 & T32 GM146694
Association between the dynamics of the gut microbiota and responsiveness to mental health therapy
Authors: Zhou Xin , Ganz Ariel B , Lu Xinrui , Li Yizhou , Snyder Michael
Published: August 2023

Growing evidence suggests that the intestinal microbiota may influence brain activity through metabolites and/or inflammation, but it is unclear whether the impact of psychological interventions such as meditation is connected to the dynamics of gut microbiota. In the presented study, we collected gut microbiota and plasma cytokine, chemokine, and growth factor data from a longitudinal cohort of 52 people (142 multi-omics samples) before, during, and after a nine-day meditation. We identified a strong relationship between the mental health status and the composition of gut microbiota. High level of bacteria genus Prevotella is correlated with improved meditation effects in stressed persons. In addition, a number of inflammatory cytokines, such as GROA, are significantly reduced in the Prevotella-dominating individuals. Following nine days of meditation, there is a decrease in pro-inflammatory cytokines, an increase in relative abundance of Prevotella, and an improvement in individual’s mental health measurements. Our study provides a novel link towards the possibility that the gut microbiota affects the level of stress through regulating cytokines.
The role of immune cells in Chlamydia spread to the gastrointestinal tract following reproductive tract infection
Authors: Latifi Flutur , Hyseni Besmir , Konjufca Vjollca
Published: August 2023

Chlamydia trachomatis is the main cause of sexually transmitted disease and preventable blindness worldwide. In both animals and humans, Chlamydia spp. infect not only the female reproductive tract (FRT), but also the gastrointestinal tract (GIT), where it persists for long periods, without causing obvious pathology. In the murine model, following FRT infection, Chlamydia spreads to the GIT in stages and relies on carriage by host cells. After initial infection of epithelial cells in the FRT, lamina propria DCs internalize Chlamydia, which triggers their migration (and thus Chlamydia carriage) to the FRT-draining iliac lymph nodes. In the ILNs, Chlamydia infects other cells, which, then guided by the sphingosine 1-phosphate (S1P) gradient, enter the circulation, and carry Chlamydia to the spleen and the GIT. Selective depletion of either CD8 T cells, or monocytes/macrophages significantly lowers Chlamydia burden in the spleen and the GIT. In contrast, depletion of CD4 T cells significantly increases Chlamydia burden in the iliac lymph nodes and the GIT. While it takes about one week for FRT to GIT Chlamydia spread, ceca of CD4 depleted mice become positive for Chlamydia by day 3 of infection. It remains to be determined whether T cells and monocytes/macrophages become infected and actively transport Chlamydia to the GIT, or simply limit (or allow) its growth at different stages of infection and thus affect Chlamydia spread. Delineating the interactions of Chlamydia with specific host cells in vivo and identifying the cells and chemokines that mediate Chlamydia spread will shed light on Chlamydia pathogenesis, and on regulation of host cell migration during Chlamydia infection.

Supported by grant from NIH (1 R21 AI159743-01)
CD8+ T cell recognition of neuronal cysts during chronic Toxoplasma gondii infection
Authors: Bunkofske Molly , Eberhard Julia , Hunter Christopher A
Published: August 2023

The parasite Toxoplasma gondii establishes persistent infection within neurons by forming cysts that harbor latent form bradyzoites. The ability to form cysts is dependent on the parasite transcription factor BFD1 and, given the presence of a thick cyst wall, it was previously thought that cysts were protected from immune recognition. However, the generation of a BAG1-OVA parasite line, in which OVA protein expression is restricted to bradyzoites, revealed that in vivo infection elicited the activation and expansion of OVA-specific OT-I CD8+ T cells. To determine whether endogenous CD8+ T cells can recognize natural cyst-derived antigens and potentially participate in cyst control, in vitro T cell recognition assays were performed using a BFD1-inducible line of parasites that form in vitro cysts. In these assays, CD8+ T cells isolated from T. gondii-infected mice recognized both infected murine fibroblasts and neurons containing cysts. These studies indicate that cyst-specific T cells are present during infection and future studies will explore if any parasite antigens transported beyond the cyst wall can be presented by neurons for T cell recognition.

Supported by NIH grant R01 AI157247-01
Plasmodium-infected mosquito bite elicits an immune response at the bite site
Authors: Rabinovich-Ernst Orna , Hopp Christine , Kanatani Sachie , Archer Nathan K , Sinnis Photini
Published: August 2023

To induce long-lasting immunity, the host’s immune system must recognize and respond to an invading pathogen upon first encounter. Plasmodium sporozoites, the infective stage of the malaria parasite, are inoculated into the skin by infected mosquitoes as they probe for blood. Only 20% of sporozoites reach the liver, while the rest either reach the draining lymph nodes (dLN) or remain in the infected area for at least 6 hours, shedding an abundant amount of their major surface protein, the circumsporozoite protein (CSP). Yet, sterile immunity against sporozoites or CSP does not occur, despite the presence of the foreign antigens in the skin and the dLN, and repeated exposures, suggesting that Plasmodium sporozoites may modulate the host immune response. In this study we investigate the immune response elicited by P. berghei-infected mosquito bite at the bite site in-vivo. Using intravital imaging of sporozoites inoculation by a mosquito bite to a LysM-CRE GFP expressing mouse, we can observe neutrophil recruitment to the bite site in real time. When investigating the expression of pro- and anti-inflammatory genes in the skin, we find a specific expression patten that is elevated in response to a P. berghei-infected mosquito bite, when compared to a bite of an uninfected mosquito. Here we show fast immune cells recruitment as well as specific immune response to Plasmodium infection, suggesting a quick recognition of infection by the immune system. Nonetheless, gene expression analysis suggests a mechanism by which Plasmodium sporozoites may suppress the immune response to their presence.

Kindly supported by grants from NIH (R01 AI132359) and JHMRI.
T-cell derived interferon gamma is required for restriction of Mycobacterium tuberculosis in the murine lung: A reappraisal of conflicting models
Authors: Maciag Karolina , Cohen Sara , Plumlee Courtney , Urdahl Kevin
Published: August 2023

An effective vaccine for Mycobacterium tuberculosis (Mtb) is urgently needed, yet its development has been hampered by incomplete understanding of immunity to Mtb. While vaccine candidates are routinely assessed by their ability to induce CD4+ T cell-derived interferon gamma (IFNγ), this marker has been unreliable in predicting vaccine protection against TB. Furthermore, T cell adoptive transfer studies in RAG1−/− mice have suggested that lung T cell-derived IFNγ only plays a minor role in pulmonary immunity against Mtb. In contrast with prior results, our experiments using IFNγ−/− T cell chimeric mice, and with TCRβ−/− δ−/− mice receiving adoptively transferred IFNγ−/− T cells, demonstrate that control of lung Mtb burden is in fact dependent on T cell-derived IFNγ. We show that this discrepancy is partially due to the confounding effects of RAG−/− vs. the more specific TCRβ−/− δ−/− hosts. Flow cytometry and RNA-seq analysis of lung cells in WT, IFNγ−/−, and TCRβ−/− δ−/− T cell-chimeric mice reveal that absence of T cell-derived IFNγ decreases recruitment of monocyte-derived macrophages to the infected lung, skews infected and bystander macrophages to an alternative M2 phenotype, and promotes neutrophil and eosinophil influx. Together, these results reassert and characterize the role of T cell-derived IFNγ in protection against pulmonary Mtb.

Supported by grants from NIH (T32 AI007044, U19 AI135976) and the Firland Foundation (Northrop-Park Fellowship).
Establishing an immunological profile of 96 cytokines, chemokines, and growth factors in human biofluids via MILLIPLEX® multiplex immunoassay
Authors: Steiner Harold L , Williams Sasha , Banerjee Munmun , Saporita Anthony , Gilliam Brooke , Qiang Xiao
Published: August 2023

Multiplex immunoassays are well-established research tools enabling the simultaneous measurement of several proteins in biological samples. In the field of immunology, these immunoassay kits have been critically important for profiling the expression of a wide array of immunomodulatory proteins, including cytokines, chemokines, and growth factors. To this end, we recently developed two MILLIPLEX® multiplex immunoassay kits, each capable of the simultaneous measurement of 48 unique proteins in human samples. In this study, our objective was to profile the relative abundance of 96 cytokines, chemokines, and growth factors in a diverse collection of human biofluids, including serum, plasma, cerebrospinal fluid, saliva, sputum, urine, tears, milk, synovial fluid, and bronchoalveolar lavage (BAL). For each analyte, the fold increase in the average sample measurement relative to the limit of detection was determined. Some biofluids were generally rich in these immunological factors, such as BAL samples, whereas others were sparse, as noted in milk samples. Additionally, the reagent compatibility of the two immunoassay kits used to interrogate the sample cohort was examined to define opportunities for developing novel custom multiplex immunoassays containing analytes from each of the parent kits. The result of this effort was the generation of a set of rules guiding the acceptable combinations amongst the 96 proteins as well as the identification of mutually exclusive protein targets.
γc receptor antagonist, EQ102, prevents the NK and T cell-mediated responses driven by IL-15 and IL-21
Authors: Tiet Phoi , Giovannone A J , Al-Mawsawi Laith , Ampudia Jeanette , Marrocco Valeria , Connelly Stephen , Ng Cherie
Published: August 2023

Synergistic signaling of IL-15 and IL-21 is important in driving pathogenic T and NK cell responses in multiple inflammatory diseases. IL-15 and IL-21 are part of the common gamma chain (γc) cytokine family, which use the shared γc receptor subunit in their signaling complexes. IL-15 and IL-21 each promote cytolytic activity and IFNγ production but can also synergistically enhance the function of other cytotoxic cytokines. Consequently, blockade of IL-15 or IL-21 alone is likely insufficient to improve disease outcome in pathologic environments. EQ102 is a multi-cytokine inhibitor that selectively blocks IL-15 and IL-21 signaling while preserving signaling of other γc family members. Here, we sought to investigate the effect of synergistic signaling from both IL-15 and IL-21 on NK and T cell activities and the ability of EQ102 to inhibit these signals and resultant cellular response.

PBMCs from healthy donors or NK-92 cells were incubated with EQ102 for 1 hour and then stimulated with IL-15, IL-21, or in combination. Following stimulation, cellular expression of transcription factors and activation markers were analyzed by flow cytometry. Cell supernatant was assessed for T and NK cell cytokines.

IL-15 stimulation increases proliferation of T and NK cells, where IL-21 had a modest effect. However, co-stimulation of IL-15 and IL-21 enhanced proliferation, activation, and upregulation of IFNγ production over single cytokine IL-15 conditions. EQ102 treatment effectively inhibits the IL-15/IL-21 co-stimulatory cytolytic responses of these cell populations. These results suggest that selective blockade of IL-15 and IL-21 by EQ102 inhibits the synergistic signaling that mediates NK and T cell responses in multiple immune disorders.

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