bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostate epithelial cell intrinsic C3 represses MYC-driven tumorigenesis
Authors: Kunz Natalia , Bobby Jack , Yamamoto Hidekazu , Merle Nicolas , Wang Luopin , Taj Md , Ma Jianping , Kolev Martin , Loetscher Jonas , Kellerhals Tamara , Balmer Maria , Chandra Ash , Hess Christoph , Afzali Behdad , Kazemian Majid , Dasgupta Prokar , West Erin
Published: November 2024

The synthesis of complement component C3 within the tumor microenvironment has been implicated as a pivotal modulator in oncogenesis and is emerging as an attractive therapeutic target. Historically, tumor-infiltrating immune cells have been recognized as principal contributors to intra-tumoral C3, with resultant activation fragments primarily facilitating tumor progression via immunosuppression and induction of angiogenesis. Despite the ubiquity of C3 expression in neoplastic tissues, the implications of cancer cell autonomous C3 during oncogenesis remain inadequately characterized. Here we show that loss of steady-state, endogenous C3 expression, C3a generation, and C3a receptor engagement in human prostate epithelial cells (PECs) mark a salient transition towards malignancy. We reveal that tonic intrinsic C3a-C3aR engagement in non-transformed PECs serves to restrain phosphoinositide 3-kinase delta activity, which inhibits the oncogenic MYC cascade and MYC-driven ribosomal biogenesis and nucleolar dynamics to maintain normal cell homeostasis. Further, comprehensive analyses show that C3 transcript and C3 protein levels are widely suppressed across human prostate cancers. Collectively, these findings ascribe a non-traditional, tumor-suppressor function to cell intrinsic C3 and advocate for dissection of the context-specific roles of complement, a strategy that may inform the development of targeted cancer therapies.
Endometrial stromal cells define the fate of decidual Dendritic Cells through endoplasmic reticulum stress transmission
Authors: Gori Soledad , Schafir Ana , Soczewski Elizabeth , Grasso Esteban , Fernández Laura , Grassi Bassino Alejandra , Blejer Jorgelina , Vota Daiana , Pérez Leirós Claudia , Ramhorst Rosanna
Published: November 2024

Endometrial stromal cells acquire a secretory profile associated with a physiological endoplasmic reticulum stress (ERS) and the onset of a sterile inflammatory response to sustain embryo implantation. Here we evaluated the impact of endometrial stromal cells on Dendritic Cells (DC) differentiation through ERS-transmission. We used an in vitro model based on differentiated-monocytes from healthy women (with rhGM-CSF+rhIL-4) cultured in the absence/presence of Conditioned Media (CM) from stressed Human Endometrial Stromal Cells (HESC stimulated with Thapsigargin). Those DC cultures differentiated with stressed-HESC showed a lower frequency of CD1a+CD14- accompanied by an increased CD86high cell-population and higher COX2 expression. We also detected an increase in IL-1β secretion and in AnnexinV+/PI+ cell-population, indicating a lytic programmed cell death. The ERS-transmission was confirmed by increased IRE1α expression on DC. In fact, IRE1α inhibitor prevented the conditioning to a pro-inflammatory DC profile. Finally, to investigate the impact of the DC-inflammatory profile on blastocyst implantation ability, we tested trophoblastic spheroids migration. Blastocyst-like spheroids displayed an altered trophoblast migration when were cultured with supernatant from stressed-DC. These results suggest that ERS might be transmitted from stromal cells to DC and its fine balance is required to maintain tolerance for successful embryo implantation and trophoblast migration.
Molecular mechanism of activation of human cardiac fibroblasts during Pseudomonas aeruginosa infection
Authors: Ellendula Maneeth , Planner Clara , Kumar Naresh , Thomas Alison , Salam Sameerul , Lafuse William , Collins Patrick , Wozniak Daniel , Rajaram Murugesan
Published: November 2024

The prevalence of cardiac dysfunction and its vast implications for human health highlight the need to investigate the mechanistic processes allowing its pathological manifestations. Studies from our lab revealed Pseudomonas aeruginosa (P.a.) infection causes severe cardiac inflammation and dysfunction, despite absence of the bacteria in the heart. However, the underlying mechanism is unknown. Thus, we hypothesize cardiac fibroblast activation by PAMPs enhances cytokine and chemokine release, driving cardiac inflammation. To test our hypothesis in vitro, we used human monocyte derived macrophages (hMDMs) and cardiac fibroblasts (HCFs). We harvested conditioned media (CM) from P.a. infected hMDMs and exposed to HCFs. RNA was extracted at different time points, next generation mRNA sequencing was performed and supernatants were harvested to determine inflammatory cytokine. Our data revealed exposing CM to HCFs upregulates cytokine and chemokine gene expression. Also, we found CCL2, TNF-α and IL-1β levels are high in supernatants harvested from HCFs. To further investigate mechanisms controlling CCL2 expression in HCFs, we identified JNK was involved in induction of CCL2 in HCF. In conclusion, activation of HCFs releases chemokines (CCL2) which recruit leukocytes into heart tissue, causing severe cardiac inflammation. Ongoing and future studies are needed to dive further into mechanisms of this pathway, showing its potential impact in driving cardiac inflammation in vivo.
Exploring the Impact of Escherichia coli TcpC, a Bacterial TIR Domain-Containing Protein, Using Planaria and Dictyostelium discoideum as Model Organisms.
Authors: Akingbehin Victoria , Snyder Michelle
Published: November 2024

An estimated 1.2 million individuals lose their lives each year due to antibiotic-resistant bacterial infections, surpassing the mortality caused by HIV/AIDS or malaria. Uropathogenic Escherichia coli (UPEC) is among the most common causative agents of urinary tract infections (UTIs). UPECs survive in hosts using various mechanisms, including by secreting a protein known as TcpC. TcpC is a Toll-like/interleukin-1 receptor (TIR) domain-containing protein that modifies mammalian innate immunity by inhibiting the host Toll-like receptor (TLR) response. We hypothesize that TcpC also affects the survival of UPECs in model systems, including the social amoeba Dictyostelium discoideum and planaria flatworms. To test this hypothesis, we are using mutant strains of the UPEC CFTO73 that have altered expression of TcpC to analyze the effects of TcpC on bacterial growth and virulence in these model organisms. Our initial tests with D. discoideum indicate that the presence of TcpC in UPEC strains may increase their resistance to grazing and phagocytosis by the social amoeba D. discoideum. We are currently testing the effects of TcpC on survival of UPEC strains in planaria. If TcpC affects survival of UPECs in these model systems, this may open new avenues for characterization of the molecular mechanisms used by bacterial TIR domains to evade mammalian innate immune cells.
Understanding variant-specific variations in neutrophil functional kinetics associated with SARS-CoV-2 infection in a feline animal model
Authors: Gunasekara Sachithra , Shatnawi Shoroq , Ludwig Breya , More Sunil , Miller Craig , Rudd Jennifer
Published: November 2024

The intricate interplay between neutrophils and diverse SARS-CoV-2 variants offers insights into immunopathogenesis and potential therapeutic interventions in SARS-CoV-2 infection. Utilizing our COVID-19 feline model, we hypothesize that neutrophil dysregulation and the release of neutrophil extracellular traps (NETs) are highly strain-dependent in domestic cats. Specific-pathogen-free cats (n=8) were inoculated with SARS-CoV-2 (n=3 B.1.617.2/Delta; n=3 XBB.1.5/Omicron) or vehicle (n=2). Plasma, bronchoalveolar lavage, and lung tissues were collected over 5 days. Flow cytometric analysis of neutrophil subsets in blood, BAL, and tissues revealed functional and phenotypic changes (CXCR2+CXCR4-CCR5+) in Delta vs. Omicron vs. vehicle. Detection of NETs using MPO-DNA ELISA indicates significant elevations at 4dpi and 5dpi in the plasma and BAL of Delta and Omicron cats when compared to vehicle. Multiplex immunoassay conducted on BAL revealed significant elevation of pro-inflammatory cytokines including TNF-α, IL-8, and IL-18 in infected cats compared to vehicle favoring delta strain. RT-PCR and western blot on lung tissues utilizing three major markers of activated neutrophils and NETS (citrullinated histone 3, MPO, and neutrophil elastase) show marked expression in Delta cats vs Omicron and vehicle. These findings underscore the versatility of domestic cats as a translational model for developing targeted interventions based on variant-dependent neutrophil population dynamics.
Roles for the chemokine receptor CCR6 in pancreatitis and pancreas cancer
Authors: Drouillard Donovan , Volkman Brian , Dwinell Michael
Published: November 2024

Pancreatic ductal adenocarcinoma (PDAC) has a poor average life expectancy of ten months, in large part due to the immunosuppression surrounding the tumor preventing effective anti-tumor immune responses. A mediator of PDAC immunosuppression is regulatory T cells (Tregs). Conversely, pancreatitis is an inflammatory disease and is the leading risk factor for developing PDAC yet is lacking in Tregs. My preliminary data show that CCR6+ Th17 T cells are enriched in pancreatitis, while CCR6+ Tregs are enriched within PDAC tumors with little Th17s. Comparison of CCR6+ Tregs to CCR6- Tregs in PDAC show upregulation of genes involved in activation and suppressive function in the CCR6+ population with reliance on the RORyt and RORA transcription factors. The data suggest that CCR6+ Tregs possess superior suppressive capability and are derived from Th17s. The only ligand for CCR6 is CCL20, which is upregulated in both pancreatitis and PDAC and is correlated with a worse prognosis in PDAC. In pancreatitis, CCL20 is produced by Th17s and macrophages, while in PDAC the CCL20 is produced largely by the basal subtype of PDAC tumor cells. Using mouse models of PDAC, we demonstrated that knock-out of CCR6 resulted in beneficial increase of anti-tumorigenic immune populations within PDAC tumors. Cumulatively, these data support the notion that CCR6 is viable as both a treatment for pancreatitis and as an adjuvant to improve the efficacy of immunotherapy in PDAC.
Endogenous ICOS signaling supports Th17 therapy efficacy
Authors: Wittling Megen , Knochelmann Hannah , Cole Anna , Wyatt Megan , Ruffin Ayana , Kumarasan Soundharya , Bennett Frances , Swisher Shannon , Paulos Chrystal , Lesinski Gregory
Published: November 2024

Objective: We sought to determine the mechanism by which antigen-specific Th17 cells are able to clear melanoma tumors more effectively than their Th1 counterparts and to expand on the role of ICOS in the success of Th17 therapy.

Methods: Antigen-specific TRP-1 CD4+ T cells were polarized to Th17 or Th1 phenotypes and were adoptively transferred into mice bearing B16F10 melanoma. The role of inducible costimulatory molecule (ICOS) in this therapy response was evaluated by antibody blockade and genetic deletion. RNA-Sequencing, flow cytometry, and tumor growth measurements were additionally used to assess differences when ICOS was blocked or not.

Summary of Results: ICOS is important for the efficacy of Th17 therapy in mice with established melanoma as when blocking ICOS signaling via antibody or genetic means, both antitumor activity and survival was diminished. By assessing the phenotype of both infused and host lymphocytes, we found that B cell proliferation and the engraftment of transferred cells decreased in the mice blocked of ICOS signaling. I also found that ICOS signaling appears to be important early on after Th17 cells infusion, as blocking ICOS at early but not later time points compromised antitumor immunity.

Conclusions: ICOS is important for long-term survival and antitumor activity of mice treated with Th17 cells targeting melanoma.
Restoring NAD+ levels sensitize checkpoint resistant tumors to PD-1 therapy mediated through macrophage-NK-T cell crosstalk.
Authors: Chakraborty Paramita , Chatterjee Shilpak , Subramanian Suganya , Parikh Rasesh , Hedley Zacharia , Vaena Silvia , Romeo Martin , Schooten Wim , Gangaraju Vamsi , Berto Stefano , Mehrotra Shikhar
Published: November 2024

Immune checkpoint blockade therapy (ICB) often targets tumoricidal capacities of T cells. However, innate immune cells present in the tumor microenvironment (TME) play a decisive role in the efficacy of ICB. Therefore, developing combination therapies that target multiple immune populations is of considerable interest. Nicotinamide adenine dinucleotide (NAD+), the crucial metabolic intermediate, acts as a cofactor in redox processes and co-substrate for multiple enzymes. Immune populations in TME express surface CD38, a NAD hydrolase that reduces the tissue NAD+ levels, impairing T cells metabolic fitness. Here we used an antibody that blocks the NAD hydrolase activity of CD38 and is non-toxic in vivo, as compared to the commercially available anti-CD38 antibody (NIMR5), that relies upon antibody-dependent cellular cytotoxicity (ADCC). We found that combined therapy with αPD-1 and αCD38 antibody significantly enhances tumor control in PD-1-resistant melanoma model. Single-cell RNA sequencing, flowcytometry analysis and cell depletion studies revealed that combination therapy leads to transcriptional reprogramming of macrophages, improves macrophage-NK-T cell crosstalk, and facilitates immune infiltration in the TME. These results highlight the importance of boosting the TME's NAD+ level in conjunction with ICB and allude to the potential of an improved endogenous immune response against cancers that are resistant to checkpoint treatment and typically regarded as incurable.
Lung homeostasis is maintained by the interactions between CX3CR1-expressing interstitial macrophage and lung commensal bacteria.
Authors: Kim Kiwook , Kim Seung Hyeon , White Zachary , Gainulina Anastasiia , Kim Jiseon , Dominguez Joseph , Kang Soeun , Hay Nissim , Chevrier Nicholas , Gunzer Matthias , Sano Teruyuki
Published: November 2024

As an anti-inflammatory cytokine, IL10 plays an important role in modulating immune responses. However, it remains relatively unknown about the importance of IL10-IL10 receptor (IL10R) signaling in maintaining lung homeostasis. Here we investigated that IL10R signaling of lung interstitial macrophages (IMs) contributes to the maintenance of tissue homeostasis by preventing fibrosing lung inflammation. We observed that IL10-deficient mice spontaneously induced lung inflammation in adulthood. In parallel, IL10R-deficiency of IMs identically induced the inflammatory phenotypes observed in IL10-deficient mice. Antibiotics treatments, 16S rRNA-Sequencing analysis, and germ-free (GF) animals allowed us to determine that the lung inflammation occurring in the absence of IL10 or IL10R was triggered by lung commensal dysbiosis. Furthermore, we identified that two distinct commensal microbes (D. Acidovorans and R. Erythropolis) dominantly colonized in the lung of mice lacking IL10R in IMs, where these commensals initiate lung inflammation. Collectively, our data highlight that IL10R signaling of IMs contributes to lung homeostasis by restricting commensal dysbiosis and prevents progressive lung fibrosis.
Identification and characterization of PD-1 agonists for the treatment of autoimmune and inflammatory diseases
Authors: Ramello Maria , Grebinoski Stephanie , Panduro-Sicheva Marisella , Borah Minasri , Vitlip Joshua , Lutz Stephen , Cianci Michael , Ramello Maria , Sharma Ishan , Wittenberg Elliott , Colthart Allison , Visweswaraiah Jyothsna , Higginson-Scott Nathan , Otipoby Kevin , Cipolletta Daniela
Published: November 2024

The development of autoimmune diseases is the result of a dysregulated immune response against self-antigens, resulting in cell dysregulation and tissue damage. Upon chronic exposure to antigens, inhibitory receptors (IR) are upregulated as a feedback mechanism to limit T-cell effector activity. IR deficiency is associated with autoimmunity in mice and humans. Similarly, blockade of IR is efficacious for the treatment of cancer but is often associated with new onset of immune-related adverse events. Therefore, if loss or blocking of IR pathways can result in autoimmunity, agonism of these pathways should restore normal immune homeostasis. However, the generation of agonistic antibodies present some challenges. For instance, to elicit strong receptor agonism, super-clustering mediated by the binding of the constant region (Fc) of an antibody to FcgRs, must occur. However, while FcgRs are widely expressed on antigen-presenting cells (APCs), they are not the same and are classified, based on their function, as activating or inhibitory. Of note, FcgRIIb is the only inhibitory Fc receptor, and APCs derived from mice deficient in this receptor exhibited exaggerated cytokine release. Most PD-1 agonist antibodies in clinical development contain a wild-type Fc and can trigger unwanted production of pro-inflammatory cytokines by engaging activating FcgRs. To mitigate such liability, we designed novel PD-1 agonistic antibodies that selectively bind FcgRIIb and prevent APC activation.
Fusion of Antigen with Efferocytic Mediators Induces Antigen-Specific Tolerance and Reduces Allergic Airway Inflammation in Mice
Authors: Reda Joseph , Budina Erica , Medellin Emiliano , Foley Colleen , Garza Camryn , Ung Trevor , Chang Kevin , Wang Thomas , Hubbell Jeffrey
Published: November 2024

We made proteins that combine efferocytic mediators, which help clear dying cells, and ovalbumin (OVA), a model antigen. We demonstrate that these fusions maintain affinity to their membrane target, phosphatidylserine, and enhance uptake by murine dendritic cells in vitro. Furthermore, we found that antigen-pulsed DCs were capable of driving proliferation of both cognate CD4+ and CD8+ T cells, demonstrating evidence of antigen degradation and presentation. Similar observations were made following adoptive transfer of OVA-specific T cells into naive mice, in which the T cells demonstrated dampened recovery and effector function. Finally, we explored GAS6-OVA, one of the biologics described in this work, in the context of an allergic airway inflammation model in mice. After being treated prophylactically with GAS6-OVA, mice demonstrated lesser eosinophilia in bronchealveolar lavage fluid and lesser mucus hypersecretion as assessed by histology following challenge with OVA. These results suggest that our proteins can induce tolerance to a specific antigen, which may be useful for diseases caused by an abnormal immune response to a known antigen. Future work will explore the potential of our proteins to treat these diseases, to induce tolerance to other antigens, and to modulate immune responses in the context of an antigen-experience immune system.
IL-6 receptor blockade prevents polarization of pro-fibrotic macrophages in myeloid-rich RA synovial tissue
Authors: Mantel Ian , Singaraju Anvita , Townsend Hope , Lakhanpal Amit , Zhang Fan , Donlin Laura
Published: November 2024

In rheumatoid arthritis (RA), macrophages represent key therapeutic targets due to their ability to drive inflammation. Interestingly, new subtypes of RA have been identified, based on distinct patterns of cell types in the joint, wherein one-third of patients demonstrate an enrichment of pro-fibrotic SPP1hiCLEC5Ahi macrophages. We set out to understand what drives the formation of these macrophages in RA joints, and how anti-inflammatory medications used to treat RA may differentially affect joints with pro-fibrotic versus pro-inflammatory macrophages. In RA joints with an abundance of SPP1hiCLEC5A+ macrophages, TGFB1 expressing endothelial cells and stromal cells expressing IL6 and CSF1 are co-enriched. In an in vitro culture system, we found that the SPP1hiCLEC5A+ macrophage phenotype could result from exposure to factors from synovial fibroblasts along with TGF-β. Blockade of the IL-6 receptor with tocilizumab inhibited the generation of the fibroblast-directed SPP1hiCLEC5A+ macrophage state. This differed from the inflammatory HBEGF+IL1B+ macrophage state, found in joints with extensive T and B cell aggregates. Interestingly, in a clinical trial that used tocilizumab, joint tissue transcriptomics demonstrated a reduction in SPP1hiCLEC5A+ cell signatures. This study highlights the importance of making treatment decisions for RA patients informed by the cellular composition of the affected tissue.
Adjuvanticity of ISA51 and ISA720 in the induction of immune tolerance
Authors: Mone Kiruthiga , Rasquinha Mahima , Sur Meghna , Steffen David , Reddy Jay
Published: November 2024

Various immune suppressive therapies have been used to treat autoimmune diseases, and the risks of immune suppressants can be mitigated using strategies that promote antigen-specific tolerance. Here, we report the utility of ISA51 and ISA720, a new generation of water-in-oil adjuvants in the induction of tolerance to proteolipid protein 139-151 (PLP) using the mouse model of experimental autoimmune encephalomyelitis (EAE) for multiple sclerosis. First, emulsions of ISA51 or ISA720 containing PLP 139-151 induced robust T-cell responses comparable to the complete Freund’s adjuvant (CFA)/PLP 139-151 group. Second, CFA/PLP 139-151, but not ISA51 or ISA720 emulsions induced EAE in SJL mice. Third, while splenocytes from the CFA/PLP 139-151 group produced IFN-γ, TNF-α, and IL-6, cells from ISA 51 or ISA720 groups had predominant production of IL-5, IL-9, and IL-22, but levels of IL-17A and IL-17F were comparable. However, if any, IL-4 and IL-10 levels were high in the CFA/PLP 139-151 group. Conversely, cytokines of lymph node cultures revealed high amounts of IFN-γ, IL-9, IL-10, IL-17A, IL-17F, IL-22, TNF-α, and IL-6 in the CFA/PLP 139-151 group relative to others. Strikingly, IL-5, IL-9, IL-17A, IL-17F, and IL-22 levels were elevated in ISA720/PLP 139-151 group as compared to ISA51/PLP 139-151 group. The data suggest that T cell tolerance can be induced with self-antigens emulsified in ISA51 or ISA720 independent of cytokines produced by specific T helper subsets.
CD4+ T cell lymphopenia decreases survival in older adults with glioblastoma
Authors: Zhai Lijie , Penco-Campillo Manon , Lauing Kristen , Wainwright Derek
Published: November 2024

OBJECTIVE: Glioblastoma (GBM) is the most common and aggressive primary brain tumor with a median age of onset at ~70 years of age. GBM patients ≥ 65 years of age have worse survival outcomes than younger counterparts (p<0.0001). Older human GBM patients with CD4+ lymphopenia show decreased survival as compared to age-matched GBM controls (p<0.05). Here, we evaluated the effects of tumor and various forms of lymphopenia in young and old mice with GBM. METHODS: Young 6-12 weeks of age or old ≥80-120 weeks of age C57BL/6 mice were treated with either IgG isotype control antibodies or with depleting antibodies against murine CD4, CD8, or NK1.1 beginning at 3 days prior to- and every 3 days post-intracranial injection of the syngeneic GBM cell lines, GL261, CT-2A, or SB28. Overall survival was compared between groups. RESULTS: Older adult mice, but not younger mice, with either intracranial GL261, CT-2A, or SB28 cell-based brain tumors have a significantly decreased overall survival when depleted for CD4+ T cells as compared to the IgG control-treated group or groups depleted for CD8+ T- or NK-cells (p<0.05). Based on the precipitous decline in overall survival due to CD4+ T cell lymphopenia, a tumor-independent effect is suspected. CONCLUSIONS: The data suggest that CD4+ T cells play a more sophisticated role in maintaining the survival of older adult subjects with GBM than in the younger subjects and that their contribution goes beyond an anti-tumor-only contribution.
Plasmid-encoded CCL27 co-delivery enhances heterologous protection from respiratory virus challenge
Authors: Gary Ebony , Tursi Nicholas , Warner Bryce , Trachtman Abigail , Patel Ami , Kobasa Darwyn , Weiner David
Published: November 2024

Many pathogens enter the host at mucosal sites. Peripheral vaccination supports robust antigen-specific cellular and humoral immunity but it requires active infection to pull antigen-specific memory cells to the mucosa and relies on passive transudation of antibodies to mucosal surfaces. We reported that co-delivery of the mucosal homing chemokine CCL27 (CTACK) with a DNA plasmid encoding parental SARS-CoV-2 spike (pS) generates antigen-specific IgA in the lung, antigen-specific CD8+ T cells at mucosal surfaces, and was 100% protective in a heterologous Delta VOC challenge model. Here, we extend this work and demonstrate that co-delivery of plasmid-encoded CTACK (+pCTACK) with pS supports protection from heterologous challenge with SARS-CoV-Omicron variants. pCTACK co-delivery significantly lowered viral loads when mice were challenged with Omicron variants BA.2 and XBB.1.5. In the context of Influenza HA DNA vaccination, co-delivery of pCTACK increased HA-specific antibodies in bronchoalveolar lavage, increased antigen-specific CD8+T cells in the lung mucosa, and altered challenge outcomes in vivo. These data have broad implications for the generation of mucosal immunity with parenteral vaccination and support the use of CCL27 to augment parenteral vaccine-induced immunity at mucosal surfaces and support improved outcomes following respiratory virus infection.
Uncovering Alterations in the Murine Interferon Response due to dsRNA Mycovirus during Aspergillus fumigatus Infection
Authors: Rapp Alexander , Obar Joshua , Cramer Robert , Wang Xi , Ross Brandon , Olive Andrew , Stajich Jason
Published: November 2024

Aspergillus fumigatus is a mold that causes an array of diseases, the most severe form being invasive aspergillosis which occurs in immunocompromised individuals. We demonstrated that tissue-resident alveolar macrophages are critical for maintaining host resistance against Aspergillus fumigatus due to their role in the early type I/III interferon response. Specifically, CD11c-Cre x Mavsfl/fl mice have a decreased interferon response and survival outcomes when challenged with A. fumigatus, which can be functionally restored via adoptive transfer of wild-type SiglecF+ alveolar macrophages. Why alveolar macrophages are potent inducers of the type I/III interferon response after Aspergillus fumigatus challenge remains elusive. The type I/III interferon responses were partially related to the ability of alveolar macrophages to recognize mycoviral double-stranded RNA (dsRNA). Aspergillus fumigatus strains infected with polymycovirus 1 (PmV1) are shown to induce a robust type I/III interferon responses both in vitro and in vivo. Critically, Mda5/MAVS-dependent interferon response can still be induced in a fungal dsRNA independent manner, which warrants further explorations. In this study we identified key traits of alveolar macrophages which enable robust Mda5/MAVS signaling necessary for initiating the Aspergillus fumigatus induced interferon response, which maintains host resistance against this ubiquitous mold, through both fungal dsRNA-dependent and -independent mechanisms.
Phenotyping the complex human peripheral blood (PB) B cell compartment using multimodal Cellular Indexing of Transcriptomes and Epitopes (CITE-seq) analysis.
Authors: Meinhardt Nathan , Veltri Anthony , Dittel Bonnie
Published: November 2024

Classifying the heterogeneous human B cell compartment into discrete subsets based on surface phenotype alone remains a major challenge in the field of B cell biology. Our lab has discovered a novel mouse IgM+IgDlow/- B cell subset (BDL) that induces Treg proliferation, making it a potentially significant therapeutic target for autoimmune diseases. But due to their rarity and lack of sufficient cell surface identifiers, a more definitive phenotype is required to track this B cell subset in humans. The objective of these studies was to characterize human IgDlow/- B cells from PB using multiomic CITE-seq analysis. Here, we adapted a standardized gating scheme (CD27xIgD → CD24xCD38) that captures all the major canonical human PB B cell subsets. This scheme allowed us to analyze FACS-purified CD19+IgM+IgDlow/- PB B cells using CITE-seq by targeting these four protein markers and recreate biaxial plots similar to our flow cytometry analyses. It is increasingly understood that mRNA levels within a cell do not correlate well with cell surface protein expression. This represents a major drawback to using a transcriptome-only scRNA-seq approach because any ‘novel’ cell populations discovered based on the upregulation of surface protein transcripts may prove impossible to validate. These studies have established a workflow for a multiomic approach to identify discrete human B cell subsets based on cell surface expression that can be analyzed for transcriptional differences.
Sub-second release of Neurotransmitters from CD4+ T lymphocytes
Authors: Seibold Jordan , Ross Ashley
Published: November 2024

The neuroimmune system is complex and is regulated by small chemical messengers released from resident immune and neuronal cells. Catecholamines like dopamine and norepinephrine are important chemical messengers responsible for initiating and propagating messages between neurons and immune cells; however, the mechanism and dynamics of this signaling are relatively unknown. The presence of the intracellular machinery to synthesize and interact with catecholamines in T lymphocytes has been known for over a decade; however, direct measurement of catecholamine secretion in real time from immune cells has not been possible. Here, we have discovered that Naïve CD4+ T cells are capable of releasing catecholamines on a sub-second time scale. We have used a revolutionary technique, often used in the neuroscience field, called fast-scan cyclic voltammetry (FSCV) to measure sub-second fluctuations of catecholamines from isolated CD4+ T cells. This work provides direct evidence that T cells are capable of releasing catecholamines; potentially as a feedback communicator to resident sympathetic neurons in host immune organs. These results also prove that immune cells can signal on the same time scale as neurons. This work will hopefully open the door to enable more sophisticated studies at the neuron-immune synapse in the future and the understanding of neuro-immune based diseases.
Induction of CXCL3 in airway epithelial cells by a novel IL-9 receptor complex
Authors: Cheung Cherry Cheuk Lam , Abdelaal Ahmed , Kaplan Mark
Published: November 2024

Cytokine signaling is critical for cell-to-cell communication in development, homeostasis and disease. Interleukin (IL)-9 signaling promotes allergic diseases by acting on both immune and structural cells. In hematopoietic cells, IL-9 receptor (IL-9R) consists of two subunits – the α-chain (IL-9Rα) and a common γ (γc) chain. However, IL-9-responsive epithelial cells express IL-9Rα but not the γc chain raising a question on how these cells respond to IL-9 in the absence of the established IL-9R complex. Here, we discovered that in airway epithelial cells, IL-9Rα pairs with IL-13Rα1, forming a novel type II IL-9R. Using proximity ligation assay, we show that the IL-9Rα /IL-13Rα1 chains are in IL-9-dependent proximity in both mouse tracheal epithelial cells (mTEC) and human small airway epithelial cells (SAEC). IL-9-induced STAT3 activation in SAEC was blocked by anti-IL-13Rα1 but not by anti-γc. Using RNA-seq, in vitro IL-9 treatment of SAEC induced the expression of genes including CXCL3, a key chemokine implicated in asthma. Importantly, intranasal administration of IL-9 increased Cxcl3 expression in mTECs and CXCL3 protein in the lung, that corresponds with an expansion in the number of CXCR2+ immune cells in the lung. Thus, we identify a novel type II IL-9R which is composed of IL-9Rα and IL-13Rα1 and show that IL-9 signaling via type II IL-9R induces CXCL3 expression in epithelial cells to contribute to airway inflammation.
Tumor expressed BCAM impedes anti-tumor T cell immunity and can be targeted therapeutically
Authors: Flies Dallas , Tian Linjie , O'Neill Rachel , Fitzgerald Daniel , Sharee Sasan , Shaik Jahangheer , Bosiacki Jason , Paucarmayta Ana , Prajapati Kushal , Langermann Solomon , Mrass Paulus
Published: November 2024

The significant albeit limited clinical benefit of therapeutic targeting of checkpoint inhibitors highlights the need for immunological understanding of cancer escape from anti-tumor immunity to develop novel and better therapeutics. We surmise that many protein-protein interactions that regulate T cell immunity have yet to be identified. Using human gain-of-function genetic screens, thousands of proteins were screened for their ability to inhibit primary human T cell function. B Cell Adhesion Molecule (BCAM) was identified as a potent inhibitor of T cell proliferation and cytotoxic T cell (CTL) ability to kill human tumors in vitro. Tumor cell surface BCAM inhibited anti-tumor immunity promoted tumor growth in vivo. Blockade with decoy soluble BCAM protein fused to a either a silenced or functional Fc domain (BCAM Fc fusion protein) was able to promote T cell anti-tumor immunity and reduce tumor growth in syngeneic and humanized CDX tumor models. Moreover, tumor BCAM interaction with known ligand Laminin-a5 (LAMA5) resulted in increased production of LAMA5 that was associated with ECM modifications and potent exclusion of T cells from the tumor microenvironment (TME). BCAM Fc disruption of BCAM-LAMA5 interactions resulted in decreased LAMA5 production, increased T cell infiltration and decreased tumor growth. These data support a multi-pronged role for BCAM in anti-tumor T cell suppression and exclusion that can be targeted therapeutically.
A protective role of CD4+ T cells during Staphylococcus aureus craniotomy infection
Authors: Kak Gunjan , Van Roy Zachary , Horn Christopher , Heim Cortney , Kielian Tammy
Published: November 2024

Craniotomy is a neurosurgery performed to resolve severe neurological complications. Despite prophylaxis, nearly 1-3% of craniotomies are complicated by infection, with approximately half attributed to Staphylococcus aureus (S. aureus). Using our novel mouse model, we established a protective role for CD4+ T cells during S. aureus craniotomy infection. Rag1 knock out (KO) mice or animals treated with anti-CD4 Ab showed significantly elevated bacterial burden, suggesting an important role for adaptive immunity in controlling infection. These findings were corroborated in mice treated with VLA-4 and LFA-1 Abs, underscoring the importance of peripheral CD4+ T cell responses. scRNA-seq revealed phenotypic heterogeneity within brain CD3+ infiltrates with CD4+ cells being most predominant that were typified by robust inflammatory and glycolytic profiles. CD4+ T cells displayed attributes of both Th1 and Th17 phenotypes, whose functional importance was validated in vivo as adoptive transfer of either Th1 or Th17 cells prevented S. aureus outgrowth in Rag1 KO mice. Interestingly, milder phenotypes were observed in IL-17A/F or IFN-γ KO mice, suggesting a redundant and cooperative role of both Th1 and Th17 driven responses. This was confirmed by treatment of IFN-γ KO mice with IL-17A/F neutralizing Ab, which recapitulated phenotypes of Rag1 KO animals. Collectively, our results implicate a critical role for CD4+ T cells in S. aureus containment during craniotomy infection.
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Authors: Jamsandekar Minal
Published: November 2024

Major histocompatibility complex (MHC) genes are among the most polymorphic and fastest evolving genes in vertebrate genomes with more than 1,000 alleles found at some human MHC loci. In this study, MHCII genes are explored in Atlantic herring, a species that comprises one of the biggest populations among vertebrates and occurs in huge shoals providing a potential target for pathogens using whole genome, long read sequencing of 14 individuals from three different geographic regions. The analysis identified a total of nine MHC class II loci present on three chromosomes. Some of the genes show extremely high nucleotide diversity (π = 10-13%) compared with the genome average (0.3%) as well as strong signatures of positive selection (dN/dS >> 1). Furthermore, we showed a highly significant nonrandom association of alpha and beta genes, strongly suggesting coevolution of alpha and beta alleles forming the peptide binding domain of MHC class II molecules. To the best of our knowledge this is the first genetic study of MHC polymorphism in a non-model organism based on whole genome, long-read sequencing. It demonstrates that the Atlantic herring MHC class II genes are among the most polymorphic so far described. The very high degree of polymorphisms is most likely explained by the combination of the large population size minimizing loss of genetic diversity due to drift and pathogen-driven selection.
Deciphering the Role of LIGHT/TNFSF14 in Driving Inflammatory Activity in Human Lung Fibroblasts
Authors: Ghosh Nandita , Gupta Rinkesh , Fung Kai , Croft Michael
Published: November 2024

LIGHT/ tumor necrosis factor superfamily member 14 (TNFSF14), is a protein that we previously found was central to lung inflammation and tissue fibrosis in mouse models of severe asthma and systemic sclerosis. We have hypothesized that one of its cellular targets involved in airway remodelling is the lung fibroblast given that both of its receptors, LTβR and HVEM are strongly expressed by these cells. With RNA-seq, we investigated the activity of LIGHT in these cells compared to other important inflammatory cytokines involved in remodeling in lung diseases, namely IL13, IL17A, and TGFβ. LIGHT induced a distinct set of inflammatory gene transcripts in human lung fibroblasts, including the adhesion molecules ICAM-1 and VCAM-1; cytokines IL32, IL33 and IL34; chemokines CXCL1, CXCL5, CXCL6 and CXCL10; and matrix metalloproteinases MMP9 and ADAM8. LIGHT with IL13 synergistically induced CCL2, CCL26, IL32 and ADAM8. LIGHT with IL17A also strongly boosted expression of several chemokines CXCL1, CXCL2, CXCL5, and CXCL6. Lastly, in fibroblasts differentiated into myofibroblasts by TGFβ pre-treatment, LIGHT enhanced the expression of many inflammatory and profibrotic transcripts such as ITGA1, ITGA11, MYH11, and Col1A1. This study highlights the unique as well as synergistic activities of LIGHT on these cells and suggests it could drive strongly inflammatory and remodeling effects in these cells relevant for several diseases dependent on the cytokine environment found in the lungs.
Immunomodulation of Dendrobium fimbriatum against blue light-induced ocular inflammation
Authors: Chang Wei-Chin , Liu Chuan-Teng , Song Ying-Chyi , Heinrich Michael , Yen Hung-Rong
Published: November 2024

As electronic device use increases, concerns about the harmful effects of blue light on eye health have grown. Past studies support the idea that Dendrobium species offer benefits such as reducing inflammation, providing antioxidants, and protecting against cataracts. We aim to investigate the immunomodulatory effect of Dendrobium fimbriatum Hook. extracts (DFs) in blue light-induced ocular damage. DFs decoctions were prepared from the fresh stems of 1-year, 2-year, and 3-year-old DFs. In vitro, cell viability, apoptosis, pro-inflammation cytokines and reactive oxygen species (ROS) were assessed. In vivo, ophthalmological and histological examination, systemic and local immunological changes were evaluated. Results showed that 2-year-old DF (DF-2yD) presents the maximal alleviation of blue light-induced cell death, apoptosis, interleukin (IL)-6 and 8, and ROS in retinal and corneal cells. In blue light-induced ocular damage mouse model, DF-2yD potently attenuated the fundus abnormality and retinal thickness. Furthermore, DF-2yD decreased IL-6 and TNF-alpha production in the draining lymph nodes. Collectively, the results indicate that DF hold promise as potential agents to counteract blue light-induced eye damage through the immunoregulation.
T cell responses in pediatric patients with severe influenza disease.
Authors: S. Menon Smrithi , Souquette Aisha , Allen E Kaitlynn , St. James Deryn , Mettelman Robert , Clark Brandi , Chase Crawford Jeremy , Novak Tanya , Randolph Adrienne , Thomas Paul
Published: November 2024

Influenza virus infections impose a high risk of disease-related complications in children leading to hospitalization, and increased mortality. However, it is still unclear why only some children become critically ill. The Pediatric Intensive Care Influenza 2 study enrolled children and young adults with influenza-related critical illness, aiming to identify predictive and prognostic biomarkers of severe disease (SD). To this end, we characterized T cell populations and functional responses to influenza, to understand how adaptive immunity fails to protect patients in this cohort from SD. Our preliminary studies indicate an increased level of ICOS expressing circulating T follicular helper cells (cTfh) in patients with SD while an elevation in the levels of effector memory CD4 and CD8 T cell populations in patients with moderate disease. Moreover, an enhanced level of IL21, CXCR3, ICOS and PD1 producing cTfH cells was observed in SD patients within the age group of 9-19 yrs. Age dependent varying expression of IL2, granzyme B producing CD4 T cells, CXCR3, ICOS producing and IL21 expressing cTfH cells was also observed. Additional T cell characterization is underway to integrate the influence of patient parameters such as vaccination status. T cell repertoire profiling and assessment of epitope-specific T cell populations using single-cell sequencing is being conducted to understand how pre-existing anti-influenza immunity affects disease severity and outcome.
IL-17A-mediated JMJD3 regulation of Cxcl1 gene expression in diabetic keratinocytes increases neutrophil recruitment and impairs wound repair.
Authors: Moon Jadie , Wolf Sonya , Audu Chris , Melvin William , Joshi Amrita , Mangum Kevin , Barrett Emily , Bauer Tyler , Shadiow James , Mazumder Purba , Rocco Sabrina , Erturk Ilkim , Saldana de Jimenez Gabriela , Moore Bethany , Davis Frank , Obi Andrea , Gallagher Katherine
Published: November 2024

Diabetic keratinocytes display an overexpression of chemokines that contribute to prolonged inflammation and non-healing. Using sequencing, we identified that Th17 cells and an IL-17A signature predominate in human diabetic wound tissue compared to non-diabetic controls. The effects of increased IL-17A in diabetic skin on keratinocyte function is unknown. We subjected isolated murine keratinocytes to a 5hr rIL-17A stimulation and performed bulk RNA-seq. We observed increased neutrophil chemoattract genes Cxcl1, Cxcl3, Cxcl5 compared to their controls. We analyzed epigenetic enzymes known to increase gene expression and identified that JMJD3, an activating demethylase, was increased both in human and murine diabetic keratinocytes. IL-17A stimulation increased Jmjd3 expression in murine keratinocytes, and keratinocytes from mice with a keratinocyte-specific IL-17RA deficiency were unable to increase Jmjd3 with rIL-17A. Treatment with a JMJD3-specific inhibitor decreased chemokine genes in IL-17A-stimulated keratinocytes. Keratinocytes from mice with a keratinocyte-specific JMJD3-deficiency displayed decreased chemokines compared to their littermate controls with rIL-17A, suggesting that JMJD3 is relevant to IL-17A-mediated regulation of chemokines. We then analyzed immune cells recruited to the wounds of normal and diabetic mice via flow cytometry and found that Ly6G+ neutrophils were significantly increased in diabetic mice on day 5, a timepoint at which IL-17A is elevated.
Cellular landscape of the esophageal epithelium in systemic sclerosis
Authors: Dapas Matthew , Clevenger Margarette , Makinde Hadijat , Therron Tyler , Wei Cenfu , Carns Mary , Aren Kathleen , Carlson Dustin , Muhammad Lutfiyya , Pandolfino John , Perlman Harris , Winter Deborah , Tetreault Marie-Pier
Published: November 2024

Systemic sclerosis (SSc) is a rare autoimmune disease characterized by vasculopathy and progressive fibrosis of the skin and internal organs. Individuals with SSc often suffer from chronic acid reflux and dysphagia due to loss of esophageal motility, but this pathogenesis is poorly understood.

Recently, studies have suggested that esophageal epithelial cells (EECs) may play a central role in the pathogenesis of SSc esophageal dysmotility. In this study, we performed a thorough transcriptomic investigation of the SSc esophageal epithelium in humans to determine whether distinct changes in EECs contribute to esophageal impairment in SSc.

We performed single-cell RNA sequencing of paired proximal and distal esophageal mucosa biopsies from 10 individuals with SSc, 4 comparator individuals with gastroesophageal reflux disease (GERD), and 6 healthy controls (HCs), yielding 230,720 EECs across 40 samples. SSc and GERD samples had significantly fewer terminally differentiated, superficial cells than HCs, and differential gene expression between conditions was primarily limited to superficial EECs. Gene dysregulation in SSc was highly correlated with GERD, but was relatively greater in the proximal region, including unique dysregulation of immune mediators that correlated with esophageal dysmotility.

This work sheds light on the cellular roots of esophageal dysfunction in SSc and serves as an atlas to guide future efforts to identify actionable targets in the esophagus in SSc.
Cross-tissue single-cell landscape of neutrophil heterogeneity and immunity in health and disease
Authors: Zheng Hong , Maestri Evan , Khatri Purvesh
Published: November 2024

Neutrophils, the most abundant leukocytes in human blood, play multifaceted roles in health and disease. They serve as essential components of immune defense against infection while also contribute to pathogenesis in a variety of diseases including cancer and autoimmunity. Many studies have identified functionally diverse neutrophil subsets in specific diseases and used varied nomenclatures. A comprehensive landscape of neutrophil heterogeneity and immunity is not yet fully elucidated.

To fully characterize neutrophils in health and disease, we collected and curated the transcriptomic profiles of 910,791 neutrophils from over 14 tissues across 24 single-cell RNA-seq datasets to generate a neutrophil single-cell compendium, which covered multiple diseases including viral and bacterial infection, cancer, diabetes, cardiovascular disease, and autoimmune diseases. We identified multiple neutrophil subsets exhibiting distinct heterogeneity along the development trajectories from pro- and immature neutrophils to mature neutrophils. We focused on pro- and immature neutrophils as well as mature neutrophil subsets displaying immunosuppressive transcriptional programs shared across diseases such as cancer and infections. We also inferred the cell-cell interaction between neutrophils and other immune cells in various disease contexts. Together, our systematic meta-analysis of neutrophils supported the development of neutrophil-based diagnostic tools and immunotherapy strategies.