bluuebunny/crossref_metadata_2025 huggingface dataset:
CD3ζ ITAM diversity determines chimeric antigen receptor force and function
Authors: Echelibe Hilda , Majumdar Shubhabrata , Liu Baoyu , Kolawole Elizabeth , Bettini Maria , Evavold Brian , Bettini Matthew
Published: August 2023

Chimeric antigen receptor (CAR) T cell immunotherapy is a powerful treatment for hematologic malignancies, however clinical responses vary depending on CAR T cell function and persistence. The optimization of CAR design has gained considerable interest with a new focus on calibrating CAR intracellular signals via the immunoreceptor tyrosine-based activation motifs (ITAMs) to increase fitness of CAR T cells. Accumulating evidence suggests that unique ITAM motifs at positions 1, 2 or 3 (ITAMs-A, B, and C respectively) of the CD3ζ signaling domain may have non-redundant roles in signal strength, T cell functionality, persistence, and memory formation. However, the individual contributions of each CD3ζ ITAM in the transmission of force and the activation of mechanosensitive signals is yet unexplored. We have found that human CARs expressing specific ITAM sequences (zeta-A only, zeta-B only or zeta-C only) generate differing amounts of Force and Bond Lifetime after interacting with CD19. Therefore, a CAR may act as a mechanosensor similar to a TCR discriminating the quality of the interaction with antigen as cellular derived forces are applied to the bond. The higher transmission of force directly correlates with the better anti-tumor efficacy in vitro and in vivo. Hence, understanding the mechanotransduction in CARs has important implications in “tuning” the activation of a CAR T cell without changing specificity.

Supported by the Seed Grant, Department of Pathology, University of Utah
CO-induced TTP improves efficacy through resetting tumor-associated macrophages toward M1 phenotype in combined CTLA-4 and PD-1 blockade immunotherapy
Authors: Gong Jeong Heon , Park Jeongmin , Joe Yeonsoo , Chung Hun Taeg
Published: August 2023

Combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 has improved the prognosis for many malignancies, but also occurred a high rate of immune-related adverse events (irAEs). The manifestation of these toxicities is an important limiting factor for the successful implementation of CICB, restraining its anti-tumor efficacy. Carbon monoxide (CO), a pivotal endogenous signaling molecule, is able to maintain cell and tissue homeostasis and could be used as a therapeutic regimen. In our previous reports, CO could be useful therapeutics for the regulation of various inflammatory diseases through the increase of tristetraprolin (TTP) that binds to the AU-rich elements in target mRNA. In this study, we observed that CO-induced TTP activation ameliorated colitis and hepatitis, and improved anti-tumor efficacy in CICB treatment. We found that the colitis and hepatitis in TTP deficient mice were further exacerbated by CICB treatment. Moreover, CO increases CICB efficacy through TAM to M1 polarization together with diminishes irAEs through TTP enhancement in several irAEs target organ. Thus, we suggest that CO might be efficiently decouples anti-tumor efficacy and toxicity of CICB through TTP enhancement.
Dissecting the lineage specific roles of BCL-XL in breast cancer metastasis
Authors: Carelock Madison Elizabeth , Jiao Mo , Zhang Weizhou
Published: August 2023

B-cell lymphoma X-large (BCL-XL) has been shown to promote breast cancer metastasis independently of its anti-apoptotic function. We recently found that proteolysis-targeting chimeras (PROTACs) against BCL-XL kill cancer cells as well as tumor-induced regulatory T cells (TI-Tregs). BCL-XL-targeting PROTACs can be potentially used to treat metastatic breast cancer – by direct killing of circulating tumor cells at their vulnerable stages and/or by eliciting anti-cancer immunity via T-reg cell depletion. Our pilot experiments showed opposing results with one model showing reduced metastasis by BCL-XL PROTAC treatment and the other model showing increased metastasis. Genetic deletion of BCL-XL (KO) in those two cell lines reproduces the effect of PROTAC on metastasis. We hypothesize that BCL-XL may play different roles in breast cancer metastasis depending on cellular cues from different cancer cells. We generated 4 other syngeneic mouse breast cancer BCL-XL-KO lines and confirmed that most of the breast cancer lines depend on BCL-XL for metastasis, whereas only 1 model exhibits an opposite phenotype. Additionally, we treated 4T1-tumor bearing mice with BCL-XL-targeting PROTAC that nearly significantly reduces metastatic spread to the lung. We also found a systematic inhibition of TI-Tregs by PROTACs indicating a lineage-specific function of BCL-XL in breast cancer metastasis. We conclude that BCL-XL is a viable therapeutic target for treating breast cancer metastasis, but further investigation into lineage-specific BCL-XL targeting is needed. Our future efforts will focus on investigating why BCL-XL inhibits metastasis in some cancer types, which can be used as an exclusion criterion for BCL-XL-targeting therapy.

Supported by grants from NIH/National Cancer Institute (R01 CA260239)
Fetal Tissues Reflect Decidual-Placental Inflammation
Authors: Vazquez Jessica , Keding Logan , Koenig Michelle R , Schotzko Michele , Antony Kathleen , Racine Jenna , Simmons Heather A , Shah Dinesh M , Wieben Oliver , Golos Thaddeus
Published: August 2023

Maternal inflammation can negatively impact the development of the fetus, leading to long term adverse health outcomes. We previously reported that injection of Tisseel (surgical glue) into the maternal-fetal interface led to necrosis and inflammation skewing the peripheral fetal immunome into a pro-inflammatory state. Here, we extend those observations and report that decidual inflammation leads to similar outcomes in fetal organs.

Tisseel or saline (n = 3) was injected directly into the placenta of pregnant (~GD90) rhesus macaques. C-sections were performed at ~GD150. Fetal tissues (lung, thymus, spleen, liver) were collected at necropsy, homogenates were prepared from all fetal tissues, and cytokine levels were measured by cytometric bead analysis. Mononuclear cell suspensions were prepared from thymus and spleen, labeled with antibodies against major immune cell lineages, and acquired using the Cytek Aurora. Data was analyzed using Specter (R), statistical significance was determined by Student’s t-test (p < 0.05).

Tisseel group fetuses had higher levels of IL17A (lung, spleen), MCP1, and IL6 (liver). Interestingly, there was a trend towards elevated IL6 in the thymus of saline group fetuses.

Additionally RORγt+ T cells were elevated in the thymus of Tisseel group fetuses, while TRegs were elevated in saline group. The spleen from Tisseel group fetuses had a higher proportion of TH1-like cells. Interestingly, T-bet+ B cells, linked to autoimmune disorders, were also elevated in the Tisseel group.

Our preliminary results suggest that decidual/placental inflammation leads to a pro-inflammatory immunome in fetal organs. Further studies are needed to determine the functional properties of these altered immune populations.
The mucosal immune responses of the human cervix are modulated by Neisseria gonorrhoeae
Authors: Song Wenxia , Dai Yiwei , Yu Qian , Edwards Vonetta , Tettelin Herve , Stein Daniel C
Published: August 2023

The cervix is the gate to the upper female reproductive tract (FRT), and the mucosal surface is the first line of defense against pathogens. The sexually transmitted pathogen Neisseria gonorrhoeae (GC) infects from the lower tract and does not induce inflammation until GC ascends through the cervix to the upper FRT, suggesting immune evasion. We examined local immune responses in the human cervix to GC infection during the first 24 hours using a human tissue explant model. GC inoculation increased the secretion of the pro-inflammatory cytokines IL-1β, TNF-α, and TNF-β, the anti-inflammatory cytokines IL-10 and LIF, and the chemokines IL-8, CXCL1, CXCL2, and CCL3. GC expressing opacity-associated protein Opa52 (binds to CEACAM) enhanced anti-inflammatory cytokines production. The elevated cytokine production was concurrent with increased nuclear staining of NF-κB p65. RNAseq analysis found increases in mRNA levels of cytokines and NF-κB pathway genes in GC-inoculated cervical tissues, compared to no GC controls. Inhibiting NF-κB activation by Bay reduced GC-induced secretion of pro-inflammatory but not anti-inflammatory cytokines. While macrophages and T-cells were abundant in the human cervix, colonization of GC expressing Opa52 did not recruit the immune cells, despite the induction of chemokines and cytokines. However, colonization of Opa-deleted GC recruited macrophages to the subepithelium of the cervical transformational zone and increased the number of CD3+ T-cells contacting Langerhans cells in the ectocervical epithelium. Together these results suggest that GC infection modulates the balance of the cervical cytokine environment towards anti-inflammatory to suppress local inflammatory responses.

Supported by grants from NIH RO1 AI141894 and RO1 AI123340
SPHK1 maintains endothelial barrier function and reanneals adherens junctions, thus hastens the recovery process
Authors: Eshetie Kirubel , Najafova Nurana , Graefen Bahar , Aliyeva Leyla , Tauseef Mohammad , Fazal Nadeem
Published: August 2023

Disruption of endothelial barrier is a crucial factor in the pathogenesis of tissue inflammation, the hallmark of inflammatory diseases such as diabetes and atherosclerosis. Increased endothelial permeability occurs because of loss of cell–cell contacts and disruption of cell–extracellular matrix (ECM) adhesions. Vascular injury is associated with activation of the coagulation cascade and release of thrombin, which increases endothelial permeability by activating endothelial cell surface thrombin receptor. This increase in endothelial permeability is typically followed by a recovery period of ≈2 hours, during which barrier integrity is restored. It has been surmised that thrombin signaling stimulates intrinsic repair mechanisms that restore barrier function. To validate our finding we determined endothelial barrier function using state of the art Electric Cell-substrate Impedance Sensing (ECIS) mechanism, a measure of trans-endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Our data show that 50mM thrombin increase endothelial permeability followed by complete recovery and reannealing of adherens junction in control endothelial cells. However, SPHK1 depleted cells showed significantly decrease in barrier disruption, and barrier failed recover completely compared to control monolayer. Upon treatment with S1P in SPHK1 depleted endothelia showed completely recovery in barrier function. In conclusion, our study for the first time shows that SPHK1-S1P-S1PR1 signaling pathway is emerging as a potential therapeutic target in improving endothelial barrier function and prevent coronary artery disease.

CTRE Seed Grant to Professor Nadeem Fazal, MD, PhD
Sex-specific and immune-cell-specific contributions of CD18 in myocardial ischemia-reperfusion injury
Authors: Fan Zhichao , Liu Wei , Cronin Chunxia G , Wen Lai , Ley Klaus , Liang Bruce T
Published: August 2023

Leukocyte recruitment and their mediated inflammatory responses are critical for cardiovascular diseases, including myocardial ischemia-reperfusion (I/R) injury. Blocking leukocyte recruitment in mouse knockouts (KO) of beta2 integrin (CD18) or blocking CD18 with antibodies in multiple animals significantly reduced infarct size after myocardial I/R injury. However, the cell-specific contribution of leukocyte CD18 to I/R injury is unknown. In this study, we used the newly established CD18flox/flox (hITGB2 KI) mice to address this knowledge gap. We crossed them to CSF1R-cre (CD115) and MRP8-cre (S100A8) and tested the KO of CD18 in different leukocytes. Interestingly, CSF1R-cre CD18flox/flox unexpectedly deleted CD18 in all peripheral blood leukocytes, including neutrophils, monocytes, CD4 T cells, CD8 T cells, B cells, and NK cells. It also elevated the cell number of these leukocytes in peripheral blood. In MRP8-cre CD18flox/flox mice, CD18 was only knocked out in neutrophils but not other peripheral blood leukocytes. And only neutrophil number was elevated in peripheral blood. After 35 minutes of myocardial ischemia and 24 hours of reperfusion, we found both CSF1R-cre CD18flox/flox and MRP8-cre CD18flox/flox mice have significantly reduced infarct size compared to cre-controls. However, if we distinguish the sex in analysis, we only found a significant alleviation in female but not male CSF1R-cre CD18flox/flox mice. In contrast, we observed a significant alleviation only in male but not female MRP8-cre CD18flox/flox mice. These results suggested sex-specific and immune-cell-specific contributions of CD18 in myocardial ischemia-reperfusion injury and provided new insights into CD18 targeting therapies.

This research was supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454, R41HL156322, and R44HL152710) and a startup fund from UConn Health.
Effects of cannabidiol on the differentiation of human monocytes into macrophages
Authors: Vigano MariaLuisa , Samarani Suzanne , Ahmad Ali , Costiniuk Cecilia Theresa
Published: August 2023

Background

Innate immune myeloid cells, such as macrophages, contribute to chronic inflammation when consistently activated. Recently, cannabidiol (CBD), an active non-psychoactive constituent of the Cannabis sativa plant (i.e., Marijuana), has sparked interest as a safe and effective anti-inflammatory and immunomodulatory agent. This study aimed to investigate the CBD’s effects on the differentiation of human macrophages and their cell surface receptors.

Methods

Peripheral blood was collected from healthy human donors. Monocytes, isolated from peripheral blood mononuclear cells, were differentiated with macrophage colony stimulating factor into monocyte-derived macrophages (MDM) with and without CBD (5 μM). Differentiated MDMs were then stained for different cell surface markers and analyzed by multicolor flow-cytometry. Proportions of anti-inflammatory (M2: CD206, CD71, CD163) and pro-inflammatory (M1: CD86, CD163) MDMs, and expression of myeloid lineage markers (CD14, CD16), chemokine receptor 5 (CCR5) and endocannabinoid type 2 receptor (CB2) were determined.

Results

CBD promoted a shift towards a greater proportion of M2-type vs. M1-type MDMs (p<0.05, Fisher’s exact test). Cells displayed a significantly reduced expression of CD14, CD163, CD86, CCR5 and CB2 (p<0.05, Student t-test).

Conclusions

CBD appears to promote differentiation of MDMs into anti-inflammatory phenotypes. Further studies are ongoing to determine the mechanistic pathways implicated in these processes, as these findings may have implications for the use of CBD in various disease conditions.
cd13 controls myeloid cell-cell fusion
Authors: Ghosh Mallika , Mcgurk Fraser , Shapiro Linda
Published: August 2023

Macrophages have the ability to undergo phenotypic and functional changes in response to microenvironmental cues. One such change is the formation of multinucleated giant cells (MGCs) via intercellular fusion triggered by certain bacterial or parasitic infections or implantation of medical devices that induces deleterious MGC formation in response to the foreign body. Despite advances in biomaterials, implants ultimately corrode to produce particles that initiate a foreign body response characterized by macrophage fusion into pro-inflammatory, Foreign Body Giant Cells that directly define the pathology of loosening of medical implants. We have shown that the transmembrane protein CD13 is a multifunctional molecule that regulates diverse processes such as cell-cell adhesion, receptor endocytosis and recycling and organization of the actin cytoskeleton, all of which are critical in cell-cell fusion. We demonstrate that subcutaneous foreign implants in CD13KO mice produce significantly more MGCs with increased levels of plasma pro-inflammatory cytokines. In vitro induction of fusion in CD13-deficient myeloid progenitors generated from bone marrow resulted in hyperfusion to generate MGCs that were considerably larger in size and contained many more nuclei than those from wild type progenitors, suggesting that CD13 regulates MGC formation at the stage of fusion. While expression of the key fusion proteins are typically downregulated in mature cells post-fusion, expression of these proteins is sustained at high levels in multinucleated cells lacking CD13. Thus, CD13 acts as a brake to prevent aberrant myeloid cell-cell fusion and may be a novel therapeutic target in pathological conditions mediated by abnormal cell fusion.

Supported by grants from NIH (1R21 AI15)
RELT is overexpressed in human inflammatory bowel disease and breast cancer
Authors: Cusick John K , Moua Pachai , Wong Stephanie , Chang Esther , Chou Emily , Shyu Yennie , Abram Alyssa , Alcaide Jessa , Talbott George , Yanumula Anusri , Qureshi Vaniya , Budhiraja Anuj , Cusick Gianne , Shi Yihui , Atapattu Dhammika , Fitzpatrick Leo
Published: August 2023

Receptor Expressed in Lymphoid Tissues (RELT) is a human TNFR member that activates NF-kB and is implicated in several cancers. We therefore sought to characterize the association of RELT with inflammatory bowel disease (IBD) and breast cancer (BC). Additionally, we further characterized the association of RELT with Filamin A (FLNA), an actin-binding protein that can promote tumorigenesis, or counteract tumors, depending on its cellular location. Female CB-17 SCID mice were injected with anti-CD40 monoclonal antibody to induce colitis and euthanized seven days later. Immunohistochemistry (IHC) was utilized to compare RELT expression in the mouse colitis model with uninjected control mice. IHC was utilized to compare the expression of RELT in biopsies of human IBD and non-inflamed tissue, as well as human BC versus non-malignant tissue. Luciferase assays and an X-gal morphology assay were utilized to examine cell death in the BC cell line MDA-MB-231 (231). RELT immunostaining is significantly increased in both mouse colitis and human IBD biopsies in comparison to that of species-matched non-inflamed tissue. An apparent association between colonic histological damage and RELT immunostaining was observed in both mice and human specimens. RELT staining was also increased in human BC biopsies versus patient-matched non-malignant tissue. RELT overexpression induced death in 231 cells, an effect that was not abrogated by a RELT mutation in the binding site for the OXSR1 kinase. However, co-expression of a S2152A mutant of FLNA did abrogate the ability of RELT to induce death in 231 cells. Collectively, these results implicate RELT as being important for human disease and further our understanding of the interaction between RELT and FLNA.
Dynamics of immune cells in the fetal brain upon viral inflammation
Authors: Arreguin Montserrat Carolina , Wu Vincent , Betts Michael , Jurado Kellie Ann
Published: August 2023

The fetal immune system undergoes many developmental milestones, with several immune cell subsets emerging during pregnancy. Recent evidence has shown that maternal infections during pregnancy can influence offspring immunity in the long term. Moreover, maternal infections are associated with an increased risk for neurodevelopmental disorders in the offspring. Yet, our knowledge of the immune cell dynamics in the fetal brain throughout gestation and their response to inflammation remains limited. We sought to characterize the phenotype of immune cells in murine implantation sites at mid-gestation, a time point equivalent to the beginning of the second trimester in humans. Our preliminary studies revealed specific and robust populations of maternal and fetal immune cells in murine fetal brains. Quite unexpectedly, one of these populations expressed NK1.1, a marker for natural killer cells and innate lymphoid cells. Given that the presence of maternal and fetal NK1.1+ cells in the fetal brains at mid-gestation has not been previously reported, our goal is to determine the phenotype and kinetics of the maternal and fetal NK1.1+ immune cells in the fetal brain during a healthy pregnancy and upon congenital infection. To address this, I will use a high-dimensional sequencing technology, CITE-seq, that allows me to simultaneously study surface markers and gene expression of cells. Maternal immune responses critically impact fetal development during gestation and have long-term health outcomes in offspring. Understanding maternal and fetal immune responses to inflammation will inform the origin of fetal inflammation-driven neurodevelopmental disorders and ultimately improve offspring’s health outcomes.

Supported by Packard Foundation Fellowship for Science and Engineering
IL-1 signaling directs the prioritization of inflammatory sites in a model of post-operative pulmonary infection
Authors: Crane Meredith , Xu Yun , Monaghan Sean F , Hall Benjamin M , Henry William L , Tran Holly L , Albina Jorge E , Jamieson Amanda
Published: August 2023

Individuals recovering from surgery are at risk of nosocomial infections such as pneumonia. In this setting, the innate immune system faces competing inflammation in the injured skin and infected lung, raising the question of how the immune response prioritizes inflammatory sites. We hypothesized that the initiation of a lung infection would disrupt wound healing. Supporting this hypothesis, data from the ACS NSQIP database showed that the rate of laparotomy dehiscence in surgical patients with pneumonia was over double the rate of those without pneumonia. We next developed mouse models of post-operative pulmonary infection to assess cellular mechanisms. Using the tail skin excision and dorsal subcutaneous PVA sponge implantation wound healing models, we found that bacterial lung infection slowed the rate of wound closure and impaired wound neutrophil, monocyte, and cytokine/chemokine responses. Lung infection quickly suppressed wound fluid IL-1b and downstream chemokine concentrations within 6 hours of inoculation. IL-1 receptor antagonist (IL-1RA), an inhibitor of IL-1 signaling, was upregulated in the bronchoalveolar lavage fluid and plasma 6 hours after lung infection. Administration of IL-1RA to the wounds of uninfected mice phenocopied the effects of lung infection. Conversely, rIL-1b rescued wound inflammation when administered to the to the wounds of mice with lung infection, although this occurred at the expense of lung bacterial clearance. These results suggest that the infected lung induces IL-1RA systemically as a protective mechanism to tune inflammation at distal sites via IL-1b suppression, allowing the innate immune system to prioritize inflammatory insults.

Supported by grants from Defense Advanced Research Projects Agency (DARPA), Brown University (Dean’s Areas of Emerging New Science Award, Carney Institute Innovation Awards), and NIH (NIES T32-ES7272 Training in Environmental Pathology; NIGMS COBRE Award P20GM10935, NHLBI R01 126887),
An invertebrate model using Galleria mellonella to study the innate immune response to Staphylococcus haemolyticus
Authors: Mares Chris Anthony , McGehee Rebecca , Smyth Davida
Published: August 2023

Staphylococcus haemolyticus is a normal inhabitant of healthy skin and is also an emerging cause of nosocomial infections in the immunocompromised. S. haemolyticus infections often lead to bacteremia, septicemia, peritonitis, endocarditis and various other outcomes. The goal of this project was to establish an invertebrate infection model for studying the innate immune response to Staphylococcus haemolyticus infections using the wax moth larva (Galleria mellonella). We have found that injecting wax worm larvae with 106 to 107 cfu/μL of S. haemolyticus consistently induced a rapid response and larval death within 24 hours at the higher dose range. We have characterized the general histopathological changes associated with infection and have started to characterize the general innate immune response as well as the hemocyte response to S. haemolyticus. Our findings support the usefulness of this model as an initial screening tool for systemic microbial infections.

Supported by grants from the Research Council and the College of Arts & Sciences at Texas A&M University-San Antonio.
Deciphering immunological mechanisms underlying the efficacy of nanoparticle-based vaccines against opioid use disorder (OUD)
Authors: Hamid Fatima Alamin Awad Alkareem , Schactler Scott , Walter Debra , Amin Hardik , Le Minh , Burkhart David , Zhang Chenming , Pravetoni Marco
Published: August 2023

The highly complex OUD and overdose epidemic poses a huge public health and economic burden, urging the need for safe and more effective therapeutic options. Vaccines offer a promising strategy for OUD treatment and prevention of overdose. Vaccinating against opioids generates drug-specific polyclonal antibodies (Ab) that selectively bind targeted opioids, blocking their distribution to the brain and thus blunting opioid induced rewards, pharmacological and side effects. Previous clinical trials of addiction vaccines showed proof of efficacy only in subjects with the highest Ab titers, highlighting the need to design more effective vaccines. The generation of a robust anti-drug polyclonal Abs response relies on CD4+ T cell-dependent B cell activation, hence dendritic cells (DC) are crucial for initiating CD4+ T cells priming and B cell responses. To improve vaccine formulation, we are exploiting a lipid-polymer hybrid nanoparticle (LPNP) platform and toll-like receptor (TLR) agonists to potentiate antigen recognition by antigen presenting cells (APCs) and subsequently the efficacy of anti-opioid conjugate vaccines. Using a flow cytometry-based in vitro activation assay, we demonstrated that LPNP-based vaccines are more effective in inducing macrophages activation marker (iNOS) and DCs co-stimulatory molecules and maturation markers (CD86, CD40, and MHC II) compared to conventional vaccines. Adding selected TLR agonists further improved nanovaccine efficacy in inducing the maturation and activation of APCs. These findings were confirmed with RT-qPCR quantifying mRNA expression of above-mentioned markers. These studies will identify key APCs subsets contributing to vaccine efficacy against OUD and other targets.

Supported by grants from NIH (UG3/UH3 DA048775)
Uncovering alterations in the murine interferon response due to dsRNA mycovirus during Aspergillus fumigatus infection
Authors: Rapp Alexander , Wang Xi , Ross Brandon , Tanner Christopher , Caffrey-Carr Alayna , Cramer Robert , Obar Joshua J , Olive Andrew , Thomas Sean
Published: August 2023

Aspergillus fumigatus is a mold that causes an array of diseases, the most severe form being invasive aspergillosis which occurs in highly 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. Here, we demonstrate that TGF-beta metabolically programs alveolar macrophages to be potent interferon inducers via enhanced dependency on oxidative phosphorylation and peroxisome biogenesis. The type I/III interferon responses were partially related to the inability 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 warrant further explorations. In this study we identified key metabolic 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.
Intravenous BCG Induction of Innate Memory in Young Pigs
Authors: Byrne Kristen A , Loving Crystal L
Published: August 2023

Exposure to vaccine strain of Mycobacterium bovis(M. bovis), Bacillus Calmette-Guerin (BCG), alters innate immune cells through epigenetic and metabolic modifications resulting in heightened responses to subsequent microbial insult (innate memory). As a biomedical model for human disease, pigs provide a critical space to study the role of innate memory in disease resistance. Young pigs were inoculated intravenous (IV) or intraperitoneal (IP) with either live or heat-inactivated BCG (IV-live, IP-live, IV-inactive, noBCG groups). At 2wks post BCG inoculation, monocyte production of IL-1β nor TNF cytokines following ex vivo stimulation with LPS was not different in any BCG group compared to noBCG, i.e. no innate memory. As dose of live BCG was lower than anticipated, at 3wks post primary inoculation, all pigs were inoculated a second time with respective preparations and routes of BCG. Ex vivo cytokine production in LPS stimulated monocytes was measured again at 5wks post primary inoculation (2wks post-secondary inoculation) and both IL-1β and TNF cytokines were elevated in IV-live (innate memory), but not IP-live or IV-inactive groups. Additionally, ex vivo stimulation of PBMC with homologous recall antigen (purified proteins from M. bovis) resulted in a 3-fold higher IFNγ response in IP-live compared to IV-live and no IFNγ production in noBCG or IV-inactive. Together these data suggest that while BCG can induce a state of innate memory in monocytes of young pigs, induction of training cannot be inferred by T cell production of IFNγ to recall protein antigen. Additionally, data continue to highlight pigs as a valuable model as multiple BCG factors (i.e. route, dose, or viability) may need to be considered for induction of innate memory.

USDA-ARS CRIS project 5030-32000-225-000D
SARS-CoV-2 and SARS-CoV-2 Spike protein S1 subunit Trigger Proinflammatory Response in Macrophages in the Absence of Productive Infection
Authors: Miller Lindsay Grace , Chiok Kim , Bose Santanu , Miura Tanya A
Published: August 2023

One of the hallmarks of critically ill COVID-19 patients infected with SARS-CoV-2 is exaggerated inflammatory response. Though macrophages mediate inflammatory responses and can produce pro-inflammatory cytokines to eliminate pathogens, infection with SARS-CoV-2 has been shown to cause immune dysfunction, leading to hyperinflammation in the lungs. To further understand the role of macrophages in hyperinflammatory responses during SARS-CoV-2 infection, we infected a THP-1 human derived macrophage cell line with SARS-CoV-2. Our results show that, though macrophages do not support viral replication, infection with SARS-CoV-2 still results in the upregulation of the mRNA of cytokines TNFα and CXCL10, which are markers of COVID-related hyperinflammation. In addition, we identified SARS-CoV-2 Spike protein S1 subunit as one viral factor involved in the upregulation of cytokines in macrophages. We show that glycosylated, soluble S1 protein can upregulate TNFα and CXCL10 mRNAs, as well as the secretion of TNFα, in macrophages in the absence of virus infection. Therefore, macrophage activation by the S1 subunit and SARS-CoV-2 infection may contribute to the hyperinflammation in the lungs seen in critically ill patients through the upregulation of proinflammatory cytokines such as TNFα and CXCL10.

Supported by NIH grant (R01 AI083387), Washington Research Foundation, and NIH NIGMS Predoctoral Biotechnology Training Grant 5T32GM008336
Ebola virus protein VP40 induces accessory cell-mediated activation of human natural killer (NK) cells
Authors: Singh Karnail , Le Hung
Published: August 2023

Natural killer (NK) cells have been implicated in Ebola virus disease (EVD) pathogenesis as well as in protection against Ebola viruses. A clear understanding of their role during EVD is needed for better exploiting their functionalities in designing effective countermeasures against Ebola viruses. Previously, we reported that Ebola virus protein VP40 induces IL-12 and IL-18 dependent activation of human NK cells that involves accessory cells. Here, we extended our studies to define the identities of the accessory cells and molecular mechanism(s) underlying their role in triggering NK cell activation. Purified CD14+ myeloid cells, CD3+ T cells or CD19+ B cells, isolated from normal human peripheral blood mononuclear cells (PBMCs), were co-cultured with purified CD56+ NK cells; cultures activated with VP40 and expression of IFN-γ, granzyme B, perforin, and surface CD107a in NK cells was measured. A small, but significant, increase in NK cell activation was observed when purified NK cells alone were incubated with VP40. Inclusion of CD14+ myeloid cells or CD3+ T cells in NK cell cultures resulted in significant additional increase in NK cell activation status. Both soluble factors and cell-cell contact dependent mechanisms were involved in VP40-induced, accessory cell dependent NK cell activation. In addition, a small subset of NK cells bound directly to VP40 and was triggered. Together, our data decipher both accessory cell dependent and independent mechanisms involved in Ebola VP40 induced NK cell activation, findings that may help in designing better anti-Ebola countermeasures.

Supported by funds from Cincinnati Children’s Research Foundation.
Neutrophilia and neutrophil-derived extracellular traps as innate immune markers of SARS-CoV-2 infection in a feline animal model
Authors: Gunasekara Sachithra J , Ludwig Breya , Cowan Shannon , Selvan Miruthula Tamil , Ritchey Jerry , Miller Craig , Rudd Jennifer
Published: August 2023

Excessive neutrophil recruitment and dysregulation contribute to worsened clinical outcomes in hospitalized COVID-19 patients. Targeting this dysregulated response offers therapeutic potential in the treatment of SARS-CoV-2 infections which can be enhanced by the availability of reliable, translational animal models for disease. Domestic cats are naturally infected and exhibit clinical disease similar to that seen in COVID-19. Utilizing our previously developed feline model for COVID-19, we hypothesize that neutrophil dysregulation and the release of neutrophil extracellular traps (NETs) significantly worsen pulmonary damage in domestic cats. Specific-pathogen-free cats (n=12) were inoculated with SARS CoV-2 (B.1.617.2) or vehicle (n=6). Plasma, bronchoalveolar lavage, and tissues were collected over 12 days. Detection of NETs using MPO-DNA ELISA and Quant-iT PicoGreen® dsDNA assay indicate significant elevations at 4dpi and 8dpi in the plasma as compared to vehicle, with the peak NETs detection at 4dpi(p<0.05). These findings were similarly evident in the BAL fluid of infected versus vehicle-inoculated cats(p<0.05). Immunohistochemistry of respiratory tissues utilizing three major markers of activated neutrophils and NETS; citrullinated histone 3, MPO, and neutrophil elastase, show marked amplifications compared to vehicle. These findings co-localize to areas of significant pulmonary damage. Elucidating these innate markers and their functional changes provides insight into understanding the immunopathogenesis and potential therapeutic interventions in SARS-CoV-2 infection. These data enforce the role of domestic cats as a flexible translational model to develop these interventions.

NIH-NIGMS (P20GM103648)
MrgprD-expressing nonpeptidergic neurons suppress mast cell responsiveness via glutamate release
Authors: Zhang Youran , Edwards Tara , Zhang Shiqun , Sumpter Tina L , Kaplan Daniel H
Published: August 2023

Cutaneous mast cells underly numerous skin inflammatory processes. Our study reveals that the ablation of a subset of sensory nonpeptidergic neurons expressing MrgprD increases the expression of a mast cell gene module, including the activating receptor, Mrgprb2, leading to increased mast cell reactivity to MrgprB2 ligands, and cutaneous inflammation in several disease models, such as irritant dermatitis and rosacea. MrgprD-expressing neurons releases glutamate to inhibit mast cell responsiveness. Reducing glutamate release from MrgprD-expressing neurons in the periphery resulted in a hyperresponsive state of mast cells. The antagonism of glutamate receptors on cultured peritoneal cavity mast cells (PCMCs) enhances MC degranulation and phenocopied the genetic state in the absence of MrgprD-expressing neurons. We subsequently identifies the kainate receptor GluR6/KA2 as the glutamate receptor subtype expressed on dermal MCs. Two non-specific agonists of GluR6, and the highly selective GluR6 agonist SYM2081 suppress the degranulation of PCMC in response to MrgprB2 activation but not FcɛRI crosslinking in a dose-dependent manner. In vivo, intradermal delivery of SYM2081 suppresses compound 48/80 induced-dermal MC activation, and attenuates croton oil-induced irritant dermatitis, but not IgE-dependent passive cutaneous anaphylaxis. Together, these data demonstrate that glutamate acting via the GluR6/KA2 receptor promotes mast cell hypo-responsiveness during homeostasis and suggests that selective agonism of this pathway has the potential to reduce mast cell activation in vivo, which would be of significant utility in the treatment of diseases where mast cells represent a key component of the pathogenic pathway.
Dysregulation of natural killer cells’ receptor expression and cytotoxic function by hydroxychloroquine
Authors: Elemam Noha M , Talaat Iman M , Maghazachi Azzam A
Published: August 2023

Background and Aim

Natural killer (NK) cells are crucial players of the innate immune system, especially in the fight against tumors and infections. Besides, NK cells are known to contribute in inflammatory processes associated with multiple diseases. The drug hydroxychloroquine (HCQ) has been repurposed to be used for the treatment of malaria, autoimmune diseases, and COVID-19 infection. However, its direct mechanism of action on NK cells remains unknown. In this study, we aimed to examine the effects of HCQ on NK cell activity and function.

Materials and Methods

The human NK cell line NK92 was cultured and treated with HCQ at 1, 5, and 10 μM for 24 and 48 hours. Then, NK92 cell viability, NKG2D and CD107a expression, as well as NK92 cell cytotoxicity were assessed. Also, IFN-γ production was measured by ELISA after HCQ treatment. Statistical analysis was performed where p<0.05 was considered statistically significant.

Results

Upon treatment with different concentrations of HCQ for 48 hours, NK92 cell viability significantly decreased in a dose-dependent manner, with the lowest viability noted at 10 μM of HCQ. Additionally, NKG2D and CD107a expression were significantly reduced upon treatment with 5 and 10 μM of HCQ. Similarly, the cytotoxicity of NK92 cells against K562 cells was reduced at both 5:1 and 10:1, effector to target (E: T) cell ratios, when 10 μM HCQ was used. In contrast, IFN-γ levels were significantly increased upon the treatment with 1, 5, and 10 μM HCQ.

Conclusion

This study highlights the effect of HCQ on activating receptor expression, cytotoxicity, and IFN-γ production of NK cells. Despite being an immunomodulatory drug, more studies are needed to further understand HCQ’s effects on NK cells and perhaps other effector cells.

None
Infiltrative monocyte derived adenosine deaminase 2 is associated with COVID-19 related acute respiratory distress syndrome
Authors: Tiwari-Heckler Silpa , Vierbaum Lisa , Merle Uta , Jiang Zhenghui Gordon
Published: August 2023

Acute respiratory distress syndrome (ARDS) is a severe complication of COVID-19 characterized by immune dysregulation. The pathogenesis for this, particularly the involvement of infiltrative monocytes, is poorly understood. Extracellular ATP released during inflammation and hypoxia is rapidly scavenged into adenosine, which is further converted into inosine by adenosine deaminase (ADA). ADA2 is an extracellular isoform of ADA secreted by activated monocytes and expressed by infiltrated CD16+CXCR3+ monocytes.

Herein, we aim to study the impact of monocyte-derived ADA2 in the pathogenesis of COVID-19 associated ARDS. Critically ill COVID-19 patients were assigned to the ARDS group based on Berlin criteria, while the non-ARDS group included COVID-19 patients without any or with low-flow oxygen therapy.

We observed elevated ADA activity in the serum of ARDS patients, reflecting increased level of the secreted ADA2 protein, when compared to the non-ARDS group. ARDS patients exhibited high level of circulating IL-6, CXCL8 and CXCL10. Notably, correlation analysis revealed a significant association between circulating ADA activity and CXCL10, a chemokine for CXCR3+ monocytes. Post-mortem bulk RNAseq analysis of lung tissues showed increased expression of the ADA2 gene, CECR1, along with CXCL10 suggesting intrapulmonary accumulation of ADA2 positive macrophages. These observations were further supported by the analysis of single-nuclear and spatial transcriptomic atlas of lung tissues of severe COVID-19 patients, which demonstrated a strong expression of ADA2 in CD16+ monocytes.

In summary, our data suggest a potential role of infiltrative monocyte-derived ADA2 in modulating the immune response in COVID-19 related ARDS.

Supported by the German Center for Infection Research (TI 07.005 to STH)
TNFα-Mediated Damaging Inflammation During Aspergillus fumigatus Pulmonary Infection is Regulated by Type I Interferon Receptor 2 (IFNAR2)
Authors: Shepardson Kelly , Roemer Julia , Benson Evelyn , Hall Monica , Rynda-Apple Agnieszka
Published: August 2023

Invasive pulmonary aspergillosis (IPA), caused by the fungus Aspergillus fumigatus, generally occurs in patients with suppressed or non-functioning immune systems. The recent increase in the number of cases of influenza and SARS-Cov-2-infected patients acquiring IPA suggests that severe respiratory viral infections may create a suppressed lung immune environment that allows for fungal infections to occur. We recently found that differential type I interferon (IFN) signaling, a classic anti-viral host response, regulates susceptibility to IPA. Specifically, we found that absence of the type I IFN receptor 2 (IFNAR2) subunit of the heterodimeric IFNAR1/2 receptor, leads to increased damage and inflammation in response to A. fumigatus lung infection, while absence of IFNAR1 did not. Although the Ifnar2−/− mice had higher morbidity, we unexpectedly found that Ifnar2−/− mice killed more A. fumigatus by 24 hours post-infection compared to WT and Ifnar1−/− mice. However, this early clearance of A. fumigatus did not prevent invasive disease from developing in the Ifnar2−/− mice as infection progressed, suggesting that the early damage responses were creating a favorable environment for invasive growth. By altering the inflamed environment of the Ifnar2−/− mice early during infection, via neutralization of TNFα, we were able to reverse the damage and morbidity in these mice back to WT levels. Further, our results suggest that altered myeloid effector cell responses in Ifnar2−/− mice during A. fumigatus infection contribute to the damage that occurs. Together, our results expand our understanding of how anti-viral mechanisms, via IFNAR2, are creating a permissive environment for fungal infections to occur through regulation of damage.

Supported by grants from NIH/NIGMS (INBRE P20GM103474), NIH/NIAID (K22AI153671), AAI Careers in Immunology Fellowship, Parker B. Francis Fellowship
Murine Eosinophils Release IL-1β via a Non-Lytic Mechanism Dependent on Gasdermin D (GSDMD) Expression but Independent of GSDMD Proteolytic Processing
Authors: Nguyen Thu R , Diaz-Aponte Jose R , Dubyak George R
Published: August 2023

Physiological roles for gasdermin D (GSDMD) in pyroptosis and IL-1β release during inflammasome signaling have been well-characterized in macrophages. This involves GSDMD cleavage by caspase-1 to generate N-GSDMD products that assemble as macropores in the plasma membrane (PM) to mediate direct IL-1β efflux and eventual pyroptosis. Granulocytes, including neutrophils and eosinophils, are also important sources of IL-1β in multiple innate immune responses. In neutrophils, we have previously reported that GSDMD expression and proteolytic processing to N-GSDMD is required for IL-1β release but is independent from accumulation of PM N-GSDMD pores and pyroptosis due to alternative trafficking of N-GSDMD to azurophilic granules and autophagosomes; this atypical GSDMD-dependent IL-1β release is mediated in part by secretory autophagy. We have now tested whether this alternative GSDMD-dependent mechanism for non-lytic IL-1β release is also operative in eosinophils using primary murine bone marrow-derived eosinophils (BMDE) from wildtype (C57Bl/6) or Gsdmd−/−mice and stimulated with activators of NLRP3 inflammasome signaling. NLRP3 inflammasome-activated BMDE rapidly process and release IL-1β via a mechanism dependent on GSDMD expression but independent of GSDMD proteolytic processing, N-GSDMD pore accumulation in the PM, and pyroptosis. This requirement for GSDMD in IL-1β release from BMDE is operative during the initial phases (~60 min) of NLRP3 inflammasome activation but is then complemented by a yet-be-defined GSDMD-independent pathway with prolonged stimulation. These findings indicate a role for GSDMD as a chaperone rather than pore-forming protein and distinct from mechanisms defined in neutrophils.

Supported by grants from (NIH P01-AI141350, R01-EY014362).
Aryl polyenes expressed by Uropathogenic Escherichia coli contribute to pathogenesis in a mouse model with an aberrant macrophage polarization
Authors: Markley R L , Johnston Isabel , Osborn Lucas J , DeLucia Beckey , Alqudah Sara , Bobba Viharika , Nia Layan Hamidi , Ouyang Kelsey , Dester Emma , Ginter-Frankovitch Maggie , Claesen Jan
Published: August 2023

Uropathogenic Escherichia coli (UPEC) is a causative agent of urinary tract infections (UTIs), and one of the most common causes of pyelonephritis. Interestingly, UPEC is often found as a non-pathobiont in the gut flora. The transition from gut flora to pathogen for UPEC is dependent upon a flexible tropism, and a number of virulence factors that aid in UPEC’s colonization and invasion of the urinary tract. We recently demonstrated a role for the E. coli aryl polyenes (APEs) as a potential UPEC fitness factor. The current hypothesis is that APEs contribute to UPEC virulence in the host system. APEs are encoded by a biosynthetic gene cluster (BGC) family identified among many gram-negative bacteria. The products of this BGC family are lipids comprised of an aryl head group and conjugated double bond system, which are structurally reminiscent of established virulence factors found in gram-positives. In addition to the structural mimicry, UPEC APE expression conferred resistance to reactive oxygen species and enhanced biofilm formation. In a UTI mouse model, infection with UPEC that constitutively expressed APEs (UPECAPE+) increased pathogenicity. Transcriptomic analyses of in vitro co-incubation experiments indicates that UPECAPE+ exposure influences the macrophage gene transcription profile differently as compared to UPECWT. Taken together, our findings suggest that APE expression aids in bacterial virulence and immune modulation during infection. Therefore, targeting the biosynthetic gene product APE may be a viable therapeutic approach to reduce fitness of pathogenic populations while leaving commensal gut microflora unharmed and minimize tissue damage.

NIAID RO1AI153173 Cleveland Clinic Foundation