Gus A. Wright

Background: Endotoxemia is a severe inflammatory condition that is characterized by acute immune responses and oxidative stress; endotoxemia can further develop into a cytokine storm and sepsis leading to severe organ damage. Our recent studies revealed that the growth hormone secretagogue receptor (GHSR) regulates macrophage polarization in obesity- and aging-associated chronic inflammation. However, its role in acute inflammation during endotoxemia remains unclear. Methods: We subjected myeloid-specific Ghsr knockout mice (LysM-Cre;Ghsr^f/f) to lipopolysaccharide (LPS)-induced endotoxemia in vivo and treated bone marrow-derived macrophages (BMDMs) with LPS in vitro. Subsequently, mouse survival rate and inflammatory signatures in the blood, peritoneal cavity, liver, and BMDM were assessed. In the ex vivo study, conditioned medium (CM) from BMDMs was applied to primary hepatocytes to assess how BMDM-derived CM influences hepatocyte inflammatory responses. Results: Myeloid-specific Ghsr knockout mice exhibited a significantly improved survival rate following LPS-induced endotoxemia, accompanied by reduced systemic inflammation, evident in the blood, peritoneal macrophages, and liver. In addition, Ghsr deficiency suppressed LPS-induced caspase-1 activation and pro-inflammatory cytokine secretion in macrophages. Consistent with these results, conditioned media from Ghsr-deficient BMDMs attenuated the inflammatory responses of primary hepatocytes. Mechanistically, LPS increased GHSR expression in BMDMs, and Ghsr-deficient BMDMs activated mitochondrial respiration and suppressed production of mitochondrial reactive oxygen species (ROS), resulting in downregulation of inflammatory activation of macrophages following LPS exposure. Conclusions: These data demonstrate that macrophage GHSR promotes systemic and tissue inflammation during endotoxemia by regulating mitochondria-associated macrophage polarization. The findings suggest that macrophage GHSR may represent a promising immunomodulatory target for acute inflammatory states, including endotoxemia and sepsis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, can infect animals by binding to the angiotensin-converting enzyme 2 (ACE2). Equine infection appears possible due to high homology (≈97%) between human and equine ACE2, evidence of in vitro infection in cell lines expressing equine ACE2, and evidence of seroconversion in horses after exposure to persons infected with SARS-CoV-2. Our objective was to examine susceptibility of cultured primary equine bronchial epithelial cells (EBECs) to a SARS-CoV-2 pseudovirus relative to human bronchial epithelial cells (HBECs; positive control). ACE2 expression in EBECs detected by immunofluorescence, western immunoblotting, and flow cytometry was lower in EBECs than in HBECs. EBECs were transduced with a lentivirus pseudotyped with the SARS-CoV-2 spike protein that binds to ACE2 and expresses the enhanced green fluorescent protein (eGFP) as a reporter. Cells were co-cultivated with the pseudovirus at a multiplicity of infection of 0.1 for 6 hours, washed, and maintained in media. After 96 hours, eGFP expression in EBECs was demonstrated by fluorescence microscopy, and mean Δ Ct values from quantitative PCR were significantly (P < 0.0001) higher in HBECs (8.78) than HBECs (3.24) indicating lower infectivity in EBECs. Equine respiratory tract cells were susceptible to infection with a SARS-CoV-2 pseudovirus. Lower replication efficiency in EBECs suggests that horses are unlikely to be an important zoonotic host of SARS-CoV-2, but viral mutations could render some strains more infectious to horses. Serological and virological monitoring of horses in contact with persons shedding SARS-CoV-2 is warranted. IMPORTANCE: This study provides the first published evidence for SARS-CoV-2 pseudovirus infection in equine airway epithelial cells, which were less susceptible to infection than cells of human origin. This was presumably due to lower ACE2 expression in equine cells, lower viral affinity for equine ACE2, or both. Our results are important considering recent evidence for asymptomatic seroconversion in horses following exposure to COVID-19 positive humans, despite this lower susceptibility, and increased affinity of viral variants of concern for equine ACE2 compared to ancestral strains. Thus, there is great need to better characterize SARS-CoV-2 susceptibility in horses for the benefit of veterinary and human health.

Colorectal cancer (CRC) is a highly complex disease with multiple risk factors. The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in several cancers and is a negative prognostic factor for cancer patient survival. Previous reports indicate a potential role for overexpression of NR4A1 in T-cell exhaustion and in this study, we aim to investigate the antitumorigenic activity of two bis-indole derived ligands (DIMs) that act as receptor antagonists. Immune competent C57BL/6 mice and mouse MC-38 colon cancer cells were used and tumor Infiltrating Lymphocytes (TILs) were isolated from mice either untreated or treated with CDIM/NR4A1 antagonists. FACS analysis and Real -Time PCR were performed to determine expression of exhaustion markers in these tumor T-cell population. 1,1-Bis(3΄-indolyl)-1-(3-bromo-5-trifluoromethoxyphenyl)methane (DIM-3-Br-5-OCF₃) and the 3,5-dichlorophenyl analog (DIM-3,5-Cl₂) at doses of 2.5 and 7.5 mg/kg/d inhibited tumor growth and downregulated expression of PD-L1, an NR4A1-regulated gene in MC-38 - derived tumors and cells. The mouse MC-38 colon cancer cell line was used in a syngeneic mouse model of colon cancer and tumor infiltrating lymphocytes (TILs) from MC-38 cell-derived colon tumors exhibited multiple markers of T-cell exhaustion in both CD8⁺ and CD4⁺ T-cells. Lymphocytes from an enlarged spleen in these tumor-bearing animals also exhibited markers of CD8⁺ and CD4⁺ T-cell exhaustion. Analysis of CD8⁺ cells from TILs showed that treatment with the NR4A1 antagonists modulated expression of several genes associated with T-cell exhaustion namely a decrease in TOX, TOX2 and NFAT mRNAs, activation of T-Bet. The percentage of CD8⁺ and CD4⁺ T-cells from tumors and spleen expressing PD-1, 2B4 and TIGIT was decreased in the treated vs control mice and TIM3 expression was also decreased in CD8⁺ (tumors and spleen) and CD4⁺ (tumors) T-cells. These findings suggest that NR4A1 antagonists are highly effective as anticancer agents in this mouse syngeneic colon tumor model by inactivating the pro-oncogenic activities of NR4A1 in tumors and by remediating NR4A1-regulated T-cell exhaustion in tumor and splenic lymphocytes. Citation Format: Kumaravel Mohankumar, Gus Wright, Subhashree Kumaravel, Rupesh Shrestha, Lei Zhang, Maen Abdelrahim, Robert S. Chapkin, Stephen Safe. Nuclear receptor 4A1 ligands target T-cell exhaustion in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 236.

Early life exposure to particulate matter (PM) air pollution negatively impacts neonatal health. The underlying mechanisms following prenatal exposure, particularly to ultrafine particles (UFP, diameter ≤ 0.1 μm), are not fully understood; To evaluate the role of Nrf2 in response to in utero UFP exposure, we exposed time-mated Nrf2-deficient (Nrf2^−/−) or wildtype (WT) mice to filtered air (FA) or 100 μg/m³ ultrafine PM daily throughout pregnancy. Offspring were evaluated for pulmonary immunophenotypes and pulmonary/systemic oxidative stress on postnatal day 5, a timepoint at which we previously demonstrated viral respiratory infection susceptibility; Nrf2^−/− offspring exposed to FA had significantly lower average body weights compared to FA-exposed WT pups. Moreover, PM-exposed Nrf2^−/− offspring weighed significantly less than PM-exposed WT pups. Notably, PM-exposed Nrf2^−/− offspring showed a decreased pulmonary Th1/Th2 ratio, indicating a Th2 bias. Th17 cells were increased in FA-exposed Nrf2^−/− neonates yet decreased in PM-exposed Nrf2^−/− neonates. Analysis of oxidative stress-related genes in lung and oxidative stress biomarkers in liver tissues did not vary significantly across exposure groups or genotypes. Collectively, these findings indicate that the lack of Nrf2 causes growth inhibitory effects in general and in response to gestational UFP exposure. Prenatal UFP exposure skews CD4+ T lymphocyte differentiation toward Th2 in neonates lacking Nrf2, signifying its importance in maternal exposure and infant immune responses.

Background: The promise of biopharmaceuticals comprising one or more binding domains motivates the development of novel methods for de novo isolation and affinity maturation of virion-binding domains. Identifying avenues for overcoming the challenges associated with using virions as screening reagents is paramount given the difficulties associated with obtaining high-purity virus-associated proteins that retain the conformation exhibited on the virion surface.Results: Fluorescence activated cell sorting (FACS) of 1.5 × 10⁷clones taken from a naïve yeast surface-displayed human fibronectin domain (Fn3) against whole virions yielded two unique binders to Zika virions. Construction and FACS of site-directed binding loop mutant libraries based on one of these binders yielded multiple progeny clones with enhanced Zika-binding affinities. These affinity-matured clones bound Zika virions with low double- or single-digit nanomolar affinity in ELISA assays, and expressed well as soluble proteins inE. colishake flask culture, with post-purification yields exceeding 10 mg/L.Conclusions: FACS of a yeast-displayed binding domain library is an efficient method for de novo isolation of virion-binding domains. Affinities of isolated virion-binding clones are readily enhanced via FACS screening of mutant progeny libraries. Given that most binding domains are compatible with yeast display, the approach taken in this work may be broadly utilized for generating virion-binding domains against many different viruses for use in passive immunotherapy and the prevention of viral infection.

Objective: To determine the ability of an intraoperative cell salvage (IOCS) system and a leukocyte reduction filter (LRF) to remove hemangiosarcoma (HSA) cells from canine blood.Study Design: Cultured HSA cells were added to canine blood to simulate intraoperative hemorrhage and address hemoabdomen from ruptured splenic HSA. The blood/HSA cell mixture was processed through an IOCS, followed by LRF processing.Sample Population: Whole blood from 3 healthy dogs combined with cultured HSA cells.Methods: The ability of quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR), multiparameter flow cytometry, and cytologic examination to detect 50 HSA cells per milliliter of culture media was confirmed. RT‐PCR, multiparameter flow cytometry, and cytologic examination were used to determine the presence of cultured HSA cells at 4 points during processing.Results: HSA cells were found in all control samples and in all samples after IOCS but prior to LRF processing with all 3 cell detection methods. HSA cells were not found after IOCS/LRF processing with all 3 cell detection methods.Conclusion: IOCS combined with LRF processing is able to remove cultured HSA cells from canine blood. The addition of LRF to IOCS may allow application of IOCS in dogs with HSA.Clinical significance: A combination of IOCS and LRF processing may provide an alternative to allogeneic blood transfusion in dogs with hemoabdomen due to HSA.

Gene therapy represents a promising therapeutic paradigm for addressing many disorders, but the absence of a vector that can be robustly and reproducibly functionalized with cell‐homing functionality to mediate the delivery of genetic cargo specifically to target cells following systemic administration has stood as a major impediment. In this study, a high‐affinity protein–protein pair comprising a splicing‐deficient naturally split intein was used as molecular Velcro to append a HER2/neu‐binding protein (DARPin) onto the surface of a binding‐deficient, fusion‐competent lentivirus. HER2/neu‐specific lentiviruses created using this in vitro pseudotyping approach were able to deliver their genetic reporter cargo specifically to cells that express the target receptor at high levels in a co‐culture. We envision that the described technology could provide a powerful, broadly applicable platform for the incorporation of cell‐targeting functionality onto viral vectors. Biotechnol. Bioeng. 2015;112: 2611–2617.

Evidence suggests that targeting cancer cell energy metabolism might be an effective therapeutic approach for selective ablation of malignancies. Using a Seahorse Extracellular Flux Analyzer, we have demonstrated that select environmental agents can alter colonic mitochondrial function by increasing respiration-induced proton leak, thereby inducing apoptosis, a marker of colon cancer risk. To further probe bioenergetics in primary intestinal cells, we developed methodology that can be modified and adapted to measure the bioenergetic profiles of colonic crypts, the basic functional unit of the colon, and colonic organoids, an ex vivo 3D culture of colonic crypts. Furthermore, in combination with the MoFlo Astrios High-Speed Cell Sorter, we were able to measure the bioenergetic profiles of colonic adult stem and daughter cells from Lgr5-EGFP-IRES-creER^T2 transgenic mice. We examined the effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), a full arylhydrocarbon receptor agonist, known to affect gastrointestinal function and cancer risk, on the bioenergetic profiles of intestinal epithelial cells. Mouse colonic crypts, organoids, or sorted single cells were seeded onto Matrigel-precoated Seahorse XF24 microplates for extracellular flux analysis. Temporal analyses revealed distinct energy metabolic profiles in crypts and organoids challenged with TCDD. Furthermore, sorted Lgr5⁺ stem cells exhibited a Warburg-like metabolic profile. This is noteworthy because perturbations in stem cell dynamics are generally believed to represent the earliest step toward colon tumorigenesis. We propose that our innovative methodology may facilitate future in vivo/ex vivo metabolic studies using environmental agents affecting colonocyte energy metabolism.

A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.