PSGL-1: a novel immune checkpoint driving T-cell dysfunction in obstructive sleep apnea.

Introduction: Although higher incidence of cancer represents a major burden for obstructive sleep apnea (OSA) patients, the molecular pathways driving this association are not completely understood. Recently, the adhesion receptor P-selectin glycoprotein-1 (PSGL 1) has been identified as a novel immune checkpoint, which are recognized major hallmarks in several types of cancer and have revolutionized cancer therapy. Methods: The expression of PSGL-1 and its ligands VISTA and SIGLEC-5 was assessed in the leucocytes of OSA patients and control subjects exploring the role of intermittent hypoxia (IH) using in vitro models. In addition, PSGL-1 impact on T-cells function was evaluated by ex vivo models. Results: Data showed PSGL-1 expression is upregulated in the T-lymphocytes from patients with severe OSA, indicating a relevant role of hypoxemia mediated by intermittent hypoxia. Besides, results suggest an inhibitory role of PSGL-1 on T-cell proliferation capacity. Finally, the expression of SIGLEC-5 but not VISTA was increased in monocytes from OSA patients, suggesting a regulatory role of intermittent hypoxia. Discussion: In conclusion, PSGL-1 might constitute an additional immune checkpoint leading to T-cell dysfunction in OSA patients, contributing to the disruption of immune surveillance, which might provide biological plausibility to the higher incidence and aggressiveness of several tumors in these patients.

Hypoxia Attenuates Colonic Innate Immune Response and Inhibits TLR4/NF-κB Signaling Pathway in Lipopolysaccharide-Induced Colonic Epithelial Injury Mice.

High altitude hypoxia can lead to a spectrum of gastrointestinal problems. As the first line of host immune defense, innate immune response in the intestinal mucosa plays a pivotal role in maintaining intestinal homeostasis and protecting against intestinal injury at high altitude. This study aimed to investigate the effect of hypoxia on the colonic mucosal barrier and toll-like receptor 4 (TLR4)-mediated innate immune responses in the colon. The mice were exposed to a hypobaric chamber to simulate a 5,000 m plateau environment for 7 days, and the colonic mucosa changes were recorded. At the same time, the inflammation model was established by lipopolysaccharide (LPS) to explore the effects of hypoxia on the TLR4/nuclear factor kappa B (NF-κB) signaling pathway and its downstream inflammatory factors [tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and interferon (IFN)-γ] in the colon. We found that hypoxic exposure caused weight loss and structural disturbance of the colonic mucosa in mice. Compared with the control group, the protein levels of TLR4 [fold change (FC) = 0.75 versus FC = 0.23], MyD88 (FC = 0.80 versus FC = 0.30), TIR-domain-containing adaptor protein inducing interferon-ß (TRIF: FC = 0.89 versus FC = 0.38), and NF-κB p65 (FC = 0.75 versus FC = 0.24) in the colon of mice in the hypobaric hypoxia group were significantly decreased. LPS-induced upregulation of the TLR4/NF-κB signaling and its downstream inflammatory factors was inhibited by hypoxia. Specifically, compared with the LPS group, the protein levels of TLR4 (FC = 1.18, FC = 0.86), MyD88 (FC = 1.20, FC = 0.80), TRIF (FC = 1.20, FC = 0.86), and NF-κB p65 (FC = 1.29, FC = 0.62) and the mRNA levels of IL-1ß (FC = 7.38, FC = 5.06), IL-6 (FC = 16.06, FC = 9.22), and IFN-γ (FC = 2.01, FC = 1.16) were reduced in the hypobaric hypoxia plus LPS group. Our findings imply that hypoxia could lead to marked damage of the colonic mucosa and a reduction of TLR4-mediated colonic innate immune responses, potentially reducing host defense responses to colonic pathogens.

Targeted inhibition of the GRK2/HIF-1α pathway is an effective strategy to alleviate synovial hypoxia and inflammation.

G-protein coupled receptor (GPCR) kinases (GRKs) and hypoxia-inducible factor-1α (HIF-1α) play key roles in rheumatoid arthritis (RA). Several studies have demonstrated that HIF-1α expression is positively regulated by GRK2, suggesting its posttranscriptional effects on HIF-1α. In this study, we review the role of HIF-1α and GRK2 in RA pathophysiology, focusing on their proinflammatory roles in immune cells and fibroblast-like synoviocytes (FLS).We then introduce several drugs that inhibit GRK2 and HIF-1α, and briefly outline their molecular mechanisms. We conclude by presenting gaps in knowledge and our prospects for the pharmacological potential of targeting these proteins and the relevant downstream signaling pathways.Future research is warranted and paramount for untangling these novel and promising roles for GRK2 and HIF-1α in RA.

ISGylation directly modifies hypoxia-inducible factor-2α and enhances its polysome association.

The hypoxia-inducible factors (HIF)-1α and HIF-2α are central regulators of transcriptional programmes in settings such as development and tumour expansion. HIF-2α moonlights as a cap-dependent translation factor. We provide new insights into how the interferon-stimulated gene 15 (ISG15), a ubiquitin-like modifier, and the HIFs regulate one another in hypoxia and interferon-induced cells. We show that upon ISGylation induction and HIF-α stabilization, both HIFs promote protein ISGylates through transcriptional and/or post-transcriptional pathways. We show the first evidence of HIF-2α modification by ISG15. ISGylation induces system-level alterations to the HIF transcriptional programme and increases the cytoplasmic/nuclear fraction and translation activity of HIF-2α. This work identifies ISG15 as a regulator of hypoxic mRNA translation, which has implications for immune processes and disease progression.

Interleukin-17 governs hypoxic adaptation of injured epithelium.

Mammalian cells autonomously activate hypoxia-inducible transcription factors (HIFs) to ensure survival in low-oxygen environments. We report here that injury-induced hypoxia is insufficient to trigger HIF1α in damaged epithelium. Instead, multimodal single-cell and spatial transcriptomics analyses and functional studies reveal that retinoic acid-related orphan receptor γt+ (RORγt+) γδ T cell-derived interleukin-17A (IL-17A) is necessary and sufficient to activate HIF1α. Protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling proximal of IL-17 receptor C (IL-17RC) activates mammalian target of rapamycin (mTOR) and consequently HIF1α. The IL-17A-HIF1α axis drives glycolysis in wound front epithelia. Epithelial-specific loss of IL-17RC, HIF1α, or blockade of glycolysis derails repair. Our findings underscore the coupling of inflammatory, metabolic, and migratory programs to expedite epithelial healing and illuminate the immune cell-derived inputs in cellular adaptation to hypoxic stress during repair.

The Effect of Hypoxia and Hypoxia-Associated Pathways in the Regulation of Antitumor Response: Friends or Foes?

Hypoxia is an environmental stressor that is instigated by low oxygen availability. It fuels the progression of solid tumors by driving tumor plasticity, heterogeneity, stemness and genomic instability. Hypoxia metabolically reprograms the tumor microenvironment (TME), adding insult to injury to the acidic, nutrient deprived and poorly vascularized conditions that act to dampen immune cell function. Through its impact on key cancer hallmarks and by creating a physical barrier conducive to tumor survival, hypoxia modulates tumor cell escape from the mounted immune response. The tumor cell-immune cell crosstalk in the context of a hypoxic TME tips the balance towards a cold and immunosuppressed microenvironment that is resistant to immune checkpoint inhibitors (ICI). Nonetheless, evidence is emerging that could make hypoxia an asset for improving response to ICI. Tackling the tumor immune contexture has taken on an in silico, digitalized approach with an increasing number of studies applying bioinformatics to deconvolute the cellular and non-cellular elements of the TME. Such approaches have additionally been combined with signature-based proxies of hypoxia to further dissect the turbulent hypoxia-immune relationship. In this review we will be highlighting the mechanisms by which hypoxia impacts immune cell functions and how that could translate to predicting response to immunotherapy in an era of machine learning and computational biology.

Cardiorespiratory anomalies and increased brainstem microglia in a rat model of neonatal opioid withdrawal syndrome.

Infants born with neonatal opioid withdrawal syndrome (NOWS) can display abnormal cardiorespiratory patterns including tachypnea, tachycardia, and impaired ventilatory responses to hypoxia (HVR) and hypercapnia (HCVR). Chronic morphine exposure is associated with increased midbrain microglial expression. Using a rat model of pre- and post-natal morphine exposure, we assessed cardiorespiratory features of NOWS (resting tachycardia and tachypnea) including the attenuated HVR and HCVR and whether they are associated with increased brainstem microglia expression. Pregnant rats (dams) received twice-daily subcutaneous injections of morphine (5 mg/kg) during the third (last) week of pregnancy to simulate 3rd trimester in utero opioid exposure. Offspring then received once-daily subcutaneous injections of morphine (0.5 mg/kg) until postnatal (P) day P10 days of age to simulate postnatal morphine therapy. Cardiorespiratory responses were assessed 24 h later (P11 days) following spontaneous withdrawal. Compared to saline-treated pups, morphine-exposed offspring exhibited tachycardia and tachypnea as well as an attenuated HVR and HCVR. Microglial cell counts were increased in the nucleus tractus solitarius (nTS), dorsal motor nucleus of the vagus (DMNV) and nucleus ambiguous (NAamb), but not the retrapezoid nucleus (RTN) or the non-cardiorespriatory region, the cuneate nucleus (CN). These data suggest that the cardiorespiratory features and autonomic dysregulation in NOWS infants may be associated with altered microglial function in specific brainstem cardiorespiratory control regions.

Effects of Intermittent Hypoxia on Cytokine Expression Involved in Insulin Resistance.

Sleep apnea syndrome (SAS) is a prevalent disorder characterized by recurrent apnea or hypoxia episodes leading to intermittent hypoxia (IH) and arousals during sleep. Currently, the relationship between SAS and metabolic diseases is being actively analyzed, and SAS is considered to be an independent risk factor for the development and progression of insulin resistance/type 2 diabetes (T2DM). Accumulating evidence suggests that the short cycles of decreased oxygen saturation and rapid reoxygenation, a typical feature of SAS, contribute to the development of glucose intolerance and insulin resistance. In addition to IH, several pathological conditions may also contribute to insulin resistance, including sympathetic nervous system hyperactivity, oxidative stress, vascular endothelial dysfunction, and the activation of inflammatory cytokines. However, the detailed mechanism by which IH induces insulin resistance in SAS patients has not been fully revealed. We have previously reported that IH stress may exacerbate insulin resistance/T2DM, especially in hepatocytes, adipocytes, and skeletal muscle cells, by causing abnormal cytokine expression/secretion from each cell. Adipose tissues, skeletal muscle, and the liver are the main endocrine organs producing hepatokines, adipokines, and myokines, respectively. In this review, we focus on the effect of IH on hepatokine, adipokine, and myokine expression.

Hypoxic Processes Induce Complement Activation via Classical Pathway in Porcine Neuroretinas.

Considering the fact that many retinal diseases are yet to be cured, the pathomechanisms of these multifactorial diseases need to be investigated in more detail. Among others, oxidative stress and hypoxia are pathomechanisms that take place in retinal diseases, such as glaucoma, age-related macular degeneration, or diabetic retinopathy. In consideration of these diseases, it is also evidenced that the immune system, including the complement system and its activation, plays an important role. Suitable models to investigate neuroretinal diseases are organ cultures of porcine retina. Based on an established model, the role of the complement system was studied after the induction of oxidative stress or hypoxia. Both stressors led to a loss of retinal ganglion cells (RGCs) accompanied by apoptosis. Hypoxia activated the complement system as noted by higher C3+ and MAC+ cell numbers. In this model, activation of the complement cascade occurred via the classical pathway and the number of C1q+ microglia was increased. In oxidative stressed retinas, the complement system had no consideration, but strong inflammation took place, with elevated TNF, IL6, and IL8 mRNA expression levels. Together, this study shows that hypoxia and oxidative stress induce different mechanisms in the porcine retina inducing either the immune response or an inflammation. Our findings support the thesis that the immune system is involved in the development of retinal diseases. Furthermore, this study is evidence that both approaches seem suitable models to investigate undergoing pathomechanisms of several neuroretinal diseases.

Elevated Monocytic Interleukin-8 Expression under Intermittent Hypoxia Condition and in Obstructive Sleep Apnea Patients.

Obstructive sleep apnea (OSA) is a disease with great cardiovascular risk. Interleukin-8 (IL-8), an important chemokine for monocyte chemotactic migration, was studied under intermittent hypoxia condition and in OSA patients. Monocytic THP-1 cells were used to investigate the effect of intermittent hypoxia on the regulation of IL-8 by an intermittent hypoxic culture system. The secreted protein and mRNA levels were studied by means of enzyme-linked immunosorbent assay and RT/real-time PCR. The chemotactic migration of monocytes toward a conditioned medium containing IL-8 was performed by means of the transwell filter migration assay. Peripheral venous blood was collected from 31 adult OSA patients and RNA was extracted from the monocytes for the analysis of IL-8 expression. The result revealed that intermittent hypoxia enhanced the monocytic THP-1 cells to actively express IL-8 at both the secreted protein and mRNA levels, which subsequently increased the migration ability of monocytes toward IL-8. The ERK, PI3K and PKC pathways were demonstrated to contribute to the activation of IL-8 expression by intermittent hypoxia. In addition, increased monocytic IL-8 expression was found in OSA patients, with disease severity dependence and diurnal changes. This study concluded the monocytic IL-8 gene expression can be activated by intermittent hypoxia and increased in OSA patients.

Tumor-Associated Macrophages and Their Functional Transformation in the Hypoxic Tumor Microenvironment.

Tumor-associated macrophages (TAMs) are some of the most abundant immune cells within tumors and perform a broad repertoire of functions via diverse phenotypes. On the basis of their functional differences in tumor growth, TAMs are usually categorized into two subsets of M1 and M2. It is well established that the tumor microenvironment (TME) is characterized by hypoxia along with tumor progression. TAMs adopt an M1-like pro-inflammatory phenotype at the early phases of oncogenesis and mediate immune response that inhibits tumor growth. As tumors progress, anabatic hypoxia of the TME gradually induces the M2-like functional transformation of TAMs by means of direct effects, metabolic influence, lactic acidosis, angiogenesis, remodeled stroma, and then urges them to participate in immunosuppression, angiogenesis and other tumor-supporting procedure. Therefore, thorough comprehension of internal mechanism of this TAM functional transformation in the hypoxic TME is of the essence, and might provide some novel insights in hypoxic tumor immunotherapeutic strategies.

Trophoblast-derived interleukin 9 mediates immune cell conversion and contributes to maternal-fetal tolerance.

In the maternal-fetal crosstalk, fetal derived trophoblast cells can secret several molecules to regulate immune tolerance such as cytokines and chemokines, besides human leukocyte antigens (HLA) providing. However, the mechanism of these factors in pregnancy is still unknown. Our previous study showed that IL9 could be secreted by trophoblasts and exerted a positive effect on trophoblasts themselves through autocrine signaling. Given the immunoregulatory function of IL9 and its expression in trophoblasts, we hypothesize that IL9 contributes to maternal-fetal tolerance by regulating immune cells, especially CD14+ dendritic cells (DCs) and naïve CD4 + T cells who have essential roles in maternal-fetal immune tolerance. We performed a series of experiments, finding that HTR8/SVneo cells could secrete IL9 in vitro, and this secretion was decreased under hypoxia; both CD14 + DCs and naïve CD4 + T cells expressed IL9 receptors, indicating potential interactions among these cells. In CD14 + DCs, trophoblast-derived IL9 promoted the immature differentiation, and induced the secretion of Th2 cytokines, including IL4 and IL10, shifting the Th1/Th2 ratio to Th2. In naïve CD4 + T cells, IL9 also increased Foxp3 expression and promoted the secretion of Treg cytokines, including TGFß and IL10, inhibiting pro-inflammatory Th17. Therefore, trophoblasts may act as fetal-derived immune cells to maintain maternal-fetal tolerance by secreting IL9. Given that trophoblast derived IL9 is decreased in preeclampsia, our study provides a new insight into maternal-fetal immunology and immunological disorders in abnormal pregnancy.

Effects of hypoxia and hyperoxia on growth parameters and transcription levels of growth, immune system and stress related genes in rainbow trout.

Hypoxia and hyperoxia are disparate stressors which can have destructive influences on fish growth and physiology. It is yet to be determined if hypoxia and hyperoxia have a cumulative effect in aquatic ecosystems that affect biological parameters in fish, and to understand if this is associated with gene expression. Here we address whether growth performance and expressions of growth, immune system and stress related genes were affected by hypoxia and hyperoxia in fish. Rainbow trout was chosen as the study organism due to its excellent service as biomonitor. After an acclimatization period, fish were exposed to hypoxia (4.0 ± 0.5 ppm O2), normoxia (7.5 ± 0.5 ppm O2) and hyperoxia (12 ± 1.2 ppm O2) for 28 days. At 6 h, 12 h, 24 h, 48 h, 72 h and 28 days, samples were collected. Hypoxia and hyperoxia negatively affected weight gain (WG), specific growth rate (SGR), survival rate (SR) and feed conversion ratio (FCR). The best WG, SGR, SR and FCR values occurred in fish exposed to normoxia, whereas hypoxia was most suppressive on growth and hyperoxia showed intermediate suppression of these parameters. Gene expression analyses were performed in liver and results revealed that long term exposure caused reduced growth hormone-I (GH-I) and insulin like growth factor I-II (IGF I-II) levels in both hypoxia and hyperoxia-treated fish. Heat shock protein (HSP70) levels increased in both hypoxia and hyperoxia treatment, and both exposures caused elevation of leptin (LEP) expression in long-term exposure. Overall data indicate that both hypoxia and hyperoxia cause stress in rainbow trout and negatively affects growth parameters.

A combined hypoxia and immune gene signature for predicting survival and risk stratification in triple-negative breast cancer.

BACKGROUND: Increasing evidence showed that the clinical significance of the interaction between hypoxia and immune status in tumor microenvironment. However, reliable biomarkers based on the hypoxia and immune status in triple-negative breast cancer (TNBC) have not been well established. This study aimed to explore a gene signature based on the hypoxia and immune status for predicting prognosis, risk stratification, and individual treatment in TNBC. METHODS: Hypoxia-related genes (HRGs) and Immune-related genes (IRGs) were identified using the weighted gene co-expression network analysis (WGCNA) method and the single-sample gene set enrichment analysis (ssGSEA Z-score) with the transcriptomic profiles from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort. Then, prognostic hypoxia and immune based genes were identified in TNBC patients from the METABRIC (N = 221), The Cancer Genome Atlas (TCGA) (N = 142), and GSE58812 (N = 107) using univariate cox regression model. A robust hypoxia-immune based gene signature for prognosis was constructed using the least absolute shrinkage and selection operator (LASSO) method. Based on the cross-cohort prognostic hypoxia-immune related gene signature, a comprehensive index of hypoxia and immune was developed and two risk groups with distinct hypoxia-immune status were identified. The prognosis value, hypoxia and immune status, and therapeutic response in different risk groups were analyzed. Furthermore, a nomogram was constructed to predict the prognosis for individual patients, and an independent cohort from the gene expression omnibus (GEO) database was used for external validation. RESULTS: Six cross-cohort prognostic hypoxia-immune related genes were identified to establish the comprehensive index of hypoxia and immune. Then, patients were clustered into high- and low-risk groups based on the hypoxia-immune status. Patients in the high-risk group showed poorer prognoses to their low-risk counterparts, and the nomogram we constructed yielded favorable performance to predict survival and risk stratification. Besides, the high-risk group had a higher expression of hypoxia-related genes and correlated with hypoxia status in tumor microenvironment. The high-risk group had lower fractions of activated immune cells, and exhibited lower expression of immune checkpoint markers. Furthermore, the ratio of complete response (CR) was greatly declined, and the ratio of breast cancer related events were significantly elevated in the high-risk group. CONCLUSION: The hypoxia-immune based gene signature we constructed for predicting prognosis was developed and validated, which may contribute to the optimization of risk stratification for prognosis and personalized treatment in TNBC patients.

Macrophage Polarization States in the Tumor Microenvironment.

The M1/M2 macrophage paradigm plays a key role in tumor progression. M1 macrophages are historically regarded as anti-tumor, while M2-polarized macrophages, commonly deemed tumor-associated macrophages (TAMs), are contributors to many pro-tumorigenic outcomes in cancer through angiogenic and lymphangiogenic regulation, immune suppression, hypoxia induction, tumor cell proliferation, and metastasis. The tumor microenvironment (TME) can influence macrophage recruitment and polarization, giving way to these pro-tumorigenic outcomes. Investigating TME-induced macrophage polarization is critical for further understanding of TAM-related pro-tumor outcomes and potential development of new therapeutic approaches. This review explores the current understanding of TME-induced macrophage polarization and the role of M2-polarized macrophages in promoting tumor progression.

A hypoxia risk signature for the tumor immune microenvironment evaluation and prognosis prediction in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most prevalent form of acute leukemia. Patients with AML often have poor clinical prognoses. Hypoxia can activate a series of immunosuppressive processes in tumors, resulting in diseases and poor clinical prognoses. However, how to evaluate the severity of hypoxia in tumor immune microenvironment remains unknown. In this study, we downloaded the profiles of RNA sequence and clinicopathological data of pediatric AML patients from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, as well as those of AML patients from Gene Expression Omnibus (GEO). In order to explore the immune microenvironment in AML, we established a risk signature to predict clinical prognosis. Our data showed that patients with high hypoxia risk score had shorter overall survival, indicating that higher hypoxia risk scores was significantly linked to immunosuppressive microenvironment in AML. Further analysis showed that the hypoxia could be used to serve as an independent prognostic indicator for AML patients. Moreover, we found gene sets enriched in high-risk AML group participated in the carcinogenesis. In summary, the established hypoxia-related risk model could act as an independent predictor for the clinical prognosis of AML, and also reflect the response intensity of the immune microenvironment in AML.

An Eight-Gene Hypoxia Signature Predicts Survival in Pancreatic Cancer and Is Associated With an Immunosuppressed Tumor Microenvironment.

Intratumoral hypoxia is a widely established element of the pancreatic tumor microenvironment (TME) promoting immune escape, tumor invasion, and progression, while contributing to treatment resistance and poor survival. Despite this critical role, hypoxia is underrepresented in molecular signatures of pancreatic ductal adenocarcinoma (PDA) and concurrent investigations into the hypoxia-immune status are lacking. In this work a literature-based approach was applied to derive an eight-gene hypoxia signature that was validated in fourteen cancer cell lines and in a cohort of PDA. The eight-gene hypoxia signature was significantly associated with overall survival in two distinct PDA datasets and showed independent prognostic value in multivariate analysis. Comparative analysis of tumors according to their hypoxia score (high versus low) determined that tumors with high hypoxia were significantly less enriched in cytotoxic T-cells, and cytolytic activity. In addition, they had lower expression of cytokines and tumor inflammatory markers, pointing to the signature's ability to discern an immune "cold", hypoxic TME. Combining the signature with an immune metric highlighted a worse survival probability in patients with high hypoxia and low immune reactivity, indicating that this approach could further refine survival estimates. Hypoxia as determined by our signature, was significantly associated with certain immune checkpoint inhibitors (ICI) biomarkers, suggesting that the signature reflects an aspect of the TME that is worth pursuing in future clinical trials. This is the first work of its kind in PDA, and our findings on the hypoxia-immune tumor contexture are not only relevant for ICI but could also guide combinatorial hypoxia-mediated therapeutic strategies in this cancer type.

Roles of lysine-specific demethylase 1 (LSD1) in homeostasis and diseases.

Lysine-specific demethylase 1 (LSD1) targets mono- or di-methylated histone H3K4 and H3K9 as well as non-histone substrates and functions in the regulation of gene expression as a transcriptional repressor or activator. This enzyme plays a pivotal role in various physiological processes, including development, differentiation, inflammation, thermogenesis, neuronal and cerebral physiology, and the maintenance of stemness in stem cells. LSD1 also participates in pathological processes, including cancer as the most representative disease. It promotes oncogenesis by facilitating the survival of cancer cells and by generating a pro-cancer microenvironment. In this review, we discuss the role of LSD1 in several aspects of cancer, such as hypoxia, epithelial-to-mesenchymal transition, stemness versus differentiation of cancer stem cells, as well as anti-tumor immunity. Additionally, the current understanding of the involvement of LSD1 in various other pathological processes is discussed.

Involvement of the VEGF signaling pathway in immunosuppression and hypoxia stress: analysis of mRNA expression in lymphocytes mediating panting in Jersey cattle under heat stress.

BACKGROUND: Extreme panting under heat stress threatens dairy cattle milk production. Previous research has revealed that the gas exchange-mediated respiratory drive in critically ill dairy cattle with low O2 saturation induces panting. Vascular endothelial growth factor (VEGF) signaling may play important roles in immunosuppression and oxidative stress during severe respiratory stress responses in heat-stressed cattle. The objectives of this study were to transcriptomically analyze mRNA expression mediating heat-induced respiratory stress-associated panting, evaluate gas exchange, screen hub genes, and verify the expression of proteins encoded by differentially expressed genes in lymphocyte pathways. RESULTS: Jersey cattle were naturally heat-exposed. Physiological data were collected for response evaluation, and blood was collected for gas exchange and gene expression assays at 06:00, 10:00 and 14:00 continuously for 1 week. Lymphocytes were isolated from whole-blood samples for mRNA-seq and expression analysis of key pathway genes/proteins. The cattle respiration rates differed with time, averaging 51 bpm at 06:00, 76 bpm at 10:00, and 121 bpm at 14:00 (p < 0.05). Gas exchange analysis showed that both pH and pCO2 differed with time: they were 7.41 and 41 mmHg at 06:00, 7.45 and 37.5 mmHg at 10:00, and 7.49 and 33 mmHg at 14:00, respectively (p < 0.01). Sixteen heat-related differentially expressed genes (DEGs; 13 upregulated and 3 downregulated) were screened between 212 DEGs and 1370 heat stress-affected genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) hub gene functional analysis annotated eleven genes to signal transduction, six genes to the immune response, and five genes to the endocrine response, including both prostaglandin-endoperoxide synthase 2 (PTGS2) and VEGF. Gene Ontology (GO) functional enrichment analysis revealed that oxygen regulation was associated with the phosphorus metabolic process, response to oxygen levels, response to decreased oxygen levels, response to hypoxia and cytokine activity terms. The main signaling pathways were the VEGF, hypoxia inducible factor-1(HIF-1), cytokine-cytokine receptor interaction and TNF pathways. Four genes involved Integrin beta 3 (ITBG3), PTGS2, VEGF, and myosin light chain 9 (MYL9) among the 16 genes related to immunosuppression, oxidative stress, and endocrine dysfunction were identified as participants in the VEGF signaling pathway and oxygenation. CONCLUSION: These findings help elucidate the underlying immune and oxygen regulation mechanisms associated with the VEGF signaling pathway in heat-stressed dairy cattle.

Studies of molecular pathways associated with blood neutrophil corticosteroid insensitivity in equine asthma.

Severe equine asthma is characterized by airway hyperresponsiveness, neutrophilic inflammation and structural alterations of the lower airways. In asthmatic horses with neutrophilic inflammation, there is insensitivity to corticosteroids characterized by the persistence of neutrophils within the airways with therapy. We hypothesized that hypoxia or oxidative stress in the microenvironment of the lung contributes to this insensitivity of neutrophils to corticosteroids in asthmatic horses. Blood neutrophils isolated from horses with severe asthma (N = 8) and from healthy controls (N = 8) were incubated under different cell culture conditions simulating hypoxia and oxidative stress and, in the presence, or absence of dexamethasone. The pro-inflammatory gene and protein expression of neutrophils were studied. In both groups, pyocyanin-induced oxidative stress increased the mRNA expression of IL-8, IL-1ß, and TNF-α. While IL-1ß and TNF-α were downregulated by dexamethasone under these conditions, IL-8 was not. Simulated hypoxic conditions did not enhance pro-inflammatory gene expression in neutrophils from either group of horses. In conclusion, oxidative stress but not hypoxia may contribute to corticosteroid insensitivity via a selective gene regulation pathway. Equine neutrophil responses were similar in both heathy and asthmatic horses, indicating that it is not specific to asthmatic inflammation.