COVID-19: disease pathways and gene expression changes predict methylprednisolone can improve outcome in severe cases.

MOTIVATION: COVID-19 has several distinct clinical phases: a viral replication phase, an inflammatory phase and in some patients, a hyper-inflammatory phase. High mortality is associated with patients developing cytokine storm syndrome. Treatment of hyper-inflammation in these patients using existing approved therapies with proven safety profiles could address the immediate need to reduce mortality. RESULTS: We analyzed the changes in the gene expression, pathways and putative mechanisms induced by SARS-CoV2 in NHBE, and A549 cells, as well as COVID-19 lung versus their respective controls. We used these changes to identify FDA approved drugs that could be repurposed to help COVID-19 patients with severe symptoms related to hyper-inflammation. We identified methylprednisolone (MP) as a potential leading therapy. The results were then confirmed in five independent validation datasets including Vero E6 cells, lung and intestinal organoids, as well as additional patient lung sample versus their respective controls. Finally, the efficacy of MP was validated in an independent clinical study. Thirty-day all-cause mortality occurred at a significantly lower rate in the MP-treated group compared to control group (29.6% versus 16.6%, P = 0.027). Clinical results confirmed the in silico prediction that MP could improve outcomes in severe cases of COVID-19. A low number needed to treat (NNT = 5) suggests MP may be more efficacious than dexamethasone or hydrocortisone. AVAILABILITY AND IMPLEMENTATION: iPathwayGuide is available at https://advaitabio.com/ipathwayguide/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Integrated physiological mechanisms of exercise performance, adaptation, and maladaptation to heat stress.

This article emphasizes significant recent advances regarding heat stress and its impact on exercise performance, adaptations, fluid electrolyte imbalances, and pathophysiology. During exercise-heat stress, the physiological burden of supporting high skin blood flow and high sweating rates can impose considerable cardiovascular strain and initiate a cascade of pathophysiological events leading to heat stroke. We examine the association between heat stress, particularly high skin temperature, on diminishing cardiovascular/aerobic reserves as well as increasing relative intensity and perceptual cues that degrade aerobic exercise performance. We discuss novel systemic (heat acclimation) and cellular (acquired thermal tolerance) adaptations that improve performance in hot and temperate environments and protect organs from heat stroke as well as other dissimilar stresses. We delineate how heat stroke evolves from gut underperfusion/ischemia causing endotoxin release or the release of mitochondrial DNA fragments in response to cell necrosis, to mediate a systemic inflammatory syndrome inducing coagulopathies, immune dysfunction, cytokine modulation, and multiorgan damage and failure. We discuss how an inflammatory response that induces simultaneous fever and/or prior exposure to a pathogen (e.g., viral infection) that deactivates molecular protective mechanisms interacts synergistically with the hyperthermia of exercise to perhaps explain heat stroke cases reported in low-risk populations performing routine activities. Importantly, we question the "traditional" notion that high core temperature is the critical mediator of exercise performance degradation and heat stroke. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.

A microarray analysis of the effects of moderate hypothermia and rewarming on gene expression by human hepatocytes (HepG2).

The gene expression changes produced by moderate hypothermia are not fully known, but appear to differ in important ways from those produced by heat shock. We examined the gene expression changes produced by moderate hypothermia and tested the hypothesis that rewarming after hypothermia approximates a heat-shock response. Six sets of human HepG2 hepatocytes were subjected to moderate hypothermia (31 degrees C for 16 h), a conventional in vitro heat shock (43 degrees C for 30 min) or control conditions (37 degrees C), then harvested immediately or allowed to recover for 3 h at 37 degrees C. Expression analysis was performed with Affymetrix U133A gene chips, using analysis of variance-based techniques. Moderate hypothermia led to distinct time-dependent expression changes, as did heat shock. Hypothermia initially caused statistically significant, greater than or equal to twofold changes in expression (relative to controls) of 409 sequences (143 increased and 266 decreased), whereas heat shock affected 71 (35 increased and 36 decreased). After 3 h of recovery, 192 sequences (83 increased, 109 decreased) were affected by hypothermia and 231 (146 increased, 85 decreased) by heat shock. Expression of many heat shock proteins was decreased by hypothermia but significantly increased after rewarming. A comparison of sequences affected by thermal stress without regard to the magnitude of change revealed that the overlap between heat and cold stress was greater after 3 h of recovery than immediately following thermal stress. Thus, while some overlap occurs (particularly after rewarming), moderate hypothermia produces extensive, time-dependent gene expression changes in HepG2 cells that differ in important ways from those induced by heat shock.

Core temperature correlates with expression of selected stress and immunomodulatory genes in febrile patients with sepsis and noninfectious SIRS.

Environmental hyperthermia and exercise produce extensive changes in gene expression in human blood cells, but it is unknown whether this also happens during febrile-range hyperthermia. We tested the hypothesis that heat shock protein (HSP) and immunomodulatory stress gene expression correlate with fever in intensive care unit patients. Whole blood messenger RNA was obtained over consecutive days from 100 hospitalized patients suffering from sepsis or noninfectious systemic inflammatory response syndrome (SIRS) as defined by conventional criteria. The most abnormal body temperature in the preceding 24 h was recorded for each sample. Expression analysis was performed using the Affymetrix U133 chip. ANCOVA followed by correlation analysis was performed on a subset of 278 prospectively identified sequences of interest. Temperature affected expression of 60 sequences, either independently or as a function of clinical diagnosis. Forty-eight of these (representing 38 genes) were affected by temperature only, including several HSPs, transcription factors heat shock factor (HSF)-1 and HSF-4, cellular adhesion molecules such as ICAM1/CD54 and JAM3, toll receptors TLR-6 and TLR-7, ribosomal proteins, and a number of molecules involved in inflammatory pathways. Twelve sequences demonstrated temperature-dependent responses that differed significantly between patients with sepsis and noninfectious SIRS: CXCL-13; heat shock proteins DNAJB12 and DNAJC4; the F11 receptor; folate hydrolase 1; HSF-2; HSP 70 proteins HSPA1A, HSPA1B, and HSPA1L; interleukin 8; lipopolysaccharide binding protein; and prostaglandin E synthase. Febrile-range temperatures achieved during sepsis and noninfectious SIRS correlate with detectable changes in stress gene expression in vivo, suggesting that fever can activate HSP gene expression and modify innate immune responses. For some genes, it appears that clinical condition can alter temperature-sensitive gene expression. Collectively, these data underscore the potential importance of body temperature in shaping the immune response to infection and injury.

Heat shock protein 90beta: a novel mediator of vitamin D action.

We investigated the role of Heat shock protein 90 (Hsp90) in vitamin D action in Caco-2 cells using geldanamycin (GA) to block Hsp90 function and RNA interference to reduce Hsp90beta expression. When cells were exposed to GA, vitamin D-mediated gene expression and transcriptional activity were inhibited by 69% and 54%, respectively. Gel shift analysis indicated that GA reduced vitamin D-mediated DNA binding activity of the vitamin D receptor (VDR). We tested the specific role of Hsp90beta by knocking down its expression with stably expressed short hairpin RNA. Vitamin D-induced gene expression and transcriptional activity were reduced by 90% and 80%, respectively, in Hsp90beta-deficient cells. Nuclear protein for VDR and RXRalpha, its heterodimer partner, were not reduced in Hsp90beta-deficient cells. These findings indicate that Hsp90beta is needed for optimal vitamin D responsiveness in the enterocyte and demonstrate a specific role for Hsp90beta in VDR signaling.

Ontogeny of the eotaxins in human lung.

The ontogeny of the C-C chemokines eotaxin-1, eotaxin-2, and eotaxin-3 has not been fully elucidated in human lung. We explored a possible role for eotaxin in developing lung by determining the ontogeny of eotaxin-1 (CCL11), eotaxin-2 (CCL24), eotaxin-3 (CCL26), and the eotaxin receptor, CCR3. We tested discarded surgical samples of developing human lung tissue using quantitative RT-PCR (QRT-PCR) and immunostaining for expression of CCL11, CCL24, CCL26, and CCR3. We assessed possible functionality of the eotaxin-CCR3 system by treating lung explant cultures with exogenous CCL11 and analyzing the cultures for evidence of changes in proliferation and activation of ERK1/2, a signaling pathway associated with CCR3. QRT-PCR analyses of 22 developing lung tissue samples with gestational ages 10-23 wk demonstrated that eotaxin-1 mRNA is most abundant in developing lung, whereas mRNAs for eotaxin-2 and eotaxin-3 are minimally detectable. CCL11 mRNA levels correlated with gestational age (P < 0.05), and immunoreactivity was localized predominantly to airway epithelial cells. QRT-PCR analysis detected CCR3 expression in 16 of 19 developing lung samples. Supporting functional capacity in the immature lung, CCL11 treatment of lung explant cultures resulted in significantly increased (P < 0.05) cell proliferation and activation of the ERK signaling pathway, which is downstream from CCR3, suggesting that proliferation was due to activation of CCR3 receptors by CCL11. We conclude that developing lung expresses the eotaxins and functional CCR3 receptor. CCL11 may promote airway epithelial proliferation in the developing lung.

Exertional heat illness and human gene expression.

Microarray analysis of gene expression at the level of RNA has generated new insights into the relationship between cellular responses to acute heat shock in vitro, exercise, and exertional heat illness. Here we discuss the systemic physiology of exertional hyperthermia and exertional heat illness, and compare the results of several recent microarray studies performed in vitro on human cells subjected to heat shock and in vivo on samples obtained from subjects performing exercise or suffering from exertional heat injury. From these comparisons, a concept of overlapping component responses emerges. Namely, some of the gene expression changes observed in peripheral blood mononuclear cells during exertional heat injury can be accounted for by normal cellular responses to heat, exercise, or both; others appear to be specific to the disease state itself. If confirmed in future studies, these component responses might provide a better understanding of adaptive and pathological responses to exercise and exercise-induced hyperthermia, help find new ways of identifying individuals at risk for exertional heat illness, and perhaps even help find rational molecular targets for therapeutic intervention.

Effect of moderate hypothermia on gene expression by THP-1 cells: a DNA microarray study.

The mechanisms by which moderate hypothermia (32 degrees C for 12-72 h) affect human cellular function are unclear. We tested the hypothesis that it produces broad changes in mRNA expression in vitro. Acute monocytic leukemia (THP-1) cells were incubated under control conditions (37 degrees C) or moderate hypothermia (32 degrees C) for 24 h. RNA was extracted, and the hypothermic response was confirmed by examining the expression of the cold-induced RNA-binding protein (CIRBP) gene by RT-PCR. Gene expression analysis was performed on seven sets of paired samples with Affymetrix U133A chips using established statistical methods. Sequences were considered affected by cold if they showed statistically significant changes in expression and also met published post hoc filter criteria (changes in geometric mean expression of > or =2-fold and expression calls of "present" or "marginal" in at least half of the experiments). Changes in the expression of selected sequences were further confirmed by PCR. Sixty-seven sequences met the criteria for increased expression (including cold-inducible genes CIRBP and RNA binding motif 3), and 100 sequences showed decreased expression as a result of hypothermia. Functional categories affected by hypothermia included genes involved in immune responses; cell growth, proliferation, and differentiation; and metabolism and biosynthesis. Several heat shock proteins (HSPs) showed decreases in expression. Moderate hypothermia produces substantial changes in gene expression, in categories potentially of systemic importance. Cold exposure without rewarming decreased the expression of several HSPs. These in vitro findings suggest that prolonged hypothermia in vivo might be capable of producing physiologically relevant changes in gene expression by circulating leukocytes.

Effects of allergen challenge on airway epithelial cell gene expression.

Allergen exposure induces the airway epithelium to produce chemoattractants, proallergic interleukins, matrix-modifying proteins, and proteins that influence the growth and activation state of airway structural cells. These proteins, in turn, contribute to the influx of inflammatory cells and changes in structure that characterize the asthmatic airway. To use the response of the airway epithelium to allergen to identify genes not previously associated with allergic responses, we compared gene expression in cytokeratin-positive cells before and after segmental allergen challenge. After challenge with concentrations of allergen in the clinically relevant range, 755 (6%) of the detectable sequences had geometric mean fold-changes in expression, with 95% confidence intervals that excluded unity. Using a prospectively defined conservative filtering algorithm, we identified 141 sequences as upregulated and eight as downregulated, with confirmation by conventional polymerase chain reaction in all 10 sequences studied. Using this approach, we identified asthma-associated sequences including interleukin (IL-)-3, IL-4, and IL-5 receptor subunits, the p65 component of nuclear factor-kappaB, and lipocortin. The genomic response of the human airway to concentrations of allergen in the clinically relevant range involves a greater number of genes than previously recognized, including many not previously associated with asthma that are differentially expressed after airway allergen exposure.

Role of prostanoid DP receptor variants in susceptibility to asthma.

BACKGROUND: Previous genetic studies have associated the region of the human genome (14q22.1) containing the gene for the prostanoid DP receptor (PTGDR) with asthma. A study of a mouse model suggests that the receptor is required for the expression of the asthma phenotype. Our associations of asthma with functional genetic variants of PTGDR link these observations. METHODS: We identified and evaluated combinations of genetic variants that influence PTGDR transcription for disease association in case-control studies of 518 white patients with asthma and 175 white controls and 80 black patients with asthma and 45 black controls. RESULTS: We identified four novel and two previously reported single-nucleotide polymorphisms (SNPs) in PTGDR and its vicinity. These define four common three-SNP haplotypes, which vary in their ability to support transcription of PTGDR and have distinct DNA-binding-protein affinity profiles. Individual PTGDR SNPs were significantly associated with asthma in both populations. Specific PTGDR haplotypes were significantly associated with a diagnosis of asthma in a large case-control study of whites (P=0.002); we confirmed these findings in a second population of blacks (P=0.01). Multivariate analysis of the haplotype combinations (diplotypes) demonstrated that both whites (odds ratio, 0.55; 95 percent confidence interval, 0.38 to 0.80; P=0.002) and blacks (odds ratio, 0.32; 95 percent confidence interval, 0.12 to 0.89; P=0.03) who had at least one copy of the haplotype with a low transcriptional efficiency had a lower risk of asthma than subjects with no copies of the haplotype. CONCLUSIONS: Our functional and genetic findings identify PTGDR as an asthma-susceptibility gene.

Nitric oxide synthase-2 down-regulates surfactant protein-B expression and enhances endotoxin-induced lung injury in mice.

Acute respiratory distress syndrome (ARDS) is a life-threatening ailment characterized by severe lung injury involving inflammatory cell recruitment to the lung, cytokine production, surfactant dysfunction, and up-regulation of nitric oxide synthase 2 (NOS2) resulting in nitric oxide (NO) production. We hypothesized that NO production from NOS2 expressed in lung parenchymal cells in a murine model of ARDS would correlate with abnormal surfactant function and reduced surfactant protein-B (SP-B) expression. Pulmonary responses to nebulized endotoxin (lipopolysaccharide, LPS) were evaluated in wild-type (WT) mice, NOS2 null (-/-) mice, and NOS2-chimeric animals derived from bone marrow transplantation. NOS2-/- animals exhibited significantly less physiologic lung dysfunction and loss of SP-B expression than did WT animals. However, lung neutrophil recruitment and bronchoalveolar lavage cytokine levels did not significantly differ between NOS2-/- and WT animals. Chimeric animals for NOS2 exhibited the phenotype of the recipient and therefore demonstrated that parenchymal production of NOS2 is critical for the development of LPS-induced lung injury. Furthermore, administration of NO donors, independent of cytokine stimulation, decreased SP-B promoter activity and mRNA expression in mouse lung epithelial cells. This study demonstrates that expression of NOS2 in lung epithelial cells is critical for the development of lung injury and mediates surfactant dysfunction independent of NOS2 inflammatory cell expression and cytokine production.

Monocyte chemotactic protein-4 (MCP-4; CCL-13): a biomarker of asthma.

Airway expression of monocyte chemotactic protein-4 (MCP-4; CCL-13) is known to be increased in asthmatic airways where it is induced by proallergic cytokines, but the relationship of its systemic expression to asthma and naturally occurring exacerbations is unknown. We determined plasma levels of MCP-4 in 356 individuals with chronic-stable asthma and 240 normal subjects and compared plasma levels of MCP-4 in 30 patients who presented for emergent treatment of asthma with levels in 90 subjects with chronic-stable asthma matched for age, gender, and ethnicity. Median plasma MCP-4 levels were higher in patients with chronic-stable asthma than in normal subjects (399 vs. 307 pg/mL) (p < 0.001). In our entire cohort (n = 596), subjects with an MCP-4 > 218 pg/mL were at increased risk of asthma (p < 0.001 odds ratio, 3.26; 95% CI, 2.22-4.79). Logistic regression identified MCP-4 as an independent predictor of asthma diagnosis. The MCP-4 levels are higher in individuals with an acute asthma exacerbation than in subjects with chronic-stable asthma (513 vs. 355 pg/mL) (p = 0.002). The MCP-4 is a systemically expressed biomarker that independently predicts susceptibility to asthma and is directly associated with exacerbations. Elevated MCP-4 levels identify a group of asthmatics with systemic evidence of allergic inflammation who may be at risk for exacerbations or may benefit from abrogation of MCP-4.

DNA microarray analysis of vitamin D-induced gene expression in a human colon carcinoma cell line.

The full extent to which 1,25-dihydroxyvitamin D(3) affects gene expression in human intestinal epithelial cells is unknown. We used oligonucleotide arrays to catalog vitamin D-induced changes in gene expression in Caco-2 cells, a human colon carcinoma cell line. Five paired sets of Caco-2 cell cultures were subjected to either control conditions or 1,25-dihydroxyvitamin D (10(-7) mol/l x 24 h), and RNA was analyzed on an Affymetrix cDNA array containing 12,625 human sequences. Only 13 sequences representing 12 distinct genes exhibited statistically significant changes in expression of twofold or greater and were also called as "present" or "marginal" by the array-reading software in all five experiments. Genes regulated by 1,25-dihydroxyvitamin D included two previously known genes (25-hydroxyvitamin D-24-hydroxylase and amphiregulin) and 10 genes (sorcin, Gem, adaptin-gamma, TIG1, CEACAM6, carbonic anhydrase XII, junB, ceruloplasmin, and two unidentified sequences) that were novel. We tested and independently confirmed the effect of 1,25-dihydroxyvitamin D on 11 of these genes by RT-PCR. Increased protein expression was tested and confirmed in two of the novel 1,25-dihydroxyvitamin D-regulated genes, ceruloplasmin and sorcin. The known function of these genes suggests that many of them could be involved in the antiproliferative effects of 1,25-dihydroxyvitamin D3.

Exertional heat injury and gene expression changes: a DNA microarray analysis study.

This study examined gene expression changes associated with exertional heat injury (EHI) in vivo and compared these changes to in vitro heat shock responses previously reported by our laboratory. Peripheral blood mononuclear cell (PBMC) RNA was obtained from four male Marine recruits (ages 17-19 yr) who presented with symptoms consistent with EHI, core temperatures ranging from 39.3 to 42.5 degrees C, and elevations in serum enzymes such as creatine kinase. Controls were age- and gender-matched Marines from whom samples were obtained before and several days after an intense field-training exercise in the heat ("The Crucible"). Expression analysis was performed on Affymetrix arrays (containing approximately 12,600 sequences) from pooled samples obtained at three times for EHI group (at presentation, 2-3 h after cooling, and 1-2 days later) and compared with control values (average signals from two chips representing pre- and post-Crucible samples). After post hoc filtering, the analysis identified 361 transcripts that had twofold or greater increases in expression at one or more of the time points assayed and 331 transcripts that had twofold or greater decreases in expression. The affected transcripts included sequences previously shown to be heat-shock responsive in PBMCs in vitro (including both heat shock proteins and non-heat shock proteins), a number of sequences whose changes in expression had not previously been noted as a result of in vitro heat shock in PBMCs (including several interferon-induced sequences), and several nonspecific stress response genes (including ubiquitin C and dual-specificity phosphatase-1). We conclude that EHI produces a broad stress response that is detectable in PBMCs and that heat stress per se can only account for some of the observed changes in transcript expression. The molecular evidence from these patients is thus consistent with the hypothesis that EHI can result from cumulative effects of multiple adverse interacting stimuli.

Effect of hypoxia on gene expression by human hepatocytes (HepG2).

The full extent to which hypoxia produces gene expression changes in human cells is unknown. We used late-generation oligonucleotide arrays to catalog hypoxia-induced changes in gene expression in HepG2 cells. Five paired sets of cultures were subjected to either control (room air-5% CO(2)) or hypoxic (1% O(2)-5% CO(2)) conditions for 24 h, and RNA was analyzed on an Affymetrix cDNA array containing approximately 12,600 sequences. A statistically significant change in expression was shown by 2,908 sequences (1,255 increased and 1,653 decreased). The observed changes were highly concordant with published literature on hypoxic stress but showed relatively little overlap (12-22%) with changes in gene expression that have been reported to occur after heat stress in other systems. Of note, of these 2,908 sequences, only 387 (213 increased and 174 decreased) both exhibited changes in expression of twofold or greater and were highly expressed in at least three of the five experiments. We conclude that the effect of hypoxia on gene expression by HepG2 cells is broad, has a significant component of downregulation, and includes a relatively small number of genes whose response is truly independent of cell and stress type.

DNA sequence variants in epithelium-specific ETS-2 and ETS-3 are not associated with asthma.

Epithelium-specific ETS-2 and ETS-3 are transcription factors that have been proposed as asthma candidate genes. To investigate the association of sequence variants in these genes with asthma, we conducted a case-control association analysis in a sample of 311 white subjects with asthma and 177 white subjects without asthma. Common polymorphisms in these genes were detected by sequencing DNA from 32 cell lines obtained from Coriel (Camden, NJ). Seven noncoding or synonymous single-nucleotide polymorphisms were detected: three in epithelium-specific ETS-2 and four in epithelium-specific ETS-3. Subjects were genotyped at all loci by mass spectroscopy. To ensure the suitability of our control subjects, we also genotyped subjects at 49 unlinked polymorphisms evenly distributed throughout the autosomes and found no evidence of population stratification. Logistic regression adjusted for age and sex suggested a weak association of one epithelium-specific ETS-2 polymorphism with asthma diagnosis (odds ratio = 1.89, 95% confidence interval = 1.13-3.18, p = 0.02). Total serum immunoglobulin E and FEV1 predicted levels were not associated with any of the polymorphisms. Extended haplotyping indicated linkage disequilibrium in these genes; however, no association or epistatic interaction was found. This study suggests that epithelium-specific ETS-2 and ETS-3 genes are unlikely to contain polymorphic loci that have a major impact on asthma susceptibility in our population.

Effect of acute heat shock on gene expression by human peripheral blood mononuclear cells.

We studied the effect of heat shock on gene expression by normal human cells. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adults. Paired samples from each subject were subjected to either 20 min of heat shock (43 degrees C) or control (37 degrees C) conditions and then returned to 37 degrees C. RNA was isolated 160 min later, and five representative samples were analyzed on Affymetrix gene chip arrays containing approximately 12,600 probes. A biologically meaningful effect was defined as a statistically significant, twofold or greater difference in expression of sequences that were detected in all five experiments under control (downregulated sequences) or heat shock (upregulated sequences) conditions. Changes occurred in 395 sequences (227 increased by heat shock, 168 decreased), representing 353 Unigene numbers, in every functional category previously implicated in the heat shock response. By RT-PCR, we confirmed the findings for one upregulated sequence (Rad, a G protein) and one downregulated sequence (osteopontin, a cytokine). We conclude that heat shock causes extensive gene expression changes in PBMCs, affecting all functional categories of the heat shock response.

Invited review: Effects of heat and cold stress on mammalian gene expression.

This review examines the effects of thermal stress on gene expression, with special emphasis on changes in the expression of genes other than heat shock proteins (HSPs). There are approximately 50 genes not traditionally considered to be HSPs that have been shown, by conventional techniques, to change expression as a result of heat stress, and there are <20 genes (including HSPs) that have been shown to be affected by cold. These numbers will likely become much larger as gene chip array and proteomic technologies are applied to the study of the cell stress response. Several mechanisms have been identified by which gene expression may be altered by heat and cold stress. The similarities and differences between the cellular responses to heat and cold may yield key insights into how cells, and by extension tissues and organisms, survive and adapt to stress.