Immunohematologic Biomarkers in COVID-19: Insights into Pathogenesis, Prognosis, and Prevention.

Coronavirus disease 2019 (COVID-19) has had profound effects on the health of individuals and on healthcare systems worldwide. While healthcare workers on the frontlines have fought to quell multiple waves of infection, the efforts of the larger research community have changed the arch of this pandemic as well. This review will focus on biomarker discovery and other efforts to identify features that predict outcomes, and in so doing, identify possible effector and passenger mechanisms of adverse outcomes. Identifying measurable soluble factors, cell-types, and clinical parameters that predict a patient's disease course will have a legacy for the study of immunologic responses, especially stimuli, which induce an overactive, yet ineffectual immune system. As prognostic biomarkers were identified, some have served to represent pathways of therapeutic interest in clinical trials. The pandemic conditions have created urgency for accelerated target identification and validation. Collectively, these COVID-19 studies of biomarkers, disease outcomes, and therapeutic efficacy have revealed that immunologic systems and responses to stimuli are more heterogeneous than previously assumed. Understanding the genetic and acquired features that mediate divergent immunologic outcomes in response to this global exposure is ongoing and will ultimately improve our preparedness for future pandemics, as well as impact preventive approaches to other immunologic diseases.

Krüppel-Like Factors Orchestrate Endothelial Gene Expression Through Redundant and Non-Redundant Enhancer Networks.

Background Proper function of endothelial cells is critical for vascular integrity and organismal survival. Studies over the past 2 decades have identified 2 members of the KLF (Krüppel-like factor) family of proteins, KLF2 and KLF4, as nodal regulators of endothelial function. Strikingly, inducible postnatal deletion of both KLF2 and KLF4 resulted in widespread vascular leak, coagulopathy, and rapid death. Importantly, while transcriptomic studies revealed profound alterations in gene expression, the molecular mechanisms underlying these changes remain poorly understood. Here, we seek to determine mechanisms of KLF2 and KLF4 transcriptional control in multiple vascular beds to further understand their roles as critical endothelial regulators. Methods and Results We integrate chromatin occupancy and transcription studies from multiple transgenic mouse models to demonstrate that KLF2 and KLF4 have overlapping yet distinct binding patterns and transcriptional targets in heart and lung endothelium. Mechanistically, KLFs use open chromatin regions in promoters and enhancers and bind in context-specific patterns that govern transcription in microvasculature. Importantly, this occurs during homeostasis in vivo without additional exogenous stimuli. Conclusions Together, this work provides mechanistic insight behind the well-described transcriptional and functional heterogeneity seen in vascular populations, while also establishing tools into exploring microvascular endothelial dynamics in vivo.

KLF15 controls brown adipose tissue transcriptional flexibility and metabolism in response to various energetic demands.

Brown adipose tissue (BAT) is a specialized metabolic organ responsible for non-shivering thermogenesis. Recently, its activity has been shown to be critical in systemic metabolic health through its utilization and consumption of macronutrients. In the face of energetically demanding states, metabolic flexibility and systemic coordination of nutrient partitioning is requisite for health and survival. In this study, we elucidate BAT's differential transcriptional adaptations in response to multiple nutrient challenges and demonstrate its context-dependent prioritization of lipid, glucose, and amino acid metabolism. We show that the transcription factor Krüppel-like factor 15 (KLF15) plays a critical role in BAT metabolic flexibility. BAT-specific loss of KLF15 results in widespread changes in circulating metabolites and severely compromised thermogenesis in response to high energy demands, indicative of impaired nutrient utilization and metabolic flexibility. Together, our data demonstrate KLF15 in BAT plays an indispensable role in partitioning resources to maintain homeostasis and ensure survival.

A targetable pathway in neutrophils mitigates both arterial and venous thrombosis.

Arterial and venous thrombosis constitutes a major source of morbidity and mortality worldwide. Long considered as distinct entities, accumulating evidence indicates that arterial and venous thrombosis can occur in the same populations, suggesting that common mechanisms are likely operative. Although hyperactivation of the immune system is a common forerunner to the genesis of thrombotic events in both vascular systems, the key molecular control points remain poorly understood. Consequently, antithrombotic therapies targeting the immune system for therapeutics gain are lacking. Here, we show that neutrophils are key effectors of both arterial and venous thrombosis and can be targeted through immunoregulatory nanoparticles. Using antiphospholipid antibody syndrome (APS) as a model for arterial and venous thrombosis, we identified the transcription factor Krüppel-like factor 2 (KLF2) as a key regulator of neutrophil activation. Upon activation through genetic loss of KLF2 or administration of antiphospholipid antibodies, neutrophils clustered P-selectin glycoprotein ligand 1 (PSGL-1) by cortical actin remodeling, thereby increasing adhesion potential at sites of thrombosis. Targeting clustered PSGL-1 using nanoparticles attenuated neutrophil-mediated thrombosis in APS and KLF2 knockout models, illustrating the importance and feasibility of targeting activated neutrophils to prevent pathological thrombosis. Together, our results demonstrate a role for activated neutrophils in both arterial and venous thrombosis and identify key molecular events that serve as potential targets for therapeutics against diverse causes of immunothrombosis.

Transcription factors KLF15 and PPARδ cooperatively orchestrate genome-wide regulation of lipid metabolism in skeletal muscle.

Skeletal muscle dynamically regulates systemic nutrient homeostasis through transcriptional adaptations to physiological cues. In response to changes in the metabolic environment (e.g., alterations in circulating glucose or lipid levels), networks of transcription factors and coregulators are recruited to specific genomic loci to fine-tune homeostatic gene regulation. Elucidating these mechanisms is of particular interest as these gene regulatory pathways can serve as potential targets to treat metabolic disease. The zinc-finger transcription factor Krüppel-like factor 15 (KLF15) is a critical regulator of metabolic homeostasis; however, its genome-wide distribution in skeletal muscle has not been previously identified. Here, we characterize the KLF15 cistrome in vivo in skeletal muscle and find that the majority of KLF15 binding is localized to distal intergenic regions and associated with genes related to circadian rhythmicity and lipid metabolism. We also identify critical interdependence between KLF15 and the nuclear receptor PPARδ in the regulation of lipid metabolic gene programs. We further demonstrate that KLF15 and PPARδ colocalize genome-wide, physically interact, and are dependent on one another to exert their transcriptional effects on target genes. These findings reveal that skeletal muscle KLF15 plays a critical role in metabolic adaptation through its direct actions on target genes and interactions with other nodal transcription factors such as PPARδ.

KLF2 regulates neutrophil activation and thrombosis in cardiac hypertrophy and heart failure progression.

It is widely recognized that inflammation plays a critical role in cardiac hypertrophy and heart failure. However, clinical trials targeting cytokines have shown equivocal effects, indicating the need for a deeper understanding of the precise role of inflammation and inflammatory cells in heart failure. Leukocytes from human subjects and a rodent model of heart failure were characterized by a marked reduction in expression of Klf2 mRNA. Using a mouse model of angiotensin II-induced nonischemic cardiac dysfunction, we showed that neutrophils played an essential role in the pathogenesis and progression of heart failure. Mechanistically, chronic angiotensin II infusion activated a neutrophil KLF2/NETosis pathway that triggered sporadic thrombosis in small myocardial vessels, leading to myocardial hypoxia, cell death, and hypertrophy. Conversely, targeting neutrophils, neutrophil extracellular traps (NETs), or thrombosis ameliorated these pathological changes and preserved cardiac dysfunction. KLF2 regulated neutrophil activation in response to angiotensin II at the molecular level, partly through crosstalk with HIF1 signaling. Taken together, our data implicate neutrophil-mediated immunothrombotic dysregulation as a critical pathogenic mechanism leading to cardiac hypertrophy and heart failure. This neutrophil KLF2-NETosis-thrombosis mechanism underlying chronic heart failure can be exploited for therapeutic gain by therapies targeting neutrophils, NETosis, or thrombosis.

Evolutionary Protection of Krüppel-Like Factors 2 and 4 in the Development of the Mature Hemovascular System.

A properly functioning hemovascular system, consisting of circulating innate immune cells and endothelial cells (ECs), is essential in the distribution of nutrients to distant tissues while ensuring protection from invading pathogens. Professional phagocytes (e.g., macrophages) and ECs have co-evolved in vertebrates to adapt to increased physiological demands. Intercellular interactions between components of the hemovascular system facilitate numerous functions in physiology and disease in part through the utilization of shared signaling pathways and factors. Krüppel-like factors (KLFs) 2 and 4 are two such transcription factors with critical roles in both cellular compartments. Decreased expression of either factor in myeloid or endothelial cells increases susceptibility to a multitude of inflammatory diseases, underscoring the essential role for their expression in maintaining cellular quiescence. Given the close evolutionary relationship between macrophages and ECs, along with their shared utilization of KLF2 and 4, we hypothesize that KLF genes evolved in such a way that protected their expression in myeloid and endothelial cells. Within this Perspective, we review the roles of KLF2 and 4 in the hemovascular system and explore evolutionary trends in their nucleotide composition that suggest a coordinated protection that corresponds with the development of mature myeloid and endothelial systems.

Muscle Krüppel-like factor 15 regulates lipid flux and systemic metabolic homeostasis.

Skeletal muscle is a major determinant of systemic metabolic homeostasis that plays a critical role in glucose metabolism and insulin sensitivity. By contrast, despite being a major user of fatty acids, and evidence that muscular disorders can lead to abnormal lipid deposition (e.g., nonalcoholic fatty liver disease in myopathies), our understanding of skeletal muscle regulation of systemic lipid homeostasis is not well understood. Here we show that skeletal muscle Krüppel-like factor 15 (KLF15) coordinates pathways central to systemic lipid homeostasis under basal conditions and in response to nutrient overload. Mice with skeletal muscle-specific KLF15 deletion demonstrated (a) reduced expression of key targets involved in lipid uptake, mitochondrial transport, and utilization, (b) elevated circulating lipids, (c) insulin resistance/glucose intolerance, and (d) increased lipid deposition in white adipose tissue and liver. Strikingly, a diet rich in short-chain fatty acids bypassed these defects in lipid flux and ameliorated aspects of metabolic dysregulation. Together, these findings establish skeletal muscle control of lipid flux as critical to systemic lipid homeostasis and metabolic health.

Myeloid Krüppel-like factor 2 is a critical regulator of metabolic inflammation.

Substantial evidence implicates crosstalk between metabolic tissues and the immune system in the inception and progression of obesity. However, molecular regulators that orchestrate metaflammation both centrally and peripherally remains incompletely understood. Here, we identify myeloid Krüppel-like factor 2 (KLF2) as an essential regulator of obesity and its sequelae. In mice and humans, consumption of a fatty diet downregulates myeloid KLF2 levels. Under basal conditions, myeloid-specific KLF2 knockout mice (K2KO) exhibit increased feeding and weight gain. High-fat diet (HFD) feeding further exacerbates the K2KO metabolic disease phenotype. Mechanistically, loss of myeloid KLF2 increases metaflammation in peripheral and central tissues. A combination of pair-feeding, bone marrow-transplant, and microglial ablation implicate central and peripheral contributions to K2KO-induced metabolic dysfunction observed. Finally, overexpression of myeloid KLF2 protects mice from HFD-induced obesity and insulin resistance. Together, these data establish myeloid KLF2 as a nodal regulator of central and peripheral metabolic inflammation in homeostasis and disease.

Serial Systemic Injections of Endotoxin (LPS) Elicit Neuroprotective Spinal Cord Microglia through IL-1-Dependent Cross Talk with Endothelial Cells.

Microglia are dynamic immunosurveillance cells in the CNS. Whether microglia are protective or pathologic is context dependent; the outcome varies as a function of time relative to the stimulus, activation state of neighboring cells in the microenvironment or within progression of a particular disease. Although brain microglia can be "primed" using bacterial lipopolysaccharide (LPS)/endotoxin, it is unknown whether LPS delivered systemically can also induce neuroprotective microglia in the spinal cord. Here, we show that serial systemic injections of LPS (1 mg/kg, i.p., daily) for 4 consecutive days (LPSx4) consistently elicit a reactive spinal cord microglia response marked by dramatic morphologic changes, increased production of IL-1, and enhanced proliferation without triggering leukocyte recruitment or overt neuropathology. Following LPSx4, reactive microglia frequently contact spinal cord endothelial cells. Targeted ablation or selective expression of IL-1 and IL-1 receptor (IL-1R) in either microglia or endothelia reveal that IL-1-dependent signaling between these cells mediates microglia activation. Using a mouse model of ischemic spinal cord injury in male and female mice, we show that preoperative LPSx4 provides complete protection from ischemia-induced neuron loss and hindlimb paralysis. Neuroprotection is partly reversed by either pharmacological elimination of microglia or selective removal of IL-1R in microglia or endothelia. These data indicate that spinal cord microglia are amenable to therapeutic reprogramming via systemic manipulation and that this potential can be harnessed to protect the spinal cord from injury.SIGNIFICANCE STATEMENT Data in this report indicate that a neuroprotective spinal cord microglia response can be triggered by daily systemic injections of LPS over a period of 4 d (LPSx4). The LPSx4 regimen induces morphologic transformation and enhances proliferation of spinal cord microglia without causing neuropathology. Using advanced transgenic mouse technology, we show that IL-1-dependent microglia-endothelia cross talk is necessary for eliciting this spinal cord microglia phenotype and also for conferring optimal protection to spinal motor neurons from ischemic spinal cord injury (ISCI). Collectively, these novel data show that it is possible to consistently elicit spinal cord microglia via systemic delivery of inflammogens to achieve a therapeutically effective neuroprotective response against ISCI.

Taking KLF9 to "Cort" for crimes against metabolism.

Glucocorticoids (GCs) are essential for proper glycemic control, but in excess, can lead to hyperglycemia and diabetes. In this issue of the JCI, Cui et al. elucidate a mechanism by which GCs regulate gluconeogenesis utilizing the transcription factor Krüppel-like factor 9 (KLF9) in physiology and disease settings. They report that KLF9 is a GC-inducible factor that ultimately increases the transcription of proliferator-activated receptor γ coactivator 1 α (PGC1α), resulting in gluconeogenesis. Given the high incidence of GC-induced diabetes, identification of this signaling axis provides, not only critical scientific insight, but also a foundation for preventative therapies for patients receiving chronic GC treatment.

KLF15 regulates endobiotic and xenobiotic metabolism.

Hepatic metabolism and elimination of endobiotics (for example, steroids, bile acids) and xenobiotics (for example, drugs, toxins) is essential for health. While the enzymatic (termed phase I-II) and transport machinery (termed phase III) controlling endobiotic and xenobiotic metabolism (EXM) is known, understanding of molecular nodal points that coordinate EXM function in physiology and disease remains incomplete. Here we show that the transcription factor Kruppel-like factor 15 (KLF15) regulates all three phases of the EXM system by direct and indirect pathways. Unbiased transcriptomic analyses coupled with validation studies in cells, human tissues, and animals, support direct transcriptional control of the EXM machinery by KLF15. Liver-specific deficiency of KLF15 (Li-KO) results in altered expression of numerous phase I-III targets, and renders animals resistant to the pathologic effects of bile acid and acetaminophen toxicity. Furthermore, Li-KO mice demonstrate enhanced degradation and elimination of endogenous steroid hormones, such as testosterone and glucocorticoid, resulting in reduced male fertility and blood glucose levels, respectively. Viral reconstitution of hepatic KLF15 expression in Li-KO mice reverses these phenotypes. Our observations identify a previously unappreciated transcriptional pathway regulating metabolism and elimination of endobiotics and xenobiotics.

The Krüppel-Like Factors and Control of Energy Homeostasis.

Nutrient handling by higher organisms is a complex process that is regulated at the transcriptional level. Studies over the past 15 years have highlighted the critical importance of a family of transcriptional regulators termed the Krüppel-like factors (KLFs) in metabolism. Within an organ, distinct KLFs direct networks of metabolic gene targets to achieve specialized functions. This regulation is often orchestrated in concert with recruitment of tissue-specific transcriptional regulators, particularly members of the nuclear receptor family. Upon nutrient entry into the intestine, gut, and liver, KLFs control a range of functions from bile synthesis to intestinal stem cell maintenance to effect nutrient acquisition. Subsequently, coordinated KLF activity across multiple organs distributes nutrients to sites of storage or liberates them for use in response to changes in nutrient status. Finally, in energy-consuming organs like cardiac and skeletal muscle, KLFs tune local metabolic programs to precisely match substrate uptake, flux, and use, particularly via mitochondrial function, with energetic demand; this is achieved in part via circulating mediators, including glucocorticoids and insulin. Here, we summarize current understanding of KLFs in regulation of nutrient absorption, interorgan circulation, and tissue-specific use.

Distinct roles of resident and nonresident macrophages in nonischemic cardiomyopathy.

Nonischemic cardiomyopathy (NICM) resulting from long-standing hypertension, valvular disease, and genetic mutations is a major cause of heart failure worldwide. Recent observations suggest that myeloid cells can impact cardiac function, but the role of tissue-intrinsic vs. tissue-extrinsic myeloid cells in NICM remains poorly understood. Here, we show that cardiac resident macrophage proliferation occurs within the first week following pressure overload hypertrophy (POH; a model of heart failure) and is requisite for the heart's adaptive response. Mechanistically, we identify Kruppel-like factor 4 (KLF4) as a key transcription factor that regulates cardiac resident macrophage proliferation and angiogenic activities. Finally, we show that blood-borne macrophages recruited in late-phase POH are detrimental, and that blockade of their infiltration improves myocardial angiogenesis and preserves cardiac function. These observations demonstrate previously unappreciated temporal and spatial roles for resident and nonresident macrophages in the development of heart failure.

Journal Club: MRI reveals acute inflammation in cortical lesions during early MS.

Early multiple sclerosis is characterized by immune-associated demyelination of CNS axons. In a recent Neurology® article, Maranzano et al. evaluated MRI scans of patients with early multiple sclerosis to study the evolution of leukocortical lesions. Their novel data suggest that acute inflammation after blood-brain barrier leakage may contribute to gray matter cortical lesions in early multiple sclerosis.

Krüppel-Like Factors in Vascular Inflammation: Mechanistic Insights and Therapeutic Potential.

The role of inflammation in vascular disease is well recognized, involving dysregulation of both circulating immune cells as well as the cells of the vessel wall itself. Unrestrained vascular inflammation leads to pathological remodeling that eventually contributes to atherothrombotic disease and its associated sequelae (e.g., myocardial/cerebral infarction, embolism, and critical limb ischemia). Signaling events during vascular inflammation orchestrate widespread transcriptional programs that affect the functions of vascular and circulating inflammatory cells. The Krüppel-like factors (KLFs) are a family of transcription factors central in regulating vascular biology in states of homeostasis and disease. Given their abundance and diversity of function in cells associated with vascular inflammation, understanding the transcriptional networks regulated by KLFs will further our understanding of the pathogenesis underlying several pervasive health concerns (e.g., atherosclerosis, stroke, etc.) and consequently inform the treatment of cardiovascular disease. Within this review, we will discuss the role of KLFs in coordinating protective and deleterious responses during vascular inflammation, while addressing the potential targeting of these critical transcription factors in future therapies.

Krüppel-like factor 15: Regulator of BCAA metabolism and circadian protein rhythmicity.

Regulation of nutrient intake, utilization, and storage exhibits a circadian rhythmicity that allows organisms to anticipate and adequately respond to changes in the environment across day/night cycles. The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are important modulators of metabolism and metabolic health - for example, their catabolism yields carbon substrates for gluconeogenesis during periods of fasting. Krüppel-like factor 15 (KLF15) has recently emerged as a critical transcriptional regulator of BCAA metabolism, and the absence of this transcription factor contributes to severe pathologies such as Duchenne muscular dystrophy and heart failure. This review highlights KLF15's role as a central regulator of BCAA metabolism during periods of fasting, throughout day/night cycles, and in experimental models of muscle disease.

Aging and the Krüppel-like factors.

The mammalian Krüppel-like factors (KLFs) are a family of zinc-finger containing transcription factors with diverse patterns of expression and a wide array of cellular functions. While their roles in mammalian physiology are well known, there is a growing appreciation for their roles in modulating the fundamental progression of aging. Here we review the current knowledge of Krüppel-like factors with a focus on their roles in processes regulating aging and age-associated diseases.

miR-155 Deletion in Mice Overcomes Neuron-Intrinsic and Neuron-Extrinsic Barriers to Spinal Cord Repair.

UNLABELLED: Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers including inflammation. microRNA (miR)-155-5p is a small, noncoding RNA that negatively regulates mRNA translation. In macrophages, miR-155-5p is induced by inflammatory stimuli and elicits a response that could be toxic after SCI. miR-155 may also independently alter expression of genes that regulate axon growth in neurons. Here, we hypothesized that miR-155 deletion would simultaneously improve axon growth and reduce neuroinflammation after SCI by acting on both neurons and macrophages. New data show that miR-155 deletion attenuates inflammatory signaling in macrophages, reduces macrophage-mediated neuron toxicity, and increases macrophage-elicited axon growth by ∼40% relative to control conditions. In addition, miR-155 deletion increases spontaneous axon growth from neurons; adult miR-155 KO dorsal root ganglion (DRG) neurons extend 44% longer neurites than WT neurons. In vivo, miR-155 deletion augments conditioning lesion-induced intraneuronal expression of SPRR1A, a regeneration-associated gene; ∼50% more injured KO DRG neurons expressed SPRR1A versus WT neurons. After dorsal column SCI, miR-155 KO mouse spinal cord has reduced neuroinflammation and increased peripheral conditioning-lesion-enhanced axon regeneration beyond the epicenter. Finally, in a model of spinal contusion injury, miR-155 deletion improves locomotor function at postinjury times corresponding with the arrival and maximal appearance of activated intraspinal macrophages. In miR-155 KO mice, improved locomotor function is associated with smaller contusion lesions and decreased accumulation of inflammatory macrophages. Collectively, these data indicate that miR-155 is a novel therapeutic target capable of simultaneously overcoming neuron-intrinsic and neuron-extrinsic barriers to repair after SCI. SIGNIFICANCE STATEMENT: Axon regeneration after spinal cord injury (SCI) fails due to neuron-intrinsic mechanisms and extracellular barriers, including inflammation. Here, new data show that deleting microRNA-155 (miR-155) affects both mechanisms and improves repair and functional recovery after SCI. Macrophages lacking miR-155 have altered inflammatory capacity, which enhances neuron survival and axon growth of cocultured neurons. In addition, independent of macrophages, adult miR-155 KO neurons show enhanced spontaneous axon growth. Using either spinal cord dorsal column crush or contusion injury models, miR-155 deletion improves indices of repair and recovery. Therefore, miR-155 has a dual role in regulating spinal cord repair and may be a novel therapeutic target for SCI and other CNS pathologies.