Neuroinflammation underlies the development of social stress induced cognitive deficit in sickle cell disease.

Cognitive deficit is a debilitating complication of SCD with multifactorial pathobiology. Here we show that neuroinflammation and dysregulation in lipidomics and transcriptomics profiles are major underlying mechanisms of social stress-induced cognitive deficit in SCD. Townes sickle cell (SS) mice and controls (AA) were exposed to social stress using the repeat social defeat (RSD) paradigm concurrently with or without treatment with minocycline. Mice were tested for cognitive deficit using novel object recognition (NOR) and fear conditioning (FC) tests. SS mice exposed to RSD without treatment had worse performance on cognitive tests compared to SS mice exposed to RSD with treatment or to AA controls, irrespective of their RSD or treatment disposition. Additionally, compared to SS mice exposed to RSD with treatment, SS mice exposed to RSD without treatment had significantly more cellular evidence of neuroinflammation coupled with a significant shift in the differentiation of neural progenitor cells towards astrogliogenesis. Additionally, brain tissue from SS mice exposed to RSD was significantly enriched for genes associated with blood-brain barrier dysfunction, neuron excitotoxicity, inflammation, and significant dysregulation in sphingolipids important to neuronal cell processes. We demonstrate in this study that neuroinflammation and lipid dysregulation are potential underlying mechanisms of social stress-related cognitive deficit in SS mice.

The inflammatory profiles of pulmonary alveolar macrophages and alveolar type 2 cells in SCD.

The lung microenvironment plays a crucial role in maintaining lung homeostasis as well as the initiation and resolution of both acute and chronic lung injury. Acute chest syndrome (ACS) is a complication of sickle cell disease (SCD) like acute lung injury. Both the endothelial cells and peripheral blood mononuclear cells are known to secrete proinflammatory cytokines elevated during ACS episodes. However, in SCD, the lung microenvironment that may favor excessive production of proinflammatory cytokines and the contribution of other lung resident cells, such as alveolar macrophages and alveolar type 2 epithelial (AT-2) cells, to ACS pathogenesis is not completely understood. Here, we sought to understand the pulmonary microenvironment and the proinflammatory profile of lung alveolar macrophages (LAMs) and AT-2 cells at steady state in Townes sickle cell (SS) mice compared to control mice (AA). In addition, we examined lung function and micromechanics molecules essential for pulmonary epithelial barrier function in these mice. Our results showed that bronchoalveolar lavage (BAL) fluid in SS mice had elevated protein levels of pro-inflammatory cytokines interleukin (IL)-1ß and IL-12 (p ⩽ 0.05) compared to AA controls. We showed for the first time, significantly increased protein levels of inflammatory mediators (Human antigen R (HuR), Toll-like receptor 4 (TLR4), MyD88, and PU.1) in AT-2 cells (1.4 to 2.2-fold) and LAM (17-21%) isolated from SS mice compared to AA control mice at steady state. There were also low levels of anti-inflammatory transcription factors (Nrf2 and PPARy) in SS mice compared to AA controls (p ⩽ 0.05). Finally, we found impaired lung function and a dysregulated composition of surfactant proteins (B and C). Our results demonstrate that SS mice at steady state had a compromised lung microenvironment with elevated expression of proinflammatory cytokines by AT-2 cells and LAM, as well as dysregulated expression of surfactant proteins necessary for maintaining the alveolar barrier integrity and lung function.

The Role of Inflammation in The Cellular and Molecular Mechanisms of Cardiopulmonary Complications of Sickle Cell Disease.

Cardiopulmonary complications remain the major cause of mortality despite newer therapies and improvements in the lifespan of patients with sickle cell disease (SCD). Inflammation has been identified as a major risk modifier in the pathogenesis of SCD-associated cardiopulmonary complications in recent mechanistic and observational studies. In this review, we discuss recent cellular and molecular mechanisms of cardiopulmonary complications in SCD and summarize the most recent evidence from clinical and laboratory studies. We emphasize the role of inflammation in the onset and progression of these complications to better understand the underlying pathobiological processes. We also discuss future basic and translational research in addressing questions about the complex role of inflammation in the development of SCD cardiopulmonary complications, which may lead to promising therapies and reduce morbidity and mortality in this vulnerable population.

A bone to pick-cellular and molecular mechanisms of bone pain in sickle cell disease.

The bone is one of the most commonly affected organs in sickle cell disease (SCD). Repeated ischemia, oxidative stress and inflammation within the bone is largely responsible for promoting bone pain. As more individuals with SCD survive into adulthood, they are likely to experience a synergistic impact of both aging and SCD on their bone health. As bone health deteriorates, bone pain will likely exacerbate. Recent mechanistic and observational studies emphasize an intricate relationship between bone remodeling and the peripheral nervous system. Under pathological conditions, abnormal bone remodeling plays a key role in the propagation of bone pain. In this review, we first summarize mechanisms and burden of select bone complications in SCD. We then discuss processes that contribute to pathological bone pain that have been described in both SCD as well as non-sickle cell animal models. We emphasize the role of bone-nervous system interactions and pitfalls when designing new therapies especially for the sickle cell population. Lastly, we also discuss future basic and translational research in addressing questions about the complex role of stress erythropoiesis and inflammation in the development of SCD bone complications, which may lead to promising therapies and reduce morbidity in this vulnerable population.

Adhesion molecules and cerebral microvascular hemodynamic abnormalities in sickle cell disease.

Cerebrovascular abnormalities are a common feature of sickle cell disease that may be associated with risk of vaso-occlusive pain crises, microinfarcts, and cognitive impairment. An activated endothelium and adhesion factors, VCAM-1 and P-selectin, are implicated in sickle cell vasculopathy, including abnormal hemodynamics and leukocyte adherence. This study examined the association between cerebral expression of these adhesion factors and cortical microvascular blood flow dynamics by using in-vivo two-photon microscopy. We also examined the impact of blood transfusion treatment on these markers of vasculopathy. Results showed that sickle cell mice had significantly higher maximum red blood cell (RBC) velocity (6.80 ± 0.25 mm/sec, p ≤ 0.01 vs. 5.35 ± 0.35 mm/sec) and more frequent blood flow reversals (18.04 ± 0.95 /min, p ≤ 0.01 vs. 13.59 ± 1.40 /min) in the cortical microvasculature compared to controls. In addition, sickle cell mice had a 2.6-fold (RFU/mm2) increase in expression of VCAM-1 and 17-fold (RFU/mm2) increase in expression of P-selectin compared to controls. This was accompanied by an increased frequency in leukocyte adherence (4.83 ± 0.57 /100 µm/min vs. 2.26 ± 0.37 /100 µm/min, p ≤ 0.001). We also found that microinfarcts identified in sickle cell mice were 50% larger than in controls. After blood transfusion, many of these parameters improved, as results demonstrated that sickle cell mice had a lower post-transfusion maximum RBC velocity (8.30 ± 0.98 mm/sec vs. 11.29 ± 0.95 mm/sec), lower frequency of blood flow reversals (12.80 ± 2.76 /min vs. 27.75 ± 2.09 /min), and fewer instances of leukocyte adherence compared to their pre-transfusion imaging time point (1.35 ± 0.32 /100 µm/min vs. 3.46 ± 0.58 /100 µm/min). Additionally, we found that blood transfusion was associated with lower expression of adhesion factors. Our results suggest that blood transfusion and adhesion factors, VCAM-1 and P-selectin, are potential therapeutic targets for addressing cerebrovascular pathology, such as vaso-occlusion, in sickle cell disease.

Heme Induces IL-6 and Cardiac Hypertrophy Genes Transcripts in Sickle Cell Mice.

Emerging data indicate that free heme promotes inflammation in many different disease settings, including in sickle cell disease (SCD). Although free heme, proinflammatory cytokines, and cardiac hypertrophy are co-existing features of SCD, no mechanistic links between these features have been demonstrated. We now report significantly higher levels of IL-6 mRNA and protein in hearts of the Townes sickle cell disease (SS) mice (2.9-fold, p ≤ 0.05) than control mice expressing normal human hemoglobin (AA). We find that experimental administration of heme 50 µmoles/kg body weight induces IL-6 expression directly in vivo and induces gene expression markers of cardiac hypertrophy in SS mice. We administered heme intravenously and found that within three hours plasma IL-6 protein significantly increased in SS mice compared to AA mice (3248 ± 275 vs. 2384 ± 255 pg/ml, p ≤ 0.05). In the heart, heme induced a 15-fold increase in IL-6 transcript in SS mice heart compared to controls. Heme simultaneously induced other markers of cardiac stress and hypertrophy, including atrial natriuretic factor (Nppa; 14-fold, p ≤ 0.05) and beta myosin heavy chain (Myh7; 8-fold, p ≤ 0.05) in SS mice. Our experiments in Nrf2-deficient mice indicate that the cardiac IL-6 response to heme does not require Nrf2, the usual mediator of transcriptional response to heme for heme detoxification by heme oxygenase-1. These data are the first to show heme-induced IL-6 expression in vivo, suggesting that hemolysis may play a role in the elevated IL-6 and cardiac hypertrophy seen in patients and mice with SCD. Our results align with published evidence from rodents and humans without SCD that suggest a causal relationship between IL-6 and cardiac hypertrophy.

Nrf2 deficiency in mice attenuates erythropoietic stress-related macrophage hypercellularity.

Erythropoiesis in the bone marrow and spleen depends on intricate interactions between the resident macrophages and erythroblasts. Our study focuses on identifying the role of nuclear factor erythroid 2-related factor 2 (Nrf2) during recovery from stress erythropoiesis. To that end, we induced stress erythropoiesis in Nrf2+/+ and Nrf2-null mice and evaluated macrophage subsets known to support erythropoiesis and erythroid cell populations. Our results confirm macrophage and erythroid hypercellularity after acute blood loss. Importantly, Nrf2 depletion results in a marked numerical reduction of F4/80+/CD169+/CD11b+ macrophages, which is more prominent under the induction of stress erythropoiesis. The observed macrophage deficiency is concomitant to a significantly impaired erythroid response to acute stress erythropoiesis in both murine bone marrow and murine spleen. Additionally, peripheral blood reticulocyte count as a response to acute blood loss is delayed in Nrf2-deficient mice compared with age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p ≤ 0.001). Interestingly, we observe macrophage hypercellularity in conjunction with erythroid hyperplasia in the bone marrow during stress erythropoiesis in Nrf2+/+ controls, with both impaired in Nrf2-/- mice. We further confirm the finding of macrophage hypercellularity in another model of erythroid hyperplasia, the transgenic sickle cell mouse, characterized by hemolytic anemia and chronic stress erythropoiesis. Our results revealed the role of Nrf2 in stress erythropoiesis in the bone marrow and that macrophage hypercellularity occurs concurrently with erythroid expansion during stress erythropoiesis. Macrophage hypercellularity is a previously underappreciated feature of stress erythropoiesis in sickle cell disease and recovery from blood loss.

The Worst Things in Life are Free: The Role of Free Heme in Sickle Cell Disease.

Hemolysis is a pathological feature of several diseases of diverse etiology such as hereditary anemias, malaria, and sepsis. A major complication of hemolysis involves the release of large quantities of hemoglobin into the blood circulation and the subsequent generation of harmful metabolites like labile heme. Protective mechanisms like haptoglobin-hemoglobin and hemopexin-heme binding, and heme oxygenase-1 enzymatic degradation of heme limit the toxicity of the hemolysis-related molecules. The capacity of these protective systems is exceeded in hemolytic diseases, resulting in high residual levels of hemolysis products in the circulation, which pose a great oxidative and proinflammatory risk. Sickle cell disease (SCD) features a prominent hemolytic anemia which impacts the phenotypic variability and disease severity. Not only is circulating heme a potent oxidative molecule, but it can act as an erythrocytic danger-associated molecular pattern (eDAMP) molecule which contributes to a proinflammatory state, promoting sickle complications such as vaso-occlusion and acute lung injury. Exposure to extracellular heme in SCD can also augment the expression of placental growth factor (PlGF) and interleukin-6 (IL-6), with important consequences to enthothelin-1 (ET-1) secretion and pulmonary hypertension, and potentially the development of renal and cardiac dysfunction. This review focuses on heme-induced mechanisms that are implicated in disease pathways, mainly in SCD. A special emphasis is given to heme-induced PlGF and IL-6 related mechanisms and their role in SCD disease progression.

Cardiac expression of HMOX1 and PGF in sickle cell mice and haem-treated wild type mice dominates organ expression profiles via Nrf2 (Nfe2l2).

Haemolysis is a major feature of sickle cell disease (SCD) that contributes to organ damage. It is well established that haem, a product of haemolysis, induces expression of the enzyme that degrades it, haem oxygenase-1 (HMOX1). We have also shown that haem induces expression of placental growth factor (PGF), but the organ specificity of these responses has not been well-defined. As expected, we found high level expression of Hmox1 and Pgf transcripts in the reticuloendothelial system organs of transgenic sickle cell mice, but surprisingly strong expression in the heart (P < 0·0001). This pattern was largely replicated in wild type mice by intravenous injection of exogenous haem. In the heart, haem induced unexpectedly strong mRNA responses for Hmox1 (18-fold), Pgf (4-fold), and the haem transporter Slc48a1 (also termed Hrg1; 2·4-fold). This was comparable to the liver, the principal known haem-detoxifying organ. The NFE2L2 (also termed NRF2) transcription factor mediated much of the haem induction of Hmox1 and Hrg1 in all organs, but less so for Pgf. Our results indicate that the heart expresses haem response pathway genes at surprisingly high basal levels and shares with the liver a similar transcriptional response to circulating haem. The role of the heart in haem response should be investigated further.

Free heme regulates placenta growth factor through NRF2-antioxidant response signaling.

Free heme activates erythroblasts to express and secrete Placenta Growth Factor (PlGF), an angiogenic peptide of the VEGF family. High circulating levels of PlGF have been associated in experimental animals and in patients with sickle cell disease with echocardiographic markers of pulmonary hypertension, a life-limiting complication associated with more intense hemolysis. We now show that the mechanism of heme regulation of PlGF requires the contribution of the key antioxidant response regulator NRF2. Mimicking the effect of heme, the NRF2 agonist sulforaphane stimulates the PlGF transcript level nearly 30-fold in cultured human erythroblastoid cells. Heme and sulforaphane also induce transcripts for NRF2 itself, its partners MAFF and MAFG, and its competitor BACH1. Furthermore, heme induction of the PlGF transcript is significantly diminished by the NRF2 inhibitor brusatol and by siRNA knockdown of the NRF2 and/or MAFG transcription factors. Chromatin immunoprecipitation experiments show that heme induces NRF2 to bind directly to the PlGF promoter region. In complementary in vivo experiments, mice injected with heme show a significant increase in their plasma PlGF protein as early as 3 h after treatment. Our results reveal an important mechanism of PlGF regulation, adding to the growing literature that supports the pivotal importance of the NRF2 axis in the pathobiology of sickle cell disease.