Combining SOS1 and MEK Inhibitors in a Murine Model of Plexiform Neurofibroma Results in Tumor Shrinkage.

Individuals with neurofibromatosis type 1 develop rat sarcoma virus (RAS)-mitogen-activated protein kinase-mitogen-activated and extracellular signal-regulated kinase (RAS-MAPK-MEK)-driven nerve tumors called neurofibromas. Although MEK inhibitors transiently reduce volumes of most plexiform neurofibromas in mouse models and in neurofibromatosis type 1 (NF1) patients, therapies that increase the efficacy of MEK inhibitors are needed. BI-3406 is a small molecule that prevents Son of Sevenless (SOS)1 interaction with Kirsten rat sarcoma viral oncoprotein (KRAS)-GDP, interfering with the RAS-MAPK cascade upstream of MEK. Single agent SOS1 inhibition had no significant effect in the DhhCre;Nf1 fl/fl mouse model of plexiform neurofibroma, but pharmacokinetics (PK)-driven combination of selumetinib with BI-3406 significantly improved tumor parameters. Tumor volumes and neurofibroma cell proliferation, reduced by MEK inhibition, were further reduced by the combination. Neurofibromas are rich in ionized calcium binding adaptor molecule 1 (Iba1)+ macrophages; combination treatment resulted in small and round macrophages, with altered cytokine expression indicative of altered activation. The significant effects of MEK inhibitor plus SOS1 inhibition in this preclinical study suggest potential clinical benefit of dual targeting of the RAS-MAPK pathway in neurofibromas. SIGNIFICANCE STATEMENT: Interfering with the RAS-mitogen-activated protein kinase (RAS-MAPK) cascade upstream of mitogen activated protein kinase kinase (MEK), together with MEK inhibition, augment effects of MEK inhibition on neurofibroma volume and tumor macrophages in a preclinical model system. This study emphasizes the critical role of the RAS-MAPK pathway in controlling tumor cell proliferation and the tumor microenvironment in benign neurofibromas.

The importance of bone marrow and the immune system in driving increases in blood pressure and sympathetic nerve activity in hypertension.

NEW FINDINGS: What is the topic of this review? This manuscript provides a review of the current understanding of the role of the sympathetic nervous system in regulation of bone marrow-derived immune cells and the effect that the infiltrating bone marrow cells may have on perpetuation of the sympathetic over-activation in hypertension. What advances does it highlight? We highlight the recent advances in understanding of the neuroimmune interactions both peripherally and centrally as they relate to blood pressure control. ABSTRACT: The sympathetic nervous system (SNS) plays a crucial role in maintaining physiological homeostasis, in part by regulating, integrating and orchestrating processes between many physiological systems, including the immune system. Sympathetic nerves innervate all primary and secondary immune organs, and all cells of the immune system express ß-adrenoreceptors. In turn, immune cells can produce cytokines, chemokines and neurotransmitters capable of modulating neuronal activity and, ultimately, SNS activity. Thus, the essential role of the SNS in the regulation of innate and adaptive immune functions is mediated, in part, via ß-adrenoreceptor-induced activation of bone marrow cells by noradrenaline. Interestingly, both central and systemic inflammation are well-established hallmarks of hypertension and its co-morbidities, including an inflammatory process involving the transmigration and infiltration of immune cells into tissues. We propose that physiological states that prolong ß-adrenoreceptor activation in bone marrow can disrupt neuroimmune homeostasis and impair communication between the immune system and SNS, leading to immune dysregulation, which, in turn, is sustained via a central mechanism involving neuroinflammation.

Elevated bone marrow sympathetic drive precedes systemic inflammation in angiotensin II hypertension.

Increased sympathetic nervous system activity is a hallmark of hypertension (HTN), and it is implicated in altered immune system responses in its pathophysiology. However, the precise mechanisms of neural-immune interaction in HTN remain elusive. We have previously shown an association between elevated sympathetic drive to the bone marrow (BM) and activated BM immune cells in rodent models of HTN. Moreover, microglial-dependent neuroinflammation is also seen in rodent models of HTN. However, the cause-effect relationship between central and systemic inflammatory responses and the sympathetic drive remains unknown. These observations led us to hypothesize that increase in the femoral BM sympathetic nerve activity (fSNA) initiates a cascade of events leading to increase in blood pressure (BP). Here, we investigated the temporal relationship between the BM sympathetic drive, activation of the central and peripheral immune system, and increase in BP in the events leading to established HTN. The present study demonstrates that central infusion of angiotensin II (ANG II) induces early microglial activation in the paraventricular nucleus of hypothalamus, which preceded increase in the fSNA. In turn, activation of fSNA correlated with the timing of increased production and release of CD4+.IL17+ T cells and other proinflammatory cells into circulation and elevation in BP, whereas infiltration of CD4+ cells to the paraventricular nucleus marked establishment of ANG II HTN. This study identifies cellular and molecular mechanisms involved in neural-immune interactions in early and established stages of rodent ANG II HTN. NEW & NOTEWORTHY Early microglia activation in paraventricular nucleus precedes sympathetic activation of the bone marrow. This leads to increased bone marrow immune cells and their release into circulation and an increase in blood pressure. Infiltration of CD4+ T cells into paraventricular nucleus paraventricular nucleus marks late hypertension.

Involvement of bone marrow cells and neuroinflammation in hypertension.

RATIONALE: Microglial activation in autonomic brain regions is a hallmark of neuroinflammation in neurogenic hypertension. Despite evidence that an impaired sympathetic nerve activity supplying the bone marrow (BM) increases inflammatory cells and decreases angiogenic cells, little is known about the reciprocal impact of BM-derived inflammatory cells on neuroinflammation in hypertension. OBJECTIVE: To test the hypothesis that proinflammatory BM cells from hypertensive animals contribute to neuroinflammation and hypertension via a brain-BM interaction. METHODS AND RESULTS: After BM ablation in spontaneously hypertensive rats, and reconstitution with normotensive Wistar Kyoto rat BM, the resultant chimeric spontaneously hypertensive rats displayed significant reduction in mean arterial pressure associated with attenuation of both central and peripheral inflammation. In contrast, an elevated mean arterial pressure along with increased central and peripheral inflammation was observed in chimeric Wistar-Kyoto rats reconstituted with spontaneously hypertensive rat BM. Oral treatment with minocycline, an inhibitor of microglial activation, attenuated hypertension in both the spontaneously hypertensive rats and the chronic angiotensin II-infused rats. This was accompanied by decreased sympathetic drive and inflammation. Furthermore, in chronic angiotensin II-infused rats, minocycline prevented extravasation of BM-derived cells to the hypothalamic paraventricular nucleus, presumably via a mechanism of decreased C-C chemokine ligand 2 levels in the cerebrospinal fluid. CONCLUSIONS: The BM contributes to hypertension by increasing peripheral inflammatory cells and their extravasation into the brain. Minocycline is an effective therapy to modify neurogenic components of hypertension. These observations support the hypothesis that BM-derived cells are involved in neuroinflammation, and targeting them may be an innovative strategy for neurogenic resistant hypertension therapy.

Loss of bone marrow adrenergic beta 1 and 2 receptors modifies transcriptional networks, reduces circulating inflammatory factors, and regulates blood pressure.

Hypertension (HTN) is a prevalent condition with complex etiology and pathophysiology. Evidence exists of significant communication between the nervous system and the immune system (IS), and there appears to be a direct role for inflammatory bone marrow (BM) cells in the pathophysiology of hypertension. However, the molecular and neural mechanisms underlying this interaction have not been characterized. Here, we transplanted whole BM cells from the beta 1 and 2 adrenergic receptor (AdrB1(tm1Bkk)AdrB2(tm1Bkk)/J) knockout (KO) mice into near lethally irradiated C57BL/6J mice to generate a BM AdrB1.B2 KO chimera. This allowed us to evaluate the role of the BM beta 1 and beta 2 adrenergic receptors in mediating BM IS homeostasis and regulating blood pressure (BP) in an otherwise intact physiological setting. Fluorescence-activated cell sorting demonstrated that a decrease in systolic and mean BP in the AdrB1.B2 KO chimera is associated with a decrease in circulating inflammatory T cells, macrophage/monocytes, and neutrophils. Transcriptomics in the BM identified 7,419 differentially expressed transcripts between the C57 and AdrB1.B2 KO chimera. Pathway analysis revealed differentially expressed transcripts related to several cell processes in the BM of C57 compared with AdrB1.B2 KO chimera, including processes related to immunity (e.g., T-cell activation, T-cell recruitment, cytokine production, leukocyte migration and function), the cardiovascular system (e.g., blood vessel development, peripheral nerve blood flow), and the brain (e.g., central nervous system development, neurite development) among others. This study generates new insight into the molecular events that underlie the interaction between the sympathetic drive and IS in modulation of BP.

C5aR plus MEK inhibition durably targets the tumor milieu and reveals tumor cell phagocytosis.

Plexiform neurofibromas (PNFs) are nerve tumors caused by loss of NF1 and dysregulation of RAS-MAPK signaling in Schwann cells. Most PNFs shrink in response to MEK inhibition, but targets with increased and durable effects are needed. We identified the anaphylatoxin C5a as increased in PNFs and expressed largely by PNF m acrophages. We defined pharmacokinetic and immunomodulatory properties of a C5aR1/2 antagonist and tested if peptide antagonists augment the effects of MEK inhibition. MEK inhibition recruited C5AR1 to the macrophage surface; short-term inhibition of C5aR elevated macrophage apoptosis and Schwann cell death, without affecting MEK-induced tumor shrinkage. PNF macrophages lacking C5aR1 increased the engulfment of dying Schwann cells, allowing their visualization. Halting combination therapy resulted in altered T-cell distribution, elevated Iba1+ and CD169+ immunoreactivity, and profoundly altered cytokine expression, but not sustained trumor shrinkage. Thus, C5aRA inhibition independently induces macrophage cell death and causes sustained and durable effects on the PNF microenvironment.

Butyrate regulates inflammatory cytokine expression without affecting oxidative respiration in primary astrocytes from spontaneously hypertensive rats.

Neurons and glia exhibit metabolic imbalances in hypertensive animal models, and loss of metabolic homeostasis can lead to neuroinflammation and oxidative stress. The objective of this study was to determine the effects of the microbial metabolite butyrate on mitochondrial bioenergetics and inflammatory markers in mixed brainstem and hypothalamic primary cultures of astrocytes between normotensive (Sprague-Dawley, S-D) and spontaneously hypertensive (SHR) rats. Bioenergetics of mitochondria in astrocytes from normotensive S-D rats were modified with butyrate, but this was not the case in astrocytes derived from SHR, suggesting aberrant mitochondrial function. Transcripts related to oxidative stress, butyrate transporters, butyrate metabolism, and neuroinflammation were quantified in astrocyte cultures treated with butyrate at 0, 200, 600, and 1000 µmol/L. Butyrate decreased catalase and monocarboxylate transporter 1 mRNA in astrocytes of S-D rats but not in the SHR. Moreover, while butyrate did not directly regulate the expression of 3-hydroxybutyrate dehydrogenase 1 and 2 in astrocytes of either strain, the expression levels for these transcripts in untreated cultures were lower in the SHR compared to S-D. We observed higher levels of specific inflammatory cytokines in astrocytes of SHR, and treatment with butyrate decreased expression of Ccl2 and Tlr4 in SHR astrocytes only. Conversely, butyrate treatment increased expression of tumor necrosis factor in astrocytes from SHR but not from the S-D rats. This study improves our understanding of the role of microbial metabolites in regulating astrocyte function, and provides support that butyrate differentially regulates both the bioenergetics and transcripts related to neuroinflammation in astrocytes from SHR versus S-D rats.

Shifts in the Gut Microbiota Composition Due to Depleted Bone Marrow Beta Adrenergic Signaling Are Associated with Suppressed Inflammatory Transcriptional Networks in the Mouse Colon.

The brain-gut axis plays a critical role in the regulation of different diseases, many of which are characterized by sympathetic dysregulation. However, a direct link between sympathetic dysregulation and gut dysbiosis remains to be illustrated. Bone marrow (BM)-derived immune cells continuously interact with the gut microbiota to maintain homeostasis in the host. Their function is largely dependent upon the sympathetic nervous system acting via adrenergic receptors present on the BM immune cells. In this study, we utilized a novel chimera mouse that lacks the expression of BM beta1/2 adrenergic receptors (b1/2-ARs) to investigate the role of the sympathetic drive to the BM in gut and microbiota homeostasis. Fecal analyses demonstrated a shift from a dominance of Firmicutes to Bacteroidetes phylum in the b1/2-ARs KO chimera, resulting in a reduction in Firmicutes/Bacteroidetes ratio. Meanwhile, a significant reduction in Proteobacteria phylum was determined. No changes in the abundance of acetate-, butyrate-, and lactate-producing bacteria, and colon pathology were observed in the b1/2-ARs KO chimera. Transcriptomic profiling in colon identified Killer Cell Lectin-Like Receptor Subfamily D, Member 1 (Klrd1), Membrane-Spanning 4-Domains Subfamily A Member 4A (Ms4a4b), and Casein Kinase 2 Alpha Prime Polypeptide (Csnk2a2) as main transcripts associated with the microbiota shifts in the b1/2-ARs KO chimera. Suppression of leukocyte-related transcriptome networks (i.e., function, differentiation, migration), classical compliment pathway, and networks associated with intestinal function, barrier integrity, and excretion was also observed in the colon of the KO chimera. Moreover, reduced expression of transcriptional networks related to intestinal diseases (i.e., ileitis, enteritis, inflammatory lesions, and stress) was noted. The observed suppressed transcriptome networks were associated with a reduction in NK cells, macrophages, and CD4+ T cells in the b1/2-ARs KO chimera colon. Thus, sympathetic regulation of BM-derived immune cells plays a significant role in modifying inflammatory networks in the colon and the gut microbiota composition. To our knowledge, this study is the first to suggest a key role of BM b1/2-ARs signaling in host-microbiota interactions, and reveals specific molecular mechanisms that may lead to generation of novel anti-inflammatory treatments for many immune and autonomic diseases as well as gut dysbiosis across the board.

Gut dysbiosis is linked to hypertension.

Emerging evidence suggests that gut microbiota is critical in the maintenance of physiological homeostasis. This study was designed to test the hypothesis that dysbiosis in gut microbiota is associated with hypertension because genetic, environmental, and dietary factors profoundly influence both gut microbiota and blood pressure. Bacterial DNA from fecal samples of 2 rat models of hypertension and a small cohort of patients was used for bacterial genomic analysis. We observed a significant decrease in microbial richness, diversity, and evenness in the spontaneously hypertensive rat, in addition to an increased Firmicutes/Bacteroidetes ratio. These changes were accompanied by decreases in acetate- and butyrate-producing bacteria. In addition, the microbiota of a small cohort of human hypertensive patients was found to follow a similar dysbiotic pattern, as it was less rich and diverse than that of control subjects. Similar changes in gut microbiota were observed in the chronic angiotensin II infusion rat model, most notably decreased microbial richness and an increased Firmicutes/Bacteroidetes ratio. In this model, we evaluated the efficacy of oral minocycline in restoring gut microbiota. In addition to attenuating high blood pressure, minocycline was able to rebalance the dysbiotic hypertension gut microbiota by reducing the Firmicutes/Bacteroidetes ratio. These observations demonstrate that high blood pressure is associated with gut microbiota dysbiosis, both in animal and human hypertension. They suggest that dietary intervention to correct gut microbiota could be an innovative nutritional therapeutic strategy for hypertension.