Homeostatic Conditions Research Articles

Ker-050, an Inhibitor of TGF- β Superfamily Signaling, Promoted Thrombopoiesis and Reversed Immune Thrombocytopenia in a Mouse Model of Disease

KER-050 is a modified ActRIIA ligand trap that is designed to inhibit the activity of the TGF-β ligands, including activin A, activin B, GDF8 and GDF11, that act through the SMAD2/3 signaling cascade. Notably, in a Phase 1 clinical study we observed that, in addition to increases in red blood cells and hemoglobin, treatment with KER-050 elicited a robust and sustained increase in platelets (PLTs) in healthy volunteers. While ActRII ligand traps have been shown to increase erythropoiesis in preclinical and clinical studies, their role in thrombopoiesis has not yet been well-elucidated.A variety of conditions exist where hematopoiesis is impaired and cytopenia persists, including comorbidities associated with aging, diseases causing ineffective hematopoiesis such as myelodysplastic syndrome (MDS) and myelofibrosis (MF), and acute bleeding. Thrombocytopenia can arise from primary or secondary causes due to multiple etiologies and treatments are limited, aiming at treating the root cause of disease or replacing PLTs through transfusion. Therefore, there is an unmet medical need for more targeted treatments to correct thrombocytopenia. Here, in a series of preclinical studies, we investigated the mechanism of action of KER-050 on thrombopoiesis and evaluated its ability to accelerate recovery from acute platelet depletion.First, we examined how RKER-050 (a research form of KER-050) affected thrombopoiesis under homeostatic conditions. We observed that a single intraperitoneal dose of RKER-050 (10 mg/kg) to 11-week-old mice resulted in a 2-fold increase in PLTs compared to vehicle-treated mice 12 hours after treatment. The timing of the effect is suggestive of a direct effect of RKER-050 on terminal platelet maturation. Additionally, there was a 35% increase in the number of bone marrow (BM) megakaryocyte (Mk) progenitors (Lin -; sca1 -; cKit -; CD41 + cells), demonstrating that RKER-050 affected earlier stages of the PLT formation process. We also evaluated the effect of RKER-050 on the polyploidization of Mks, a hallmark of Mk differentiation. At 24 hours after treatment with RKER-050, there was an increase in BM CD41 + cells with ploidy greater than 16N compared to vehicle-treated mice, demonstrating that RKER-050 treatment resulted in a greater number of Mk that are potentially primed for platelet production. Taken together, these data are consistent with RKER-050 affecting multiple stages of thrombopoiesis in a preclinical model.We next tested whether RKER-050 affects PLTs in a mouse model of immune thrombocytopenia (IT) where antibodies directed against mouse GPIbα result in acutely reduced PLT numbers. In this model, mice receiving anti-GPIbα had a 25% reduction in PLT number at 4 days post-dose compared to IgG control-treated mice. At this point, the anti-GPIba cohort was divided into receiving either a single dose of vehicle or RKER-050. On day 7 following anti-GPIbα treatment, PLT counts in vehicle-treated mice were 62% lower compared to IgG-control-treated mice. In contrast, the GPIbα-mediated effect on PLT count was stabilized in the anti-GPIbα + RKER-050 group, which had 55% more platelets compared to the anti-GPIba + vehicle group. These data suggest that RKER-050 promoted thrombopoiesis in mice even under conditions when the system is acutely challenged and potentially could promote faster recovery from thrombocytopenia. Additionally, we observed a 25% increase in the number of CD41 + cells in the BM of the RKER-050-treated group compared to the vehicle-treated group at day 10 after PLT depletion, suggesting that under acute thrombocytopenia, RKER-050 treatment promoted differentiation of Mks as a mechanism of the accelerated recovery in platelet-depleted mice.In summary, our preclinical data demonstrate a potentially novel effect of RKER-050 on thrombopoiesis. RKER-050 treatment resulted in a rapid increase in PLTs, consistent with an effect on terminal maturation. Treatment also increased the number of Mk progenitors and increased the number of polyploid Mks, demonstrating an effect on early stages of thrombopoiesis. These findings support that RKER-050-targeted ligands regulate multiple stages of the thrombopoiesis pathway in mice. Additionally, our data demonstrate that KER-050 has the potential to accelerate the rate of PLT recovery due to acute depletion, and could represent a potential novel treatment option for thrombocytopenia in patients with MDS, MF and IT. DisclosuresFeigenson: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Nathan: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lamora: Keros Therapeutics: Current Employment. Fisher: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tseng: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

The Tetraspanin CD53 Protects Hematopoietic Stem Cells Following Cellular Stress through Induction of DREAM Complex-Mediated Quiescence

Maintenance of quiescence is necessary for optimal hematopoietic stem cell (HSC) function. The absence of fine-tuned cycling regulation of HSCs can result in impaired hematopoiesis, bone marrow (BM) failure, or malignant transformation. While various factors, including inflammatory cytokines and chemotherapy, have been identified to induce HSC cycling, the mechanisms regulating HSC return to quiescence are unclear.Herein, we profiled HSCs in response to a variety of inflammatory stressors (cytokines, toll-like receptor agonists, mobilizing agents, and chemotherapeutics) and found markedly upregulated expression of CD53 in both mouse and human HSCs, in some cases by as much as 70-fold higher expression over vehicle-treated controls. CD53 is a tetraspanin, a type of transmembrane protein involved in plasma membrane organization and regulation of processes such as cellular migration, adhesion, and signaling. CD53 has been shown to be asymmetrically segregated in HSCs, with CD53-enriched HSCs believed to be more stem-like; however, there is no current proposed mechanism to explain the association between CD53 and stem cell quality.To understand the role of CD53 in HSC quality, we generated a CD53 knockout mouse, and profiled HSC phenotype and function. Under homeostatic conditions, Cd53 -/- HSC number and frequency are normal as compared to wild type (WT) mice. However, we found Cd53 -/-HSCs to have significantly impaired function, particularly in response to inflammatory stimuli. Cd53 -/- BM failed to engraft as well as WT BM (45% chimerism vs 63%, p<0.05) in competitive transplants, and this deficit was exacerbated when G-CSF-mobilized HSCs from the spleen were transplanted (47% vs 77%, p<0.0001). Analysis of cycling status during and after G-CSF stimulation found that Cd53 -/- HSCs, particularly those that mobilize to the spleen, undergo cell division twice as frequently as WT HSCs (22% vs 10%, p<0.05). Treatment of WT and Cd53 -/- mice with serial doses of the chemotherapeutic 5-fluorouracil resulted in significantly accelerated death in Cd53 -/- mice (median survival 14 days vs 28 days, p<0.0001), supporting that loss of CD53 is associated with dysregulated HSC quiescence.Transcriptomic sequencing revealed significant upregulation of genes associated with cell cycling and division in G-CSF-treated Cd53 -/- HSCs compared to WT controls. Notably, these differentially expressed genes are targets of the dimerization partner, RB-like, E2F and multi-vulval class B (DREAM) complex, a newly described transcriptional regulator that represses cell cycling-associated genes, suggesting that CD53 promotes DREAM-mediated quiescence in stressed HSCs. We performed CUT&Tag profiling of Rbl2/p130, a DREAM complex subunit, in HSCs and found that loss of CD53 was associated with decreased complex binding (consistent with enhanced transcription of cell cycle genes). Proximity labeling studies demonstrated that CD53 interacts with cell cycle machinery proteins, including Rbl2/p130, and Western blots of sorted HSCs from Cd53 -/- and WT mice treated with G-CSF showed decreased expression of DREAM complex components (Rbl1/p107 and Rbl2/p130) in the absence of CD53. Finally, enforced activation of DREAM and HSC quiescence using the CDK4/6 inhibitor palbociclib rescued the Cd53 -/-HSC repopulating defect. Together, these data derive a novel mechanism whereby CD53 regulates HSC quiescence through regulating DREAM complex binding during stress-induced cycling. DisclosuresNo relevant conflicts of interest to declare.

HMGA2 Maintains Hematopoietic Stem Cell Via Pleiotropic Regulation of the Transcription in Stress Conditions

High-mobility group AT-hook 2 (Hmga2), an epigenetic modifier, opens the chromatin and modulate the transcription. Hmga2 is highly expressed in fetal and adult hematopoietic stem cells (HSCs). Hmga2 over-expression has been shown to promote self-renewal of HSC, however the molecular mechanism of how Hmga2 enhanced the self-renewal of HSC is still unclear. In this study, we assessed the function of Hmga2 in HSCs in steady and stress conditions by utilizing new Hmga2 conditional knock-in (KI) mouse and Hmga2 conditional knock-out mouse, which were crossed with either Cre-ERT2 mouse or Vav1-iCre mouse. Hmga2 KI mice showed a mild elevation in platelet counts, but did not develop malignancies in one year observation period. We performed a competitive transplantation assay by using purified HSCs, and found that wild-type HSCs diminished the repopulating capacity at the tertiary transplantation, Hmga2 KI HSCs maintained higher chimerism in myeloid cells and platelets in the PB and HSCs in the BM. We found that Hmga2 KO cells reduced the repopulating capacity, compared to wild-type cells. Thus, the expression of Hmga2 is critical for the self-renewal of HSC upon the transplantation. By performing RNA-sequencing of HSCs in homeostatic condition, we found that Hmga2 KI HSCs showed positive enrichments in cell cycle and proliferative signature, but maintained a stem cell signature, compared to wild-type HSCs. Since Hmga2 has been shown to globally open the chromatin in neural stem cells, we performed ATAC-sequence analysis in HSCs. Notably, Hmga2 KI HSCs showed 539 opened and 387 closed chromatin in H3K27ac-marked active regulatory regions, compared to wild-type HSCs. Among these opened genes by Hmga2, we generated a virus vector for fifteen genes, which were highly expressed in Hmga2 KI HSCs, and found that ectopic expression of Igf2bp2, an RNA binding protein, increased self-renewal capacity of HSC, but did not induce the enhanced production of myeloid cells and platelets that were observed in Hmga2 KI cells, in in vitro and in vivo settings. Indeed, Hmga2-ChIP-sequencing revealed that Hmga2 was directly bound to a proximal region of the Igf2bp2 gene, and CRISPR/Cas9-mediated deletion of the Igf2bp2 gene canceled the enhanced self-renewal capacity of Hmga2 KI HSCs, indicating that the Hmga2-Igf2bp2 axis is critical for the self-renewal of HSC.We next assessed function of Hmga2 in stress hematopoiesis after in vivo treatment of 5-FU. Hmga2 KI mice showed faster recoveries of reduction of platelets in the PB and increased CD41+HSCs and megakaryocyte progenitors in the BM in twelve days, in which WT mice reduced numbers of those cells in this condition. RNA-sequencing revealed that Hmga2 KI HSCs maintained expression levels of genes in stem cell- and proliferation-signatures at 3 days and 6 days post 5-FU injection, compared to WT HSCs that reduced expression of stem cell genes but activated inflammatory response genes. In contrast, Hgma2 KO HSCs enhanced expression of inflammatory response genes post the 5-FU injection, indicating Hmga2 represses expression of inflammatory response genes in the stress condition. Indeed, Hmga2-ChIP-sequencing revealed that Hmga2 was bound to larger numbers of genes involved in inflammatory responses in Kit+ cells post the 5-FU treatment from those in the control cells. Given increased expression of and a post-translational modification of Hmga2 protein in Kit+ cells post the 5-FU treatment, the remodeling of Hmga2 binding regions was appeared to depend on the modification of Hmga2 at downstream of the stress signal. Thus, Hmga2 directly activates Igf2bp2 to enhance the self-renew of HSC, but also represses the inflammatory response, leading to the enhanced megakaryopoiesis in the stress condition. DisclosuresNo relevant conflicts of interest to declare.

Utilizing a reductionist model to study host-microbe interactions in intestinal inflammation

BackgroundThe gut microbiome is altered in patients with inflammatory bowel disease, yet how these alterations contribute to intestinal inflammation is poorly understood. Murine models have demonstrated the importance of the microbiome in colitis since colitis fails to develop in many genetically susceptible animal models when re-derived into germ-free environments. We have previously shown that Wiskott-Aldrich syndrome protein (WASP)-deficient mice (Was−/−) develop spontaneous colitis, similar to human patients with loss-of-function mutations in WAS. Furthermore, we showed that the development of colitis in Was−/− mice is Helicobacter dependent. Here, we utilized a reductionist model coupled with multi-omics approaches to study the role of host-microbe interactions in intestinal inflammation.ResultsWas−/− mice colonized with both altered Schaedler flora (ASF) and Helicobacter developed colitis, while those colonized with either ASF or Helicobacter alone did not. In Was−/− mice, Helicobacter relative abundance was positively correlated with fecal lipocalin-2 (LCN2), a marker of intestinal inflammation. In contrast, WT mice colonized with ASF and Helicobacter were free of inflammation and strikingly, Helicobacter relative abundance was negatively correlated with LCN2. In Was−/− colons, bacteria breach the mucus layer, and the mucosal relative abundance of ASF457 Mucispirillum schaedleri was positively correlated with fecal LCN2. Meta-transcriptomic analyses revealed that ASF457 had higher expression of genes predicted to enhance fitness and immunogenicity in Was−/− compared to WT mice. In contrast, ASF519 Parabacteroides goldsteinii’s relative abundance was negatively correlated with LCN2 in Was−/− mice, and transcriptional analyses showed lower expression of genes predicted to facilitate stress adaptation by ASF519 in Was−/−compared to WT mice.ConclusionsThese studies indicate that the effect of a microbe on the immune system can be context dependent, with the same bacteria eliciting a tolerogenic response under homeostatic conditions but promoting inflammation in immune-dysregulated hosts. Furthermore, in inflamed environments, some bacteria up-regulate genes that enhance their fitness and immunogenicity, while other bacteria are less able to adapt and decrease in abundance. These findings highlight the importance of studying host-microbe interactions in different contexts and considering how the transcriptional profile and fitness of bacteria may change in different hosts when developing microbiota-based therapeutics.EtQioPqacpXxmFoWy2LpqYVideo abstract

Microbiota regulates the turnover kinetics of gut macrophages in health and inflammation.

The gut immune system has evolved to co-exist in a mutually beneficial symbiotic relationship with its microflora. Here, using a germ-free fate-mapping mouse model, we provide clear insight into how the enteric commensals determine the kinetics of macrophage turnover. The microbiome density along the gastrointestinal tract defines the persistence of ontogenically diverse macrophages, with the highest numbers of the long-lived F4/80hiTim4+ macrophage subset in the less densely colonized small intestine. Furthermore, the microbiome contributes to a tightly regulated monocyte-dependent replenishment of both long- and short-lived F4/80hi macrophages under homeostatic and inflammatory conditions. In the latter situation, the commensals regulate rapid replenishment of the depleted macrophage niche caused by the intestinal inflammation. The microbial ecosystem imprints a favorable cytokine microenvironment in the intestine to support macrophage survival and monocyte-dependent replenishment. Therefore, the host immune system-commensal cross-talk provides an efficient strategy to assure intestinal homeostasis.

Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors.

Age‐related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen‐associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen‐associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely‐tuned mechanism regulating directional apical:basal sorting and secretion of drusen‐associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE‐derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.

Chromatin-state barriers enforce an irreversible mammalian cell fate decision.

Stem and progenitor cells have the capacity to balance self-renewal and differentiation. Hematopoietic myeloid progenitors replenish more than 25 billion terminally differentiated neutrophils every day under homeostatic conditions and can increase this output in response to stress or infection. At what point along the spectrum of maturation do progenitors lose capacity for self-renewal and become irreversibly committed to differentiation? Using a system of conditional myeloid development that can be toggled between self-renewal and differentiation, we interrogate determinants of this "point of no return" in differentiation commitment. Irreversible commitment is due primarily to loss of open regulatory site access and disruption of a positive feedback transcription factor activation loop. Restoration of the transcription factor feedback loop extends the window of cell plasticity and alters the point of no return. These findings demonstrate how the chromatin state enforces and perpetuates cell fate and identify potential avenues for manipulating cell identity.

COMMD10 is critical for Kupffer cell survival and controls Ly6Chi monocyte differentiation and inflammation in the injured liver.

Liver-resident macrophages Kupffer cells (KCs) and infiltrating Ly6Chi monocytes both contribute to liver tissue regeneration in various pathologies but also to disease progression upon disruption of orderly consecutive regeneration cascades. Little is known about molecular pathways that regulate their differentiation, maintenance, or inflammatory behavior during injury. Here, we show that copper metabolism MURR1 domain (COMMD)10-deficient KCs adopt liver-specific identity. Strikingly, COMMD10 deficiency in KCs and in other tissue-resident macrophages impedes their homeostatic survival, leading to their continuous replacement by Ly6Chi monocytes. While COMMD10 deficiency in KCs mildly worsens acetaminophen-induced liver injury (AILI), its deficiency in Ly6Chi monocytes results in exacerbated and sustained hepatic damage. Monocytes display unleashed inflammasome activation and a reduced type I interferon response and acquire "neutrophil-like" and lipid-associated macrophage differentiation fates. Collectively, COMMD10 appears indispensable for KC and other tissue-resident macrophage survival and is an important regulator of Ly6Chi monocyte fate decisions and reparative behavior in the diseased liver.

Arginase Signalling as a Key Player in Chronic Wound Pathophysiology and Healing.

Arginase (ARG) represents an important evolutionarily conserved enzyme that is expressed by multiple cell types in the skin. Arg acts as the mediator of the last step of the urea cycle, thus providing protection against excessive ammonia under homeostatic conditions through the production of L-ornithine and urea. L-ornithine represents the intersection point between the ARG-dependent pathways and the urea cycle, therefore contributing to cell detoxification, proliferation and collagen production. The ARG pathways help balance pro- and anti-inflammatory responses in the context of wound healing. However, local and systemic dysfunctionalities of the ARG pathways have been shown to contribute to the hindrance of the healing process and the occurrence of chronic wounds. This review discusses the functions of ARG in macrophages and fibroblasts while detailing the deleterious implications of a malfunctioning ARG enzyme in chronic skin conditions such as leg ulcers. The review also highlights how ARG links with the microbiota and how this impacts on infected chronic wounds. Lastly, the review depicts chronic wound treatments targeting the ARG pathway, alongside future diagnosis and treatment perspectives.

Sox9+ cells are required for salivary gland regeneration after radiation damage via the Wnt/β-catenin pathway

Radiotherapy for head and neck cancer can cause serious side effects, including severe damage to the salivary glands, resulting in symptoms such as xerostomia, dental caries, and oral infection. Because of the lack of long-term treatment for the symptoms of xerostomia, current research has focused on finding endogenous stem cells that can differentiate into various cell lineages to replace lost tissues and restore functions. Here, we report that Sox9+ cells can differentiate into various salivary epithelial cell lineages under homeostatic conditions. After ablating Sox9+ cells, the salivary glands of irradiated mice showed more severe phenotypes and the reduced proliferative capacity. Analysis of online single-cell RNA-sequencing data reveals the enrichment of the Wnt/β-catenin pathway in the Sox9+ cell population. Furthermore, treatment with a Wnt/β-catenin inhibitor in irradiated mice inhibits the regenerative capability of Sox9+ cells. Finally, we show that Sox9+ cells are capable of forming organoids in vitro and that transplanting these organoids into salivary glands after radiation partially restored salivary gland functions. These results suggest that regenerative therapy targeting Sox9+ cells is a promising approach to treat radiation-induced salivary gland injury.

Amelioration of enteric dysbiosis by polyoxotungstates in mice gut

Here we show that Preyssler-type polyoxotungstates (Preyssler-type POTs, [NaP5W30O110]−14) complexed with peptides can prevent the dysbiotic expansion of anaerobic bacteria of the Enterobacteriaceae family. In a dextran sulfate sodium (DSS)-induced colitis model, symptom remission of C57BL/6 J mice with colitis is achieved by orally treated with POT complexes. Ten days of daily administration of POT complexes reduces 5% body weight loss and the mRNA levels of proinflammatory markers (77% reduction for Il6, 73% reduction for Tnf, 91% reduction for Cxcl1) in the caecum and proximal colon. Bacterial population analysis reveals that these Enterobacteriaceae population in the caecal content decline by one order of magnitude after administration of POT complexes. POT complexes exert anti-inflammatory effects indirectly on the host immune system by inhibition of malignant expansion of anaerobic Enterobacteriaceae during gut inflammation. Furthermore, POTs show negligible effect on bacterial growth in vitro, healthy mice and their microbiota composition under homeostatic conditions. Rationally designed POT complexes will provide distinctive approach to improve enteric bacteria dysbiosis-associated gut inflammation by balancing bacterial communities.

Role of Eosinophils in Intestinal Inflammation and Fibrosis in Inflammatory Bowel Disease: An Overlooked Villain?

Eosinophils are leukocytes which reside in the gastrointestinal tract under homeostatic conditions, except for the esophagus which is normally devoid of eosinophils. Research on eosinophils has primarily focused on anti-helminth responses and type 2 immune disorders. In contrast, the search for a role of eosinophils in chronic intestinal inflammation and fibrosis has been limited. With a shift in research focus from adaptive to innate immunity and the fact that the eosinophilic granules are filled with inflammatory mediators, eosinophils are becoming a point of interest in inflammatory bowel diseases. In the current review we summarize eosinophil characteristics and recruitment as well as the current knowledge on presence, inflammatory and pro-fibrotic functions of eosinophils in inflammatory bowel disease and other chronic inflammatory conditions, and we identify research gaps which should be covered in the future.

Pharmacological Inhibition of the VCP/Proteasome Axis Rescues Photoreceptor Degeneration in RHOP23H Rat Retinal Explants.

Rhodopsin (RHO) misfolding mutations are a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). The most prevalent mutation, RHOP23H, results in its misfolding and retention in the endoplasmic reticulum (ER). Under homeostatic conditions, misfolded proteins are selectively identified, retained at the ER, and cleared via ER-associated degradation (ERAD). Overload of these degradation processes for a prolonged period leads to imbalanced proteostasis and may eventually result in cell death. ERAD of misfolded proteins, such as RHOP23H, includes the subsequent steps of protein recognition, targeting for ERAD, retrotranslocation, and proteasomal degradation. In the present study, we investigated and compared pharmacological modulation of ERAD at these four different major steps. We show that inhibition of the VCP/proteasome activity favors cell survival and suppresses P23H-mediated retinal degeneration in RHOP23H rat retinal explants. We suggest targeting this activity as a therapeutic approach for patients with currently untreatable adRP.

Microglia Regulate Neuronal Circuits in Homeostatic and High-Fat Diet-Induced Inflammatory Conditions.

Microglia are brain resident macrophages, which actively survey the surrounding microenvironment and promote tissue homeostasis under physiological conditions. During this process, microglia participate in synaptic remodeling, neurogenesis, elimination of unwanted neurons and cellular debris. The complex interplay between microglia and neurons drives the formation of functional neuronal connections and maintains an optimal neural network. However, activation of microglia induced by chronic inflammation increases synaptic phagocytosis and leads to neuronal impairment or death. Microglial dysfunction is implicated in almost all brain diseases and leads to long-lasting functional deficiency, such as hippocampus-related cognitive decline and hypothalamus-associated energy imbalance (i.e., obesity). High-fat diet (HFD) consumption triggers mediobasal hypothalamic microglial activation and inflammation. Moreover, HFD-induced inflammation results in cognitive deficits by triggering hippocampal microglial activation. Here, we have summarized the current knowledge of microglial characteristics and biological functions and also reviewed the molecular mechanism of microglia in shaping neural circuitries mainly related to cognition and energy balance in homeostatic and diet-induced inflammatory conditions.

Immunoregulation by type I interferons in the peritoneal cavity.

The peritoneal cavity, a fluid-containing potential space surrounding the abdominal and pelvic organs, is home to a rich network of immune cells that maintain tissue homeostasis and provide protection against infection. However, under pathological conditions such as peritonitis, endometriosis, and peritoneal carcinomatosis, the peritoneal immune system can become dysregulated, resulting in nonresolving inflammation and disease progression. An enhanced understanding of the factors that regulate peritoneal immune cells under both homeostatic conditions and in disease contexts is therefore required to identify new treatment strategies for these often life-limiting peritoneal pathologies. Type I interferons (T1IFNs) are a family of cytokines with broad immunoregulatory functions, which provide defense against viruses, bacteria, and cancer. There have been numerous reports of immunoregulation by T1IFNs within the peritoneal cavity, which can contribute to both the resolution or propagation of peritoneal disease states, depending on the specifics of the disease setting and local environment. In this review, we provide an overview of the major immune cell populations that reside in the peritoneal cavity (or infiltrate it under inflammatory conditions) and highlight their contribution to the initiation, progression, or resolution of peritoneal diseases. Additionally, we will discuss the role of T1IFNs in the regulation of peritoneal immune cells, and summarize the results of laboratory studies and clinical trials which have investigated T1IFNs in peritonitis/sepsis, endometriosis, and peritoneal carcinomatosis.

P-092: Multiple Myeloma cells depend on the DDI2/NRF1-mediated proteasome stress response for survival

<h3>Background</h3> Multiple myeloma (MM) cells depend on the ubiquitin-proteasome system for survival. Proteasome inhibitors (PIs) are FDA-approved in MM and have radically improved patient survival. However, acquisition of resistance is inevitable. The proteasome stress response (PSR) contributes to proteostasis via de novo proteasome biogenesis. The PSR master regulator NRF1 is constitutively degraded by the proteasome in homeostatic conditions, but in face of proteasomal insufficiency, it is deglycosylated by NGLY1, cleaved by the aspartic protease DDI2, and translocates to the nucleus, to induce proteasome subunit gene transcription. Pharmacological targeting of NGLY1 was recently shown to sensitize cells to PIs. <h3>Hypothesis</h3> We hypothesize that blocking DDI2 or NRF1 could target an intrinsic vulnerability of MM and represent an innovative therapeutic strategy to overcome PI resistance. <h3>Methods</h3> We used MM cell lines with distinct baseline sensitivity to PIs, including AMO1-VR, an isogenic AMO1 cell line adapted to grow in continuous bortezomib. NRF1 cleavage and nuclear localization were assessed via western blotting. We used CRISPR-Cas9 to knock out (KO) DDI2/NRF1. Cell viability or tumor growth of DDI2/NRF1 KO versus non-targeting gRNA-edited cells were compared in in vitro and in vivo growth competition studies, respectively. Addback studies were performed by stably expressing WT or aspartic protease dead DDI2 in DDI2 KO AMO1-VR monoclones. Chymotryptic-like proteasome activity was measured via cleavage of a fluorescent substrate. Proteasome subunit PSMA7, PSMB5, PSMB6 and PSMD11 transcription was evaluated via real time PCR. <h3>Results</h3> Full length and cleaved NRF1 is detectable in MM cell lines and positively correlates with polyubiquitinated proteins, suggesting constitutively active PSR in MM. DDI2/NRF1 KO is cytotoxic alone or in combination with PI carfilzomib in MM cells with distinct PI sensitivity, including de-novo PI-resistant KMS20. In vivo, DDI2 KO leads to reduced plasmacytoma formation in NSG mice and results in prolongation of animal survival. DDI2 KO in AMO1-VR sensitizes to carfilzomib. Further, DDI2 KO blocks NRF1 cleavage and nuclear import, thereby impairing proteasome subunit transcription and CT-L proteasome activity in baseline conditions and following carfilzomib treatment, resulting in increased sensitivity to PI. Wild-type, but not catalytically-dead DDI2 addback rescues these phenotypes, confirming a causative link. <h3>Conclusions</h3> MM cells exhibit baseline activation of NRF1 and are dependent upon DDI2 for survival. DDI2 KO blocks NRF1 cleavage and nuclear translocation, causing impaired proteasome subunit biogenesis and recovery of CT-L proteasome activity, thereby increasing sensitivity to PI. Add-back of wild-type, but not of catalytically-dead DDI2 fully rescues these phenotypes. Our study provides the preclinical rationale for development of novel therapeutics targeting DDI2/NRF1 in MM.

P53-dependent induction of P2X7 on hematopoietic stem and progenitor cells regulates hematopoietic response to genotoxic stress

Stem and progenitor cells are the main mediators of tissue renewal and repair, both under homeostatic conditions and in response to physiological stress and injury. Hematopoietic system is responsible for the regeneration of blood and immune cells and is maintained by bone marrow-resident hematopoietic stem and progenitor cells (HSPCs). Hematopoietic system is particularly susceptible to injury in response to genotoxic stress, resulting in the risk of bone marrow failure and secondary malignancies in cancer patients undergoing radiotherapy. Here we analyze the in vivo transcriptional response of HSPCs to genotoxic stress in a mouse whole-body irradiation model and, together with p53 ChIP-Seq and studies in p53-knockout (p53KO) mice, characterize the p53-dependent and p53-independent branches of this transcriptional response. Our work demonstrates the p53-independent induction of inflammatory transcriptional signatures in HSPCs in response to genotoxic stress and identifies multiple novel p53-target genes induced in HSPCs in response to whole-body irradiation. In particular, we establish the direct p53-mediated induction of P2X7 expression on HSCs and HSPCs in response to genotoxic stress. We further demonstrate the role of P2X7 in hematopoietic response to acute genotoxic stress, with P2X7 deficiency significantly extending mouse survival in irradiation-induced hematopoietic failure. We also demonstrate the role of P2X7 in the context of long-term HSC regenerative fitness following sublethal irradiation. Overall our studies provide important insights into the mechanisms of HSC response to genotoxic stress and further suggest P2X7 as a target for pharmacological modulation of HSC fitness and hematopoietic response to genotoxic injury.

193 Different Skin Regions are Characterized by Distinct Epimmunome Patterns

In recent years, we provided the first evidence that in parallel with their distinct microbial and chemical milieu, topographically distinct skin areas show different immune tuning. In the skin, keratinocytes (KCs) produce epimmunomes which molecules can instruct the innate and adaptive immune cells under both homeostatic and inflammatory conditions. In our current study, we aimed to investigate at the molecular level whether epimmunome molecules (IL-25, IL-33, IL-23, IL-8, IL-18, IL-6, IL-24, IL-17C, IL-1α, IL-1β, IL-36, IL-37, IL-38) show different expression levels in distinct healthy skin regions, namely in gland poor (GP), sebaceous gland rich (SGR), and apocrine gland rich (AGR) areas. The expression of epimmunomes were analyzed both at the mRNA and protein levels by RT-qPCR and IHC. According to our results, GP skin is characterized by a pro-Th2 epimmunome milieu with a significantly elevated expression of IL-25, IL-33 at the protein and gene level, while the IL-18, IL-36RA, IL-37 and IL-38 molecules were upregulated at the gene level compared to SGR and AGR regions. On the other hand, both SGR and AGR areas were characterized by a pro-Th17 epimmunome milieu with a significantly higher expression level of IL-17C protein, whereas, in SGR region, IL-1α was upregulated at the protein level while IL-1β and IL-8 at the mRNA level. Our results indicate that distinct skin regions have a unique epimmunome composition even under steady-state. Our findings may further explain why some outside-in skin diseases prefer to be localized to certain skin regions. We propose that the non-inflammatory pro-Th2 immune milieu of GP area predisposes it for the development of Th2-driven atopic dermatitis. On the contrary, the homeostatic pro-Th17 immune milieu makes AGR and SGR areas susceptible for the development of their characteristic Th1/Th17-driven region-specific diseases, hidradenitis suppurativa and rosacea, respectively.

Hormones regulating energy homeostasis in breastfeeding versus formula feeding mothers.

To meet energy demands for lactogenesis and to sustain homeostatic conditions post-partum, the organism of breastfeeding mother undergoes combined endocrine and metabolic regulation. The main objective of this study was to determine basal serum concentrations of hormones involved in the maintenance and defense of energy balance in breastfeeding (BF) and formula feeding (FF) mothers. Twenty healthy exclusively breastfeeding mothers at 3rd month of lactation (EBF3), 17 healthy partially breastfeeding at 6th month of lactation (PB6) and 17 healthy FF mothers participated in this study. Fasting serum prolactin (PRL), acylated ghrelin (aGhr), total ghrelin (tGhr), leptin, adiponectin, insulin, and cortisol were determined for all study participants and correlations between studied parameters were calculated for BF women. We found significantly lower basal insulin (p = 0.0048) and cortisol (p = 0.0002) and significantly elevated basal prolactin (p = 0.0020) and leptin (p = 0.0416) in BF when compared with FF women. The differences were not associated with the duration of lactation (3 vs. 6 months), except for PRL, which was highest in EBF3. Levels of Ghr and adiponectin did not differ between study groups. In the BF group, the negative correlations were found between: aGhr and insulin, aGhr and adiponectin, leptin and cortisol, leptin and adiponectin, insulin and adiponectin, cortisol and adiponectin. Positive associations were noted between: insulin and leptin, leptin and aGhr, PRL and leptin, PRL and aGhr. Leptin and insulin correlated positively, whereas adiponectin negatively with BMI. These data may suggest that EBF3 and PB6 as compared with FF mothers, exhibit hormonal regulation which tends to be more advantageous for their metabolic profile and is not related to the duration of breastfeeding within the first 6 months of lactation.

The modified mitochondrial outer membrane carrier MTCH2 links mitochondrial fusion to lipogenesis.

Mitochondrial function is integrated with cellular status through the regulation of opposing mitochondrial fusion and division events. Here we uncover a link between mitochondrial dynamics and lipid metabolism by examining the cellular role of mitochondrial carrier homologue 2 (MTCH2). MTCH2 is a modified outer mitochondrial membrane carrier protein implicated in intrinsic cell death and in the in vivo regulation of fatty acid metabolism. Our data indicate that MTCH2 is a selective effector of starvation-induced mitochondrial hyperfusion, a cytoprotective response to nutrient deprivation. We find that MTCH2 stimulates mitochondrial fusion in a manner dependent on the bioactive lipogenesis intermediate lysophosphatidic acid. We propose that MTCH2 monitors flux through the lipogenesis pathway and transmits this information to the mitochondrial fusion machinery to promote mitochondrial elongation, enhanced energy production, and cellular survival under homeostatic and starvation conditions. These findings will help resolve the roles of MTCH2 and mitochondria in tissue-specific lipid metabolism in animals.