L-fucose and fucoidan alleviate high-salt diet-promoted acute inflammation.

Excessive salt intake is a widespread health issue observed in almost every country around the world. A high salt diet (HSD) has a strong correlation with numerous diseases, including hypertension, chronic kidney disease, and autoimmune disorders. However, the mechanisms underlying HSD-promotion of inflammation and exacerbation of these diseases are not fully understood. In this study, we observed that HSD consumption reduced the abundance of the gut microbial metabolite L-fucose, leading to a more substantial inflammatory response in mice. A HSD led to increased peritonitis incidence in mice, as evidenced by the increased accumulation of inflammatory cells and elevated levels of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and monocyte chemotactic protein-1 (MCP-1, also known as C-C motif chemokine ligand 2 or CCL2), in peritoneal lavage fluid. Following the administration of broad-spectrum antibiotics, HSD-induced inflammation was abolished, indicating that the proinflammatory effects of HSD were not due to the direct effect of sodium, but rather to HSD-induced alterations in the composition of the gut microbiota. By using untargeted metabolomics techniques, we determined that the levels of the gut microbial metabolite L-fucose were reduced by a HSD. Moreover, the administration of L-fucose or fucoidan, a compound derived from brown that is rich in L-fucose, normalized the level of inflammation in mice following HSD induction. In addition, both L-fucose and fucoidan inhibited LPS-induced macrophage activation in vitro. In summary, our research showed that reduced L-fucose levels in the gut contributed to HSD-exacerbated acute inflammation in mice; these results indicate that L-fucose and fucoidan could interfere with HSD-promotion of the inflammatory response.

Erythropoietin mediates re-programming of endotoxin-tolerant macrophages through PI3K/AKT signaling and protects mice against secondary infection.

Initial lipopolysaccharide (LPS) exposure leads to a hypo-responsive state by macrophages to a secondary stimulation of LPS, known as endotoxin tolerance. However, recent findings show that functions of endotoxin-tolerant macrophages are not completely suppressed, whereas they undergo a functional re-programming process with upregulation of a panel of molecules leading to enhanced protective functions including antimicrobial and tissue-remodeling activities. However, the underlying molecular mechanisms are still elusive. Erythropoietin (EPO), a glycoprotein regulated by hypoxia-inducible factor 1α (HIF-1α), exerts anti-inflammatory and tissue-protective activities. Nevertheless, the potential effects of EPO on functional re-programming of endotoxin-tolerant macrophages have not been investigated yet. Here, we found that initial LPS exposure led to upregulation of HIF-1α/EPO in macrophages and that EPO enhanced tolerance in tolerized macrophages and mice as demonstrated by suppressed proinflammatory genes such as Il1b, Il6, and Tnfa after secondary LPS stimulation. Moreover, we showed that EPO improved host protective genes in endotoxin-tolerant macrophages and mice, such as the anti-bacterial genes coding for cathelicidin-related antimicrobial peptide (Cnlp) and macrophage receptor with collagenous structure (Marco), and the tissue-repairing gene vascular endothelial growth factor C (Vegfc). Therefore, our findings indicate that EPO mediates the functional re-programming of endotoxin-tolerant macrophages. Mechanistically, we found that PI3K/AKT signaling contributed to EPO-mediated re-programming through upregulation of Irak3 and Wdr5 expression. Specifically, IL-1 receptor-associated kinase 3 (IRAK3) was responsible for inhibiting proinflammatory genes Il1b, Il6, and Tnfa in tolerized macrophages after LPS rechallenge, whereas WDR5 contributed to the upregulation of host beneficial genes including Cnlp, Marco, and Vegfc. In a septic model of mice, EPO pretreatment significantly promoted endotoxin-tolerant re-programming, alleviated lung injury, enhanced bacterial clearance, and decreased mortality in LPS-tolerized mice after secondary infection of Escherichia coli. Collectively, our results reveal a novel role for EPO in mediating functional re-programming of endotoxin-tolerant macrophages; thus, targeting EPO appears to be a new therapeutic option in sepsis and other inflammatory disorders.

Erythropoietin Promotes Infection Resolution and Lowers Antibiotic Requirements in E. coli- and S. aureus-Initiated Infections.

Endogenous mechanisms underlying bacterial infection resolution are essential for the development of novel therapies for the treatment of inflammation caused by infection without unwanted side effects. Herein, we found that erythropoietin (EPO) promoted the resolution and enhanced antibiotic actions in Escherichia coli (E. coli)- and Staphylococcus aureus (S. aureus)-initiated infections. Levels of peritoneal EPO and macrophage erythropoietin receptor (EPOR) were elevated in self-limited E. coli-initiated peritonitis. Myeloid-specific EPOR-deficient mice exhibited an impaired inflammatory resolution and exogenous EPO enhanced this resolution in self-limited infections. Mechanistically, EPO increased macrophage clearance of bacteria via peroxisome proliferator-activated receptor γ (PPARγ)-induced CD36. Moreover, EPO ameliorated inflammation and increased the actions of ciprofloxacin and vancomycin in resolution-delayed E. coli- and S. aureus-initiated infections. Collectively, macrophage EPO signaling is temporally induced during infections. EPO is anti-phlogistic, increases engulfment, promotes infection resolution, and lowers antibiotic requirements.

Pentose Phosphate Pathway Regulates Tolerogenic Apoptotic Cell Clearance and Immune Tolerance.

The efficient removal of apoptotic cells (ACs), a process termed as efferocytosis, is essential for immune homeostasis. While recent work has established an important interplay between efferocytosis and cellular metabolic changing, underlying mechanisms remain poorly known. Here, we discovered that pentose phosphate pathway (PPP) regulates tolerogenic ACs clearance and immune tolerance. ACs decreased levels of PPP-related genes and metabolites in macrophages. AG1, the agonist of PPP, increased the activity of PPP but greatly reduced macrophage phagocytosis of ACs and enhanced the inflammatory response during efferocytosis. miR-323-5p regulated the expression of PPP-related genes and its levels increased during efferocytosis. miR-323-5p inhibitor greatly promoted levels of PPP-related genes, reduced the macrophage phagocytosis of ACs, and increased inflammatory response during efferocytosis, suggesting that miR-323-5p was essential in regulating PPP activity and ACs clearance in macrophages. Correspondingly, the PPP agonist AG1 exacerbated the lupus-like symptoms in the AC-induced systemic lupus erythematosus (SLE) model. Our study reveals that regulating PPP-dependent metabolic reprogramming is critical for tolerogenic ACs phagocytosis and immune tolerance.

The deficiency of macrophage erythropoietin signaling contributes to delayed acute inflammation resolution in diet-induced obese mice.

Obesity has been linked with altered acute inflammation resolution which contributes to obesity-related clinical complications; however, the mechanisms that contribute to obesity-related unresolved inflammation are not fully known. Here we demonstrated that the deficiency of macrophage erythropoietin (EPO) signaling contributed to delayed acute inflammation resolution in diet-induced obese mice. In zymosan-induced acute peritonitis, in line with the delayed resolution of inflammation, the induction of macrophage EPO signaling was significantly reduced in obese mice relative to normal mice. Exogenous EPO induced macrophage EPO signaling and promoted acute inflammation resolution in obese mice. Efferocytosis of apoptotic cells by macrophages which is central in inflammation resolution was impaired in obese mice and restored by exogenous EPO. Mechanistically, macrophage peroxisome proliferator-activated receptor-γ (PPARγ) was greatly reduced in obese mice and EPO increased macrophage PPARγ to promote efferocytosis in obese mice. Together, our results identify an important mechanism underlying aberrant acute inflammation resolution in obesity, with important implications for regulating unresolved acute inflammation and normalizing macrophage defects in obese and diabetic individuals.

Non-erythropoietic erythropoietin-derived peptide protects mice from systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, which results in various organ pathologies. However, current treatment towards SLE is suboptimal. Erythropoietin (EPO) has been shown to promote SLE recovery, but clinical application can be limited by its haematopoiesis-stimulating effects. EPO-derived helix-B peptide (ARA290) is non-erythrogenic but has been reported to retain the anti-inflammatory and tissue-protective functions of EPO. Therefore, here we investigated the effects and potential mechanisms of ARA290 on SLE. The administration of ARA290 to pristane-induced SLE and MRL/lpr mice significantly suppressed the level of serum antinuclear autoantibodies (ANAs) and anti-dsDNA autoantibodies, reduced the deposition of IgG and C3, and ameliorated the nephritis symptoms. Moreover, the serum concentrations of inflammatory cytokine IL-6, MCP-1 and TNF-α in SLE mice were reduced by ARA290. Further, ARA290 decreased the number of apoptotic cells in kidney. In vitro experiment revealed that ARA290 inhibited the inflammatory activation of macrophages and promoted the phagocytotic function of macrophages to apoptotic cells. Finally, ARA290 did not induce haematopoiesis during treatment. In conclusion, ARA290 ameliorated SLE, which at least could be partly due to its anti-inflammatory and apoptotic cell clearance promoting effects, without stimulating haematopoiesis, suggesting that ARA290 could be a hopeful candidate for SLE treatment.

Resolvin D1 Programs Inflammation Resolution by Increasing TGF-ß Expression Induced by Dying Cell Clearance in Experimental Autoimmune Neuritis.

UNLABELLED: Experimental autoimmune neuritis (EAN) is the animal model of human acute inflammatory demyelinating polyradiculoneuropathies (AIDP), an auto-immune inflammatory demyelination disease of the peripheral nervous system (PNS) and the world's leading cause of acute autoimmune neuromuscular paralysis. EAN and AIDP are characterized by self-limitation with spontaneous recovery; however, endogenous pathways that regulate inflammation resolution in EAN and AIDP remain elusive. A pathway of endogenous mediators, especially resolvins and clearance of apoptotic cells, may be involved. Here, we determined that resolvin D1 (RvD1), its synthetic enzyme, and its receptor were greatly increased in PNS during the recovery stage of EAN. Both endogenous and exogenous RvD1 increased regulatory T (Treg) cell and anti-inflammatory macrophage counts in PNS, enhanced inflammation resolution, and promoted disease recovery in EAN rats. Moreover, RvD1 upregulated the transforming growth factor-ß (TGF-ß) level and pharmacologic inhibition of TGF-ß signaling suppressed RvD1-induced Treg cell counts, but not anti-inflammatory macrophage counts, and RvD1-improved inflammation resolution and disease recovery in EAN rats. Mechanistically, the RvD1-enhanced macrophage phagocytosis of apoptotic T cells leading to reduced apoptotic T-cell accumulation in PNS induced TGF-ß production and caused Treg cells to promote inflammation resolution and disease recovery in EAN. Therefore, these data highlight the crucial role of RvD1 as an important pro-resolving molecule in EAN and suggest its potential as a therapeutic target in human neuropathies. SIGNIFICANCE STATEMENT: Experimental autoimmune neuritis (EAN) is the animal model of human acute inflammatory demyelinating polyradiculoneuropathies, an auto-immune inflammatory demyelination disease of the peripheral nervous system (PNS) and the world's leading cause of acute autoimmune neuromuscular paralysis. Here, we demonstrated that resolvin D1 (RvD1) promoted macrophage phagocytosis of apoptotic T cells in PNS, thereby upregulating transforming growth factor-ß by macrophages, increased local Treg cell counts, and finally promoted inflammation resolution and disease recovery in EAN. These data highlight the crucial role of RvD1 as an important pro-resolving molecule in EAN and suggest that it has potential as a therapeutic target in human neuritis.

Phagocyte respiratory burst activates macrophage erythropoietin signalling to promote acute inflammation resolution.

Inflammation resolution is an active process, the failure of which causes uncontrolled inflammation which underlies many chronic diseases. Therefore, endogenous pathways that regulate inflammation resolution are fundamental and of wide interest. Here, we demonstrate that phagocyte respiratory burst-induced hypoxia activates macrophage erythropoietin signalling to promote acute inflammation resolution. This signalling is activated following acute but not chronic inflammation. Pharmacological or genetical inhibition of the respiratory burst suppresses hypoxia and macrophage erythropoietin signalling. Macrophage-specific erythropoietin receptor-deficient mice and chronic granulomatous disease (CGD) mice, which lack the capacity for respiratory burst, display impaired inflammation resolution, and exogenous erythropoietin enhances this resolution in WT and CGD mice. Mechanistically, erythropoietin increases macrophage engulfment of apoptotic neutrophils via PPARγ, promotes macrophage removal of debris and enhances macrophage migration to draining lymph nodes. Together, our results provide evidences of an endogenous pathway that regulates inflammation resolution, with important implications for treating inflammatory conditions.

Erythropoeitin Signaling in Macrophages Promotes Dying Cell Clearance and Immune Tolerance.

The failure of apoptotic cell clearance is linked to autoimmune diseases, nonresolving inflammation, and developmental abnormalities; however, pathways that regulate phagocytes for efficient apoptotic cell clearance remain poorly known. Apoptotic cells release find-me signals to recruit phagocytes to initiate their clearance. Here we found that find-me signal sphingosine 1-phosphate (S1P) activated macrophage erythropoietin (EPO) signaling promoted apoptotic cell clearance and immune tolerance. Dying cell-released S1P activated macrophage EPO signaling. Erythropoietin receptor (EPOR)-deficient macrophages exhibited impaired apoptotic cell phagocytosis. EPO enhanced apoptotic cell clearance through peroxisome proliferator activated receptor-γ (PPARγ). Moreover, macrophage-specific Epor(-/-) mice developed lupus-like symptoms, and interference in EPO signaling ameliorated the disease progression in lupus-like mice. Thus, we have identified a pathway that regulates macrophages to clear dying cells, uncovered the priming function of find-me signal S1P, and found a role of the erythropoiesis regulator EPO in apoptotic cell disposal, with implications for harnessing dying cell clearance.

Nonerythropoietic Erythropoietin-Derived Peptide Suppresses Adipogenesis, Inflammation, Obesity and Insulin Resistance.

Erythropoietin (EPO) has been identified as being crucial for obesity modulation; however, its erythropoietic activity may limit its clinical application. EPO-derived Helix B-surface peptide (pHBSP) is nonerythrogenic but has been reported to retain other functions of EPO. The current study aimed to evaluate the effects and potential mechanisms of pHBSP in obesity modulation. We found that pHBSP suppressed adipogenesis, adipokine expression and peroxisome proliferator-activated receptor γ (PPARγ) levels during 3T3-L1 preadipocyte maturation through the EPO receptor (EPOR). In addition, also through EPOR, pHBSP attenuated macrophage inflammatory activation and promoted PPARγ expression. Furthermore, PPARγ deficiency partly ablated the anti-inflammatory activity of pHBSP in macrophages. Correspondingly, pHBSP administration to high-fat diet (HFD)-fed mice significantly improved obesity, insulin resistance (IR) and adipose tissue inflammation without stimulating hematopoiesis. Therefore, pHBSP can significantly protect against obesity and IR partly by inhibiting adipogenesis and inflammation. These findings have therapeutic implications for metabolic disorders, such as obesity and diabetes.

Accumulation of connective tissue growth factor+ cells during the early phase of rat traumatic brain injury.

BACKGROUND: Glial scar formation is a common histopathological feature of traumatic brain injury (TBI). Astrogliosis and expression of transforming growth factor beta (TGF-ß) are key components of scar formation and blood-brain barrier modulation. Connective tissue growth factor (CTGF) is considered a cytokine mediating the effects of TGF-ß. METHODS: Here, we studied the CTGF expression in an open-skull weight-drop-induced TBI, with a focus on the early phase, most amenable to therapy. RESULTS: In normal rat brains of our study, CTGF+ cells were rarely observed. Significant parenchymal accumulation of CTGF+ non-neuron cells was observed 72 h post-TBI and increased continuously during the investigating time. We also observed that the accumulated CTGF+ non-neuron cells were mainly distributed in the perilesional areas and showed activated astrocyte phenotypes with typical stellate morphologic characteristics. CONCLUSION: Our observations demonstrated the time-dependent and lesion-associated accumulation of cellular CTGF expression in TBI, suggesting a pathological role of CTGF in TBI. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3963462091241165.

Erythropoietin-derived nonerythropoietic peptide ameliorates experimental autoimmune neuritis by inflammation suppression and tissue protection.

Experimental autoimmune neuritis (EAN) is an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system. Erythropoietin (EPO) has been known to promote EAN recovery but its haematopoiesis stimulating effects may limit its clinic application. Here we investigated the effects and potential mechanisms of an EPO-derived nonerythropoietic peptide, ARA 290, in EAN. Exogenous ARA 290 intervention greatly improved EAN recovery, improved nerve regeneration and remyelination, and suppressed nerve inflammation. Furthermore, haematopoiesis was not induced by ARA 290 during EAN treatment. ARA 290 intervention suppressed lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3+/CD4+ regulatory T cells and IL-4+/CD4+ Th2 cells and decrease of IFN-γ+/CD4+ Th1 cells in EAN. In addition, ARA 290 inhibited inflammatory macrophage activation and promoted its phagocytic activity. In vitro, ARA 290 was shown to promote Schwann cell proliferation and inhibit its inflammatory activation. In summary, our data demonstrated that ARA 290 could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that ARA 290 could be a potent candidate for treatment of autoimmune neuropathies.

Therapeutic effects of nonerythropoietic erythropoietin analog ARA290 in experimental autoimmune encephalomyelitis rat.

ARA290 is a nonerythropoietic analog of erythropoietin (EPO) containing 11 amino acids which provides the anti-inflammatory and neuroprotective effects of EPO without stimulating hematopoiesis. Here we studied the therapeutic effects of ARA290 in experimental autoimmune encephalomyelitis (EAE) Lewis rats. Therapeutic (from Day 7 to Day 18 or from Day 9 to Day 19) administration of ARA290 (35, 70 µg/kg, intra-peritoneal) to EAE rats once daily significantly reduced the severity and shortened the duration of clinical score, reduced the accumulation of inflammatory cells in EAE spinal cords and suppressed mRNA levels of interleukin-1ß (IL-1ß), IL-17, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), matrix metalloproteinase 9 (MMP9) and transcription factor T-bet in spinal cords of EAE rats. Furthermore, ARA290 treatment reduced the helper T cell number in lymph nodes and circulation in EAE. In vitro study showed that ARA290 dose-dependently inhibited antigen specific- and antigen non-specific-lymphocyte proliferation as well. In addition, ARA290 altered the cytokine milieu to favor the polarization of Th2 and regulatory T (Treg) cells but suppressed the polarization of Th1 and Th17 cells in EAE lymph nodes. In summary, our study here showed that ARA290 could alter T cell function to suppress inflammation to ameliorate EAE, suggesting that ARA290 may be a new therapeutic candidate for multiple sclerosis.

Curcumin ameliorates rat experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN) is a helper T cell-mediated autoimmune demyelinating inflammatory disease of the peripheral nervous system that serves as an animal model for human Guillain-Barre syndrome. Curcumin, a naturally occurring polyphenolic phytochemical isolated from the medicinal plant Curcuma longa, has anti-inflammatory activities. Here we investigated the therapeutic effects and potential mechanisms of curcumin in EAN rats. Exogenous curcumin treatment (100 mg/kg/day) significantly delayed the onset of EAN neurological signs, ameliorated EAN neurological severity, and reduced body weight loss of EAN rats. In EAN sciatic nerves, curcumin treatment suppressed the inflammatory cell accumulation and the expression of interferon (IFN)-γ, tumor necrosis factor-α, interleukin (IL)-1ß, and IL-17. Furthermore, curcumin treatment significantly decreased the percentage of CD4(+) T helper cells in EAN spleen and suppressed concanavalin A-induced lymphocyte proliferation in vitro. In addition, curcumin altered helper T cell differentiation by decreasing IFN-γ(+) CD4(+) Th1 cells in EAN lymph node and spleen. In summary, our data demonstrate that curcumin could effectively suppress EAN by attenuating inflammation, indicating that curcumin might be a candidate for treatment of autoimmune neuropathies.

Erythropoietin is a hypoxia inducible factor-induced protective molecule in experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN), an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system, is characterized by self-limitation. Here we investigated the regulation and contribution of erythropoietin (EPO) in EAN self-limitation. In EAN sciatic nerves, hypoxia, and protein and mRNA levels of hypoxia-inducible factor 1α (HIF-1α), HIF-2α, EPO and EPO receptor (EPOR) were induced in parallel at disease peak phase but reduced at recovery periods. Further, the deactivation of HIF reduced EAN-induced EPO/EPOR upregulation in EAN, suggesting the central contribution of HIF to EPO/EPOR induction. The deactivation of EPOR signalling exacerbated EAN progression, implying that endogenous EPO contributed to EAN recovery. Exogenous EPO treatment greatly improved EAN recovery. In addition, EPO was shown to promote Schwann cell survival and myelin production. In EAN, EPO treatment inhibited lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3(+)/CD4(+) regulatory T cells and decrease of IFN-γ(+)/CD4(+) Th1 cells. Furthermore, EPO inhibited inflammatory macrophage activation and promoted its phagocytic activity. In summary, our data demonstrated that EPO was induced in EAN by HIF and contributed to EAN recovery, and endogenous and exogenous EPO could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that EPO contributes to the self-recovery of EAN and could be a potent candidate for treatment of autoimmune neuropathies.

Lesional accumulation of heme oxygenase-1+ microglia/macrophages in rat traumatic brain injury.

Heme oxygenase-1 (HO-1) is an inducible rate-limiting enzyme for heme degradation. Here, we studied the HO-1 expression in an open-skull weight-drop-induced traumatic brain injury, with a focus on the early phase, most amenable to therapy. In normal rat brains of our study, HO-1 cells were rarely observed. Significant parenchymal accumulation of HO-1 non-neuron cells was observed 18 h post-traumatic brain injury and increased continuously during the investigating time. We also observed that the accumulated HO-1 non-neuron cells were mainly distributed in the perilesional areas and showed activated microglia/macrophage phenotypes with ramified or amoeboid morphologic characteristics. Further double-labeling experiments showed that most HO-1 non-neuron cells coexpressed CD68 and CD163, but not glial fibrillary acid protein. Our data suggest that HO-1 expression defines a subtype of activated microglia/macrophages involved in the early processes following traumatic brain injury.