Combination of inflammation-related cytokines promotes long-term muscle stem cell expansion

Muscle stem cells (MuSCs, satellite cells) are the major contributor to muscle regeneration. Like most adult stem cells, long-term expansion of MuSCs in vitro is difficult. The in vivo muscle regeneration abilities of MuSCs are quickly lost after culturing in vitro, which prevents the potential applications of MuSCs in cell-based therapies. Here, we establish a system to serially expand MuSCs in vitro for over 20 passages by mimicking the endogenous microenvironment. We identified that the combination of four pro-inflammatory cytokines, IL-1α, IL-13, TNF-α, and IFN-γ, secreted by T cells was able to stimulate MuSC proliferation in vivo upon injury and promote serial expansion of MuSCs in vitro. The expanded MuSCs can replenish the endogenous stem cell pool and are capable of repairing multiple rounds of muscle injuries in vivo after a single transplantation. The establishment of the in vitro system provides us a powerful method to expand functional MuSCs to repair muscle injuries.     

Requirement of Plasminogen Binding to Its Cell-Surface Receptor α-Enolase for Efficient Regeneration of Normal and Dystrophic Skeletal Muscle

Adult regenerative myogenesis is central for restoring normal tissue structure and function after muscle damage. In muscle repair after injury, as in severe myopathies, damaged and necrotic fibers are removed by infiltrating inflammatory cells and then replaced by muscle stem cells or satellite cells, which will fuse to form new myofibers. Extracellular proteolysis mediated by uPA-generated plasmin plays a critical role in controlling inflammation and satellite-cell-dependent myogenesis. α-enolase has been described as plasminogen receptor in several cell types, where it acts concentrating plasmin proteolytic activity on the cell surface. In this study, we investigated whether α-enolase plasminogen receptor plays a regulatory role during the muscular repair process. Inhibitors of α-enolase/plasminogen binding: MAb11G1 (a monoclonal antibody against α-enolase) and ε-aminocaproic acid, EACA (a lysine analogue) inhibited the myogenic abilities of satellite cells-derived myoblasts. Furthermore, knockdown of α-enolase decreased myogenic fusion of myoblasts. Injured wild-type mice and dystrophic mdx mice were also treated with MAb11G1 and EACA. These treatments had negative impacts on muscle repair impairing satellite cell functions in vitro in agreement with blunted growth of new myofibers in vivo. Furthermore, both MAb11G1 and EACA treatments impaired adequate inflammatory cell infiltration and promoted extracellular matrix deposition in vivo, which resulted in persistent degeneration. These results demonstrate the novel requirement of α-enolase for restoring homeostasis of injured muscle tissue, by controlling the pericellular localization of plasmin activity.     

Reovirus Induction of and Sensitivity to Beta Interferon in Cardiac Myocyte Cultures Correlate with Induction of Myocarditis and Are Determined by Viral Core Proteins

Reovirus-induced acute myocarditis in mice serves as a model to investigate non-immune-mediated mechanisms of viral myocarditis. We have used primary cardiac myocyte cultures infected with a large panel of myocarditic and nonmyocarditic reassortant reoviruses to identify determinants of viral myocarditic potential. Here, we report that while both myocarditic and nonmyocarditic reoviruses kill cardiac myocytes, viral myocarditic potential correlates with viral spread through cardiac myocyte cultures and with cumulative cell death. To address the role of secreted interferon (IFN), we added anti-IFN-α/β antibody to infected cardiac myocyte cultures. Antibody benefited nonmyocarditic more than myocarditic virus spread (P < 0.001), and this benefit was associated with the reovirus M1 and L2 genes. There was no benefit for a differentiated skeletal muscle cell line culture (C2C12 cells), suggesting cell type specificity. IFN-β induction in reovirus-infected cardiac myocyte cultures correlated with viral myocarditic potential (P = 0.006) and was associated with the reovirus M1, S2, and L2 genes. Sensitivity to the antiviral effects of IFN-α/β added to cardiac myocyte cultures also correlated with viral myocarditic potential (P = 0.004) and was associated with the same reovirus genes. Several reoviruses induced IFN-β levels discordant with their myocarditic phenotypes, and for those tested, sensitivity to IFN-α/β compensated for the anomalous induction levels. Thus, the combination of induction of and sensitivity to IFN-α/β is a determinant of reovirus myocarditic potential. Finally, a nonmyocarditic reovirus induced cardiac lesions in mice depleted of IFN-α/β, demonstrating that IFN-α/β is a determinant of reovirus-induced myocarditis. This provides the first identification of reovirus genes associated with IFN induction and sensitivity and provides the first evidence that IFN-β can be a determinant of viral myocarditis and reovirus disease.     

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs’ program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.     

Regulatory T cells in skeletal muscle repair and regeneration: recent insights

Skeletal muscle repair and regeneration after injury is a multi-stage process, involving a dynamic inflammatory microenvironment consisting of a complex network formed by the interaction of immune cells and their secreted cytokines. The homeostasis of the inflammatory microenvironment determines whether skeletal muscle repair tissues will ultimately form scar tissue or regenerative tissue. Regulatory T cells (Tregs) regulate homeostasis within the immune system and self-immune tolerance, and play a crucial role in skeletal muscle repair and regeneration. Dysregulated Tregs function leads to abnormal repair. In this review, we discuss the role and mechanisms of Tregs in skeletal muscle repair and regeneration after injury and provide new strategies for Treg immunotherapy in skeletal muscle diseases.Subject terms: Trauma, Immunotherapy     

Macrophages commit postnatal endothelium-derived progenitors to angiogenesis and restrict endothelial to mesenchymal transition during muscle regeneration

The damage of the skeletal muscle prompts a complex and coordinated response that involves the interactions of many different cell populations and promotes inflammation, vascular remodeling and finally muscle regeneration. Muscle disorders exist in which the irreversible loss of tissue integrity and function is linked to defective neo-angiogenesis with persistence of tissue necrosis and inflammation. Here we show that macrophages (MPs) are necessary for efficient vascular remodeling in the injured muscle. In particular, MPs sustain the differentiation of endothelial-derived progenitors to contribute to neo-capillary formation, by secreting pro-angiogenic growth factors. When phagocyte infiltration is compromised endothelial-derived progenitors undergo a significant endothelial to mesenchymal transition (EndoMT), possibly triggered by the activation of transforming growth factor-β/bone morphogenetic protein signaling, collagen accumulates and the muscle is replaced by fibrotic tissue. Our findings provide new insights in EndoMT in the adult skeletal muscle, and suggest that endothelial cells in the skeletal muscle may represent a new target for therapeutic intervention in fibrotic diseases.     

FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration

Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2^-/- γ-chain^-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4⁺CD25⁺FOXP3⁺ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue.     

Activation of Olfactory Receptors on Mouse Pulmonary Macrophages Promotes Monocyte Chemotactic Protein-1 Production

Background

Emerging evidence suggests that non-olfactory tissues and cells can express olfactory receptors (ORs), however, the exact function of ectopic OR expression remains unknown. We have previously shown in mouse models that a unique cooperation between interferon-γ (IFN-γ) and lipopolysaccharide (LPS) drives the activation of pulmonary macrophages and leads to the induction of pathogenic responses in the respiratory tract. Further, through gene array studies, we have shown that activation of macrophages by these molecules results in the selective expression of a number of ORs. In this study, we validated the expression of these ORs in mouse airway and pulmonary macrophages in response to IFN-γ and LPS (γ/LPS) stimulation, and further explored the effect of odorant stimulation on macrophage function.

Methodology/Principal Findings

OR expression in airway and pulmonary macrophages in response to IFN-γ, LPS or γ/LPS treatments was assessed by microarray and validated by q-PCR. OR expression (e.g. OR622) on macrophages was confirmed by visualization in immunofluoresence assays. Functional responses to odorants were assessed by quantifying inflammatory cytokine and chemokine expression using q-PCR and cell migration was assessed by a modified Boyden chamber migration assay. Our results demonstrate that eight ORs are expressed at basal levels in both airway and pulmonary macrophages, and that γ/LPS stimulation cooperatively increased this expression. Pulmonary macrophages exposed to the combined treatment of γ/LPS+octanal (an odorant) exhibited a 3-fold increase in MCP-1 protein production, compared to cells treated with γ/LPS alone. Supernatants from γ/LPS+octanal exposed macrophages also increased macrophage migration in vitro.

Conclusions/Significance

Eight different ORs are expressed at basal levels in pulmonary macrophages and expression is upregulated by the synergistic action of γ/LPS. Octanal stimulation further increased MCP-1 production and the motility of macrophages. Our results suggest that ORs may mediate macrophage function by regulating MCP-1 production and cell migration.     

Key Role of Splenic Myeloid DCs in the IFN-αβ Response to Adenoviruses In Vivo

The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR^−/− mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.     

Modelling Neuroinflammation In Vitro: A Tool to Test the Potential Neuroprotective Effect of Anti-Inflammatory Agents

Neuron-microglia co-cultures treated with pro-inflammatory agents are a useful tool to study neuroinflammation in vitro, where to test the potential neuroprotective effect of anti-inflammatory compounds. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. We compared the use of neuron-primary microglia and neuron-BV2 cells (a microglial cell line) co-cultures, using different neuron:microglia ratios, treatments and time post-treatment to induce glial activation and derived neurotoxicity. We show that each model requires different experimental conditions, but that both neuron-BV2 and neuron-primary microglia LPS/IFN-γ-treated co-cultures are good to study the potential neuroprotective effect of anti-inflammatory agents. The contribution of different pro-inflammatory parameters in the neurotoxicity induced by reactive microglial cells was determined. IL-10 pre-treatment completely inhibited LPS/IFN-γ-induced TNF-α and IL-6 release, and COX-2 expression both in BV2 and primary microglial cultures, but not NO production and iNOS expression. However, LPS/IFN-γ induced neurotoxicity was not inhibited in IL-10 pre-treated co-cultures. The inhibition of NO production using the specific iNOS inhibitor 1400 W totally abolished the neurotoxic effect of LPS/IFN-γ, suggesting a major role for NO in the neurotoxic effect of activated microglia. Consequently, among the anti-inflammatory agents, special attention should be paid to compounds that inhibit NO production.     

Muscle Regeneration with Intermuscular Adipose Tissue (IMAT) Accumulation Is Modulated by Mechanical Constraints

Sports trauma are able to induce muscle injury with fibrosis and accumulation of intermuscular adipose tissue (IMAT), which affect muscle function. This study was designed to investigate whether hypoactivity would influence IMAT accumulation in regenerating mouse skeletal muscle using the glycerol model of muscle regeneration. The animals were immediately hindlimb unloaded for 21 days after glycerol injection into the tibialis anterior (TA) muscle. Muscle fiber and adipocyte cross-sectional area (CSA) and IMAT accumulation were determined by histomorphometric analysis. Adipogenesis during regenerative processes was examined using RT-qPCR and Western blot quantification. Twenty-one days of hindlimb unloading resulted in decreases of 38% and 50.6% in the muscle weight/body weight ratio and CSA, respectively, in soleus muscle. Glycerol injection into TA induced IMAT accumulation, reaching 3% of control normal-loading muscle area. This IMAT accumulation was largely inhibited in unloading conditions (0.09%) and concomitant with a marked reduction in perilipin and FABP4 protein content, two key markers of mature adipocytes. Induction of PPARγ and C/EBPα mRNA, two markers of adipogenesis, was also decreased. Furthermore, the protein expression of PDGFRα, a cell surface marker of fibro/adipogenic progenitors, was much lower in regenerating TA from the unloaded group. Exposure of regenerating muscle to hypoactivity severely reduces IMAT development and accumulation. These results provide new insight into the mechanisms regulating IMAT development in skeletal muscle and highlight the importance of taking into account the level of mechanical constraint imposed on skeletal muscle during the regeneration processes.     

Eccentric exercise facilitates mesenchymal stem cell appearance in skeletal muscle

Eccentric, or lengthening, contractions result in injury and subsequently stimulate the activation and proliferation of satellite stem cells which are important for skeletal muscle regeneration. The discovery of alternative myogenic progenitors in skeletal muscle raises the question as to whether stem cells other than satellite cells accumulate in muscle in response to exercise and contribute to post-exercise repair and/or growth. In this study, stem cell antigen-1 (Sca-1) positive, non-hematopoetic (CD45^-) cells were evaluated in wild type (WT) and α7 integrin transgenic (α7Tg) mouse muscle, which is resistant to injury yet liable to strain, 24 hr following a single bout of eccentric exercise. Sca-1⁺CD45⁻ stem cells were increased 2-fold in WT muscle post-exercise. The α7 integrin regulated the presence of Sca-1⁺ cells, with expansion occurring in α7Tg muscle and minimal cells present in muscle lacking the α7 integrin. Sca-1⁺CD45⁻ cells isolated from α7Tg muscle following exercise were characterized as mesenchymal-like stem cells (mMSCs), predominantly pericytes. In vitro multiaxial strain upregulated mMSC stem cells markers in the presence of laminin, but not gelatin, identifying a potential mechanistic basis for the accumulation of these cells in muscle following exercise. Transplantation of DiI-labeled mMSCs into WT muscle increased Pax7⁺ cells and facilitated formation of eMHC⁺DiI⁻ fibers. This study provides the first demonstration that mMSCs rapidly appear in skeletal muscle in an α7 integrin dependent manner post-exercise, revealing an early event that may be necessary for effective repair and/or growth following exercise. The results from this study also support a role for the α7 integrin and/or mMSCs in molecular- and cellular-based therapeutic strategies that can effectively combat disuse muscle atrophy.     

AMP-activated Protein Kinase Stimulates Warburg-like Glycolysis and Activation of Satellite Cells during Muscle Regeneration

Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells, and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPKα1 knock-out. Taken together, AMPKα1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration.     

Gαi2 Signaling Is Required for Skeletal Muscle Growth,Regeneration, and Satellite Cell Proliferation and Differentiation

We have previously shown that activation of Gαi2, an α subunit of the heterotrimeric G protein complex, induces skeletal muscle hypertrophy and myoblast differentiation. To determine whether Gαi2 is required for skeletal muscle growth or regeneration, Gαi2-null mice were analyzed. Gαi2 knockout mice display decreased lean body mass, reduced muscle size, and impaired skeletal muscle regeneration after cardiotoxin-induced injury. Short hairpin RNA (shRNA)-mediated knockdown of Gαi2 in satellite cells (SCs) leads to defective satellite cell proliferation, fusion, and differentiation ex vivo. The impaired differentiation is consistent with the observation that the myogenic regulatory factors MyoD and Myf5 are downregulated upon knockdown of Gαi2. Interestingly, the expression of microRNA 1 (miR-1), miR-27b, and miR-206, three microRNAs that have been shown to regulate SC proliferation and differentiation, is increased by a constitutively active mutant of Gαi2 [Gαi2(Q205L)] and counterregulated by Gαi2 knockdown. As for the mechanism, this study demonstrates that Gαi2(Q205L) regulates satellite cell differentiation into myotubes in a protein kinase C (PKC)- and histone deacetylase (HDAC)-dependent manner.     

Leishmania Eukaryotic Initiation Factor (LeIF) Inhibits Parasite Growth in Murine Macrophages

The leishmaniases constitute neglected global public health problems that require adequate control measures, prophylactic clinical vaccines and effective and non-toxic drug treatments. In this study, we explored the potential of Leishmania infantum eukaryotic initiation factor (LieIF), an exosomal protein, as a novel anti-infective therapeutic molecule. More specifically, we assessed the efficacy of recombinant LieIF, in combination with recombinant IFN-γ, in eliminating intracellular L. donovani parasites in an in vitro macrophage model. J774A.1 macrophages were initially treated with LieIF/IFN-γ prior to in vitro infection with L. donovani stationary phase promastigotes (pre-infection treatment), and resistance to infection was observed 72 h after infection. J774A.1 macrophages were also treated with LieIF/IFN-γ after L. donovani infection (post-infection treatment), and resistance to infection was also observed at both time points tested (19 h and 72 h) after infection. To elucidate the LieIF/IFN-γ-induced mechanism(s) that mediate the reduction of intracellular parasite growth, we examined the generation of potent microbicidal molecules, such as nitric oxide (NO) and reactive oxygen species (ROS), within infected macrophages. Furthermore, macrophages pre-treated with LieIF/IFN-γ showed a clear up-regulation in macrophage inflammatory protein 1α (MIP-1α) as well as tumor necrosis factor alpha (TNF-α) expression. However, significant different protein levels were not detected. In addition, macrophages pre-treated with LieIF/IFN-γ combined with anti-TNF-α monoclonal antibody produced significantly lower amounts of ROS. These data suggest that during the pre-treatment state, LieIF induces intramacrophage parasite growth inhibition through the production of TNF-α, which induces microbicidal activity by stimulating NO and ROS production. The mechanisms of NO and ROS production when macrophages are treated with LieIF after infection are probably different. Overall, these results indicate that LieIF is a good candidate for use as an anti-leishmanial molecule.     

Abnormal Skeletal Muscle Regeneration plus Mild Alterations in Mature Fiber Type Specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice

Glycosylated α-dystroglycan provides an essential link between extracellular matrix proteins, like laminin, and the cellular cytoskeleton via the dystrophin-glycoprotein complex. In secondary dystroglycanopathy muscular dystrophy, glycosylation abnormalities disrupt a complex O-mannose glycan necessary for muscle structural integrity and signaling. Fktn-deficient dystroglycanopathy mice develop moderate to severe muscular dystrophy with skeletal muscle developmental and/or regeneration defects. To gain insight into the role of glycosylated α-dystroglycan in these processes, we performed muscle fiber typing in young (2, 4 and 8 week old) and regenerated muscle. In mice with Fktn disruption during skeletal muscle specification (Myf5/Fktn KO), newly regenerated fibers (embryonic myosin heavy chain positive) peaked at 4 weeks old, while total regenerated fibers (centrally nucleated) were highest at 8 weeks old in tibialis anterior (TA) and iliopsoas, indicating peak degeneration/regeneration activity around 4 weeks of age. In contrast, mature fiber type specification at 2, 4 and 8 weeks old was relatively unchanged. Fourteen days after necrotic toxin-induced injury, there was a divergence in muscle fiber types between Myf5/Fktn KO (skeletal-muscle specific) and whole animal knockout induced with tamoxifen post-development (Tam/Fktn KO) despite equivalent time after gene deletion. Notably, Tam/Fktn KO retained higher levels of embryonic myosin heavy chain expression after injury, suggesting a delay or abnormality in differentiation programs. In mature fiber type specification post-injury, there were significant interactions between genotype and toxin parameters for type 1, 2a, and 2x fibers, and a difference between Myf5/Fktn and Tam/Fktn study groups in type 2b fibers. These data suggest that functionally glycosylated α-dystroglycan has a unique role in muscle regeneration and may influence fiber type specification post-injury.     

Yohimbine Enhances Protection of Berberine against LPS-Induced Mouse Lethality through Multiple Mechanisms

Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.