Deficient Skeletal Muscle Regeneration after Injury Induced by a Clostridium perfringens Strain Associated with Gas Gangrene

ABSTRACT Gas gangrene, or clostridial myonecrosis, is usually caused by Clostridium perfringens and may occur spontaneously in association with diabetes mellitus, peripheral vascular disease, or some malignancies but more often after contamination of a deep surgical or traumatic lesion. If not controlled, clostridial myonecrosis results in multiorgan failure, shock, and death, but very little is known about the muscle regeneration process that follows myonecrosis when the infection is controlled. In this study, we characterized the muscle regeneration process after myonecrosis caused in a murine experimental infection with a sublethal inoculum of C. perfringens vegetative cells. The results show that myonecrosis occurs concomitantly with significant vascular injury, which limits the migration of inflammatory cells. A significant increase in cytokines that promote inflammation explains the presence of an inflammatory infiltrate; however, impaired interferon gamma (IFN-γ) expression, a reduced number of M1 macrophages, deficient phagocytic activity, and a prolongation of the permanence of inflammatory cells lead to deficient muscle regeneration. The expression of transforming growth factor β1 (TGF-β1) agrees with the consequent accumulation of collagen in the muscle, i.e., fibrosis observed 30 days after infection. These results provide new information on the pathogenesis of gas gangrene caused by C. perfringens, shed light on the basis of the deficient muscle regenerative activity, and may open new perspectives for the development of novel therapies for patients suffering from this disease.

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Deficient skeletal muscle regeneration after injury induced by a Clostridium perfringens strain associated with gas gangrene

10.1101/441204 ◽

2018 ◽

Author(s):

Ana Mariel Zúñiga-Pereira ◽

Carlos Carlos Santamaría ◽

José María Gutiérrez ◽

Alberto Alape-Girón ◽

Marietta Flores-Díaz

Keyword(s):

Muscle Regeneration ◽

Inflammatory Infiltrate ◽

Inflammatory Cells ◽

Gas Gangrene ◽

Skeletal Muscle Regeneration ◽

The Poor ◽

New Information ◽

Regenerative Activity ◽

Muscle Necrosis ◽

Shed Light

AbstractVery little is known about the muscle regeneration process that follows myonecrosis induced by C. perfringens, the main agent of gas gangrene. This study revealed that, in a murine model of the infection with a sublethal inoculum of C. perfringens, muscle necrosis occurs concomitantly with significant vascular damage, which limits the migration of inflammatory cells. A significant increase in cytokines that promote inflammation explains the presence of inflammatory infiltrate; however, an impaired IFNγ expression, a reduced number of Ml macrophages, a deficient phagocytic activity, and the prolongation of the permanence of inflammatory cells, lead to deficient muscle regeneration. The expression of TGFβ1 and the consequent accumulation of collagen in the muscle, likely contribute to the fibrosis observed 30 days after infection. These results provide new information on the pathogenesis of gas gangrene caused by C. perfringens, shed light on the basis of the poor muscle regenerative activity, and may open new perspectives for the development of novel therapies for patients suffering this disease.

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Perfringolysin O Expression in Clostridium perfringens Is Independent of the Upstream pfoR Gene

Journal of Bacteriology ◽

10.1128/jb.184.7.2034-2038.2002 ◽

2002 ◽

Vol 184 (7) ◽

pp. 2034-2038 ◽

Cited By ~ 5

Author(s):

Milena M. Awad ◽

Julian I. Rood

Keyword(s):

Escherichia Coli ◽

Homologous Recombination ◽

Gene Product ◽

Structural Gene ◽

Clostridium Perfringens ◽

Deletion Mutant ◽

Gas Gangrene ◽

Alpha Toxin ◽

Clostridial Myonecrosis ◽

Perfringolysin O

ABSTRACT The pathogenesis of Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis involves the extracellular toxins alpha-toxin and perfringolysin O. Previous studies (T. Shimizu, A. Okabe, J. Minami, and H. Hayashi, Infect. Immun. 59:137-142, 1991) carried out with Escherichia coli suggested that the perfringolysin O structural gene, pfoA, was positively regulated by the product of the upstream pfoR gene. In an attempt to confirm this hypothesis in C. perfringens, a pfoR-pfoA deletion mutant was complemented with isogenic pfoA+ shuttle plasmids that varied only in their ability to encode an intact pfoR gene. No difference in the ability to produce perfringolysin O was observed for C. perfringens strains carrying these plasmids. In addition, chromosomal pfoR mutants were constructed by homologous recombination in C. perfringens. Again no difference in perfringolysin O activity was observed. Since it was not possible to alter perfringolysin O expression by mutation of pfoR, it was concluded that the pfoR gene product is unlikely to have a role in the regulation of pfoA expression in C. perfringens.

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The VirS/VirR Two-Component System Regulates the Anaerobic Cytotoxicity, Intestinal Pathogenicity, and Enterotoxemic Lethality of Clostridium perfringens Type C Isolate CN3685

mBio ◽

10.1128/mbio.00338-10 ◽

2011 ◽

Vol 2 (1) ◽

Cited By ~ 24

Author(s):

Menglin Ma ◽

Jorge Vidal ◽

Juliann Saputo ◽

Bruce A. McClane ◽

Francisco Uzal

Keyword(s):

Clostridium Perfringens ◽

Regulatory System ◽

Gas Gangrene ◽

Necrotic Enteritis ◽

Vegetative Cells ◽

Content Type ◽

Two Component ◽

Type C ◽

Beta Toxin

ABSTRACT Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells. IMPORTANCE Clostridium perfringens is an important human and veterinary pathogen. C. perfringens vegetative cells cause both histotoxic infections, e.g., traumatic gas gangrene, and infections originating when this bacterium grows in the intestines. The VirS/VirR two-component regulatory system has been shown to control the pathogenicity of C. perfringens type A strains in a mouse gas gangrene model, but there is no understanding of pathogenicity regulation when C. perfringens vegetative cells cause disease originating in the complex intestinal environment. The current study establishes that VirS/VirR controls vegetative cell pathogenicity when C. perfringens type C isolates cause hemorrhagic necrotic enteritis and lethal enterotoxemia (i.e., toxin absorption from the intestines into the circulation, allowing targeting of internal organs). This effect involves VirS/VirR-mediated regulation of beta toxin production in vivo. Therefore, VirS/VirR is the first identified global in vivo regulator controlling the ability of C. perfringens vegetative cells to cause gas gangrene and, at least some, intestinal infections.

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Synergistic Effects of Alpha-Toxin and Perfringolysin O in Clostridium perfringens-Mediated Gas Gangrene

Infection and Immunity ◽

10.1128/iai.69.12.7904-7910.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7904-7910 ◽

Cited By ~ 119

Author(s):

Milena M. Awad ◽

Darren M. Ellemor ◽

Richard L. Boyd ◽

John J. Emmins ◽

Julian I. Rood

Keyword(s):

Structural Gene ◽

Clostridium Perfringens ◽

Synergistic Effects ◽

Gas Gangrene ◽

Alpha Toxin ◽

Coagulative Necrosis ◽

Clostridial Myonecrosis ◽

Perfringolysin O ◽

Vascular Disruption ◽

Rapid Spread

ABSTRACT To examine the synergistic effects of alpha-toxin and perfringolysin O in clostridial myonecrosis, homologous recombination was used to construct an alpha-toxin deficient derivative of a perfringolysin O mutant of Clostridium perfringens. The subsequent strain was complemented with separate plasmids that carried the alpha-toxin structural gene (plc), the perfringolysin O gene (pfoA), or both toxin genes, and the resultant isogenic strains were examined in a mouse myonecrosis model. Synergistic effects were clearly observed in these experiments. Infection with the control strain, which did not produce either toxin, resulted in very minimal gross pathological changes, whereas the isogenic strain that was reconstituted for both toxins produced a pathology that was clearly more severe than when alpha-toxin alone was reconstituted. These changes were most apparent in the rapid spread of the disease, the gross pathology of the footpad and in the rate at which the mice had to be euthanatized for ethical reasons. Elimination of both alpha-toxin and perfringolysin O production removed most of the histopathological features typical of clostridial myonecrosis. These effects were restored when the mutant was complemented with the alpha-toxin structural gene, but reconstituting only perfringolysin O activity produced vastly different results, with regions of coagulative necrosis, apparently enhanced by vascular disruption, being observed. Reconstitution of both alpha-toxin and perfringolysin O activity produced histopathology most similar to that observed with the alpha-toxin reconstituted strain. The spreading of myonecrosis was very rapid in these tissues, and coagulative necrosis appeared to be restricted to the lumen of the blood vessels. The results of these virulence experiments clearly support the hypothesis that alpha-toxin and perfringolysin O have a synergistic effect in the pathology of gas gangrene.

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Use of Genetically Manipulated Strains of Clostridium perfringens Reveals that Both Alpha-Toxin and Theta-Toxin Are Required for Vascular Leukostasis To Occur in Experimental Gas Gangrene

Infection and Immunity ◽

10.1128/iai.67.9.4902-4907.1999 ◽

1999 ◽

Vol 67 (9) ◽

pp. 4902-4907 ◽

Cited By ~ 55

Author(s):

Darren M. Ellemor ◽

Rebecca N. Baird ◽

Milena M. Awad ◽

Richard L. Boyd ◽

Julian I. Rood ◽

...

Keyword(s):

Clostridium Perfringens ◽

Type A ◽

Toxin Production ◽

Gas Gangrene ◽

Necrotic Tissue ◽

Alpha Toxin ◽

Tissue Necrosis ◽

Clostridial Myonecrosis ◽

Leukocyte Aggregation ◽

Genetically Manipulated

ABSTRACT A hallmark of gas gangrene (clostridial myonecrosis) pathology is a paucity of leukocytes infiltrating the necrotic tissue. The cause of this paucity most likely relates to the observation of leukocyte aggregates at the border of the area of tissue necrosis, often within the microvasculature itself. Infecting mice with genetically manipulated strains of Clostridium perfringens type A (deficient in either alpha-toxin or theta-toxin production) resulted in significantly reduced leukocyte aggregation when alpha-toxin was absent and complete abrogation of leukocyte aggregation when theta-toxin was absent. Thus, both alpha-toxin and theta-toxin are necessary for the characteristic vascular leukostasis observed in clostridial myonecrosis.

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P0707UREMIC TOXINS IMPAIR SKELETAL MUSCLE REGENERATION PROCESS INDUCING CELL CYCLE ARREST AND APOPTOSIS IN CULTURED MYOBLASTS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0707 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Elena Alcalde-Estévez ◽

Ana Asenjo-Bueno ◽

Patricia Sosa ◽

Patricia Plaza ◽

Diego Rodríguez-Puyol ◽

...

Keyword(s):

Cell Cycle ◽

Muscle Regeneration ◽

Uremic Toxins ◽

C2c12 Cells ◽

Cyclin B ◽

Skeletal Muscle Regeneration ◽

Regeneration Process ◽

Cycle Arrest ◽

Advanced Stages ◽

And Function

Abstract Background and Aims The loss of muscle mass and function has been related to chronic kidney disease (CKD). About 37% of dialysis patients show symptoms of sarcopenia and this has been related to an increased risk of mortality. Changes in sarcopenic muscle include the loss of its regenerative capacity due to a reduction in the number and function of satellite cells, the muscle stem cells. The concentration of serum uremic toxins (UT) increases in parallel to a decline in the glomerular filtration rate in patients with CKD and this uremia may be involved in the development of sarcopenia. Previous studies showed as serum concentration of UT found in the early stages of CKD inhibits myogenic differentiation of cultured myoblasts. Nevertheless, the effect of those concentrations found in the advanced stages of CKD has not been described. The study aimed to analyse whether UT affect the muscular regeneration process by modifying the proliferation capacity of myoblasts (activated satellite cells). Method Cultured mouse myoblasts C2C12 cells were used for all experiments. Cells were grown with 0% or 10% FBS culture media in the presence or absence of indoxyl sulphate and para-cresol at doses of 100µg/ml each one, which are similar to ones found in the advanced stages of CKD. Proliferation was evaluated by scratch wound healing and cell cycle by flow cytometry with propidium iodide and the fluorescent probe CFSE, an intracellular protein binding dye that is divided equally between daughter cells, allowing the discrimination of successive rounds of cell division. Chromosome condensation was assessed by immunofluorescence staining by confocal microscopy. Apoptosis was analysed by annexin V staining. Results C2C12 cells treated with UT shown a significant decrease in the proliferation rate. A significant delay in wound closure was observed in cells treated with UT compared to control cells. Myoblasts treated with UT suffered a significant decrease in the proliferation rate since the probe remained higher than in the vehicle-treated cells. Proliferating cells treated with UT suffered a dramatic cell cycle arrest between the phases S and G2/M. Chromosome condensation was also analysed, finding that in the presence of colcemid, vehicle-treated cells condensed their chromosomes, as expected, whereas UT-treated cells did not, suggesting that UT stop the cell cycle at any point before the entry of cells in the mitosis phase. Besides, there was strong phosphorylation of cdc2 in the presence of UT indicating that cdc2 and the complex cdc2-cyclin B were inactive. This result explains why cells did not enter in the mitosis phase under UT exposition. Finally, UT induced the death of proliferating C2C12 cells by apoptosis. Conclusion In the advanced stages of CKD, uremic toxins concentration increases, thereby inducing a dramatic arrest in the cell cycle of myoblasts, inactivating the cdc2-cyclin B complex, interrupting their proliferation and leading them towards cell apoptosis. These results point to a role of uremic toxins impairing the skeletal muscle regeneration process, which could be involved in CKD-related sarcopenia and frailty.

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The COX-2 pathway is essential during early stages of skeletal muscle regeneration

AJP Cell Physiology ◽

10.1152/ajpcell.00088.2004 ◽

2004 ◽

Vol 287 (2) ◽

pp. C475-C483 ◽

Cited By ~ 189

Author(s):

Brenda A. Bondesen ◽

Stephen T. Mills ◽

Kristy M. Kegley ◽

Grace K. Pavlath

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Inflammatory Cells ◽

Skeletal Muscle Regeneration ◽

Cellular Processes ◽

Early Stages ◽

Inflammatory Drugs ◽

Cox 2 ◽

Anti Inflammatory

Skeletal muscle regeneration comprises several overlapping cellular processes, including inflammation and myogenesis. Prostaglandins (PGs) may regulate muscle regeneration, because they modulate inflammation and are involved in various stages of myogenesis in vitro. PG synthesis is catalyzed by different isoforms of cyclooxygenase (COX), which are inhibited by nonsteroidal anti-inflammatory drugs. Although experiments employing nonsteroidal anti-inflammatory drugs have implicated PGs in tissue repair, how PGs regulate muscle regeneration remains unclear, and the potentially distinct roles of different COX isoforms have not been investigated. To address these questions, a localized freeze injury was induced in the tibialis anterior muscles of mice chronically treated with either a COX-1- or COX-2-selective inhibitor (SC-560 and SC-236, respectively), starting before injury. The size of regenerating myofibers was analyzed at time points up to 5 wk after injury and found to be decreased by SC-236 and in COX-2−/− muscles, but unaffected by SC-560. In contrast, SC-236 had no effect on myofiber growth when administered starting 7 days after injury. The attenuation of myofiber growth by SC-236 treatment and in COX-2−/− muscles is associated with decreases in the number of myoblasts and intramuscular inflammatory cells at early times after injury. Together, these data suggest that COX-2-dependent PG synthesis is required during early stages of muscle regeneration and thus raise caution about the use of COX-2-selective inhibitors in patients with muscle injury or disease.

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Macrophages Are Key Regulators of Stem Cells during Skeletal Muscle Regeneration and Diseases

Stem Cells International ◽

10.1155/2019/4761427 ◽

2019 ◽

Vol 2019 ◽

pp. 1-20 ◽

Cited By ~ 20

Author(s):

Junio Dort ◽

Paul Fabre ◽

Thomas Molina ◽

Nicolas A. Dumont

Keyword(s):

Skeletal Muscle ◽

Muscle Regeneration ◽

Inflammatory Cells ◽

Cell Types ◽

Skeletal Muscle Regeneration ◽

Cellular Interactions ◽

Muscle Disorders ◽

New Therapeutic Approaches ◽

Inflammatory Phenotype ◽

Muscle Homeostasis

Muscle regeneration is a closely regulated process that involves a variety of cell types such as satellite cells, myofibers, fibroadipogenic progenitors, endothelial cells, and inflammatory cells. Among these different cell types, macrophages emerged as a central actor coordinating the different cellular interactions and biological processes. Particularly, the transition of macrophages from their proinflammatory to their anti-inflammatory phenotype was shown to regulate inflammation, myogenesis, fibrosis, vascularization, and return to homeostasis. On the other hand, deregulation of macrophage accumulation or polarization in chronic degenerative muscle disorders was shown to impair muscle regeneration. Considering the key roles of macrophages in skeletal muscle, they represent an attractive target for new therapeutic approaches aiming at mitigating various muscle disorders. This review aims at summarizing the novel insights into macrophage heterogeneity, plasticity, and functions in skeletal muscle homeostasis, regeneration, and disease.

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The Agr-Like Quorum-Sensing System Is Important for Clostridium perfringens Type A Strain ATCC 3624 To Cause Gas Gangrene in a Mouse Model

mSphere ◽

10.1128/msphere.00500-20 ◽

2020 ◽

Vol 5 (3) ◽

Author(s):

Mauricio A. Navarro ◽

Jihong Li ◽

Juliann Beingesser ◽

Bruce A. McClane ◽

Francisco A. Uzal

Keyword(s):

Quorum Sensing ◽

Mouse Model ◽

Clostridium Perfringens ◽

Regulatory System ◽

Type A ◽

Gas Gangrene ◽

Null Mutant ◽

Wild Type ◽

Content Type

ABSTRACT Clostridium perfringens type A is involved in gas gangrene in humans and animals. Following a traumatic injury, rapid bacterial proliferation and exotoxin production result in severe myonecrosis. C. perfringens alpha toxin (CPA) and perfringolysin (PFO) are the main virulence factors responsible for the disease. Recent in vitro studies have identified an Agr-like quorum-sensing (QS) system in C. perfringens that regulates the production of both toxins. The system is composed of an AgrB membrane transporter and an AgrD peptide that interacts with a two-component regulatory system in response to fluctuations in the cell population density. In addition, a synthetic peptide named 6-R has been shown to interfere with this signaling mechanism, affecting the function of the Agr-like QS system in vitro. In the present study, C. perfringens type A strain ATCC 3624 and an isogenic agrB-null mutant were tested in a mouse model of gas gangrene. When mice were intramuscularly challenged with 106 CFU of wild-type ATCC 3624, severe myonecrosis and leukocyte aggregation occurred by 4 h. Similar numbers of an agrB-null mutant strain produced significantly less severe changes in the skeletal muscle of challenged mice. Complementation of the mutant to regain agrB expression restored virulence to wild-type levels. The burdens of all three C. perfringens strains in infected muscle were similar. In addition, animals injected intramuscularly with wild-type ATCC 3624 coincubated with the 6-R peptide developed less severe microscopic changes. This study provides the first in vivo evidence that the Agr-like QS system is important for C. perfringens type A-mediated gas gangrene. IMPORTANCE Clostridium perfringens type A strains produce toxins that are responsible for clostridial myonecrosis, also known as gas gangrene. Toxin production is regulated by an Agr-like quorum-sensing (QS) system that responds to changes in cell population density. In this study, we investigated the importance of this QS system in a mouse model of gas gangrene. Mice challenged with a C. perfringens strain with a nonfunctional regulatory system developed less severe changes in the injected skeletal muscle compared to animals receiving the wild-type strain. In addition, a synthetic peptide was able to decrease the effects of the QS in this disease model. These studies provide new understanding of the pathogenesis of gas gangrene and identified a potential therapeutic target to prevent the disease.

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Cytokines and immunoregulatory molecules in malignant glial neoplasms

Journal of Neurosurgery ◽

10.3171/jns.1992.77.2.0265 ◽

1992 ◽

Vol 77 (2) ◽

pp. 265-273 ◽

Cited By ~ 67

Author(s):

John Schneider ◽

Florence M. Hofman ◽

Michael L. J. Apuzzo ◽

David R. Hinton

Keyword(s):

Transforming Growth Factor ◽

Polyclonal Antibodies ◽

Malignant Gliomas ◽

Interleukin 2 ◽

Inflammatory Cells ◽

Transforming Growth Factor Β ◽

Content Type ◽

Cd8 Cells ◽

Tnf Α ◽

Factor Α

✓ Cytokines are important regulatory proteins controlling growth and differentiation of normal and malignant glial cells. Astrocytes and microglial cells produce and respond to many of the same cytokines employed by cells of the immune system. The authors have analyzed 15 histologically confirmed malignant glial neoplasms for the presence of infiltrating lymphocytes, macrophages, cytokines, and other immunoregulatory molecules using a panel of specific monoclonal and polyclonal antibodies on frozen-tissue sections. All neoplasms showed focal T-cell infiltration with CD8 cells predominating. Infiltration of activated macrophages (positive for CD11c, class II, and interleukin-2 receptor) was marked in all tumors. Within the neoplasm, tumor necrosis factor-α (TNF-α)- and interleukin (IL)-6-positive macrophages were prominent in five cases, while the tumor cells themselves were only weakly positive. In the other 10 cases, the numerous infiltrating macrophages were only rarely immunoreactive for TNF-α or IL-6. Transforming growth factor-β (TGF-β) immunoreactivity was most prominent in those tumors with little TNF-α-positive macrophage infiltration, although intratumoral variability was present. This study suggests that, in malignant gliomas, the cytokines TNF-α and IL-6, although weakly present in neoplastic cells, are most prominent in infiltrating macrophages and in those regions of the tumors that show little immunoreactivity for TGF-β. The important interactions among neoplastic, reactive glial, and inflammatory cells, which regulate tumor growth, are likely to be in part mediated through these molecules.

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