Mouse Peritonitis Model Research Articles

Pristimerin protects against inflammation and metabolic disorder in mice through inhibition of NLRP3 inflammasome activation.

Excessive activation of NLRP3 inflammasome is associated with the pathogenesis of inflammatory diseases. Pristimerin (Pri) is a quinonoid triterpene derived from traditional Chinese medical herb Celastraceae and Hippocrateaceae. Pri has shown antifungal, antibacterial, antioxidant, and anticancer activities. In this study we investigated whether NLRP3 inflammasome was associated with the anti-inflammatory activity of Pri. We showed that Pri (0.1-0.4 μM) dose-dependently blocked caspase-1 activation and IL-1β maturation in LPS-primed mouse bone-marrow-derived macrophages (BMDMs). Pri specifically inhibited NLRP3 inflammasome activation, had no visible effects on NLRC4 and AIM2 inflammasome activation. Furthermore, we demonstrated that Pri blocked the assembly of the NLRP3 inflammasome via disturbing the interaction between NEK7 and NLRP3; the α, β-unsaturated carbonyl moiety of Pri was essential for NLRP3 inflammasome inactivation. In LPS-induced systemic inflammation mouse model and MSU-induced mouse peritonitis model, preinjection of Pri (500 μg/kg, ip) produced remarkable therapeutic effects via inhibition of NLRP3 inflammasome in vivo. In HFD-induced diabetic mouse model, administration of Pri (100 μg· kg-1 ·d-1, ip, for 6 weeks) reversed HFD-induced metabolic disorders via suppression of NLRP3 inflammasome activation. Taken together, our results demonstrate that Pri acts as a NLRP3 inhibitor, suggesting that Pri might be useful for the treatment of NLRP3-associated diseases.

Arctigenin protects mice from thioglycollate-induced acute peritonitis.

Acute peritonitis is an acute inflammatory response of the peritoneal cavity to physical injury and chemical stimulation. Timely resolution of this response is critical to prevent further damage to the body, which can eventually lead to more severe chronic inflammation. Arctigenin (ATG) is the main active ingredient of the Chinese medicine Arctium lappa. In recent years, there have been an increasing number of studies on the anti‐inflammatory effect of ATG, but there have been few studies on the effect of ATG on acute inflammation, especially in acute peritonitis, which has not been reported. In this study, a mouse model of experimental acute peritonitis induced by thioglycolate (TG) solution was used to study the protective anti‐inflammatory effect of ATG against acute peritonitis and the relevant mechanism. Our results showed that, after 12 hours of TG treatment, ATG significantly reduced inflammatory cell infiltration in mouse tissues and inhibited the secretion and expression of interleukin‐6 (IL‐6) and tumor necrosis factor alpha (TNF‐α) in mice. ATG significantly reduced the percentage of CD11b+Ly6G+ neutrophils and F4/80+ macrophages in the spleen and peritoneal exudate. In addition, ATG significantly inhibited the expression of the chemokines CCL3 and CCL4 and the adhesion molecule CD62L on the surface of CD11b‐positive monocytes. ATG was observed to inhibit the phosphorylation of p65 and p38 in LPS‐stimulated RAW264.7 cells. In conclusion, ATG can improve the symptoms of TG‐induced acute peritonitis through immune regulation. ATG can reduce the inflammatory response in TG‐induced acute peritonitis in mice.

Bitis arietans Snake Venom Induces an Inflammatory Response Which Is Partially Dependent on Lipid Mediators.

Bitis arietans is a snake of medical importance, as it is responsible for more accidents in humans and domestic animals than all other African snakes put together. The accidents are characterized by local and systemic alterations, such as inflammation, cardiovascular and hemostatic disturbances, which can lead victims to death or permanent disability. However, little is known about the envenomation mechanism, especially regarding the inflammatory response, which is related to severe clinical conditions triggered by the venom. Therefore, the aim of the present study was to evaluate the inflammatory response related to the B. arietans envenomation using a peritonitis mice model. By pharmacological interventions and use of mice genetically deficient of the 5-lipoxygenase enzyme (5-LO−/−) or platelet-activating factor (PAF) receptor (PAFR−/− the participation of eicosanoids and PAF in this response was also investigated. The obtained results demonstrated that the venom induces an in vivo inflammatory response, characterized by an early increased vascular permeability, followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity, accompanied by the production of the eicosanoids LTB4, LTC4, TXB2 and PGE2, as well as the local and systemic production of IL-6 and MCP-1. These inflammatory events were attenuated by the pre-treatment with anti-inflammatory drugs that interfere in lipid mediators’ functions. However, 5-LO−/− mice did not show a reduction of inflammatory response induced by the venom, while PAFR−/− mice showed a reduction in both the PMN leukocytes number and the local and systemic production of IL-6 and MCP-1. This study demonstrated that the Bitis arietans venom contains toxins that trigger an inflammatory process, which is partially dependent on lipid mediators, and may contribute to the envenomation pathology.

Synthetic vitamin K analogs inhibit inflammation by targeting the NLRP3 inflammasome.

Vitamin K refers to a group of structurally similar vitamins that are essential for proper blood coagulation, as well as bone and cardiovascular health. Previous studies have indicated that vitamin K may also have anti-inflammatory properties, although the underlying mechanisms of its anti-inflammatory effects remain unclear. The NLRP3 inflammasome is a multiprotein complex, and its activation leads to IL-1β and IL-18 secretion and contributes to the pathogenesis of various human inflammatory diseases. Here, we show that synthetic vitamins K3 and K4 are selective, potent inhibitors of the NLRP3 inflammasome and specifically block the interaction between NLRP3 and ASC, thereby inhibiting NLRP3 inflammasome assembly. Moreover, we show that treatment with vitamin K3 or K4 attenuates the severity of inflammation in a mouse model of peritonitis. Our results demonstrate that vitamins K3 and K4 exert their anti-inflammatory effects by inhibiting NLRP3 inflammasome activation and indicate that vitamin K supplementation may be a treatment option for NLRP3-associated inflammatory diseases.

Design of a Sea Snake Antimicrobial Peptide Derivative with Therapeutic Potential against Drug-Resistant Bacterial Infection.

Infections caused by drug-resistant pathogens are a worldwide challenge for public health. Antimicrobial peptides (AMPs) are regarded as promising antibiotic alternatives for the treatment of drug-resistant infections. In the present study, a series of small peptides were designed based on our previously reported sea snake AMP Hc-CATH. From them, the lead peptide HC1-D2, a truncated peptide entirely substituted by d-amino acids, was selected. HC1-D2 exhibited significantly improved stability and antibiofilm and anti-inflammatory activities. Meanwhile, HC1-D2 retained potent, broad-spectrum, and rapid antimicrobial properties against bacteria and fungi, especially drug-resistant bacteria. Moreover, HC1-D2 showed low propensity to induce bacterial resistance and low cytotoxicity and hemolytic activity. Notably, HC1-D2 showed potent in vivo anti-infective ability in mouse peritonitis models infected by both standard and drug-resistant bacteria. It significantly decreased the bacterial counts in the abdominal cavity and spleen of mice and apparently increased the survival rates of the mice. Acting through the MAPKs inflammatory pathway, HC1-D2 selectively induced the production of chemokine and the subsequent immune cell recruitment to the infection site, while inhibiting the production of pro-inflammatory cytokines with undesirable toxicities. These much improved properties make HC1-D2 a promising candidate for the development of novel peptide anti-infective agents against drug-resistant infections.

Repurposing Antispasmodic Agent Otilonium Bromide for Treatment of Staphylococcus aureus Infections.

Recently, the problem of bacterial resistance has been brought into focus, which makes the development of new antibiotics become a necessity. Compared with traditional development approaches, drug repurposing provides a faster and more effective approach to find new antimicrobial agents. In this study, we found that antispasmodic agent otilonium bromide had strong antibacterial ability and bactericidal activity against Staphylococcus aureus, with minimal inhibitory concentrations (MICs) of 4–8 μg/ml, and bacteria could be killed completely after treatment with 2× MIC of otilonium bromide for 5 h. Furthermore, it had a potent effect on eradicating biofilm at concentrations ranging from 16 to 64 μg/ml. At the same time, it had low tendency to develop resistance and possessed limited cytotoxicity. In the methicillin-resistant S. aureus–infected mouse peritonitis model, it was also effective to cure mice and improve their survival rate. In addition, we observed that otilonium bromide changed the permeability of bacterial membrane and caused membrane damage, and it is probably the antibacterial mechanism of otilonium bromide. Taken together, our results indicated that otilonium bromide could be a new antimicrobial agent to treat S. aureus infections more safely and efficiently.

CaMKII-dependent ryanodine receptor phosphorylation mediates sepsis-induced cardiomyocyte apoptosis.

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis‐induced apoptosis. Wild‐type (WT) CASP mice hearts showed an increase in apoptosis respect to WT‐Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3‐I) were protected against sepsis‐induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca2+ release, prevented apoptosis in WT‐CASP. To examine whether CaMKII‐dependent RyR2 phosphorylation mediates diastolic Ca2+ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation‐dependent enhancement in diastolic SR Ca2+ release leading to mitochondrial Ca2+ overload, mitochondrial Ca2+ retention capacity was reduced in mitochondria isolated from WT‐CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene‐treated mice. We conclude that in sepsis, CaMKII‐dependent RyR2 phosphorylation results in diastolic Ca2+ release from SR which leads to mitochondrial Ca2+ overload and apoptosis.

Expression of CTX-M-15 limits the efficacy of ceftolozane/tazobactam against Escherichia coli in a high-inoculum murine peritonitis model

ObjectivesAmong carbapenem-sparing therapies, ceftolozane/tazobactam has been proposed for the treatment of infections due to CTX-M-15-producing Escherichia coli. However, few data exist on its in vivo activity in infections associated with a high bacterial inoculum. MethodsWe analysed ceftolozane/tazobactam activity against susceptible E. coli CFT073-RR and its CTX-M-15-producing transconjugant E. coli CFT073-RR Tc blaCTX-M-15, in vitro at low and high inocula, and in a high-inoculum murine model of peritonitis. ResultsAgainst E. coli CFT073-RR Tc blaCTX-M-15, ceftolozane/tazobactam bactericidal effect was impaired in vitro with only a minor inoculum effect; this translated into reduced activity compared with imipenem in the mouse peritonitis model. ConclusionsCombination of extended spectrum β-lactamase expression and high inoculum size may be a clinical situation at risk of reduced bactericidal activity of ceftolozane/tazobactam.

An amphipathic peptide with antibiotic activity against multidrug-resistant Gram-negative bacteria

ABSTRACTPeptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3–4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the ‘no observable adverse effect level’ (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.

A single dose of eHSP72 attenuates sepsis severity in mice

High levels of extracellular 72 kDa heat shock protein (eHSP72) can be detected in the serum of septic patients and are associated with increased oxidative profiles and elevated rates of mortality among these patients. However, a possible immunomodulatory role for this protein, resulting in tissue protection during sepsis, has never been assessed. In this study, we investigated whether eHSP72 administration could attenuate the severity of sepsis in a mouse peritonitis model. Animals (90-day-old male C57BL/6J mice) were divided into Sepsis (n = 8) and Sepsis + eHSP72 (n = 9) groups, which both received injections of 20% fecal solution [1 mg/g body weight (wt), intraperitoneal (i.p.)], to trigger peritonitis induced-sepsis, whereas a Control group (n = 7) received a saline injection. eHSP72 was administered (1.33 ng/g body wt) to the Sepsis+eHSP72 group, 12 h after sepsis induction. All animals were evaluated for murine sepsis score (MSS), hemogram, core temperature, and glycemia (before and 4, 12, and 24 h after sepsis induction). Treatment with eHSP72 promoted reduced sepsis severity 24 h after sepsis induction, based on MSS scores (Control = 1.14 ± 1.02; Sepsis = 11.07 ± 7.24, and Sepsis + eHSP72 = 5.62 ± 1.72, P < 0.001) and core temperatures (°C; Control = 37.48 ± 0.58; Sepsis = 35.17 ± 2.88, and Sepsis + eHSP72 = 36.94 ± 2.02; P = 0.006). eHSP72 treatment also limited the oxidative profile and respiratory dysfunction in mice with sepsis. Although sepsis modified glycemic levels and white and red blood cell counts, these variables were not influenced by eHSP72 treatment (P > 0.05). Finally, eHSP72 improved the survival rate after sepsis (P = 0.0371). Together, our results indicated that eHSP72 may ameliorate sepsis severity and possibly improve some sepsis indices in mice.

Platensimycin-Encapsulated Poly(lactic-co-glycolic acid) and Poly(amidoamine) Dendrimers Nanoparticles with Enhanced Anti-Staphylococcal Activity in Vivo.

Serious bacterial infections by multi-drug-resistant pathogens lead to human losses and endanger public health. The discovery of antibiotics with new modes of action, in combination with nanotechnology, might offer a promising route to combat multi-drug-resistant pathogens. Platensimycin (PTM), a potent inhibitor of FabB/FabF for bacterial fatty acid biosynthesis, is a promising drug lead against many drug-resistant bacteria. However, the clinical development of PTM is hampered by its poor pharmacokinetics. Herein, we report a nanostrategy that encapsulated PTM in two types of nanoparticles (NPs) poly(lactic-co-glycolic acid) (PLGA) and poly(amidoamine) (PAMAM) dendrimer to enhance its antibacterial activity in vitro and in vivo. The PTM-encapsulated NPs were effective to inhibit Staphylococcus aureus biofilm formation, and killed more S. aureus in a macrophage cell infection model over free PTM. The pharmacokinetic studies showed that PTM-loaded PLGA and PAMAM NPs exhibited increased AUC0-t (area under the curve) (∼4- and 2-fold) over free PTM. In a mouse peritonitis model, treatment of methicillin-resistant S. aureus infected mice using both PTM-loaded NPs (10 mg/kg) by intraperitoneal injection led to their full survival, while all infected mice died when treated by free PTM (10 mg/kg). These results not only suggest that PTM-loaded NPs may hold great potential to improve the poor pharmacokinetic properties of PTM, but support the rationale to develop bacterial fatty acid synthase inhibitors as promising antibiotics against drug-resistant pathogens.

Loratadine, an antihistamine drug, exhibits anti-inflammatory activity through suppression of the NF-kB pathway

Loratadine is an antihistamine drug that shows promise as an anti-inflammatory drug, but supportive studies are lacking. We elucidated the effects and mechanisms by which loratadine inhibits inflammatory responses. Molecular components were evaluated in macrophages by nitric oxide assay, polymerase chain reaction, luciferase assay, immunoblotting, overexpression strategies and cellular thermal shift assay. At the molecular level, loratadine reduced the levels of nitric oxide, iNOS, IL-1β, TNF-α, IL-6, and COX-2 in RAW264.7 cells treated with lipopolysaccharide. Loratadine also specifically inhibited the NF-kB pathway, targeting the Syk and Src proteins. Furthermore, loratadine bound Src in the bridge between SH2 and SH3, and bound Syk in the protein tyrosine kinase domain. The NF-kB signaling pathway was assessed along with putative binding sites through a docking approach. The anti-inflammatory effect of loratadine was tested using mouse models of gastritis, hepatitis, colitis, and peritonitis. Stomach tissue histopathology, liver morphology, and colon length in the loratadine group were improved over the group without loratadine treatment. Taken together, loratadine inhibited the inflammatory response through the NF-kB pathway by binding with the Syk and Src proteins.

Near-Infrared Chemiluminescent Carbon Nanodots and Their Application in Reactive Oxygen Species Bioimaging.

Reactive oxygen species (ROS) are generated in the body and related to many pathophysiological processes. Hence, detection of ROS is indispensable in understanding, diagnosis, and treatment of many diseases. Here, near‐infrared (NIR) chemiluminescent (CL) carbon nanodots (CDs) are fabricated for the first time and their CL quantum yield can reach 9.98 × 10−3 einstein mol−1, which is the highest value ever reported for CDs until now. Nanointegration of NIR CDs and peroxalate (P‐CDs) through the bridging effect of amphiphilic triblock copolymer can serve as turn‐on probes for the detection and imaging of hydrogen peroxide (H2O2). Considering high efficiency and large penetration depth of NIR photons, the P‐CDs are employed in bioimaging H2O2 in vitro and in vivo, and the detection limit can reach 5 × 10−9 m, among the best reported of CDs‐based sensors. Moreover, imaging of inflammatory H2O2 in a mouse model of peritonitis is achieved by employing the P‐CDs as sensors. The results may provide a clue for the diagnosis and treatment of inflammation or cancers employing CL CDs as sensors.

Neutrophils Enhance Their Own Influx to Sites of Bacterial Infection via Endosomal TLR-Dependent Cxcl2 Production.

The influx of neutrophils to infection sites is a fundamental step in host defenses against the frequent human pathogen group B Streptococcus (GBS) and other extracellular bacteria. Using a mouse model of GBS-induced peritonitis, we show in this study that the chemokines Cxcl1 and Cxcl2 play distinctive roles in enhancing the recruitment and the antibacterial activities of neutrophils in a manner that is linked to differences in the cellular sources of these mediators. Cell depletion experiments demonstrated that neutrophils make a significant contribution to the in vivo production of Cxcl2 but not Cxcl1. In vitro, neutrophils responded weakly to LPS but released high levels of Cxcl2 after stimulation with GBS or other bacteria. Neutrophil-derived Cxcl2 acted in an autocrinous manner to increase its own production and to enhance antibacterial activities, including the release of oxygen radicals. In both neutrophils and macrophages, the production of Cxcl1/2 largely required the presence of functional UNC93B1, a chaperone protein involved in signaling by endosomal TLRs. Moreover, the phenotype of UNC93B1-defective phagocytes could be recapitulated by the simultaneous absence of TLR7, 9, and 13 but not by the absence of individual TLRs. Collectively, our data show that neutrophils recognize Gram-positive and Gram-negative bacteria by means of multiple phagosomal TLRs, resulting in de novo synthesis of Cxcl2, amplification of neutrophil recruitment, and potentiation of their antibacterial activities. These data may be useful to devise alternative therapeutic strategies aimed at enhancing the recruitment and the functional activities of polymorphonuclear leukocytes during infections caused by antibiotic-resistant bacteria.

Synthetic 4-Hydroxy Auxarconjugatin B, a Novel Autophagy Inducer, Attenuates Gouty Inflammation by Inhibiting the NLRP3 Inflammasome.

Gouty arthritis results from the generation of uric acid crystals within the joints. These uric acid crystals activate the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, which is involved in chronic inflammatory diseases, including gouty arthritis. This study identified the polyenylpyrrole derivative 4-hydroxy auxarconjugatin B (4-HAB), a novel autophagy inducer, which attenuated uric acid crystals-mediated activation of the NLRP3 inflammasome in vitro and in vivo. 4-HAB dose-dependently reduced the release of interleukin (IL)-1β, IL-18, active caspase-1 and apoptosis-associated speck-like protein (ASC) in uric acid crystals-activated macrophages. In a mechanistic study, 4-HAB was shown to inhibit uric acid crystals-induced mitochondrial damage, lysosomal rupture and ASC oligomerization. Additionally, 4-HAB inhibited the NLRP3 inflammasome through Sirt1-dependent autophagy induction. Furthermore, the anti-inflammatory properties of 4-HAB were confirmed in a mouse model of uric acid crystals-mediated peritonitis by the reduced levels of neutrophil influx, IL-1β, active caspase-1, IL-6 and MCP-1 in lavage fluids. In conclusion, 4-HAB attenuates gouty inflammation, in part by attenuating activation of the NLRP3 inflammasome through the Sirt1/autophagy induction pathway.

Ginsenoside metabolite 20(S)-protopanaxatriol from Panax ginseng attenuates inflammation-mediated NLRP3 inflammasome activation.

Panax ginseng C.A. Meyer (Araliaceae), has been used in traditional medicine for preventive and therapeutic purposes in Asian countries. One of the active ginsenoside metabolites, 20(S)-Protopanaxatriol (PPT), has been associated with diverse pharmacological effects, including anti-inflammatory properties. Although the capacity of PPT as an anti-inflammatory agent has been studied, this study aimed to explore the intrinsic mechanism of PPT in regulating inflammasome activation-mediated inflammatory responses in experimental models. Lipopolysaccharide (LPS)-primed peritoneal macrophages in vitro was used to study the role of PPT on inflammasome activation. LPS-induced septic shock and monosodium urate (MSU)-induced murine peritonitis models were employed for in vivo evaluations. PPT attenuated NLRP3 inflammasome activation and also reduced ASC oligomerization, leading to attenuation of interleukin (IL)-1β secretion. Further, PPT inhibited IL-1β secretion in both LPS-induced septic shock and MSU-induced mouse peritonitis models. This study revealed that ginsenoside metabolite PPT, inhibits inflammation-mediated inflammasome activation and supported the traditional use of ginseng in treating various inflammatory disorders.

Tannic acid-based nanogel as an efficient anti-inflammatory agent.

Biologically produced reactive oxygen species (ROS) are important signaling molecules in the human body. Despite their importance under normal conditions, abnormal overproduction of ROS under unbalanced or irregular homeostasis can cause severe inflammatory diseases. Various antioxidants have been developed in the biomedical field to resolve high levels of ROS; however, high doses of natural antioxidants such as polyphenol can induce side effects on health. Further, synthetic antioxidants are still controversial in regards to their safety and their complicated synthesis. Inspired from our previous work, a nitric oxide-scavenging nanogel designed for treating rheumatoid arthritis, we report herein a biocompatible tannic acid (TA)-based nanogel as an effective ROS scavenger. A polymeric phenylboronic acid-tannic acid nanogel (PTNG) was prepared by simply mixing through to the formation of phenylboronic ester bonds between polymeric phenylboronate and TA. We focused on the reaction of phenylboronic ester with H2O2, which readily consumes H2O2 molecules, and applied it as an antioxidant. In addition, TA is a well-known antioxidant, specifically a free radical scavenger; thus, we expected combinatory ROS scavenging effects for PTNG. Various ROS scavenging assays revealed the significant antioxidant effects of PTNG. Under an induced inflammation model in vitro, our PTNG showed high biocompatibility as well as strong anti-inflammatory effects. Furthermore, in the zymosan-induced peritonitis mouse model, a representative acute inflammation model in vivo, PTNG reduced significant neutrophil recruitment and pro-inflammatory cytokines, indicating successful alleviation of inflammation. On the basis of these results, we suggest that PTNG has great potential as an antioxidant and should find application in the treatment of further ROS-overproducing inflammatory diseases.

Biological role of GITR/GITRL in attributes and immune responses of macrophage.

Glucocorticoid-induced tumor necrosis factor receptor family-related protein ligand (GITRL), a member of the tumor necrosis factor superfamily, is expressed in APCs and acts as a costimulatory molecule in the immune system. Although the glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR)/GITRL system has been modulated to promote or decrease T cell-related responses in multiple diseases, studies in macrophages are limited. To address this issue, we compared the expression of GITRL in various types of macrophages and analyzed whether GITRL can affect the fundamental properties and major functions of these cells. Our results demonstrated that M1 polarized macrophages had the highest GITRL levels. Furthermore, GITRL overexpression skewed macrophage polarization toward the M1 phenotype, accelerating proliferation and migration and regulating phagocytosis and killing function. Moreover, GITRL-silenced cells showed a loss of these functions, further confirming its vital role. We also developed an acute peritonitis mouse model, in which macrophages were driven to differentiate into a proinflammatory phenotype with GITRL up-regulation, triggering a positive feedback loop. Our results provide molecular insight into how the GITR/GITRL system modulates innate immune responses, suggesting that manipulation of the GITR/GITRL system to treat diseases depends not only on T cell regulation but also on macrophage participation.

Prophylactic potential of cytolethal distending toxin B (CdtB) subunit of typhoid toxin against Typhoid fever

Typhoid fever caused by Salmonella enterica serovar Typhi (S.Typhi) continues to be a major problem, especially in developing countries. Due to the rapid emergence of multi-drug-resistant (MDR) strains, which limits the efficacy of conventional antibiotics as well as problems associated with the existing vaccines, efforts are being made to develop effective prophylactic agents. CdtB subunit of typhoid toxin was selected for assessing its vaccine potential due to its high conservation throughout the Typhi strains. In-vitro assessment of DNase activity of cloned and purified CdtB protein showed a significant decrease in the band intensity of DNA. The measure of metabolic activity and morphological alterations assessed using different cell lines in the presence of CdtB protein showed no significant signs of toxicity. These observations were further strengthened by cell cycle analysis, assessed by flow cytometry. Keeping these observations in mind, the immunoprotective potential of CdtB was assessed using S.Typhi induced mouse peritonitis model. A significant titer of IgG antibodies (>128000) against CdtB protein was recorded in the immunized mice by enzyme-linked immunosorbent assay (ELISA), which was also validated by immunoblotting. Active immunization with the protein protected 75% mice against a lethal dose of S.Typhi Ty2. The data indicated a significant (up to 5 log) reduction in the bacterial load in the spleen and liver of immunized-infected mice compared to control (unimmunized-infected) mice which might have resulted in the modulation of histoarchitecture of spleen and liver and the levels of cytokines (IL-6, TNF-α and IL-10) production; thereby indicating the effectiveness of the subunit. The observations deduced from the study give the proof of concept of immunogenic potential of protein. However, further studies involving the immunoreactivity of CdtB with the statistically significant number of sera samples obtained from the human patients would be helpful in establishing the relevance of CdtB protein in humans and for making the strategies to develop it as an effective vaccine candidate.

T6SS and ExoA of flesh-eating Aeromonas hydrophila in peritonitis and necrotizing fasciitis during mono- and polymicrobial infections

An earlier report described a human case of necrotizing fasciitis (NF) caused by mixed infection with 4 Aeromonas hydrophila strains (NF1-NF4). While the NF2, NF3, and NF4 strains were clonal and possessed exotoxin A (ExoA), the NF1 strain was determined to be phylogenetically distinct, harboring a unique type 6 secretion system (T6SS) effector (TseC). During NF1 and NF2 mixed infection, only NF1 disseminated, while NF2 was rapidly killed by a contact-dependent mechanism and macrophage phagocytosis, as was demonstrated by using in vitro models. To confirm these findings, we developed 2 NF1 mutants (NF1ΔtseC and NF1ΔvasK); vasK encodes an essential T6SS structural component. NF1 VasK and TseC were proven to be involved in contact-dependent killing of NF2 in vitro, as well as in its elimination at the intramuscular injection site in vivo during mixed infection, with overall reduced mouse mortality. ExoA was shown to have an important role in NF by both NF1-exoA (with cis exoA) and NF2 during monomicrobial infection. However, the contribution of ExoA was more important for NF2 than NF1 in the murine peritonitis model. The NF2∆exoA mutant did not significantly alter animal mortality or NF1 dissemination during mixed infection in the NF model, suggesting that the ExoA activity was significant at the injection site. Immunization of mice to ExoA protected animals from NF2 monomicrobial challenge, but not from polymicrobial infection because of NF2 clearance. This study clarified the roles of T6SS and ExoA in pathogenesis caused by A. hydrophila NF strains in both mouse peritonitis and NF models in monomicrobial and polymicrobial infections.