TLR4/TNFR1 blockade suppresses STAT1/STAT3 expression and increases SOCS3 expression in modulation of LPS-induced macrophage responses.

Due to the urgent need to create appropriate treatment techniques, which are currently unavailable, LPS-induced sepsis has become a serious concern on a global scale. The primary active component in the pathophysiology of inflammatory diseases such as sepsis is the Gram-negative bacterial lipopolysaccharide (LPS). LPS interacts with cell surface TLR4 in macrophages, causing the formation of reactive oxygen species (ROS), TNF-α, IL-1ß and oxidative stress. It also significantly activates the MAPKs and NF-κB pathway. Excessive production of pro-inflammatory cytokines is one of the primary characteristic features in the onset and progression of inflammation. Cytokines mainly signal through the JAK/STAT pathway. We hypothesize that blocking of TLR4 along with TNFR1 might be beneficial in suppressing the effects of STAT1/STAT3 due to the stimulation of SOCS3 proteins. Prior to the LPS challenge, the macrophages were treated with antibodies against TLR4 and TNFR1 either individually or in combination. On analysis of the macrophage populations by flowcytometry, it was seen that receptor blockade facilitated the phenotypic shift of the M1 macrophages towards M2 resulting in lowered oxidative stress. Blocking of TLR4/TNFR1 upregulated the SOCS3 and mTOR expressions that enabled the transition of inflammatory M1 macrophages towards the anti-inflammatory M2 phenotype, which might be crucial in curbing the inflammatory responses. Also the reduction in the production of inflammatory cytokines such as IL-6, IL-1ß due to the reduction in the activation of the STAT1 and STAT3 molecules was observed in our combination treatment group. All these results indicated that neutralization of both TLR4 and TNFR1 might provide new insights in establishing an alternative therapeutic strategy for LPS-sepsis.

Verapamil and tangeretin enhances the M1 macrophages to M2 type in lipopolysaccharide-treated mice and inhibits the P-glycoprotein expression by downregulating STAT1/STAT3 and upregulating SOCS3.

LPS induced sepsis is a complex process involving various immune cells and signaling molecules. Dysregulation of macrophage polarization and ROS production contributed to the pathogenesis of sepsis. PGP is a transmembrane transporter responsible for the efflux of a number of drugs and also expressed in murine macrophages. Natural products have been shown to decrease inflammation and expression of efflux transporters. However, no treatment is currently available to treat LPS induced sepsis. Verapamil and Tangeretin also reported to attenuate lipopolysaccharide-induced inflammation. However, the effects of verapamil or tangeretin on lipopolysaccharide (LPS)-induced sepsis and its detailed anti-inflammatory mechanism have not been reported. Here, we have determined that verapamil and tangeretin protects against LPS-induced sepsis by suppressing M1 macrophages populations and also through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression in macrophages. An hour before LPS (10 mg/kg) was administered; mice were given intraperitoneal injections of either verapamil (5 mg/kg) or tangeretin (5 mg/kg). The peritoneal macrophages from different experimental groups of mice were isolated. Hepatic, pulmonary and splenic morphometric analyses revealed that verapamil and tangeretin decreased the infiltration of neutrophils into the tissues. Verapamil and tangeritin also enhanced the activity of SOD, CAT, GRX and GSH level in all the tissues tested. verapamil or tangeretin pre-treated mice shifted M1 macrophages to M2 type possibly through the inhibition of P-glycoprotein expression via downregulating STAT1/STAT3 and upregulating SOCS3 expression. Hence, both these drugs have shown protective effects in sepsis via suppressing iNOS, COX-2, oxidative stress and NF-κB signaling in macrophages. Therefore, in our study we can summarize that mice were treated with either Vera or Tan before LPS administration cause an elevated IL-10 by the macrophages which enhances the SOCS3 expression, and thereby able to limits STAT1/STAT3 inter-conversion in the macrophages. As a result, NF-κB activity is also getting down regulated and ultimately mitigating the adverse effect of inflammation caused by LPS in resident macrophages. Whether verapamil or tangeretin offers such protection possibly through the inhibition of P-glycoprotein expression in macrophages needs clarification with the bio availability of these drugs under PGP inhibited conditions is a limitation of this study.

Vitexin along with verapamil downregulates efflux pump P-glycoprotein in macrophages and potentiate M1 to M2 switching via TLR4-NF-κB-TNFR2 pathway in lipopolysaccharide treated mice.

The lipopolysaccharide, a microbial toxin, is one of the major causative agents of sepsis. P-gp expression and its functions are altered during inflammation. LPS has been known to impair the functions of P-gp, an efflux transporter. But the effect of LPS on P-gp expression in murine peritoneal macrophages is poorly understood. Molecular docking studies reveal that vitexin is a potent substrate and verapamil a potent inhibitor of P-gp. In the present experimental study, the curative potential of vitexin as a fruit component and verapamil treated as a control inhibitor of P-gp was examined in a murine LPS sepsis model. The effects of vitexin and verapamil on P-gp expression in macrophages correlating with changes in macrophage polarization and associated functional responses during LPS induced sepsis were studied. Peritoneal macrophages of LPS (10 mg/kg body weight) challenged mice exhibited elevated levels of H2O2, superoxide, and NO in parallel with lower antioxidant activity. LPS treatment increased P-gp expression through increased TLR4/expression. However, LPS challenged mice treated with vitexin (5 mg/kg body weight) + verapamil (5 mg/kg body weight) showed higher anti-oxidant enzyme activity (SOD, CAT and GRx) resulting in reduced oxidative stress. This combination treatment also elevated TNFR2, concomitant with down-regulation of TLR4, NF-κB and P-gp expression in murine peritoneal macrophages, resulting in a switch from M1 to M2 polarisation of macrophages and reduced inflammatory responses. In conclusion, combined vitexin and verapamil treatment could be used as a promising therapy to regulate P-gp expression and protection against LPS mediated sepsis and inflammatory damages.

Endogenous blocking of TLR2 along with TNF-α and IL-1ß ameliorates the severity of the S. aureus arthritis via modulating STAT3/SOCS3 expressions in tissue resident macrophages.

In vivo studies identifying a role of TLR2 in septic arthritis models are lacking. TNF-α played as the most important proinflammatory cytokine, and connected directly to the pathogenesis of bacterial arthritis. IL-1ß is another central mediator cytokine in arthritis. It is therefore reasonable to question the role of neutralization of endogenous TNF-α and IL-1ß along with TLR2 and associated downstream signaling as crucial mediators in the S. aureus -induced inflammatory arthritis. In reaction to an injury or a pathogen encounter, innate immune cells serve as the initial line of defense. TLR2 mediated entry of S. aureus into macrophage cells initiates an array of inflammatory cascades. After macrophage cell gets activated at the site inflammation, they generate elevated number of cytokines which includes TNF-α, IL-1ß. This cytokines signals through STAT1/STAT3 mediated pathways. Thus, aim of this study was to discover how This bone damage could be altered by altering the STAT/STAT3/SOCS3 ratio by blocking TLR2, a particular S. aureus binding site, in conjunction with the use of IL-1 and TNF- antibodies for neutralizing endogenous IL-1ß and TNF-α. Additionally, the role of local macrophages in therapy of arthritis was investigated in synovial and Splenic tissue. To comprehend the inflammatory milieu within the system, ROS and other antioxidant enzymes, along with the expression of mTOR in macrophage cells, were also taken into consideration. The detrimental impact of bacterial burden on synovial joints was reduced by simultaneously inhibiting TLR2, TNF-α, and IL-1ß. Lowered IFN-γ decreases its sensitivity to STAT1 and lowered IL-6 reduces STAT3 expressions. Whereas, elevated IL-10 enhances SOSC3 expression, which thereby able to limits STAT1/STAT3 inter-conversion. As a result, NF-κB activity was downregulated.

Impact of dual neutralization of TNF-α and IL-1ß along with Gentamicin treatment on the functions of blood and splenic neutrophils and its role on improvement of S. aureus induced septic arthritis.

Researches of recent past years have emphasized potential of antibiotics to improve septic arthritis but as multi-drug resistant strains like MRSA are emerging fast, new alternative therapeutic advances are high in demand. This study aims to figure out the role of neutrophils in regulating inflammatory responses of S. aureus induced septic arthritis while using TNF-α Ab or IL-1ß Ab along with antibiotic gentamicin or both in combination. In this study, role of anti-oxidant enzymes were investigated and correlated with generated ROS level. While expression of TLR2, TNFR2, MMP2, RANKL, SAPK/JNK in the spleen were evaluated through western blot. Serum activity of IL-8, IL-10, IL-12, OPG, OPN, CRP was assessed using ELISA. Flow cytometry study evaluated inflamed neutrophil population. Results have shown TNF-α neutralization along with gentamicin was able to reduce arthritic swelling prominently. While combination therapy effectively reduced blood neutrophil ROS activity, arginase activity, MPO activity along with spleen bacterial burden. Serum OPG, CRP, IL-10 level got reduced while serum OPN, IL-8 and IL-12 level enhanced in treatment groups, showing mitigation of inflammatory damage. Overall, it is a novel work that observed how antibiotic and antibody therapy enhanced neutrophil function positively to combat sepsis. This study may not be fully applicable in clinical trials as it is performed with animal model. Clinical trials include crystalline and inflammatory arthritides, trauma, neoplasm. Interdisciplinary collaboration between radiology, orthopaedic surgery and knowledge of animal system responses may give better idea to find proper therapeutic approach in future research works.

Exogenous IL-10 posttreatment along with TLR4 and TNFR1 blockade improves tissue antioxidant status by modulating sepsis-induced macrophage polarization.

Multi-organ dysfunction is one of the major reasons behind the high mortality of sepsis throughout the world. With the pathophysiology of sepsis remaining largely unknown, the uncontrolled reactive oxygen species (ROS) production along with the decreased antioxidants contributes to the progression toward septic shock. Being the effector cells of the innate immunity system, macrophages secrete both pro-inflammatory and anti-inflammatory mediators during inflammation. Lipopolysaccharide (LPS) binding to toll-like receptor 4 (TLR4) releases TNF-α, which initiates pro-inflammatory events through tumor necrosis factor receptor 1 (TNFR1) signaling. However, it is counteracted by the anti-inflammatory interleukin 10 (IL-10) causing decreased oxidative stress. Our study thus aimed to assess the effects of exogenous IL-10 treatment post-neutralization of TLR4 and TNFR1 (by anti-TLR4 antibody and anti-TNFR1 antibody, respectively) in an in vivo murine model of LPS-sepsis. We have also examined the tissue-specific antioxidant status in the spleen, liver, and lungs along with the serum cytokine levels in adult male Swiss albino mice to determine the functional association with the disease. The results showed that administration of recombinant IL-10 post-neutralization of the receptors was beneficial in shifting the macrophage polarization to the anti-inflammatory M2 phenotype. IL-10 treatment significantly downregulated the free radicals production resulting in diminished lipid peroxidase (LPO) levels. The increased antioxidant activities of superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GRX ) conferred protection against LPS-induced sepsis. Western blot data further confirmed diminished expressions of TLR4 and TNFR1 along with suppressed stress-activated protein kinases/Jun amino-terminal kinases (SAPK/JNK) and increased SOD and CAT expressions, which altogether indicated that neutralization of TLR4 and TNFR1 along with IL-10 posttreatment might be a potential therapeutic measure for the treatment of sepsis.

IL-10 in combination with IL-12 and TNF-α attenuates CXCL8/CXCR1 axis in peritoneal macrophages of mice infected with Staphylococcus aureus through the TNFR1-IL-1R-NF-κB pathway.

Overexpression of Staphylococcus aureus mediated CXCL8/CXCR1 axis is a major cause of sepsis and severe inflammatory diseases. This chemokine acts conjointly with various pro-inflammatory and anti-inflammatory cytokines that govern the severity of inflammation. The effects of different combinations of exogenous cytokines on CXCR1 expression in macrophages remain undetermined. Exogenous cytokine and anti-inflammatory cytokine therapy had been used to modulate CXCL8 and CXCR1 expression in peritoneal macrophages. Male Swiss albino mice were inoculated with live S. aureus (106 cells/ mouse) for the development of infection. Exogenous cytokines (TNF-α, IL-12, IFN-γ and IL-10) were administered intraperitoneally (single or combination) 24 h post S. aureus infection. The mice were sacrificed and peritoneal macrophages were isolated three days post infection. CXCL8, IL-12, IL-10 secretion, ROS generation and the bacterial phagocytic process had been evaluated. Western blot was used to study the expressions of TNFR1, IL-1R, CXCR1 and NF-κB. TNF-α, IL-12 and IFN-γ treatments aggravated CXCL8 and CXCR1 expression in the macrophages of infected mice. TNF-α + IFN-γ treatment was a major inducer of nitric oxide release and mediated maximum bacterial killing. IL-12 + TNF-α treatment was most potent in increasing ROS, CXCL8/CXCR1 expression through increased levels of TNFR1, IL-1R and NF-κB activation. IL-10 reversed the effects of exogenous cytokines but also impaired the bacterial clearance phenomenon in peritoneal lavage. Treatment with IL-12 + TNF-α + IL-10 was most effective in ameliorating oxidative stress, reduced CXCL8 release and expression levels of TNFR1, IL-1R, and NF-κB. Concludingly, IL-12 + TNF-α + IL-10 treatment mitigated CXCL8/CXCR1 expression and inflammatory signalling via downregulation of TNFR1-IL-1R-NF-κB pathway in peritoneal macrophages and inflammatory sequelae during S. aureus infection.

Synovial macrophages of rheumatoid arthritic mice protectively responded by altered M1/M2 differentiation after antibody blocking of TNFR1 and IL-1R.

Rheumatoid arthritis (RA) primarily affecting the synovial tissue, has emerged as a major concern leading to the pressing need to develop effective treatment strategies. In the affected synovial tissue, resident macrophages play a pivotal role in the pathogenesis of RA. TNF-α and IL-1ß released from pro-inflammatory M1 synovial macrophages are the master regulators of chronic joint inflammation. In this study collagen-induced rheumatoid arthritis model was developed in mice and post isolation, macrophages were subjected to administration with neutralizing antibodies IL1R and TNFR1 either alone or in combination. Flow cytometric analysis followed by Western blots, ROS, and IL-1ß, TNF-α release assays were performed. Outcomes suggested that post-dual blockade of IL1R and TNFR1 arthritic synovial macrophages showed a shifting of the M1 towards the anti-inflammatory M2 phenotype. Moreover, the switch towards the M2 phenotype might be responsible for decreased levels of IL-1ß,TNF-α, and ROS and simultaneous elevation in the activity of antioxidant enzymes like SOD, CAT, and GPX content in the isolated macrophages. Simultaneous blocking of both IL1R and TNFR1 also showed a sharp reduction in the expression of NF-κB and SAPK-JNK. The elevated arginase and GRX activity further confirmed the polarization towards M2. Moreover, bioinformatics analysis was performed,and it was found that blocking TNFR1 with an antibody could hamper the binding of TNF to TNFR1 in the TNF-TNFR1 pathway. Thus, it may be inferred that dual blockade of IL1R and TNFR1 and a suitable antibody blocking of TNFR1 might be alternative therapeutic approaches for the regulation of RA-induced inflammation in the future.

Simultaneous neutralization of TGF-ß and IL-6 attenuates Staphylococcus aureus-induced arthritic inflammation through differential modulation of splenic and synovial macrophages.

Septic arthritis is a joint disease caused by Staphylococcus aureus. Different macrophage populations contribute in various ways to control blood-borne infections and induce inflammatory responses. Macrophage tissue-resident niche is necessary for the suppression of chronic inflammation and may contribute to the pathogenesis of septic arthritis. Thus, to obtain a resolution of the disease and restoration of synovial homeostasis, it needs the activation of macrophages that further regulate the inflammatory consequences. The aim of this study was to find out the mechanism by which neutralization of transforming growth factor-beta (TGF-ß) and/or interleukin (IL)-6 after induction of septic arthritis could alter the specific macrophage responses in spleen and synovial joints via different cytokines (osteoprotegerin (OPG), osteopontin (OPN), IL-10, IL-12 and CXCL8) cross-talking, and how the response could be modulated by reactive oxygen species vs antioxidant enzyme activities. Dual neutralization of TGF-ß and IL-6 is notably effective in eliciting splenic and synovial tissue-resident macrophage responses. Synovial macrophage-derived IL-10 can elicit protection against septic arthritis via regulating receptor-activated nuclear factor Kappa-B ligand (RANKL)/OPG interaction. They also reduced oxidative stress by increasing the activity of antioxidant enzymes including SOD and catalase. Histopathological analysis revealed that dual neutralization of TGF-ß and IL-6 prevented bone destruction and osteoclastic activity in septic arthritis by promoting the differential functional response of the splenic and synovial macrophages. Additionally, the macrophage-derived IL-10 can elicit protection against S. aureus-induced septic arthritis via regulating RANKL/OPG interaction. Further studies on STAT3 and STAT4 are needed for the understanding of such cross-talking in resident macrophages of arthritic mice.

Endogenous neutralization of TGF-ß and IL-6 ameliorates septic arthritis by altering RANKL/OPG interaction in lymphocytes.

Septic arthritis is an inflammatory joint disease caused by S. aureus. Hematogenous entry of the bacteria to the synovium produces pro-inflammatory cytokines TGF-ß and IL-6, which alter the Th17/Treg balance. Hence, targeting TGF-ß and IL-6 could be beneficial in ameliorating arthritis. Antibody neutralization of TGF-ß and IL-6 to modulate Th17/Treg homeostasis and RANKL/OPG ratio are not investigated so far in S. aureus-induced septic arthritis. Contribution of synovial lymphocyte-derived cytokines IL-10, IL-12, and CXCL-8; along with OPN, OPG, CRP, cellular ROS, antioxidant enzymes, and the expressions of RANKL, SAPK-JNK, MMP2, SOD, CAT, GPx, TGF-ß and IL-6 were studied in lymphocytes of blood, spleen and synovial tissues of mice treated with antibody against of TGF-ß and IL-6 after induction of septic arthritis. Dual neutralization of TGF-ß and IL-6 is effective in shifting the Th17 cell into immunosuppressive Treg cell of the arthritic mice and enhances the RANKL/OPG interaction leading to the down-regulation of osteoclastic activity and reduces the production of OPN, IL-12, CXCL-8, and CRP. Additionally, it reduces oxidative stress via enhancing the activities of antioxidant enzymes including SOD, catalase, and GPx in lymphocytes. Thus it can be concluded that dual endogenous neutralization of TGF-ß and IL-6 may be chosen as an alternative therapeutic approach for controlling the severity of septic arthritis through Treg-derived IL-10 that could ameliorate the inflammatory consequences of septic arthritis via influencing RANKL/OPG interaction in lymphocytes.

Neutralization of IL-17 and treatment with IL-2 protects septic arthritis by regulating free radical production and antioxidant enzymes in Th17 and Tregs: An immunomodulatory TLR2 versus TNFR response.

Septic arthritis is a destructive joint disease caused by Staphylococcus aureus. Synovial inflammation involved Th17 proliferation and down regulation of Treg population, thus resolution of inflammation targeting IL-17 may be important to control arthritis. Endogenous inhibition of IL-17 to regulate arthritic inflammation correlating with Th17/Treg cells TLR2 and TNFRs are not done. The role of SOD, CAT and GRx in relation to ROS production during arthritis along with expression of TLR2, TNFR1/TNFR2 in Th17/Treg cells of mice treated with IL-17A Ab/ IL-2 were studied. Increased ROS, reduced antioxidant enzyme activity was found in Th17 cells of SA infected mice whereas Treg cells of IL-17A Ab/ IL-2 treated group showed opposite effects. Neutralization of IL-17 after arthritis cause decreased TNFR1 and increased TNFR2 expression in Treg cells. Thus, neutralization of IL-17 or IL-2 treatment regulates septic arthritis by enhancing anti-inflammatory properties of Treg via antioxidant balance and modulating TLR2/TNFR response.

TLR4 and TNFR1 blockade dampen M1 macrophage activation and shifts them towards an M2 phenotype.

The Gram-negative bacterial lipopolysaccharide (LPS)-induced sepsis has emerged as major concern worldwide due to the pressing need to develop its effective treatment strategies which is not available yet. LPS is the major causative agent in the pathogenesis of septic shock. In macrophages, LPS interacts with cell surface TLR4 leading to reactive oxygen species (ROS), TNF-α, IL-1ß production, oxidative stress and markedly activated the MAPKs and NF-kB pathway. Post cell isolation, the macrophages were subjected to administration with neutralizing antibodies to TLR4 and TNFR1 either alone or in combination prior to LPS challenge. Subsequently, we performed flow cytometric analysis along with Western blots, reactive oxygen species production, and TNF-α, IL-1ß release. Outcomes suggested that the dual blockade of TLR4 and TNFR1 was indeed beneficial in shifting the LPS-induced M1 polarization towards M2. Both TLR4 and TNFR1 exhibited dependency during LPS stimulation. Furthermore, the switch towards the M2 phenotype might be responsible for the decreased levels of TNF-α, IL-1ß, NO, and superoxide anion and the simultaneous elevation in the activity level of anti-oxidant enzymes like SOD, CAT (catalase), and GSH content in the isolated peritoneal macrophages. Simultaneous blocking of both TLR4 and TNFR1 also showed reduced expression of NF-kB, JNK, and COX-2 by promoting TNFR2-mediated TNF-α signaling. The increased arginase activity further confirmed the polarization towards M2. Thus it may be inferred that dual blockade of TLR4 and TNFR1 might be an alternative therapeutic approach for regulating of sepsis in future.

Neutralization of TNF-α and IL-1ß Regulates CXCL8 Production through CXCL8/CXCR1 Axis in Macrophages during Staphylococcus aureus Infection.

Anti-cytokine therapy is widely acknowledged as an anti-inflammatory technique to treat varied infectious diseases. TNF-α and IL-1ß are major cytokines that regulate every aspect of the inflammatory process. However, the effects of single or dual cytokine neutralization on S. aureus mediated CXCL8 secretion and CXCR1 expression in murine peritoneal macrophages remained noninvestigated. Thus we aimed to explore the effects of kinetic-dose dependent neutralization of TNF-α and IL-1ß using specific anti-cytokine antibodies and its influential impact on the CXCL8/CXCR1 axis at different stages of S. aureus (30, 60, and 90 min) infection. The murine peritoneal macrophages were isolated and infected with viable S. aureus followed by subsequent addition of anti-TNF-α and anti-IL-1ß into the medium. The treated cells were centrifuged and lysate and supernatant collected for various experiments. The ROS generation was measured and cytokine production was estimated by ELISA. The expression of TNFR1, IL-1R, CXCR1, signaling molecules (NF-κB and JNK) were evaluated by Western blot. The role of single or dual cytokine neutralization on intracellular bacterial phagocytosis had also been analyzed by confocal microscopy. Dual cytokine neutralization significantly suppressed ROS, cytokines, CXCL8 secretion, and intracellular bacterial count compared to single cytokine neutralization and it was more apparent at 90 min post S. aureus infection. There was a drastic reduction in TNFR1, IL-1R, and CXCR1 expression on macrophage surface due to reduced expression of downstream signaling molecules, NF-κB and JNK. Hence dual cytokine neutralization was more effectual compared to single cytokine neutralization in the downregulation of S. aureus induced CXCR1 expression.

Role of different Th17 and Treg downstream signalling pathways in the pathogenesis of Staphylococcus aureus infection induced septic arthritis in mice.

Septic arthritis is a condition of bone disorder caused predominantly by Staphylococcus aureus. Following the bacterial entry activated immune cells specially macrophages and dendritic cells release pro-inflammatory mediators such as IL-6, TNF-α, IL-1ß etc., which not only create an inflammatory microenvironment but also play crucial roles in the proliferation of different CD+ T cell subsets. Among them, Th17 and Tregs are of major concern in recent times because of their potential roles in regulating the ongoing inflammation in many diseases including experimental arthritis. But the downstream signalling mechanism of these cells in regulating the severity of inflammation in case of septic arthritis is not known yet. So, here we have established a murine model of S. aureus induced septic arthritis and kept the animal upto 15 days post-infection. To examine the signalling mechanism, Th17 and Treg cells were isolated from blood, spleen and synovial joints of control and infected mice and observed the expression of JNK, NFκB and RANKL in the lysate of isolated Th17 and Tregs. We have also estimated the levels of serum IL-21 and TGF-ß. NFκB, JNK and RANKL expression was found to be higher at 3 and 15 days post-infection along with serum IL-21 levels. On the other hand, maximum TGF-ß level was observed at 9 days post-infection along with increased Treg population. In conclusion it was hypothesized that bone resorption is related with downstream signalling pathways of Th17 cells, which stimulate osteoclast generation via NFκB/JNK-RANKL axis and helps in the persistence of the disease.

Etoposide-mediated depletion of peripheral blood monocytes post s.aureus infection attenuates septic arthritis by modulating macrophage-derived factors responsible for inflammatory destruction.

S.aureus induced septic arthritis remains a serious medical concern due to its rapidly progressive disease profile. The multidrug resistant nature of S.aureus demands the development of new strategies for the treatment of S.aureus arthritis. Since monocyte/macrophage population has been recognized as an important axis in joint inflammation and destruction, selective depletion of peripheral blood monocytes might influence the outcome and progression of the disease. Therefore, in this study we have put forward the concept of monocyte depletion by using etoposide, a drug that selectively depletes the monocyte/macrophage population. Mice were inoculated with live S.aureus for the development of septic arthritis. Post S.aureus infection, etoposide was subcutaneously injected. The severity of arthritis was found to be significantly low in the etoposide treated mice throughout the course. Arthritis index, histopathological analysis and TRAP staining images confirmed effectiveness of etoposide treatment in regulating inflammation and bone cartilage destruction. Lower levels of inflammatory cytokines, ROS, MMP-2, RANKL, OPN and plasmin reflected less severe arthritic destruction after etoposide treatment in arthritic mice. The bacterial load was not increased after etoposide treatment. Together, the presented data suggested that monocyte depletion by etoposide might represent an alternative therapeutic strategy for the treatment of S.aureus arthritis.

Potential anti-arthritic and anti-inflammatory effects of TNF-α processing inhibitor-1 (TAPI-1): A new approach to the treatment of S. aureus arthritis.

Treatment of septic arthritis has become more challenging due to the rise of multidrug resistant strains of Staphylococcus aureus (S. aureus) in recent years. Failure of antibiotic therapies has compelled to initiate the search for new alternatives. This study aimed to unveil the potential anti-arthritic effects of TAPI-1 (TNF-α processing inhibitor-1), an inhibitor that inhibits TACE (TNF-α converting enzyme) mediated release of soluble TNF-α and its receptors along with attenuation of other inflammatory and joint destructive factors responsible for the progression of arthritis. Male Swiss albino mice were inoculated with live S. aureus (5 × 106 cells/mouse) for the development of septic arthritis. TAPI-1 was administered intraperitoneally (10 mg/kg body weight) post S. aureus infection at regular intervals. Throughout the experiment, the severity of arthritis was obtained to be significantly low after TAPI-1 administration. Arthritis index and histopathology confirmed effectiveness of TAPI-1 in mitigating inflammation induced paw swelling and less bone-cartilage destruction in the arthritic knee joints. Lower levels of soluble tumor necrosis factor alpha (sTNF-α) and soluble tumor necrosis factor alpha receptor-1 (sTNFR-1) were detected in the TAPI-1 treated group suggesting TAPI-1 mediated blocking of TACE with subsequent inhibition of TNF-α signalling. Treatment with TAPI-1 lowered the levels of reactive species; matrix metalloproteinase-2 (MMP-2), receptor activator of nuclear factor kappa-B ligand (RANKL) and osteopontin (OPN) denoting less matrix degradation and less osteoclastic bone resorption. Together, this experimental work authenticates TAPI-1 as an alternative therapeutic intervention for the treatment of S. aureus arthritis.

Regulation of Staphylococcus aureus-induced CXCR1 expression via inhibition of receptor mobilization and receptor shedding during dual receptor (TNFR1 and IL-1R) neutralization.

Our earlier studies proposed a radically new idea suggesting interdependency between TNF-α/TNFR1 and IL-1ß/IL-1R pathways in modulation of Staphylococcus aureus-induced CXCL8/CXCR1 axis. However, the effects of inhibition of cytokine receptor mobilization at intracellular level and surface TNFR1 and IL-1R shedding on S. aureus-induced CXCR1 expression have not been studied so far in peritoneal macrophages. This study aimed to investigate the role of inhibition of receptor mobilization from the intracellular pool (using brefeldin A) and surface receptor shedding (using TAPI-1) on CXCR1 expression during dual receptor (TNFR1 plus IL-1R) neutralization in peritoneal macrophages isolated from wild-type Swiss Albino mice. Release of superoxide anion, nitric oxide, and hydrogen peroxide was measured and cytokine production was done by ELISA. Expression of surface receptors (TNFR1, IL-1R, and CXCR1) and inflammatory mediators was studied by Western blot. It was observed that S. aureus-infected macrophages showed elevated ROS production, secretion of TNF-α, IL-1ß, and CXCL8, along with increased expression of surface receptors (TNFR1, IL-1R, and CXCR1), and inflammatory markers (iNOS and COX-2) compared with control or treated groups (p < 0.05). However, prior treatment of macrophages with BFA or TAPI-1 in the presence of anti-TNFR1 antibody and IRAP during S. aureus infection showed significant reduction of all these parameters (p < 0.05). We can conclude that targeting of TNFR1 and IL-1R (with major focus on surface expression study) either through blockage of intracellular receptor trafficking pathway or via surface receptor shedding diminishes TNFR1/IL-1R interaction and consequently downregulates CXCR1 expression along with inflammatory signalling pathways during bacterial infections.

Neutralization of TNFR-1 and TNFR-2 modulates S. aureus induced septic arthritis by regulating the levels of pro inflammatory and anti inflammatory cytokines during the progression of the disease.

Staphylococcal septic arthritis remains a serious medical concern due to rapid and sustained production of inflammatory cytokines that leads to progressive and irreversible joint destruction with high mortality rate in patients despite adequate antibiotics treatment. TNF-α signalling via TNFR-1 contributes to arthritic destruction by aggravating inflammation. Impact of TNFR-2 signalling is not well established in this aspect. Hence the objective of our study was to evaluate the role of dual neutralization TNFR-1 and TNFR-2 in the pathogenesis of S. aureus infection induced septic arthritis. Mice were infected with live S. aureus (5 × 106 cells/ml) followed by administration of TNFR-1and TNFR-2 neutralizing antibody. To measure arthritis index and osteoclastogenesis, histology result in joint tissue and TRAP staining images of arthritis joints have been performed respectively. Maximum reduction in the joint and paw swelling was observed in infected mice treated with both TNFR-1 and TNFR-2 antibody. NF-κB signalling was found to be mainly regulated by TNFR-1 whereas TNFR-2 significantly modulated JNK pathway. Lowest levels of inflammatory cytokines like TNF-α, IL-1ß, IL-6, and IFN-γ were observed in both serum and synovial tissues signifying maximum protection in S. aureus arthritis during combination treatment. However IFN-γ and IL-10 levels were significantly altered by TNFR-2 neutralization that indicates both pro and anti inflammatory role of TNFR-2 respectively. Highest decrement in ROS concentration, iNOS expression with least MPO and lysozyme activity was detected in case of combined neutralization. During the early phase of infection all the aforesaid inflammatory parameters remained elevated due to lack of IL-10 as a result of TNFR-2 neutralization as IL-10 negatively modulates pro inflammatory cytokines. Increase in inflammatory cytokines during early phase might also be responsible for decreased bacterial count in TNFR-2 neutralized groups. Thus it can be suggested that combined administration of TNFR-1 and TNFR-2 antibody has a beneficial effect against the severity of S. aureus induced arthritis.

Dual neutralization of TNFR-2 and MMP-2 regulates the severity of S. aureus induced septic arthritis correlating alteration in the level of interferon gamma and interleukin-10 in terms of TNFR2 blocking.

Severity of S. aureus septic arthritis is correlated to prolonged inflammation by inflammatory cytokines like TNF-α, IL-1ß, and IL-6 even after successful elimination of bacteria. Role of TNF-α via TNFR2 is not well established in this aspect. IFN-γ induces TNF-α release from the macrophages augmenting the inflammatory arthritis. IL-10 modulates the levels of pro-inflammatory cytokines promoting resolution of inflammation. TNF-α-TNFR2 signaling upregulates both of these cytokines. Higher level of MMP-2 induction by inflammatory cytokines during arthritis promotes tissue destruction. Whether dual neutralization of TNFR-2 and MMP-2 regulates the severity of S. aureus arthritis by modulating local and systemic cytokine milieu mainly due to TNFR-2 blocking was an obvious question. Here, we attempted the effects of neutralization of MMP-2 and TNFR2 on S. aureus arthritis and its impact on pro-inflammatory cytokines and some other parameters related to tissue destruction. Reduction in arthritis index was noticed in infected mice treated with both MMP-2 inhibitor and TNFR2 antibody. Lowest levels of inflammatory cytokines, iNOS, RANKL, NF-κb, JNK kinase, ROS, and MPO, and lysozyme activity were observed in combined neutralization group at 9 and 15 dpi, but at 3 dpi, most of the above parameters remained elevated due to TNFR2 neutralization. Diminished IL-10 and IFN-γ levels as a result of TNFR2 neutralization at early and later phase of infection respectively might be responsible for these contrasting effects. Overall, it can be suggested that administration of MMP-2 inhibitor and TNFR2 antibody in combination is protective against the inflammation and tissue destruction associated with S. aureus infection during the arthritic episode.

Clitoria ternatea flower petals: Effect on TNFR1 neutralization via downregulation of synovial matrix metalloproteases.

ETHNOPHARMACOLOGICAL RELEVANCE: Clitoria ternatea Linn. (C. ternatea) is a traditionally used herb in arthritis, and its anti-arthritic activity has been attributed to polyphenols (e.g. quercetins) from its flower petal. AIM OF THE STUDY: The present study was designed to investigate whether C. ternatea or quercetin-3ß-D-glucoside (QG) support the antibody mediated TNFα-receptor 1 (TNFR1) neutralization to ameliorate arthritis in mice. MATERIALS AND METHODS: Development of collagen-induced arthritis (CIA) in male Swiss mice (20-22g, 3-4 weeks of age) was followed by estimation of synovial polymorphonuclear cell (PMN) accumulation (in terms of myeloperoxidase activity), synovial and systemic release of cytokines, chemokines and C-reactive protein (CRP) by enzyme-linked immunosorbent assay (ELISA), biochemical estimation of synovial free radical generation and antioxidant status, as well as immunoblot assessment of synovial TNFR1, toll-like receptor 2(TLR2), cyclooxygenase-2(COX-2) and inducible nitric oxide synthase (iNOS) expression; and zymographic analysis of synovial matrix-metalloprotease-2 (MMP-2) activity. RESULTS: CIA was induced from day 2 post-secondary immunizations as evidenced from arthritic scores and joint swelling in parallel to increased inflammatory and oxidative stress parameters in synovial joints. Long term supplementation with extract from Clitoria ternatea flower petals CTE (50mg/kg) and QG (2.5mg/kg) upto 24 days post booster immunization augmented anti-arthritic potential of TNFR1 neutralization with anti-TNFR1 antibody (10µg per mice) in terms of reduced MPO activity, decrease in release of pro-inflammatory cytokines, chemokines, reactive oxygen species (ROS)/ reactive nitrogen species (RNS) production in parallel to significant (p<0.05) reduction in TNFR1, TLR2, iNOS, COX-2 and MMP-2 expression. CONCLUSION: CTE and QG possess potential anti-arthritic activity which targets synovial MMP-2 in arthritic joints and TNFR1 targeting followed by CTE or QG treatment might become a combinatorial approach in future therapeutic research in treatment of arthritis.