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.

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.