Toll like receptor 4 (TLR4), a pathogen-associated molecular pattern (PAMP) receptor, is known to exert inflammation in various cases of microbial infection, cancer and autoimmune disorders. However, any such involvement of TLR4 in Chikungunya virus (CHIKV) infection is yet to be explored. Accordingly, the role of TLR4 was investigated towards CHIKV infection and modulation of host immune responses in the current study using mice macrophage cell line RAW264.7, primary macrophage cells of different origins and in vivo mice model. The findings suggest that TLR4 inhibition using TAK-242 (a specific pharmacological inhibitor) reduces viral copy number as well as reduces the CHIKV-E2 protein level significantly using p38 and JNK-MAPK pathways. Moreover, this led to reduced expression of macrophage activation markers like CD14, CD86, MHC-II and pro-inflammatory cytokines (TNF, IL-6, MCP-1) significantly in both the mouse primary macrophages and RAW264.7 cell line, in vitro. Additionally, TAK-242-directed TLR4 inhibition demonstrated a significant reduction of percent E2-positive cells, viral titre and TNF expression in hPBMC-derived macrophages, in vitro. These observations were further validated in TLR4-knockout (KO) RAW cells. Furthermore, the interaction between CHIKV-E2 and TLR4 was demonstrated by immuno-precipitation studies, in vitro and supported by molecular docking analysis, in silico. TLR4-dependent viral entry was further validated by an anti-TLR4 antibody-mediated blocking experiment. It was noticed that TLR4 is necessary for the early events of viral infection, especially during the attachment and entry stages. Interestingly, it was also observed that TLR4 is not involved in the post-entry stages of CHIKV infection in host macrophages. The administration of TAK-242 decreased CHIKV infection significantly by reducing disease manifestations, improving survivability (around 75%) and reducing inflammation in mice model. Collectively, for the first time, this study reports TLR4 as one of the novel receptors to facilitate the attachment and entry of CHIKV in host macrophages, the TLR4-CHIKV-E2 interactions are essential for efficient viral entry and modulation of infection-induced pro-inflammatory responses in host macrophages, which might have translational implication for designing future therapeutics to regulate the CHIKV infection.
Copyright © 2023 Mahish, De, Chatterjee, Ghosh, Keshry, Mukherjee, Khamaru, Tung, Subudhi, Chattopadhyay and Chattopadhyay.

The cell membrane-coated nanoparticles (MNPs) showed great potential in treating infectious disease due to their superior biofunctions in improving biocompatibility of nanoparticles and neutralization of pathogen or toxins. However, bone infection is accompanied with severe inflammation and bone loss, which also requires anti-inflammatory and osteoconductive treatment. The conventional membrane coating method has to undergo ultrasonication and extrusion procedures, which reduces the functionality of cell membrane and limits the choice of nanoparticles. In this study, we proposed an electroporation-based membrane coating strategy to facilitate the synthesis of MNPs to tackle those problems. Methods: Magnetic composite nanoparticles with osteoconductive Ca3(PO4)2 and bactericidal TiO2 were assembled into macrophages through phagocytosis and then collected to expose in electric field for obtaining macrophage membrane-coating nanoparticles. By using molecular dynamics simulation and materials characterizations, the cell membrane coating efficiency was confirmed. The in vitro anti-bacterial and anti-inflammatory abilities were tested by bacteria culturing and immune cells activation. Then drug-resistant bacteria induced bone infection model was established to verify its in vivo therapeutic effects. Results: The coated membrane prepared through electroporation reserved the integrality of membrane structure and right-sidedness, with more functional proteins. Those led to the superior properties of recognition and adsorption with bacteria, toxins and inflammatory cytokines. Owing to the benefits of electroporation, the MNPs exhibited significant better antibacterial and anti-inflammatory abilities for enhancing the tissue repair process. Conclusion: This study provides a novel self-assembly cell membrane coating strategy by electroporation to construct multifunctional membrane-coating nanoparticles for bone infection treatment. This strategy not only improves the functions of coated membrane, but is also proved to be universal for varies nanoparticles or cells, indicating a great potential for future applications in the bioengineering field.
© The author(s).

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB40, binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB40 a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB40 conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB40 (RI-MUB40) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB40-Cy5 using live cell microscopy. Systemically administered RI-MUB40-Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB40 marker will aid the study of broad ranges of inflammatory diseases.
Copyright © 2018 Elsevier Ltd. All rights reserved.

Vaccinia virus (VACV) stimulates long-term immunity against highly pathogenic orthopoxvirus infection of humans (smallpox) and mice (mousepox [ectromelia virus {ECTV}]) despite the lack of a natural host-pathogen relationship with either of these species. Previous research revealed that VACV is able to induce polyfunctional CD8(+) T-cell responses after immunization of humans. However, the degree to which the functional profile of T cells induced by VACV is similar to that generated during natural poxvirus infection remains unknown. In this study, we monitored virus-specific T-cell responses following the dermal infection of C57BL/6 mice with ECTV or VACV. Using polychromatic flow cytometry, we measured levels of degranulation, cytokine expression (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]), and the cytolytic mediator granzyme B. We observed that the functional capacities of T cells induced by VACV and ECTV were of a similar quality in spite of the markedly different replication abilities and pathogenic outcomes of these viruses. In general, a significant fraction (≥50%) of all T-cell responses were positive for at least three functions both during acute infection and into the memory phase. In vivo killing assays revealed that CD8(+) T cells specific for both viruses were equally cytolytic (∼80% target cell lysis after 4 h), consistent with the similar levels of granzyme B and degranulation detected among these cells. Collectively, these data provide a mechanism to explain the ability of VACV to induce protective T-cell responses against pathogenic poxviruses in their natural hosts and provide further support for the use of VACV as a vaccine platform able to induce polyfunctional T cells.