Open Access

Natural killer (NK) cells play key roles in eliminating pathogen-infected cells. Verbena officinalis (V. officinalis) has been used as a medical plant in traditional and modern medicine for its anti-tumor and anti-inflammatory activities, but its effects on immune responses remain largely elusive. This study aimed to investigate the potential of V. officinalis extract (VO extract) to regulate inflammation and NK cell functions. We examined the effects of VO extract on lung injury in a mouse model of influenza virus infection. We also investigated the impact of five bioactive components of VO extract on NK killing functions using primary human NK cells. Our results showed that oral administration of VO extract reduced lung injury, promoted the maturation and activation of NK cells in the lung, and decreased the levels of inflammatory cytokines (IL-6, TNF-α and IL-1β) in the serum. Among five bioactive components of VO extract, Verbenalin significantly enhanced NK killing efficiency in vitro, as determined by real-time killing assays based on plate-reader or high-content live-cell imaging in 3D using primary human NK cells. Further investigation showed that treatment of Verbenalin accelerated the killing process by reducing the contact time of NK cells with their target cells without affecting NK cell proliferation, expression of cytotoxic proteins, or lytic granule degranulation. Together, our findings suggest that VO extract has a satisfactory anti-inflammatory effect against viral infection in vivo, and regulates the activation, maturation, and killing functions of NK cells. Verbenalin from V. officinalis enhances NK killing efficiency, suggesting its potential as a promising therapeutic to fight viral infection.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC), which is characterized by the total absence of human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER) expression. Cinobufacini injection (CI) is the aqueous extract from the dry skin of Bufo gargarizans, which is broadly used for the treatment of malignant tumors. However, the potential mechanism of CI against TNBC has not been fully revealed. In this study, we found that CI inhibited the proliferation of MDA-MB-231 and 4T1 cells in a time- and dose-dependent manner. RNA-seq data showed that downregulated and upregulated genes were mainly enriched in biological processes related to tumor cell proliferation, including cell cycle arrest and regulation of apoptosis signaling pathways. Indeed, after CI treatment, the protein level of CDK1 and Bcl-2/Bax decreased, indicating that CI induced the cell cycle of MDA-MB-231 arrest in the G2/M phase and increased the rate of apoptosis. Meanwhile, CI significantly inhibited the growth of tumor in vivo, and RNA-seq data showed that the TAZ signaling pathway played a vital role after CI treatment. Both immunohistochemistry and Western blot analysis confirmed the downregulation of Pin1 and TAZ, caused by CI treatment. Furthermore, the bioinformatics analysis indicated that Pin1 and TAZ were indeed elevated in TNBC patients, with poor staging, classification, and patient survival rate. In conclusion, CI effectively inhibited the proliferation of TNBC in vitro and in vivo and induced their apoptosis and cycle arrest through the Pin1-TAZ pathway.

The immune system provides our defense against pathogens and aberrant cells, including tumorigenic and infected cells. Motility is one of the fundamental characteristics that enable immune cells to find invading pathogens, control tissue damage, and eliminate primary developing tumors, even in the absence of external treatments. These processes are termed “immune surveillance.” Migration disorders of immune cells are related to autoimmune diseases, chronic inflammation, and tumor evasion. It is therefore essential to characterize immune cell motility in different physiologically and pathologically relevant scenarios to understand the regulatory mechanisms of functionality of immune responses. This review is focused on immune cell migration, to define the underlying mechanisms and the corresponding investigative approaches. We highlight the challenges that immune cells encounter in vivo, and the microfabrication methods to mimic particular aspects of their microenvironment. We discuss the advantages and disadvantages of the proposed tools, and provide information on how to access them. Furthermore, we summarize the directional cues that regulate individual immune cell migration, and discuss the behavior of immune cells in a complex environment composed of multiple directional cues.

CD4+ T cells are essential players in orchestrating the specific immune response against intracellular pathogens, and in inhibiting tumor development in an early stage. The activation of T cells is triggered by engagement of T cell receptors (TCRs). Here, CD3 and CD28 molecules are key factors, (co)stimulating signaling pathways essential for activation and proliferation of CD4+ T cells. T cell activation induces the formation of a tight mechanical bond between T cell and target cell, the so-called immunological synapse (IS). Due to this, mechanical cell properties, including stiffness, play a significant role in modulating cell functions. In the past, many approaches were made to investigate mechanical properties of immune cells, including micropipette aspiration, microplate-based rheometry, techniques based on deformation during cytometry, or the use of optical tweezers. However, the stiffness of T lymphocytes at a subcellular level at the IS still remains largely elusive.With this protocol, we introduce a method based on atomic force microscopy (AFM), to investigate the local cellular stiffness of T cells on functionalized glass/Polydimethylsiloxan (PDMS) surfaces, which mimicks focal stimulation of target cells inducing IS formation by T cells. By applying the peak force nanomechanical mapping (QNM) technique, cellular surface structures and the local stiffness are determined simultaneously, with a resolution of approximately 60 nm. This protocol can be easily adapted to investigate the mechanical impact of numerous factors influencing IS formation and T cell activation.Graphical abstract: Overview of the experimental workflow.Individual experimental steps are shown on the left, hands on and incubation times for each step are shown right.

In saliva and gingival crevicular fluid (GCF) soluble factors such as cytokines, chemokines and growth factors have shown a great potential serving as biomarkers for early detection and/or diagnosis of oral and systemic diseases. However, GCF and saliva, which one is a better source is still under debate. This study aimed to gain an overview of cytokines, chemokines and growth factors in saliva and GCF to pave the way for selecting suitable oral fluids for oral and systemic diseases. Multiplex cytokine assay was conducted to determine concentrations of cytokines, chemokines and growth factors in saliva and GCF samples from healthy subjects. The protocol for sample collection was carefully optimized. Stabilization, repeatability, and donor variation of the profiles were analyzed. We found that for different donors, cytokine and chemokine profiles showed unique patterns in saliva but similar patterns in GCF. In terms of growth factors, the profiles were individualized in saliva and GCF. All profiles stayed stable for the same healthy individual. In saliva, profiles of cytokines, chemokines and growth factors are individualized for different donors. In GCF, profiles of cytokines and chemokines are similar. Other factors, such as growth factors and T helper-related cytokines, are highly variable in donors. Profiles of soluble factors are not correlated in saliva and GCF. The comprehensive cytokine profiles in saliva and GCF reported in this work would serve as a good base for choosing promising cytokines for developing biomarkers in oral fluids.Competing Interest StatementThe authors have declared no competing interest.