Open Access

Background Natural killer (NK) cells play a key role in eliminating tumorigenic and pathogen-infected cells. Verbena officinalis (V. officinalis) has been used as a medical plant in traditional and modern medicine, exhibiting anti-tumor and anti-inflammation activity.Purpose The impact of bioactive constituents of V. officinalis on immune responses still remains largely elusive. In this work we investigated the potential targets of V. officinalis and focused on killing efficiency and related functions of NK cells regulated by bioactive constituents of V. officinalis.Study design/methods We used primary human NK cells from peripheral blood mononuclear cells. Potential regulatory roles of selected compounds were analyzed by network pharmacology approaches. Killing efficiency was determined with real-time killing assay and live-cell imaging in 3D. Proliferation was examined by CFSE staining. Expression of cytotoxic proteins was analyzed using flow cytometry. Lytic granule release was quantified by CD107a degranulation assay. Contact time required for killing and determination of serial killers were analyzed using live cell imaging results. Results: Using network pharmacology approaches, we analyzed potential regulatory roles of five compounds (Acteoside, Apigenin, Kaempferol, Verbenalin and Hastatoside) from V. officinalis on immune cell functions and revealed NK cells as a major target. The effect of these compounds on NK killing efficiency was examined with real-time killing assay, and Verbenalin enhanced NK killing efficiency significantly. Further investigation showed that Verbenalin did not affect proliferation, expression of cytotoxic proteins, or lytic granule degranulation, but rather reduced contact time required for killing therefore enhancing total killing events per NK cell, suggestively via inhibition of inhibitory receptors as determined by docking assay.Conclusions Our findings reveal the underlying mechanisms how V. officinalis regulates functions of immune cells, especially NK cells, suggesting Verbenalin from V. officinalis as a promising therapeutic reagent to fight cancer and infection.Competing Interest StatementThe authors have declared no competing interest.

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.

Our work presents a high-throughput kinetic killing assay in the 3D matrix using high-content imaging that is a robust and powerful cytotoxicity assay for evaluating the killing efficiency of immune killer cells or conducting drug screening under physiologically and pathologically relevant scenarios, particularly in the context of solid tumors.

Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three-dimensional (3D) environments is critical for their functions under physiological conditions. Recent studies have shown that NK cell activation is regulated by substrate stiffness. However, the role of mechanosensing in regulating NK cell killing efficiency in physiologically relevant scenarios is poorly understood. In this study, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK cell killing efficiency in 3D is impaired against softened tumor cells, while it is enhanced against stiffened tumor cells. Notably, the durations required for NK cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel in NK cells. Perturbation of PIEZO1 by GsMTx4 abolishes stiffness-dependent NK cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1-mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK cell killing efficiency under physiological conditions and the influence of environmental physical cues on NK cell functions.

NK cells are one of the major immune killer cell types exhibiting anti-tumor activity. During immune surveillance NK cells infiltrate into tissues and come in contact with cells and organs with varied stiffness. It has been shown that tumor cells with a lower elasticity modulus than their counterparts to have a higher metastatic potential. Whether the change in tumor cell stiffness affects the functionality of NK cells is not well understood. In this work, to test the effect of substrate stiffness on NK responses, PAAm-co-AA hydrogels of varied stiffness (2 kPa,12 kPa, 50 kPa) functionalized with biotinylated NKp46 activating antibody, prepared by Dr. Jingnan Zhang (research group of Prof. del Campo, INM-Leibniz Institute for New Materials) were used. Stiffness of substrate indeed played a huge role in modulating NK responses with stiffer substrates (12 kPa, 50 kPa) eliciting a stronger response in most donors whereas the soft substrates (2 kPa) failed to do so. To further test the impact of target cell stiffness on NK cytolytic activity, stiffness of target cells was altered using blebbistatin (made cells stiffer) and DMSO (made cells softer). Cytotoxicity of NK cells was boosted against stiffened tumor cells and impaired against softened tumor cells. In addition, the time required for NK cell to detach from the stiffened target cell after apoptosis or necrosis of the latter was significantly shorter, also contributing to a more effective cytotoxicity. To further decipher the role of mechanosensing in killing processes, functions of mechanosensitive ion channels was blocked using unspecific antagonizers (gadolinium and nifedipine), and it was found that blockage of mechanosensing substantially impaired NK mediated cytotoxicity as determined by 2D and 3D killing assays. Regarding the responsible mechanosensor, we have identified from the microarray data of our lab (by Dr. Eva Schwarz) that PIEZO1 are the predominately expressed mechanosensitive ion channels in NK cells. Blockade of PIEZO1 in NK cells by GsMTx4 impaired NK mediated cytotoxicity and activation of PIEZO1, using its specific agonist Yoda-1, potentiated NK mediated killing of the target cells. Blockade of PIEZO1 was shown to decrease the infiltration of NK cells into 3D collagen matrices, and activation of PIEZO1 boosted the infiltration of NK cells into 3D collagen matrix. As the role of PIEZO1 to be a major mechanosensor in NK cells was established, its role in sensing the stiffness of substrates was explored. Perturbation of PIEZO1 channels abrogated NK responses to substrate stiffness. Together, these data emphasize the role of mechanosensing in regulating NK cytotoxicity and the central role of tumor cell stiffness in evading immune surveillance. To fight cancer and other infective diseases, living therapeutic materials (LTMs) offer possibilities to release therapeutics in a sustainable and tunable manner. LTMs contain genetically engineered biological component encapsulated in a polymeric material such as hydrogels to contain their exposure in the host. LTMs are being extensively researched for their use in treatment of cancer with many studies reinforcing the beneficial effects of using smart materials. To contain the exposure of living component such as bacteria and to protect it from adverse environmental conditions of the host and to avoid a direct contact with immune cells, they are often encapsulated. However, one major concern for LTMs is that they may trigger an immune response and create a pro-inflammatory milieu in the host which could lead to critical situations if unregulated. So, the second part of my thesis is to characterize the immune response of PBMCs to PluDA hydrogel encapsulated E.coli and ClearColi bacteria. This work was carried out in collaboration with the group of Dr. Shrikrishnan Sankaran, Bioprogramable materials, INM-Leibniz Institute for New Materials, Saarbrücken. The ClearColi strain was produced from E.coli after genetically deleting LPS. ClearColi was encapsulated in Pluronic F127-based hydrogels (PluDA). It has to be noted that the bacteria were not in direct contact with the host so any immune reaction elicited would be due to the release of soluble factors and metabolites. The release of pro-inflammatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, TNFα and IFNγ) and cytotoxic proteins (granzyme A, granzyme B, perforin, granulysin, sFas, and sFasL) by PBMCs in response to bacteria and bacteria encapsulated gels was checked along with its influence on immune killer cells’ subtypes. Interestingly, PBMCs from the blood donors could be grouped in to two groups, donors with low spontaneous IL-2 and high spontaneous IL-2 release, based on the IL-2 release when PBMCs were cultured alone. Our results show that co-incubation of PluDA blank gels with PBMCs did not alter their profiles of cytokines and cytotoxic proteins, and had no influence on differentiation of NK cells, CD4+ and CD8+ T cells in donors with low spontaneous release of IL-2. ClearColi elicited release of IL-6 and IFNγ from PBMCs. Interestingly, the predominantly released cytokine was IL-6 for low spontaneous IL-2 release donors, but IFNγ for high spontaneous IL-2 release donors. Both the transwell condition and the encapsulated gel condition showed the same tendency. When the bacteria were in direct contact with the PBMCs they triggered the apoptosis of PBMCs on day 3 but encapsulation of the bacteria in PluDA gels completely abolished this effect.