*3.4. Mac-1 and TLR2 Surface Neutrophil Receptors Are Involved in Farnesol-Induced Activation of Netosis*

Many of the neutrophil surface receptors are involved in the recognition of molecules derived from pathogenic microorganisms, which leads to activation of the netosis signaling pathway and the NET release. We selected six receptors known to be able to mediate the release of NETs and checked their role in FOH-induced netosis. The receptors were blocked with specific antibodies, and the neutrophils were stimulated with selected concentrations of FOH for three hours. A decrease in the level of fluorescence of Sytox Green-stained DNA suggests the role of this receptor in the activation of netosis. Among selected receptors, we identified that the inhibition of CD11b and CD18 caused a ca. 2.5-fold decrease in the level of released NETs (Figure 6). This result strongly suggests the important role of these receptors in the recognition of FOH and activation of netosis. Moreover, inhibition of the TLR2 receptor caused a reduction of the level of NETs to about 50% of PMA-treated response, also indicating the participation of this receptor in netosis. A small contribution to the activation of

FOH-induced netosis could also be assigned to CD11a receptor whose blockade caused a 20% decrease in the amount of released DNA. Other receptors tested—CD16 and TLR4—did not seem to be essential for FOH-induced netosis.

**Figure 6.** The participation of selected neutrophil receptors in FOH-activated netosis. Neutrophils (2.2 <sup>×</sup> 105) were preincubated with antibodies (ab, 1 <sup>μ</sup>g/mL) against the selected neutrophil receptors and then netosis was induced for 3 h by 25 nM PMA, 100 μM FOH, and 200 μM FOH. IgG antibody was used as an isotype control. Released NETs were digested with MNase, and collected supernatants were stained with Sytox Green, followed by fluorescence measurements. The results are the means of two replicates ± S.E.M., represented as a percentage relative to the PMA-treated control. ANOVA and Dunnett's post-tests were used. Asterisks denote statistical significance (*p* > 0.1234 ns, \*\* *p* ≤ 0.0021, \*\*\* *p* ≤ 0.0002, \*\*\*\* *p* < 0.0001).

#### *3.5. Farnesol Leads to the Activation of Protein Kinase C and ERK1*/*2*

PKC plays a crucial role in the netosis signal pathway. We checked the level of PKC activation in lysates of neutrophils previously treated with two concentrations of FOH or FA for one hour. After pre-purification of the lysates on DEAE cellulose, the kinase activity was determined based on the phosphorylation of the synthetic peptide, which was then subjected to electrophoretic separation. Figure 7a presents an electrophoretic separation of a synthetic peptide whose amount in the phosphorylated form determines the level of PKC activation. Figure 7b shows quantitative PKC activity in assayed samples corresponding to the densitometrically analyzed bands of the phosphorylated form of the peptide on an electrophoretic gel.

The results showed that PKC activity in neutrophils treated with FOH increased rapidly, reaching a two-fold higher level than in unstimulated cells, comparable to that obtained for chemically induced (PMA) activation of this enzyme. The PKC activity leads to the activation of subsequent netosis mediators. On the other hand, PMNs treated with FA did not show any changes in PKC activity. The lack of PKC activation by FA confirms that neutrophils do not release NETs in the presence of this QSM.

**Figure 7.** Activation of Protein Kinase C (PKC) in QSM–treated neutrophils. Neutrophils (5 <sup>×</sup> 105) were stimulated with selected concentrations of FOH, FA, and 25 nM PMA for 1 h. Then, cell lysate was added to the PKC substrate solution-PepTag® C1 Peptide, and after 30 min separated electrophoretically on an agarose gel. Unstimulated cells were negative cellular control, and PMA-treated cells were positive cellular control. Purified PKC enzyme was used for an assay positive control (control of C1 Peptide phosphorylation), negative control was C1 peptide without any PKC enzyme. (**a**) PKC activation as a percentage of phosphorylated C1 peptide, a substrate for PKC. The values represent the densitometrically-analyzed bands on the electrophoretic gel, assigned as "Active" in (**b**).

The results of the analysis of ERK1/2 kinase activation in neutrophils treated with FOH are shown on Figure 8. The activation of ERK1/2 by 100 μM FOH is two-fold higher than in unstimulated cells, but 200 μM FOH caused three-fold greater phosphorylation of this enzyme. These findings suggest that the activation of the signaling pathway by FOH in neutrophils involving ERK1/2 in a dose-dependent manner.

**Figure 8.** Activation of ERK1/2 in FOH-treated neutrophils. Neutrophil cells (1 <sup>×</sup> 106 cells/well) were suspended in PBS in 12-well microplate and stimulated with FOH at concentrations of 100 μM and 200 μM. Unstimulated cells served as a negative control, and PMA-treated cells were a positive control. After 1 h, the cells were lysed, and the amount of total and phosphorylated ERK1/2 was quantified using ELISA Kit SimpleStep (Abcam). Data represent the mean values of absorbance ± S.E.M. from two replicates as the percentage ratio of phosphorylated ERK1/2 to total ERK1/2. ANOVA and Tukey post-tests were used. Asterisks denote statistical significance (*p* > 0.1234–ns, \* *p* ≤ 0.0332, \*\* *p* ≤ 0.0021).

#### *3.6. The ROS-Dependent Netosis Pathway is Activated by Farnesol*

Stimulation of surface receptors involved in the induction of netosis leads to the activation of selected mediators of the signaling pathway. We checked the role of the five primary mediators involved in ROS-dependent and ROS-independent netosis pathways. For this purpose, selected mediators were blocked in PMNs using specific inhibitors before neutrophil activation with 200 μM FOH. Fluorescence of Sytox Green-stained extracellular DNA was used to determine NET release.

The results (Figure 9) indicate that each of the tested mediators was involved in FOH-induced netosis, but with a relative variable contribution. Syk and Src kinases co-operated with neutrophil surface receptors, and their involvement in netosis activation appeared to be significant. The inhibition of these proteins caused a 50% decrease in the amount of released NETs. In turn, the role of PI3K in FOH-induced netosis seemed to be less important. PI3K probably played a significant role in the ROS-independent netosis signaling pathway. In our experiments, blockade of this kinase caused a decrease in the amount of extracellular DNA by 20% relative to the control.

**Figure 9.** Role of selected signal mediators in activation of netosis by FOH. Neutrophils (2.2 <sup>×</sup> 105) were preincubated with inhibitors of the indicated signaling mediators: Syk—30 μM piceatannol, Src—10 μM PP2, PI3K—25 μM LY29004, ERK1/2—10 μM U0126, NADPH oxidase—5 μM DPI. Cells were then incubated with FOH at 200 μM concentration for 3 h to induce netosis. Neutrophils not treated with inhibitors but stimulated with FOH served as a control. The data are presented as means ± S.E.M. from two replicates and are expressed as a percentage ratio relative to the control. ANOVA and Tukey post-tests were used. Asterisks denote statistical significance (*p* > 0.1234 ns, \* *p* ≤ 0.0332, \*\* *p* ≤ 0.0021, \*\*\* *p* ≤ 0.0002).

ERK1/2 and NADPH oxidase also participate in the activation of the ROS-dependent netosis pathway. The release of NETs depending on ROS production appears to be the primary netosis mechanism involved in neutrophil responses to FOH, as the FOH stimulation of PMNs with blocked ERK1/2 resulted in a 50% reduction of the amount of released NETs. Moreover, NADPH oxidase, which is responsible for the production of ROS in the cells, seems to play the most significant role in the netosis process induced with FOH. Only 20% of DNA was released by neutrophils with the blocked activity of NADPH oxidase compared to the control cells. This result and data showing increased ROS production by neutrophils treated with farnesol confirm that FOH-induced netosis is a ROS-dependent process.

#### *3.7. Farnesol Is a Chemoattractant for Neutrophils*

Additionally, we showed that FOH may be a chemoattractant for PMNs. To support this hypothesis, the neutrophils were placed in chambers to measure chemotactic ability, and then the chambers were transferred to the FOH solutions at the concentration within a range of 50 to 400 μM. As the negative control, PBS was used, whereas the positive control was represented by the responses of neutrophils to 1 μM solution of fMLP. The number of cells that passed through the membrane according to the factor gradient was counted after one hour of treatment. The obtained results (Figure 10) showed that FOH is recognized by neutrophils as a chemoattractant, causing the PMN movement toward the concentration gradient. The number of cells that passed through the membrane was proportional to FOH concentration in the range of 50 to 200 μM, that corresponds to the concentration of FOH detected under in vivo conditions [23]. The higher concentrations of FOH did not cause any further increase in neutrophil migration, but anyway the maximal level of neutrophil chemotaxis upon FOH treatment reached 60% of the response to fMLP, the positive control. These results suggest that neutrophils can migrate to the *Candida* infection sites in response to QSMs released by the yeast.

**Figure 10.** Chemotactic activity of FOH-treated neutrophils. CellTracker Red labeled neutrophil cells (1 <sup>×</sup> 106 cells/well) were placed into chemotaxis chambers being in contact with different concentration of FOH or 1 μM fML (a positive control). Negative control was PBS. After 1 h of incubation, the chambers were removed, and migrated cells were counted. Data represent the mean number of cells ± S.E.M. from three replicates. ANOVA and Dunnett's multiple comparisons post-tests were used. Asterisks denote statistical significance (*p* > 0.1234 ns, \*\*\*\* *p* < 0.0001).

#### **4. Discussion**

Quorum sensing molecules are essential virulence factors of many pathogenic microorganisms-bacteria as well as fungi. The release of QSMs ensures the fast local communication between cells in the infected area, growth synchronization, as well as response to environmental changes of temperature and pH, the presence of biocidal compounds, etc. [34,35].

*C. albicans* yeast mainly use QSMs to regulate growth, change of morphological form and also create biofilms [36]. However, these molecules have an impact on many host cells, like immune cells, such as macrophages, DCs, and neutrophils [20,23].

Our results presented in this paper indicated an important role of neutrophils in the response to QSMs produced by *C. albicans*, which is based on the release of NETs. We showed that farnesol, one of the three compounds identified as *C. albicans* QSMs (FOH, FA and TR) was responsible for activating the netosis, a finding that had not been presented in the literature previously.

The role of FOH released by *C. albicans*is still not fully understood, but it is known that the presence of this compound inhibits the growth of fungal biofilm, and also blocks the change in the morphological transition of fungal cells from blastospores to filamentous forms. Also, high concentrations of FOH can stimulate the yeast to reverse switch from invasive filamentous hyphae back to blastospore [37]. As we presented in the current work, FOH produced at different local concentrations at the site of infection caused the neutrophil response involving NET release. The finding is important for the host defense against *C. albicans* cells because current knowledge has suggested that the morphological form of the fungus determines the mechanism of neutrophil response [28,30]. Some findings indicated that the size of the fungal cells determines the release of NETs, suggesting that the large, filamentous form of *C. albicans* is responsible for activating the netosis [28]. Other studies indicated that the composition of the cell wall of the filamentous form, as well as the released proteolytic enzymes are crucial for the stimulation of neutrophils to NET production [30,38]. Regardless of the identified NET triggers, all studies pointed that *C. albicans* blastospores generate much smaller amounts of NETs, suggesting that this morphological form of *C. albicans* cells can "hide" from neutrophils and avoid the killing. Perhaps, fungi use FOH to inhibit their cell filamentation and progress of infection, just to survive in milieu

infiltrated by neutrophils This situation is dangerous for the host because the 'invisible' to neutrophils intruder cells can survive and under favorable conditions, develop the difficult to treat, secondary infections [39]. Therefore, the ability of neutrophils to recognize FOH, the essential QSM molecule of the fungus, and release NETs in the response to its production at the place of infection, can significantly affect the effectiveness of host defense against *C. albicans* presence. We confirmed the recognition of FOH by neutrophils using chemotaxis analysis, showing that FOH is an efficient chemotactic factor for neutrophils. To date, there is no literature data showing neutrophil migration caused by FOH. However, FOH is known to be a chemoattractant for macrophages [40].

Moreover, given that the microbiocidal activity of NETs covers a certain area of infection, a direct identification of fungal cells by neutrophils is not required, and the presence of FOH may be sufficient.

Studies performed by Leonhardt et al. regarding the effect of FOH on neutrophils did not show any increasing ability of these host cells to phagocyte and kill *C. albicans* cells. However, the viability of *C. albicans* was analyzed after 1 h of contact, while the classic ROS-dependent netosis pathway leads to NET release after about 2–3 h of neutrophil activation [23]. The short time period used in Leonhard's analysis may explain the lack of changes in the fungal cell vitality. The same studies showed an increased FOH concentration-dependent release of elastase, myeloperoxidase, and lactoferrin–granule proteins identified in NETs [23,29]. Thus, our observations of NET release may explain the presence of granule proteins upon neutrophil treatment with FOH in Leonhardt's studies.

Activation of netosis by QSMs has been described in only one case—the *Pseudomonas aeruginosa* bacterium. It was shown that mutations in the quorum-sensing regulatory gene lasR affect the amount and structure of released NETs. In addition, stimulation of neutrophils with purified protein, recombinant endotoxin-free LasA induced NETs in a concentration-dependent manner [41].

The other two analyzed QSMs-FA and TR–did not activate netosis. There are also no literature reports on the impact of these compounds on the functioning of neutrophils. Only TR was identified as a protective agent against phagocytic killing by neutrophils, however, the mechanism of its action is still unknown. One study showed the antioxidant activity of TR [42], which could influence the neutrophil response by blocking ROS production, required for proper functioning of netosis mechanisms. However, other studies showed no antioxidant activity of TR, thus leaving the mechanism of its action on neutrophils unrecognized [18].

The production of extracellular DNA but not apoptosis was demonstrated in this work for neutrophils treated with FOH at the range of concentrations observed locally at the site of *C. albicans* infection. This result is consistent with the observation of Leonhardt et al. [23]. In contrary, we presented that FA is active inducer of neutrophil apoptosis in a dose-dependent manner, the finding that can explain the lack of netosis in neutrophil response to this QSM. These results were confirmed by the cytometric analysis showing the morphological changes of neutrophil cells observed during the FOH-induced netosis process but absent upon treatment with FA.

During the contact of *C. albicans* cells with neutrophils, resulting in NET production, the more common ROS-dependent mechanism of netosis was adopted. The analysis of ROS production by PMNs stimulated with FOH showed that neutrophils release ROS in just a few minutes after activation confirming the ROS-dependent netosis pathway used by neutrophils in response to FOH. In addition, Gilbert et al., identified farnesyl thiotriazole (FTT), a FOH precursor as an activator of NADPH oxidase in neutrophils [43]. However, the production of ROS in response to FA and TR was not observed, confirming a lack of response in the netosis way.

Knowing that FOH is a chemoattractant for neutrophils, and also activates the ROS-dependent netosis pathway, the role of selected surface receptors in activation of netosis by this QSM was analyzed. The results pointed the CD11b/CD18 and TLR2 receptors as being involved in the recognition of FOH and further activation of neutrophils. The CD11b/CD18 receptor engagement in neutrophil chemotaxis and netosis responses to fungal infection was previously demonstrated [30]. Also Leonhardt et al. observed that during stimulation of PMNs by FOH, the level of CD11b receptor on the cell surface increased, while the amount of CD16 receptor decreased [23]. However, Ghosh et al. showed increased

TLR2 and Dectin-1 expression in FOH-treated macrophages [22]. No analogous analysis was performed for neutrophils, however, the effect of FOH on TLR2 may also be significant in PMNs.

Further, the role of known mediators of netosis signaling pathway was verified upon neutrophil treatment with FOH. We showed FOH-induced phosphorylation of PKC, an important signal transducer of netosis. Activation of PKC results in the activation of NADPH oxidase and the production of significant amounts of ROS [44]. This result confirms and explains the role of ROS produced by neutrophils in contact with FOH. There is no literature data on the activation of PKC by FOH in neutrophils; however, in human acute leukemia CEM-C1 cells, the synthesis of diacylglycerol (DAG), a PKC activator, was observed in response to 20 μM FOH [45]. In addition, FTT seems to be also a PKC activator [43].

Inhibition of netosis pathway mediators such as Syk, Src, PI3K, and ERK1/2 caused a decrease in the amount of released NETs. Previously, only the pro-apoptotic activity of FOH was known, leading to reduction of PI3K expression in cells and ERK1/2 in HeLa and DU145 prostate cancer cells [46,47]. In turn, Joo et al. also pointed to the pro-apoptotic effect of FOH on cells, however, by activation of ERK1/2 [48]. However, our results did not show the inhibition of these mediators. In contrary, ERK1/2 kinase activity showed a two-fold increase at the activation of the netosis mechanism during which the apoptosis pathway in the PMNs was blocked.

Our results showed for the first time the role of FOH in the neutrophil recognition of fungal infection at an early stage of microbial invasion. It seems that the regulation of fungal cell morphology by FOH during the progress of fungal infection can also be sensed by the host. Neutrophils, the cells of the first line of host defense can "eavesdrop" the fungal cell communication that uses the quorum sensing molecule and then quickly migrate to the pathogen's habitat and kill or limit its spread by using NETs. The discovery of QSMs that can activate netosis can be of great importance for the developing of effective therapy against early candidiasis.

**Author Contributions:** Conceptualization, M.R.-K. and M.Z.; methodology, M.R.-K. and M.Z.; conceived and designed the experiments, M.R.-K. and M.Z.; all experiments performed by M.Z., except the data shown in Figure 1b, prepared by K.W.; writing—original draft preparation, M.R.-K. and M.Z.; writing—review and editing, M.R.-K. and M.Z.; funding acquisition M.R.-K.

**Funding:** This work was funded by a grant from the National Science Centre of Poland (grant number 2015/17/B/NZ6/02078 to MRK).

**Acknowledgments:** Thanks to Andrzej Kozik for critical discussion during the manuscript preparation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Perspective*
