One of the basic conditions of the human body physiological state is the redox balance observed both at the level of cells and tissues, including body fluids such as blood. This is due to the fact that the physiological generation of ROS is balanced by effective enzymatic and non-enzymatic antioxidants [23]. This is conducive to the proper functioning of the human body, which requires strict control of metabolic processes, including those related to the transmission of signals from the environment, e.g. by pathogens. One of the basic elements of the humans body response to the invasion of pathogens is the found increased activity of pro-oxidative enzymes in leukocytes, which play a key role in the immune defense of the host against pathogens [24]. This response is confirmed by previous studies of blood plasma of patients with both TBEV infection and co-infections with bacteria such as B. burgdorferi s.l. and A. phagocytophilum [4]. Enhanced generation of ROS is also conducive to modulation of the signal transduction cascade and strengthening the immune function of lymphocytes in the patient's body [25]. Infection with pathogens is usually accompanied by changes in the effectiveness of endogenous antioxidant mechanisms that are responsible for maintaining the redox balance in physiological conditions [4]. This situation is consistent with the significantly reduced plasma total antioxidant status (TAS) of patients with TBEV infection and bacterial co-infections observed in this study. It is worth emphasizing that after pharmacotherapy the plasma TAS level in patients with TBE and co-infections increases, but still remains statistically significantly lower (in both groups of patients) compared to the values ​​in healthy people. This clearly indicates that standard therapy in the case of TBE, and especially bacterial co-infections, is not able to restore the patient's organism into a physiological state.

The results of previous [4] as well as this study indicate that both TBE alone and together with bacterial co-infections promote the formation of oxidative stress in the patient's body, and a similar situation applies to many diseases, including those caused by both viruses and bacteria [1]. Although oxidative stress plays a dual role in infectious diseases, ROS modify the susceptibility of the host organism to infection, as well as ROS become harmful to both the pathogen and host [26], which promotes oxidative modifications of basic components of the human body, including crucial for the host's immune response lipids and proteins [27].

In tick-borne encephalitis, the pathogen interferes both at the level of the central nervous system (CNS) and at the systemic level [28]. Because the membranes of nerve cells are exceptionally rich in phospholipids containing large amounts of PUFAs, including arachidonic and docosahexaenoic acids, which, due to the large number of unsaturated bonds, are extremely susceptible to ROS action. As a result, reactive aldehydes, including MDA, are formed, which are products of oxidative fragmentation phospholipid arachidonic acid, as well as prostaglandin derivatives—products of oxidative cyclization of hydrocarbon chains of fatty acids [29]. Due to the large amount of docosahexaenoic acid in neurons and its greater susceptibility to oxidation than arachidonic acid, neuroprostanes are formed—cyclization products of this acid (DHA), whose level changes can also be observed in the blood. Literature data indicate that in patients with TBE as a result of CNS infection, the blood–brain barrier is damaged [30], which, combined with generalized increased oxidative stress, promotes lipid peroxidation, which may explain their approximately fourfold increase of neuroprostanes in plasma concentration, and at the same time, a significant decrease in their level after pharmacotherapy. Similar situation was observed for isoprostanes [4]. In turn, the increase in the level of MDA is mainly associated with the oxidative modification of arachidonic acid, the level of which in the plasma of patients is significantly reduced. Thus, changes in the level of fatty acids and lipid peroxidation products confirm generalized oxidative stress in the body of patients with TBE and bacterial co-infections. Statistically significant decrease in the level of MDA and neuroprostanes in patients with TBE after pharmacotherapy indicates an improvement in the systemic metabolism of phospholipids and free PUFAs in this group of patients. However, in the case of TBE with bacterial co-infections, a significant reduction in the level is observed only in the case of MDA. Therefore, it can be suggested that pro-oxidative conditions still prevail in the patient's body and/or the state of the blood–brain barrier has not completely improved.

The metabolism of phospholipids is modified not only as a result of the direct reaction of PUFAs with ROS, but is also controlled by enzymes metabolizing phospholipids and free PUFAs, the activity of which is increased under conditions of oxidative stress [31]. Therefore, in states of viral infection (TBE) and bacterial co-infection (TBE + LB/HGA), PLA2 is activated, which promotes the release of both fatty acids and their cyclization products from lipid structures. There is also increased activity of COX1/2 and lipoxygenase, including LOX5, which metabolize free PUFA into lipid mediators [32]. This situation favors reducing the level of fatty acids (AA and DHA). However, previous reports have shown that in HCV infection PUFAs, mainly AA, may enhance the antiviral effect of IFN-α and contribute to the antipathogenic effect [33]. However, in TBE infection, the level of free AA does not change, while it decreases in co-infection, especially after treatment, which may be due to increased lipid peroxidation. On the other hand, a decrease in the level of AA after the end of therapy may suggest the depletion of antiviral resources in the patients' organism. On the other hand, the increase in free DHA in the blood plasma of patients with TBE may indicate the accumulation of this acid coming from both CNS and blood levels. Literature data also show that the inducible COX2 isoform is upregulated during influenza virus infection, contributing to the excessive cytokine response observed in severe H5N1 infections [34]. As a consequence, it can be suggested that a significant increase in the activity of enzymes metabolizing PUFAs may be an offensive reaction of the body to infection.

Another consequence of the increased activity of enzymes responsible for PUFA metabolism is an increased level of some lipid mediators, including endocannabinoids and eicosanoids, whose metabolic effects are mainly the result of activation of membrane receptors of blood cells [35, 36]. Among the endocannabinoids presented in this paper, AEA and OEA are characterized by a significant increase in plasma levels as a result of infection, but only AEA as a result of co-infection. It can be suggested that the increased level of endocannabinoids is mainly a consequence of the inhibition of the activity of enzymes degrading these lipid mediators in conditions of infection [37]. Literature data also indicate that in viral infections caused by HIV or Theiler virus, AEA may participate in the regulation of inflammation by lowering the level of pro-inflammatory cytokines and activating anti-inflammatory pathways in immunosuppressive cells [38]. OEA also has anti-inflammatory and antioxidant properties, which prevents damage to the vascular endothelium [39].

The consequence of increased activity of COXs and LOXs as a result of infection is increased enzymatic metabolism of free PUFAs to other lipid mediators, such as eicosanoids, which may reveal both pro- and anti-inflammatory effects. Literature data clearly indicate that viral infection increases generation of 15-HETE and 13-HODE, which belong to the pathways that modulate inflammation [35, 40]. It is known that eicosanoids act mainly through membrane receptors and that both 13-HODE and 15-HETE are agonists of PPARγ receptors, thus promoting the resolution phase of inflammation [35] and in addition, 13-HODE is also a ligand that activates the TRPV1 receptor [40], whose increased activation is observed in granulocytes of patients with both TBE and bacterial co-infections. Thus, the increase in 13-HODE concentration observed after pharmacotherapy of patients with TBE and bacterial co-infections (TBE + LB/HGA) may confirm the resolution of the disease process. In addition, 13-HODE inhibits platelet adhesion and macrophage apoptosis [35], and is also a ligand that activates the TRPV1 receptor [40], whose increased activation is observed in the granulocytes of patients with both TBE and bacterial co-infections.

However, the level of 15-HETE does not show a tendency to increase, which may be due to the fact that 15-HETE is further metabolized as a precursor of the biosynthesis of lipoxin LXB4, which is an important agonist of inflammation resolution [41]. Thus, such a change in the level of these mediators may indicate a tendency to resolution of inflammation. The results of this study indicate the same direction of changes in PLA2 and 15-HETE activity, which may also confirm their mutual interaction in the case of TBE. However, the reduced level of anti-inflammatory 15-PGJ2 observed in the plasma of patients with both TBE and bacterial co-infections, which reduces the activation of the pro-inflammatory transcription factor NFκB [42], indicates the presence of inflammation in patients with both TBE and co-infections.

Moreover, in the early stages of TBE and viral-bacterial co-infections, elevated levels of leukotriene B4 (derived from AA) are observed in the plasma of patients, which are involved in the early stages of inflammation during infection [43]. It is known that LTB4 promotes the migration and survival of neutrophils, increases the secretion of enzymes from neutrophil granules and the production of superoxide anion radicals [44]. These leukotrienes may also participate in the recruitment of T lymphocytes to sites of inflammation [45]. In addition, LTB4 has been shown to induce the release of an antimicrobial peptide from neutrophils and, in some cases, inhibit viral replication [44]. It can therefore be suggested that the several-fold increase in LTB4 observed at the onset of TBEV infection stimulates the antiviral response against pathogens. On the other hand, in viral-bacterial co-infection, there is a much smaller increase in LTB4 levels and a smaller reduction after treatment. However, studies on the metabolic consequences of Mycobacterium tuberculosis infection have shown that the correct LTB4/PGE2 ratio is essential in controlling the host immune response, suggesting that optimal PGE2 production is also required for an effective immune response [46]. This may suggest that the situation may be similar for other infections, including bacterial co-infections. Thus, in the case of bacterial co-infections, no changes in the level of this prostaglandin are observed, while in the plasma of patients with TBE, reduced levels of PGE2 are observed before treatment, which may indicate a weakened immune response.

It is known, however, that in addition to the role of modulators in the central nervous system, endocannabinoids as well as eicosanoids are also peripheral immune mediators, with anti-inflammatory or pro-inflammatory effects depending on the representative and the receptors activated by it. These lipid mediators are known to act as agonists of the G protein-bound receptors, including CB1, CB2, TRPV1 and PPARγ receptors, the increased expression of which is observed in the present study in the granulocytes of patients with TBE and bacterial co-infection. However G protein-bound receptors are involved in the regulation of redox balance and inflammation [47] and activation of CB1 receptors increases the generation of ROS and the pro-inflammatory factor TNF-α, while activation of CB2 receptors promotes the reduction of ROS and TNF-α generation [48], which may contribute to the reduction of the pro-inflammatory and stress response in viral and bacterial infection. Such a multidirectional effect is indicated by a strong activation of CB1 receptors and at the same time a much lower activation of CB2 receptors in the granulocytes of patients with co-infections (KZM + LB/HGA). In addition, CB2 receptors not only reduce oxidative stress and inflammation, but also regulate mitogen-activated protein kinases (MAPK) and indirectly stimulate phospholipases, including PLA2 [49, 50], which may indicate a feedback loop and further intensification of phospholipid metabolism, as may indicate reduced levels of free PUFAs, especially after co-infection. It is also known that by activating the PPARγ family of receptors, endocannabinoids inhibit inflammatory responses and enhance the host response to inflammation [51]. This seems to be confirmed by the results of the present study, which indicate significantly less activation of PPARγ in the case of co-infection than in the case of TBEV infection alone. In contrast to other viral diseases such as COVID-19, it has been suggested that OEA, which is also upregulated only in TBE patients, is responsible for the downregulation of NFκB-related gene expression [52] and also modulates the effects partially mediated by PPARα [53]. In addition, in patients with COVID-19, it was found that activation of the endocannabinoid system reduces viral replication and the level of pro-inflammatory cytokines [54], hence a similar response of the endocannabinoid system, especially in the case of TBE, but also partially in co-infections, can be considered beneficial and even pro- survival effect on the infected organism.

Taking into account changes in the level of lipid mediators, both from the group of endocannabinoids and eicosanoids, and the granulocyte receptors they activate, which may be activated as a result of interaction with various lipid mediators, it is not possible to clearly link and compare changes in the levels of lipid mediators and activated receptors with the course of TBE and bacterial co-infections and with clear effects of therapy.