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Two Balkan Peninsula endemics, Nepeta rtanjensis and N. argolica subsp. argolica, both characterized by specialized metabolite profiles predominated by iridoids and phenolics, are differentiated according to the stereochemistry of major iridoid aglycone nepetalactone (NL). For the first time, the present study provides a comparative analysis of antimicrobial and immunomodulating activities of the two Nepeta species and their major iridoids isolated from natural sources—cis,trans-NL, trans,cis-NL, and 1,5,9-epideoxyloganic acid (1,5,9-eDLA), as well as of phenolic acid rosmarinic acid (RA). Methanol extracts and pure iridoids displayed excellent antimicrobial activity against eight strains of bacteria and seven strains of fungi. They were especially potent against food-borne pathogens such as L. monocytogenes, E. coli, S. aureus, Penicillium sp., and Aspergillus sp. Targeted iridoids were efficient agents in preventing biofilm formation of resistant P. aeruginosa strain, and they displayed additive antimicrobial interaction. Iridoids are, to a great extent, responsible for the prominent antimicrobial activities of the two Nepeta species, although are probably minor contributors to the moderate immunomodulatory effects. The analyzed iridoids and RA, individually or in mixtures, have the potential to be used in the pharmaceutical industry as potent antimicrobials, and in the food industry to increase the shelf life and safety of food products.

Significance: Autoimmune diseases are progressively affecting westernized societies, as the proportion of individuals suffering from autoimmunity is steadily increasing over the past decades. Understanding the role of reactive oxygen species (ROS) in modulation of the immune response in the pathogenesis of autoimmune disorders is of utmost importance. The focus of this review is the regulation of ROS production within tolerogenic dendritic cells (tolDC) and regulatory T (Treg) cells that have the essential role in the prevention of autoimmune diseases and significant potency in their therapy. Recent Advances: It is now clear that ROS are extremely important for the proper function of both DC and T cells. Antigen processing/presentation and the ability of DC to activate T cells depend upon the ROS availability. Treg differentiation, suppressive function and stability are profoundly influenced by ROS presence. Critical Issues: Although a plethora of results on the relation between ROS and immune cells exists, it remains unclear whether ROS modulation is a productive way for skewing T cells and DC towards a tolerogenic phenotype. Also, the possibility of ROS modulation for enhancement of regulatory properties of DC and Treg during their preparation for use in cellular therapy has to be clarified. Future Directions: Studies of DC and T cell redox regulation should allow for the improvement of the therapy of autoimmune diseases. This could be achieved through the direct therapeutic application of ROS modulators in autoimmunity or indirectly, through ROS-dependent enhancement of tolDC and Treg preparation for cell-based immunotherapy.

Gut microbiota dysbiosis has been considered the essential element in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Antibiotics were administered orally to Dark Agouti (DA) rats early in their life with the aim of perturbing gut microbiota and investigating the effects of such intervention on the course of EAE. As a result, the diversity of the gut microbiota was reduced under the influence of antibiotics. Mainly, Firmicutes and Actinobacteria were replaced by Proteobacteria and Bacteroidetes, while decreased proportions of Clostridia and Bacilli classes were accompanied by an increase in Gamma-Proteobacteria in antibiotic-treated animals. Interestingly, a notable decrease in the Helicobacteraceae, Spirochaetaceae and Turicibacteriaceae was scored in antibiotic-treated groups. Also, levels of short chain fatty acids were reduced in the faeces of antibiotic-treated rats. Consequently, aggravation of EAE, paralleled with stronger immune response in lymph nodes draining the site of immunization, and increased inflammation within the CNS, were observed in antibiotic-treated DA rats. Thus, the alteration of gut microbiota leads to an escalation of CNS-directed autoimmunity in DA rats. The results of this study indicate that antibiotic use in early life may have subsequent unfavourable effects on the regulation of the immune system.

Dendritic cells (DC) are responsible for the initiation and shaping of the adaptive immune response and are in the focus of autoimmunity research. We were interested in comparison of DC obtained from autoimmunity-prone Dark Agouti (DA) rats and autoimmunity-resistant Albino Oxford (AO) rats. DC were generated from bone marrow precursors and matured (mDC) by lipopolysaccharide. Tolerogenic DC (tolDC) obtained by vitamin D3 treatment were studied in parallel. Profile of cytokine production was different in AO and DA mDC and tolDC. Expression of MHC class II molecules and CD86 were higher in DA DC, while vitamin D3 reduced their expression in dendritic cells of both strains. Allogeneic proliferation of CD4(+) T cells was reduced by AO tolDC, but not with DA tolDC in comparison to respective mDC. Finally, expression of various genes identified as differentially expressed in human mDC and tolDC was also analyzed in AO and DA DC. Again, AO and DA DC differed in the expression of the analyzed genes. To conclude, AO and DA DC differ in production of cytokines, expression of antigen presentation-related molecules and in regulation of CD4(+) T proliferation. The difference is valuable for understanding the divergence of the strains in their susceptibility to autoimmunity.

Eksperimentalni autoimunski encefalomijelitis (EAE) je model multiple skleroze (MS), hronične inflamatorne bolesti centralnog nervnog sistema (CNS). Osnovne patogene populacije u EAE su efektorski T limfociti koji produkuju interferon-γ (Th1) ili interleukin-17 (Th17) i aktivirani makrofagi koji dovode do oštećenja CNS. Imajući u vidu da krastavac (Cucumis sativus) predstavlja bogat izvor sekundarnih metabolita kao što su polifenoli i kukurbitacini, u ovoj studiji je po prvi put ispitivano dejstvo različitih ekstrakata krastavca na encefalitogeni potencijal T limfocita, efektorske funkcije makrofaga i antigen-prezentujuću aktivnost dendritskih ćelija (DĆ). Rezultati su pokazali da ekstrakt lista krastavca (ELK) ostvaruje najpotentnije dejstvo, inhibirajući produkciju IFN-γ i IL-17 od strane encefalitogenih T limfocita, modulišući signalne puteve Nf-κB i p38 (MAPK). Pored toga, ELK je ostvario inhibitorno dejstvo na efektorske funkcije makrofaga i antigen-prezentujuću aktivnost DĆ u koncentracijama koje nisu pokazale citotoksičnost na embrione zebrice (Danio rerio) in vivo. Dalje, ELK je uspešno inhibirao encefalitogene T limfocite nakon in vivo primene u EAE. Kvantitativna i kvalitativna analiza ELK metodom masene spektrometrije potvrdila je prisustvo 37 različitih jedinjenja od kojih su kukurbitacini bili najzastupljenija jedinjenja. Shodno tome, ispitana su i imunomodulacijska dejstva kukurbitacina B i kukurbitacina E na glavnim imunskim ćelijama uključenim u patogenezu EAE. Ova ispitivanja su potvrdila dejstva ELK, sugerišući da su kukurbitacini, kao glavni konstituenti ovog ekstrakta, barem delimično odgovorni za ostvarena dejstva ELK. Rezultati ove doktorske disertacije ukazuju na to da ekstrakt lista krastavca i kukurbitacini ostvaruju snažne antiencefalitogene efekte u modelu EAE i ukazuju na važnost dodatnih istraživanja u cilju pronalaženja odgovarajućih tretmana autoimunosti CNS., Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS). T lymphocytes, which produce interferon-γ (Th1) or interleukin-17 (Th17), and activated macrophages, that lead to CNS damage, are the major pathogenic cell populations in EAE. Having in mind that cucumber (Cucumis sativus) is a rich source of secondary metabolites such as polyphenols and cucurbitacins, in this study, for the first time, the effects of various cucumber extracts were investigated on encephalytogenic T lymphocytes, effector function of macrophages and antigen-presenting activities of dendritic cells (DC). The results show that cucumber leaf extract (CLE) expresses the most potent effect by inhibiting the production of IFN-γ and IL-17 in encephalytogenic T lymphocytes, through modulation of signaling pathways Nf-κB and p38 (MAPK). Additionally, CLE imposes an inhibitory effect on the effector functions of macrophages and antigen-presenting activity of DC in concentrations that do not show cytotoxicity in zebrafish (Danio rerio) in vivo. Also, CLE inhibits generation of encephalitogenic cells in vivo. Phytochemical analysis of CLE, characterized by mass spectrometry, confirmed the presence of 37 different compounds, among which cucurbitacins were the most abundant compounds. Hence, the immunomodulatory effects of cucurbitacin B and cucurbitacin E were also examined on the major immune cells involved in EAE pathogenesis. The obtained results confirm the effects of CLE, suggesting that cucurbitacins are, at least partially, responsible for its effects. The results of this doctoral dissertation indicate that the cucumber leaf extract and its major constituents cucurbitacins have anti-encephalitogenic effects in the EAE model, Moreover, they suggest the importance of additional research towards novel highly efficient CNS autoimmunity treatments.

Albino Oxford (AO) rats are extremely resistant to induction of experimental autoimmune encephalomyelitis (EAE). EAE is an animal model of multiple sclerosis, a chronic inflammatory disease of the central nervous system (CNS), with established autoimmune pathogenesis. The autoimmune response against the antigens of the CNS is initiated in the peripheral lymphoid tissues after immunization of AO rats with CNS antigens. Subsequently, limited infiltration of the CNS occurs, yet without clinical sequels. It has recently become increasingly appreciated that gut-associated lymphoid tissues (GALT) and gut microbiota play an important role in regulation and propagation of encephalitogenic immune response. Therefore, modulation of AO gut microbiota by antibiotics was performed in this study. The treatment altered composition of gut microbiota in AO rats and led to a reduction in the proportion of regulatory T cells in Peyer's patches, mesenteric lymph nodes, and in lymph nodes draining the site of immunization. Upregulation of interferon-. and interleukin (IL)-17 production was observed in the draining lymph nodes. The treatment led to clinically manifested EAE in AO rats with more numerous infiltrates and higher production of IL-17 observed in the CNS. Importantly, transfer of AO gut microbiota into EAE-prone Dark Agouti rats ameliorated the disease. These results clearly imply that gut microbiota is an important factor in AO rat resistance to EAE and that gut microbiota transfer is an efficacious way to treat CNS autoimmunity. These findings also support the idea that gut microbiota modulation has a potential as a future treatment of multiple sclerosis.

Cucurbitacin E (CucE) is a highly oxidized steroid consisting of a tetracyclic triterpene. It is a member of a Cucurbitacin family of biomolecules that are predominantly found in Cucurbitaceae plants. CucE has already been identified as a potent anti-inflammatory compound. Here, its effects on CD4(+) T helper (Th) cells and macrophages, as the major encephalitogenic cells in the autoimmunity of the central nervous system, were investigated. Production of major pathogenic Th cell cytokines: interferon-gamma and interleukin-17 were inhibited under the influence of CucE. The effects of CucE on CD4+ T cells were mediated through the modulation of aryl hydrocarbon receptor, STAT3, Na kappa B, p38 MAPK, and miR-146 signaling. Further, production of nitric oxide and reactive oxygen species, as well as phagocytic ability, were inhibited in macrophages treated with CucE. These results imply that CucE possesses powerful antiencephalitogenic activity.

MicroRNAs (miR) are small non-coding RNAs involved in the immune response regulation. miR-155 has been attributed a major pro-inflammatory role in the pathogenesis of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Here, a role of miR-155 in re-activation of encephalitogenic CD4(+) T cells was investigated. Dark Agouti rats were immunized with myelin basic protein (MBP) emulsified in complete Freund's adjuvant. CD4(+) T cells were purified from draining lymph node cells (DLNC) obtained in the inductive phase and from spinal cord immune cells (SCIC) isolated at the peak of EAE. CD4(+) T cells obtained from SCIC (i.e., in vivo re-activated cells) had markedly higher expression of miR-155 in comparison to those purified from DLNC (not re-activated). Likewise, in vitro re-activation of DLNC with MBP led to increase in miR-155 expression. Further, DLNC and DLNC CD4(+) T cells were transfected with an inhibitor of miR-155 during in vitro re-activation. As a result, expression of important CD4(+) T cell effector cytokines IFN-gamma and IL-17, but not of regulatory cytokines IL-10 and TGF-beta, was reduced. These results imply that miR-155 supports re-activation of encephalitogenic CD4+ T cells. Our results contribute to a view that miR-155 might be a valuable target in multiple sclerosis therapy.