Detection of Interleukin-19-like protein and its role in immune regulation strengthens Galleria mellonella as a model for host-pathogen interactions

This is a preprint and has not been peer reviewed. Data may be preliminary. Detection of Interleukin-19-like protein and its role in immune regulation strengthens Galleria mellonella as a model for host-pathogen interactions Abstract Galleria mellonella is an established invertebrate model for studying host-pathogen interactions and immune responses. Although cytokine-analogous proteins are crucial for immune regulation in both vertebrates and invertebrates, their roles in insects remain poorly understood. This study examines the involvement of an IL-19-like protein in G. mellonella immunity, particularly in response to fungal infection and immune modulation. It uses proteomic analysis, fluorescence microscopy, flow cytometry and ELISA to determine the presence of IL-19-like protein in G. mellonella hemocytes following Conidiobolus coronatus infection and exposure to the fungal metabolites harman and norharman. It also assesses the effects of recombinant IL-19 on selected functions of wax moth hemocytes. This study provides the first proteomic confirmation of an IL-19-like protein in G. mellonella, and confirms its increased expression after C. coronatus infection. Harman and norharman treatment further elevated IL-19-like protein levels in hemocytes. Functionally, IL-19 promoted hemocyte migration, network formation, and wound healing while inhibiting ROS production but did not affect apoptosis, phagocytosis or fungal susceptibility. IL-19 also upregulated Jak1- and Jak2-like proteins, indicating its involvement in JAK-STAT signaling. Our findings highlight the evolutionary conservation of cytokine-like proteins and their immunomodulatory roles in insects. They reinforce the utility of G. mellonella as a model for cytokine research and fungal pathogenesis, and provide new insights into IL-19-like proteins in immune regulation. 1. Introduction The greater wax moth ( Galleria mellonella ) has emerged as a valuable alternative model organism in biomedical research, especially for studying host-pathogen interaction and immune responses [1]. G. mellonella offers considerable ethical and logistical benefits compared with classical vertebrate models, including low maintenance costs, ease of experimentation, and freedom from the legal restrictions imposed by animal welfare legislation. Using the wax moth model highlights the importance of following principles of the ‘3Rs’ viz. replacement, reduction and refinement, when working with animals. It can be used to reduce the need for mammalian species in experiments, thus promoting ethical standards without sacrificing scientific value [2]. G. mellonella larvae are large enough to allow precise experimental manipulations and can thrive at human body temperature (37°C), making them an ideal model for investigating human pathogens and testing antimicrobial agents [3]. G. mellonella is of particular value as a model organism thanks to its characteristic immune system, which is especially relevant in studies regarding host-pathogen interactions and their immune mechanisms [4]. While not possessing an adaptive immune response, G. mellonella does possess an innate immune system sharing striking structural and functional similarities with that of vertebrates, such as both cellular and humoral mechanisms that allow the insect to develop effective defenses against microbial pathogens [5]. Similar to mammalian phagocytes, hemocytes mediate the cellular responses, being involved in essential processes including phagocytosis, encapsulation, and nodulation of pathogens [6]. Simultaneously, humoral responses are dependent on antimicrobial peptides (AMPs), opsonins and the phenoloxidase cascade, which leads to melanization; a process that immobilizes and neutralizes invading microorganisms [7]. G. mellonella display an entirely conserved innate immune system which shares features with mammalian innate immunity, making it suitable for a wealth of infectious disease research with fungal systems. Both types of system depend upon conserved pathogen recognition pathways that involve pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and C-type lectins (CLRs) that detect fungal constituents including β-glucans and mannans. Upon recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), various sensors, such as toll-like receptors (TLRs), activate downstream pathways such as the NF-κB signaling pathway. They also encourage the production of immune effectors such as antimicrobials and reactive oxygen species (ROS) [8]. In mammals, NF-κB activation induces the production of pro-inflammatory cytokines, such as TNF, IL-6, and IL-12, leading to oxidative bursts and tissue damage [9]. Similarly, in Lepidoptera, fungal infection triggers insect cytokines, such as plasmatocyte-spreading peptide (PSP), which promote immune cell activation and eicosanoid production [10]. One of the most striking parallels between G. mellonella and the mammalian immune response is that of complement-like pathways, such as opsonins and mediators of pathogen clearance. In mammals, the complement system elicits an immune response via classical, lectin, and alternative pathways to create a membrane attack complex (MAC) [11]. Likewise, G. mellonella utilizes thioester-containing proteins (TEPs) and phenoloxidase-mediated melanization to identify, opsonize, and neutralize fungal pathogens, demonstrating functional conservation despite evolutionary divergence. Moreover, production of antifungal peptides such as gallerimycin and lysozyme, in Lepidoptera parallels the activity of β-defensins and other antimicrobial molecules such as histatin-5, lactoferrin and protegrins in mammals [12]. The evolutionary conservation of innate immunity mechanisms between G. mellonella and vertebrates allows for the exploration of more detailed immunological synchronisms, including the role of highly-conserved regulatory molecules, such as cytokines. Cytokines play essential roles in orchestrating immune responses in vertebrates [13] and might also occupy analogous positions during fungal infection in G. mellonella . Our previous studies identified eighteen cytokine-like molecules in G. mellonella hemocytes during infection with entomopathogenic fungus Conidiobolus coronatus . Notably, hemocytes from infected larvae exhibited increased fluorescence intensity for six cytokine-like proteins, (GM-CSF, M-CSF, IL-3, IL-15, IL-1β and IL-19), compared to untreated controls. ELISA analysis further confirmed significantly elevated levels of IL-3, IL-15, M-CSF, IL-1α, and IL-19 in the hemolymph post-infection, accompanied by reduced concentrations of TNF-β and G-CSF [14]. Additionally, the presence of IFN-γ-like protein was confirmed by proteomic analysis, which showed 33% homology with human IFN-γ [15]. Furthermore, the cytokine was found to affect several elements of immune response in the wax moth, including enhanced wound healing, chemotaxis activity and hemocyte impedance, while reducing hemocyte phagocytosis and oxidative stress in cultured immunocompetent cells; it also appears to increase the levels of Jak-2- and NF-κB-like molecules in hemocytes [16]. C. coronatus can kill insects by multiple mechanisms, including the generation of toxic metabolites [17-20]. It is known to produce two β-carboline alkaloids: harman and norharman [21]. These alkaloids cause several adverse changes in hemocytes, including inhibited immunocompetent cell network formation, irregular hemocyte shape, naked nuclei and cell aggregations; they also disrupt the cell membranes, inducing hemocyte fragmentation and actin condensation [21, 22]. They also influence IFN-γ levels in hemocytes, indicating the importance of harman and norharman in infection [16]. Cytokines are evolutionarily conserved between species, and some may be more capable of supporting both invertebrate and vertebrate immunity. As such, G. mellonella is a versatile model organism that can allow the identification of key immune mechanisms and the testing of therapeutic agents without having to rely on mammal models. The aim of this study was to determine the multifaceted effects of IL-19 treatment on G. mellonella larvae and their immunocompetent cells. Our hypothesis proposes that IL-19 application affects the immune system of insects. 2. Methods 2.1. Insects The greater wax moth, Galleria mellonella, was reared in a controlled environment maintained at 30°C and 70% relative humidity, under constant darkness, and fed an artificial diet [23]. Fully matured larvae, collected before pupation, were surface sterilized and homogenized to prepare supplements for fungal culture studies. For experiments investigating the effects of fungal infections and the influence of harman, norharman, and IL-19 on insect hemocytes, three-day-old larvae in the final instar stage were used. 2.2. Fungus The Conidiobolus coronatus isolate (number 3491) used in the study, originally obtained from Dendrolaelaps spp., was sourced from Prof. Bałazy’s collection at the Polish Academy of Sciences, Research Center for Agricultural and Forest Environment in Poznań. This isolate was cultivated on Sabouraud agar medium (SAM) enriched with homogenized G. mellonella larvae (10% wet weight) in 90 mm Petri dishes at 20°C under a 12:12 light–dark cycle. The inclusion of homogenized G. mellonella larvae markedly improved sporulation and increased the virulence of C. coronatus cultures grown on SAM. 2.3. Infection of insects with C. coronatus For the detection of IL-19 in the hemolymph of G. mellonella, three-day-old last instar larvae were exposed for 24 hours to fully grown, seven-day-old sporulating colonies of C. coronatus . Twenty larvae were placed in each Petri dish for the exposure period. A control group was exposed to a sterile Sabouraud agar medium under identical conditions for 24 hours. After the 24-hour exposure to the fungus, one group of larvae was immediately collected for analysis (F24 group), while the remaining larvae were held with an artificial diet [23] and maintained at 20°C for an additional 24 hours before collection (F48 group). To investigate the influence of IL-19 on susceptibility to fungal infection, wax moth larvae were injected with IL-19 (72 pg/ml or 1.2 ng/ml) and subsequently exposed to fully sporulating colonies of C. coronatus for 24 hours at 20°C, ten larvae per Petri dish, with three repetitions. Three control groups were included: (1) larvae exposed to sterile Sabouraud agar medium (C1) for 24 hours, (2) larvae exposed to C. coronatus for 24 hours without injection (C2), and (3) larvae injected with PBS and then exposed to the fungus for 24 hours (C3). After the exposure period, larvae were transferred to fresh Petri dishes containing an artificial diet [23] and maintained at 20°C for an additional 48 hours. Mortality rates were recorded for each group. 2.4. Application of harman and norharman to wax moth larvae Harman and norharman (both crystallin) were obtained from Sigma Aldrich (product number 41467 and N6252; CAS Number 486-84-0 and 244-63-3, respectively). They were administered to wax moth larvae either topically or through their diet, at concentrations outlined by Wrońska et al. [21]. For topical application, the alkaloids were dissolved in acetone (Chempur), and each larva received 5 µl of a solution containing 750, 1000, or 1250 ppm of the respective compound. Two groups of larvae were used as controls: the first received solvent and the second was not treated. For dietary administration, the alkaloids were dissolved in 5% methanol (Chempur) in distilled water, and 5 ml of each solution at concentrations of 750, 1000, or 1250 ppm was mixed with 5 g of insect food. To remove the solvent, the treated food was placed in an oven at 35°C for 48 hours. Before adding the alkaloid solutions, the food was baked at 180°C for one hour to dry it and eliminate any potential bacterial contaminants. The food was prepared in advance for the duration of the experiment, and larvae continuously consumed the treated diet. Two control groups were included: (1) larvae fed with food treated with the solvent only, and (2) larvae fed with untreated food. All treatments with harman and norharman, whether topical or dietary, were applied to three-day-old seventh instar larvae. Each experiment was performed in three independent replicates. 2.5. Application of IL-19 standard to wax moth larvae A standard solution of recombinant human IL-19 (PeproTech, Gibco) was prepared by dissolving the protein in either double-distilled water (ddH₂O) for in vitro experiments with freshly collected G. mellonella hemocytes, or in phosphate-buffered saline (PBS; Merck) for in vivo administration via injection. Recombinant human IL-19 was applied both in vivo via injection into larvae and in vitro directly to hemocyte cultures. Two concentrations, 72 pg/ml and 1.2 ng/ml (final concentration in well, diluted in water), were selected based on levels observed in immunocompetent wax moth cells following fungal infection [14] and the ED50 ( Effective Dose 50) value based on literature data [24]. In the in vitro experiments, the cytokine standard was added to freshly-collected hemolymph, which was cultured overnight. For in vivo studies, 5 µl of the cytokine solution was injected into larvae, which were monitored through to adult eclosion to assess the effects on insect development. The experiment included three replicates, with 10 larvae per replicate. Control groups consisted of untreated insects (C1) and larvae injected with PBS (C2). To evaluate the impact of cytokine injection on fungal infection, larvae were treated with IL-19 and incubated for 24 hours before being exposed to C. coronatus . Each experiment included three replicates, with 10 larvae per replicate. Control groups consisted of untreated insects (C1) and larvae injected with PBS (C2). 2.6. Hemolymph collection and preparation from G. mellonella larvae Larvae were disinfected with 70% ethanol and rinsed with distilled water to minimize contamination before collection. Hemolymph was taken via an incision in the last proleg, and the preparation method varied depending on the intended analysis. Proteomic analysis: Hemolymph from 30 larvae was mixed with 100 µl of supplemented GIM with phenylthiourea (PTU; 0.1 mM; Sigma-Aldrich) and centrifuged at 10,000×g for 10 minutes at 4°C. Pellets were stored at −20°C. Hemocyte culture: Fresh hemolymph (100 µl) from five larvae was mixed with 350 µl of Grace’s Insect Medium (GIM; Invitrogen) supplemented with gentamicin (10 mg/ml; Gibco), amphotericin B (250 µg/ml; Gibco), and phenylthiourea (PTU; 0.1 mM; Sigma-Aldrich). The mixture was distributed into a six-channel µ-Slide IV 0.4 (IBIDI), with 100 µl added per channel. Impact of IL-19 application: Recombinant human IL-19 standard was added to freshly collected hemolymph at final concentrations of 72 pg/ml and 1.2 ng/ml per well. Cultures were incubated at 27°C for 24 hours. Flow cytometry analysis: Hemolymph from 20 larvae was suspended in 100 µl of GIM containing 10 mM EDTA (ethylenediaminetetraacetic acid) and 30 mM sodium citrate. The suspension was centrifuged at 300×g at 4°C for 5 minutes, and the resulting pellet was collected for analysis. ELISA test: Hemolymph from 35 larvae was mixed with 100 µl of supplemented GIM, sonicated at 20 kHz for 3 minutes to lyse the cells, and centrifuged at 10,000×g for 10 minutes at 4°C. Supernatants were transferred to new tubes and stored at −20°C. Chemotaxis analysis: Freshly-collected hemolymph (100 µl) from five larvae was combined with 300 µl of supplemented GIM. A 6 µl aliquot of the suspension was added to the cell channel of the µ-Slide Chemotaxis (IBIDI) and incubated at 27°C for 30 minutes. Wound healing assay: Hemolymph (200 µl) from 10 larvae was mixed with 500 µl of supplemented GIM. A 50 µl suspension (containing 3 × 10⁵ cells/ml) was added to each well of a 35 mm µ-Dish with Culture-Insert 2 Well (IBIDI) and incubated for 2 hours at 27°C. Electric Cell-Substrate Impedance Sensing (ECIS): Hemolymph (140 µl) from seven larvae was mixed with 350 µl of Schneider medium supplemented with 10% penicillin/streptomycin (10,000 units/ml/10,000 µg/ml; Gibco) and PTU. 2.7. Detection of IL-19 in G. mellonella hemocytes and in full hemolymph 2.7.1. Proteomic analysis in G. mellonella hemolymph after fungal infection Mass spectrometry experiments were performed at the Mass Spectrometry Laboratory at the Institute of Biochemistry and Biophysics PAS. The pellet samples ware resuspended in 100mM ammonium bicarbonate, pH7.5 buffor. Next, the cysteines were reduced by 1 hour incubation with 20 mM tris(2-carboxyethyl)phosphine (TCEP) at 37oC followed by 10 min incubation at a room temperature with 50 mM methyl methanethiosulfonate (MMTS). Digestion was performed overnight using 2 µg of trypsin (Promega GmbH, Mannheim, Germany) at 37 o C. After digestion, peptides were acidified with 0.1% formic acid. Samples were analysed using LC-MS system composed of Evosep One (Evosep Biosystems, Odense, Denmark) coupled to a Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) via Flex nanoESI ion source (Thermo Fisher Scientific, Bremen, Germany) with ion mobility separation carried out with FAIMSpro interface (Thermo Fisher Scientific). Samples were loaded onto disposable Evotips C18 trap columns (Evosep Biosystems, Odense, Denmark) according to the manufacturer protocol with minor modifications. Briefly, Evotips were activated with 25 µl of 80% solution solvent B and 20% solvent A (solvent B: 0.1% formic acid in acetonitrile) by 1 min centrifugation at 600 g followed by 2 min incubation in 2-propanol. After equilibration with 25 µl of solvent A, 20 µl of each sample solution was loaded onto the solid phase. Bound peptides were washed with 50 µl and covered with 200 µl of solvent A. Chromatography was carried out at a flow rate 220 nl/min using the 88 min (15 samples per day) preformed gradient on EV1106 analytical column (Dr Maisch C18 AQ, 1.9 µm beads, 150 µm ID, 15 cm long, Evosep Biosystems, Odense, Denmark). Each sample was measured four times, using single-CV methods (CV -40, -60 or -80) or multi-CV method (CVs -45, -65 and -85). Single-CV methods data was acquired in positive mode with a data-dependent method using the following parameters. MS1 resolution was set at 60 000 with a normalized AGC target 300%, Auto maximum inject time and a scan range of 300 to 1600 m/z. For MS2, resolution was set at 15 000 with a Standard normalized AGC target, Auto maximum inject time and top 40 precursors within an isolation window of 1.6 m/z considered for MS/MS analysis. Dynamic exclusion was set at 20 s with allowed mass tolerance of ±10 ppm and the precursor intensity threshold at 5e3. Precursor were fragmented in HCD mode with normalized collision energy of 30%. Spray voltage was set to 2.1 kV, funnel RF level at 40, and heated capillary temperature at 275 °C. For multi-CV method the cycle time was set to 1 s for each CV value. Peptide and protein identification was performed using the Proteome Discoverer software with a fixed PSM validator (without the Percolator function). The search was performed against the Eukaryote Swissprot database. The search parameters were as follows: enzyme - Trypsin; missed cleavages - 1; modifications - Methylthio (C) and Oxidation (M). Subsequent FAIMS runs were treated as fractions. 2.7.2. Immunodetection of IL-19 in immunocompetent cells collected from larvae treated with harman or norharman Fluorescence microscopy The presence of IL-19 in hemolymph collected from insects treated with harman or norharman was assessed by immunocytochemistry. Cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) in PBS, permeabilized using 0.1% Triton X-100 (Sigma-Aldrich; in PBS) and then incubated overnight at 4°C with a primary rabbit anti-IL-19 polyclonal antibody (Invitrogen, 1mg/ml) diluted 1:60 in PBS. Subsequently, the cells were incubated at room temperature for two hours with a secondary antibody, Goat anti-Rabbit IgG (H+L) DyLight 488 (1 mg/ml; Invitrogen), at a final concentration of 2 µg/ml in each well. Actin fibers were stained with ActinRed™ 555 ReadyProbes™ Reagent (Invitrogen), while cell nuclei were labeled using NucBlue™ Fixed Cell ReadyProbes™ Reagent (Invitrogen). Fluorescence signals were visualized using an Axio Vert.A1 fluorescence microscope equipped with an Axio Cam ICc 5 camera (both Zeiss). Flow cytometry Hemocytes were fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich) prepared in PBS and permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) in PBS. A primary antibody (diluted 1:100) was applied to the hemocytes, and the samples were incubated overnight at 4°C. The same primary rabbit anti-IL-19 polyclonal antibody used as for the previous IL-19-like protein immunolocalization (Invitrogen, 1mg/ml). Following three PBS washes, the cells were incubated with a secondary antibody (DyLight 488 Goat Anti-Rabbit IgG, Invitrogen, diluted 1:100) for 1.5 hours at room temperature [15]. Analysis was conducted using a CyFlow Cube 8 flow cytometer (Sysmex), and data processing was performed with FCS Express 6 software (DeNovo Software). For each experimental condition, 100 µl of sample was analyzed, with any IL-19-like protein detected using a 488 nm laser on the FL-1 channel. The results were visualized as dot plots comparing forward scatter (FSC) with side scatter (SSC). ELISA tests The concentration of IL-19-like protein in immunocompetent cells of G. mellonella larvae treated with harman or norharman in vivo was measured using an ELISA assay, performed according to the manufacturer’s protocol (Wuhan Fine Biotech Co., Ltd). 2.8. Changes in G. mellonella hemocytes after IL-19 treatment 2.8.1. Hemocyte morphology Immunocompetent cells were cultured for 24 and 48 hours with IL-19 and visualized using an Axio Vert.A1 microscope equipped with an Axio Cam ICc 5 camera (both Zeiss). 2.8.2. The wound healing assay Hemocytes were seeded into a 35 mm µ-Dish with Culture-Insert 2 Well (IBIDI), and the chamber was carefully removed using sterile tweezers. The cultured cells were rinsed with supplemented GIM to remove debris and unattached cells. The µ-Dish was then filled with 600 µl of cell-free medium supplemented with recombinant human IL-19 at final concentrations of 72 pg/ml or 1.2 ng/ml. Wound healing progress was monitored over 48 hours and documented using a phase-contrast fluorescence microscope (Axio Vert.A1) equipped with an Axio Cam ICC5 camera and analyzed with Zen 12 software (Zeiss). 2.9.3. Electric cell-substrate impedance Sensing (ECIS) The impedance of the cellular monolayer was measured using an Electric Cell-Substrate Impedance Sensing (ECIS) system (IBIDI) with an 8W1E array electrode chamber (IBIDI) based on previous protocol [16]. Each of the eight measuring electrodes, with a diameter of 250 µm, was pre-treated for one hour at room temperature with 5 µl of 20 µg/ml concanavalin A (Thermo Scientific, diluted in 0.15 M NaCl, as per literature [25]), followed by overnight coating at room temperature with 200 µl of 10 mM L-cysteine (Thermo Scientific, dissolved in double-distilled water). The arrays were stabilized with Schneider medium for one hour before cells were seeded onto the electrodes (400 µl of cells prepared as previously described). Impedance measurements were recorded over time at eleven frequencies (62.5, 125, 250, 500, 1000, 2000, 4000, 8000, 16,000, 32,000, and 64,000 Hz) using the cell-covered 8W1E arrays, with data collected every 2.6 minutes. 2.8.4. Chemotaxis Assays After 30 minutes of hemocytes incubation, 65 µl of GIM or chemoattractant was introduced into the chamber. Three experimental setups were applied: (1) control, with GIM in both chambers; (2) GIM in one chamber and 72 pg/ml of IL-19 in the other; and (3) GIM in one chamber and 1.2 ng/ml of IL-19 in the other. Cell migration was monitored using an Axio Vert.A1 microscope (Zeiss) equipped with an Axio Cam 305 color camera (Zeiss) at ×20 magnification. Time-lapse imaging captured cell movements over 20 hours, with a 20-minute interval between frames. Data analysis was performed using AI Fast Track software (MetaVi Labs). 2.8.5. Immunodetection of JAK-STAT and NF-κB molecules Hemocyte cultures were established as described earlier. For immunocytochemical analysis, including the JAK-STAT and NF-κB pathways, the cells were first incubated for 24 hours. Subsequently, they were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) in PBS and permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) in PBS. The cells were then incubated overnight at 4 °C with primary rabbit antibodies: NF-κB p65 (520 µg/ml), Jak1 (1000 µg/ml), Jak3 (1000 µg/ml, all from Protein Intertech), and Jak2 (1000 µg/ml) from Sigma Aldrich; antibodies were dissolved in PBS (1:60). Following this, the hemocytes were incubated at RT for a further two hours with secondary antibody (Goat anti-Rabbit IgG (H + L) Secondary Antibody DyLight 488; 1 mg/ml; Invitrogen). The concentration of the secondary antibody was 2 μg/ml (final concentration in well). Actin fibers were stained with ActinRed™ 555 ReadyProbes™ Reagent (Invitrogen), while cell nuclei were labeled with NucBlue™ Fixed Cell ReadyProbes™ Reagent (Invitrogen). Fluorescence signals were observed using an Axio Vert.A1 fluorescence microscope equipped with an Axio Cam ICc 5 camera (both from Zeiss). 2.8.6. Detection of oxidative stress Oxidative stress induced by IL-19 was evaluated using CellROX™ Green Reagent (Thermo Fisher Scientific). Permeabilized cells were exposed to CellROX™ Green Reagent at a final concentration of 5 µM per well and incubated for 30 minutes at 37°C. After incubation, the cells were rinsed three times with PBS and analyzed using fluorescence microscopy. Menadione (Vitamin K₃; Sigma-Aldrich) served as a positive control. The compound was dissolved in dimethyl sulfoxide (DMSO; POL-AURA) to create a 40 mM stock solution, which was then applied to untreated hemocyte cultures at final concentrations of 100 µM and 8 µM per well, based on the CellROX™ Green Reagent manual and literature references [26]. Cultures were incubated for one hour at 37°C before being permeabilized and treated with CellROX™ Green Reagent following the same protocol. 2.8.7. Determination of hemocyte viability Cell viability was evaluated using the ReadyProbes™ Cell Viability Imaging Kit, Blue/Green (Invitrogen). Hemocytes were cultured overnight with the IL-19 standard, and the assay was then performed following the manufacturer’s protocol with slight modifications. Live cultures were treated with NucBlue® Live, which stains all nuclei, and NucGreen® Dead, which selectively stains the nuclei of dead cells (two drops/ml in PBS), then incubated for 45 minutes. Fluorescence detection was carried out using a standard DAPI filter to visualize all cell nuclei and an FITC channel to detect nuclei of dead cells. For the positive control, hemocytes were exposed to UV-C radiation (λ = 254 nm) for 15 minutes prior to staining. 2.8.8. Apoptosis/ necrosis detection Apoptosis and necrosis were assessed using the GFP CERTIFIED™ Apoptosis/Necrosis Detection Kit (Enzo Life Sciences). Live cell cultures were incubated for 24 hours, after which adherent cells were rinsed twice with PBS. A mixture containing 5 µl of Apoptosis Detection Reagent – Annexin V Enzo Gold, 5 µl of Necrosis Detection Reagent, and 500 µl of Binding Buffer was prepared, with 100 µl added to each channel. Samples were protected from light and incubated at room temperature for 15 minutes. Following incubation, the cells were washed three times with PBS before analysis. 2.8.9. Changes in phagocytic activity of hemocytes Phagocytic activity was analyzed in immunocompetent cells from G. mellonella larvae cultured with recombinant IL-19 in vitro . Phagocytic interactions were assessed using fluorescein-conjugated Escherichia coli (K-12 strain) BioParticles™ (Invitrogen). Following a 24-hour incubation with the E. coli bioparticles, the cells were fixed and permeabilized using previously-described protocols [22]. 2.9. Statistical analysis Data distribution was evaluated using the Kolmogorov-Smirnov (K-S) test. Since the data followed a normal distribution, results were expressed as mean ± standard deviation (SD), and parametric tests were employed for analysis. The Student’s t-test or one-way ANOVA with Tukey’s post hoc test was used to compare parameters. Statistical significance was set at 95% (p < 0.05). Calculations were performed using STATISTICA software (StatSoft Polska). 3. Results 3.1. Detection of IL-19-like protein in G. mellonella hemocytes and hemolymph. 3.1.1. Proteomic identification of IL-19-like protein after C. coronatus infection Previous research based on IL-19 immunodetection using fluorescence microscopy, flow cytometry, and ELISA assays has demonstrated that infection of larvae with the C. coronatus leads to an increase in IL-19 in hemocytes and hemolymph. In the present study, protein profiling using the FAIMS method confirmed the presence of an IL-19-like protein in G. mellonella hemolymph samples collected 24- and 48-hours post-infection, and that it was absent in control samples from healthy insects. Analysis performed using Proteome Discoverer software and the Eukaryota SwissProt database revealed a 23% sequence homology between the detected protein and IL-19 from Homo sapiens . 3.1.2. Immunodetection of IL-19-like protein after harman and norharman application Immunodetection of IL-19-like protein in G. mellonella hemocyte cultures using fluorescence microscopy revealed distinct expression patterns dependent on both the concentration and type of treatment (harman or norharman), with significant differences observed between the two compounds. Fluorescence microscopy images are presented in Figure 1: part A shows the results after topical administration of the tested alkaloids and part B with food treatment. Immunodetection also revealed a concentration-dependent increase in IL-19-like protein (green fluorescence) in hemocytes collected from larvae treated topically with harman. At lower concentrations, signal intensity was moderate and primarily localized to the perinuclear regions. Higher concentrations resulted in robust fluorescence with widespread distribution across the cytoplasm. Norharman treatments elicited a similar concentration-dependent increase; however, the intensity and distribution patterns were generally more diffuse compared to equivalent concentrations of harman, suggesting that the two compounds demonstrated distinct cellular processing pathways. The highest level of green fluorescence was observed at the highest concentrations of harman (1250ppm). Control samples showed minimal green fluorescence, confirming the specificity of IL-19-like protein induction following exposure to harman and norharman. Dietary exposure to harman (775 ppm and 1000 ppm) also led to a concentration-dependent increase in IL-19-like protein immunodetection. Lower concentrations produced moderate fluorescence with a focus near the nucleus, while higher concentrations showed a marked increase in intensity and a shift toward more uniform cytoplasmic distribution. In contrast, hemocytes from norharman-fed larvae displayed consistently lower fluorescence intensity than hemocytes from harman-fed insects at concentrations 775ppm and 1000ppm, although these values were still significantly elevated relative to controls. Notably, at higher norharman concentrations, fluorescence patterns suggested aggregation or compartmentalization within cytoplasmic regions; this was less pronounced in the harman-treated cells. As in topical treatments, control groups demonstrated negligible IL-19-like protein fluorescence, serving as a baseline for comparison. Flow cytometry analysis verified the presence of an IL-19-like protein in immunocompetent cells from both control G. mellonella and those treated with alkaloids, as illustrated in Fig. 2A; this was also indicated by raw data regarding the percentage of cells in which the tested cytokine was detected (S2 Table). In the group of insects subjected to topical alkaloid administration, the highest percentage of hemocytes positive for IL-19-like protein was observed following treatment with the highest concentration of harman. Conversely, in insects receiving dietary administration, the most pronounced effect was noted in those receiving norharman at concentrations of 1000 ppm and 1250 ppm. Statistical analyses revealed a significant elevation in the percentage of hemocytes exhibiting the target cytokine in the hemolymph of larvae after topical application of harman at concentrations of 1000 ppm and 1250 ppm, as well as norharman at 1250 ppm. Similarly, dietary exposure to both alkaloids at all tested concentrations resulted in a marked increase in cytokine-positive hemocytes compared to controls. The concentration of IL-19-like protein in G. mellonella hemolymph was assessed using an ELISA test (Fig.2B) following treatment with various concentrations of harman and norharman, applied either topically or with food (750, 1000, and 1250 ppm). Significant changes in IL-19-like protein levels were observed between the control and treated groups, as well as between the two modes of application. The analysis revealed that both harman and norharman significantly increased protein concentrations compared to the control group, with statistically significant differences (p < 0.05, one-way ANOVA, Tukey’s HSD test) observed between the mode of application. The highest concentration of IL-19-like protein was detected in hemolymph from insects treated with norharman with food at 1250 ppm; in contrast, the lowest concentrations of protein among the treated groups were observed in hemolymph from insects after topical application of harman at 750 ppm. 3.2. Impact of IL-19 injection on G. mellonella development G. mellonella larvae were injected with 5 μL of IL-19 standard at two concentrations: 1.2 ng/mL and 72 pg/mL. Two control groups were included: untreated insects (C1) and insects injected with the solvent (PBS; C2). Following injection, the larvae were monitored over 25 days, and key parameters including survival rate, percentage of pupation and emergence, mean time to pupation, and adult eclosion were recorded. The results are summarized in Table 1. The injection of IL-19 into larvae had no significant impact on survival, time to pupation, or the percentage of pupae; however, a statistically significant reduction in the mean time to emergence was observed at both cytokine concentrations compared to the control. Additionally, administration of IL-19 at a concentration of 1.2 ng/mL resulted in a significant decrease in the percentage of insects reaching the imago stage. 3.3. The impact of IL-19 injection on the susceptibility of wax moth larvae to C. coronatus infection The administration of IL-19 had no effect on the susceptibility of wax moth larvae to C. coronatus infection (S1 Fugure). Notable differences in survival were observed between the control group and those exposed to the fungus. Furthermore, insects treated with the fungus exhibited comparable mortality rates to those injected with either the cytokine standard or the solvent. 3.4. Changes in G. mellonella hemocytes after IL-19 treatment To investigate the impact of IL-19 on G. mellonella hemocytes, the cytokine was administered in vitro to cell cultures at concentrations of 72 pg/mL and 1.2 ng/mL. A series of experiments were performed to assess its effects on hemocyte morphology, cellular motility and wound-healing capacity, cell viability, oxidative stress levels, and activation of the JAK/STAT signaling pathway. 3.4.1. The effect of IL-19 on hemocytes morphology and wound-healing Treatment with IL-19 did not exhibit any noticeable effect on hemocyte morphology in vitro when compared to untreated hemocytes (Fig. 3A). After 24 hours of incubation, hemocyte cultures displayed widespread distribution across the well, with plasmatocytes forming a network that facilitated granulocyte adhesion. The morphology of the immunocompetent cells in insects remained unchanged following IL-19 treatment. Interestingly, hemocytes treated with the tested cytokine demonstrated enhanced overgrowth on the culture plate and formed more extensive networks compared to the control cells (Fig. 3B). The hemocytes demonstrated an enhanced capacity to form networks following IL-19 administration, indicated by an improved ability to repair an artificially-induced wound in vitro (Fig. 3C, D). Compared to control cells, IL-19-treated hemocytes exhibited more robust intercellular connections and more effectively filled the open space on the culture plate. Plasmatocytes played a central role in this process. 3.4.2. IL-19 as a chemoattractant for G. mellonella hemocytes The influence of 72 pg/ml or 1.2 ng/ml IL-19 on the migration of G. mellonella hemocytes was investigated using a µ-slide (IBIDI) chemotaxis assay. The results are shown in Fig. 4 (raw data are in Table S3). The hemocyte trajectories were recorded over a 20-hour observation period (Fig. 4B). Cells exposed to IL-19 demonstrated a more directed movement toward the IL-19 reservoir compared to the control, which was indicative of chemotactic activity. The trajectories illustrated greater directional persistence when exposed to higher concentrations of IL-19. End-point tracking confirmed significant hemocyte accumulation near the IL-19 gradient, indicating an effective chemotactic response to IL-19, with a dose-dependent increase in cell recruitment (Fig. 4C). The displacement of hemocytes’ center of mass along the gradient direction was quantified, with the cells in the IL-19 gradients exhibiting substantial COMD (Center of Mass Displacements) compared to the control group (Fig. 4D). IL-19 exposure also increased the average migration speed of hemocytes: dose-dependent analysis showed that higher IL-19 concentrations elicited faster cell migration rates compared to the control (Fig. 4E). The data regarding the accumulated migration distance further corroborates the role of IL-19 in enhancing hemocyte motility. Cells in IL-19-supplemented conditions exhibited longer cumulative paths compared to those in the GIM-only medium (Fig. 4F). 3.4.3. Electric cell-substrate impedance sensing (ECIS) The ECIS analysis, evaluating the total impedance, barrier resistance and cellular capacitance of G. mellonella hemocytes, found that IL-19 had a positive impact on cell motility, network formation, and monolayer development (Fig 5). In both control and cytokine-treated cultures, hemocytes reached the plateau phase approximately two hours after incubation (Fig. 5C). Impedance measurements revealed that hemocytes cultured with IL-19 formed and maintained a stronger, more confluent, monolayer over time compared to untreated cells. The addition of IL-19 increased resistance relative to control cells (Fig. 5A), likely due to reduced cell permeability stemming from the development of tighter and more robust cell barriers. Furthermore, IL-19 treatment led to enhanced electrode coverage compared to untreated cells (Fig. 5B). 3.4.4. The effect of IL-19 on hemocytes viability IL-19 treatment showed no discernible effect on hemocyte viability (Fig. 6A). After 24 hours of cultivation with the cytokine standard, no increase in green fluorescence intensity was noted on the FITC channel, indicating that the number of dead cells remained comparable between the negative control (untreated cells) and treated groups. IL-19 treatment did not appear to influence apoptosis or necrosis in the tested cells (Fig. 6B). Both forms of cell death were observed in insect cells following treatment with staurosporine, and the presence of IL-19 did not affect their incidence. 3.4.5. The impact of IL-19 standard on hemocyte phagocytic activity The phagocytic activity of hemocytes (Fig. 6 C) was evaluated using fluorescein-labeled E. coli (K-12 strain) BioParticles™ (Invitrogen). The analysis revealed no significant difference in the intensity or distribution of green fluorescence between hemocytes treated with IL-19 and control hemocytes. Both groups exhibited similar levels of green fluorescence, indicating comparable phagocytic activity. These findings suggest that IL-19 does not modulate the phagocytic activity of hemocytes under the experimental conditions tested. 3.4.6. The impact of IL-19 standard on oxidative stress in G. mellonella hemocyte The impact of IL-19 treatment on oxidative stress in G. mellonella hemocytes is shown in Fig. 6D. The results demonstrate that vitamin K3 exerted a similar positive effect on ROS production in hemocytes as observed in mammalian cells [27]. Conversely, IL-19 treatment inhibited ROS production, evidenced by a reduction in green fluorescence on the FITC channel at both tested concentrations. 3.4.7. The effect of IL-19 on immunodetection of Jak- and NF-κB-like molecules Fig.7 presents the immunodetection of Jak1-, Jak2-, Jak3-, and NF-κB p65-like proteins in G. mellonella hemocytes exposed to IL-19 under in vitro conditions. A slight increase in green fluorescence intensity was observed for the Jak1- and Jak2-like proteins in IL-19-treated hemocytes compared to the control. In contrast, no significant changes in green fluorescence were detected for the Jak3- and NF-κB p65-like proteins between treated and untreated samples. Notably, the Jak1- and Jak3- proteins exhibited a perinuclear localization pattern, suggesting their close association with the nuclear region in response to IL-19 exposure. Jak1-like protein displayed a diffuse cytoplasmic distribution, while NF-κB p65-like protein was similarly localized within the cytoplasm without any perinuclear enrichment. 4. Discussion Cytokines mediate communication between immune cells and orchestrate host defense mechanisms against fungi. They are important mediators of inter alia inflammation, phagocytosis, and the generation of antifungal peptides that allow the host to respond effectively to pathogenic fungi [28]. The discovery of cytokine-like proteins in G. mellonella during infection confirms their evolutionary conservation and importance in immune regulation. Understanding how these molecules work is important not just for understanding insect immunity itself, but for forming general insights into host-pathogen interactions and possible analogies between immune systems across species. Our results demonstrate that IL-19 has a multifaceted role in the immune response of G. mellonella . Preliminary studies based on fluorescence microscopy, flow cytometry and ELISA established that the levels of IL-19 in hemocytes were significantly increased in response to C. coronatus infection [14]. Our present findings indicate that harman and norharman, two metabolites of C. coronatus, applied topically or via diet, significantly increase the detection and concentration of IL-19 in wax moth immunocompetent cells. This suggests that IL-19 plays an important role in the defense against fungal infections by G. mellonella . Moreover, the metabolites of entomopathogenic fungi can also modulate the cytokine-like protein pathways in the host, thus altering the immune response. This observation, along with the ability of harman and norharman to modulate IL-19 levels in G. mellonella, confirms the duality of these fungal metabolites as both virulence factors as potential immune modulators. Our current proteomic analysis is the first to confirm the presence of an IL-19-like protein in the hemolymph of G. mellonella larvae infected with Conidiobolus coronatus . FAIMS analysis found that IL-19-like protein was detectable in samples taken 24 and 48 hours after infection, while no such protein was found in hemolymph from healthy control insects. Analysis with Proteome Discoverer software and the Eukaryota SwissProt database, identified a 23% homology to the sequence of human IL-19, indicating the evolutionary conservation of this cytokine-like protein and a potential role in the immune response in G. mellonella. Overall, these results provide strong support for the hypothesis that fungal infection drives the production of IL-19-like proteins in wax moth hemolymph. Furthermore, these results are consistent with our own previous proteomic analyses, which identified an IFN-γ-like protein in G. mellonella hemolymph that shared 33% sequence coverage with human IFN-γ [15]. Taken together, these studies emphasize that cytokine-like proteins are evolutionarily conserved between species, and play important roles in immune responses against pathogenic infections. The present study builds upon these findings by adding 1.2 ng/mL and 72 pg/mL cytokine standard to hemocyte cultures to explore the functional implications of IL-19 in G. mellonella in vitro . IL-19 treatment improved the network formation and barrier integrity of hemocytes in the G. mellonella, confirming its functional activity. After IL-19 treatment, the hemocytes exhibited an increased ability in forming intercellular connections, thereby promoting enhanced repair of artificial wounds in vitro . This process, driven primarily by plasmatocytes, resulted in robust filling of open spaces on the culture plate. The formation and maintenance of a stronger and more confluent IL-19-treated hemocyte monolayer was confirmed by complementary impedance measurements: the tests indicated greater resistance and enhanced electrode coverage, suggesting that IL-19 promoted the development of tighter, more cohesive cell barriers, likely by reducing cell permeability and strengthening intercellular adhesion. The idea that IL-19 promotes wound healing is in line with previous studies in mammals, where IL-19 has been shown to increase skin fibroblast keratinocyte growth factor expression to stimulate wound healing [29]. One of the critical aspects of the insect cellular immune response is the ability of hemocytes to adhere to surfaces and form monolayers, which often involves the creation of robust intercellular connections [30]. These spreading behaviours are commonly used as measures of immune fitness, as they directly correlate with the ability of hemocytes to dynamically respond to infection. Previous studies in a range of insect models have also shown that some infections, especially with viral, fungal and bacterial pathogens, support hemocyte phenotypes with increased spreading ability [31-33]. These hyper-spreading cells, characterized by extreme surface area expansion, appear early during fungal infections, likely as part of the cellular immune defense involved in processes such as nodule formation [34]. Our present findings demonstrate that IL-19 is an effective chemoattractant for G. mellonella hemocytes, directing migration to the IL-19 gradients in a dose-dependent manner. Hemocytes exposed to IL-19 exhibited enhanced directional persistence, increased migration speed, and longer cumulative migration paths compared to controls. The observed displacement of the center of mass of the hemocytes, and their significant accumulation near the IL-19 gradient further underscore its role in recruiting immune cells to sites of simulated immune activation. These findings align with those observed in mammalian systems in which IL-19 induced chemotaxis of neutrophils and increased the recruitment of immune cells. Moreover, studies in lung epithelial cells showed that IL-19 decreases neutrophil apoptosis, which suggests IL-19 may regulate the immune response and neutrophil lifespan [35]. The parallels between insect and vertebrate systems suggest that the chemotactic properties of IL-19 are evolutionarily conserved. Although IL-19 has been shown to inhibit apoptosis in non-immune cells, such as synovial cells [36] and vascular smooth muscle cells [37], our results indicate that the cytokine does not affect hemocyte viability in G. mellonella . Following 24-hour incubation with recombinant IL-19, no significant differences in apoptosis or necrosis were found between treated and control groups. It appears that IL-19 may have a pro- or anti-apoptotic depending on the type of immune system cell, indicating that its role in cell apoptosis is context specific, and should be further examined across various cell types. These data show that IL-19 does not influence the phagocytic activity of G. mellonella hemocytes, the IL-19-treated and control cells demonstrated comparable fluorescence intensity and distribution. No differences were observed in the levels of phagocytosis between the groups, indicating that the cytokine does not affect this particular immune function in the evaluated conditions. In addition, there is no supporting evidence in the literature that IL-19 moderates the phagocytic capacity of mammalian immune cells. Unlike its inability to affect hemocyte phagocytosis, IL-19 treatment was found to suppress ROS production in G. mellonella hemocytes. Previous studies have noted an inverse correlation of IL-19 with ROS in mammalian systems; IL-19 reduced ROS levels in cultured vascular smooth muscle cells [37]. On the other hand, ROS production is vital for combating fungal infections: fungal pathogens encounter such highly-reactive molecules during their interaction with phagocytic cells, which is one of the first lines of immune defense [38]. Oxidative stress has been implicated in the antifungal response of G. mellonella to C. coronatus [39]. However, IL-19 treatment did not lead to an elevation in ROS levels, indicating that its response to fungal infection is not associated with increased oxidative stress. Instead, ROS production is probably driven by other aspects of the immune response, and IL-19 may have a different immunomodulatory effect. The JAK-STAT pathways of insects play a similar functional role as the mammalian interferon system in mammalian immune signaling and cellular responses to infections. Our findings indicate IL-19 to be a cytokine-like molecule that induced an increase in Jak1- and Jak2-like protein levels in G. mellonella hemocytes; this suggests that the JAK-STAT pathway may be engaged in immune regulation in this species. A subfamily of IL-10 cytokines, IL-19, in mammals, act as a ligand for Jak1 and Jak2, which, in turn, activates the transcription factor STAT3 [40]. Additionally, previous research indicates that IL-4 signals through the JAK-STAT pathway to regulate IL-19 expression in keratinocytes, highlighting the complex interplay between cytokine signaling networks [41]. Our findings provide valuable insights regarding our understanding of the role of cytokine-like molecules in the immune response to fungi by G. mellonella . Most strikingly, the study offers direct proteomic evidence of an IL-19-like protein in the wax moth hemolymph, and shows that its expression is significantly up-regulated in response to C. coronatus challenge. These results demonstrate that IL-19-like molecules exist in insects, and that they may have similar functions to those of their vertebrate counterparts. The study provides a new perspective on the intricate immune system of G. mellonella, confirming that the identified cytokine modulates various aspects of hemocyte behavior, including chemotaxis, wound healing, and monolayer formation, while also modulating oxidative stress JAK-STAT pathway activation. Our findings support the growing belief that G. mellonella is a powerful and many-faceted model for host-pathogen studies, not least when considering fungi. These results broaden our knowledge of cytokine-mediated immune regulation in invertebrates and provide avenues for future comparative immunological studies, as well as potential insights and translational applications in biomedical and immunological systems. 5. References 1. 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The impact of IL-19 injection on wax moth larvae survival and development. | C1 | 0 µg/ml (0µl) | 28 | 93.33±4.71 | 3.22±0.40 | 93.33±4.71 | 15.83±0.46 A B | 80.00±21.60 A | | | C2 | 0 µg/ml (5µl) | 29 | 88.89±15.71 | 3.96±0.51 | 88.89±15.71 | 16.47±0.08 C D | 78.89±9.56 B | | | IL-19 injection | 72 pg/ml (5µl) | 30 | 96.67±4.71 | 3.00±0.09 | 96.67±4.71 | 14.00±0.00 A C | 70.00±0.00 | | | 1.2 ng/ml (5µl) | 30 | 96.67±4.71 | 2.93±0.11 | 93.33±4.71 | 14.00±0.00 B D | 33.33±4.71 A B | Three-day-old last instar larvae were used. C1—untreated control; C2–solvent (PBS) injection; N—total number of individuals; SD—standard deviation; as a 100 % was counting the number of examined larvae; A – Statistically-significant differences between control and insects injected with PBS or IL-19 (one-way ANOVA, Tukey’s HSD test, p < 0.05). Figure legends Fig 1. Immunodetection of IL-19-like protein in G. mellonella hemocyte cultures following harman and norharman exposure. (A) Topical application of harman and norharman. Hemocytes were collected from larvae subjected to topical treatment. (B) Food application of harman and norharman. Hemocytes were derived from larvae exposed to the compounds via feeding. Cell nuclei (blue) were stained with NucBlue™ Fixed Cell ReadyProbes™ Reagent (Invitrogen), and β-actin (orange) was visualized using ActinRed™ 555 ReadyProbes™ Reagent (Invitrogen). IL-19-like protein was detected using a primary rabbit anti-IL-19 polyclonal antibody (Invitrogen) and Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 488 (1 mg/ml; Invitrogen). Scale bar: 25 μm. Fig 2. Immunodetection of IL-19-like protein in freshly-collected G. mellonella hemolymph. (A) Flow cytometry analysis. IL-19-like protein was detected in hemocytes following topical and food exposure to harman and norharman. Results are presented as FSC vs. SSC plots. Detection utilized a primary rabbit anti-IL-19 polyclonal antibody (Invitrogen, 1:100 dilution) and a DyLight 488-conjugated Goat Anti-Rabbit IgG secondary antibody (Abbkine, 1:100 dilution). FITC (green) gating identifies cells containing IL-19-like protein. (B) ELISA analysis. Changes in IL-19-like protein concentration in hemocytes after harman or norharman treatment were measured using an ELISA test. Statistically significant differences between control and treated groups are indicated by shared letters (one-way ANOVA, Tukey’s HSD test, p < 0.05). Fig 3. Effect of IL-19 treatment on G. mellonella hemocyte cultures. (A) Impact of IL-19 on hemocyte morphology. Hemocyte morphology was observed using a µ-Slide VI 0.4 (IBIDI) following in vitro application of IL-19 at concentrations of 72 pg/ml and 1.2 ng/ml. Observations were conducted at 0 h, 24 h, and 48 h. Scale bar: 25 μm. (B) Percentage of plate coverage by cells. The proportion of the plate covered by hemocytes was quantified at 0 h, 24 h, and 48 h. Data were analyzed in ImageJ software. (C) Impact of IL-19 on wound healing. A wound healing assay was performed using a μ-Dish with Culture-Insert 2 Well (IBIDI), with IL-19 applied in vitro at concentrations of 72 pg/ml and 1.2 ng/ml. Observations were made at 0 h, 24 h, and 48 h. Scale bar: 25 μm. (D) Percentage of wound closure by cells. The extent of wound closure by hemocytes was measured at the three observation time points (0 h, 24 h, and 48 h). Data were analyzed in ImageJ software. Fig 4. Chemotactic activity of IL-19 on G. mellonella hemocytes. (A) Diagram of the μ-slide (IBIDI) chemotaxis assay setup. Hemocytes were seeded in the 1 mm-wide central gap between the reservoirs (middle compartment). The reservoirs on either side (depicted as trapezoids) were filled with either growth medium (GIM) (-) or IL-19 dissolved in medium (+). (B) Cell trajectories recorded over 20 hours; (C) Final cell positions tracked at the endpoint; (D) Displacement of the average cell position along the gradient direction (center of mass displacement, COMD); (E) Average cell migration speed; (F) Total accumulated migration distance. Bar graphs represent data as mean ± standard deviation (SD); significance levels: *p<0.05, **p<0.001 (one-way ANOVA with Tukey’s HSD post hoc test). Fig 5. The real - time analysis of barrier resistance (A), cell capacitance (B), and overall impedance (C) of G. mellonella larvae exposed to two concentrations of IL-19 standard: (1) 72 pg/ml and (2) 1.2 ng/ml. The changes in resistance, capacity, and impedance of hemocytes treated with cytokine and control versus time, were measured at a frequency of 4000 Hz for 12 hours. Fig. 6 Impact of IL-19 on G. mellonella hemocytes under in vitro conditions. (A) Effect of IL-19 on hemocyte viability. Hemocyte viability was assessed using the ReadyProbes™ Cell Viability Imaging Kit, Blue/Green (Invitrogen), following 24 hours of cultivation with IL-19 standard. Scale bar: 25 μm. (B) Effect of IL-19 on apoptosis and necrosis in hemocytes. Apoptosis (green) and necrosis (red) were detected using the GFP CERTIFIED™ Apoptosis/Necrosis Detection Kit (Enzo Life Sciences). Scale bar: 25 μm. (C) Effect of IL-19 on hemocyte phagocytic activity. Phagocytic activity was evaluated using fluorescein-labeled Escherichia coli (K-12 strain) BioParticles™ (Invitrogen). β-actin (red) was stained with ActinRed™ 555 ReadyProbes™ Reagent (Invitrogen), and cell nuclei (blue) were stained with NucBlue™ Fixed Cell ReadyProbes™ Reagent (Invitrogen). Scale bar: 25 μm. (D) Effect of IL-19 on oxidative stress in hemocytes. Oxidative stress (green) was detected using CellROX™ Green Reagent (Thermo Fisher Scientific), while cell nuclei (blue) were stained with NucBlue™ Fixed Cell ReadyProbes™ Reagent (Invitrogen). Scale bar: 25 μm. Fig. 7. Effect of IL-19 standard on the immunodetection of Jak1-, Jak2-, Jak3-, and NF-κB p65-like proteins under in vitro conditions. (A) Immunodetection of Jak1-like protein; (B) Jak2-like protein; (C) Jak3-like protein; (D) NF-κB p65-like protein. β-Actin (red) was visualized using ActinRed™ 555 ReadyProbes™ Reagent (Invitrogen), while cell nuclei (blue) were stained with NucBlue™ Fixed Cell ReadyProbes™ Reagent (Invitrogen). Jak1-, Jak2-, and Jak3-like proteins, as well as NF-κB p65-like protein, were detected using specific primary rabbit antibodies (Jak proteins: 1000 μg/ml; NF-κB p65: 520 μg/ml, Protein Intertech) and Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 488 (1 mg/ml, Invitrogen). Scale bar: 25 μm. Supporting informations S1 Figure. The impact of IL-19 on the effectiveness of C. coronatus infection in G. mellonella larvae. Three-day-old last instar larvae were used. Control – larvae exposed for 24 hours to sterile Sabouraud agar medium, control of infection – larvae exposed for 24 hours to fully-grown and sporulating C. coronatus colonies, PBS injection – larvae injected with solvent. F24 larvae were sampled immediately after 24-hour exposure to fungal infection; F48 larvae were sampled 24 hours after 24-hour exposure Statistically significant differences were found between insects exposed to fungus and unexposed controls (one-way ANOVA, Tukey’s HSD test, p < 0.001). S2 Table. Immunodetection of IL-19-like protein in freshly collected G. mellonella hemolymph using flow cytometry – raw data S3 Table. Chemotactic activity of IL-19 on G. mellonella hemocytes – raw data Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Metrics & Citations Metrics Article Usage 300views 116downloads Citations Download citation Anna Wrońska, Agata Kaczmarek, Justyna Sobich, et al. Detection of Interleukin-19-like protein and its role in immune regulation strengthens Galleria mellonella as a model for host-pathogen interactions. Authorea. 06 March 2025. DOI: https://doi.org/10.22541/au.174122118.87425979/v1 DOI: https://doi.org/10.22541/au.174122118.87425979/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.