ORIGINAL RESEARCH published: 12 November 2020 doi: 10.3389/fimmu.2020.544718 Non-Specific Antibodies Induce Lysosomal Activation in Atlantic Salmon Macrophages Infected by Piscirickettsia salmonis Diego Pérez-Stuardo 1, Allison Espinoza 1, Sebastián Tapia 1,2, Jonathan Morales-Reyes 2, Claudio Barrientos 2, Eva Vallejos-Vidal 3,4, Ana M. Sandino 2,4, Eugenio Spencer 2,4, Daniela Toro-Ascuy 5, J. Andrés Rivas-Pardo 1,6, Felipe E. Reyes-López 3 and Sebastián Reyes-Cerpa 1,6* 1 Centro de Genómica y Bioinformática, Facultad de Ciencias, Universidad Mayor, Santiago, Chile, 2 Consorcio Tecnológico Edited by: de Sanidad Acuı´cola, Ictio Biotechnologies S.A., Santiago, Chile, 3 Department of Cell Biology, Physiology and Immunology, Kim Dawn Thompson, Universitat Autonoma de Barcelona, Bellaterra, Spain, 4 Centro de Biotecnologı´a Acuı´cola, Facultad de Quı´mica y Biologı´a, Moredun Research Institute, Universidad de Santiago de Chile, Santiago, Chile, 5 Laboratorio de Virologı´a, Instituto de Ciencias Biomedicas, Facultad de United Kingdom Ciencias de la Salud, Universidad Autónoma de Chile, Santiago, Chile, 6 Escuela de Biotecnologı´a, Facultad de Ciencias, Reviewed by: Universidad Mayor, Santiago, Chile Irene Cano Cejas, Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of United Kingdom salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that Javier Santander, generates high mortalities and large losses in Chilean salmon farming, threatening the Memorial University of Newfoundland, Canada sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to *Correspondence: survive and replicate in salmonid macrophages, inducing an anti-inflammatory Sebastián Reyes-Cerpa environment and a limited lysosomal response that may be associated with host sebastian.reyes@umayor.cl immune evasion mechanisms favoring bacterial survival. Current control and Specialty section: prophylaxis strategies against P. salmonis (based on the use of antibiotics and This article was submitted to vaccines) have not had the expected success against infection. This makes it urgent to Comparative Immunology, a section of the journal unravel the host-pathogen interaction to develop more effective therapeutic strategies. In Frontiers in Immunology this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Received: 22 March 2020 Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing Accepted: 14 October 2020 the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM- Published: 12 November 2020 beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by Citation: Pérez-Stuardo D, Espinoza A, Tapia S, quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Morales-Reyes J, Barrientos C, Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, Vallejos-Vidal E, Sandino AM, Spencer E, Toro-Ascuy D, and counting of colony-forming units (CFU) present in the extracellular and intracellular Rivas-Pardo JA, Reyes-López FE environment. Our results suggest that stimulation with antibodies promotes lysosomal and Reyes-Cerpa S (2020) Non- activity by lowering lysosomal pH and increasing the proteolytic activity within this Specific Antibodies Induce Lysosomal Activation in Atlantic organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial- Salmon Macrophages Infected induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial by Piscirickettsia salmonis. Front. Immunol. 11:544718. load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM- doi: 10.3389/fimmu.2020.544718 beads reverses the modulation of the lysosomal activity induced by bacterial infection, Frontiers in Immunology | www.frontiersin.org 1 November 2020 | Volume 11 | Article 544718 Pérez-Stuardo et al. IgM-Beads Induce Lysosomal Activity promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS. Keywords: macrophages, Atlantic salmon, IgM, P. salmonis, lysosome activity INTRODUCTION pathogens, such as Legionella pneumophila, for which erythromycin (antibiotic most commonly used for 25 years to Piscirickettsia salmonis is the etiological agent of piscirickettsiosis treat infections with this bacterium) is no longer effective in in vivo or salmonid rickettsial septicemia (SRS), which mostly affects models (18). In this scenario, the passive immunization strategy farmed salmonid species (1, 2). Piscirickettsia salmonis is a against infection with L. pneumophila proposed by Joller et al. (19) Gram-negative, non-motile, unencapsulated, pleomorphic, and offers new prospects in the development of antimicrobials against usually coccoid bacterium, between 0.2 and 1.5 mm in diameter intracellular pathogens. Joller et al. used non-specific antibodies (1, 3, 4). It is an intracellular pathogen, classified phylogenetically that form IgG-beads that stimulate the intracellular immune as a Gammaproteobacteria in the family Piscirickettsiaceae, and response in macrophages infected with L. pneumophila, closely related order to Legionella, Francisella, and Coxiella (1). promoting their degradation within lysosomes of infected cells. In Chile, the National Fisheries Service (SERNAPESCA, Servicio The underlying mechanism is suggested to involve induction of Nacional de Pesca) has identified SRS as the most serious health the signaling cascade that counteracts the modulation of the problem facing the Chilean salmon industry (5) owing to its highly endocytic vacuolar traffic (19). The activation of the response of aggressive nature, recurrent outbreaks, and widespread cells infected by L. pneumophila due to the IgG-beads stimulation transmission among other cultivated salmonid species (6–9). In is dependent on the interaction of the Fc region of non-specific 2018, mortalities associated with P. salmonis represented 54.7% and a n t i b o d i e s wi t h th e F c r e c ep t o r ( F c R ) p r e s e n t i n 83.3% of the total mortalities attributed to infectious causes in macrophages (20). Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus In salmonids, antibody-mediated opsonization of particles mykiss), respectively (9). The control and prophylactic strategies enhances phagocytosis and respiratory burst by phagocytes, against P. salmonis have relied on antibiotics and vaccines to date; providing functional evidence for the presence of antibody however, both are inadequate. Antibiotics have been used receptors on leukocytes (21–24). In Atlantic salmon, this indiscriminately to control outbreaks of infection. In 2018, the mechanism is potentially mediated by receptors that may be Chilean aquaculture industry alone utilized over 322 tons of similar to Fc receptors, which are widely distributed on antibiotics, mainly florfenicol and oxytetracycline (10). Moreover, leukocytes in mammals (25, 26). The utilization of antibodies infected salmonids respond poorly to these treatments, likely bound to antigens as a passive immunization strategy against because of the intracellular characteristics of the infective cycle of infectious diseases has been poorly explored. In the context of P. salmonis and the insufficient concentrations of antibiotics that strategies against bacteria such as Flavobacterium psychrophilum reach the intracellular niche to eliminate the bacterium (11). This and Vibrio anguillarum in rainbow trout, the delivery of specific situation is further complicated by the lack of effective vaccines antibodies attenuates the mortality rates and aggressiveness against P. salmonis (12), because prophylactic vaccines do not associated with infectious outbreaks, suggesting a protective provide acceptable levels of protection (11). role and a putative immune stimulatory effect (27, 28). Despite the severe impact of P. salmonis on the Chilean Previously, our group reported that P. salmonis survives at least aquaculture industry, key aspects of this bacterium, such as its 120 h within Atlantic salmon macrophages-enriched cell cultures. life cycle and pathogenic mechanisms, are poorly characterized During this time, the intracellular bacterial load rises, as evidenced (13). It is reported that P. salmonis survives and replicates inside by an increase in the number of copies of the bacterial 16S rDNA macrophage vacuoles (14) that do not mature to fuse them with ribosomal gene. Additionally, we suggest that the survival of the lysosomes to degrade the pathogen (13, 15). Recently, we P. salmonis is favored by perturbations in lysosomal activity, as observed that macrophages infected by P. salmonis have a evidenced by a limited lysosomal proteolytic activity observed in lower number of lysosomes in comparison with those infected cells (16). The aim of this study was to evaluate whether incubated with inactivated P. salmonis; as a consequence, these the use of non-specific antibodies forming IgM-beads activate the infected macrophages exhibited fewer proteolytic foci and also lysosomal response of macrophage-enriched cell cultures infected had a pH close to neutral (16). Moreover, it has been suggested by P. salmonis. The effectiveness of the treatment was analyzed in that P. salmonis induces an anti-inflammatory milieu when it terms of the effect that non-specific antibodies forming IgM- infects macrophages, by manipulating the host cytokine profile beads would have on the modulation of both lysosomal to promote an environment that is favorable for its survival and acidification and proteolytic activity induced by the bacterium replication in salmonid macrophage-like cells (17). when it infects macrophages-like cells of Atlantic salmon. The low effectiveness of antibiotic treatment is not only Additionally, we evaluated the effect on bacterial load and observed against P. salmonis, but also against other intracellular survival of infected macrophages. Frontiers in Immunology | www.frontiersin.org 2 November 2020 | Volume 11 | Article 544718 Pérez-Stuardo et al. IgM-Beads Induce Lysosomal Activity MATERIALS AND METHODS 214 (ATCC N°CRL-1682), as previously described by Fryer et al., 1992 (30). The CHSE-214 cell line was maintained in minimal Experimental Fish essential medium (MEM; Corning) with supplement 5 Atlantic salmon with an average weight of 55 ± 15 g were (Supplementary Table 1) at 16°C. The infected cells were obtained from a local farm and maintained in tanks with observed using conventional inverted light microscopy (Motic freshwater at a biomass of 10–12 kg/m3, with controlled AE31E, Leica Microsystems, Wetzlar, Germany) after 4 to 6 days temperature (14–16°C) and continuous aeration. Water quality post-infection (dpi) to observe any cytopathic effect (CPE) (31). parameters, such as oxygen, pH, and levels of nitrogen Piscirickettsia salmonis was extracted from the supernatant of compounds (i.e., nitrate, nitrite, and ammonia) were infected CHSE-214 cells displaying CPE. Bacterial quantification monitored daily and maintained at constant values. The fish was performed in a Petroff-Hausser chamber (Hausser Scientific, were fed with commercial pellet twice daily (Golden Optima, PA, USA) according to the instructions provided by the Biomar, Puerto Montt, Chile), and acclimatized for 3 weeks prior manufacturer. Cellular debris was eliminated through to the experiments. The maintenance of fish was performed in centrifugation for 5 min at 500 g. Subsequently, P. salmonis accordance with the ethical standards of the Institutional Ethics was centrifuged at 7,500 g for 15 min at 16°C. Macrophage- Committee of Universidad de Santiago de Chile (approved in enriched cell cultures from Atlantic salmon head kidneys were internal report n°364) and the relevant legislation in force. seeded at 6,000 cells/cm2 in six-well flat bottom plates and were incubated for 2 h with P. salmonis at a multiplicity of infection Isolation of Macrophages and Cell (MOI) of 10 bacteria/cell in L-15 medium with supplement 3 Cultures (Supplementary Table 1). Infection was synchronized by Macrophage-enriched cell cultures were obtained from Atlantic centrifugation at 150 g for 3 min at 16°C, and the infected cells salmon head kidneys, as described by Braun-Nesje et al. (29) and were incubated at 16°C. Then, cells were washed twice with 1× by our group in Pé r ez-Stuardo et al. (16) with slight PBS and incubated with L-15 medium with supplement 3 modifications. Briefly, Atlantic salmon head kidney were (Supplementary Table 1). As a control, P. salmonis was aseptically extracted and disaggregated using a cell strainer inactivated by incubation of the pellet in 4% paraformaldehyde (pore size: 70 µm) (BD Falcon, Seaton Delaval, England) (PFA; Sigma Aldrich, MO, USA), diluted in 1× PBS and suspended in Leibovitz-15 medium (L-15; Corning, New York, incubated overnight at 4°C. The bacterial suspension was USA) with supplement 1 (Supplementary Table 1). Mechanical centrifuged for 15 min at 7,500 g at 16°C; then, the disaggregation was performed until a homogeneous cell supernatant was discarded and the pellet was washed three suspension was obtained. The leukocyte fraction was isolated times with 1× PBS. Finally, the bacterial suspension was through a discontinuous gradient in densities of 34 and 51% of centrifuged for 15 min at 7,500 g at 16°C; the supernatant was Percoll (GE Healthcare) diluted in miliQ water, supplemented discarded and the pellet was resuspended in L-15 medium with with 1× Hank’s Balanced Salt Solution (HBSS, Gibco). The cell supplement 3 (Supplementary Table 1). suspension was placed on the Percoll gradient, and centrifuged at 400 g for 40 min at 16°C. The leukocyte interface was collected Evaluation of Lysosomal Acidification and resuspended in L-15 medium with supplement 1 Evaluation of lysosomal acidification in macrophage-enriched cell (Supplementary Table 1). To eliminate the traces of Percoll, cultures was performed by fluorescence analysis using the the cell suspension was centrifuged twice for 7 min at 250 g at 16°C. LysoSensor™ Yellow/Blue probe (LSYB; Thermo Scientific). This The cell suspension was seeded at 40,000 cells/cm2 in L-15 medium ratiometric probe can be used to measure the pH of acidic with supplement 2 (Supplementary Table 1) at 16°C. organelles. The LysoSensor™ dye produces blue and yellow To enrich the primary cell culture with monocytic/ fluorescence in neutral and acidic environments, respectively. macrophage adherent cell population, the primary culture was Therefore, a fluorescence shift from blue (maximum emission at washed the following day with three washes of 1× phosphate- 430 nm) to yellow (maximum emission at 535 nm) indicates a buffered saline (PBS) at pH 7.4. Non-adherent cells were decrease in lysosomal pH (16, 32). discarded and the remaining cells were cultivated with fresh The IgM-beads were prepared with latex beads coated with L-15 medium with supplement 2 (Supplementary Table 1). At 3, immunoglobulin M (IgM). IgM was purified from the serum of 5, and 7 days of cultivation, the cells were washed three times Atlantic salmon (weighing 80 to 100 g) by an affinity protein A with 1× PBS. Non-adherent cells were discarded, and fresh L-15 sepharose column. The presence of IgM was confirmed by medium with supplement 3 (Supplementary Table 1) was added western blotting and ELISA based on a monoclonal antibody to the cultures. (I-14 hybridoma) (33). Additionally, the homogeneity of the IgM SHK-1 cells (Salmo salar; ATCC, American Type Culture preparation was evaluated by SDS-PAGE. In order to assess the Collection, Manassas, VA, USA), which are described as short-term effect of the IgM-beads on lysosomal acidification, the macrophage-like cells (30), were grown at 16°C in L-15 medium macrophage-enriched cell cultures were seeded at 6,000 cells/cm2 with supplement 4 (Supplementary Table 1). in 12-well flat bottom plates with glass coverslips and infected with P. salmonis at MOI 10 for 2 h, as previously described. Piscirickettsia salmonis and Infection Then, infected macrophage-enriched cell culture was incubated Culture and propagation of P. salmonis (strain 9734, ETECMA, for 1 h with 30 beads/cell. After incubation, the cells were washed Puerto Montt, Chile) was performed in salmonid cell line CHSE- twice with 1× PBS and cultivated in fresh L-15 medium with Frontiers in Immunology | www.frontiersin.org 3 November 2020 | Volume 11 | Article 544718 Pérez-Stuardo et al. IgM-Beads Induce Lysosomal Activity supplement 3 (Supplementary Table 1). The analysis of fluorescent products are observed. The cleavage of DQ™ Green lysosomal acidification was performed at 1 and 3 h post- BSA results in the release of fragments with maximum excitation treatment (hpt), according to the described by Pé rez-Stuardo and emission at 505 and 515 nm, respectively (35–37). et al. (16). Additionally, we evaluated the effect of Bovine Serum The macrophage-enriched cell cultures were established at Albumin (BSA)-beads in infected cells as protein-beads control. 6,000 cells/cm2 in 12-well flat bottom plates with glass coverslips To evaluate the lysosomal acidification in macrophage-enriched and infected with P. salmonis, as described in the previous cell cultures infected by P. salmonis, we incubated the cells with section. Subsequently, analysis of lysosomal activity was bacteria for 2 h as was previously described and maintained in performed at 1 and 3 hpt from IgM-beads incubation. The L-15 medium with supplement 3 for 3 and 5 h post-infection macrophage-enriched cells were treated with IgM-beads (or (hpi) (equivalent to 1 and 3 hpt). Similarly, as a control for BSA-beads as control) in the same way as described in the lysosomal activation, macrophage-enriched cell cultures were previous section. Lysosomal activity was also evaluated in incubated with inactivated P. salmonis and evaluated at the infected macrophage-enriched cell cultures incubated with same times as the experimental group. BSA-beads. To evaluate the lysosomal activity in macrophage- At each time point, the infected cells were incubated for 5 min enriched cell cultures infected by P. salmonis, we incubated the with 10 µM LSYB. Subsequently, the cells were washed three cells with bacteria for 2 h as was previously described and times with 1× PBS and fixed for 10 min with 4% (w/v) PFA maintained in L-15 medium containing supplement 3 for 3 (Sigma Aldrich), followed by three additional washes with 1× and 5 hpi (equivalent to 1 and 3 hpt). As positive control for PBS. Finally, the cells were stained by incubating for 1 min with lysosomal activation, macrophage-enriched cell cultures were 1 mg/mL propidium iodide (PI); then, the cells were washed twice incubated with inactivated P. salmonis, and lysosomal activity with 1× PBS and once with MilliQ water to remove the residual was evaluated at the same times as in the experimental group. salts. The samples were mounted on slides with Fluoromount™ Two hours prior to each time point for IgM-beads incubation, (Sigma Aldrich), and images were obtained using a Leica SP8 DQ™ Green BSA 10 µg/mL in L-15 medium containing confocal microscope (Leica, Wetzlar, Germany). Results were supplement 3 (Supplementary Table 1) was added to the obtained by the division of the emission spectrum of the LSYB evaluated cells. Then, the cells were washed three times with probe, specifically to obtain an acidic indicator channel (acquired 1× PBS and fixed using 4% (w/v) PFA, followed by three between 500 and 580 nm) and a neutral-basic indicator channel additional washes with 1× PBS. The cells were stained with (acquired between 450 and 495 nm). According to instructions 1 mg/mL PI as previously described. The samples were mounted provided by the manufacturer. The fluorescence intensity on slides using the mounting solution Fluoromount™ (Thermo obtained in the acidic indicator channel had to be ≥2-fold that Scientific). Micrographs were obtained using a Leica SP 8 obtained in the neutral-basic indicator channel for a lysosome to confocal microscope, and processed and analyzed using Fiji be considered as acidic. The analysis of lysosomal acidity was software (ImageJ 1.52g) (34). The analysis was performed by performed using the software LAS X (version 3.3.0). Briefly, the counting each positive event per cell due to the fluorescence of average fluorescence intensity for each indicator channel was DQ™ Green BSA, from four micrographs, with a merge of the obtained for each lysosome present in the micrographs obtained images from z-stack, containing an average of 30 cells per from the samples. Subsequently, the ratio between the micrograph for each experimental condition. The data were fluorescence intensity values obtained from the two indicator normalized to the number of cells on the micrograph, and the channels was used as the acidity index. An acidic index of x≥2 results are represented as the number of proteolytic events/cell. denoted an acidic pH, while an acidity index between 0