Murine and Human Cathelicidins Contribute Differently to Hallmarks of Mastitis Induced by Pathogenic Prototheca bovis Algae

Prototheca bovis (formerly P. zopfii genotype-II) is an opportunistic, achlorophyllous alga that causes mastitis in cows and skin disease in cats and dogs, as well as cutaneous lesions in both immunocompetent and immunosuppressed humans. Antifungal medications are commonly ineffective. This study aimed to investigate innate immune responses contributed by cathelicidins to P. bovis in the mammary gland using a mastitis model in mice deficient in the sole murine cathelicidin (Camp). We determined P. bovis caused acute mastitis in mice and induced Camp gene transcription. Whereas, Camp−/− and Camp+/+ littermates had similar local algae burden, Camp+/+ mice produced more pro-inflammatory cytokines, TNF-α, and Cxcl-1. Likewise, Camp+/+ bone marrow-derived macrophages were more responsive to P. bovis, producing more TNF-α and Cxcl-1. Human cathelicidin (LL-37) exhibited a different effect against P. bovis; it had direct algicidal activity against P. bovis and lowered TNF-α, Cxcl-1, and IL-1β production in both cultured murine macrophages and mammary epithelial cells exposed to the pathogenic algae. In conclusion, cathelicidins were involved in protothecosis pathogenesis, with unique roles among the diverse peptide family. Whereas, endogenous cathelicidin (Camp) was key in mammary gland innate defense against P. bovis, human LL-37 had algicidal and immunomodulatory functions.


INTRODUCTION
Prototheca species are unicellular achlorophyllous algae, 3-30 µm in diameter, that lack a specific glucosamine cell wall or chloroplasts (Baudelet et al., 2017). Their reproduction is asexual, with endospores being released from sporangia (Jagielski and Lagneau, 2007). Prototheca spp. are widely distributed in the environment, particularly in organic matter with a high moisture content (Scaccabarozzi et al., 2008). Prototheca spp. are also pathogenic and provoke a variety of maladies in animals. In dogs and cats, infection with Prototheca spp. causes either cutaneous lesions or systemic disease with hemorrhagic enteritis and progressive retinal degradation (Huth et al., 2015;Carfora et al., 2017). In humans, Prototheca spp. causes rare chronic skin or articular infections (Seok et al., 2013) or disseminated infections mostly in immunodeficient patients (Tello-Zavala et al., 2013).
Among Prototheca species, P. bovis has been proposed as a major cause of chronic mastitis in cattle. Traditional taxonomic studies on Prototheca species identified two genotypes (GT): P. zopfii GT-I and P. bovis (formerly P. zopfii GT-II) (Roesler et al., 2006). P. bovis was isolated from the milk of cows with mastitis and identified as the causative agent of bovine mastitis (Jagielski et al., 2012(Jagielski et al., , 2019aBozzo et al., 2014). Prototheca zopfii GT-I is commonly isolated from the environment and occasionally causes granulomatous lesions in experimentally infected bovine udders (Ito et al., 2011) and protothecosis in humans (Hirose et al., 2018). Clinical signs of protothecal mastitis in cattle can include fever, pain, edema, anorexia, and lethargy, although it is most commonly subclinical, causing decreased milk production (Wawron et al., 2013). Bovine mastitis caused by P. bovis is refractory to most therapeutic agents (Jagielski et al., 2012) and has a very low rate of spontaneous remission (Janosi et al., 2001). Virulence factors, including intracellular heat shock proteins (Hsp70) and proteins that augment peroxisome metabolism to resist harsh environments (e.g., macrophage phagolysosomes) are mostly present in pathogenic P. bovis (Irrgang et al., 2015;Zeng et al., 2019). From an epidemiological perspective, cattle infected with Prototheca spp. are a common source of Prototheca spp. for humans, with immunocompromised farmers at highest risk (Lass-Florl and Mayr, 2007). Since Prototheca spp. may survive chlorination by forming biofilms (Kwiecinski, 2015), and be returned to the environment via sewage effluent and household waste (Wirth et al., 1999), cows with mastitis caused by P. bovis are a risk factor in epidemiology of protothecosis (Chao et al., 2002).
Little is known about innate mammary gland defenses during P. bovis infection. However, cultured mammary epithelial cells from cattle and mice undergo oxidative stress and apoptosis when exposed to P. bovis (Shahid et al., , 2017a. Moreover, bovine mammary epithelial cells respond to P. bovis infection with upregulated expression of Toll Like Receptors (TLRs)−2 and−4, pro-inflammatory cytokines (TNF-α, IL-1β, IL-8) and β-defensin-5 (Deng et al., 2016). To further understand innate mammary defenses in protothecosis, we focused on cathelicidins. Cathelicidins are cationic amphipathic peptides with an N-terminal domain containing a signal peptide, a well-conserved central cathelin domain, and a variable Cterminal domain (Schauber et al., 2003). The C-terminal domain is cleaved to produce the peptide with antimicrobial and antifungal activity (Ordonez et al., 2014). In humans, a single cathelicidin gene (cathelicidin antimicrobial peptide, Camp) yields this C-terminal active peptide termed leucineleucine with 37 amino acid residues (LL-37) (Agerberth et al., 1995). In mice, the functional homologous peptide is cathelicidin-related-antimicrobial-peptide (Cramp) encoded by the gene Camp (Nizet et al., 2001). Cathelicidins are abundantly expressed in mammalian cells, mainly neutrophils and epithelial cells and thus, are present in various organs/tissues, including skin, eyes, mouth, lungs, intestine, and mammary gland (Zanetti, 2005;Cobo et al., 2012Cobo et al., , 2017. Cathelicidins have been involved in various host responses against bacterial and parasitic intracellular pathogens through activation of cytokines/chemokine secretion (Boucher et al., 2018;Cirone et al., 2019;Marin et al., 2019). In sheep and murine mammary glands and in human milk, cathelicidins also have antimicrobial and anti-inflammatory effects (Murakami et al., 2005;Addis et al., 2013). Moreover, cathelicidins increased in bovine mammary epithelial cells exposed to a main mastitis pathogen, Staphylococcus aureus (Ibeagha-Awemu et al., 2010). Thus, we hypothesized that cathelicidins produced by the mammary gland contribute to host innate defense against P. bovis infection. Using genetically mutant mice that lack cathelicidin (Camp −/− ) and cultured murine macrophages and mammary epithelial cells, we demonstrated that endogenous cathelicidins regulate mammary gland inflammation and macrophage activity against P. bovis, whereas exogenous human cathelicidins (LL-37) downregulated epithelial and macrophage responses and had algicidal activities.

Ethics Statement
Animal experiments were conducted in accordance with Canadian Guidelines for Animal Welfare (CGAW) and the University of Calgary Animal Care Committee (Animal Protocol AC16-0061).

Experimental Induction of Murine Mastitis by Prototheca bovis
C57BL/6 (6-8 wk. old) lactating female wild-type Camp +/+ and cathelicidin-null Camp −/− C57BL/6 mice (B6.129X1-Camp tm1Rlg/J ; The Jackson Laboratory) were housed in a specific pathogen-free environment with ad libitum access to feed and water (University of Calgary). In these Camp −/− mice, there is deletion of exons 3 and 4 of the cathelicidin gene Camp; whereas some gene portions could be detected, they do not produce functional cathelicidin peptides (Nizet et al., 2001). Camp −/− and Camp +/+ mice were infected intramammary (10-14 days after parturition) with either P. bovis (50 µL containing 1 × 10 5 colony forming units (CFU)/mL) or an equal volume of phosphate buffered saline (PBS) (control) in the left 4th and right 4th mammary glands (L4 and R4) (n: 4 per group). Mice were euthanized by carbon dioxide followed by cervical dislocation at 4 days post-infection when they had indications of clinical mastitis. This termination point was chosen based on preliminary studies where mice become lethargic and before humane end points associated with longer intervals.
Immediately after euthanasia, all mammary glands were excised, weighed, and collected as follows: A portion was incubated into Trizol (Invitrogen) for gene quantification and another fixed in formalin (10%) solution, embedded in paraffin wax, sectioning (5 µm) and stained with hematoxylin and eosin (H&E) or toluidine blue for histological examination (Seok et al., 2013). Prototheca organisms were identified by specific staining, using periodic acid-Schiff (PAS) and Grocott methenamine silver (GMS).

Determination of Prototheca bovis in Infected Mammary Tissue
Prototheca infection in mammary tissues was assessed by histological counting of Prototheca sporangia and Prototheca culture. For counting, sporongia of Prototheca spp. (7-30 µM in diameter) were counted in 10 images of the mammary gland per mouse, individually and randomly captured at 40x magnifications. For culturing, the entire mammary gland was homogenized in sterile tubes containing 1 mL of 0.025% Triton X-100 in PBS. 10-fold homogenate dilutions were spread onto duplicate plates of Sabouraud dextrose agar (Sigma-Aldrich). Number of colonies, typically with a granular serrated shape, grayish white and with a central protrusion, was determined after 2-3 days of incubation (Gogoi-Tiwari et al., 2017).

Direct Algicidal Activity of Cathelicidin Against P. bovis
For killing assays, P. bovis grown into Sabouraud in dextrose broth (Sigma-Aldrich) (37 • C, 2 days on a rotary shaker) was poured into 96 wells plates (1 × 10 5 CFU/mL in logarithmic growth phase) and simultaneously incubated with LL-37 peptides (H-6224; Bachem) (1 and 2 µM diluted in distilled water) (37 • C, up to 1 day). Final Prototheca spp. concentration was determined by CFU/mL and plotted after conversion to logarithm (log 10 ) using a standard curve. P. bovis and media only were used as controls.

Statistical Analyses
Normality was assessed using D'Agostino & Pearson omnibus normality or Shapiro-Wilk (Royston) tests. Analytical data represented as histograms were recorded as mean values with bars representing standard errors of the mean (SEM) from a minimum of two independent experiments, with data obtained in triplicate, unless otherwise stated. All statistical comparisons were performed using one-way analysis of variance (ANOVA) with a post hoc Bonferroni correction for multiple group comparisons (Graph Pad Prism, 5.0). A p-value was assigned to each experimental group with reference to a control group. A p-value of <0.05 was considered significant. Microscopically, uninfected control mice had normal mammary gland architecture whereas both Camp +/+ and Camp −/− infected mice developed severe acute mastitis ( Figure 1A). Mammary epithelial lobules, interstitial fibrovascular tissue and interlobular adipose tissue in both Camp +/+ and Camp −/− infected mice had infiltration of a mixed population of inflammatory cells, including lymphocytes, plasma cells, neutrophils, macrophages (Figures 1A,B) and rare mast cells ( Figure 1C). More macrophages infiltrated the mammary in P. bovis infected Camp −/− mice (p < 0.05, Figure 1D). Neutrophils were numerous in P. bovis infected mice, but there was no difference between Camp −/− and Camp +/+ mice (p > 0.05, Figure 1E).

P. bovis-Challenged
Mammary infection with P. bovis was confirmed microscopically in Camp +/+ and Camp −/− mice 4 days post-infection. Using PAS and GMS staining, round to oval sporangia with internal divisions compatible with Prototheca spp. were visible both free within alveolar lumen and throughout the mammary gland interstitium (Figure 2A). The grade of infection with P. bovis did not differ between Camp +/+ and Camp −/− mice at 4 days post-infection, as determined by histological counting of sporangia and Prototheca spp. culture ( Figure 2B).
The general process of mastitis is characterized by upregulated synthesis of certain pro-inflammatory cytokines, such as TNFα and IL-1β (Bannerman et al., 2004). P. bovis induced mRNA synthesis and protein secretion of TNF-α in mammary glands from both Camp +/+ and Camp −/− mice infected with P. bovis, with the highest concentration in Camp +/+ mice (p < 0.05, Figures 3A,B).
Cxcl-1 chemoattracts leukocytes to sites of infection (De Filippo et al., 2013); recruitment of neutrophils and monocytes/macrophages from blood to milk compartments is a critical defense mechanism in bovine mastitis . In our study, Cxcl-1 mRNA was upregulated in P. bovis infected mammary glands of Camp +/+ relative to Camp −/− mice (p < 0.05, Figure 3C). Secretion of Cxcl-1 increased in mammary glands from both Camp +/+ and Camp −/− infected mice relative to non-infected controls but, the highest level was in Camp −/− infected mammary glands (p < 0.05, Figure 3D). Transcriptional IL-1β gene levels were increased after P. bovis challenge, but not different between Camp +/+ and Camp −/− infected mammary glands (p > 0.05, Figure 3E). Transcriptomic gene expression of host defense peptides in mammary tissue revealed that β-defensin-4 was not increased after P. bovis infection in mammary glands of Camp −/− and Camp +/+ mice (p > 0.05, Figure 3F), whereas Camp mRNA was increased in infected mammary glands of Camp +/+ mice compared to uninfected Camp +/+ mice (p < 0.05, Figure 3G). No Camp gene expression was detected in Camp −/− mice (data not shown). Taken together, P. bovis provoked acute mastitis in experimentally challenged mice. Furthermore, endogenous production of cathelicidins, induced by the algae, was associated with increased synthesis of pro-inflammatory TNF-α and Cxcl-1.

Human Cathelicidin LL-37 Inhibited Growth of P. bovis and Decreased Pro-inflammatory Responses in Infected Macrophages and Mammary Epithelial Cells
To mechanistically explore the role of cathelicidin in P. bovis mastitis, we studied whether exogenous cathelicidin exert algicidal or immunomodulatory effects in key cellular components in mastitis: macrophages and mammary epithelium. In regard to direct killing, synthetic human cathelicidin LL-37 (1 and 2 µM) decreased in vitro growth of P. bovis up to 8 h, with some inhibitory activity still present at 24 h (p < 0.05, Figure 5). In reference to the immunomodulatory role of LL-37, P. bovis upregulated mRNA expression of TNF-α, Cxcl-1, and IL-1β in murine phagocytic J774.A1 cells (at 2 and 8 h post-infection), whereas co-stimulation with LL-37 decreased concentrations of TNF-α, Cxcl-1, and IL-1β mRNA expression (p < 0.05, Figures 6A-C). Next, the immunoregulatory role of LL-37 was assessed in murine mammary epithelial cells (HC11) capable of producing milk casein protein. Challenge of HC11 cells with P. bovis induced early (2 h) TNF-α, Cxcl-1 and IL-1β gene expression (p < 0.05, Figures 6D-F), whereas costimulation with LL-37 lowered these mRNA TNF-α, Cxcl-1, and IL-1β responses. Addition of LL-37 to the mammary epithelium stimulated transcriptional expression of IL-1β and TNF-α at a later point (8 h post-infection) (p < 0.05, Figures 6D-F). Thus, human cathelicidin LL-37 reduced synthesis of proinflammatory cytokines in murine macrophages and mammary epithelium, whereas it had some direct killing effects on P. bovis.

DISCUSSION
Although P. bovis is a major cause of insidious and chronic (>30 days) mastitis in cattle (Moller et al., 2007;Wawron et al., FIGURE 5 | Synthetic human cathelicidin LL-37 inhibited in vitro Prototheca bovis. Prototheca bovis (1 × 10 5 CFU/mL in logarithmic growth phase) was incubated with synthetic human cathelicidin LL-37 peptides (up to 2 µM for up 12 h at 37 • C) into Sabouraud dextrose broth and algae concentration were determined using a standard curve, with only P. bovis and expressed as log CFU/mL. Histogram of remaining P. bovis after peptide treatment. Data are shown as means ± SEM (n = 3 independent experiments done in triplicate). *p < 0.05, **p < 0.01 (one-way ANOVA post hoc Bonferroni correction) was considered significant.
2013; Bozzo et al., 2014;Jagielski et al., 2019b), the udder innate immune response to pathogenic Prototheca spp. remains poorly understood. In this study, P. bovis triggered severe acute mastitis in mice and naturally occurring cathelicidins aided in establishing local inflammation by promoting synthesis of pro-inflammatory cytokines. Two main lineages of Prototheca spp. have been relevant in public health: a dominant species typically associated with dairy cattle, namely P. ciferrii (formerly P. zopfii GT-I), P. blaschkeae, and P. bovis (formerly P. zopfii GT-II) and others human-associated Prototheca spp. (i.e., P. wickerhamii, P. cutis, P. miyajii) (Jagielski et al., 2019a). In this study, we used a Prototheca spp. identified as P. bovis following a taxonomic approach commonly accepted for Prototheca (Roesler et al., 2006) and a cytb-based genotyping used for unambiguous Prototheca spp. identification (Jagielski et al., 2018) based on the protothecal phylogeny (Jagielski et al., 2019a).
Although Camp −/− mice had increased infiltration of macrophages in infected mammary glands, they also produced less local expression and secretion of TNF-α and Cxcl-1. Moreover, BMDMs isolated from Camp −/− mice had lower in vitro synthesis of pro-inflammatory TNF-α, IL-1β, and Cxcl-1 in response to P. bovis. That Cxcl-1 secretion in infected Camp −/− mice ended up higher than in wild type mice denoted a lack of association between Cxcl-1 mRNA and protein kinetics. Such differences in cytokine mRNA/protein expressions are usually due to post-transcriptional mechanisms (Greenbaum et al., 2003) or perhaps, Cxcl-1 gene transcription is augmented only at early points by cathelicidins or the Cxcl-1 protein half-life is longer compared with mRNA. Alternatively, increased Cxcl-1 in infected Camp −/− mice may correspond to an overwhelming and generalized inflammation, higher than any cathelicidin effect in the wild type. Overall, the observed role of endogenous cathelicidin promoting Cxcl-1 in protothecal mastitis could be critical in attracting leukocytes early to sites of infection and retain macrophages into the udder during the innate immune response against pathogenic algae. In agreement, murine cathelicidin Cramp attracted in vitro T-cells, macrophages, neutrophils, eosinophils and mast cells via an FPRL-1 receptor (Yang et al., 2000;Kurosaka et al., 2005;Tjabringa et al., 2006) whereas LL-37 chemoattracted leukocytes via G-protein coupled receptors (Vandamme et al., 2012). Cathelicidins could also chemoattract neutrophils indirectly, by inducing chemokines. In this regard, cathelicidins induced CXCL8 transcription alone and in synergy with TNFα though Src family kinase pathways downstream of P2X7R in keratinocytes (Nijnik et al., 2012;Chen et al., 2013) and gingival fibroblasts (Montreekachon et al., 2011) and in an EGFR-dependent manner in skin (Wu et al., 2010) and airway epithelial cells (Tjabringa et al., 2006). Whereas, the influx of leukocytes in the mammary gland during P. bovis infection could be functionally regulated by cathelicidin, other cathelicidin-independent chemoattractive effects cannot be disregarded. For instance, cell-surface glycoproteins involved in cell-cell interaction, such as CD44, which mediates specific adhesion of bovine blood polymorphonuclears to mammary epithelial cells (Gonen et al., 2008), could initiate recruitment of inflammatory cells during mastitis.
Our study demonstrated that a cathelicidin can exert an immunosuppressive effect in another species; in particular, human LL-37 mitigated production of pro-inflammatory TNFα, IL-1β, and Cxcl-1 in murine macrophages and mammary epithelial cells challenged with P. bovis. This immunomodulatory role of human cathelicidin LL-37 was not surprising, as LL-37 reduced secretion of IL-1β, IL-6, IL-8, and TNF-α in human and murine neutrophils exposed to heat-inactivated Pseudomonas aeruginosa or Staphylococcus aureus (Alalwani et al., 2010). Likewise, human or murine macrophage-like cells had reduced expression of TNF-α and nitric oxide (NO) in the presence of LL-37 when stimulated with bacterial lipooligosaccharide or LPS (Scott et al., 2002;Zughaier et al., 2005). Mechanistically, LL-37 could directly bind LPS in macrophages, thereby suppressing IL-6, IL-1β, and TNF-α synthesis Tomasinsig et al., 2010).
In terms of algicidal effects, there was no differences between Camp +/+ and Camp −/− infected mice in P. bovis burden in mammary glands at an early point of infection (4 days). In contrast, human cathelicidin LL-37 in vitro directly reduced number of P. bovis in a dose-dependent manner. These killing properties of cathelicidins, LL-37 in this case, are related with the cationic peptide capacity to bind and disrupt negatively charged microbial membranes, leading to cell death due to destabilization of plasma membranes and efflux of ATP and proteins (Tsai et al., 2014). Cathelicidins can also cross membranes and disrupt intracellular processes, including RNA and DNA synthesis and protein degradation (Mansour et al., 2014). Although the role of cathelicidins in fungal/algal infectious is not fully explored, there is increasing evidence of protective effects. Human LL-37 has anti-fungus and algae activity against Candida albicans, Malassezia furfur, FIGURE 6 | Synthetic human cathelicidin LL-37 mitigated production of pro-inflammatory cytokines in murine macrophages and mammary epithelia infected with Prototheca bovis. Murine phagocytic (J774.A1) macrophages (A-C) and mammary epithelial (HC-11) cells (D-F) were challenged with P. bovis (1 × 10 5 cfu/mL) ± synthetic human cathelicidin LL-37 (2 µM) (37 • C with 5% CO 2 ). Transcriptomic expression of (A,D) TNF-α, (B,E) Cxcl-1 and (C,F) IL-1β were determined at 2 and 8 h post-infection. mRNA synthesis was quantified using RT qPCR. Data are shown as means ± SEM (n = 3 independent experiments done in triplicate). *p < 0.05, **p < 0.01 (one-way ANOVA post hoc Bonferroni correction) was considered significant. Ns, not significant.
Trichophyton rubrum and Trichophyton. mentagrophytes (Lopez-Garcia et al., 2006;Tomasinsig et al., 2012;Tsai et al., 2014). Cattle have a vast repertoire of cathelicidins, with at least 8 naturally occurring  and direct killing effects on P. bovis observed in cathelicidins from cattle origin. Bovine myeloid antimicrobial peptide (BMAP)-28 displayed quick anti-Prototheca activity (<1 h), with extensive surface blebbing and release of intracellular material due to cell permeabilization (Tomasinsig et al., 2012) whereas other bovine host defense peptides (bactericin 5 and lingual anticrobial peptide; LAP) also had anti Prototheca activity, albeit through non-lytic mechanisms (Tomasinsig et al., 2012). Conversely, a different susceptibility of P. bovis to cathelicidins of diverse origins agrees with studies in bacteria. Human LL-37 and murine Cramp had dissimilar minimum inhibitory concentrations (MICs) against Citrobacter rodentium, Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Salmonella spp. and Helicobacter pylori (Iimura et al., 2005;Coorens et al., 2017;Marin et al., 2019). In addition, endogenous Cramp had a pro-inflammatory role in murine mastitis induced by P. bovis, whereas in contrast, LL-37 reduced synthesis of proinflammatory cytokines in macrophages and mammary epithelia. Although human LL-37 and murine Cramp are considered homologous based on broadly similar and interspecies functions (antimicrobial and immunomodulatory) (Barlow et al., 2011) and structural properties (α-helical and net charge of +6), they actually share <70% sequence identity in their active peptide (Nizet et al., 2001;Tomasinsig and Zanetti, 2005). This may explain singular activities for each peptide and indeed, human LL-37 binded dsRNA and activated TLR3 signaling in early endosomes but murine Cramp did not (Singh et al., 2013). Likewise, LL-37 was more effective in reducing S. aureus proliferation in vitro when compared to Cramp (Coorens et al., 2017). In our study, murine cathelicidin Cramp may not have killing properties against P. bovis or perhaps acts later on the pathogen burden, when immune defenses are developed and more Cramp is available. Alternatively, Cramp may have indirect antimicrobial effects, promoting neutrophil killing by increasing production of reactive oxygen species (ROS) and enhancing phagocytic activity (Alalwani et al., 2010). In all cases, cathelicidin LL 37 appeared to be a good model of cathelicidins and potential target molecule for developing therapeutics, although comparative studies with various cathelicidins and their derivatives may offer distinctive immunomodulatory advantages.
Mast cells in mammary glands infected with P. bovis were an unexpected finding. Mast cells are immune effectors that degranulate pro-inflammatory granules rich in histamine and heparin upon activation. LL-37 stimulated degranulation of mast cells (Niyonsaba et al., 2010) through MAP kinases p38 and ERK phosphorylation, increasing vascular permeability in skin (Chen et al., 2006). Thus, such release of histamine, proteoglycans, serotonin and serine proteases from degranulated mast cells, likely regulated by cathelicidin, may be important in pathogenesis of P. bovis and other mastitis pathogens (e.g., Staphyloccocus aureus, Pseudomonas) by augmenting inflammation (e.g., increasing permeability of capillaries to permit leukocyte extravasation).
In summary, pathogenesis of P. bovis, a globally ubiquitous and environmental (grass, water, trees) algae (Rösler et al., 2003) capable of infecting various vertebrate hosts (Seok et al., 2013;Capra et al., 2014;Carfora et al., 2017) remains poorly understood. Therapeutic control for human and animal protothecosis is on demand. Whereas, a human cathelicidin (LL-37) could kill P. bovis, endogenous cathelicidin (Cramp) contributed to the inflammatory response during P. bovisinduced mastitis, likely recruiting early leukocytes, thereby aiding pathogen control. Whereas, we focused on early steps in protothecal mastitis, cathelicidins could have other effects through later stages of inflammation due to known interactions with many cell types, including endothelial cells (Koczulla et al., 2003), epithelial cells (Tjabringa et al., 2003), mast cells  and macrophages (Scott et al., 2002). Cathelicidins were pleitrophic, either preventing (Kirikae et al., 1998) or augmenting inflammatory reactions, e.g., skin with rosacea (Yamasaki et al., 2007). Therefore, this study represented a proof-of-concept regarding the role of innate immune responses contributed by cathelicidins in protothecal mastitis; therefore, we propose LL-37 has potential as a therapeutic compound for controlling pathogenic algae. More studies are needed to decipher the kinetics of myeloid and non-myeloid cells and cytokine milieu that regulate specific functions of cathelicidins in mastitis.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal experiments were conducted in accordance with Canadian Guidelines for Animal Welfare (CGAW) and the University of Calgary Animal Care Committee.

AUTHOR CONTRIBUTIONS
MS, PC, and JG performed the in vitro and in vivo experiments. CK conducted the histopathologic analysis. MS and JG performed the analysis of data and prepared the figures. MS, BH, HB, and EC conceived this research, designed the experiments, and wrote the manuscript. All authors reviewed the manuscript.