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A portrait of the immune response to proliferative kidney disease (PKD) in rainbow trout

Abstract Proliferative kidney disease (PKD), caused by the myxozoan Tetracapsuloides bryosalmonae, is one of the most serious parasitic diseases of salmonids in which outbreaks cause severe economic constraints for the aquaculture industry and declines of wild species throughout Europe and North America. Given that rainbow trout (Oncorhynchus mykiss) is one of the most widely farmed freshwater fish and an important model species for fish immunology, most of the knowledge on how the fish immune response is affected during PKD is from this organism. Once rainbow trout are infected, PKD pathogenesis results in a chronic kidney immunopathology mediated by decreasing myeloid cells and increasing lymphocytes. Transcriptional studies have revealed the regulation of essential genes related to T‐helper (Th)‐like functions and a dysregulated B‐cell antibody type response. Recent reports have discovered unique details of teleost B‐cell differentiation and functionality and characterized the differential immunoglobulin (Ig)‐mediated response. These studies have solidified the rainbow trout T. bryosalmonae system as a sophisticated disease model capable of feeding key advances into mainstream immunology and have contributed essential information to design novel parasite disease prevention strategies. In our following perspective, we summarize these efforts to evaluate the immune mechanisms of rainbow trout during PKD pathogenesis.

and, therefore, arguably the most substantive parasitic diseases in aquaculture. Notable outbreaks and economic constraints caused by other parasitic infections that may have previously gone unreported are also suggested to be on the rise. 11 Fish are one of the largest groups of vertebrates and the first animal group exhibiting both innate and adaptive immunity. 12 While many immunological elements of the innate and adaptive immune system are common to mammals and fish, there are several differences concerning both elements and functionality. For example, unlike mammals, in teleost fish the kidney is the main hematopoietic tissue in the absence of bone marrow with the spleen representing the only systemic secondary lymphoid organ in the absence of lymph nodes. While as in mammals, fish possess numerous local mucosal-associated lymphoid tissues (gills, nares, gut and skin) as well as lymphoid tissue associated with the liver and thymus. Following the two rounds of whole-genome duplication (WGD) that occurred in the common ancestor of vertebrates, a third genome duplication occurred in the stem lineage of teleost fishes, which largely accounts for fish-specific evolutionary trajectories in both innate and adaptive immunity. 13 Thus, as a consequence, many of the genes involved in immunity are at least duplicated in salmonids. 13 Additionally, many aspects of adaptive immune function appear to have evolved independently in fish, with numerous teleost immune genes being at least duplicated. 13,14 In the past years, a great effort has been made to expand our knowledge of the evolution and diversification of vertebrate immune systems as well as identifying important targets for disease prevention across many species. In light of these advances, how immunity against parasites is organized and regulated is being explored with great progress.

| Proliferative kidney disease: Impact and life cycle
Proliferative kidney disease (PKD) is one of the most serious parasitic diseases of fish in which outbreaks are linked to global warming, given that incidence and severity of PKD has increased largely owing to seasonal increases in water temperatures. 15,16 PKD outbreaks have resulted in severe economic constraints for freshwater fish farmers throughout Europe and North America. Economic losses in rainbow trout (Oncorhynchus mykiss) production attributed to the disease in 2004 in the UK alone were estimated at £2.5 million (~£4 million in the current market). 17 During severe outbreaks, mortality can reach up to 95%-100%. 15,18 PKD has also been implicated in the longterm decline of wild brown trout (Salmo trutta) populations in Switzerland. 19,20 Additionally, the parasite has been observed in an increasing number of European countries, reaching as far north as Iceland. [21][22][23] Recently, the disease was also identified as the cause of a mass mortality event of fish during a heat wave in North America. 24 PKD is caused by the myxozoan parasite Tetracapsuloides bryosalmonae. The T.bryosalmonae life cycle exploits two hosts, salmonid fish, the vertebrae host and freshwater bryozoans, the invertebrate host. Infective T. bryosalmonae spores are released from bryozoans, into the water, infecting the fish host via the gills. Following attachment of a spore to the fish host, a single sporoplasm invades primarily the skin epithelium. Subsequent parasite stages migrate, via the vascular system, to the primary target organ, the kidney. 25 Additionally, parasites can also colonize other organs, including the spleen and liver. The trout kidney is located ventral to the backbone and has two main regions extending from the base of the cranium (anterior kidney) to the caudal region (posterior kidney). The anterior kidney is interdigitated with adrenal-like tissue, has no renal function and lacks nephrons and is the primary site for lymphohaematopoiesis where B cells develop and where most proliferating B-cell precursors are located. 26 The posterior kidney possesses both renal and immune tissues, hosting substantial populations of partially activated B cells and plasmablasts. 26 T. bryosalmonae mature as sporogonic stages in the kidney tubules and collecting ducts (coelozoic sporulation) with mature spores, infective to bryozoans, only released in the urine of native brown trout (Salmo trutta) and brook trout (Salvelinus fontinalis). 27 In Europe, non-native rainbow trout develop severe PKD clinical signs. They do not shed spores infective to bryozoans, which precludes the completion of the parasite life cycle and are, thus, considered as dead-end hosts. 27 The immunosuppressive nature of PKD can increase fish susceptibility to secondary infections. 28 Despite high mortalities in naïve fish stocks, depending on both biotic and abiotic factors, severely infected rainbow trout can on occasion survive the disease, clear the parasite burden, restore kidney structure and develop a form of protective immunity against re-exposure. 29,30

| Pathological and histological changes during PKD pathogenesis
In the kidney, T. bryosalmonae penetrates the interstitial tissue, multiplies and differentiates from extrasporogonic to renal sporogenic stages. Due to the immune nature of the organ, parasite development provokes a chronic immunopathology characterized by a lymphocytic hyperplasia, hyperimmunoglobulinaemia and renal atrophy. 15,16 Histopathological changes in the kidney can include thrombus formation, necrotizing vasculitis, a strong hyperplastic response, proliferative and diffuse granulomatous nephrosis, leading to the deterioration of renal tubules. 31,32 Eventually, renal lesions are contained, and proliferation and infiltration in the interstitium become displaced by fibrotic tissue followed by tissue regeneration. 29 Apart from a marked kidney swelling, other gross pathological changes include ascites, gill and liver pallor, systemic anaemia, sub-capsular renal oedema, liver and splenic discolouration and enlargement. 15,16 A similar type of cellular reaction to the kidney can occur in the spleen, and in heavily parasitized fish, a progression to extensive collagen formation has been reported. Furthermore, in cases when the spleen has been heavily enlarged, patches of greyish mottling beneath the capsule and throughout the stroma can be seen. 18 Despite this strong host reaction in the spleen, almost all immunological studies of PKD pathogenesis have been focused on the kidney and to a much lesser extent on the liver and spleen despite the presence of the parasite in these organs.

| General fish host immune response to T. bryosalmonae
A decrease in myeloid cells and the in situ proliferation of lymphocytes are the most notable cellular aspects of PKD pathogenesis. 33,34 Comprehensive transcriptional studies have revealed the regulation of a number of important genes related to T-helper (Th)-like functions, a dysregulated B-cell antibody type response and also the further inhibition of mechanisms of the innate immune response. [34][35][36] Recent reports have also disentangled several unique details of rainbow trout B-cell differentiation and functionality, characterizing the differential immunoglobulin (Ig)-mediated response in T.
bryosalmonae-infected fish. 37 Additional studies have described the role cytokines of the tumour necrosis factor (tnf) superfamily, such as B-cell activation factor (baff) and a proliferation-inducing ligand (april), 38 which are now also known to play a major role in B-cell differentiation and survival in rainbow trout. 39 An overview of these mechanisms as well as those presented in this review is provided in Table 1. Collectively, these studies have solidified the rainbow trout-T bryosalmonae system as a sophisticated immunological model for teleost B-cell research capable of feeding major advances into mainstream immunology.
Apart from investigations of rainbow trout, a few transcriptional reports have described the immune response during T. bryosalmonae infection of brown trout. For instance, in an earlier study Ig light chain (igl) and igm sec (secretory form) transcripts were found to be upregulated in the kidney of infected brown trout. 40,41 While a recent study by Bailey et al. 42 expanded on this to measure a panel of genes encoding for Th-1 and Th-2-like cytokines and markers of myeloid, natural killer and B-and T-cell subsets, their findings also detailed a strong B-cell antibody type response in brown trout. However, the authors did describe some subtle differences in sequential aspects of the immune response and of immune genes that correlated with parasite intensity for brown trout, in contrast to rainbow trout, in terms of the B-cell response and Th-like transcripts that may be linked to PKD pathogenesis. Still, further research especially at the functional level is required as nothing currently exists of this in brown trout to clarify whether there are actually specific differences in B-cell diversity in comparison with rainbow trout. 42 Thus, the present review places the emphasis on the efforts to evaluate the cells and pathways involved in the immune response of rainbow trout during PKD pathogenesis. Along the way, we explore the capabilities and discuss possibilities for future research using this disease model.

| Toll-like receptors
The innate immune response is the first line of defence against invading pathogens, recognizing highly conserved microbial structures known as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) through pattern recognition receptors (PPRs). PRRs and their signalling pathways have essential roles in triggering inflammatory responses that eliminate invading microorganisms and damaged cells. 43 transcripts were also upregulated at grade 1, in addition to the higher grades. However, tlr18 and tlr21 were downregulated at every grade investigated, with tlr3, tlr8b2 and tlr9 expression inhibited at only the more advanced disease stages. Concerning the nonmammalian TLRs that were elevated during PKD pathogenesis, tlr19 and tlr20 have also been reported to play a role in the fish host immune response to ectoparasites. For example, tlr20 was significantly upregulated in the gill, liver, head kidney, tail fin and spleen of goldfish (Carassius auratus) challenged with Dactylogyrus intermedius, 47 while tlr19, in channel catfish (Ictalurus punctatus) challenged with Ichthyophthirius multifiliis, was significantly elevated in the skin and gill tissues relative to the controls. 48 The collective expression patterns of nonmammalian tlr19 and tlr20 during PKD promote the idea of investigating these molecules at an initial stage at the port of entry for T. bryosalmonae, the gills. Additionally, examining markers for DAMPs during host recovery from clinical infection in the kidney, in which fibroblasts and extracellular matrix components as well as dying or stressed immune cells may secrete DAMPs. This represents an important area for future research to determine whether homeostatic recovery processes correlate with parasite clearance and tissue resolution.

BOX 1 Experimental techniques to study PKD pathogensis
(1) Natural exposure-Sampling infected fish from a natural outbreak source in either a fish farm, lake or river. 35,36,49 Infected fish are maintained and studied in aquarium facilities, or by taking fish directly at a T. bryosalmonae-infected fish farm thus transporting them once infected to an aquarium facility. 25,50 Or by exposing specific pathogen-free fish to such enzootic waters. In this context, samples might not always be studied chronologically as it is difficult to determine the precise start of exposure, and immune parameters are often TA B L E 1 An overview of the main immune features described in the present review focusing on the immune mechanisms investigated in rainbow trout during PKD pathogensis. The abbreviations in the table are consistent with those mentioned in the text

Immune mechanism Section
Brief summary of response during

T.bryosalmonae infection in rainbow trout Study conditions
Supporting literature  36 In addition, there is a risk of coinfections using these techniques.
(2) Laboratory controlled exposure-This is carried out using parasites cultured in the laboratory obtained from either maintaining T. bryosalmonae-infected bryozoa 27 or from parasites collected directly from the field (via sampling infected bryozoa) for infection. 34 Either exposure procedure requires an initial source of infected bryozoans and also the methodology to confirm and quantify parasite stock for infection. Successful fish infection is confirmed by the presence of the parasite in the host tissues using qPCR or histology.
The laboratory-based infection model enables the same evaluation by infection read outs as the natural exposure approach mentioned above, but also allows a clearly defined sequential analysis in terms of parameters taken at a precise post-exposure time period.
However, an earlier study performed a successful exposure with an experimental intraperitoneal (i.p.) injection with parasitic cells from infected donor fish. 33

Advantages and disadvantages
Both technical approaches result in clinical PKD in fish; the major difference is that fish sampled at the infection source are constantly exposed to other pathogens primary or opportunistic or an additional stressor in the environment (dependant on the source) vs. a once-off exposure in a controlled laboratory environment. This provides a major advantage for the laboratory-based study as it allows to compare with true control noninfected fish under the same conditions, from the same fish stock. Alternatively, it could be also argued that fish exposed in the laboratory are not a true reflection of what happens in nature to fish that are continuously exposed to the parasite. Moreover, in an initial study by

| Pro-inflammatory cytokines and antimicrobial peptides
After an invading pathogen is recognized, PRRs activate signalling cascades leading to induction of pro-inflammatory cytokines, antimicrobial responses and chemotactic cytokines. 36 or downregulation of pro-inflammatory cytokines as part of a global downregulation of innate response genes (summarized in 53 ).
In contrast, several anti-microbial peptides (AMPs), considered as key components of innate immunity (cathelicidins-cath-1, cath-2, hepcidin-1 and liver-enriched antimicrobial peptide-2 (leap-2) were upregulated at all kidney swelling grades during T. bryosalmonae infection. In particular, cath-1 and cath-2 were strongly upregulated. 26 Given that AMPs also have an immunoregulatory function preventing an excessive inflammatory response, they might also contribute to the anti-inflammatory phenotype observed during the course of PKD progression. 36,54,55

| Macrophage activity
Further evidence for the lack of a pro-inflammatory response to T.
bryosalmonae is generated by the downregulation and general unresponsiveness of macrophage marker genes such as macrophage stimulating colony factor (mcsf), its receptor (mcsf-r) and arginase paralogues. 34,36,56 Exemplifying this is a study in which arginase 1 phenotype. 58 However, arginase gene expression has only been investigated from early (grade 1) to advanced (grade 3) stages of disease pathology. Thus, given that M2-like macrophages have a role in tissue repair, an fuller assessment of M2 macrophage marker expression would be needed that would cover advanced to resolving stages of disease pathogenesis. It must also be noted that two trout recombinant il-12 molecules have been characterized, namely p35p40b and p35p40c. Importantly, during PKD pathogenesis only a moderate (ca. 4-to 7-fold) transcriptional increase in the p35 and p40c subunits reported with p40b transcription remaining unaffected. 58 Thus, limited expression, especially of the p35 subunit, may not translate into an increase in il-12 at the protein level.

| Chemokines
In igt + B cells) as well as antigen-presenting cells (APCs) (major histocompatibility complex class II -mhc ii + cells) in rainbow trout blood, spleen and kidney, 69 whereas ck12 has been revealed to recruit igm + B cells within the rainbow trout spleen. 70 Finally, it would also be interesting to determine the functional role of chemokines in PKD pathogenesis, particularly ck11 that is known to have antimicrobial activity to a wide range of pathogens, including parasites. 65 For example, Toxoplasma gondii (socs1) 75 and Leishmania donovani (socs3) 76 induce SOCS expression that subsequently inhibits interferon gamma (ifnγ)-mediated macrophage activation as an immune evasion tactic. In T. bryosalmonae-infected rainbow trout, although there is a lack of macrophage response during PKD pathogenesis, ifnγ expression was only moderately upregulated at all swelling grades in a natural outbreak study in the posterior kidney. 36 Furthermore, ifnγ was downregulated in the anterior kidney at weeks 4, 6 and 7

| SOCS: suppressors of chemokine signalling
post-exposure (PE) correlating with a significant decrease in myeloid cells. 34 Thus, while macrophage activity may be suppressed during PKD pathogenesis, whether this down-regulation is related to ifnγ production requires further investigation.  Given that PKD pathogenesis is a chronic immunopathological disorder which resembles classical aspects of a lymphoproliferative autoimmune disease, a mode of complete Th1-or Th2-like polarization may not take place as seen in other immune responses that are not directly against a pathogen, such as those against autoantigens and environmental antigens. 86 Alternatively, an aspect of parasite virulence could be to prevent a clear Th polarization that enables effective parasite clearance.

| Th17/Treg-associated transcripts
Th17 cells and Treg cells share a common precursor cell and require a common transforming growth factor β (tgfβ) signal for their initial differentiation, although they fulfil almost opposite functions. 87 Many features of mammalian Th17 cell differentiation have been described in fish, for example il-6, tgf-β-1, il -21, il-22, il-23, il-17, and the Th17 master transcription factor Retinoic acid-related orphan receptor-gamma (rorγ). 78 All of these genes have been investigated in rainbow trout during T. bryosalmonae infection. 34,36,88 il-17 family members il-17a/f2a, il-17c-2 and il-17d exhibited weak or no correlation with parasite burden and no correlation with kidney swelling in the posterior kidney. 36 In contrast, il-17c-1 was shown to be significantly upregulated at early clinical stages in several studies. 88

| BAFF/APRIL axis
In mammals, cytokines of the tnf superfamily play a key role in the modulation of B-cell responses. 77 Among them, baff and april produced by cells of the innate immune system are known to modulate the homoeostasis, activation and differentiation of B cells. 90 In fish, an additional baff and april-like molecule (balm) closely related to both april and baff is also present in this family. 29 As B-cell function appears dysregulated during PKD, Granja et al. 38

| Immunoglobulins
Fish have three major Ig isotypes, igm, igd and igt. [91][92][93] igm and igd are equivalent to the mammalian counterparts. Thus, igm is the main Ig produced and is known to be the primary responding Ig at a systemic level. igt, on the other hand, is a fish-specific Ig, expressed by an independent B-cell lineage, that is considered to be specialized in mucosal immunity. 94  bryosalmonae and viral haemorrhagic septicaemia virus (VHSV). In this study, a more pronounced upregulation pro-inflammatory and Th1-like cytokines, as well several antiviral marker genes, were observed in these fish. 28 This was in contrast to single infected fish and in contrast to what has been reported within this review. Given the drastic immunosuppressive effects on myeloid cells and the apparent dysregulation of lymphoid cells that takes place during PKD, these findings suggest that the way in which T. bryosalmonae interacts with other pathogens could be specific to the type of secondary agent, with different consequences in each case.

| Temperature
Elevated water temperatures can increase PKD incidence and severity but can also modulate the rainbow trout immune response during T. bryosalmonae infection. Bailey et al. 34 investigated whether a subtle change in water temperature, reflecting a realistic environmental scenario, could influence the immune response of rainbow trout exposed to T. bryosalmonae. At the lower temperature (12°C), fish exhibited an asymptomatic version of the disease and lower parasite burden. This was associated with a cytotoxic-like immune response characterized by a subtle increase in lymphocytes, expression of Th1-like cytokines and strong upregulation of natural killer enhancement factor (nkef). 34 At the warmer temperature (15°C), T bryosalmonae infection was consistent with the phenotype reported within this review, a chronic immunopathology driven by increasing lymphocytes/decreasing myeloid cells in both the anterior and posterior kidneys. Furthermore, a strong B-cell antibody response and a dysregulated Th1/Th2-like cytokine response were observed. 34 The increase in only 3°C appeared to impact parasite proliferation and the biological and biochemical processes of the host, with the lower temperature being less detrimental to host immunocompetence. 34

| Anthropogenic contaminants
Given the strong interference of human activities on freshwater fish, the impact of anthropogenic factors on T bryosalmonae infection has also been studied and shown to modulate the host response. Earlier reports described the impact of wastewater effluents upon T bryosalmonae infection of salmonids. 102 in T. bryosalmonae-infected fish relative to T. bryosalmonae-infected EE2-exposed fish at the plateau of parasite intensity. 105 As expected, however, hepatic vitellogenin (vtg) mRNA levels, commonly used to assess exposure of fish to oestrogens, were significantly elevated in fish exposed to T. bryosalmonae and EE2 relative to T bryosalmonae only infected fish. In addition, the liver somatic index was increased and levels of insulin-like growth factor (igf) decreased in the same organ, indicating the impact of EE2 in these fish. 105 Bailey et al., 106 as part of a long-term chronic study (130 days), used RNA-Seq to assess the posterior kidney transcriptome in rainbow trout exposed to T bryosalmonae and EE2 and all combinations thereof. This study compared the following four Additionally, in the study by Bailey et al. 106 several markers associated with various aspects of T-cell immunity (t-bet, cd8α, foxp3a, foxp3b, cd3 and mhcii) were significantly downregulated in the multiple stressor group. This might indicate that T. bryosalmonae and EE2 combined could be having a greater immunosuppressing role than in T. bryosalmonae-infected fish in the absence of EE2.
Specifically, reduced PKD-associated immunopathology and attenuated disease impact. Alternatively, since it was observed that a greater number of immune genes were upregulated in these fish, this may be consistent with decreased parasite intensity and reduced pathological alterations. 106 Thus, while EE2 modulates the immune response during infection, the effects appear somewhat paradoxical in that they exert both immunosuppressive and immunoenhancing actions as reported in mammals. 106

| CON CLUD ING REMARK S AND FUTURE AVEN UE S FOR RE S E ARCH
A plethora of studies have contributed to this review of the rainbow trout immune response during PKD pathogenesis. It must also be mentioned that beyond the scope of this review, several reports exist regarding brown trout immune responses. [40][41][42] Several studies have adopted broader techniques such as predictive modelling, 110

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest regarding the publication of this paper.

AUTH O R CO NTR I B UTI O N S
CB and CT wrote the main body of the manuscript, with contributions from JWH and CJS. All authors reviewed and approved the final version.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analysed in this study.